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The Biologic Basis of Tissue Transplantation
The Biologic Basis of Tissue Transplantation WILLIAM T. NEWTON, M.D., F.A.C.S.*
This is a short summary of modern advances in transplantation ' biology. It is written for that busy practitioner who, in a reasonable economy of time, wishes to become intelligently conversant in this field. And, to this end, literary citations of reviews and summations of work are preferred over documentation of original sources. The interested student will wish to consult the volume by Woodruff 81 and the current scholarly review by Russell and Monaco. 63 It will shortly become clear that the failure of one individual to accept and maintain a tissue graft from another is an immune response on the part of the host. Consequently, this author chooses to deviate from the pattern of other published reviews and to examine the phenomena of homograft sensitivity against a background review of the general nature of immune responses. INTRODUCTION
Progress in organ homotransplantation required that three basic questions be answered. How mayan excised organ be joined to a new host? By what process does a temporarily successful union of an individual's organ to another host almost invariably fail? Can the processes that have directed the transplantation failure be governed? The first question was answered almost in entirety in the decade from 1902 to 1912 by the studies of Carrel and Guthrie." Satisfactory surgical union of major blood vessels allowed the transplantation of almost every organ of the body to new sites in new hosts. Yet the transplant, after functioning quite well for a few days, invariably failed. It remained for Medawar" to provide experimental From the Department of Surgery, Washington University School of Medicine and the Veterans Administration Hospital, St. Louis, Missouri * Associate Professor of Surgery, Washington University School of Medicine; Chief of Surgery, John Cochran Veterans Administration Hospital; Staff Physician, Barnes Hospital
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evidence in answer to the second question, the means by which destruction of the graft is mediated. He observed that a second graft of skin from one rabbit to another, after a first graft had been rejected, was now rejected in a (more rapid and intense fashion. He concluded that the host had been immunized by the first graft, and that the rejection of grafts was an immune response on the part of the host. The third question, regarding the control of immune processes, had already been demonstrated in some immune systems. Transplantation research provided the impetus to rapid development of immunosuppressive techniques. The answer to the first question made it obvious that any organ could be transplanted. Certain technical features of transplantation of the liver" and heart" required refinement, but success was almost guaranteed after successful union of blood vessels had been achieved. The major contributions of the surgeon as a technician came to an end in the first decade of this century.
Terminology The terms used to designate various types of grafts first proposed by Medawar" have been widely used, although Gorer'" has pointed out etymological inconsistencies. A graft of tissue from one part of an individual to another part of the same individual is an autograft. A transplantation from one individual to another of identical genetic constitution, such as in identical twins or a member of the same inbred animal strain, is an isograft. Transplants between individual members of the same species, but of different genetic make-up, are homografts. Transplants from one species to a member of another species are heterografts. In the newer terminology proposed by Gorer, autografts and isografts remain unchanged and are adjectively referred to as autologous and isogeneic (or syngeneic) transplantations. The homograft becomes an allogeneic transplantation of an allograft. Heterotransplants become xenogeneic transplantations of xenografts.
THE NATURE OF THE HOMOGRAFT REACTION
Biologic Observations A transplant of skin from one rabbit to another heals in well and has the same appearance as a skin autograft. Capillaries from the host grow across the graft bed and link with those of the graft or invade the graft. There are no significant collections of cells associated with inflammation. There is no pre-existing incompaiibilitu of host and graft. The incompatibility develops after the graft has been performed. Between the fourth and fifteenth day after grafting, biopsies of the graft show collections of lymphocytes, plasma cells, and perhaps eosinophils in the graft-host junction. Shortly thereafter, observation through a dissecting microscope will show a slowing
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of circulation in some of the capillaries and formation of microthrombi. 74 Suddenly, the circulation ceases completely, and the graft becomes black. If, after rejection of such a skin graft, another graft is attempted between the same original donor and host, the rejection occurs after only three to six days and is associated with a more intense inflammatory process, the "second-set" reaction. In fact, the graft may not heal in at all-the "white graft" type of second-set reaction. Similar reactions have been found in almost all vertebrates studied, from fishes" to man. Aside from certain differences in tempo and pathology, the reaction has been noted in tissues or organs derived from all three germ layers. The reaction has been noted in such disparate tissues as kidney.P liver," lung," gut/o nerve" and bone." In all cases there is a brief period of apparent acceptance followed by rejection associated with inflammation and vascular occlusion. There may be reservations regarding this generality in the case of endocrine tissues. 