The Regulatory Influence of Activated T Cells on B Cell Responses to Antigen

The Regulatory Influence of Activated T Cells on B Cell Responses to Antigen

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DAVID H KATZ AND BARUJ BENACERRAF Deportmenf of Pofhology. Harvord Medical School. Borfon. Morrochuretts

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I . Introduction I1 Specific Cells of the Immune System I11. Requirement of Two Distinct Lymphoid Cell Types in the Development of Humoral Immune Responses . . . . . . . . . A . Response to Foreign Erythrocyte and Protein Antigens B. “Carrier Effect” and Cooperative Interactions Specific for Different Determinants on the Same Antigen IV. Nature of the Regulatory Influence of Activated T Cells on Antibody Responses by B Cells A . Stimulation of B Cells in the Absence of T-cell Regulation . . B. Effect of T-cell Activity on the Class of Immunoglobulin Synthesized C. Role of T-cell Regulation in the Selective Pressure by Antigen on B Cells . . . . . . . . . . . . . V. Immunological Specificity and Properties of T and B Cells Concerned . . . . . . . . with Cooperation Phenomena . A . Immunological Specificity of T and B Cells B. Antigen Receptors on T and B Cells C. Recognition of Hapten and Carrier Determinants by T and B Cells . D Sensitivity and Resistance of T and B Cell Function to X-Irradiation and Corticosteroids VI. Mechanism of Regulation of B Cell Function by T Cells A . Transfer of Genetic Information B. Antigen Presentation and Concentration . . . . . . C Regulation of B Cell Function in Antibody Production by Mediators Produced and Secreted by T Cells VII . Suppressive Effects of T Cells on Antibody Synthesis . . . . A . Enhancement of Immune Responses by Depletion of T Cells . . B. Suppression of Antibody Responses by the Administration of More . . . . Than One Antigen (Antigenic Competition) . VIII. Functions of T and B Lymphocytes in Various Immunological Phenomena A . Immunological Tolerance . . . . . . . . . B. Immunological Memory . . . . . . . . . . C Immunological Adjuvants . . . . . . . . . D . Cell-Mediated Immunity . . . . . . . . . . IX . Biological and Pathophysiological Significance of the Regulatory In. . . . . . fluence of T Cells on Antibody Production . References

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14 23 23 25 26 28 28 32 33 37 42 42 43 47 62 62 65 67 67 75 79 81 82 85

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DAVID H.KATZ AND BARU J BENACERRAF

I. Introduction

The clonal selection theory of Burnet and his postulate that antigenreactive precursors of antibody-secreting cells bear antibody receptors of unique specificity (1-3) have been largely substantiated in the past decade ( 4 3 ) . Another major advance in immunobiology has been the recognition of two pathways for the differentiation of antigen-reactive cells. It is generally accepted that a class of bone marrow lymphocytes migrates to the thymus where the cells develop the ability to respond to antigen. These thymus-derived lymphocytes, generally referred to as T cells, are responsible for the various phenomena of cell-mediated immunity: delayed sensitivity, homograft, and graft-versus-host reactions. The second lymphocyte cell type arises also in the bone marrow and settles ultimately in distinct anatomical sites in peripheral lymphoid tissues where these cells give rise to the precursors of antibody-secreting cells, B cells (9-11). The most recent discoveries in immunobiology concern the realization that the differentiation of antigen-stimulated specific B cells into antibody-secreting cells depends, for most antigens, on the concomitant activity of specifically stimulated T cells. The original observations established the requirements of specifically activated T cells for the antibody response by B cells to antigen in vivo and in witro in various systems and clarified the relationship between hapten determinants and carrier function originally introduced by Landsteiner ( 1 2 ) . It was later recognized that the effect of stimulated T cells on the response of B cells to antigen is more complex and affects also ( a ) the switch from the production of IgM to IgG antibodies and ( b ) the rate of selection of specific cells by antigen in the immune response as reflected in the change in affinity of humoral antibody with time. It was further shown that the activity of histocompatibility-linked, specific, immune response ( Ir ) genes in T cells is essential for all these phenomena triggered by antigen. More recently, it is becoming apparent also that regulatory effects of activated T cells on antibody responses by B cells may be suppressive under certain conditions, whereas under other conditions, as stated above, they are stimulatory, which may explain the well-known phenomenon of antigenic competition. In fact, what appeared at first as an important and essential cooperation phenomenon between two specific cell types and antigen to trigger effective antibody responses is now more appropriately interpreted as the expression of a fundamental regulatory function of activated T cells on B cell responses. The present review first describes the experimental data on which these statements are based and relates how insight into these fundamen-

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tal and fascinating phenomena was achieved. The topics discussed also include intimate mechanisms of the regulation of antibody responses by T cells, the signifbance of these phenomena for the regulatory processes of the immune system, and their possible implication for the pathogenesis of various immunopathological states. II. Specific Cells of the Immune System

The immunocompetent lymphocytes can be divided into two general types on the basis of functional differences: (1) T cells-small lymphocytes that have adapted to certain specific immune functions by virtue of some as yet undefined influence of the thymus (thymus-derived); and ( 2 ) B cells-small lymphocytes that have not been directly influenced by the thymus and which are the progenitors of mature antibody-producing plasma cells. Experimental evidence weighs heavily in favor of the concept that unipotential cells which populate the various hematopoietic tissues of fully developed individuals are derived from common pluripotential stem cells (for review, see 9). Ontogenically, stem cells originate in the embryonic yolk sac and primitive blood islands, migrating later to hematopoietic colonies in fetal liver and bone marrow. Further migration occurs via the bloodstream to various tissues of the hematopoietic system where further differentiation occurs ( 13, 1 4 ) . Differentiation to unipotential progenitor cells of either lymphoid or myeloid lines is signaled by inductive factors, presumably existing in the microenvironment of the different hematopoietic organs of the individual ( 1 5 ) . We shall limit our considerations here to the lymphoid cell lines. Stem cells differentiate into unipotential progenitor lymphoid cells under the microenvironmental influences of the primary lymphoid organs ( 1 4 ) . Avian lymphoid systems have been shown to consist of two distinct primary lymphoid organs-the bursa of Fabricius and the thymus-the influence of which on the differentiation of the stem cells that have migrated to them is clearly distinguishable on the basis of the functional differences of such cells in the immune system (16-20). Surgical extirpation of the bursa from a newly hatched chick results in depression of serum immunoglobulin levels and marked diminution in the capacity to develop humoral antibody responses to antigen stimulation, but has little effect on the ability to reject tissue allografts (20-22); in contrast, early removal of the thymus diminishes the capacity to develop delayed hypersensitivity and impairs allograft rejection (20-25). In mammals, it is now also clearly established that there exists two distinct lymphoid systems responsible for differentiation of immunocompetent cells. One is clearly thymus-influenced, but the other is not. Hence, neonatal thymec-

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DAVID H. KATZ AND BARUJ BENACERRAF

tomy of rats or mice virtually abrogates their capacity to reject tissue allografts specifically (26-29), and humans with congenital absence of the thymus are markedly impaired in their ability to develop delayed hypersensitivity reactions or manifest allograft immunity ( 30734). Such individuals have normal levels of serum immunoglobulins and can respond to certain antigens with production of humoral antibody. The analog of the bursa of Fabricius in mammals has not been discovered in any discrete lymphoid organ, and it is quite likely that it does not exist as such. In this review, we adhere to popular terminology to refer to the two distinct classes of immunocompetent lymphocytes. (1) The T cells are lymphocytes that have differentiated under the influence of the thymus and are responsible for mediating cellular immune reactions such as delayed hypersensitivity and transplantation reactions. These cells participate in the development of humoral immunity, as will be detailed below, but are not capable of secreting humoral antibodies. Since T cells stimulated by antigen respond, on the one hand, by a clonal expansion and differentiation, and, on the other hand, by being activated to perform their specific function (i.e., helper cells, target cell killers, etc.), we have elected to refer to the former as “educated T cells, and the latter as “activated T cells. As will be discussed in this review, activated T cells may result from stimulation by agents other than specific antigen, ( 2 ) The B cells, are lymphocytes that have differentiated under the influence of the bursa or its analog in mammals and ultimately become the effector cells in humoral immunity by virtue of synthesizing and secreting immunoglobulin antibodies. Ill. Requirement of Two Distinct Lymphoid Cell Types in the Development of Humoral Immune Responses

A. RESPONSETO FOREIGN ERYTHROCYTE AND PROTEINANTIGENS

1. In Vivo The compartmentalization of immunocompetent lymphocytes into T and B classes was recognized as an efficient product of evolution in the development of more sophisticated and complex immune systems in higher animals. In recent years, however, it has been realized and now firmly established that these distinct lymphoid cell lines not only perform different roles in the generation of different forms of immunity but also interact with one another in the development of certain immune responses, notably the humoral immune response to various antigens.

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In retrospect, the requirement of T and B cell interaction in eliciting antibody responses is suggested by results obtained from studies in neonatally thymectomized mice 10 years ago (26, 35-37). These studies demonstrated that neonatal thymectomy in mice prevented the normal development of immune responsiveness. The effect was particularly marked with respect to cellular immunity exemplified by the homograft reaction. It was also noted, however, that the ability of such mice to develop antibody responses to certain antigens, such as sheep erythrocytes or foreign serum proteins, was diminished or absent (26, 3 5 ) , but the response to some bacterial and viral antigens was normal. Moreover, the defect of neonatally thymectomized animals could be partially restored to normal by implantation of a thymus graft (26, 35, 3 8 4 0 ) . Furthermore, it was found that lethally irradiated C57B1 mice which were reconstituted with syngeneic bone marrow cells recovered full inimunoconipetence only if the thymus was present (41 ) . The initial understanding of thymic function in the immune system was based on the findings of Osoba and Miller ( 4 2 ) that the immunological competence of neonatally thymectomized mice could be partially restored by implantation of thymus grafts enclosed in Millipore chambers. This observation suggested that the thymus can function as an endocrine organ liberating a humoral factor which can potentiate antibody responses. Arguments against this interpretation have been based on the fact that the level of immunological restoration by chamberenclosed thymus grafts was less than that obtained with nonencapsulated grafts. Moreover, full restoration of peripheral blood lymphocyte counts was not achieved with chamber-enclosed thymus grafts. However, it does not seem likely that enclosing a thymus graft in such chambers would create a favorable milieu for full functional expression. Interest in thymic function soon shifted from considerations of endocrine-like activity to more careful scrutiny of the behavior of cells derived from this organ. Using an irradiation-induced chromosome abnormality as a cytological marker ( T6T6in CBA/H mice), it was found that in neonatally (35, 43) or adult ( 4 0 ) thymectomized mice reconstituted with T6-marked thymus grafts, a small number of the T, thymus cells underwent mitosis in the host spleen. Although the significance of these dividing cells was not at first appreciated ( 4 3 ) ,it was subsequently demonstrated that they were sensitive to antigen-induced mitosis following intraperitoneal administration of sheep erythrocytes ( 4447). Thus, Davies et al. ( 4 5 ) used T6T, thymus grafts to reconstitute thymectomized syngeneic radiation chimeras and demonstrated a well-defined mitotic pattern of response of the thymus cells to either sheep red blood cells (SRBC) or skin homografts.

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DAVID H. KATZ AND BARUJ BENACERRAF

Another interesting observation which, retrospectively, illustrates the requirement of T and B cell interaction was described by Kennedy et aZ. ( 48). Their experiments were designed to enumerate cells in normal mouse spleen which were specifically committed to recognize and respond to sheep erythrocyte antigens. Lethally irradiated mice were injected intravenously with normal mouse lymphoid cells and SRBC. Eight days later, thin serial slices of recipients’ spleen were cut and embedded in agar. Incubation of the spleen slices with SRBC in the presence of complement at 37°C permitted the development of hemolytic foci or discrete clusters of plaque-forming cells (PFC), the number of which were linearly related to the number of cells in the orginal donor inoculum. The latter point suggested that each cluster of PFC reflected the activity of a single SRBC-sensitive cell in the original inoculum which had lodged in the recipient spleen and proliferated and differentiated in response to antigenic stimulation. The development of such hemolytic foci occurred regularly when normal mouse spleen or lymph node cells were used as the original inoculum, but neither thymus cells alone nor bone marrow cells alone were capable of initiating hemolytic foci in recipient spleens. Several other observations indirectly suggested the requirement of cell interactions in antibody responses. Thus, Celada (49) examined the relation between the transfer secondary response to human serum albumin (HSA) and the number of syngeneic spleen cells transferred and found that over a restricted range of cell numbers the response increased disproportionately to the increase in cell dose. He referred to this phenomenon as the “premium effect.” Later, Gregory and Lajtha (50) compared the numbers of hemolytic foci and PFC developing in spleens of irradiated mice injected with syngeneic spleen cells and SRBC. They found that, although the number of foci was linearly related to number of spleen cells transferred (slope of l), the number of PFC increased disproportionately (slope of 2 ) . Such results can be explained if the development of a hemolytic focus depends upon the presence of only one cell type, whereas the development of antibody-secreting PFC requires an interaction between two cells neither of which is present in overwhelming excess. Bussard and Lurie ( 5 1 ) observed a similar effect in in vitro cultures of peritoneal cells in presence of SRBC. The first direct evidence for T and B cell interaction was provided by Claman and co-workers in studies of the humoral response to SRBC in mice (52). These investigations were based on a very simple design to test for the existence of potentially immunocompetent cells in the adult mouse thymus using the hemolytic focus assay system (48,53). Thus, lethally irradiated mice ( 650-750 R) were injected intravenously with varying numbers of either spleen, thymus, bone marrow, or thymus

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plus bone marrow cells from normal or immune syngeneic donors. The cell transfer was followed by antigenic challenge with SRBC. Spleens of recipients were assayed for hemolytic focus activity at various times after cell transfer. The results were strikingly clear in showing the dissociation of immune responsiveness among the various cell populations. Thus, neither normal marrow nor normal or SRBC-immune thymus contained cells which alone could give a hemolysin response. However, when thymus and marrow cells were combined in the same recipient a marked synergy, which was linearly related to number of thymus and marrow cells in the mixture, was observed ( 5 2 ) . The authors postulated from these results that the marrow population contained “effector cells’’ capable of producing antibody, but only in the presence of “auxiliary cells” present in the thymus population. Support for this interpretation was forthcoming from subsequent studies carried out in two independent laboratories. Davies et al. ( 5 4 ) transferred spleen cells from donors, which had been immunized with SRBC 1 month after irradiation and thymus-grafting, to irradiated recipients which had been presensitized against either the thymus-derived cells or the marrow-derived cells. If the donor marrow cells were rejected, the secondary response to SRBC was abolished, whereas rejection of the thymus cells only diminished the response, suggesting that antibody production was by marrow cells and not by thymus cells, although the latter cells made a most vigorous mitotic response to antigen. Moreover, the highest antibody response occurred when both cell populations were allowed to react to antigen. Miller and Mitchell ( 5 5 ) reported that bone marrow of neonatally thymectomized mice was as effective as that of normal mice in restoring immunological responsiveness to heavily irradiated mice with intact thymuses. Mitchell and Miller ( 5 6 ) further showed that in neonatally thymectomized mice given allogeneic thoracic duct or thymus lymphocytes to restore the response to SRBC, the PFC could be inhibited with anti-H-2 sera against host cell antigens but not with anti-H-2 sera against donor thoracic duct or thymus cells. Moreover, in an adoptive transfer system in irradiated hosts in which thymus cells were temporarily separated from bone marrow cells, it was shown that the thymus lymphocytes had to react first with the specific antigen before interaction with bone marrow cells could produce a significant anti-SRBC response ( 5 6 ) . These observations established that the antibody-forming cell precursor is marrow-derived and that thymus cells recognize and react specifically with antigen. In 1968, a series of elegant experiments were published by Miller, Mitchell, and associates from the Walter and Eliza Hall Institute (57-59). Their results provided the first clear elucidation of the occurrence of

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DAVID H. KATZ AND BARUJ BENACERRAF

specific cell interactions in the humoral immune response to sheep erythrocytes in mice and deserve, therefore, detailed analysis. The first series of experiments (57) examined the capacity of various cell types to restore the yM hemolysin response of neonatally thymectomized CBA mice and these results are summarized in Table I. In the absence of any treatment, such mice gave a very meager response to SRBC, as measured by the number of PFC in their spleens (2300) compared to that of sham-operated littermates (32,000 PFC/spleen). Transfer of 10 million viable syngeneic thymus or thoracic duct cells increased the PFC response of neonatally thymectomized mice to 20,000, and transfer of 50 million viable thymus cells restored their response to normal ( >32,000 PFC). Furthermore, 10 million viable semiallogeneic ( CBA X C57B1)F, thoracic duct or thymus cells or allogeneic C57B1 thoracic duct cells were equivalent to syngeneic cells in restorative capacity. Allogeneic thymus cells could restore but were slightly less effective. In contrast, these authors observed no restoration following transfer of syngeneic bone marrow cells, thymus, or thoracic duct cells which had been exposed to 1000 R in vitro X-irradiation or to thymus extracts. Thoracic duct cells obtained from syngeneic donors made tolerant to SRBC were markedly reduced in their capacity to restore immunocompetence in neonatally thymectomized mice. The most important and crucial denionstration was that the hemolysin response of neonatally thymectomized mice given thymus or thoracic duct cells reflected antibody production by cells of the host. This was accomplished by Miller and Mitchell by employing anti-H-2 isoantisera (57) and confirmed by Nossal et al. (59) using the TBchromosome marker. Thus, in neonatally thymectomized CBA recipients of semiallogeneic F, or allogeneic C57B1 thymus or thoracic duct cells plus SRBC, the anti-SRBC PFC could be specifically reduced by more than 90%following treatment of their spleen cells with anti-CBA serum in the presence of complement but not by treatment with anti-C57B1 serum. Similarly, by the use of neonatally thymectomized CBA mice possessing the T, chromosome marker as hosts for syngeneic thymus or thoracic duct cells, it was possible to demonstrate that all of the PFC in mitosis were of host rather than donor origin (59). In the second series of experiments ( 5 8 ) , a study was made of the capacity of various cell types to restore the yM hemolysin response to SRBC of irradiated or thymectomized and irradiated adult mice. In lethally irradiated mice, a synergistic effect in the response to SRBC was obtained when both syngeneic thoracic duct and.bone marrow cells were used for reconstitutions. In adult thymectomized, lethally irradiated mice protected with syngeneic bone marrow, a similar, although less

TABLE I DIRECTPLAQUE-FORMING CELLSPRODUCED IN SPLEENSOF NEONATALLY THYMECTOMIZED MICE AFTER INJECTION OF SHEEPERYTHROCYTES AND THYMUS OR THORACIC DUCTLYMPHOCYTES FROM SYNGENEIC, SEMIALLOGENEIC OR ALLOGENEIC DONOR SO^^ Inhibition of P F C by anti-H-2 serum (yo)

Avg. No. P F C per spleen ( fSE) Anti-C6,Bl

Cells inoculated A. Sham-thymectomized recipients None SRBC B. Thymectomized recipients SRBC SRBC’ 50 x 106 CBA thymocytes 10 x 106 CBA thymocytes SRBC 10 x 106 CBA TDL SRBC 10 x 108 F1 thymocytes SRBC 10 X 106 F1 T D L SRBC 50 X 106 C57B1 thymocytes SRBC 10 X 108 C57B1 TDL 50 x 106 X-irradiated CBA SRBC thymocytes 10 x 106 X-irradiated CBA TDL SRBC 10 x 106 X-irradiated F1 TDL SRBC Thymus extract SRBC 10 x 106 T D L from control cyclophosphamide-treated CBA donors SRBC 10 X 106 TDL from SRBC-tolerant CBA donors (treated with SRBC and cyclophosphamide)

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+

+ +

+

+

+

+

+

+

+

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Anti-CBA

123 f 29 32,177 f 3550 2,356 f 537 38,855 f 7448 19,160 f 3840 20,254 f 3646 22,380 f 6285 24,537 f 5519 17,448 f 3907 30,120 f 7791 1,160 f 615

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-

6 0-17 8-12

97 89-100 90-92

0-1

86-96

-

-

3,776 f 979 4,344 f 2540 1,802 f 1155 41,600 f 13,436 7,331 f 2276

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We thank Dr. J. F. A. P. Miller for permission to reproduce some pertinent data from Miller and Mitchell (67). b This table shows the antibody response to sheep erythrocytes (SRBC) of neonatally thymectomized CBA mice reconstituted intravenously with varying numbers of syngeneic, semiallogeneic (CBA X C57Bl)E’I, or allogeneic C57Bl thymus or thoracic duct lymphocytes (TDL). Results obtained in sham-thymectomized controls are included for comparison. The data are expressed as the average number of plaque-forming cells (PFC) per spleen detected on days 4-5 after inoculation with donor cells and SRBC. Recipient groups ranged from 4 to 36 mice each. The data obtained in studies of the source of P F C by incubation of spleen cells with anti-H-2 serum plus complement are included in the far right columns expressed as percent inhibition. X-Irradiated donor cells were exposed to 1000 R in vitro immediately prior to transfer. Thymus extract equivalent to 100 x lo6 thymus cells per recipient WBS injected intravenously with SRBC. Mice were rendered tolerant to SRBC by treatment with cyclophosphamide and SRBC and used BS donors of TDL 23-26 days after completion of tolerance induction. a

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DAVID H. KATZ AND BARUJ BENACERRAF

marked, synergism was observed in the response to SRBC following transfer of F, thoracic duct cells provided the latter were not administered until 2 weeks after the syngeneic bone marrow cells. Employing anti-H-2 sera (58) or the T, chromosome marker ( 5 9 ) , it was firmly established that the source of the PFC in this system was the bone marrow lymphocyte population. Such studies made it clear, therefore, that the unicellular concept of antigen recognition and antibody formation did not reflect the true state of affairs, at least with respect to the sheep erythrocyte antigen. Thus, at least two functionally distinct but morphologically indistinguishable lymphoid cells apparently must engage in some interplay to effect in wivo antibody production. One cell, derived from the thymus and also present in thoracic duct lymph, thereby placing it in the class of lymphocytes comprising the recirculating lymphocyte pool ( 60), is stimulated by antigen to undergo mitosis (44-47) but is incapable of secreting antibodies (48, 52, 54, 5 6 5 9 ) ; the other cell, derived from bone marrow, is the precursor of the antibody-secreting cell but requires an interaction of some sort with the thymus-derived cell before it can perform its function (9-11,52, 5 7 5 9 ) . The development of these new concepts concerning the immune system not only stimulated a reappraisal of some earlier phenomena of immunology and their explanations, but also resulted in a virtual avalanche of experiments designed to probe the nature of cell interactions and the cell types involved, and their significance in normal and abnormal immunc reactions. Indeed, since the time of the initial studies demonstrating the requirement for cell interactions in the sheep erythrocyte system, analogous interactions have been demonstrated in the in vivo humoral response to protein antigens (61, 62) and to haptencarrier conjugates (63-74), which will be reviewed in detail in Section II1,B. Other studies illustrated some of the properties of the interacting T and B cells. First, Claman and co-workers (75, 76) showed that, in their single transfer system in lethally irradiated recipient mice, only viable syngeneic thymus cells could interact synergistically with marrow cells in the response to SRBC. Hence, no cooperative interaction was obtained with sonicated or minced thymus cells. Second, irradiated thymus cells or thymus cells from xenogeneic or semiallogeneic donors did not collaborate with marrow cells. Third, the T cells had specificity; using a double transfer system similar to that used earlier by Mitchell and Miller (56), Claman and Chaperon ( 1 1 ) attempted to dissect further their model of synergism. The experimental design consisted of transferring thymus cells with or without SRBC to a lethally irradiated first host and after 6 to 8 days transferring the spleen cells of this first

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host to a second irradiated syngeneic recipient together with syngeneic marrow cells. Six days later the spleen of the second recipient were assayed for y M anti-SRBC PFC. They found that thymus-marrow synergy in the second host occurred only if the original thymus cell inoculum was exposed to SRBC antigen in the first host for more than 2 days, confirming the earlier finding of Mitchell and Miller (56). Moreover, exposure of the transferred thymus cells to lOOOR X-irradiation in situ, immediately prior to transfer to the second host, abrogated their capacity to cooperate synergistically with marrow cells, thus confirming earlier observations from their laboratory (77) in their single transfer system. Shortly after the initial studies with erythrocyte antigens were reported, analogous observations were made in responses of mice to protein antigens. Thus, Taylor ( 6 1 ) and Chiller et d. (62) demonstrated synergism between thymus and bone marrow lymphocytes in antibody responses to bovine serum albumin (BSA) and human y-globulin (HGG), respectively. Similar findings have been reported by Miller and Sprent (78) in the response of mice to fowl 7-globulin ( FyG). A crucial question naturally is raised in the light of two-cell models for hunioral immunity, namely, What relevance do these required interactions have in the context of Burnet’s dogma of clonal selection? Now we are faced with the problem of not just one cell type precommitted in its antigen specificity, but at least two. In this sense, a serious threat to any theory of clonal selection is readily seen when some thought is given to the probability of two functionally distinct cells with the same specificity characteristics meeting at random to carry out a necessary interaction. The cells involved are functionally distinct and may be distinct in their determinant specificities as well; this latter point is exemplified by models of cooperative cell interactions in hapten-carrier systems to be discussed. The distinction in determinant specificities, we feel, probably exists in the erythrocyte antigen system also but the very nature of the antigen makes this difficult to appreciate. There is, nevertheless, a body of evidence in the SRBC system which supports clonal selection in a two-cell model. Shearer et al. (79, 80) transferred unprimed spleen cells as precursors of immunocompetent cells with SRBC to irradiated syngeneic recipients and measured the development of three types of antibody-producing cells-IgM and IgG hemolysin ( PFC ) , and hemagglutinin-producing cluster-forming cells ( CFC )-in the recipients’ spleens. By employing graded numbers of transferred donor spleen cells, they could limit to one or a few the number of precursor cells or antigen-sensitive units ( ASU) reaching the recipient spleen.

