The 2nd International Lymphokine Workshop

CELLULAR

IMMUNOLOGY

48,

427-432

(1979)

MEETING The 2nd International

REPORT Lymphokine

Workshop

The 2nd International Lymphokine Workshop brought 140 specialists and generalists to the historic Schloss Wolfsberg at Ermatingen, Switzerland, overlooking Lake Constance. In the course of 3 days (May 28-30,1979) a total of 26 major reports were presented and actively discussed, while a much larger number of brief communications were given and discussed in workshop-poster sessions. This fresh experimental evidence of great strides in lymphokine research, together with an edited version of the extensive lively discussions, will be published by Academic Press early in 1980. The sheer number of lymphokines, many of which display apleiotropic pattern of activity, has long prevented their integration into a conceptual framework. This workshop on lymphokines marked a turning point in that phenomenological descriptions of leukocyte-derived factors are gradually being supplanted by incorporation of a large amount of new and old data into a coherent scheme. This trend has been facilitated by a wisely programmed succession of discussion topics in which each of the principal lymphokines was considered in terms of its molecular characterization, its source of production, its target, and its mode of action. The most comprehensive analysis was on those lymphokines whose identification and purification could be achieved by means of serological markers. Aided by the recent development in cell hybridization technology, the availability of lymphokine-producing cell lines has already been achieved; to an increasing degree this approach will facilitate rapid progress in lymphokine research. Such technical improvement has yielded significant advances in the characterization of non-antigen-specific lymphokines and monokines and in the molecular description of antigen-specific lymphokines such as T-cell-derived helper and suppressor factors as well. Information gained from experimental work on antigen-specific helper factor has in the past often been received with caution by the scientific community mainly because of problems in reproducibility of data. Helper factor-producing hybrid cell lines and improvements of tissue culture conditions have aided in solving the problem. Such technical advances have already permitted the generation of antigen-specific T-cell-derived helper factor from human peripheral lymphocytes cultured in vitro. One of the enigmas of T-B cell cooperation concerns the controversial issue of allogeneic restriction of such interaction. The primary induction of B cells by allogeneically restricted Ia-positive carrier-specific helper factor was reported as generating hapten-specific IgM antibody-forming cells. Factor-B cell interaction critically depended on the presence of radioresistant adherent cells. Since these 427 0008-8749/79/140427-06$02.00/O Copyright Q 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