39 Homografts of one tissue will so sensitize a recipient as to show an accelerated second-set reaction to a subsequent graft of an entirely different tissue. Thus, the sensitivity is individual specific, not organ specific. However, there is considerable variation in the duration of the sensitive state. Homografts of skin must follow grafts of lymphoid tissues by only a few days in order to show second-set skin graft rejections. On the other hand, renal homografts will show accelerated rejection if they are performed many weeks after a skin homograft rejection. Red blood cells are very poor sensitizers except in those species with nucleated red blood cells. The route of immunization has a significant role in these variations. For example, dissociated epidermal cells are more effective sensitizers injected intracutaneously than subcutaneously, and, if injected intravenously, a delay in second-set rejection may occur. 7 There exist certain peculiar sites where grafts may exist apparently invulnerable to the processes of rejection. Particularly the anterior chamber of the eye 28 and the cerebral hemispheres" are sites where tissue transplants may survive for long periods without evidence of rejection. These sites lack lymphatic drainage. The cheek pouch of the hamster" and the allantoic membrane of the hen's egg" are sites where even heterografts may be grown under favorable conditions. The Homograft Reaction as an Immune Response
Biologically, the homograft reaction appears to be but one other of a group of phenomena termed immune responses. Evidence for this statement is the lag period required for the development of the first-set reaction, the intensification of the reaction in hosts subjected to a prior exposure to donor tissue, the relative specificity of the reaction for one donor over others (there is some cross-reactivity of a sensitive host to the tissues of a population of donors"), and, finally, the demonstration by Mitchison'" that the sensitive state could be transferred from one host to another by isolated
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lymph node cells. It is appropriate, then, to examine the nature of immune responses in general and their application to allograft (homograft) sensitivity. Immune responses are characteristic of the vertebrate portion of the animal kingdom." The immune response is a result of reaction of an antigen with an immunologically competent cell.
Antigens Systematic study begins with the antigen, that is, a substance which excites an immunologically competent cell to undergo an immune response. Antigens are large molecules of greater than, say, 10,000 molecular weight, typically proteins or polysaccharides. Normally, a given host appears to recognize its own proteins as "self" and does not develop an immune response to them. The reason for this failure to react to "self" proteins that are quite antigenic in another species is the subject of much speculation'"- 31, 77 but remains in question. The immune apparatus develops late in embryogenesis. It appears that there is suppression of the growth and differentiation of those immune cells with propensity to react to antigens in the environment at that time. In exceptional circumstances, a host may react to its own antigens to produce an autoimmune reaction. Perhaps, under these circumstances, there has been a mutation of an antigenic determinant or a hapten substitution (vide infra) to produce a new antigen. In spite of the requirement that an antigen be a large molecule, the area of antigenic specificity of the molecule may be rather small, of the order of only one or two amino acid residues. And there may be many of these areas or "antigenic determinants" in a single antigen molecule. Artificial antigens have been created." Copolymers of glutamic acid, alanine and lysine are particularly effective antigens." However, polymers of a single amino acid, polylysine or polyglutamic acids have not been found to be antigenic." Antigenic specificity may be created by the chemical substitution of a simple chemical on the protein. For example, substitution of the terminal epsilon amino group of lysine in a protein.P or even in polylysine.i" by 2,4-dinitrophenyl groups produces an antigen whose specificity seems directed toward dinitrophenyllysine. Other simple chemicals may act similarly; toluene sulfonic acid, arsanilic acid, and certain azo compounds are examples. The simple chemical is called a hapten when its conjugation with protein or polyamino acid produces an antigen with specificity directed toward the substituted group. There have been many attempts to isolate a single molecular species from tissues that might represent an allograft antigen. Cell free materials have been found that have antigenic properties.P: 32, 37 The antigenic material seems to be a lipoprotein, perhaps associated with cell membranes or the enveloping membrane of cell microsomes. The lipid portion of the
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antigen may have haptenic properties in the determination of specificity, but chemical analyses of the lipoproteins isolated so far has shown a wide variety of lipids in any given preparation. There is evidence that the allograft antigen, in vivo, remains closely associated with donor cells. Algire'' constructed small diffusion chambers of a ring of lucite with circles of millipore filters of O.45JL pore size glued to the two edges of the ring. Bits of tissue could be placed within these chambers, and the whole could be implanted in the peritoneal cavity of a host. The filter pore size allowed the exchange of fluids and proteins but excluded host cells from entry and graft cells from exit. The grafts survived for long periods under these circumstances, and, pertinent to the present discussion, the hosts did not seem to be sensitized to subsequent test grafts from the original tissue donor. Antigen apparently did not pass from the cells within the chamber through the pores and to the site of reaction , with the host immune apparatus. Questions have been raised recently as to the permeability of these chambers to antibody and complement, and perhaps to antigen also.