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By applying this model to thymus-marrow cell interactions, Shearer and Cudkowicz (81) demonstrated that when graded numbers of bone marrow cells ( spleen cells of bone marrow-reconstituted X-irradiated mice ) were transferred to an irradiated recipient with constant numbers of thymus cells (from intact donors) plus SRBC, the production of IgM or IgG PFC or CFC (agglutinins) varied independently of each other but in relation to the number of grafted marrow cells. This suggests that there is specialization with respect to class and type of antibody produced within the precursors in the marrow population prior to antigenic stimulation. The question of whether thymus cells restrict the potential antibody class of ASUs was investigated by transferring thymocytes in limiting dilutions with constant numbers of marrow cells (plus SRBC) (82). Under these conditions, the frequency of formation of IgM and IgG PFC were not independent, thus indicating that the thymic-reactive cells were not themselves specialized nor did they determine directly the molecular class of antibody produced after interaction with marrow cells. This is consistent with our own findings (66) and of Mitchison et al. (65) in the hapten-carrier models. 2. In Vitro The development of methodology by Mishell and Dutton (83, 8 4 ) and by Marbrook (85) to obtain in vitro immune responses has offered another highly useful approach to the study of specific cell interactions in responses to erythrocyte antigens. Mosier and Coppleson ( 86) separated mouse spleen cells into nonadherent and adherent ( macrophage ) populations and cultured serial dilutions of one population in the presence of an excess of the other. The order of cell interactions required to produce the primary anti-SRBC response was predicted from regression line slopes derived by plotting the log of the limiting cell dose against the log of the antibody-forming cell response. They suggested from such data that one adherent and two nonadherent cells must interact for the immune response to develop and that the likely candidates for the two interacting nonadherent cells are T and B cells. The latter point was confirmed in subsequent studies using spleen cells from thymectomized mice ( 87). Hence, spleen cells from adult thymectomized, lethally irradiated mice protected with syngeneic bone marrow gave no primary in vitro response to SRBC. However, when such mice were reconstituted with a thymus graft, the in vitro response was normal. The adherent cells of spleen of thymus-deprived mice were comparable to those of thymusreconstituted mice in supporting the in vitro response of nonadherent spleen cells from normal mice. However, nonadherent spleen cells of thymus-deprived mice, were unable to respond in the presence of adherent normal spleen cells, whereas nonadherent cells from thymus-

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reconstituted mice could respond. These observations clearly placed the cellular deficit in thymectomized mice in the nonadherent populations of lymphocytes but did not firmly establish the cell type involved. Working independently at the same time, Munro and Hunter (88) restored to normal the in vitro primary response to SRBC of spleen cells from adult thymectomized, irradiated, bone marrow-protected CBA mice by the addition of small numbers of normal BALB/C spleen cells. Using anti-H-2 sera, they showed that it was, indeed, the CBA cells, not the BALB/ C cells, which produced the anti-SRBC antibody. Irradiation of the BALB/C donor with 1000 R immediately before sacrifice diminished, but did not abrogate, the capacity of such cells to restore the response of the thymus-deprived CBA cells. It was not clear, however, whether or not this reflected radiation resistance in the macrophage population which was a documented property of such cells (89). Hirst and Dutton (90) restored the primary in vitro anti-SRBC response of spleen cells from neonatally thymectomized mice with subpopulations of spleen cells from normal mice. They showed that small numbers of nonadherent normal spleen cells irradiated with 1100 R in vitro restored or enhanced the in vitro response and that this enhancement was most marked when the nonadherent cells came from allogeneic donors. Furthermore, the primary in vitro response of spleen cells from normal mice could be enhanced by the addition of small numbers of irradiated allogeneic nonadherent cells. This observation extended to the SRBC system the finding of Katz et al. (68) that the “helper” activity of the T cell in immune responses to hapten-carrier conjugates is radiation-resistant in contrast to the earlier conclusions of Clainan and co-workers ( 1 1 , 77) and Miller and Mitchell (9, 57). The studies cited above clearly infer the required role of the T cell in the primary in vitro anti-SRBC response. More direct evidence, however, comes from studies in which thymus cells themselves are employed to restore the response or when specific elimination of such cells by treating a normal spleen cell population with anti4 serum and complement abrogates the response. Despite the many investigations, the first approach has not been as easy to accomplish as one might expect (88, 90-94). Indeed, there is only one report by Doria et al. (95) in which spleen cells from neonatally or adult thymectomized, irradiated bone marrow chimeras could be restored by the addition of normal syngeneic thymus cells to respond to SRBC in vitro. Recently, Schimpl and Wecker ( 96) have reported that thymocytes from cortisone-treated syngeneic donors could restore the primary in uitro anti-SRBC response of anti-6’ serum-treated mouse spleen cells. In general, however, thymus cells must first be injected into irradiated recipients with SRBC and then recovered from the spleens of such hosts before they will optimally re-

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DAVID H. KATZ AND BARUJ BENACERRAF

store the in vitro response (91, 92). This is exemplified by Hartmann (91 ), who studied the in vitro response of bone marrow-derived cells, obtained from spleens of thymectomized, irradiated recipients %4 weeks after injection of syngeneic bone marrow. Such cells, which alone were unable to develop an in vitro primary response could be restored to respond by addition of thymus-derived cells obtained from spleens of irradiated recipients 8 days after transfer of thymocytes. The T cells had to be “educated,” (that is, the SRBC antigen had to be given to the irradiated host) in order to cooperate with the B cells in culture. Uneducated T cells did not support the response, a finding which corroborates the observations of Mitchell and Miller ( 5 6 ) and Claman and Chaperon (11) in their in vivo systems. When synergy was obtained with allogeneic mixtures of B cells and educated T cells, use of anti-H-2 sera showed that the PFC were derived from the B cell population. Moreover, the synergy was dependent on specific T cell education, i.e., SRBC-educated T cells cooperated with B cells in response to SRBC but not to horse red blood cells (HRBC) used as the in vitro immunogen. The second approach, employing the effects of treatment with antiserum and complement on the capacity of spleen cells to respond in uitro, has been studied by several investigators. Schimpl and Wecker (93) demonstrated that treatment of spleen cells from normal mice with anti-9 antibodies in the presence of complement regularly reduced the primary in vitro anti-SRBC response of such cells by 80%. Chan et aZ. (92) found that treatment of spleen cells from normal or SRBC-primed mice with anti-9 serum plus complement abrogated the primary yM and secondary yG in uitro responses, respectively, These responses could be restored by adding, to anti-&treated cells, educated T cells from spleen of irradiated thymus-infused syngeneic or semisyngeneic donors. In the latter case, the precursors of PFC were shown by anti-H-2 antisera to derive from the anti-&treated cell population. The educated T cells were likewise shown to be sensitive to anti4 antibody and complement. The major points to come out of such studies on the in vitro antiSRBC immune response can be summarized, therefore, as follows: (1) the requirement for cell interactions between T and B cells is mirrored in both in vivo and in vitro systems; and ( 2 ) the T cell component appears to become optimally functional after it has peripheralized to secondary lymphoid organs and undergone specific antigenic stimulation.

EFFECT” AND COOPERATIVE B. “CARRIER INTERACTIONS SPECIFICFOR DIFFERENT DETEFLMINANTS ON THE SAMEANTIGEN The introduction of defined haptenic determinants onto immunogenic carriers by Landsteiner ( 1 2 ) has provided a powerful tool for the analysis

REGULATORY INFLUENCE OF ACTIVATED T CELLS

15

of specific interactions between antigens and specific cells of the immune system. Considerable evidence has been obtained that cellular immune reactions to hapten-protein conjugates [delayed sensitivity (97-101 ), stimulation of deoxyribonucleic acid (DNA) synthesis by antigen (102, 103), and hapten-specific secondary responses (104106)ldisplay a significant although variable degree of carrier specificity. Such carrier specificity of hapten-specific cellular reactions was interpreted to reflect the partial specificity of the antigen-binding receptors of specific cells for the carrier molecule (107-111), exceeding the real but modest contribution in energetic terms of the carrier to the specificity of antihapten humoral antibodies (111-113). This interpretation of carrier function, however, is not able to explain several essential characteristics of hapten-specific humoral immune responses: 1. Hapten conjugates of immunogenic molecules are required to elicit strong antihapten antibody responses; nonimmunogenic substances serve only poorly, or not at all, as carriers for haptens (114116). 2. Optimal hapten-specific secondary responses require challenge with the hapten-carrier conjugate used for primary immunization (104). 3. Induction of immunological unresponsiveness to the carrier molecule results in partial or total suppression of the responses to haptens on the tolerated proteins (117-122). Assuming that the specificity of serum antibody accurately expresses the specificity of the antigen-binding receptor molecules on the precursors of antibody-forming cells, then these findings suggest the operation of an additional recognition mechanism for the carrier molecule, This interpretation finds validity in recent demonstrations that cooperative interactions between carrier-specific and hapten-specific lymphoid cells are essential for the development of antihapten immune responses. Two basic in vivo experimental models have been employed to establish this latter point: (1) the adoptive secondary antihapten response following transfer of hapten-primed and carrier-primed cells into irradiated recipient mice; and ( 2 ) the use of preimmunization or supplemental immunization with free carrier to enhance primary and secondary antihapten antibody responses in guinea pigs and rabbits. The phenomenon has also recently been investigated in in vitro systems as will be discussed.

1. In Vim Models of Cooperative Interactions in Responses to Hapten-Carrier Conjugates Even before direct evidence for cooperative interactions in immune responses to hapten carriers was obtained, there were several observa-

16

DAVID H. KATZ AND BARUJ BENACERRAF

tions which clearly, in retrospect, hinted that this be the case. These observations derived from genetic differences among certain animals in their capacity to develop immune responses to distinct antigenic determinants. Thus, one strain of inbred guinea pigs, strain 2, is genetically capable of responding to poly-L-lysine (PLL), whereas strain 13 is not ( 123). These latter “nonresponders’’ also fail to make an anti-2,4-dinitrophenyl (DNP) humoral response to D’NP-PLL under normal circumstances, However, when DNP-PLL is electrostatically complexed to methylated BSA, the nonresponder animals develop hapten-specific antibody responses, provided they have not previously been made tolerant to BSA. The second example is the response of rabbits to the tetrameric isoenzymes of lactic dehydrogenase ( LDH) (120). Some rabbits respond perfectly well to both Type I and Type V isoenzymes, whereas others respond poorly to Type I. If a hybrid molecule consisting of both Type I and V subunits is used to immunize the poor responder rabbits, they develop antibodies specific for both subunit types, suggesting that one subunit has served as a carrier for the other to which they are normally unresponsive. Both of these genetic models clearly indicated that recognition of both hapten and carrier determinants must occur before an antihapten response will develop. A somewhat analogous interpretation can explain the observation of Schierman and McBride (119) in which chickens developed enhanced primary antibody responses to weak erythrocyte antigens when highly immunogenic isoantigens were also present on the same erythrocyte. It was Mitchison ( 6 3 ) , however, who, several years ago, obtained the first direct evidence for cooperative participation of two cells with distinct determinant specificities in the humoral response to haptencarrier conjugates. By employing an adoptive transfer system in irradiated recipient mice, he made the following observations. Spleen cells from syngeneic donor mice, which had been immunized with 4-hydroxy5-iodo-3-nitrophenacetyl ( NIP )-ovalbumin ( OVA ) , injected into irradiated recipients made a secondary anti-NIP response following challenge with the homologous conjugate, NIP-OVA, but, as expected in the light of the earlier findings of Ovary and Benacerraf (104), not to a heterologous conjugate, NIP-BSA. However, when spleen cells from donors immunized with NIP-OVA were injected together with spleen cells from donors immunized with BSA, a perfectly good secondary response was made to the heterologous conjugate NIP-BSA. Hence, addition of cells specific for the heterologous carrier, BSA, permits the haptenprimed cells to make a secondary anti-NIP response to NIP-BSA. This basic in vivo observation has been confirmed by many investigators (65, 69, 70, 74, 124-127). This experiment demonstrates that in antihapten antibody responses

17

RECULATORY INFLUENCE OF ACTIVATED T CELLS

an interaction of carrier-specific cells with the hapten-carrier conjugate is required for maximal stimulation of the precursors of antihapten antibody-producing cells. The analogy to thymus-marrow cells cooperation in the response to sheep erythrocytes in mice is obvious and is now firmly established. Raff ( 1 2 6 ) showed that the carrier-specific cooperating cells, or “helper” cells are, indeed, thymus-derived, whereas the antihapten antibody-forming cell precursors are not. The results of this experiment are summarized in Table 11. By using the adoptive transfer system in mice, he found that treatment of spleen cells from donors primed to the second carrier (BSA) with anti-6’ antiserum and complement abrogated the capacity of such cells to cooperate with haptenprimed [NIP-chicken y-globulin (CGG)] spleen cells in the adoptive secondary response to NIP-BSA. Treatment of the NIP-CGG-primed spleen cells with anti-0 antibody, however, did not affect the capacity TABLE I1 EFFECT OF ANTI-BSERAO N CARRIER-PHIMED (BSA)

A N D HAPTEN-PRIMED SECONDARY (NIP-CGG) SPLEENCELLSIN COOPERATIVE ANTIHAPTEN RESPONSETO NIP-BSAasb

Boost NIPwith CGGBSANIPprimed (In vitro primed (Invitro BSA cells treatment) cells treatment) (100 pg.)

++ 0 + + +

+

+

+0 + +

0

0 0 0 0

+

+

0

+

Anti-8 GPC NMS+ GPC

+

0 0 0 0 Anti-8 GPC NMS+ GPC

+

0

+

0 ~~

+0 + ++

+ + +

Anti-NIP response on day 10 Exp. 1

Exp. 2

*

-0.91 f 0.20 -0.78 0.32 0.45 f 0.25 0.27 f 0.48 -0.77 f 0.33 Not done Notdone 1.0 f 0.15 -0.27 f 0.54 0.7 f 0.29