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cells could be either syngeneic or allogeneic with the B cells or the T cells from which helper factor was derived, it was concluded that allogeneic restriction occurs at the level of helper factor-B cell interaction and is not mediated by adherent cells. There is little doubt that the requirement for carrier-specific T-cell-derived help is absolute. The extent to which nonspecific help derived from specifically or nonspecifically stimulated T cells constitutes an essential element in the triggering of an immunocompetent effector cell in cell-mediated or humoral immunity is less clear. One such T helper cell replacing factor (TRF) derived from a specifically induced Ly-l+ FCR- T cell induces the B cells to undergo differentiation into antibody-producing cells, affirming the concept that proliferation and differentiation may be triggered by two separate signals, the first delivered by specific, the second by nonspecific factor. TRF may also be part of lymphocyte differentiation factor (LDF) that is claimed to deliver differentiation signals to dextran-primed B cells but appears to be associated with RNA. The immunopotentiating effect of TRF, as far as B-cell differentiation is concerned, may be inhibited with a T-cell-derived immunoglobulin binding factor (IBF) of as yet unknown physiological significance. This factor is believed to represent F,y receptors that are shed from alloantigen-activated T cells. Nonspecific but antigen-dependent amplification of effector cell function has also been demonstrated for the generation of cytotoxic responses. For example, Ly- I+ T cells have been shown to amplify the cytotoxic response of Ly-2+ T cells via a soluble lymphokine. A factor with similar properties obtained from Con-Astimulated Ly-lf T cells is capable of stimulating alloantigen-primed Ly-2,3+ T cells to proliferate and differentiate into cytotoxic effector cells in the absence of antigen. Allogeneic effect factor (AEF) has also been shown to help generate cytotoxic T lymphocytes. These and other closely related if not identical factors have been characterized as being able to replace helper T cells in both humoral and cell-mediated immunity and synergize with T-cell mitogens and with a B-cell differentiation factor derived from macrophages. What has been described as T-cell growth factor (TCGF), costimulator, and thymocyte stimulating factor (TSF) may also belong to this category of lymphokines. The problems in assessing the physiological significance of those lymphokines ambiguously defined as enhancing or suppressing factors influencing some parameters of the immune response in vitro were amply documented. It is thus often difficult to distinguish between specifically target cell-oriented mechanisms of action and simply cytotrophic or cytotoxic effects of a lymphokine under study. Certain monocyte-macrophage derived factors with the property of enhancing or inhibiting T- and B-cell mitogenic and antigen-specific responses or regulating their own activity in vitro may fall into this category. Nevertheless, some macrophagederived lymphokines have been described which have a clearly defined target cell and an absolute requirement for the target cell response to take place. The most notable of these is GRF (genetically related macrophage factor), responsible for the antigen-dependent induction of T cells that amplify the maturation of T, helper cell precursors. GRF is described as a molecular complex between an Ia antigencontaining unit and a noncovalently bound fragment of the immunogen. Interaction of GRF-with the T, cell is allogeneically restricted and controlled by the I-A subregion of the MHC. Identity of GRF with the Ir-gene product has been postulated

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on the grounds that GRF derived from macrophages of nonresponder mice to (T, @-A-L fails to stimulate responder T cells to become helper cells. In accord with the notion that the macrophage holds a key position within the triggering scheme of immunocompetent T and B cells is the description of yet another macrophagederived factor (MMF) that is believed to act on the mature T, lymphocyte. Another lymphokine derived from mononuclear cells and found to be associated with two molecular species of esterases is lymphocyte inhibitory factor (LIF) which appears to be subject to regulatory control by cGMP. It has also been shown to induce the release of a neutrophil inhibitory factor (NIF), a glycoprotein derived from T cells. The question remains as to the chemical nature of other macrophage-derived factors such as macrophage helper factor (MHF) which is said to directly and specifically enhance B-cell antibody responses. Monocytes and macrophages are also known to produce components of the complement system; for example, the release of C2 by human monocytes has been shown to be controlled by a T-lymphocyte-derived monocyte complement stimulator (MCS). It was not very long ago that the macrophage was regarded by immunologists as a mere scavenger cell incapable of participating in activities as sophisticated as the induction and control of the immune response. Today the cellular immunology literature abounds with experimental evidence that assigns the macrophage a central role in immunoregulatory processes. Reports on macrophage-derived mediators provided encouraging evidence that the scope of the research on this subject is already extending from the cellular to the subcellular level of investigation. Immunology may in the future be recognized less by the beauty of its intellectual concepts than by the clinical implications of research on the control of lymphokines such as macrophage migration inhibitory factor (MIF), macrophage cytotoxic factor (MCF), macrophage activating factor (MAF), lymphocyte chemotactic factor (CTF), lymphotoxic factor (LTF), fibroblast activating factor (FAF), osteoclast activating factor (OAF), prostaglandin (PG), interferon (IF), and others. An impressive degree of lymphokine purification, stemming from the availability of factor-producing cell lines and of factor-specific antisera, has dramatically increased our knowledge of MIF. While its source of production in situ is less clear, its site of action has been defined as a macrophage surface-located receptor complex containing L-fucose and N-acetyl-D-glucosamine. The identity of these substances with MIF binding receptor function has been inferred from the fact that antibody raised against them inhibits binding of MIF to its target cell. MIF has been shown to be separate and distinguishable from interferon. Its activity appears to depend on the presence of a dialyzable cofactor now reported to be functionally deficient in mouse strains that are nonproducers of MIF. One of the effects of MIF on cellular organelles of the macrophage involves the assembly and organization of microtubular protein. This may in part explain the dramatic changes observed in the surface morphology of macrophages after interaction with MIF. Circumstantial evidence suggests similar effects of MIF on basophils. Extensive discussion on the biochemical definition of MIF emphasized the difficulty of assigning MIF activity to a single molecule. Evidence, yet to be confirmed by amino acid sequence analysis, suggests that MIF consists of covalently linked subunits. Factor-specific antibodies for the purpose of separating MIF by serological markers from other lymphokines such as lymphocyte inhibition factor (LIF), lymphocyte activating factor (LAF),