The Immunologically Competent Cell Antigen, allowed to pierce the skin barrier, may either make its way in solution to a regional lymph node through the lymphatic drainage, or it may be carried there by a cell. There is evidence that a preliminary processing of antigen by macrophages is a prerequisite to the induction of the immune response." Other evidence points to the small lymphocyte as the wandering cell that carries antigen to the lymph node. The cell on arrival at the lymph node either passes on the antigenic information to other cells, or itself becomes localized to the node and differentiates into cells productive of immune responses. Particularly, the development of large lymphocytes and, later, plasma cells" seems associated with immune responses. The stimulated lymph node shows changes perhaps related to an increase in protein synthesis. An increase in the mass of the node and a greater relative increase in ribonucleic acid than in deoxyribonucleic acid occurs. Microsections of the nodes show reticular hyperplasia with engorgement of the medullary sinuses with lymphocytes, histocytes, and plasma cells. All these changes are seen in lymph nodes regional to the injection site of a potent purified antigen and also in nodes regional to rejection sites of tissue allografts;" 'The cellular participation in immune responses is the subject of comprehensive reviews.": 80 Cells teased from stimulated lymph nodes have the capacity to continue to evoke immune responses when they are transferred to another host of the same species. These cells continue to produce antibodies" or participate in delayed hypersensitivity reactions, as the case may be. As noted earlier, allograft sensitivity can be transferred in this fashion. Evidence indicates that the lymphocyte and plasma cell of the lymph node
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are able to produce antibodies and mediate delayed hypersensitivity reactions. In fact, nonsensitized lymph node cells or small lymphocytes from the thoracic duct may be sensitized in vitro by exposure to antigen, washed free of antigen, and produce an immune response when transferred to a host rendered incapable of an immune response of its own by virtue of total body irradiation." The course of events of these cells may be followed by a tritiated thymidine label. Thymidine is incorporated only into deoxyribonucleic acid of cells, and, since the turnover rate of deoxyribonucleic acid is very slow, the radioactive thymidine becomes a good label of the whole cell. Labeled cells from tuberculin sensitive hosts on transfer to a new host are found to congregate at the site of an injection of tuberculin in the new host." On the contrary, labeled cells from allograft sensitive donors do not appear at allograft rejection sites in new hosts in any great number." In summary, it can be said that the allograft reaction requires a cell for its genesis and its mediation. On the other hand, the reaction does not have all the characteristics of the known cell-associated, delayed hypersensitivity reactions. The Immune Response The biologic phenomena of immune responses are essentially of two varieties: (1) responses mediated by a humoral factor in the serum of an immunized host and capable of being transferred to another host by injecting serum of the immunized host into the second host, and (2) responses in which our imperfect techniques do not allow the demonstration of humoral antibodies and which can only be transferred to another host by living lymph node cells. The greater mass of immune responses are of the first type with demonstrable antibodies in the serum. The second types are restricted to a group of reactions termed delayed hypersentiv1."ty reactions. An individual with humoral antibody in his serum responds to an intracutaneous injection of antigen with the development of a wheal within a few minutes followed by surrounding erythema. This is an immediate reaction. The delayed type is characterized by the absence of immediate reaction and development over a period of twelve to 24 hours of an indurated erythematous reaction without a wheal. The classic example is the tuberculin reaction. The contact skin sensitivity associated with certain protein reactive chemicals is a delayed hypersensitivity reaction. 17, 40 Immunization with very small amounts of antigen or of antigen-antibody complex may be followed by a temporary state of delayed hypersensitivity to the antigen, superseded eventually by serum antibody formation and immediate skin reactivity." Delayed hypersensitivity reactions are associated only with protein antigenic stimulation; polysaccharide antigens have been ineffective. The antibodies that mediate the majority of immune responses are a
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subgroup of serum proteins, the "immune globulins," of which three or four types can be separated in mammals." The principal separatory process is related to the electrophoretic migration of proteins in an electric field. Ordinarily, all serum proteins are negatively charged at pH 8.6 and migrate toward the positively charged anode in an electric field. The most rapid migration is by the serum albumins followed by the alpha, beta and gamma globulins, in that order. Each of the globulin peaks can be shown to consist of subgroups on precise analysis. Antibody activity has been found to be associated with the {32A globulins, and with the 1'1 and 1'2 subpeaks of the gamma globulin fraction. Further, the 1'1 fraction can be separated by ultracentrifugation, or by molecular sieve action, into a heavy ('Ylm) fraction of over one million molecular weight with sedimentation velocity of over 18 Svedberg units, and a light (1'188) fraction of approximately 160,000 molecular weight and 7S sedimentation. The 1'2 antibody is also of the 7S , variety. Varying biologic activity has been found for the different antibody fractions, although all four antibody types ({32A, 'Y1m, 1'188 and 1'2) may be produced in response to a single antigenic stimulus. Allergic states, such as ragweed sensitivity, may be associated only with {32A antibody." Gamma-j, antibodies are apt to be produced early in an immune response4 • 78 with the 7S antibodies appearing later. Guinea pig 1'188 antibodies are incapable of binding