* 0.25

1.05 f 0.42

1.79

0.99 f 0.43

1.78 f 0.18

1.08 f 0.16

1.92 f 0.12

~~~~

~

We thank Dr. Raff for permission to use these data from Ref. (126).BSA, bovine serum albumin; NIP, 4-hydroxy-5-iodo-3-nitrophenacetyl; CGG, chicken 7-globulin. * The protocol of these experiments consists of adoptive intraperitoneal transfer of spleen cells from mice primed with NIP-CGG either alone or mixed with spleen cells from mice primed with BSA in irradiated syngeneic recipients. The respective cell populations were exposed prior to transfer to in uitro treatment with either: ( 1 ) nothing, ( 2 ) anti4 serum plus guinea pig complement (GPC), or ( 3 )normal mouse serum (NMS) plus GPC. Secondary boost with 100 pg. of NIP-BSA was administered intraperitoneally 1 day after cell transfer, and recipients were bled 10 days later. The anti-NIP antibody response is expressed as the loglo molar binding capacity ( x 10-8 M ) .

18

DAVID H. KATZ AND BARUJ BENACERRAF

of such cells to produce anti-NIP antibodies when transferred together with BSA-primed spleen cells and challenged with NIP-BSA ( 126). The same cooperation phenomenon between carrier-specific and hapten-specific cells has been demonstrated in guinea pigs and rabbits by immunization with free carrier. Rajewsky et al. ( 6 4 ) showed that rabbits immunized with a p-azobenzenesulfonic acid ( sulfanil) derivative of BSA made significant secondary antisulfanil antibody responses to sulfanil-HGG if they had received a supplemental intervening immunization with the free unconjugated carrier HGG. These observations have been confirmed and extended in our own laboratory (66, 67) in both rabbits and inbred guinea pigs. Guinea pigs or rabbits which have been primed with DNP-OVA fail to respond to a secondary immunization with a heterologous conjugate, DNP-BGG. However, if DNP-OVAprimed animals receive an intervening supplemental immunization with unconjugated BGG they not only develop secondary anti-DNP antibody responses to DNP-BGG, but the magnitude of such responses may be significantly greater than those elicited by secondary challenge with the original immunizing conjugate DNP-OVA (Fig. 1). Moreover, this phenomenon is not restricted to secondary responses (66). Under appropriate conditions of dose and timing, guinea pigs or rabbits that have been preimmunized with free carrier BGG manifest enhanced primary anti-DNP antibody responses following primary immunization with DNP-BGG. The kinetics as well as the magnitude of anti-DNP antibody production are sharply augmented under such conditions. Additional examples of augmentation of a response to a given antigenic determinant as a result of a concomitant, or prior, immune response to another determinant on the same antigen have been described (97,119,124,128,129). The failure of several other investigators (130-133) to obtain evidence of enhanced primary antihapten responses as a consequence of carrier preimmunization most likely reflects the conditions employed for immunization. Such studies clearly establish the operation of distinct recognition units for carrier and haptenic determinants. The carrier recognition unit is clearly not classic serum antibody. Thus, the capacity to enhance primary or secondary anti-DNP antibody responses in guinea pigs and rabbits by carrier preimmunization or supplemental immunization could not be supplanted by the intravenous infusion of small or large quantities of homologous anticarrier serum either of low or high affinity (66). Similar findings have been made in the adoptive transfer system of Mitchison in mice (65, 7 0 ) and in the supplemental carrier immunization model of Rajewsky in rabbits (65, 134). Moreover, such observations are in accord with the findings of Mitchison (70) and Kontiainen (124)

REGULATORY INFLUENCE OF ACTIVATED T CELLS

STRAIN 2 GUINEA PIGS

700 -

a

19

600 -

W

E

n

z a

2 400 z

0

a I-

W

5

8 0

4200 3

a

DNP-OVA BGG 5 0 p g

+ E

* >

DNP-BGG

\

m

0

0

I

1

50F-0:r O

I

I

I

,

I

,

DNP-OVA ;~

0 1 2 3 4 WEEKS AFTER PRIMARY

/ "'

0 4 7 DAYS AFTER 2* CHALLENGE

FIG. 1. Enhancement of hapten-specific anamnestic responses by carrier preimmunization in guinea pigs. Primary immunization with 3.0 mg of 2,4-dinitrophenyl ( DNP ) ?-ovalbumin ( OVA), administered intraperitoneally in saline, was performed at week 0. One week later supplemental immunization with either 50 pg. of bovine y-globulin (BGG) emulsified in CFA or with a saline-CFA emulsion was carried out. Four weeks after primary immunization, the animals were challenged with 1.0 mg. of either DNPZS-BGG or DNPrOVA in saline. Serum anti-DNP antibody concentration just prior to challenge and on days 4 and 7 are illustrated. The numbers in parentheses refer to the numbers of animals in the given groups. The lowermost panel illustrates the normal secondary response of DNP-OVA-primed animals to DNP-OVA challenge; the middle panel shows the absence of a secondary response to DNP-BGG in DNP-OVA-primed animals and furthermore demonstrates the failure of transfused anti-BGG serum to stimulate a response. The uppermost panel presents the enhancement of the secondary response to DNP-BGG in DNP-OVA-primed animals which have been supplementally immunized with BGG. [These data from our laboratory appeared in 1. Exp. Med. 132, 261, 1970 (reference 66).]

20

DAVID H. KATZ AND BARU J BENACERRAF

CFA immune ce/ls 0-- 4 BGG immune ce/ls

l.0-/.6x/O9 LYMPH NODE CELLS

1

DNP-OVA

0

,

I

IDNP+;

I 1 2 3 4 WEEKS AFTER PRIMARY

I

I I I

I

I

I

I

I

I

I

I

I

I

I ( 4 7 DAYS AFTER SECONDARY

0

FIG.2. Ability of bovine y-globulin (BGG)-specific lymphoid cells to prepare 2,4-dinitrophenyl ( DNP)-ovalbumin ( OVA) -immunized guinea pigs for an enhanced response to DNP-BGG. Primary immunization of recipients was performed at week 0 with 3.0 mg. of DNPT-OVA administered intraperitoneally in saline. Three weeks later the guinea pigs were transfused with 1.0-1.6 x los lymphoid cells from syngeneic donors which had been immunized with either 50 pg. of BGG in CFA or saline in CFA 3 weeks earlier. Six days after cell transfer, the recipients were boosted with 1.0 mg. of DNP2,BGG in saline. Serum anti-DNP antibody concentration just prior to challenge and on days 4 and 7 are illustrated. [These data from our laboratory appeared in J. E q . Med. 132, 283, 1970 (reference 67) .]

that the cooperative function of carrier-primed spleen cells reaches a maximum earlier after immunization than their capacity to produce carrier-specific humoral antibodies. Contrasting with the inability of antibodies to do so, lymphoid cells from syngeneic animals immunized to the second heterologous carrier will transfer enhanced antihapten responsiveness to recipient animals which themselves have not been exposed to supplemental carrier immunization. This has been demonstrated by the studies of Mitchison (63, 65, 7 0 ) in mice and of Paul et al. (67) in inbred guinea pigs, as shown in Fig. 2. 2. In Vitro Models of Cooperatiw Interactions in Antibody Responses to HaptenXarrier Conjugates The development of a system for obtaining immune responses in oitro (83, 8 4 ) has offered certain advantages over in vioo experiments in

REGULATORY INFLUENCE OF ACTIVATED T CELLS

21

the analysis of cellular events in the immune response. Since such analyses are less restricted with studies employing well-defined antigenic determinants in the form of hapten-carrier complexes, it was a logical extension of the system to develop methodology to obtain in vitro immune responses to such immunogens. This was greatly facilitated by the discovery of Rittenberg and Pratt ( 1 3 5 ) of a rapid and simple method for coupling the chemical hapten trinitrophenyl ( TNP) directly to erythrocyte membranes. Based on this method with some original modifications, Kettman and Dutton (136) were successful in obtaining a TNPspecific, primary, in vitro response of mouse spleen cells cultured with TNP-coupled erythrocytes. Shortly thereafter, investigators in three different laboratories, working independently, presented evidence for cooperation between carrierspecific and hapten-specific cells in the in vitro primary immune response. Katz et al. ( 1 3 7 ) found that spleen cells from mice which had been primed several days earlier with free carrier (either burro erythrocytes or ax174 phage) developed enhanced primary anti-TNP responses in vitro when cultured with a TNP conjugate of the carrier used for priming (Table 111). Essentially identical observations in the primary in vitro response to NIP-coupled erythrocytes were reported at the same time by Trowbridge et al. ( 1 3 8 ) . The phenomenon was studied in considerably greater detail by Dutton and his colleagues (139, 140) who also obtained enhanced primary in vitro anti-TNP antibody responses in cultures of carrier-primed spleen cells. In addition, they demonstrated that the primary anti-TNP response of normal spleen cells could be enhanced by the addition of carrier-primed spleen cells which had been exposed to in vitro X-irradiation ( 139, 140). The radioresistant cell functioning in this helper capacity is, indeed, a thymus-derived cell as shown by the following observations (139): ( 1) the activity of the irradiated, carrier-primed spleen cells can be abolished by treatment with anti-8 serum and complement, and anti-8 serum-treated normal spleen cells can be restored by the addition of irradiated, carrier-primed spleen cells; ( 2 ) the helper effect of irradiated, carrier-primed spleen cells can be replaced by thymus-derived cells obtained from spleens of irradiated mice which had been injected with thymocytes plus SRBC. By employing a different in vitro culture system, based on a Millipore filter well technique for spleen organ fragments (141 ), Kunin et al. ( 142) corroborated these findings. The phenomenon of cooperative cell interactions in the secondary response to hapten-carrier conjugates in vitro has been less well studied. One such study has been recently reported by Cheers et aE. (143) and confirms the essential features of the phenomenon which occurs in vivo.

22

DAVID H. KATZ AND BARUJ BENACERRAF

TABLE 111 AUGMENTED in Vitro PRIMARY ANTITRINITROPHENYL RESPONSESOF @ X ~ ~ ~ - P R I M E D SPLEENCELLSSTIMULATED WITH TRINITROPHENYL-CONJUGATED ax1740 Direct PFC/IOBrecovered spleen cells Anti-TNP SRBCd

Protocol An tigenb TNP4X174 (1 x 108) ax174 (1 x 109

BALB/c spleen cellsc Normal ax174 7-day primed Normal ax174 7-day primed

Total

Above unstimulated

555 1163

25 1 876

304 287

-

-

Data taken from Katz et al. (137). Numbers in parentheses refer to quantity in plaque-forming units of trinitrophenyl (TNP)-substituted phage added to each culture. Cell density, 10 x 106 cells/ml. Primed mice received 2 X looplaque-forming units of +X174 intraperitoneally 7 days before spleen cells were cultured. The number of anti-TNP plaque-forming cells (PFC) presented have been corrected for background plaques against unconjugat,ed sheep red blood cells (SRBC). The column headed “above unstimulated” represents the number of anti-TNP PFC attributable to in vitro antigen stimulation (Le., total PFC in hapten-stimulated cultures less total PFC in hapten-unstimulated cultures of the same cells).

The hapten employed was 3,5-dinitro-4-hydroxyphenylacetic acid ( NNP ) coupled to either FyG or OVA as carriers. The secondary in vitro antiNNP response of spleen cells from mice primed with NNP carrier was optimal when the homologous NNP carrier was used for the in vitro immunogen. However, cells from an NNP-OVA-primed donor would respond in vitro to NNP-FyG by adding to the culture spleen cells from other mice primed with the carrier FyG. When semiallogeneic carrierprimed cells were used to enhance the secondary anti-NNP response it was shown by anti-H-2 serum that the majority of anti-NNP PFC were derived from the hapten-primed cell population (not the carrier-primed cells). Furthermore, activated thymus cells obtained from spleens of irradiated mice injected with thymus cells plus FyG could substitute for whole spleen cells from FyG-primed mice in enhancing the in uitm secondary anti-NNP response of NNP-OVA primed cells. The effect was specific since thymus cells activated to FyG enhanced the response to NNP-FyG, whereas thymus cells activated to BSA did not.

REGULATORY INFLUENCE OF ACTIVATED T CELLS

23

IV. Nature of the Regulatory Influence of Activated T Cells on Antibody Responses by B Cells

In an assessment of the physiological significance of T cell activity in the regulation of antibody responses by B cells, an important fact must be recognized initially: T cell activity is not an absolute requirement for the induction of antibody synthesis. There is a class of antigen which is considered to be thymus-independent. Moreover, even thymusdependent antigens in a narrow concentration range can elicit low level antibody synthesis, generally confined to the IgM class in the absence of T cell activity. It seems, therefore, appropriate to view the role of activated cells in antibody responses to be, in large part, regulatory in character in many aspects of B cell function. The evolutionary advantages of such a mechanism are readily apparent. Since the two lymphocyte types are both specific and need to be activated by antigen for antibody synthesis by B cells to develop opitmally, important control mechanisms, concerning antigen recognition and tolerance, should have evolved primarily in the T cell population. This is consistent with the body of evidence identifying T cells as the site where specific histocompatibility linked Ir genes are expressed, immunogenicity is recognized, and tolerance most readily induced. In this section, we shall discuss the evidence for the general regulatory role of T cell activity on various aspects of the antibody response. A major consequence of T cell activity, as will be shown, is the facilitation of the selective pressure exerted by antigen on the proliferation and differentiation of B lymphocytes and, thereby, on the emergence of adequate populations of memory cells. In addition, the most dramatic effects of T cell activity on antibody production by B cells concern the synthesis of immunoglobulin other than IgM; in fact, there is increasing evidence from experiments in genetically controlled systems that the switch from IgM to IgG is largely dependent on the regulatory effect of appropriately activated T cells. A. STIMULATION OF B CELLS IN

ABSENCEOF T-CELLREGULATION It is now well established that optimal specific humoral immune responses to certain antigens can develop in the absence of T cell participation. Humphrey et ul. (144) some years ago showed that neonatally thymectomized mice were capable of developing normal antibody responses to pneumococcal polysaccharide-an observation confirmed recently by others (145,146). Armstrong et ul. ( 147) found that bone marrow-reconstituted, lethally irradiated mice could develop normal humoral THE

24

DAVID H. KATZ AND BARU J BENACERRAF

responses to purified polymerized flagellin (POL) of SuZmoneZZu u&Zui& without the addition of thymic lymphocytes. (Indeed, the addition of thymocytes in this situation depressed the response to POL, a finding not explained by the authors that we shall consider in a later section.) Similar T cell independence has been observed with other antigens such as Escherichiu coZi polysaccharide ( 148, 149), polyvinylpyrrolidone (PVP) (149), and MS2 phage (150). Two points of considerable importance with respect to thymus-independent antigens are ( I ) the nature of their physicochemical structure and ( 2 ) the nature of the classrestricted antibody response that they induce, i.e., predominantly yM antibody responses. These features are discussed below. An important feature shared in common by the antigens discussed above is their structure which consists of identical units arranged in a more-or-less linear repetitive sequence. Their unique three-dimensional structural characteristics appear to favor a positive immunogenic signal upon direct interaction with specific receptor sites on B cells. This is to be contrasted with molecules not possessing these structural features which, in the absence of T cell function, generally result in either transient stimulation, no stimulation, or even a negative tolerogenic signal upon direct interaction with B cell receptors. The latter point will be developed in more detail in the later section on tolerance. The question is raised, then, about the physicochemical properties of receptor molecules on B cells which create this marked distinction between various antigenic structures insofar as their capacity to trigger indirectly or directly the specific immune response of these cells. There is no hard evidence from which to draw conclusions concerning this question. However, it is clear that a crucial relationship exists between the structural presentation of antigen and the ability to trigger B cells. This is exemplified by recent studies of Feldmann and Basten (151) . These investigators studied the primary in vitro antibody response of spleen cells from thymus-deprived mice to antigens of various physical forms and showed that the ability of such cells to develop a primary anti-DNP response in culture was related to the thymus-dependence of the carrier molecule employed. Thus, when POL was used as carrier, a normal anti-DNP response could be obtained, whereas little, if any, response was obtained with a conjugate of DNP-erythrocytes in which case the carrier is T cell-dependent. This observation has been interpreted as evidence for the notion that the predominant T cell function is related to modulating, in some way, the presentation of antigenic determinants to B cell receptors for appropriate immune induction to occur (74, 151). A word of caution is appropriate in this regard, although a T cellindependent antigen, such as DNP-POL, will induce an antibody re-

REGULATORY INFLUENCE OF ACTIVATED T CELLS

25

sponse in one dose range, the very same DNP-POL readily induces DNPspecific tolerance in vitro in slightly higher doses ( 1 5 2 ) . Taken together with other recent observations concerning hapten-specific tolerance induction in vivo, where it has been found that this can be readily obtained with hapten-conjugated to nonimmunogenic carriers (for which presumably no or few specific T cells exist) (153-155), the implication is clear that direct interaction of antigen with B cell receptors may favor a tolerogenic signal, even where the antigen is clearly capable of providing an immunogenic signal, in the absence of T cells in the appropriate concentration. We feel that these points suggest the operation of more than just presentation of antigenic determinants as the major T cell function in the regulation of B cell responses to antigen (see Section V1,C). In our opinion, it is more appropriate to consider that the stimulation of specific B cells by thymus-independent polymeric antigens in the narrow dose range where it can be achieved represents the limited response which B cells can display in the absence of T cell function. This interpretation is strengthened by the consideration that thymus-independent antigens elicit antibody responses generally restricted to the IgM class as will be discussed in Section IV,B.

B. EFFECTOF T-CELLACTIVITY ON SYNTHESIZED

THE

CLASSOF IMMUNOGLOBULIN

Thymus-independent antigens elicit antibody responses predominantly, if not solely, of the IgM class. This is a well-known feature of the immune response to pneumococcal polysaccharide (156) and has also been shown to be the case with Escherichia coli lipopolysaccharide ( 1 5 7 ) and PVP (149). This is particularly relevant in light of reported observations that even thymus-dependent antigens can elicit, under appropriate conditions in thymus-deprived mice, low antibody responses of the IgM class. Thus, Taylor and Wortis (158) have shown that IgM antibody production was less thymus-dependent than the production of IgG, when thymectomized, irradiated mice were given increasing doses of SRBC. Aird (159) has recently reported that thymus-deprived mice could be made to develop anti-NIP antibody responses with high doses of a polyvalent conjugate of NIP-BSA. The antibodies produced were exclusively of the IgM type. Moreover, monovalent NIP-BSA failed to elicit any response even in very high doses. This observation confirmed the findings of Makela ( 160) that polyvalent hapten-carrier conjugates give rise to higher proportions of IgM antibodies than monovalent or mainly monovalent conjugates. Similarly, Hamaoka ( 161 ) has recently observed that the antihapten response elicited in primed mice with high

26

DAVID H. KATZ AND BARU J BENACERRAF

doses of a heterologous carrier-hapten conjugate consists exclusively of IgM antibodies. The influence of T cells on production of IgM versus IgG antibodies is particularly apparent in the genetically controlled, H-Zlinked responses of mice to (T,G,)-A--L (162). It has recently been demonstrated by Grumet (163) that nonresponder mice develop early IgM antibody responses to 10 pg. of aqueous (T,G,)-A--L comparable in titer to those of responder mice. Extending this observation, Mitchell et al. (164) compared the antibody responses to ( T,G, ) -A--L of thymectomized responder and nonresponder mice to those of control mice of each type. Early IgM antibody titers were comparable among all the groups, whereas additional antigenic challenge induced IgG antibody production in only the group of responder mice which had not been thymectomized. These observations indicate that one of the requirements for a T cell regulatory influence in antibody production is related to the development of IgG antibodies more so than IgM. Thus, it may well be that the nature of the cell receptors together with physicochemical properties of the antigenic determinants, such as valency and size, are major determining factors as to whether or not B cells can be triggered to antibody production in the absence of T cell function. Moreover, it appears that where T cells participate in antibody responses, a definite consequence of their activity is related to selective forces relevant to the switch from IgM antibody synthesis to synthesis of IgG antibodies. C. ROLEOF T-CELLREGULATION IN THE SELECTIVE PRESSURE BY ANTIGEN ON B CELLS One of the regulatory functions of T cells in antibody responses appears to be the exertion of some selective pressure either directly or indirectly on the precursor B cell population. The absence of such effects most likely explains the occurrence of predominantly yM antibody responses to thymus-independent antigens ( see Section IV,B ). Our own attempts to demonstrate a selective pressure with respect to antibody class by carrier-prinicd T cells on hapten-specific B cell precursors in guinea pigs were complicated by the fact that this species makes little, if any, yM antibody to hapten-carrier conjugates (66). Others (65, 82, 1 3 4 ) , however, have failed to observe an effect of T cells on B cells as concerns the class of antibody produced in rabbits and mice. Recently, however, Miller et al. ( 7 4 ) have reported that they observed a marked shift in the class of anti-NNP antibodies produced in response to NNPHRBC by mice preimmunized to the carrier, HRBC, alone. Thus, in contrast to noninmuiie mice which developed predominantly yM antiNNP antibodies within 4 days aftcr immunization with NNP-HRBC,

REGULATORY INFLUENCE OF ACTIVATED T CELLS

27

mice preimmunized to the carrier 4 days before primary immunization with NNP-HRBC developed predominantly yG antibodies in the same time interval. It is noteworthy that carrier-primed mice produced equivalent yM responses to the nonprimed group which indicates that carrier priming did not truly “shift” the antibody class response but rather enhanced the kinetics of yG antibody-forming cell expression. The latter point is relevant when considering the failure of others to observe such an effect in different systems (65, 82, 134) where the factor of timing in the experimental protocol may be crucial. Thus, a selective pressure by T cells on antibody class might not be expected to be readily obvious at a time after immunization when yM and yG antibodies are both being produced. Perhaps the best illustrations of the participation of T cell function in the exertion of selective pressures on B cells derives from the following two recent studies. The first is the experiment of Mitchell et al. (164) on the effects of thymus deprivation on antibody responses of nonresponder and responder mice to (T,G)-A--L which we already described (see Section IV,B ) . The second is a recent study of the effect of T cells on affinity of antihapten antibodies ( 165). The experimental model employed adult thymectomized, lethally irradiated, bone marrow-reconstituted mice as thymus-deprived recipients of syngeneic T cells administered in various quantities. Such mice were then immunized with DNP-BSA or DNPkeyhole limpet hemocyanin (KLH), and the affinities of the anti-DNP antibodies produced were determined. In this system, Gershon and Paul (165) found that the affinity of anti-DNP antibody produced depended on the nature of the carrier molecule and on the number of T cells possessed by the immunized animal. Thus, the affinity as well as amount of antibody produced by thymus-deprived mice challenged with DNPKLH could be restored to normal with 0.33 X lo5 syngeneic T cells, whereas the response to DNP-BSA was not fully restored with even 1 x lo5T cells. The difference in numbers of T cells required for restoration of antibody probably reflects a difference in numbers of cells specific for KLH and BSA in nonimmunized T lymphocyte populations. These observations point clearly to an important role for T cells in the regulation of precursor B cells with respect to emergence of cells bearing highaffinity receptors. Moreover, the data argue somewhat against a restricted role for T cells of antigen presentation or concentration by virtue of the following reasoning. If T cell function were limited to presenting antigen to B cells in an effective concentration for stimulation to occur, then the diminution or absence of specific T cells would be likely to favor production of high-affinity antibodies, since only those B cells bearing high-

28

DAVID H. KATZ AND BARU J BENACERRAF

affinity receptors for the hapten would be expected to be stimulated. If one assumes, as we do, a more sophisticated role for T cells in the regulation of B cell function, it is not at all surprising that Gershon and Paul (165) found low-affinity antibody production by B cell descendants in the relative absence of T cells. The manner in which T cells exert selectional pressures on B cells could be either by direct or by indirect means. Inasmuch as a direct effect implies that T cells may regulate selection of B cell precursors of antibody-forming cells irrespective of an antigen-driven mechanism, we favor the concept of an indirect role of T cells in this regard. Thus, T cells may indirectly influence B cell selection by increasing the rate of proliferation, induced by antigen, of specific B cell precursors of antibody-forming cells. In this situation, one can easily imagine a more rapid change in the B cell population upon which selective pressure by antigen is being exerted, thus leading, through differentiative events, to more rapid appearance of B cells bearing high-affinity receptors and ultimately to their progeny synthesizing and secreting high-affinity antibodies. V. Immunological Specificity and Properties of T and B Cells Concerned with Cooperation Phenomena

The establishment of the requirement for the participation of two distinct populations of lymphocytes in the induction of immune responses to various antigens raised questions concerning the properties of such cells. Foremost among these are questions concerning the immunological specificity of each cell type. Inherent in such considerations is the question of receptor specificity among T and B lymphocytes. In this section we shall deal with these questions and consider also certain distinctive features of T and B cells. We shall describe the available evidence showing that ( 1 ) immunological specificity is a property of both B and T cells; ( 2 ) receptor specificity exists among both B and T cells; ( 3 ) B and T cells manifest distinct differences in their capacity to recognize hapten and carrier determinants; and ( 4 ) B and T cells are functionally distinguishable in their sensitivity to X-irradiation and corticosteroids. The major distinguishing features of T and B lymphocytes are summarized in Table IV.

A. IMMUNOLOGICAL SPECIFICITYOF T AND B CELLS There is little doubt that both T and B cells manifest immunological specificity. This is exemplified by the fact that specificity exists in immune responses characteristic of each cell population. Thus, cell-mediated immunity, such as delayed hypersensitivity which is predominantly a function of T cells, and antibody production which is mediated by B cells,

REGULATORY INFLUENCE OF ACTIVATED T CELLS

29