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mitogenic factor (MF), and macrophage agglutinating factor (MAggF) offer an invaluable advantage over purely physical methods of purification. Such technical improvements parallel the need for standardization of in vitro assay systems from the point of view of MIF induction, statistical evaluation of data, and the source and degree of uniformity of the target cells. Lymphokines of potential importance in immune surveillance have been described in partially purified form as activators of macrophages (MAF) and of promonocytes which then kill tumor target cells. One of the lymphokines known to induce promonocytes to generate natural killer cell-like cytotoxicity has been identified as interferon. It is noteworthy in this respect that interferon has been found to be produced by cells identical or closely related to natural killer cells. In addition to interferon, natural killer cell activity is also claimed to be controlled by prostaglandin, an effect analogous to prostaglandin-induced regulation of the PHA mitogenic response that appears to be mediated by a prostaglandin-sensitive suppressor cell. Whether or not the correlation between interferon levels and cell-mediated immunity in vivo indirectly reflects an interferon-dependent monocyte-macrophage mediated regulatory mechanism remains unclear. On the other hand, variations in the in vivo titers of interferon do not correlate with variations in cytotoxic activity, a notion in agreement with another report pointing out that a genetically determined difference in protection ri-om virus infections is due to variations in the animals’ cellular responsiveness to the effect of interferon. This genetically determined susceptibility to the immune potentiating effect of interferon (dominant gene Mx in mice) contrasts with the mouse strain-dependent capacity to produce either high or low levels of interferon in response to LPS, a trait controlled by a single autosomal dominant gene. Chemotactic factors controlling the migration of neutrophils and eosinophils are known to involve various components of the complement system. On the other hand, migratory control of DTH-reactive lymphocytes is exerted by lymphotactin (CTF) which is specific for T but not B cells. The significance of chemotactic and MIF-active lymphokines in granuloma formation has impressively been demonstrated in vivo. These lymphokines bound to Sepharose beads and injected intravenously into mice were shown to act as focal points of pulmonary granuloma formation. A phenomenon commonly associated with chronic inflammation is the fibrotic reaction that seems to depend upon the release from antigen or LPS-stimulated T cells or macrophages, respectively, of fibroblast activating factor (FAF). This factor causes the cells to proliferate, to increase protein synthesis, and to release prostaglandin. It may be akin to what has been described as mononuclear cell factor (MCF) which stimulates the production of prostaglandin E, in association with inflammatory joint disease. The prostaglandin-dependent release of osteoclast activating factor (OAF) from activated lymphocytes may also be controlled by a monokine, although no such relationship has as yet been demonstrated. It has been suggested that certain pathways of the host’s inflammatory response such as fibrin gel production may be utilized by tumor cells so as to surround themselves with a protective zone. It has indeed been found that malignant tumors are capable of secreting a number of lymphokines associated with inflammatory responses such as vascular permeability factor (VPF), a procoagulant (PC), plasminogen activator (PA), MIF, and a newly described soluble immunosuppres-