complement" as opposed to the 1'2 type. The definition of chemical structure of these antibodies and its relationship to biologic activity is a fermentive field of research today, and these relationships may have significant impact on the allograft sensitivity problem. Since all known immunologic processes that injure cells appear to be mediated by a binding of complement to an antigen-antibody complex, it is feasible that the 1'1 antibodies or digested fragments of antibodies that are incapable of binding complement" might be protective rather than destructive when they join a tissue graft. A reasonable explanation of the phenomenon of tumor transplant enhancement," might be offered through this mechanism. In this phenomenon, immunization to certain tumors across strains in mice produces an ability to accept tumor transplants rather than increased resistance. The protection can be shown to be associated with serum l' globulin. Early failures to transfer allograft sensitivity by serum from animals that had rejected tissue allografts, and the ability to transfer the sensitivity with lymph node cells compelled many to consider the allograft reaction as a cell-bound antibody response of the delayed hypersensitivity type. Controversy was aroused when it was shown that allograft sensitivity could be transferred in some experiments by serum from hyperimmunized mice.?' Furthermore, regular transfer of sensitivity has recently been obtained with extracts of sensitive lymph node cells." The active material in these extracts seems to be a gamma globulin. This controversy is the subject of a comprehensive current review. 71
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SUPPRESSION OF IMMUNE RESPONSE
The reaction of a population of animals to the same antigenic stimulation varies widely. In some cases one genetic strain of animals may react intensely to a given antigen, but another strain may react poorly if at all. Until the resurgence of interest in tissue transplantation, immunologists had been more interested in the intensification of immune responses than in their suppression. However, some indication of the possibility of immune suppression came from Felton's observations on "immune paralysis.i'" He observed that mice immunized with very large amounts of pneumococcal polysaccharide not only failed to develop serum antibody to the polysaccharide but were refractory for long periods thereafter to subsequent attempts to immunize the mice with the usual amounts. Subsequently, the suppressive effect on immune responses of large antigen excesses has been confirmed in a number of immune systems. Martinez et al. 43 have demonstrated that the phenomenon applies to tissue transplantation. Mice given large amounts of tissue brei from another strain show a significantly prolonged survival of test skin grafts from that strain. Application of these findings to organ transplantation in man is tempered by the observation that animals "paralyzed" by large antigen excesses may eventually develop immunity to the antigen after long periods. It was also known that total body irradiation'! and the alkylating nitrogen mustards" that destroyed lymphoid tissue preferentially could inhibit both the induction and expression of immune responses. It was known that cortisone administration caused an abrupt decline in circulating lymphocytes and inhibition of the intensity of immune responses. 75 Chronologically, the next major advance in immune suppression was the discovery of neonatal tolerance by Owens" and Billingham et al." after the predictions of Burnet.!" Owens' basic observation involved freemartin cattle. Frequently, in bovine twin births, the twins have shared placental vascular anastomoses, with the blood of each twin perfusing the other. Under these circumstances the twins may become chimeras with cells of both genotypes circulating in their blood in later life. These twins will accept grafts of skin from one another. It was reasoned that exposure to the cells of one twin before the immune apparatus had been developed and subsequent persistence of these cells had caused the immune apparatus of the second twin to recognize these antigens as "self" in later life. Billingham et al. showed that injection of cells from one animal strain into embryos or the newborn of another strain produced a state of acquired tolerance to tissue grafts from the donor strain. However, if the cells injected into the fetus or newborn were in themselves immunologically competent (lymph node cells or spleen cells) a high proportion of the recipients developed "runt disease."! This general wasting and inanition was later shown to be mediated by the transfer of small lymphocytes'"
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and the development of a "graft versus host" reaction, the development of immunity by the transferred cells to the tissues of the host. For maintenance of the tolerant state the antigen must replicate and persist. Acquired tolerance to noncellular antigens eventually disappears. The discovery of acquired tolerance led to intensified research in the development of immune capacity in neonatal life and the rediscovery that the thymus has a regulatory influence over the development of immune maturity in mammals." Thymectomy in newborn mice produces a state of immune unresponsiveness as the animal matures and the ability to accept test skin grafts from other strains. Thymectomy later in life is ineffective except when the skin donor and recipient bear only weak histocompatibility differences." Since thymus tissue implanted in cell-impenetrable diffusion chambers in thymectomized animals allows the development of immune , capacity, it is assumed that the regulation is humoral in nature rather than by population of the reticuloendothelial system by cells born in the thymus. In the rare state of congenital agammaglobulinemia, skin homografts have been observed to survive for long periods." These children do have the capacity to develop delayed hypersensitivity to tuberculin. Schwartz and Damashek'" reasoned that if the introduction of antigen to an inactive, immature lymphoid system in the newborn produced tolerance to the antigen, it might be possible to produce tolerance in the
Table 1. Immunosuppressive Techniques TECHNIQUE
Antigen excess
REFERENCE
.