are specific events in the immune system. In immune responses to T cellindependent antigens (see Section IV,A) in which B cells alone develop antibody responses, such cells truly reflect specificity for the antigens involved. The situation becomes more complex in humoral immune responses to antigens where T and B cell cooperative interactions are required. The question arises as to which of these cells has the predominant role in dictating the specificity of the response. The existence of specificity among T cells has been demonstrated on the basis of (1) classic cell-mediated immunity experiments (Section V,C); ( 2 ) the ability to induce specific immunological tolerance in T lymphocytes (Section VII1,A); and ( 3 ) the capacity of T cells to be specifically activated by antigen as shown by several investigators. Thus, in the studies of Mitchell and Miller (56) using a double transfer system in mice, the ability of thymus-derived cells from the first host to cooperate with bone marrow cells in response to SRBC in a second irradiated host required specific antigen activation of the thymus cells in the first host. Activation of thymus cells in the first host by non-crossreacting erythrocytes, such as rabbit or horse, did not permit such cells to cooperate with bone marrow cells in response to SRBC in the second host. The requirement for exposure of thymus cells to antigen in the first host of the double transfer system was confirmed by Claman and Chaperon ( 1 1 ) and by Shearer and Cudkowicz (166). Similar observations have been made by a number of investigators (91, 92, 9 4 ) studying the reconstitution of in vitro responses to SRBC as pointed out in a previous section. Recently, Miller et al. ( 7 4 ) have extended their studies of specificity among T cells. Again employing a double transfer system they have found that T cells activated in a first irradiated host with FyG cooperate specifically with bone marrow cells in a second irradiated host in the response to FyG. The T cells activated by BSA in the first host failed to cooperate with bone marrow in the response to FyG. Unfortunately, these studies lack a very crucial additional control, namely, the response to FyG in the second host which received BSA-activated T cells if BSA was given as well. The significance of this observation would have a direct bearing on the specificity requirements of the actual cooperative interaction between T and B cells and will be considered in detail in a later section. The existence of specificity among B cells is most readily exemplified by the fact that it is this population, and not the T cell population, which gives rise to cells ultimately synthesizing and secreting specific antibodies. Furthermore, B cells possess immunoglobulin receptor molecules on their surface with antigen-specific binding properties both in nonimmune

w

0

TABLE IV DISTINGUISHING FEATURES OF B AND T LYMPHOCYTES Parameters

B Lymphocytes (Refs.p

T Lymphocytes (Refs.p

1. Surface antigenic markers 2. Surface immunoglobulin determinants

Mouse B lymphocyte antigen (1) High density of immunoglobulin (106 molecules) (4-8)

3. Surface recept.ors

Specific antigen receptors (9-15) C3 receptor (16) Receptors for immune complexes

0 Antigen (2,3) Not readily detect.able as classic Ig either because of low density (103 molecules or less) or absence (4-7) Specific antigen receptors (18-20) No C3 receptors (16) No receptors for immune complexes (17)

4. Peripheral localization (as % of lymphocytes) (a) Blood (b) Thoracic duct (c) Lymphnode (d) Spleen 5. Functional sensitivity to X-irradiation and corticosteroids

(17) 10% or less (3) 15-20% (3) 25% (3) 6 0 4 5 % (3) Sensitive to X-irradiation (21, 22); sensitive to corticosteroids after peripheralization, resistant before leaving marrow (28, 29)

90% or more (3) 8 0 4 5 % (3) 75% (3) 3 5 4 0 % (3) Functionally resistant to Xirradiation with respect to helper activity (22-24), cytotoxic activity (25) and delayed hypersensitivity (26, 27); corticosteroids distinguish 2 populations of T cells-a sensitive population (95%) located in cortex of thymus and a resistant population (5%) in the medulla (30); cortisone-resistant T cells are capable of performing all T cell functions (29, 31-35)

t!

"3 *s

-E U

c

z W

P q

6. Response to ant,igen (see text)

a

Key to references: 1. Raff et al. (3.45) 2. Reif and Allen (346) 3. Raff and Wortis (347) 4. Unanue et al. (174) 5 . Rabellino et a/. (173) 6. Raff et al. (171) 7. Pernis et al. (172) 8. Coombs et al. (176) 9. Naor and Suliteeanu (167) 10. Humphrey and Keller (170) 11. Davie and Paul (181)

Differentiate into antibodyproducing cells; usually require T cell influence to accomplish this. Not specifically involved in cell-mediated immune reactions. Establish immunological memory and can be rendered specifically tolerant

Recognize and bind antigen; undergo mitotic proliferation; exert regulatory influence on B cells, but do not synthesize and secrete classic antibodies. Specifically involved in cell-mediated immune reactions. Establish immunological memory and can be rendered specifically tolerant

5 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Byrt and Ada (177) Dwyer and Mackay (178) Unanue (180) Warner et a / . (173) Nussenzweig et al. (348) Basten et al. (349) Basten et al. (186) Roelants and Askonas (125) Engers and Unanue (350) Claman and Chaperon (11) Kate et al. (68) Dutton et al. (139)

24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35.

Kettman and I h t t o n (140) Moller and Moller (201) Asherson and Loewi (202) Feldman (203) Levine and Claman (211) Cohen and Claman (212) Ishidate and Metcalf (207) Warner (19) Blomgren and Andersson (209) Cohen et al. (210) Andersson and Blomgren (213) Cohen and Claman (214)

8 4

a

8 4

8 4

H

M

U

H

$3r

c

32

DAVID H. KATZ AND BARUJ BENACERRAF

and specifically immunized animals (167-185). The fact that B cells express specific immunological memory and can be rendered specifically tolerant is further confirmation of their specificity; these will be dealt with in subsequent sections.

B. ANTIGENRECEPTORSON T

AND

B CELLS

In recent years a considerable body of evidence has accumulated demonstrating the existence of specific receptors for antigen on lymphocytes. Now it is unequivocally established by direct methodology that the receptors on B lymphocytes are immunoglobulin in nature (for review, see ref. 8 ) . Coombs et al. (176) have shown that immunoglobulincoated erythrocytes will form specific rosettes with normal lymphocytes in the presence of anti-immunoglobulin antiserum. Using refined techniques of immunofluorescence microscopy, several investigators have directly demonstrated the presence of immunoglobulin determinants on the surface of live B lymphocytes (171-175). The receptor function of these immunoglobulin determinants is based on radioautographic observations of specific binding of highly .radioactive antigen to B lymphocyte surface membranes (167,170,177-182) and the inhibition of such specific antigen-binding by preincubation of the cells with anti-immunoglobulin antisera (177, 178, 180-182). Finally, it has been well demonstrated that antigen-specific B lymphocyte precursors of antibody-forming cells can be selectively depleted from a heterogeneous lymphocyte population by passage through antigen-coated bead columns (169, 181, 183, 184) or by antigen-induced suicide which follows specific binding of radioactivelabeled antigen of very high specific activity (168, 170, 180, 185, 186). Direct evidence for the presence of immunoglobulin receptors on T cells has been more difficult to obtain, possibly because the quantity of T cell receptors is significantly lower than the sensitivity which present technology will permit to observe in a reproducible manner. In fact, the number of Ig receptors on B cells has been determined to be approximately lo5,whereas if they exist on the T cell they could not exceed lo3 and remain undetectable by direct techniques (174). Certain observations demonstrating the inhibition of some T cell functions by antibodies directed against immunoglobulins or their subunits have been interpreted to indicate that T cell receptors consist, at least, of subunits of classically defined immunoglobulin structures ( 186-191 ) , However, these observations are not without some controversy. Other investigators have tried and failed to confirm some of these experimental results. Indeed, a very critical source of error and variability in such studies, which must be taken into account, concerns the quality and specificity of the anti-immunoglobulin reagents employed by different

REGULATORY INFLUENCE OF ACTIVATED T CELLS

33

investigators, Furthermore, interpretations based on inhibition studies are subject to several alternative explanations. Thus, the inhibitory effects of anti-K L-chain reagents could also obtain if such determinants were, indeed, present on the surface of T cells but bore no functional relation to the true antigen-specific receptor, Inhibition may then occur as a result of steric considerations of the L chain determinants with respect to the actual receptor. This would offer one explanation for the failure to observe inhibitory effects with class-specific anti-H chain sera. The alternative consideration is that T cell receptors either consist solely of L polypeptide chains or that the L chains are present in association with a unique class of H chain (IgX) not found in secreted immunoglobulin (188). Thus, it is established that B lymphocytes possess antigen-specific receptors which are immunoglobulin in nature. The T lymphocytes clearly appear to possess antigen-specific receptors, but it is not yet definitely established whether or not these are immunoglobulin. TWO important points concerning T and B cell receptors are germane to developing an understanding of T and B cell interaction. a. Both T and B cells possess antigen-specific receptors which bear a functionally important relationship to the immunological activity of such cells. This is well exemplified by studies of Basten et al. ( 1 8 6 ) using the technique of radioactive antigen-induced suicide. Under appropriate conditions, incubation of either thymus lymphocytes or bone marrowderived splenic lymphocytes with high specific activity '"I-FyG abrogated the cooperative adoptive transfer response to FyG. The specificity of the suicide was shown by the ability of such treated cells to cooperate in response to another unrelated antigen, HRBC. Roelants and Askonas (125) have made similar observations in the adoptive transfer haptencarrier system in mice. Thus, incubation of Maiu squinado hemocyanin ( MSH) -primed spleen cells with high specific activity 1251-MSH abolished their helper effect with DNP-OVA-primed spleen cells in the adoptive secondary response to DNP-MSH. These data very clearly illustrate not only the receptor specificity of both T and B cells but the functional relevance of such receptors as well. b. Although both T and B cells possess receptors, the specificity restrictions of such receptors may manifest considerable differences in T cells compared to B cells, as discussed below. C. RECOGNITION OF HAPTEN AND CARRIER DETERMINANT^ BY T B CELLS

AND

Studies on the specificities of T and B cells have been best carried out with responses to hapten-carrier conjugates and have been elegantly

34

DAVID H. KATZ AND BARU J BENACERRAF

reviewed recently by Paul (8). Indications from such studies, in general terms, are that T cells participating in cellular immune reactions to hapten-carrier conjugates have specificity characteristics different from that of antihapten antibody and, therefore, by extension, different from the specificity characteristics of the hapten-specific precursor B cells. In this context, it is relevant to note that in responses to hapten-carrier conjugates, haptens do not, in themselves, constitute determinants capable of being recognized by cells concerned with carrier function or, alternatively, of stimulating such cells to perform this function under usual conditions of immunization. Haptens may, however, contribute to such determinants. Evidence for this statement derives from the following studies carried out in our laboratory (67). Sensitization of animals to haptens by preimmunization with haptenprotein conjugates failed to prepare them for enhanced primary or secondary responses to other determinants associated with that hapten on a different carrier (67). Thus, guinea pigs immunized with DNPBGG in complete Freund's adjuvant did not manifest an enhanced primary anti-OVA response upon challenge with DNP-OVA. Similarly, DNP-OVA-primed rabbits which received a supplemental immunization with sulfanil-azo ( SULF) -BGG in complete Freunds adjuvant failed to display enhanced secondary anti-DNP responses upon challenge with the double hapten conjugate of the protein carrier glucose oxidase ( GO), DNP-SULF-GO. Finally, SULF-BGG-primed guinea pigs which received a supplemental immunization with DNP-guinea pig albumin (GPA) in complete Freund's adjuvant did not make enhanced secondary anti-SULF antibody responses to a challenge with SULF-DNP-GO. However, the finding that increased anti-SULF titers resulted from secondary challenge of such animals with SULF-DNP-GPA but not with SULF-GPA indicates that the DNP determinant, indeed, contributes to the carrier determinants recognized by helper T cells. It may be argued that the latter finding may result simply from a general alteration in GPA imposed by substituting many groups on the lysine side chains, However, the failure of SULF-dimethylaminonaphtalene sulfonyl ( DANSYL )GPA to elicit a secondary anti-SULF response in the above experiment makes this explanation unlikely ( since DANSYL also binds covalently to lysines). It may likewise be argued that the capacity to enhance secondary antihapten responses by preimmunization with a protein carrier but the failure to prepare for enhanced responsiveness by preimmunization with a hapten, conjugated to a protein not used again for immunization, could be explained by the great diversity of antigenic determinants on proteins in contradistinction to the large number of similar haptenic determinants

REGULATORY INFLUENCE OF ACTIVATED T CELLS

35

in hapten conjugates. If this were the case, then it may be expected that relatively simple immunogenic polypeptides, which are limited in antigenic heterogeneity, would be marginal in their capacity to function as cooperating carrier determinants. In order to test this hypothesis, we compared the relative efficacy of a hapten, DANSYL, and a very simple carrier immunogen, PLL, in enhancing secondary anti-DNP antibody responses (67). Strain 2 guinea pigs were primed with DNP-OVA and then given a supplemental immunization of DANSYGPLL in complete Freunds adjuvant. The effectiveness of PLL as a supplement was tested by challenging one group of animals with DNP-PLL; another group was challenged with a double hapten conjugate of poly-D-lysine (PDL), DNP-DANSYGPDL, to test the effectiveness of DANSYL as a supplement. We employed PDL as a nonimmunogenic or weakly immunogenic carrier for the two haptens because its physical properties are similar to those of PLL. Whereas the animals challenged with DNP-PLL dispIayed enhanced secondary anti-DNP responses, no secondary anti-DNP responses were observed in those challenged with DNP-DANSYL-PDL despite the fact that it was considerably more heavily substituted with respect to both DNP (174 groups/mole) and DANSYL (81 groups/ mole) than the corresponding L-polymer ( 35 DNP groups/mole), which might have been expected to favor its binding by hapten-specific cells. Thus, even a molecule with restricted determinant heterogeneity, such as PLL, can function quite effectively as a carrier or helper molecule, provided it can stimulate T cells, whereas a univalent haptenic determinant, such as DANSYL, either fails to do so or does so very poorly. This evidence argues against the notion that properties simply related to possession of multiple determinants on the same backbone are relevant in determining the capacity of molecules to express carrier function. Indeed, it has recently been shown that even a nona-L-lysine can effectively exert a helper function in anti-DNP responses ( 1 8 7 ~ )We . should stress again, however, that carrier-reactive helper T cells can recognize the existence of haptenic determinants on a functional carrier. This was illustrated in the experiments employing DNP-GPA described earlier and was also observed, even more strikingly, in the later-described studies using DANSYL-PLL. Hence, although enhanced secondary antiDNP responses were obtained upon challenge with DNP-PLL, secondary challenge with a double hapten conjugate of PLL, DNP-DANSYL-PLL, resulted in considerably greater ( twelve-fold ) secondary anti-DNP antibody responses. This implies, therefore, that the relevant T lymphocytes can distinguish between PLL and DANSYL-PLL, illustrating that carrier-specific helper cells have equivalent specificities to cells mediating cellular immune reactions.

36

DAVID H. KATZ AND BARUJ BENACERRAF

We wish to make it perfectly clear that the observations described above are not interpreted by us to rule out definitively the existence of T cells with hapten specificity. It is, indeed, possible that such cells exist. Nevertheless, the existence of hapten-specific T cells need not imply that they manifest an analogous role to that of carrier-reactive helper cells, or, alternatively, if they do, their functional effectiveness may be marginal under ordinary circumstances. Mitchison ( 65, 73, 192) has reported marginal enhancement in a hapten preimmunization system in mice which may argue in favor of the existence of hapten-specific T cells expressing helper function. Thus, the adoptive secondary response of spleen cells obtained from donor mice primed with a protein BSA could be suppressed by treatment of the donors with antilymphocyte serum (ALS). However, the antibody response to BSA could be partially restored by transferring also cells from donors primed with DNP-CGG and then challenging with DNP-BSA ( 73). Mitchison interprets the observation as evidence for T cells specific for DNP which perform a helper function to enable BSA-specific B cell precursors of antibody-forming cells to develop an anti-BSA response. Several points are noteworthy regarding these experiments: (1) the effect could only be observed when donors of BSA-primed cells were subjected to immunosuppression by ALS, implying that in the presence of a normal complement of BSA-specific T cells, help from hapten-specific T cells is an irrelevant issue; and (2) the effect was obtained using extremely high doses of DNP-BSA ( loo0 pg. ) for secondary challenge. Iverson (193) and Taylor and Iverson (194) have reported similar observations using skin-painted mice as donors of hapten-specific helper cells. Unfortunately, these reports fail to mention whether or not transfer of passive antihapten antibody could substitute for primed cells in mediating the hapten-specific helper effect. This point is of considerable importance in interpreting these data in light of the recent observation of Janeway and Paul ( 195) that passively transferred anti-DNP antibody prepared BALB/ C mice for an anti-idiotype antibody response to a DNP derivative of isologous yza myeloma protein (LPC-1). This enhancement by serum antibody is not, however, comparable in magnitude to the helper effect obtained in other systems with carrierspecific T cells as described in Section II1,B. Nevertheless, the very existence of carrier specificity in hapten-specific antibody responses tells us that the expression of carrier function is not a property that can be effectively ascribed to haptens. Why, then, do haptenic determinants fail to express carrier function, or do so only marginally, although they can participate in the specificity of the cells that mediate carrier function? Three general explanations may be entertained.

~ G ~ ~ . A T O INFLUENCE R Y OF ACTIVATED T CELLS

37

1. Cells mediating carrier function ( T cells), as a class, have antigensensitive receptors generally similar to those of the precursors of antibody-forming cells ( B cells); however, the receptors of T cells differ from those of B cells in their range of specificity, inasmuch as they are specialized to recognize the determinants of native and of altered proteins and polypeptides. 2. The T cells mediating carrier function generally possess receptors of low affinity, relative to the concentration conditions in their environment, and can bind and be activated most efficiently by exposure to a total antigenic determinant and only rarely by the partial determinant which haptens constitute. 3. The T cells mediating carrier function are specialized either in the possession of an entirely different class of recognition units or in the normal binding of antigen followed by an additional operation which can only be performed on substrates of given structure. At the present time there is no hard evidence to allow a definitive choice between the above alternatives. Indeed, they are not mutually exclusive possibilities.

D. SENSITIVITY AND RESISTANCEOF T AND B CELLFUNCTION TO

X-IRRADIATION AND CORTICOSTEROIDS

A working knowledge of T and B cell functions in the immune system must take into consideration a variety of parameters. Among these are considerations of functional and receptor specificity which have been discussed in previous sections. In recent years, considerable attention has been given to the development of methodology to isolate or deplete selectively one population or the other in relatively pure terms, and a variety of techniques are now described to accomplish this end. Thus, in vitro treatment of mouse lymphoid organs with anti4 serum plus complement depletes the great majority of T cells, leaving a relatively pure population of B cells (196). Other techniques have been described but it is not relevant to discuss them at length here [for review, see Miller et al. ( 7 4 ) ] . It is of considerable interest, however, that the two cell types are functionally distinguishable on the basis of their sensitivity or resistance to X-irradiation and certain drugs. It is crucial to understand these differences in B and T cells in order to develop a reasonable clue to the nature of the interaction which occurs between them. In this section we shall present evidence demonstrating that differentiated T cells are functionally resistant with respect to helper activity to X-irradiation and corticosteroids, whereas B cells are functionally sensitive to such agents.

38

DAVID H. KATZ AND UARUJ BENACERRAF

1. X-Zrradiution The general sensitivity of the immune system to X-irradiation is a phenomenon recognized for many years. Indeed, it is on this basis that experimental models for successful adoptive transfer of immunity with lymphoid cells have been developed. Thus, X-irradiation of a host animal, under appropriate conditions, abrogates the capacity of its own lymphoid cells to engage in an immune response such that the response of normally competent transferred lymphoid cells can be assessed. In recent years, it became recognized that the functional role of macrophages in immune responses was unaffected by exposure to X-irradiation ( 8 9 ) , but until very recently it was generally believed that immunocompetent lymphocytes were all radiosensitive. Our present state of knowledge, however, permits us to make a functional distinction between T and B lymphocytes on the basis of their respective resistance and sensitivity to X-irradiation. The functional resistance to X-irradiation of T cells concerned with helper activity was first clearly established by investigators working independently in two laboratories using different cell cooperation systems. Katz et al. (68) found that carrier-primed lymphoid cells which had been exposed to high doses of X-irradiation in vitro were, nevertheless, capable of performing a helper function in enhancing secondary anti-DNP antibody responses of syngeneic guinea pig recipients to which they had been transferred. Exposure of the carrier-specific BGGprimed lymphocytes to X-irradiation in vitro prior to transfer into DNP-OVA-primed recipients did not affect the capacity of such cells to perform a helper function in the secondary anti-DNP response to DNP-BGG. This was true over a wide dose range of X-irradiation ( 1OOO-5OOO R). Furthermore, a clear functional distinction between T and B cells, with respect to radioresistance, was established in these experiments. Thus, the functional capacity of B cells in the BGG-primed lymphoid cell population not exposed to in vitro X-irradiation was shown to be intact by the presence of anti-BGG antibodies in the serum of recipients of such cells prior to DNP-BGG challenge and a clear anamnestic anti-BGG antibody response following DNP-BGG challenge. In contrast, no anti-BGG antibody could be detected either before or after DNP-BGG challenge in the sera of recipients of BGG-primed cells which had been exposed to in uitro X-irradiation ( 68). Almost simultaneously, investigators in Dutton’s laboratory found that educated helper T cells in the in uitro primary responses to SRBC and to trinitrophenyl (TNP)-erythrocytes were radioresistant (90,139, 1 4 ) . Thus, the in uitro primary anti-TNP response of normal mouse spleen

REGULATORY INFLUENCE OF ACTIVATED T CELLS

39

cells to TNP-SRBC was markedly enhanced by the addition of spleen cells from carrier (SRBC)-primed mice (139, 140). Exposure of the carrier-primed spleen cells to in vitro X-irradiation (as much as 4000 R ) completely inhibited the capacity of such cells to develop an antibody response but did not prevent their ability to function as helpers for the anti-TNP response of the normal spleen cells. Moreover, the radioresistant helper cell was shown to be a T cell (139, 197). In their studies of reconstitution of the primary in vitro anti-SRBC response of spleen cells from thyniectomized mice, Munro and Hunter (88) observed that X-irradiation ( l W R ) of the allogeneic donor of normal spleen cells immediately prior to sacrifice diminished, but did not abrogate the cooperating effect of such cells. Observations of other investigators can be interpreted as consistent with the above results ( 198-200). However, early studies of thymus-marrow synergism in mice had suggested that both T and B cell components were sensitive to X-irradiation. In the studies of Claman and co-workers (11, 77) and Miller and Mitchell (9, 57) the functional capacity of thymus-derived cells to cooperate with B cells was abrogated by X-irradiation of such cells either in situ or in vitro. It would appear, therefore, that a contradiction exists between these observations and those of Katz et al. (68) and Kettman and Dutton (140) cited above. We feel, however, that a valid explanation may be offered for the conflicting observations. The experimental protocols employed by Claman (11,77) and Miller (9, 57) and their associates required a crucial period of proliferation by T cells in the presence of antigen, as shown by their own data, before such cells could effectively facilitate the antigen-induced activity of normal B cells. That this is, indeed, the case is also illustrated by the findings of Claman and Chaperon ( 77) and Shearer and Cudkowicz ( 1 6 6 ) in the double transfer thymus-marrow synergy model that a crucial 2-3 day period of interaction between SRBC and T cells in the first host was required before such T cells could cooperate with B cells in a second host. Under these circumstances, any inhibitory influence on mitotic activity during this crucial period by X-irradiation or antimitotic drugs would understandably abolish the helper function of the T cells. In contrast, under conditions where T cell proliferation is not required, because the cell population has already been expanded and differentiated, the helper function of the cells is not affected by X-irradiation. This is well exemplified in our in v h o model where the crucial proliferative response of the helper T cells had presumably occurred early following BGG priming (68). It is noteworthy here to make the following additional points: (1) the capacity of such irradiated carrier-primed

40

D A W H. KATZ AND BARU J BENACERRAF

cells to respond to antigen in vitro with DNA synthesis was 90% inhibited with relatively low doses of in vitro X-irradiation ( W R ) and totally abolished at doses above 1500 R; and (2)the facilitative capacity of irradiated carrier cells was intact after the 6-day interval between the transfer of cells and the subsequent secondary antigenic challenge (68). These points are of major importance in developing an understanding of the T cell function in cooperative interactions with B cells. It appears, therefore, that having successfully undergone whatever degree of proliferation and differentiation is occasioned by antigenic stimulation, the T cell can perform its helper function in the absence of any further proliferation. From a purely functional standpoint this would imply that ( 1 ) the mature antigen-specific T cell cooperatively interacts with B cells in a way that requires little or no further division; ( 2 ) the heavily irradiated helper T cell can survive and perform its specific function in vivo for at least as long as 6 days; or, alternatively, the helper T cell, once fully differentiated, can perform its role through interaction with other cells prior to subsequent immunization and, therefore, its presence is not required at the precise time of antigenic stimulation. There have been several previous demonstrations of functional radioresistance among T cells participating in cell-mediated immune reactions. Thus, Moller and Moller (201) observed that in vitro killing of target cells by mouse lymphoid cells was not inhibited by exposure of the latter to 1500 R in vitro. Asherson and Loewi (202) found that delayed hypersensitivity reactions in guinea pigs could be transferred with donor cells exposed in vitro to lo00 R. Feldman (203)was able passively to transfer delayed hypersensitivity in rats with donor lymphocytes which had been exposed in vitro to 6000 R X-irradiation. It seems likely, therefore, that T cell function, in cellular immunity as well as in antibody production, does not require proliferation once the initial antigen-induced process of differentiation and clonal expansion has occurred. 2. Corticosteroids It has been known for quite some time that cortisone has a general suppressive effect on immune responses in some species (204, 205), presumably reflecting the rapid atrophy of lymphoid structures which follows administration of large doses of the drug. This effect is particularly striking in the thymus where approximately 95%of the total thymic lymphocyte population is depleted by cortisone ( 2 0 6 ) .It is the 5%of thymocytes which remain after such treatment which has attracted renewed interest in the effects of cortisone on immunocompetent cells in the past few years. The cortisone-resistant thymocytes appear to be located primarily in the medulla, whereas the sensitive cells are predominantly in