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sive factor (SIF). Whether such lymphokine activity is linked with the observed suppressed levels of CAMP in spleen lymphocytes of immunosuppressed animals during enhanced tumor growth remains unanswered. Cells other than those of the RES may, under certain circumstances, such as after viral infection, produce lymphokines. A lymphokine of debatable significance in vivo , at least in extracellular form, is lymphotoxin (LTF) as produced by PHA-stimulated lymphocytes and by lymphoid cell lines. LTF exerts nonspecific cytotoxicity against a Variety of target Cells in vitro, A factor similar to LTF in its susceptibility to the enhancing effect of actinomycin D has been described as tumor necrosis factor. It is discriminately toxic for tumors and transformed cells in vitro and in vivo but does not affect primary mouse embryo fibroblasts. Biochemical evidence suggests that LTF is a multicomponent lymphokine that is distinct from CTF and from IF in spite of overlapping biological activities with the latter in its ability to inhibit cell proliferation. Circumstantial evidence suggests that LTF production results from a cooperative event between an adherent and a nonadherent cell. It is possible that LTF secreted in vitro is in fact identical with the lytic effector substance that controls contact-mediated cytotoxicity. A lymphokine with a very broad spectrum of effector function and comprising a variety of molecular components is interferon (IF). With the help of monospecific antisera it has been possible to distinguish between Le-IF and F-IF, both forms being produced by lymphoid as well as nonlymphoid cells. The two forms differ in antiviral activity (Le for high, F for low) and show different biophysical properties such as hydrophobicity, molecular weight, and isoelectric point. A third form of IF called T is produced by T cells and is the least efficient in antiviral potency. The old classification distinguishing between Type I and Type II IF is likely to be replaced by a more sophisticated classification based strictly upon serological and biophysical criteria. The steady progress toward a biochemical analysis of IF is made possible by an increasingly improved methodology of IF production in vitro which takes into account the choice of the IF-producing cell and the nature of the IF-inducing agent. A significant development in the molecular analysis of IF has been its production by cell free synthesis, allowing the characterization of IF mRNA. Purification of one species of IF yielded a single active protein of MW 17,500. Colony stimulating factors have, for some time, provided an invaluable tool to study the ontogenic development of hemopoietic and lymphopoietic cell lines in vitro. However, the high degree of molecular heterogeneity, and many different sources of origin, and the dependency on a variety of stimulating agents have made it difficult to assess their identity with the corresponding lymphokines that are physiologically relevant in situ. The establishment of factor-producing cell lines, combined with the production of factor-specific antisera, should soon resolve this ambiguity. In addition to colony growth stimulation by lymphokines of exogenous source, the colony’s own product may enhance its own proliferative capacity. Thus T lymphocytes were shown to secrete their own lymphocyte colony enhancing factor (LCEF). Colony growth was inhibited by a monocyte-macrophage derived lymphocyte colony inhibiting factor (LCIF) that was identified as prostaglandins E 1 and E2. Similarly, a low molecular weight (< 1000) peptide derived from granulocytes has been described to inhibit the proliferation of lymphocytes as well as that of myelopoietic progenitor cells. A thymus-derived factor of equally low

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molecular weight (1400) has been described that inhibits T-lymphocyte colony growth. Many lymphokine specialists themselves, like many a reader of this report, may well feel bewildered by the number and the diversity of lymphokines. Their number will surely decrease as chemical identities between differently named factors are uncovered. It has become evident that lymphokines and monokines play a significant role in controlling both humoral and cell-mediated immunity in vivo, the production of which requires the presence of antigen, adjuvant, and hormones. Because the biological effects of lymphokines are generally observed in vitro, it will bear increasingly on individual investigators to demonstrate their physiological relevance. Emphasis has traditionally been on those areas of immunological research where experimental parameters are clearly defined, such as in cell cooperation models. Although this had briefly occasioned less interest in lymphokines, the situation is rapidly changing, now that these agents are being better characterized at the biological and the biochemical level. ERWIN Department of Immunology The University of Alberta Edmonton, Alberta, Canada

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