22
PROPOSED MECHANISM OF ACTION
Inhibition of both development and manifestation of immune response in presence of excess antigen Total body irradiation . 14 Destruction of lymphoid tissues Reticuloendothelial blockade. Engorgement of phagocytic capacity of reticuloendothelial system by Trypan Blue or Thorotrast Thymectomy . 25,49 Removal of regulatory center for development and differentiation of immune apparatus Antipurine chemotherapy .... 1,59,66,83 Competitive inhibition of ribonucleic acid synthesis in lymphoid tissues Corticoids . Inhibition of development of primary im82 mune reactions and inhibition of inflammatory effector cells Local irradiation of graft . 41 Destruction of effector cells Thoracic duct drainage . 16,42,46 Depletion of lymphoid cells Alpha globulin administration Coating of reactive sites on target cells 52 Amino acid administration ... Competition with other amino acids for 64 entry into antibody forming cells Agammaglobulinemia . 26 Failure to make antibody Nutritional deficiency, uremia Nonspecific inhibition of antibody synthesis
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adult if the antigen were introduced after the immune system had been drastically suppressed by chemotherapy. They found that adult rabbits given large doses of 6-mercaptopurine could accept skin homografts for long periods. Graft acceptance continued only so long as the .drug was administered. The effect of a variety of drugs on the survival time of kidney homografts was investigated.' Work of this type laid the basis for current protocols in human renal homotransplantation. These investigations together with those of Zukoski et al. 83 and of Pierce and Varc0 59 indicated that renal homotransplantation in dogs might be successful for long periods if antipurines were given continuously. Subsequently, the administration of corticoids" and local irradiation" of a graft have allowed long-term survival of a few dogs. These modalities have become widely used techniques in suppression of renal homograft rejection crises in man. The current techniques for immune suppression for organ transplantation in man are dangerous; large number of organ recipients continue to die of the toxicity of antimetabolite administration, both directly and through the development of uncontrollable infections. Examination of other immunosuppressive techniques is needed. Fortunately, a number of promising leads are open. McGregor and Gowans" have shown that depletion of small lymphocytes from the bodies of rats can be achieved by a few day's drainage of the thoracic duct. These animals do not develop primary immune responses but appear to be fully competent with regard to anamnestic booster responses. Prolongation of skin and renal homograft survival time in dogs and rats occurs when thoracic duct drainage is instituted at the same time the graft is performed." but there appears to be little prolongation if the drainage is done before the graft is placed." It is more difficult to deplete man of lymphoid tissue." In one personal case, drainage of about 20 billion lymphocytes from the recipient prior to a cadaver renal transplantation failed to alter the nature and severity of the rejection crisis 16 days later. Other immunosuppressive techniques are currently in laboratory stages of investigation. Administration of large amounts of an a globulin of serum seems to suppress primary immune responses and to increase the survival time of homografts.P Administration of large doses of the amino acid, phenylalanine;" seems to delay immune responses. The Relation of Host and the "Accepted" Allograft The greatest immediate threat to the individual who has undergone an organ homotransplantation is the forthcoming rejection crisis. Control with antipurines, prednisone, local graft irradiation or other immunosuppressive measures leads to a state of apparent tolerance, perhaps interspersed with milder rejection crises. The nature of this state is not clear at present. Is there complete immune suppression in the host maintained on antipurines? Has the graft taken on the antigenic identity of the host? Is there a state of tolerance on the part of the host to the tissues of the
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donor? The group at the Peter Bent Brigham Hospital have considered these questions in a series of incisive experiments." They found, as have others, that an occasional dog recipient of a kidney homograft can be taken off antipurines without damage to the graft. Both those animals taken off drugs and others still receiving drugs are immunologically competent; they have the capacity to reject skin grafts from indifferent donors. Further, these conditioned animals may reject a second kidney transplant from the original donor while the first transplant continues to function undisturbed! The "conditioned" graft may be returned to its original host with normal function, indicating that it has not taken on the antigenic features of the host in which it was accepted. And, if this "conditioned" homograft is transplanted to a third indifferent dog, it undergoes the usual homograft rejection indicating that it has not lost antigenic specificity. Thus, an allograft that is antigenic exists and functions in an immunologically competent animal. Clearly, under these circumstances, the transplanted tissue has undergone some type of adaptation to its new environment. The nature of this adaptation defies precise explanation in terms of known biologic mechanisms. It is appropriate to end this review on this note of uncertainty with its implication of the experimental character of tissue transplantation between separate individuals. The evidence cited here has provided the basis for clinical experimentation in man.