REGULATORY INFLUENCE OF ACTIVATED T CELLS

41

the cortex of the thymus ( 2 0 7 ) .These cortisone-resistant thymocytes are larger and possess more histocompatibility antigens on their membranes than the sensitive thymocytes ( 2 0 8 ) . The basis for such renewed interest in this area, in light of the establishment of the two-cell concept of immunity, was an observation made by Warner ( 1 9 ) some years ago that the few remaining thymocytes of cortisone-treated chickens were capable of expressing graft-versus-host (GVH) reactivity comparable in magnitude to normal chicken thymus. Recently, Blomgren and Anderson ( 2 0 9 ) found that thymus cells obtained from cortisone-treated donor mice were of the order of ten-fold more reactive in inducing the GVH response than whole thymus from untreated donors. Cohen et al. ( 2 1 0 ) similarly observed that cells in mouse spleen, bone marrow, and thymus capable of initiating GVH reactivity were cortisone-resistant. Such observations clearly established that the T cells responsible for cell-mediated immunity were resistant to the effects of cortisone. Since administration of corticosteroids to mice at the same time as primary SRBC immunization markedly suppressed the humoral response (205), the question was raised as to whether a differential effect on T and B lymphocytes could be established. Levine and Claman (211) found that spleens from cortisone-treated donor mice were significantly decreased in capacity to transfer adoptively anti-SRBC responses to irradiated recipients; bone marrow cells from cortisone-treated donors, however, were perfectly capable of cooperating with normal thymocytes in the adoptive anti-SRBC response. Subsequent studies by Cohen and Claman ( 2 1 2 ) established that the cortisone-sensitive spleen cells were the bone marrow-derived antibody-forming cell precursors, and that the T cells responsible for helper function in the anti-SRBC system were cortisoneresistant. Similar observations were made by Anderson and Blomgren (213) in humoral responses of mice to SRBC, BSA, and the NIP hapten. Thus, adult thymectomized, irradiated, bone marrow-reconstituted mice which were unable to develop humoral immune responses to any of these antigens could be restored by injection of cortisone-resistant thymus cells from syngeneic donors. Thus, it appears that cortisone exerts a differential effect on immunocompetent lymphocytes in the following ways. (1) On the one hand, B cell precursors of antibody-forming cells which have migrated to peripheral lymphoid organs are cortisone-sensitive; those which have not yet left the bone marrow, on the other hand, appear to be cortisone-resistant. Whether this finding reflects a true difference in sensitivity based on qualitative differences in the maturation stage of the cell or merely reflects differences in effective doses of cortisone reaching the cell in bone

42

DAVID H. KATZ AND BARUJ BENACERRAF

marrow versus periphery is not known. ( 2 ) The T cells which are predominantly located in the thymic cortex and presumably not functionally mature are cortisone-sensitive; T cells of the thymic medulla and those that have migrated to peripheral lymphoid organs and are, therefore, fully differentiated to participate in humoral and cellular immune reactions are functionally resistant to the effects of cortisone. The nature of the maturational differences between sensitive and resistant T cells is not established. Inasmuch as it may be tempting to ascribe cortisone sensitivity to those cells undergoing mitotic turnover, it is noteworthy that cortisone administration during antigen-induced mitosis of T cells does not have a suppressive effect on the capacity of such cells to participate in either humoral or cell-mediated immune responses (212,214). VI. Mechanism of Regulation of B Cell Function by T Cells

When considering the cellular events critical to the expression of a regulatory function by T cells on the response to antigen of B cells, several possibilities have been entertained by various workers in the field. These will be considered, in order, in our opinion, of their increasing relevance as follows: ( 1) transfer of genetic information; (2) antigen concentration or presentation; ( 3 ) regulation of B cell response to antigen by products of activated T cells. A. TRANSFER OF GENETIC INFORMATION There is no evidence whatever that transfer of genetic information could explain the regulatory role of T cells on the response of B cells to certain antigens. Nonetheless, from a purely hypothetical standpoint, any consideration of cell interactions must include the possibility that a signal from one cell type, in the form of genetic information, may be transferred to another cell type leading to a fundamental change in the specificity and/ or function of the second cell. This possibility was entertained in early studies of Mitchell and Miller ( 5 6 ) . If this were a relevant consideration with respect to T and B cell interaction, then one would not expect to find the high level of specificity restriction existing, that, indeed, exists among B cells in the nonimniune animal (see Section V,A ) . Moreover, the possibility of genetic information transfer from T cells to B cells of functional significance has been recently definitively excluded on the basis of the experiments described below. Mitchison et al. (65, 7 0 ) showed in the adoptive transfer response to hapten-carrier conjugates in mice that, if the donors of carrier-primed cells had immunoglobulin allotype markers different from donors of hapten-primed cells, then the antihapten antibodies produced after secondary challenge with hapten carrier were of the allotype of the hapten-

REGULATORY INFLUENCE OF ACI’NATED T CELLS

43

primed cell donors. Similarly, Jacobson et al. (215) using congenic mice as donors of T and B cells in the adoptive transfer response to SRBC observed that all of the anti-SRBC PFC were of the allotype of the B cell donors. Since the allotypic markers are located on the nonvariable Fc portion of the heavy chains of immunoglobulin molecules (216), it may be considered that these observations are not completely convincing arguments against the possibility of genetic information transfer. However, studies of Katz et al. (66) have shown that the affinity of antihapten antibodies produced during enhanced primary and secondary antihapten responses in guinea pigs and rabbits is determined by the mode and time of immunization with the hapten-carrier conjugate and not by the mode and time of immunization with the free carrier (see Section 111,B). Since antibody affinity is a marker of the variable region of the immunoglobulin molecule, these findings taken together with the allotype data cited above definitively exclude the possibility of T cell regulation of B cell function by virtue of macromolecular information transfer.

B. ANTIGENPRESENTATION AND CONCENTRATION The hypothesis of “antigen focusing” has been, perhaps, the earliest and most popular explanation for the nature of T and B cell interaction in antibody responses. This concept, first proposed by Mitchison (63, 65), assumes a totally passive role for the specific helper T cell, whereby antigen is recognized and bound by such cells, transported to sites in lymphoid organs where B lymphocytes reside, and then presented to specific B cells in a concentration appropriate to stimulate them. The antigen-focusing hypothesis is immediately appealing in that it has a certain degree of elegance in its simplicity and can be used to explain many established features of T and B cell interactions. Thus, T cell specificity is required for recognition and binding of antigen by certain of its determinants, not necessarily and, indeed, probably not, the same as those determinants recognized by specific B cell receptors. The antigen would thereby be “bridged between the two cells having different determinant specificities for distinct moieties of the complex antigen. Since T cells comprise the major portion of the recirculating small lymphocyte population (217), they can be considered to be particularly well-suited for the role of binding antigen and concentrating it at sites where B cells reside. There are alternative ways in which T cells may serve in antigen concentration and presentation other than forming an antigen bridge with B cells. One such possibility is that T cells elaborate a unique class of antibody ( IgX) with specific binding capacity for certain determinants of the antigen which it then concentrates either onto the B cell itself or

44

DAVID H. KATZ AND BARU J BENACERRAF

onto macrophages ( 7 4 ) .The advantage to the B cell of an antigen concentration mechanism, either through bridging or IgX, is felt to be related to presentation of antigenic determinants in such a way as to favor multivalent binding of these determinants by the B cell receptors. The argument for the antigen concentration hypothesis rests mainly on logical speculation and circumstantial evidence. Direct evidence that T cells serve predominantly in an antigen presentation role is largely lacking. Indeed, more evidence exists in favor of a more sophisticated role for T cells in antibody responses as will be discussed in Section V1,C. There are, nevertheless, certain observations which are more easily explained by an antigen concentration scheme. Mitchison ( 7 0 ) , in the adoptive transfer of secondary antihapten responses in mice, has shown that the hapten must be present on the carrier molecule in order to elicit a cooperative effect. When NIP-OVA-primed cells are transferred together with BSA-primed cells to irradiated recipients, a cooperative response is obtained to challenge with NIP-BSA but not if an unrelated NIP carrier is administered simultaneously with free BSA. Similar observations were reported by Kettman and Dutton (140) in studies of primary in oitru antihapten antibody responses. Thus, carrier-primed spleen cells develop enhanced primary in uitro anti-TNP responses to a TNP conjugate of the same carrier used for priming, but not to a TNP conjugate of another carrier even if the original carrier used for priming is simultaneously present, in unconjugated form, in the culture. Although these observations can be interpreted as evidence for an antigen-focusing hypothesis, i.e., to explain the requirement for hapten and carrier to be together in the same molecule, a word of caution should be added concerning the experimental protocols employed. It is possible that the design of Mitchison’s (70) and of Kettman and Dutton’s (140)experiments inadvertently favored a negative result for reasons other than a requirement for hapten to be present on the carrier molecule. We refer to the possibility of antigenic competition at the level of the helper T cell developing under the conditions employed, inasmuch as we shall present compelling evidence in later ( Section VI1,B) that such competition does exist with respect to helper function in antihapten antibody responses. It may be alternatively considered, at least with respect to the in uiuo situation, that a negative result in such experiments may reflect a lack of close proximity between the responding T and B cells. Even a mechanism, such as we shall propose in the next section, whereby stimulated T cells may regulate B cell function via release of soluble mediators may require a reasonably close association between the respective cells, especially if such mediators are relatively fast-acting and short-lived. If the helper function of T cells reflected simply a passive role of

REGULATORY INFLUENCE OF ACTIVATED T CELLS

45

these cells as vehicles of antigen presentation and/ or concentration to B cells, then it should be possible to supplant the need for T cells in antibody production by the use of inert antigen-coated particles or cells. Several attempts to accomplish this have been unsuccessful in our own laboratory and in those of others. Thus, Unanue (218) has shown that thymectomized mice are markedly depressed in their capacity to develop antibody responses to KLH. Moreover, he failed to restore their responsiveness by administering the KLH bound to live macrophages. Since the macrophages localized in the vicinity of B cells, this result indicates that more than antigen presentation is required for B cell stimulation. Katz et al. (219), using the model of enhanced secondary anti-DNP responses in guinea pig recipients of syngeneic carrier-primed lymphoid cells ( 6 7 ) , were unable to observe a cooperative effect when anticarrier antibody-coated polyacrylic resin particles were substituted for carrierprimed lymphoid cells. Polyacrylic resin particles coated with highaffinity guinea pig anti-BGG immunoglobulin were injected intravenously in various quantities into groups of DNP-OVA-primed guinea pigs. Similarly, primed control groups received either syngeneic BGG-primed lymphoid cells or lymphoid cells from nonprimed donors. All groups were then challenged with a heterologous DNP-BGG conjugate. The recipients of BGG-primed lymphoid cells displayed enhanced secondary anti-DNP responses to DNP-BGG challenge, whereas the other groups made no secondary response. Had the BGG-specific lymphoid cells simply picked up the DNP-BGG via specific receptors and concentrated it at sites of B cells, we may have expected a similar passive effect of the antiBGG-coated particles. The argument from such an experiment, we realize, is far from compelling because, among other reasons, although particles such as those we used may be the same size as lymphoid cells, they are not lymphoid cells and, therefore, make any physiological interpretation fruitless. The best approach to the problem, obviously, should employ lymphoid cells as the antigen-coated particles. Recently, Miller et al. (220) tested the possibility that T cells cooperated with B cells in antibody responses by acting as passive carriers of antigen. This was done using nonreactive T or B lymphocytes artificially coated with the antigen, FyG, in an adoptive transfer system in mice (Table V ) . They found that T cells from FyG-tolerant donors which were coated with FyG failed to cooperate with B cells in irradiated recipients in response to FyG, although such cells could cooperate in response to HRBC. However, FyGcoated T cells from nontolerant donors were capable of cooperating with B cells in response to FyG. Similar observations were obtained using antigen-coated B cells (220). Thus, B cells were incubated in oitm with

TABLE V ANTI-FyG PFC RESPONSE IN SPLEENS OF T X BM CBA MICE RECEIVINQ FALG-INCUBATED TDL FROM NONTOLERANT OR FyG-TOLERANT (CBA X C57Bl)Fl MICE'.~

TDL given None 2 X 10' FALG- coated cells from FyGtolerant mice 2 X lo7 FALG- coated cells from cyclophosphamidepretreakd mice

Antigen challenge in viva

+

No. of mice in groupc

Peak anti-FyG PFC per spleend

Reduction of anti-FyG with

19SPFC

7 S PFC

CBA antiC57B1 serum

2(4-1)

-

500 pg. F r G AP 4 X 108 HRBC 2 wk postirradiation 4 X 108HRBC

16

510 (700-370)

16

140 (160-120)'

4 X 108 HRBC

16

2(4-1p

7690 14,730 (8890-6640)e(16,640-13,040)6

-

20 (19 S) 0 (7 S)

C57B1 anti-CBA serum

Peak 19 S anti-HRBC PFC

a

390 (690-230)

x

32,230 (40,640-25,560) 99 (19 S) 29,210 99 (7 S) (38,680-22,070)

We thank Dr. J. F. A. P. Miller for permission to reproduce these data from Miller et al. (127). This experiment illustrates the inability of antigen-coated tolerant thoracic duct lymphocytes (TDL) to induce a response to fowl -,-globulin (FyG) in adult, thymectomized, irradiated, bone marrow-protected (T X BM) recipient mice. The TDL from nontolerant or FyG-tolerant Fl donor mice (tolerance induced by cyclophosphamide method) were reacted in oitra with fowl antimouse lymphocyte globulin (FALG) and then transferred to T X BM CBA mice. As control, these mice also received antigen challenge with human red blood cells (HRBC) int.ravenously.Control T X BM mice received no TDL but were challenged intravenously with alum-precipitated FyG plus pertussis and HRBC. A t the times indicated after transfer and challenge the numbers of 19 and 7 S anti-FyG plaque-forming cells (PFC) and 19 S anti-HRBC PFC in spleens of recipient mice were determined. Half the mice were killed at 5 to 6 days to determine 19 S anti-FyG PFC, the other half at 7 days to assay 7 S anti-FyG PFC and 19 S anti-HRBC PFC. Geometric means (upper and lower limits of SE). e P values < 0.005.

%

u

*

E

;?

3

5

m cl

z

m

n

8%

REGULATORY INFLUENCE OF ACTIVATED T CELLS

47

an antigen-antibody complex consisting of NIP-FyG-anti-FyG, and the coated B cells were transferred with or without normal T cells into irradiated hosts. A primary anti-NIP response occurred only if both B and T cells were given. Furthermore, transfer of such coated B cells together with spleen cells from NIP-HGG-primed donors resulted in a substantial secondary anti-NIP response; this effect was abolished by treatment of the NIP-HGG-primed spleen cells with anti-8 serum and complement. These observations can only be interpreted in one way: antigen-coated T or B lymphocytes cannot substitute for specific T cells in either primary or secondary antibody responses. Since Miller et al. ( 220) determined that the artificially antigen-coated cells they employed followed normal homing patterns to the lymphoid organs, the data argue strongly against a passive antigen-focusing role for T lymphocytes in antibody responses. Finally, in this context it would be well to recall the observations of Gershon and Paul ( 1 6 5 ) concerning the relationship of T cell activity to affinity of antibodies produced. Reiterating what we said earlier (Section IV,C), if the predominant role of T cells in antibody production was a relatively passive one serving to localize and increase the effective antigen concentration in the microenvironment of B cell receptors, then one would expect that with decreased number of T cells, antigen would most likely stimulate B cells with high-affinity receptors. The resulting serum antibodies should, therefore, be of high average affinity. In contrast, Gershon and Paul (165) found just the opposite, i.e., thymusdeprived animals produced low-affinity antibodies and the addition of T cells led to production of antibodies of increasing affinity. Their results imply a more complex and sophisticated role of T cells in the regulation of B cell function in antibody production. OF B CELL FUNCTION IN ANTIBODYPRODUC~ION BY C. REGULATION MEDIATORS PRODUCED AND SECRETED BY T CELLS

The last possibility regarding T cell function in humoral immune responses, and the one we favor, is that T cells play an active regulatory role on B cell precursors of antibody-forming cells reacting with antigen. The expression of this regulatory effect would be mediated through soluble factors synthesized and secreted by T cells following antigenic stimulation. It may well be that all differentiated T cells responsible for a variety of cellular immune reactions mediate their function in large part through the release of nonspecific soluble factors. The elaboration of migration inhibitory factor (MIF) by T cells involved in delayed hypersensitivity reactions, which has been so elegantly demonstrated by David (221, 222) and Bloom and Bennett (223, 224), is exemplary of

48

DAVID H. KATZ AND BARUJ BENACERRAF

this point. We feel that elaboration of soluble mediators capable of affecting, in some way, the function of other cells of the immune system is a general property of specific T cells in contrast to the general property of B cells which is to differentiate into antibody-secreting cells. Since, as yet, no one has isolated or characterized a soluble factor produced by T cells which can be definitively shown to express all aspects of cooperative T cell function with respect to B cell responses to antigen, this discussion must rest to a certain extent on logical speculation. Nevertheless, the evidence which we interpret to argue in favor of this concept of T cell function in humoral immunity is rapidly mounting in a very compelling way. In many instances, certain experimental observations which support the concept were either puzzling to the reporting investigator( s ) or went unnoticed by them. We have taken obvious liberties, therefore, in placing our own interpretations on them. We have attempted to bring together appropriately related observations and/or experimental systems, and, therefore, the discussion has been divided in subsections covering the following: (1) the enhancement of immunocompetent cell function by GVH or comparable reactions, i.e., the “allogeneic effect”; (2) enhancement of immune responses by soluble factors released by T cells in culture; and ( 3 ) enhancement of antibody responses following nonspecific T cell stimulation.

I . Allogeneic Effect In the course of our studies of cell interactions in hapten-specific immune responses in guinea pigs, we noted that induction of a transient GVH reaction in otherwise perfectly normal and fully immunocompetent adult animals had a rather remarkable effect on several parameters of immune function (153, 225, 226). This phenomenon, which we have termed “allogeneic effect,” appears, in our opinion, to have great general relevance to the nature of the regulatory function of T cell activity on other cells, either B cells or other T cells, of the immune system. The characteristics of the allogeneic effect will be reviewed in some detail here. In the initial studies (225), it was observed that intravenous injection of lymphoid cells from inbred strain 2 guinea pig donors into DNP-OVAprimed allogeneic strain 13 recipients resulted in a significant rise in the levels of both anti-DNP and anti-OVA antibodies in the recipients’ serum (Fig. 3). This rise in antibody titers, which occurred spontaneously in the absence of any further exogeneous antigen challenge, began between the sixth and tenth day after allogeneic cell transfer, reached a peak by day 13 and then gradually diminished thereafter. An increase in total serum 7-globulins which was also observed during this time was of rela-

REGULATORY INFLUENCE OF ACI'IVATED T CELLS

200

1

'

I

I

1

'

1

'

1

'

SERUM ANTI-DNP ANTIBODY

I

'

1

I

'

I

is g 100Q

'

1

'

1

Stroin 2 Donors Stroin 13 Recipients-No Boost

P

a

1

TOTAL GAMMA GLOBULINS

d % - 140E \ p 120-

s

'

1

8)

80-

z

SERUM ANTIANTIBODY OVA

I

-

,? 60-

I= 2

a

40 -

-

20 0; '

h ' 11' L ' b

'

I d ' 16'

Ib' I;' DAYS AFTER TRANSFER

u 6

10

14

FIG. 3. Stimulation of antibody production as a result of transfer of immunocompetent strain 2 cells into primed strain 13 recipients in the absence of secondary antigenic challenge. 200 x 10" Strain 2 lymph node and spleen cells were injected intravenously into strain 13 recipients primed 3 weeks earlier with 2,4-dinitrophenyl ( DNP)-ovalbumin (OVA), Recipients were bled at various times after cell transfer without a subsequent antigenic challenge, and their sera were analyzed for anti-DNP and anti-OVA antibody and total y-globulin concentrations. The numbers in parentheses refer to the number of recipients analyzed at a given time after transfer. [These data from our laboratory appeared in J . Ex&. Med. 133, 169, 1971 ( reference 225) .]

tively small magnitude in comparison to the increase in specific antibodies. In addition to the spontaneous synthesis of both anti-DNP and antiOVA antibodies by such recipients in the absence of antigenic challenge, a striking secondary anti-DNP antibody response could be elicited by an appropriately timed, secondary challenge with DNP-BGG ( Fig. 4 ) (225). The crucial feature of the latter effect is that the requirement for carrier specificity is completely abrogated by the allogeneic effect. In a syngeneic cell transfer system, we had previously shown that DNP-OVAprimed guinea pigs developed enhanced secondary anti-DNP antibody responses to challenge with DNP-BGG provided they had received BGGprimed lymphoid cells from syngeneic donors; transfer of nonprimed

50

DAVID H. KATZ AND BARUJ BENACERRAF

STRAIN 13 RECIPIENTS -Strain

2 CFA Cells, No Boost

-

Strain 2 BGG Cells, No Boost

600 U

5

500-

6 400-

8'

88

z

2 0

300-

1

I

U

I-

I

5 200W

I

I

I I

W

c3

I

I

100 -

T

DAYS AFTER TRANSFER

FIG.4. Stimulation of anti-2,4-dinitrophenyl ( DNP) antibody production as a result of transfer of immunocompetent strain 2 cells into primed strain 13 recipients, showing the effect of secondary antigenic challenge with a DNP-heterologous conjugate 6 days after transfer. About 200 X loe strain 2 lymph node and spleen [ (CFA) or bovine y-globulin (BGG)] cells were injected intravenously into strain 13 recipients primed 3 weeks earlier with DNP-ovalbumin (OVA). Recipients were either not boosted or boosted with DNP-BGG 6 days after transfer. The numbers in parentheses refer to the number of recipients in a given group. The left panel illustrates the anti-DNP responses of recipients of CFA cells, whereas the right panel illustrates the anti-DNP responses of recipients of BGG-specific cells. [These data from our laboratory appeared in J . Exptl. Med. 133, 169, 1971 (reference 225) .I

lymphoid cells was ineffective ( 6 7 ) . In marked contrast, DNP-OVAprimed guinea pigs displayed dramatic secondary anti-DNP responses to DNP-BGG challenge administered 6 days following transfer of allogeneic lymphoid cells, irrespective of whether or not the allogeneic cell donors were primed with BGG (225). Hence, we have a situation in which the need for carrier-specific T cells has been apparently abolished. Indeed, this has been conclusively demonstrated in subsequent studies by Katz et al. (153)in which a secondary anti-DNP response has been elicited in DNP-OVA-primed guinea pig recipients of allogeneic cells following

51

REGULATORY INFLUENCE OF ACTIVATED T CELLS

challenge with a nonimmunogenic DNP copolymer. This compound, a DNP conjugate of the copolymer of D-glutamic acid and D-lysine (DNPD-GL), is normally very highly tolerogenic in guinea pigs, but in the presence of the allogeneic effect, it is capable of eliciting a secondary response comparable to that induced with DNP-BGG (Fig. 5). Since it is fair to assume that T cells specific for D-GL either do not exist or are not functionally significant, the allogeneic effect appears to have permitted the direct triggering of DNP-specific B cell precursors of antibody-forming cells by the nonimmunogenic DNP-D-GL. The mechanism of the allogeneic effect has been clearly established by our studies to be the result of a specific immunological attack of T ' 1779)

u

E

-

Strain 13 Recipients Strain 2 Donors MDNP-BGG Secondary Immunization *--b DNP-

D -GL Secondary Immunization

I

WEEKS AFTER PRIMARY IMMUNIZATION

7

1

II

DAYS AFTER SECONDARY IMMUNIZATION

FIG. 5. Stimulation of anti-2,4-dinitrophenyl ( DNP ) antibody production as a result of transfer of immunocompetent strain 2 cells into primed strain 13 recipients, showing the effect of secondary antigenic challenge with ( DNP)-bovine y-globulin (BCG) and DNP-copolymer of D-glutamic acid and n-lysine ( D-GL), About 550 X 10" strain 2 lymph node and spleen cells were injected intravenously into strain 13 recipients primed 3 weeks earlier with DNP-ovalbumin (OVA). Recipients were either not boosted or boosted with 1.0 mg. of DNP-BGG or DNP-D-GL in saline 6 days after transfer. Serum anti-DNP antibody concentrations just prior to secondary immunization and on days 7 and 11 are illustrated. The numbers in parentheses refer to the numbers of animals in the given groups. [These data from our laboratory appeared in J. Erptl. Med. 134, 201, 1971 (reference 153).]

52

DAVID H. KATZ AND BARUJ BENACERRAF

grafted cells on host cells (225, 226). This conclusion is based on the following observations. 1. Transfer of lymphoid cells from allogeneic or semiallogeneic donors under circumstances where the transferred cells are incapable of reacting against tissue antigens of the host fails to elicit the allogeneic effect. This is true despite the fact that the transferred cells, by virtue of possessing foreign histocompatibility antigen, elicit a specific rejection response on the part of the host. These points have been demonstrated by transferring into DNP-OVA-primed strain 13 recipients the following lymphoid cells (225): ( a ) L,C leukemia lymphocytes from strain 2 guinea pigs-these leukemia cells possess the strain 2 histocompatibility antigens but are immunologically incompetent; ( b ) strain 2 lymphoid cells which have been rendered immunologically incompetent by exposure to in vitro X-irradiation (3000 R ) ; and ( c ) lymphoid cells from ( 2 X 13)F, hybrid donors-these cells are immunologically competent but cannot mount a rejection response against the parental recipients. 2. Transfer of lymphoid cells from parental strain 2 donors elicits the allogeneic effect in ( 2 X 13)F, hybrid recipients which have been primed with DNP-OVA. In this situation, the parental lymphoid cells can mount a GVH reaction against the F, recipients, but the hosts are incapable of rejecting the parental cells (226). The data of these experiments are summarized in Table VI. Thus, the allogeneic effect phenomenon reflects the development in the lymphoid organs of the host of a specific GVH reaction. Moreover, the phenomenon is expressed irrespective of the concomitant development of a host rejection response. The degree to which the phenomenon influences antibody production is related to the following crucial factors : 1. The host must be primed with the antigen in question before the transfer of allogeneic cells. We have made several unsuccessful attempts to enhance primary anti-DNP responses in guinea pigs inoculated with allogeneic cells at various times prior to primary immunization (153,225, 226). This raises the possibility that a critical change related to antigenic stimulation must occur in the specific B cell population before such cells can be affected by the allogeneic effect. 2. The intensity of the allogeneic effect on antibody synthesis is related to the intensity of the GVH reaction induced. In guinea pigs, transfer of as little as 50 x lo6 allogeneic lymphoid cells enhanced the production of anti-DNP antibodies in recipients, but an almost linear increase in magnitude of the phenomenon was observed with progressively higher numbers of cells transferred (225). We also observed that donor cell inocula, consisting of both lymph node and spleen cells, were more potent in mediating the effect than an equivalent number of spleen cells