REFERENCES 1. Alexandre, G. P. J., Murray, J. E., Dammin, G. J. and Nolan, B.: Immunosuppressive drug therapy in canine renal and skin homografts. Transplantation 1: 432-461, 1963. 2. Algire, G. H., Weaver, J. M. and Prehn, R. T.: Studies on tissue homotransplantation in mice, using diffusion-chamber methods. Ann. N ew York Acad. Sc. 64: 1009-1012, 1957. 3. Barnes, B. A. and Flax, M. H.: Experimental pulmonary homo grafts in dog. I. Morphological studies. Transplantation 1: 351-364, 1963. 4. Bauer, D. C. and Stavitsky, A. B.: On the different molecular forms of antibody synthesized by rabbits during the early response to a single injection of protein and cellular antigens. Proc. Nat. Acad. Sc. 47: 1667-1680, 1961. 5. Billingham, R. E., Brent, L. and Medawar, P. B.: Actively acquired tolerance of foreign cells. Nature 172: 603-606, 1953. 6. Billingham, R. E., Defendi, V., Silvers, W. K. and Steinmuller, D.: Quantitative studies on the induction of tolerance to skin homografts and on runt disease in neonatal rats. J. Nat. Cancer Inst. 28: 365-435, 1962. 7. Billingham, R. E. and Sparrow, E. M.: Studies on the nature of immunity to homologous grafted skin, with special reference to the use of pure epidermal grafts. J. Exper. BioI. 31: 16-39, 1954. 8. Bloch, K. J., Kourilsky, F. M., Ovary, Z. and Benacerraf, B.: Properties of guinea pig 7S antibodies. III. Identification of antibodies involved in complement fixation and hemolysis. J. Exper. Med. 117: 965-981, 1963. 9. Bonfiglio, M., Jeter, W. S. and Smith, C. L.: The immune concept: its relation to bone transplantation. Ann. New York Acad. Sc. 59: 417-433,1955.
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10. Burnet, M.: The Clonal Selection Theory of Acquired Immunity. Nashville, Tenn., Vanderbilt Univ. Press, 1959, 209 pp. 11. Dagg, C. P., Karnofsky, D. A. and Roddy, J.: Growth of transplantable human tumors in the chick embryo and hatched chick. Cancer Res. 16: 589-594, 1956. 12. Davies, D. A. L.: The isolation of mouse antigens carrying H-2 histocompatibility specificity: Some preliminary studies. Biochem, J. 84: 307-317, 1962. 13. Dempster, W. J.: Kidney homotransplantation. Brit. J. Surge 40: 447-465, 1953. 14. Dixon, F. J., Talmage, W. and Maurer, P. H.: Radiosensitive and radioresistant phases in antibody response. J. Immunol. 68: 693-700, 1952. 15. Dong, E., Hurley, E. J., Lower, R. R. and Shumway, N. E.: Performance of the heart two years after autotransplantation. Surgery 56: 270-273, 1964. 16. Dumont, A. E., Mayer, D. J. and Mulholland, J. H.: The suppression of thoracic duct lymph. Ann. Surge 160: 373-382, 1964. 17. Eisen, H. N.: Hypersensitivity to simple chemicals. In Cellular and Humoral Aspects of the Hypersensitive States (H. S. Lawrence, Ed.). New York, P. B. Hoeber, 1959, pp. 89-119. 18. Eisen, H. N., Carsten, M. and Belman, S.: Studies of hypersensitivity to low molecular weight substances. III. The 2,4-dinitrophenyl group as a determinant in the precipitin reaction. J. Immunol. 73: 296-308, 1954. 19. Fagraeus, A.: Antibody production in relation to the development of plasma cells. Acta med. Scandinav. (Suppl.) 204: 5-122, 1948. 20. Fahey, J. L.: Heterogeneity of -v-globulins. In Advances in Immunology, Vol. 2 (W. H. Taliaferro and J. H. Humphrey, Eds.). New York, Academic Press, 1962, pp.41-109. 21. Favour, C. B.: Comparative immunology and the phylogeny of homotransplantation. Ann. New York Acad. Sc. 73: 590-598,1958. 22. Felton, L. D.: Significance of antigen in animal tissues. J. Immunol. 61: 10t-117, 1949. 23. Fireman, P., Vannier, W. E. and Goodman, H. C.: Association of skin-sensitizing antibody with the ~2A-globu1insin sera from ragweed-sensitive patients. J. Exper. Med. 117: 603-619, 1963. 24. Fishman, M. and Adler, F. L.: Antibody formation initiated in vitro. II. Antibody synthesis in x-irradiated recipients of diffusion chambers containing nucleic acid derived from macrophages incubated with antigen. J. Exper. Med. 117: 595-602, 1963. 25. Good, R. A., Dalmasso, A. P., Martinez, C., Archer, O. K., Pierce, J. C. and Papermaster, B. W.: Role of the thymus in development of immunologic capacity in rabbits and mice. J. Exper. Med. 116: 773-796,1962. 26. Good, R. A. and Varco, R. L.: Successful homograft of skin in a child with agammaglobulinemia. J.A.M.A. 157: 713-716, 1955. 27. Gorer, P. A., Loutit, J. F. and Micklem, H. S.: Proposed revisions of "Transplantese." Nature 189: 1024-1025, 1961. 28. Greene, H. S. N.: Compatibility and noncompatibility. Ann. New York Acad. Sc. 59: 311-318, 1955. 29. Harbison, S~ P.: Origins of vascular surgery: The Carrel-Guthrie letters. Surgery 52: 406-418, 1962. 30. Harris, S. and Harris, T. N.: Studies on the transfer of lymph node cells. V. Transfer of cells incubated in vitro with suspensions of Shigella paradysenteriae. J. Immunol. 74: 318-328, 1954. 31. Haurowitz, F.: The mechanism of the immunological response. BioI. Rev. 27: 247280,1952. 32. Herzenberg, L. A. and Herzenberg, L. A.: Association of H-2 antigens with the cell membrane fraction of mouse liver. Proc. Nat. Acad. Sc. 47: 762-767, 1961. 33. Hildemann, W. H.: Scale homotransplantation in goldfish (Carassius auratus). Ann. New York Acad. Sc. 64: 775-790,1957. 34. Immunology Symposium: Immunologic phenomena in cold-blooded vertebrates. InFed.Proc. 22: 1131-1155, 1963. 35. Ishizaka, K., Ishizaka, T. and Sugahara, T.: Biological activity of soluble antigenantibody complexes. VII. Role of an antibody fragment in the induction of biologic activities. J. Immunol. 88: 690-701, 1962.
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36. Kaliss, N.: Immunological enhancement of tumor homografts in mice: Review. Cancer Res. 18: 992-1003, 1958. 37. Kandutsch, A. A. and Stimpfling, J. H.: Partial purification of tissue isoantigens from a mouse sarcoma. Transplantation 1: 201-216, 1963. 38. Kantor, F. S., Ojeda, A. and Benacerraf, B.: Studies on artificial antigens. I. Antigenicity of DNP-polylysine and DNP copolymer of lysine and glutamic acid in guinea pigs. J. Expel'. l\1ed. 117: 55-69, 1963. 39. Krohn, P. L.: Ovarian homotransplantation. Ann. New York Acad. Sc. 59: 443447,1955. 40. Landsteiner, K. and Chase, M. W.: Experiments on transfer of cutaneous sensitivity to simple compounds. Proc. Soc. Expel'. BioI. & Med. 49: 688-690, 1942. 41. Lee, H. M., Kauffman, H. M., Cleveland, R. J. and Hume, D. M.: Selective immunosuppression by local radiation of renal homografts. S. Forum 15: 158-160, 1964. 42. McGregor, D. D. and Gowans, J. L.: Antibody response of rats depleted of lymphocytes by chronic drainage from the thoracic duct. J. Expel'. Med. 117: 303-320, 1963. 43. Martinez, C., Smith, J. M., Blaese, M. and Good, R. A.: Production of immunological tolerance in mice after repeated injections of disrupted spleen cells. J. Exper. Med. 118: 743-757, 1963. 44. Maurer, P. H.: Attempts to produce antibodies to a preparation of polyglutamic acid. Proc. Soc. Expel'. BioI. & Med. 96: 394-396, 1957. 45. Maurer, P. H., Gerulat, B. F. and Pinchuck, P.: Antigenicity of synthetic polypeptides (abstr.). Fed. Proc. 22: 555, 1963. 46. Mayer, D. J. and Dumont, A. E.: Prolonged survival of skin homografts following diversion of thoracic duct lymph. S. Forum 14: 194-195, 1963. 47. Medawar, P. B.: Behaviour and fate of skin autografts and skin homografts. J. Anat. 78: 176-199, 1944. 