REGULATORY INFLUENCE OF ACTIVATED T CELLS

53

TABLE VI REACTION AS THE UNDERLYING MECHANISM OF ALLOGENEIC EFFECT IN GUINEA

GRAFT-VERSUS-HOST THE

No. and strain of DNP-OVA-primed recipien t s b

Type of donor lymphoid cells transferred

5 Strain 13

Nonirradiated strain 2 Nonirradiated strain 2 Irradiated (3000 R) strain 2 Strain 2 leukemia, nonirradiated (2 X 13)F1 hybrid, nonirradiated

5 (2 X 13)Fi 5 Strain 13 5 Strain 13

5 Strain 13

Increase in geometric mean anti-DNP antibody concentrations (pg/ml) from days 6 to 13 after cell transfer 728.4 461.3 -5.8

-23.1 -7.0

a This table represents a composite of dat,a from several studies of Katz et al. (2259 226). * These observations demonstrate that the allogeneic effect is mediated through the development of a graft-versus-host reaction and does not require the concomitant existence of a host rejection response. Strain 13 and (2 X 13)R hybrid guinea pigs were immunized intraperitoneally with 2,Cdinitrophenyl (DNP)-ovalbumin (OVA) in saline. Three weeks later allogeneic lymphoid cells of the types indicated were injected intravenously into individual recipients. Six days later, all recipients were boosted with DNP-bovine r-globulin (BGG). The data are expressed as the increase in geometric mean anti-DNP antibody concentrations from the day of secondary boost (day 6 after transfer) to 7 days later (day 13).

alone ( 2 2 5 ) . This superiority of lymph node cells is consistent with observations in the mouse that fewer lymph node cells than spleen cells are required to obtain a GVH reaction of a given magnitude ( 2 2 7 ) . Finally, the relationship between intensity of the GVH reaction and the magnitude of the allogeneic effect can be illustrated by varying the strength of histocompatibility differences between the strains of guinea pigs employed. Thus, when lymphoid cells from a random-bred line of guinea pigs, the NIH strain, are used to elicit the allogeneic effect in strain 2 recipients, a much higher cell number (1.0 X lo9) is required to obtain the same magnitude elicited by a moderate number (0.200 X lo9) of lymphoid cells from strain 13 donors ( 2 2 8 ) . 3. A very critical time relationship exists between the transfer of allogeneic cells and the administration of a secondary antigenic challenge with respect to the elicitation of an enhanced secondary antibody response. The optimal time interval between cell transfer and challenge is

54

DAVID H. KATZ AND BARU J BENACERRAF

6 days. Administration of antigen either at earlier or later times after transfer may result in little or no increase, or even some suppression in antibody production (225, 226). The relevance of the allogeneic effect with respect to understanding the regulatory function of T cells in antibody production derives from the observation that during the peak period of the GVH reaction the operation of normal helper T cell function is no longer required for anti-DNP secondary responses. Two possible explanations for this phenomenon may be entertained: (1) the GVH reaction may lead to a general proliferative response within the host T cell population among which are contained normally occurring carrier-specific helper cells, and active proliferation of these cells as a result of the GVH reaction could result in sufficient numbers of helpers available at the time of the secondary DNP challenge; ( 2 ) the GVH reaction may exert a facilitative effect of some sort on B cell precursors of antibody-forming cells and, perhaps, on antibody-forming cells themselves, which results in a fundamental change in such cells with respect to their ability to respond or, alternatively, on the nature of their response to antigen. It should be noted that we refer to B cell precursors of host origin, not of donor origin, since it has been established that the anti-DNP antibodies are made by cells of the host (226). It is the second of these possible explanations which is most consistent with the data and which places the greatest relevance on the allogeneic effect phenomenon insofar as normal regulatory function of T cells is concerned. The strongest evidence in support of an effect of the GVH reaction on B cells derives from the following two observations: ( 1 ) haptenspecific memory, presumably reflecting a B cell memory, is markedly enhanced when antigenic challenge is appropriately timed during later stages of the GVH reaction (226); and ( 2 ) antibody responses can be elicited with antigens for which presumably no T cells exist (or are nonfunctional ) when such antigens are administered during the GVH reaction (153). The first observation was made in the guinea pig allogeneic transfer system (226). As we mentioned earlier, secondary DNP-BGG challenge at very early or later (than 6 days) times after allogeneic cell transfer consistently resulted in little or no secondary anti-DNP response. Nevertheless, a striking change had occurred in the memory cell population; when such animals were subjected to a final antigenic challenge with the original immunizing antigen, DNP-OVA, the magnitude of anti-DNP antibody responses was always higher in recipients of allogeneic cells than respective controls (not receiving allogeneic cells ) . Hence, it appears that the allogeneic effect enhances not only antibody production

REGULATORY INFLUENCE OF ACTIVATED T CELLS

55

but also the development of memory in the host B lymphocyte population as well. The second observation concerns the induction of anti-DNP responses with DNP-D-GL (153).As described earlier, the DNP conjugate of this copolymer is nonimmunogenic and, indeed, highly tolerogenic in both strain 13 and strain 2 guinea pigs. Yet, when DNP-OVA-primed strain 13 guinea pigs were given allogeneic lymphoid cells and then challenged 6 days later with DNP-D-GLor DNP-BGG, striking secondary anti-DNP responses were obtained in both cases (Fig. 5). It is a fair assumption that, particularly insofar as strain 13 guinea pigs are concerned, few or no D-GL-specific T cells capable of performing helper function exist in these animals. Any manipulation which would permit the development of an anti-DNP antibody response to DNP-D-GL must reflect the exertion of some influence on B cells or their progeny, and this is precisely what we interpret to occur during the allogeneic effect. The capacity of B cells to be triggered by DNP-D-GL in the presence of the allogeneic effect exemplifies, therefore, the case we are making for T cell regulation of B cell function via nonspecific mediators. Since administration of DNP-D-GL to guinea pigs ( 153) or mice (228) not undergoing a GVH reaction leads to profound DNP-specific tolerance, it would appear that the T cell mediators play a very crucial role in B cell triggering. The allogeneic effect phenomenon is not restricted to guinea pigs. Hirst and Dutton (90)have reported that small numbers of allogeneic nonadherent spleen cells from normal donors enhanced the primary in uitro anti-SRBC response of spleen cells from neonatally thymectomized or normal mice. Schimpl and Wecker (96) have found that mouse spleen cells treated with anti4 serum and complement can be restored to respond in uitro to SRBC by addition of allogeneic thymocytes capable of recognizing the T-deprived spleen cells as histoincompatible. EkpahaMensah and Kennedy (229) reported enhancement of the primary in vitro anti-SRBC response of normal mouse spleen cells by the addition of irradiated allogeneic spleen cells. Recently, we have elicited an in vivo allogeneic effect in mice ( 2 3 0 ) .Thus, DNP-KLH-primed CAF, mice injected intravenously with spleen cells from normal parental A strain donors displayed markedly enhanced secondary anti-DNP antibody responses to a challenge with DNP-BGG administered 6 days after cell transfer. The phenomenon has also recently been demonstrated in mice by Ordal and Grumet (231) working with C,H( Hzklk), congenic C,H * Q( HDq/q),and F, ( Hzk/q) mice which are all genetic nonresponders to (T,G)-A--L. These nonresponders normally produce only 19 S antibodies to one or more challenges with (T,G)-A--L (163). However, non-

56

DAVID H. KATZ AND BARUJ BENACERRAF

responder F, ( HZk/q)mice displayed enhanced antibody responses, and of the 7 S class, when nonresponder parental C,H( HzkIk)lymph node and spleen cells were injected intravenously on the same day as antigen administration ( 231 ). This observation precisely confirms our findings with DNP-D-GL in guinea pigs (153) and reflects the release of T cell mediators during the GVH reaction which permit or facilitate triggering of B cells by antigen. There are other recently reported findings which we interpret as reflecting phenomena related to the allogeneic effect insofar as the concept of T cell factors regulating B cell function is concerned. The one which is most relevant in this regard is that of Hartmann (91 ) in studies of the in vitro antierythrocyte response of mouse bone marrow-derived cells. The experimental design has been described in detail in Section II1,A. In brief, he found that the capacity of B cells to respond in vitro to SRBC could be restored by the addition of educated T cells. When only one in uitro immunogen was employed, the response of B cells occurred only if the T cells had been specifically educated to that immunogen. Thus, B cells developed an in uitro antibody response to SRBC when SRBC-educated T cells were added but not when HRBC-educated T cells were used instead. However, if both SRBC and HRBC were added as in uitro immunogens to mixtures of B cells with SRBC- or HRBCeducated T cells, then PFC specific for both erythrocytes were produced. Hence, T cell stimulation by the specific antigen to which the population had been primed permits triggering of B cells to develop an antibody response to another, non-cross-reacting antigen. We consider this observation to be analogous in interpretation to the allogeneic effect phenomenon. Other studies which we interpret in a similar fashion include: (1) the observation by Sulica et aE. (232) that suspension cell cultures of spleen cells from rabbits immunized with DNP-BSA produced antiDNP antibodies when exposed in vitro to the carrier, BSA, alone; ( 2 ) the report by Rubin and Coons (233) that spleen cells from mice which had previously been immunized with an antigen, such as tetanus toxoid or ovalbumin, developed significantly enhanced primary in vitro antibody responses to SRBC if the priming antigen was also added to the culture; ( 3 ) the observation by Storb and Weiser (234) that adoptively transferred spleen cells developed higher anti-SRBC responses in irradiated allogeneic recipients than in irradiated syngeneic recipients; and (4)the studies of Moller (235) on the suppressive effect of the GVH reaction on primary anti-SRBC responses in mice, showing that, in certain H-2 histocompatibility combinations, transfer of parental spleen cells into adult F, recipients resulted in enhanced rather than suppressed antibody responses.

REGULATORY INFLUENCE OF ACXIVATED T CELLS

57

In conclusion, we propose that the normal role of T cells in antibody responses is to exert a regulatory influence on B cell precursors of antibody-forming cells with respect to their reactivity to antigen. This regulatory influence is required or not required depending on the physicochemical state and quantity of the antigenic moiety when it is capable of interacting with the specific B cell. Most of the antigens with which we are familiar require the existence of a regulatory influence for optimal, or any, B cell response to become manifest; only a few molecules with unique physicochemical structural features appear to act on B cells independently of such a regulatory influence (see Section IV,A). The regulatory influence of T cells on B cells is, we believe, mediated through soluble factors which are not yet defined. These soluble factors are not antigen-specific and most likely act rapidly and have a short half-life. Although the factors themselves may be nonspecific, the T cells which synthesize and secrete them are, indeed, antigen-specific. We propose that under normal circumstances specific antigenic stimulation of T cells must occur to signal the elaboration of mediators by such cells. The release of such factors in the microenvironment of specific B cells exerts a regulatory influence on these cells insofar as their immediate response to antigen is concerned. For the sake of simplicity of the model, we shall consider, at this point, only a positive regulatory effect of such factors on B cells, i.e., the factor facilitates or permits triggering of the B cell by antigen. The possible existence of T cell mediators with the opposite effect will be dealt with in a later section. This model is particularly appealing to us because it allows for specificity restriction among T cells and does not create an unlikely or unique functional requirement upon such cells, i.e., as discussed earlier, we know that T cells responsible for other immune reactions perform their role via released mediators. It is most logical then to assume a similar situation exists for T cells performing helper function in antibody responses. Indeed, in a recent paper Jimenez et al. (235a) suggest that carrier-specific helper T cells are identical to delayed-type hypersensitivity T cells. Furthermore, the model relieves the T cell from an unlikely passive participation serving only to concentrate and present antigen to B cells, which, in essence, represents a wasteful duplication of macrophage function. 2. Soluble Factors Released by T Cells in Culture Several investigators have reported findings suggesting the release of a soluble factor from T cells active in antibody production. Haskill et al. (200) studying in vitro anti-SRBC responses of density-gradientseparated mouse spleen cells found that cells of the light-density region

58

DAVID H. KATZ AND BARUJ BENACERRAF

of the gradient could be stimulated to respond to SRBC in culture by addition of cells from the heavier-density region or by thymus cells even when the two populations were separated by a dialysis or Nucleopore membrane. The effect, however, was quite variable. Kennedy et al. (236) reported that a supernatant obtained from peritoneal exudate lymphocytes exposed to gentle heating was active in permitting B cells to develop an antiburro erythrocyte (BRBC) response upon adoptive transfer to irradiated recipient mice. This heated cell supernatant was antigenspecific but not species-specific, i.e., the supernatant was active only if obtained from cells of donors specifically immunized with BRBC, but either mice or rats could serve as peritoneal exudate cell donors. Furthermore, the supernatant itself was not immunogenic. The authors suggest that the active factor is most likely some form of antibody, but no studies have been reported on physicochemical characterization of the supernatant. Dona et al. (237) were able to demonstrate that a cell-free medium of thymus cell cultures were effective in restoring the primary in vitro anti-SRBC response of spleen cells from neonatally thymectomized mice. Thus, thymus cells were incubated alone or in the presence of SRBC for 24 to 40 hours; the medium obtained from such cultures was separated from all cells by centrifugation and then added to spleen cells of thymectomized mice in culture with SRBC. The capacity of such cells to respond to SRBC was restored by addition of the T cell medium irrespective of whether or not T cells had been exposed to SRBC in vitro. Gorczynski et al. (238) have also successfully restored the capacity of T cell-depleted mouse spleen cells to develop primary in vitro anti-SRBC responses with a soluble factor released by cultured T cells. In their system, SRBC-educated T cells (derived from spleens of irradiated recipients of syngeneic thymus and SRBC) were cultured for 3 days with SRBC, after which they were gently heated (48°C X 30 minutes) and removed by centrifugation. This heated cell culture supernatant was active in reconstituting the response of mouse spleen cells treated with anti-8 serum and complement to approximately 50%of the normal control response. In contrast to the results of Doria et al. (237), active supernatants were obtained only from T cell cultures containing SRBC. Moreover, the active factor was not dialyzable in contrast to results of Haskill et al. (200). In studies of the enhancement of in vitro antibody responses by the allogeneic effect, investigators in at least three laboratories have preliminary evidence for a soluble factor active in these systems, Thus, Dutton et al. (239) have obtained a cell-free supernatant from 24-hour mixtures of allogeneic spleen cells which can enhance the in vitro anti-

REGULATORY INFLUENCE OF ACTIVATED T CELLS

59

SRBC response of both normal and T cell-deprived spleen cells. EkpahaMensah and Kennedy (229) reported enhancement of primary in vitro anti-SRBC responses of normal mouse spleen cells by addition of irradiated histoincompatible spleen cells. Moreover, they state that the anti-SRBC response of spleen cells can be stimulated by mixtures of allogeneic lymphoid cells on the opposite side of a 0.5-pm. Nucleopore membrane. Similar preliminary observations have been made in our laboratory in studies of the secondary in vitro anti-TNP antibody response of mouse spleen cells cultured in double-chamber vessels separated by 0.2-p. Millipore filters ( 2 2 8 ) .Thus, the anti-TNP secondary response of spleen cells on one side of the vessel can be enhanced by the appropriate mixture of allogeneic cells on the opposite side of the Millipore filter (Table VII). It must be stressed that all of the above observations should be interpreted with some degree of caution. In vitro antibody responses may be subject to wide variability determined by many unknown factors concerning culture conditions. 3. Nonspecific Stimulution of T Cell Function

In this category we shall discuss the agents that exert a stimulatory influence on T cells irrespective of the antigen specificity of their receptors. A variety of substances are known to do this, perhaps chief among them being the mitogenic agents phytohemagglutinin (PHA ), pokeweed mitogen, and concanavalin A (Con A). It is known that some plant mitogens can induce in lymphoid cells many of the morphological changes characteristic of antigenic stimulation (240, 241 ). It is generally accepted that specific receptors exist for these substances on the surface membrane of T lymphocytes. However, in contrast to antigen-specific receptors, no clonal restriction exists for these mitogens and, therefore, most if not all T cells can be stimulated by them. There are several reports in the literature demonstrating an enhancing effect of mitogens in antibody production. Tao (242) observed that lymph node fragments from rabbits primed with BSA or human chorionic gonadotropin developed anamnestic antibody responses in vitro when exposed transiently to PHA at the beginning of culture. Makela and Pasanen (243) found that NIP-CGG primed mouse spleen cells adoptively transferred to syngeneic irradiated recipients could be stimulated to some extent to produce anti-NIP antibodies by the intraperitoneal administration of pokeweed mitogen. The degree of stimulation by the mitogen, although only 1/ 1500 the magnitude of the antigen-induced secondary response, was significantly higher than nonstimulated control values. It is highly possible that in both of these models, small traces of priming antigen were carried over to either the in vitro culture or to the

6,

0

EFFECT O F ALLOGENEIC INTER-4CTIONS No antigen chamber

Spleen cellsb

Anti-TNP PFC/IOO (direct)

TNP-burro

731 970 598

3

TNP-bumo

526 684 557

5

TNP-burro C57B1

220 93

1

+

TABLE VII O N HAPTEN-SPECIFIC I M M U N E RESPONSES in vitrOD

Geom. Ave. 751+--

TNP-Burro RBCc chamber

Spleen cells

2

0

585 +--+

4

TNP-burro

1.43

6

TNP-bumo

-

Anti-TNP PFC/lW

Geom. Ave.

4

0 0 0

0

1480 1320 928

1219

2440 2110

U

sU

9 w >

2253

~

This table represents data from our laboratory (228) which illustrates the capacity of allogeneic cell interactions on one side of a cellimpermeable membrane (chamber 5) to enhance the specific antihapten response of cells on the other side of the membrane (chamber 6) most probably as a result of active soluble mediators. Cells were cultured in doublechambered vessels separated by ultrathin Millipore filters (pore size, 0.20 p ) . Opposing chambers are indicated by arrows (-). Numbers of anti-trinitrophenyl (TNP) plaque-forming cells (PFC) in individual cultures and geometric averages of respective culture groups are presented. Shtistical analysis by student’s t test: comparison of chambers 4 and 6 yielded a P value of 0.05 > P > 0.02. * Dissociated spleen cells were obtained from BALB/C mice primed 7 days earlier with 1 X lo8 heavily conjugated (TNP)-burro erythrocytes and from nonimmune C57B1/6N mice; 1 X lo7 cells/ml. were cultured in each chamber. Chamber 5 contained 0.5 x 10’ of each cell type. Chambers 1, 3, and 5 contained primed cells without in vitro antigen, whereas chambers 2,4, and 6 contained primed cells plus in vitro immunogen. In vitro immunogen was 1 X 105 heavily conjugated TNP-burro erythrocytes.

8

4 2: tr

m

$

REGULATORY INFLUENCE OF ACTIVATED T CELLS

61

irradiated recipient. Such small quantities of antigen, although insufficient on their own to induce antibody formation, can do so in the presence of a sufficient concomitant T cell stimulation which is exerted by the added nonspecific mitogen. An intriguing observation of Makela and Pasanen ( 2 4 3 ) which they could not explain was that incubation of primed donor cells at 37°C in vitro for a short time before adoptive transfer resulted in a significant synthesis of anti-NIP antibodies in recipients in the absence of any antigenic or mitogenic stimulation; this did not occur if the cells were kept at 0°C in vitro. In the context of recent observations on movement of B cell membrane receptors (244,245), it is likely that at 37°C stimulation of B cells by small traces of antigen in the suspension could have occurred in vitro prior to transfer. More recently, Rich and Pierce ( 2 4 6 ) have significantly enhanced in vitro responses to SRBC with Con A. Thus, submitogenic doses of Con A either administered in vivo to donor mice or added to spleen cell suspensions in vitro results in an increase up to tenfold in the magnitude of the in vitro anti-SRBC response. Recent observations of Katz and Unanue ( 2 4 7 ) are also relevant in this regard. When spleen cells from HRBC-primed mice are cultured in vitro in the presence of small quantities of ALS they develop antigenspecific yM and yG PFC in vitro responses in the presence of small quantities of soluble HRBC immunogen. In addition, there is also an enhancement of the secondary in vitro response of such cells to particulate HRBC antigen when cultured in presence of ALS. The action of ALS appears to be a stimulatory influence on T cells since the effect is abrogated by treatment of donor spleen cells with anti4 serum and complement. A further observation of considerable interest is that transient exposure of the HRBC-primed spleen cells to anti-Ig or anti-Fab serum prior to culture significantly increases the magnitude of both soluble antigen-induced antibody production and enhanced particulate antigen-induced secondary response resulting from ALS activity. However, exposure of the cells to only anti-Ig or anti-Fab has no effect ( 2 4 7 ) . We interpret the above data ( 2 4 7 ) as suggesting the following sequence of cellular events leading to antibody production: T and B lymphocytes can be stimulated to perform their respective functions in antibody production by an appropriate immune reaction on their surface membranes, usually related to the clonally-restricted antigen-specificity of their receptors. However, an additional immune reaction of anti-Ig or anti-Fab with Ig receptors on B cells may significantly enhance the triggering event of such cells to produce their specific antibodies, provided an essential regulatory influence exerted by mediators elaborated by T cells is operating. It would appear also that T cells can be stimulated to

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DAVID H. KATZ AND BARU J BENACERRAF

release their mediators by the occurrence of a similar reaction on their membrane as shown by the enhancing activity of ALS on these cells. VII. Suppressive Effects of

T Cells on Antibody Synthesis

Heretofore our discussion has been concerned with the positive regulatory influence of T cell function of B cell precursors of antibodyforming cells. It is rapidly becoming clear that the regulatory function of T cells consists also of a negative aspect with regard to antibody production. This need not imply the existence of distinct T cells responsible for the respective roles of either enhancing or suppressing B cell function. It is equally possible that the same T cell may express either of these regulatory influences via the same or distinct mediators, the difference in outcome being determined by (1) the relative concentration of T cell products released in the microenvironment of B cells, ( 2 ) the range at which these mediators are active and their respective half-lives, and (3) the amount and physicochemical properties of effective antigen. In this section we shall consider the evidence suggesting the existence of a suppressive T cell influence on antibody formation under the following experimental conditions: ( 1) enhancement of immune responses by measures which diminish the number of T cells and ( 2 ) suppression of antibody responses by the administration of more than one antigen (antigenic competition).

A. ENHANCEMENT OF IMMUNE RESPONSES BY DEPLETION OF T CELLS Several studies have clearly demonstrated that in vivo administration of ALS or antilymphocyte globulin ( ALG) may cause enhanced antibody responses to subsequent immunization with antigen. Baum et al. (248) found that rats pretreated with lymphocytopenic doses of ALG developed primary antibody responses to KLH that were sixteen-fold higher in magnitude than saline or normal rabbit globulin-treated controls. In contrast, the same dose of ALG abrogated the capacity of another experimental group to develop primary anti-SRBC responses. Baker et d.(156) reported that mice treated with ALS developed primary PFC responses to Type 111 pneumococcal polysaccharide ( SIII) which were ten-fold higher in magnitude than controls not treated with ALS, although there was no significant difference in serum hemagglutinin titers between the nontreated and ALS-treated animals. Previous studies have demonstrated that the response of mice to SIIIconsists of the development of two types of y M PFC in equal numbers, i.e., direct PFC and indirect PFC which can be developed only with appropriate anti-yM sera ( 249). It is interesting that Baker et al. (156) found that the increased PFC responses in ALS-treated mice were restricted to the direct PFC.

REGULATORY INFLUENCE OF ACTIVATED T CELLS

63

The usual effect of ALS treatment in vivo is to depress the humoral response to a variety of antigens (250). Its immunosuppressive activity has been shown to be due to a preferential elimination of thymus-derived recirculating small lymphocytes ( 251 ) . The implication of the studies cited above (156, 248) is that under certain circumstances removal or diminution of T cells permits greater antibody production that occurs in their presence. This reasoning is easily extended to the concept that T cells exist which exert a suppressive regulatory influence on B cells. Alternatively, we must consider also the possibility that the enhanced antibody responses observed following ALS treatment reflects the release from destroyed cells of large amounts of nucleic acids which may then cause an adjuvant effect such as we shall discuss in Section VII1,C with respect to synthetic polynucleotides. This possibility has been cited to explain the enhanced antibody production sometimes observed following whole-body X-irradiation ( 252, 253) or the administration of cytotoxic agents ( 254-256). A final possibility to explain the enhancing effect of ALS is that this reagent may be exerting a stimulatory influence on T cell function. It is well established that in the appropriate dose range ALS may have a marked stimulatory effect on lymphoid cells. Many studies have shown that ALS stimulates blast transformation and DNA synthesis of lymphoid cells in culture (257-261 ). Moreover, ALS has been found to enhance response of lymphoid cells to other antigens in culture. Thus, Greaves et al. (261) observed that addition of submitogenic doses of ALS to cultures of PPD-sensitive human leukocytes significantly enhanced the DNA synthetic response of such cells to PPD. More recently, as reported in Section VI,C, Katz and Unanue (247) have demonstrated that the response of primed mouse spleen cells to antigen in vitro can be markedly enhanced by the exposure of such cells to small quantities of ALS and antiimmunoglobulin. It is possible that a similar mechanism operating in vivo could explain the results of Baum et aZ. (248) and Baker et al. (156). However, we are inclined to believe that this is not the explanation for their findings and favor the explanation based on elimination of T cell function. Recent observations which support this explanation will now be given. Baker et aZ. (262) have recently extended their observations of ALS enhancement of antibody responses to SrIr in mice. The experiment was designed to study the effect of various types of passively transferred syngeneic lymphoid cells on the enhanced responses to SrII displayed hy ALS-treated mice. They found that passively transferred thymocytes suppressed the anti-SIIIresponse, whereas peripheral blood lymphocytes further enhanced the response. Spleen cells had no effect. The authors

64

DAVID H. KATZ AND BARUJ BENACERRAF

interpret these data as indicating the existence of two functionally distinct cells-one in the thymus which suppressed the response and another in peripheral blood which amplifies the response. Perhaps analogous is the observation of Armstrong et al. (147) who found that the addition of thymic lymphocytes to thymus-deprived mice depressed the antibody responses of such mice to the thymus-independent antigen, POL. Perhaps more intriguing are the observations of Okumura and Tada (263) in studies on the formation of homocytotropic antibody (HTA) in rats immunized with DNP-Ascaris (As). These investigators found that thymectomy or splenectomy in adults 3-10 days before primary immunization with DNP-As resulted in greatly enhanced and prolonged HTA titers in such rats compared to controls, A similar result was obtained with ALS administration ( 2 6 4 ) .These manipulations had no effect on the yM and yG anti-DNP antibody responses, however. The data have added relevance inasmuch as neonatally thymectomized rats are markedly reduced in their capacity to develop anti-DNP HTA responses to DNP-As ( 2 6 3 ) .In the same system, it has also been observed that totalbody X-irradiation (265) and certain immunosuppressive drugs (266) cause enhanced HTA production, although the interpretation of these data is complicated by additional considerations concerning the effects of such treatments. The enhancing effects of thymectomy provide the clearest support of all for the possible existence of a suppressive regulatory T cell function. This is further strengthened by published observations of Okumura and Tada (267) that passively transferred thymocytes from hyperimmune donor rats terminate preexisting anti-DNP HTA production in recipients. Finally, Kerbel and Eidinger (268) recently reported results of a very detailed study on the effects of adult thymectomy and/or ALS administration on primary antibody responses of mice to a variety of antigens. On the one hand, the antibody responses to erythrocyte antigens of mice treated with ALS alone or by a combination of adult thymectomy plus ALS were markedly suppressed; the responses to KLH and PVP, on the other hand, were considerably enhanced by treatment with ALS alone or combined with adult thymectorny. These authors monitored the serum antibody levels over 25 days, in contrast to Baum et at. (248), and observed that after 15 days the treated mice manifested a suppression in their anti-KLH titers as compared to controls. Moreover, the enhancement of antibody production by ALS alone or in combination with thymectomy was restricted to anti-KLH antibodies of the yM class ( 2 6 8 ) . Indeed, as the antibody response shifted from the yM to the yG phase, an actual suppression could be attributed to the treatment. This brings up a very important point to consider inasmuch as all of the examples of

REGULATORY INFLUENCE OF ACTIVATED T CELLS

65

ALS enhancement described above, with the exception of HTA formation in rats, have the common feature of being related to synthesis of antibodies predominantly of the yM class. It may well be that the suppressive regulatory T cell function is related to promotion of early yG antibody synthesis which, in turn, exerts a feedback type suppression on yM antibody synthesis. OF ANTIBODYRESPONSES BY THE ADMINISTRATION OF B. SUPPRESSION MORETHANONEANTIGEN ( ANTIGENICCOMPETITION )

The phenomenon of antigenic competition, first recognized by Michaelis (269) three-quarters of a century ago, occurs when an animal is immunized with two antigens simultaneously or in close sequence resulting in a depressed antibody response to one or both antigens [for review, see Adler (270)l. The underlying mechanism has been the subject of much study but is still not known. However, recent investigations have provided evidence which favors the existence of soluble factors acting in this phenomenon. It is relevant, therefore, to consider certain aspects of the phenomenon in this review, since we believe that such soluble inhibitory factors responsible for antigenic competition are most likely elaborated by T cells and are related, if not identical, to the T cell mediators active in other regulatory influences on antibody production. Evidence for the existence of a soluble factor active in antigenic competition was first advanced by the studies of Radovich and Talmage (271). They demonstrated that injection of mice with HRBC several days before primary immunization with SRBC resulted in a suppressed anti-SRBC response; no suppression occurred when the two antigens were administered simultaneously. Furthermore, they demonstrated that competition between antigens given to irradiated recipients of spleen cells increased proportionately with the number of spleen cells transferred. Moller and Sjoberg (272) confirmed and extended these observations. Employing an adoptive transfer system in mice, they found that spleen cells from HRBC-primed donors developed adoptive primary anti-SRBC responses in irradiated recipients which were comparable in magnitude to the responses obtained with normal donor cells, indicating that the number of antigen-sensitive cells to SRBC was not appreciably affected by the HRBC priming. They also demonstrated that adoptively transferred normal spleen cells into irradiated recipients which had previously been primed with HRBC resulted in marked suppression of the response to SRBC given at the same time as cell transfer (272). Analogous results were obtained when the transferred spleen cells were derived from donors which had been preimmunized with SRBC. These findings indicate that differentiation and proliferation of precursors into antibody-

66

DAVID H. KATZ AND BARUJ BENACERRAF

forming cells have been impaired, The same observation has been reported by Waterston (273). Brody and Siskind (274) studied the phenomenon in a haptenic system in rabbits. They found that competition between two haptens was equally pronounced whether haptens were attached to the same or different carriers-this argues against competition for a common antigensensitive cell. Moreover, the affinity of the antibody produced in antigenic competition is approximately equal to the antibody produced by animals immunized with only one antigen, suggesting that neither tolerance nor antibody suppression operated in the phenomenon. They also observed that antigenic competition only occurred when both antigens are injected so as to drain into the same regional lymph node (274). The latter point is of some controversy since it superficially conflicts with results of other investigators (275-277). However, differences in route and mode of immunization may explain the discrepancies in some instances (275), whereas the method of determining the immune response may explain others (276,277). Hence, Fauci and Johnson (276, 277) studied antigen competition by determining the PFC response in regional lymph nodes of rabbits. They found that local lymph node PFC responses to p-arsanilic acid ( An)-KLH injected into front and hind footpads on one side were significantly suppressed by simultaneous injection of TNP-KLH into the contralateral footpads. They interpreted these observations as indicating the presence of a circulating inhibitory factor. It is not possible, however, to determine whether or not their data reflect a migration of the specific PFC out of the regional lymph nodes. They further observed a diminished anti-TNP PFC response in local lymph nodes of rabbits that had been preimmunized with soluble KLH prior to footpad immunization with TNP-KLH (277). This finding may be analogous to the “preemption” phenomenon of O’Toole and Davies (278) whereby mice immunized with SRBC intraperitoneally manifest anti-SRBC PFC responses which were depressed in the local lymph nodes draining the site of a subsequent subcutaneous injection of SRBC. In our own laboratory, we have recently observed marked inhibition of secondary anti-DNP responses in guinea pigs which have been simultaneously immunized with two unrelated carrier molecules (279). Thus, DNP-OVA-primed guinea pigs supplementally immunized with BGG in adjuvant develop markedly enhanced secondary anti-DNP responses to DNP-BGG secondary immunization. However, this enhanced secondary response can be either diminished or abolished by the following manipulations of this system: ( 1 ) simultaneously administering KLH with BGG in the supplemental immunization diminishes the anti-DNP response by 91%; ( 2) administering soluble BGG intraperitoneally 1-2 days before secondary DNP-BGG challenge completely abolished the response re-

REGULATORY INFLUENCE OF ACTIVATED T CELLS

67

gardless of whether BGG alone or BGG together with KLH were used in the supplemental immunization; ( 3 ) administering soluble KLH 1-2 days before secondary challenge with DNP-BGG inhibits the response by 98%,provided KLH was administered simultaneously with BGG in the supplemental immunization. Perhaps most relevant was the finding that a nonimmunogenic amino acid copolymer, D-GL, failed to cause any inhibition of the anti-DNP response as was observed with the immunogens KLH and BGG ( 2 7 9 ) . We interpret these observations to indicate the existence of soluble inhibitory mediators in the phenomenon and, furthermore, that such mediators are most likely elaborated by carrier-specific T cells. The fact that the greatest degree of suppression occurred when the specific carrier (BGG) was used may argue against an explanation based on a general shift in frequency of specific helper T cells such as recently proposed by Kerbel and Eidinger ( 2 8 0 ) . The best evidence for a critical role of T cell-released inhibitory substance derives from the recent studies of Gershon and Kondo (281, 282). These authors found that the phenomenon of antigenic competition was thymus-dependent. Thus, antigenic competition elicited by sequential immunizations with SRBC and HRBC did not occur in thymus-deprived mice. Moreover, passively transferred normal thymocytes restored the competition phenomenon and the magnitude of inhibition was related to the numbers of thymocytes transferred (281).They also observed that antigenic competition developed irrespective of circulating antibodies specific for the competing antigen ( 2 8 2 ) .They interpreted their findings as strongly suggestive of an inhibitory factor released by T cells which seems valid in our opinion. There is even some very recent evidence that the inhibitory effect can be overcome to some extent by administration of polynucleotides ( 2 8 3 ) . VIII. Functions of T and B lymphocytes in Various Immunological Phenomena

A. IMMUNOLOGICAL TOLERANCE The establishment of T and B cell interactions in humoral immune responses demanded a reappraisal of the conceptual and experimental approach to the study of immunological tolerance. Since this review cannot consider at length the various parameters and details of tolerance induction, the reader is referred to a recent review on the subject by Dresser and Mitchison ( 2 8 4 ) .

1. Target Cells for Tolerance Induction The most immediate question concerns which cell, T or B lymphocyte, is the target for tolerance induction. It was clear very early that T cells could manifest a specific tolerant state and, thereby, a specificity restric-

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tion in this population is shown. Thus, Isakovic et al. (285) demonstrated that thymus grafts from BGG-tolerant rats transferred the specific tolerance to thymectomized irradiated recipients. In mice rendered tolerant by repeated injections of SRBC, Gershon et al. (286) found a reduced T cell mitotic response to SRBC. Taylor (61, 287) demonstrated that T cells of mice made tolerant to BSA were unable to cooperate with normal B cells in an adoptive transfer response in irradiated recipients. Miller and Mitchell (288) induced tolerance to SRBC in mice with cyclophosphamide and observed that recirculating T cells in the thoracic duct lymph (but not in thymus) were specifically tolerant. Paul et al. (67) demonstrated that carrier-specific T cells could be rendered specifically tolerant thereby abolishing their helper function in secondary antihapten antibody responses. Whereas tolerance in T lymphocytes could be readily demonstrated, it was much more difficult to establish the existence of tolerance in B lymphocytes. In the experiments above of Taylor (61, 287) and Miller and Mitchell (288), B cells from tolerant animals were able to cooperate with normal T cells in adoptive transfer responses. Playfair (289) used higher doses of SRBC plus cyclophosamide to induce tolerance in mice and found that B cells were transiently tolerant. Gershon and Kondo (290) observed that B cells could be rendered tolerant by repeated injections of SRBC. Chiller et al. (62) found that tolerance existed in both B and T cells of mice rendered tolerant by a single injection of deaggregated HGG. More recently, techniques have been devised to induce a high level of hapten-specific tolerance which probably reflects a selective state of B cell tolerance (153-155, 291). The very elegant experiments of Chiller et al. (292) have elucidated the critical kinetic differences in tolerance induction of T and B lymphocytes and have placed a much clearer perspective on this matter. Their experimental design was based on the adoptive transfer response to HGG in irradiated syngeneic recipients with T and B lymphocytes from normal donors and/or donors made tolerant to HGG by a single injection of deaggregated HGG. They found a marked difference between T and B lymphocytes with respect to both kinetics of tolerance induction and the dose of tolerogen required. As shown in Fig. 6, T cells were rendered tolerant very early ( 2 days) after administration of the tolerogen and remained tolerant for a very long period (77 days), In contrast, B cells did not exhibit the tolerant state until later (11 days) after tolerance induction and recovered much earlier (49 days) than T cells. The crucial point is that the immune state of the whole animal reflected the immune state of the respective T cells. In other words, although at a given time the B cells were normally immunocompetent, the existence of tolerance in

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-

69

100

n n Y

.-n c Y

-

0 Y n

c

= E

30

, i A-A

1

14

21

DH66 Injecttd Donor

28

35

42

49

Days Following Tolerogon

FIG.6. Kinetics of tolerance induction and spontaneous loss of unresponsiveness in thymus and bone marrow cells. Mice were rendered tolerant with deaggregated human y-globulin (DHGG) administered as a single intravenous dose of 2.5 mg. At various times thereafter, groups were sacrificed and suspensions of their thymus or bone marrow cells were injected intravenously along with normal bone marrow cells or thymus cells, respectively, into lethally irradiated recipients. At the same time and again 10 days later these recipients were challenged with 0.4 mg. of aggregated HGG, and 5 days after the second injection their spleens were assayed for plaque-forming cells to HGG. The results are presented as percent unresponsiveness in the respective cell types. [Taken from Chiller et al., Science 171, 813, 1971 (reference 292); we thank Drs. Chiller, Habicht, and Weigle for permission to reproduce this figure which they made available to us.]

the T cell population reffected a tolerant state of the whole animal. Moreover, tolerance induction was found to occur at much lower doses of tolerogen in the T cell population than in the B cell population (Fig. 7 ) . These observations (292) clearly show that both B and T cells can be rendered immunologically tolerant and also explain the apparent discrepancies in earlier studies.

2. Fate of Tolerant Cells What happens to a tolerant cell? Does it exist in a functionally unresponsive or unrecognizable state or is it eliminated from the system? Here again, the available evidence may appear conflicting in that some investigators have demonstrated specific antigen-binding cells still present in relatively normal amounts in tolerant animals (7, 293-295), whereas others have observed diminished numbers of such cells (153, 170, 296). Perhaps the most logical explanation for these differences can be based on the different target cells involved in the various systems studied and the degree of specific tolerance existing at the time cells are

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DAVID H. KA'IZ AND BARUJ BENACERRAF

0

1

WT

NBM

I

TT

NBM

NT TIM

FIG.7. The effect of varying the dose of tolerogen on the induction of unresponsiveness in thymus and bone marrow cells. Each point represents the arithmetic mean of the individual response [plaque-forming cells ( P F C ) ] obtained in 6 mice. NT, NBM-thymus and bone marrow, respectively, obtained from normal donors; TT, TBM-thymus and bone marrow, respectively, obtained from tolerogen-injected 0.5 mg. ( B ) , or 2.5 mg. ( A )of deaggregated donors which received 0.1 mg. ,).( Both thymus and bone marhuman gamma globulin 11 days prior to sacrifice. (0) row cells obtained from normal donors. [Taken from Chiller et al., Science 171, 813, 1971 (293); we thank Drs. Chiller, Habicht, and Weigle for permission to reproduce this figure which they made available to us.]

examined. Hence, where tolerance exists predominantly among T cells, it would be expected to find relatively normal numbers of specific antigen-binding cells (presumably representing B cell precursors of antibody-forming cells), Another important factor is the existence of antigenbinding cells in a tolerant animal in which some degree of B cell tolerance exists; in such a circumstance the antigen-binding cells may be predominantly of low-affinity receptor type, whereas the high-affinity cells may be significantly diminished. Previous studies have demonstrated, indeed, that induction of tolerance results in preferential diminution of high-affinity antibodies (297, 298). Ideally, studies of this type should be employed in a model where tolerance is more or less restricted to the B cell population. Since the threshold of tolerance induction ir, T cells is indeed lower than it is in B cells (292), at least insofar as protein antigens are concerned, it is difficult to obtain a selective B cell tolerance in vivo once the T cells have already been tolerized. Recently, however, several investigators have reported the successful induction of hapten-specific tolerance which may reflect such a state of restricted B cell tolerance (153-155, 291, 299). We define hapten-specific tolerance as the specific suppression of antihapten antibody responses to conjugates of a given hapten resulting from

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the prior admininstration of the haptenic determinant alone or of the hapten conjugated to an unrelated carrier. The induction of tolerance to hapten-carrier conjugates has been described by several investigators ( 117, 300, 301 ) where, in general, impaired antihapten antibody responses were restricted to the hapten-carrier conjugates used for tolerance induction. The tolerant animals displayed either no suppression or only transient decrease of antihapten responses when challenged with the hapten coupled to a carrier unrelated to that used in the paralysisinducing regimen. Such experiments (117, 300, 301 ), therefore, cannot be considered examples of hapten-specific tolerance since unresponsiveness in these circumstances may reflect tolerance in either or both T and B lymphocytes as has been shown recently (302). As we define it, haptenspecific tolerance should reflect a selective B cell tolerance. Induction of hapten-specific tolerance in vivo has been accomplished by administering hapten conjugates of essentially nonimmunogenic carrier molecules, at least as far as the T cells are concerned. Thus, Bore1 (291) induced DNP-specific tolerance in mice by administering DNP coupled to mouse serum proteins. Later experiments demonstrated that DNP-mouse 7-globulin was the tolerogenic moiety (154), confirming earlier studies of Havas (155). Borek and Battisto (299) induced tolerance to the p-azobenzenearsonate hapten in guinea pigs by administering the hapten coupled to autologous red cells. Also in guinea pigs, Katz et al. (153) found that administration of DNP coupled to nonimmunogenic amino acid copolymers resulted in a profound state of DNP-specific tolerance. This was achieved with DNP-D-GL in both strain 2 and strain 13 guinea pigs and with DNP-L-GL in the PLL nonresponder strain 13 animals. Thus, administration of DNP-D-GL in soluble form intraperitoneally to nonimmune guinea pigs resulted in a significantly depressed anti-DNP antibody response to a subsequent challenge to DNP-OVA. Moreover, administration of DNP-D-GL to guinea pigs that had been previously primed with DNP-OVA and were actively producing antiDNP antibodies resulted in abrogation of antibody synthesis and their capacity to respond to a secondary challenge with DNP-OVA (Fig. 8 ) . In both situations, the tolerance was hapten-specific as evidenced by normal anti-OVA antibody responses. Recently, several investigators have induced hapten-specific tolerance in vitro. Feldmann (152) induced DNP-specific tolerance in mouse spleen cells exposed in vitro to high concentrations of DNP-POL. Moreover, induction of tolerance was related to the degree of DNP molar substitution ( a high degree being required for tolerance induction). Rittenberg and Bullock (303) induced TNP-specific tolerance in primed mouse spleen cells with high doses of TNP-KLH in uitro. Naor and

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J C ~ U MnNTI-DNP

-

e-+

5a

ANTIBODY

No Intervening Immunization DNP-D-GL DNP-L-GL

0 1 2 3 4 WEEKS AFTER PRIMARY IMMUNIZATION

0

2

4

!I,

SERUM ANTI-OVA ANTIBODY

300

6

DAYS AFTER SECCNDARY IMMUNIZATION WITH DNP-OVA

FIG.8. 2,4-Dinitrophenyl-specifictolerance induced in DNP-ovalbumin ( OVA ) primed guinea pigs as a result of an intervening treatment with DNP-copolymer of D-glutamic acid and D-lysine (D-GL) or DNP-copolymer of L-glutamic acid and L-lysine ( L-GL). Strain 13 guinea pigs received a primary immunization with 3.0 mg. of DNP-OVA administered intraperitoneally in saline at week 0. Two weeks later, an intervening treatment with 3.0 mg. of either DNP-D-GL or DNP-L-GL in saline or saline alone was carried out. Four weeks after primary immunization, the animals were challenged with 1.0 mg. of DNP-OVA in saline. Serum anti-DNP and anti-OVA antibody concentrations just prior to challenge and on day 7 are illustrated. The numbers in parentheses refer to the number of animals in the given groups. [These data from our laboratory appeared in J . Erptl. Med. 134, 201, 1971 (reference 153).]

Mishell (304) specifically inhibited in vitro antihapten responses of mouse spleen cells to TNP-SRBC or penicillin-SRBC by the addition of the respective hapten coupled to isologous (mouse) erythrocytes in relative excess. The mechanism of DNP-specific tolerance in guinea pigs in our hands is a central one as evidenced (153) by (1) a significant diminution in DNP-specific PFC in the spleen and ( 2 ) a marked depression in the number of DNP-specific antigen-binding cells in peripheral blood, lymph nodes, and spleen. More recent extension of these studies of antigenbinding cells (296) have revealed that in DNP-tolerant animals there is a reduction predominantly of high-affinity B cell precursors and antibody-forming cells. Hence, although low-affinity cells are diminished earIy in the tolerant state, they reappear during recovery from tolerance much sooner than high-affinity cells (296). We should make perfectly clear that the observed diminution in numbers of DNP-specific antigenbinding cells does not necessarily mean that such B cell precursors are eliminated from the system. They may, indeed, be lost as a result of

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tolerance induction or, alternatively, the failure to detect them in the antigen-binding assay may reflect a change in the capacity of these specific precursors to express their surface receptors, i.e., the receptor molecules may be pinocytized or no longer synthesized, or both. Hence, it is not possible at this time to know whether tolerance in specific B cells means that such cells are lost or whether they are still present but no longer express detectable surface receptor molecule and are, therefore, nonfunctional immunologically. The above discussion dealt with B cells. What about T cells in the tolerant state? The situation is, indeed, more complicated here because of the great difficulty in directly detecting antigen-specific T cells as compared to B cells. These technical problems render the determination of the existence of specifically tclerant T cells and their fate in vivo difficult to resolve at the present time. However, some recent observations provide indirect evidence that specifically tolerant T cells may exist (74, 290, 305, 306). 3. Mechanism

The studies cited above and others reported in recent years have increased our understanding of the kinetics and the cells involved in tolerance induction. Insofar as the precise cellular events resulting in specific unresponsiveness are concerned, however, we still have no definitive answers. Nonetheless, certain observations permit a few general predictions to be made. First, it is now readily apparent that the interaction of antigenic determinants and receptors of B cell precursors of antibody-forming cells may lead to very different outcomes determined by the differences in form and concentration in which the antigen is present or, particularly, by the state of the cell at the time of interaction. Hence, as shown recently by Klinman (307),spleen fragments from mice primed with DNPKLH could be stimulated to produce secondary anti-DNP responses in vitro with DNP-KLH over a wide range of molar DNP concentrations. When the spleen fragments were exposed to DNP conjugates of nonhomologous or nonimmunogenic carriers, he found that weak stimulation occurred only at very low molar DNP concentrations, whereas at higher concentrations such complexes had an inhibitory effect on anti-DNP antibody synthesis. This suggests that in the presence of a T cell regulatory influence both low and high determinant densities at the surface receptors provide a stimulatory signal to B cells, whereas in the absence of T cell regulation, low determinant density only provides a weak stimulatory signal and high density provides a tolerogenic signal. The same interpretation may be given to observations of Diener and Arm-

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strong (308) and Feldmann (152) who found that POL and DNP-POL, respectively, induced antibody production by mouse spleen cells exposed in uitro to low concentrations of antigen, whereas high concentrations induced specific tolerance. It is noteworthy that POL is a thymus-independent antigen (147).However, even in the presence of T cell activity, decreased B cell responsiveness may result from exposure to very high concentrations of antigen. The observation of Bullock and Rittenberg (309) that, with increasing time after in vivo priming with TNP-KLH, successively lower doses of TNP-KLH stimulated secondary anti-TNP responses in vitro whereas higher doses (which were stimulatory to cells obtained at earlier times after priming) suppressed the anti-TNP response can be interpreted in the same manner. The operational significance of determinant-B cell receptor interaction in the presence or absence of T cell regulation may be exemplified by some recent observations of our own (153).As we have mentioned before, administration of DNP-D-GL, a nonimmunogen, to normal or DNP-OVA-primed guinea pigs results in profound DNP-specific tolerance manifested by depressed antibody production and diminished numbers of detectable DNP-specific antigen-binding cells and anti-DNP antibody-producing cells. We have interpreted the ease of tolerance induction with these and other nonimmunogenic or weakly immunogenic substances (154,155, 291), for which few or no specific helper T cells exist, to result from direct interaction in the appropriate dose range with specific precursors of antibody-forming cells without intervening helper cells. This concept may explain the puzzling phenomenon described by some investigators studying the “termination” of tolerance to protein antigens by administration of modified or cross-reactive proteins (310-312). In those experiments, the simultaneous administration of even very small quantities of the tolerated protein prevented termination of the unresponsive state. It has been postulated that the mechanism in the termination of tolerance involves the presentation of the determinant of the tolerated protein to precursors of antibody-forming cells through the action of helper T cells specific for the cross-reactive or altered protein (292). In this set of circumstances, injection of the tolerated protein simultaneously with the cross-reactive protein should lead to a potent suppressive effect on the precursors of antibody-forming cells by direct interaction with their receptors in the absence of helper cells specific for the tolerated substance. If this reasoning is correct and if a suitable substitute for specific helper T cell function exists, then it should be possible to convert a normally tolerogenic signal into an immunogenic one. We have tested this hypothesis by employing the potent nonspecific stimulatory influence

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of the allogeneic effect ( 153). Thus, DNP-OVA-primed guinea pigs were injected intravenously with allogeneic lymphoid cells and, 6 days later, challenged with either the immunogenic DNP-BGG conjugate or the normally tolerogenic conjugate DNP-D-GL. A comparable secondary anti-DNP response was obtained with both conjugates with respect to serum anti-DNP antibodies as well as anti-DNP PFC in the spleens of such animals (Fig. 5 ) . As discussed in Section V I , C , the elicitation of a GVH reaction renders the primed recipient, therefore, refractory to tolerance induction by DNP-D-GL and permits this molecule to behave as an immunogen capable of stimulating a strong anti-DNP secondary response. The observation of McCullagh (313) that adult rats tolerant to SRBC, nevertheless, are stimulated to form an anti-SRBC antibody response by administration of allogeneic immunocompetent cells and SRBC is analogous to the allogeneic effect described above. A second general prediction concerning tolerance induction can be made in light of the very elegant recent studies of Feldmann and Diener ( 314-316) concerning antibody-mediated suppression of in vitro immune responses to polymeric and monomeric flagellin. These investigators have provided impressive evidence that, in the absence of T cell function, mixtures of antigen and specific antibody in a very critical ratio lead to a profound state of specific tolerance in spleen cells exposed to the mixture in vitro. The authors interpret their data as suggesting that a lattice of antigen-antibody complexes on top of the specific antigen recognition receptor of immunocompetent cells provides the tolerogenic signal. Further experimentation is necessary to determine whether or not this interpretation is correct. Nevertheless, their observations clearly suggest a critical relationship between the extent and valency of determinant binding and the possible interpretation of the event by the specific cell, i.e., as a tolerogenic or as an immunogenic signal. If, as we postulate, it is at this level that the regulatory role of T cell mediators is effective, the prediction may be made that the tolerogenic conditions used by Feldmann and Diener should result in an antibody response in the presence of activated T cells.

B. IMMUNOLOGICAL MEMORY

The existence of specificity among both B and T cell populations implies that both cell types are capable of expressing immunological memory. This is, indeed, the case, as will be shown in this section. The existence of memory in the T cell population is implicit in observations made in the double transfer system response to SRBC in which T cells were specifically activated in the primary host (11,56, 74, 166) and in in vitro systems where activated T cells were employed (91, 92,

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DAVID H. KATZ AND BARUJ BENACERRAF

94). Attempts to demonstrate B cell memory in the SRBC system, however, gave some conflicting results. The results of some investigators clearly indicate that T cells carry memory, whereas B cell memory was not so evident (166, 317, 318). Thus, Shearer and Cudkowicz ( l 6 6 ) , using the double transfer system in mice, found that activation of T cells in the first host by SRBC was necessary to obtain synergy with B cells in the second host, but the quality of the antibody response was the same whether or not the B cells had been exposed to SRBC in the primary host. Takahashi et al. (318) studied the adoptive secondary response to SRBC in mice and found that treatment of immune spleen cells with anti-8 serum and complement abolished the adoptive second response and that addition of normal thymus or spleen cells failed to restore the response. Addition of immune spleen cells to anti-0-treated spleen cells restored the adoptive second response and this was interpreted as evidence for memory being the responsibility of the @-bearingT cell population. However, by using as T and B cell donors congenic mice differing genetically only at the loci coding for immunoglobulin allotype, Jacobson et al. (215) showed that all of the yG anti-SRBC PFC produced in the adoptive transfer response to a second antigenic challenge were of the B cell allotype, thus indicating memory in the B cell population. Kennedy et al. (236) found that a synergistic effect between adoptively transaddition of normal thymus or spleen cells failed to restore the response. to burro erythrocytes was optimal when both cell populations came from donors primed to burro red blood cells. This also supports the existence of specific memory in the B cell population. Recently, Jehn and Karlin (319) have shown that both thymocytes and bone marrow cells could adoptively transfer memory in the response to SRBC. Whereas the demonstration of B cell memory is not without complicating problems in studies of the anti-SRBC response, the use of different cooperating systems has led to a clearer definition of immunological memory in both T and B cells. In the models of cooperative cell interactions in the immune response to hapten-carrier conjugates, the existence of T and B cell memory is well established. This is so because the carrier-specific helper cell has been demonstrated to be a T cell (126, 1 4 3 ) , and it is quite clear that carrier priming leads to enhanced hapten-specific antibody responses in viuo (63, 64, 6 6 ) , and in uitro ( 137-140). Moreover, the development of heightened antihapten antibody production on the part of the B cell population, as a result of prior immunization in such systems, indicates the existence of immunological memory in the B cell. This conclusion is further strengthened by the following observations. 1. In the hapten-carrier cooperating systems involving free carrier

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immunization, the class of antihapten antibody produced is determined by the conditions employed for sensitizing the animal to the hapten (not to the secondaiy carrier). This has been observed in the enhanced secondary anti-SULF response in rabbits (65,134) and in both enhanced primary and secondary anti-DNP responses in guinea pigs (66). These findings demonstrate that the class of primary or secondary antihapten antibodies is predetermined by the hapten-primed memory cells of the B type and not by the carrier-specific helpers of the T population. 2. In the adoptive transfer system, Mitchison et al. (65,70)have used mice differing in immunoglobulin allotype as donors of carrier-primed ( BSA) or hapten-primed (NIP-OVA) spleen cells. Following transfer of the donor cell mixture into irradiated recipients the antihapten antibody produced upon challenge with NIP-BSA bore the allotype of the haptenprimed donor spleen cells. This observation clearly illustrates specific immunological memory among the hapten-specific B cell population. 3. In the hapten-carrier cooperating system in which free carrier preimmunization or supplemental immunization leads to enhanced primary or secondary antihapten responses, respectively, Katz et al. (66) have shown that the affinity of antihapten antibodies is characteristic of the mode and time of immunization with the hapten-carrier conjugate and not the mode and time of immunization with free carrier. This finding can only be explained by the existence of specific immunological memory in the hapten-primed B cell population. Recently, Miller and Sprent ( 78 ) have provided conclusive evidence for the existence of immunological memory in both B and T cells in a cooperating system not involving hapten-carrier antibody responses, By making use of their observation that thoracic duct lymphocytes (TDL) consist of both T and B cells ( 2 1 7 ) , they reconstituted neonatally thymectomized CBA mice with thymus cells from (CBA X C57B1)F1 donors and analyzed the capacity of TDL obtained from such reconstituted mice to transfer humoral responses adoptively to FyG in irradiated recipients. Such TDL obtained from reconstituted mice primed with FyG produced excellent adoptive memory responses to FyG. The PFC in the irradiated recipient spleen were shown by anti-H-2 serum to be derived from the thymectoinized CBA host, not from the F, cells. By treating the TDL with anti-C57B1 serum and complement, they were able to remove selectively the F, T cell component of the TDL. This permitted precise analysis of memory in each cell population and enabled them to show specific memory in both T and B cells. Thus, T-depleted TDL which failed to transfer the response adoptively to FyG were optimally restored by addition of TDL from F y G-primed mice demonstrating memory in the T cell population. The response of irradiated recipients

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DAVID H. KATZ AND BARUJ BENACERRAF

of primed T cells to FyG could only be enhanced by the addition of FyG-primed B cells and not with unprimed B cells, thus, demonstrating, the existence of memory in B cells. The question of whether memory is an expression of a qualitative or quantitative change in specific T and B cells is less clearly defined. Moreover, there is no reason to discount the possibility that memory in the two cell populations reflect distinctly different cellular events. It is clearly acceptable to consider that immunization bears a definite selective pressure on specific precursors of antibody-forming cells ( B cells) leading to production of antibodies of progressively higher affinity depending on time and antigen dose (298). This selective pressure is evident in the studies described above dealing with class and affinity of antihapten antibodies (65,66,134).These must be considered as qualitative changes in the B cell population. We do not know whether such B cell memory reflects an important quantitative change as well. The observation of Miller and Sprent (78) that unprimed B cells, even in very large numbers, could not substitute for primed B cells in enhancing the adoptive transfer response of primed T cells to FyG suggests that B cell memory reflects more complex events than mere quantitative considerations would explain. The T cell memory is more readily explicable in quantitative terms. The enhancement of primary or secondary antihapten antibody responses by free carrier immunization or by the adoptive transfer of carrierprimed cells can easily be thought of in terms of merely increasing the number of carrier-specific helper cells as a result of mitotic events in the T cell population occasioned by antigenic stimulation. Also relevant in this respect is the observation of Miller and Sprent (78) that very large numbers of unprimed T cells could substitute for FyG-primed T cells in enhancing the adoptive transfer response of primed B cells. Nevertheless, it is important to bear in mind that we do not have, at present, the methodology, such as we have for B cells, to delineate qualitative changes in the T cell population which may reflect selective pressures by antigen. Studies of the adoptive transfer of antihapten antibody responses in mice which have demonstrated that helper T cell activity is greatest at earlier rather than later times after immunization (65, 70, 124) may argue against the existence of antigen-related selective pressure comparable to that which exists for B cell precursors (298). The argument is strengthened by the fact that the specificity characteristics of T cells mediating delayed hypersensitivity reactions do not change significantly during the course of immunization (8, 103). The lack of selective pressure by antigen on T cells, if true, does not imply a lack of heterogeneity among such cells with respect to their receptor specificities. Indeed, that

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such heterogeneity in affinity of antigen-binding receptors among T lymphocytes exists is well illustrated by the studies of Paul et al. ( 1 0 3 ) . They showed that lymphocytes obtained from DNP-GPA-primed guinea pigs manifest a DNA synthetic response in uitm to the immunizing antigen over a wide dose range response, in contrast to a narrow dose range response to a nonspecific mitogen, clearly reflecting heterogeneity among the specific T cell population. It is clear, therefore, that both B and T cells are capable of expressing specific immunological memory. What memory precisely reflects is uncertain. It is likely that development of memory in B cells requires distinct qualitative changes in this population either with or without quantitative changes. Development of memory in T cells involves an increase in the number of antigen-specific reactive cells, but the possibility of a concomitant qualitative change of significance in these cells is not definitively ruled out. C. IMMUNOLOGICAL ADJWANTS Studies on the immunoenhancing effects of synthetic polynucleotides has become an increasingly active area of investigation in recent years. It is not within the scope of this review to consider this subject in any detail, and the reader is referred to Braun et al. (320) and Johnson et al. (321) for recent reviews. It is relevant to note that double-stranded polynucleotides such as poly A poly U, when administered with antigen, can exert a marked stimulatory effect on antibody formation and cellmediated immune responses in normal animals. Moreover, administration of such agents to genetic low responder, thymectomized or aged animals restores their deficient antibody responses to normal levels (320, 321 ). These agents appear, therefore, to act in such a way as to stimulate or replace the normal regulatory influence of T cells on antibody production. The literature is somewhat confusing at present as to which alternative is correct. Thus, Cone and Johnson (322) have recently presented evidence which they interpret as demonstrating that poly A-poly U stimulates T cell activity, whereas Campbell and Kind (323) interpret results obtained in a somewhat different system as indicating that poly Aapoly U acts directly on B cells (thereby replacing T cell function). Perhaps the clearest studies in this regard are those of Diamantstein et al. ( 3 2 4 ) who found that adult, thymectomized, irradiated, bone marrow-reconstituted mice could be restored to develop both yM and yG anti-SRBC responses by the administration of polyanionic substances such as dextran sulfate and polyacrylic acid. These observations, if -true, indicate that the polyanions are capable of replacing T cell function in antibody responses.

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What is, perhaps, most intriguing to consider in light of the biological relevance of these findings is the possibility that their effect is mediated through a cyclic 3’,5’-adenosinemonophosphate ( cyclic AMP) mechanism. It has been shown that cyclic AMP can enhance both in vivo and in vitro anti-SRBC responses in mice (325). More recently, Braun and his associates ( 326-329) have obtained results indicating that enhancement of immune responses by synthetic polynucleotides is related to the capacity of such substances to enhance formation of cyclic AMP in immunocompetent lymphocytes. It is attractive to consider that some of the mediators, which we propose are elaborated by T cells, may have similar physicochemical properties to the agents discussed above and that they exert their regulatory influence on B cell function through a cyclic AMP mechanism. Such agents, indeed, fill certain criteria that we have suggested earlier, namely they are rapidly acting and short-lived. It is likely that this will be definitively elucidated in the next few years. Before concluding this section, we should briefly consider the action of other immunological adjuvants in this same context. For many years it has been an unanswered puzzle to immunologists how classic adjuvants exert their enhancing properties on the immune system. It appears that adjuvants exert their effect to some extent on macrophage function. This follows from the observation of Unanue et al. (330) that nonparticulate or particulate adjuvants taken up by macrophages in vitro and injected into syngeneic mice increased the antibody response of the recipients to Maia squiwdo hemocyanin; adjuvants taken up by lymphoid cells, in contrast, had no similar enhancing effect. The first direct evidence that T cell function is required for potentiation of antibody responses by some adjuvants was obtained by Unanue (218). He found that beryllium, a very potent adjuvant in normal animals, had no enhancing effect on the antibody responses of thymectomized mice immunized with KLH. Recently, Allison and Davies (331) also examined the effect of thymus deprivation on the capacity of several adjuvants to enhance antibody responses of mice to BSA. Their findings confilmed those of Unanue ( 2 1 8 ) in demonstrating that the potentiation of antibody formation by adjuvants, such as Escherichia coli lipopolysaccharide ( endotoxin ) and Bordetellu pertussis vaccine require the presence of T cells. Thus, thymectomized, irradiated, bone marrow-reconstituted mice failed to respond to antigen plus adjuvant unless additional reconstitution with a thymus graft is made. It appears, therefore, that regardless of the effects of adjuvants on macrophage function, stimulation of T cells is an important aspect of the mechanism of action of at least some adjuvants. This does not exclude the possibility that other adjuvants may have a

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direct effect on B cell function, i.e., if one considers the polyanionic substances in the category of adjuvants ( 3 2 4 ) .

D. CELL-MEDIATED IMMUNITY Recognition of T and B lymphocyte interactions in humoral immune

responses prompted studies to determine whether analogous interactions existed in the ontogeny of cell-mediated immunity. Globerson and Auerbach (332) found that in vitro GVH reactivity occurred only if both thymocytes and bone marrow cells were present. Barchilon and Gershon (333) and Hilgard (334) reported observations of synergy between thymus and bone marrow cells in the production of GVH splenomegaly in X-irradiated F, recipients of parental donor cells. Inasmuch as splenomegaly in GVH reactions is predominantly a host cell proliferative response (335) and the bone marrow is the source of this host cell proliferation ( 3 3 4 ) ,these findings are hardly analogous to T and B interaction in induction of humoral responses, Very recently, Eidinger and Ackerman (336) have reported evidence for a synergism between T and B cells in the development of delayed hypersensitivity responses in mice. Considerable interest presently exists in the question of whether or not cooperative interactions between T cells are essential for the development of cell-mediated immune reactions. Several lines of investigation have provided indirect evidence for T-T cell interaction in cellmediated immunity. Thus, Cantor and Asofsky (337) reported synergy between mouse lymphoid cell populations from different sources in the production of GVH splenomegaly in F, hosts. Recent studies suggest that, at least in their system, two classes of T cells may be involved: one class consisting of precursors af the effector cells and the other serving to amplify the activity of the former ( 3 3 8 ) .Studies in our own laboratory (339,340) have shown that potent nonspecific stimulation of the immune system by induction of transient GVH reactions affords significant protection to inbred guinea pigs against lethal inocula of lymphatic leukemia cells. Since tumor immunity is generally a cell-mediated phenomenon, we have interpreted these findings as indicating that the allogeneic effect can enhance cellular as well as humoral immunological mechanisms (339, 340). Indeed, preliminary studies suggest the heightened development of delayed hypersensitivity in guinea pigs as a result of the allogeneic effect ( 3 4 1 ) .This is consistent with observations of McBride et a2. (342) made in chickens several years ago. The latter studies (342) demonstrated that induction of a GVH reaction in chick embryos resulted in accelerated maturation of immunocompetence in such chick embryos as manifested by their precocious capacity to develop specific cellular immune responsiveness.

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The above observations indicate that the activity of some T cells may enhance the development of cell-mediated immune reactions by effector T cells. Although evidence is accumulating for the existence of functional heterogeneity among T cell population (343, 344), it is premature to draw any conclusions, at this time, as to whether such cells are truly distinct in the functional sense or rather that they reflect different stages of maturation within the same cell line or class. IX. Biological and Pathophysiological Significance of the Regulatory Influence of T Cells on Antibody Production

As shown in the preceding sections, the division of the immune system into two classes of lymphocytes, T and B cells, differentiated to mediate, respectively, the two fundamental expressions of immunity, cellular or humoral immune responses, although basically correct, was recognized to account only in part for the complex events of antibody responses, It has become abundantly clear that the specific response of B cells to antigen is affected to a considerable extent by the concomitant activity of differentiated T cells. The cooperative interaction between specific T and B cells and antigen which has been discussed at length in this review may be most appropriately interpreted as a regulatory function of activated T cells on the conditions and manner in which B cells respond to the antigen bound to their immunoglobulin receptors. This interpretation takes into account that specific B cells may be stimulated to differentiate and synthesize IgM antibodies, under certain limits, by appropriate polymeric antigens in a narrow concentration range. It stresses also, however, that the contribution of stimulated T cells is required for the following essential activities of B cells: ( 1) the antibody response by B cells at a high, otherwise tolerogenic, antigen (or epitope) concentration (which results in a marked increase, over several logs, in the antigen concentration range immunogenic for B cells); ( 2 ) the enhancement of B cell antibody responses of all classes, but particularly in the IgG classes and the switch from IgM to IgG; ( 3 ) the emergence of B cell memory populations in all classes; (4)the effective selective pressure by antigen on B cell proliferation and differentiation resulting in increase of antibody affinity with time of immunization; ( 5 ) the suppressive effect of antibody synthesis under certain conditions heretofore observed in antigen competition phenomena. The considerable importance of the regulatory function of T cell activity for the antibody response of B cells to antigen is dramatized by the explosion of excellent work in immunology which the recognition of the various aspects of this phenomenon has stimulated in a short space of time as reviewed here. Although the mechanism or mechanisms of the

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various effects of T cell activity on B cell function listed above are far from completely understood, it is our opinion that most of these phenomena have been conclusively shown to result from the active participation of antigen-stimulated T cells. Furthermore, there is excellent evidence that this activity is mediated by distinct factors capable of affecting B cell function, secreted by T cells for this purpose. Rather than relating again the data in support of these interpretations, we propose to discuss briefly in this last section the biological significance of these regulatory mechanisms in the evolution of antibody responses and in the pathogenesis of selected pathological conditions. Since the regulatory activity of T cells is both specific and antigen-dependent, the dual specificity mechanism evolved in T and B cells insures several levels of control of effective specific humoral responses. The T cell level of control is all the more relevant, since the range of specificity which can activate helper T cells is in some way considerably more restricted than the range of antibody specificities which can be synthesized by B cells. There is ample evidence that this is precisely the case, as (1) based on studies of specific immune responses controlled by histocompatibility-linked Ir genes in both guinea pigs and mice (162, 351) and ( 2 ) based also on the evidence, discussed at length in Section V,A, that haptenic determinants, for which there exist many specific B cells, are not able to activate helper T cells unless they are part of a molecule recognized by T cells as immunogenic on a genetic basis. The evolutionary advantage of a T cell level of specificity control for the stimulation of antibody synthesis can be viewed as providing a safety mechanism against the possibility that antibody synthesis will be triggered indiscriminately by molecules with little biological relevance but which are, nevertheless, capable of binding to B cell receptors. This is a possibility which must be considered very seriously as the immunoglobulin receptors of B cells encompass indeed an extremely wide range of specificity, not matched by the range of antigens capable of stimulating T cells. In addition, as specific tolerance is achieved more easily with lower antigen concentrations and for longer periods of time in the T cell than in the B cell population, as shown in Section VIII,A, the specificity control at the T cell level must be of the greatest importance in the prevention of autoimmune responses. Self-tolerance may, therefore, reflect a rigid state of tolerance to self-antigens in the T cell population. The B cells expressing receptor specificity for self-antigens may be continuously generated from the stem cell pool, but in the absence of T cell activity, such B cells are subjected to a constant tolerizing signal following direct interaction with the self-antigenic determinants readily available to them.

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This mechanism insures self-tolerance at the level of both potentially responsive cells-a situation analogous to one in which two different keys must be used to open the same lock. How then is self-tolerance lost in certain pathological states? An analogy is perhaps found in the experiments where the allogeneic effect permitted B cells to develop antibody responses to a normally tolerogenic substance (153) or terminated a preexisting state of tolerance to SRBC ( Section VII1,A) ( 3 1 3 ) .The implication of these findings is that in the presence of sufficiently strong T cell activity (regardless of the specificity of the T cells involved), B cells can be triggered to respond to determinants that otherwise would have turned them off. It is conceivable that similarly potent T cell stimulation can follow certain infectious or otherwise toxic insults of exogenous origin which may permit a B cell response to self-antigens, leading to transient or prolonged autoantibody formation which may or may not have pathogenetic consequences. Aside from the possible relevance to the maintenance of self-tolerance, a two-cell mechanism of antibody production may offer definite advantages in terms of host defense mechanisms, If, as we have proposed, T cells exert a regulatory influence on the rate of B cell differentiation in the presence of antigen-induced selective pressures, the advantages to the host which has been invaded, for example, with bacteria or viruses are obvious. The kinetics of optimal antibody production are increased, the response longer sustained, and the development of specific memory is most likely greatly enhanced by the presence of T cell activity. Simultaneously, of course, T cell memory has developed among those cells destined to perform their function in cell-mediated immune reactions. In this regard, there is already evidence to suggest that the existence of the two-cell mechanism can be appropriately manipulated to permit enhanced antibody production to certain bacterial antigens characteristically weak in immunogenicity. Thus, we have recently elicited considerably enhanced antibody responses in rabbits to SIIIby administering it covalently bound to BGG ( 3 5 2 ) . If augmented antibody responses can be elicited in this manner and can be shown to be saciently protective against subsequent infection by virulent organisms, then such an approach to relatively safe human immunization against a variety of bacterial polysaccharides would be feasible. Finally, the possibility that some T cell function may regulate the activity of differentiated effector T cells in cellular immune reactions may have considerable relevance and therapeutic potential in problems of cancer immunity. This is particularly true inasmuch as the major obstacles to be dealt with in this area concern the following two points: ( 1) most tumor-specific transplantation antigens (TSTA) appear to be

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relatively weak, thereby eliciting weak or ineffective cellular immunity; and ( 2 ) antibodies produced against TSTA are in many, if not all, instances protective for the tumor cell against potential cytotoxic T cells (353). It is highly probable that both such obstacles can be overcome under appropriate conditions whereby selective heightened T cell activation may occur. In this instance, the provocateur of T cell activation need not reflect specificity related to TSTA provided the nonspecific stimulation is of sufficient magnitude. This is, perhaps, exemplified by the capacity of the allogeneic effect to confer significant protection in guinea pigs with highly fatal lymphatic leukemia transplants (339, 340) (Section VII1,D).

ACKNOWLEDGMENTS We are deeply indebted to Dr. William E. Paul for his active contribution to all of our studies on hapten-carrier cooperation phenomena. We thank Drs. J. F. A. P. Miller, Martin C. Raff, and William 0. Weigle and their colleagues who generously permitted us to reproduce some of their published data, and those investigators who provided us with unpublished observations. The excellent assistance of Miss Karen Ellis in the preparation of the manuscript is greatly appreciated.

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