48. Medawar, P. B.: Immunity to homologous grafted skin. III. The fate of skin homografts transplanted to the brain, to subcutaneous tissue, and to the anterior chamber of the eye. Brit. J. Expel'. Path. 29: 58-69, 1948. 49. Miller, J. F. A. P., Marshall, A. H. E. and White, R. G.: The immunological significance of the thymus. In Advances in Immunology, Vol. 2 (W. H. Taliaferro and J. H. Humphrey, Eds.). New York, Academic Press, 1962, pp. 111-162. 50. Mitchinson, N. A.: Passive transfer of transplantation immunity. Nature 171: 267-268, 1953. 51. Moore, F. D., Wheeler, H. B., Demissianos, H. V., Smith, L. L., Balankura, 0., Abel, K., Greenberg, J. B. and Dammin, G. J.: Experimental whole-organ transplantation of the liver and of the spleen. Ann. Surge 152: 374-385, 1960. 52. Mowbray, J. F.: Effect of large doses of an a2 glycoprotein fraction on the survival of rat skin homografts. Transplantation 1: 15-20, 1963. 53. Murray, J. E., Sheil, A. G. R., Moseley, R., Knight, P., McGavic, J. D. and Dammin, G. J.: Analysis of mechanism of immunosuppressive drugs in renal homotransplantation. Ann. Surge 160: 449-470, 1964. 54. Najarian, J. S. and Feldman, J. D.: Passive transfer of tuberculin sensitivity by tritiated thymidine-labeled lymphoid cells. J. Expel'. Med. 114: 779-789, 1961. 55. Najarian, J. S. and Feldman, J. D.: Passive transfer of transplantation immunity. III. Inbred guinea pigs. J. Exper. Med. 117: 449-456, 1963. 56. Najarian, J. S. and Feldman, J. D.: Passive transfer of transplantation immunity. IV. Transplantation antibody from extracts of sensitized lymphoid cells. J. Expel'. Med. 118:759-766,1963. 57. N ossal, G. J. V.: Cellular genetics of immune responses. In Advances in Immunology, Vol. 2 (W. H. Taliaferro and J. H. Humphrey, Eds.). New York, Academic Press, 1962, pp. 163-204. 58. Owens, R. D.: Immunogenetic consequences of vascular anastomoses between bovine twins. Science 102: 400-401, 1945. 59. Pierce, J. C. and Varco, R. L.: Effects of long-term 6-mercaptopurine treatment upon kidney homotransplants in dogs. Surgery 54: 124-134, 1963. 60. Porter, K. A. and Cooper, E. H.: Transformation of adult allogeneic smalllymphocytes after transfusion into newborn rats. J. Expel'. Med. 115: 997-1008, 1962. 61. Rappaport, F. T., Lawrence, H. S., Thomas, L., Converse, J. M., Tillett, W. S. and
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Mulholland, J. H.: Cross-reactions to skin homografts in man. J. Clin. Invest. 41: 2166-2172, 1962. Roberts, J. C. and Dixon, F. J.: The transfer of lymph node cells in the study of the immune response to foreign proteins. J. Exper. Med. 102: 379-392, 1955. Russell, P. S. and Monaco, A. P.: The biology of tissue transplantation. New England J. Med. 271: 502 ff., 1964. Ryan, W. L. and Lorincz, A. B.: Inhibition of the immune response by phenylalanine and its application to skin transplants (abstr.). J.A.M.A. 188: 442, 1964. Sanders, F. K.: Preservation of nerve grafts. In Preservation and Transplantation of Normal Tissues. Ciba Foundation Symposium (G. E. W. Wolstenholme and M. P. Cameron, Eds.). Boston, Little, Brown & Co., 1954, 236 pp. Schwartz, R. and Damashek, W.: Drug-induced immunological tolerance. Nature 183: 1682-1683, 1959. Scothorne, R. J.: Studies on the response of the regional lymph node to skin homografts. Ann. New York Acad. Sc. 64: 1028-1038,1957. Sela, M. and Arnon, R.: Studies on the chemical basis of the antigenicity of proteins. I. Antigenicity of polypeptidyl gelatins. Biochem. J. 75: 91-102, 1960. Spurr, C. L.: Influence of nitrogen mustards on antibody response. Proc. Soc. Exper. BioI. & Med. 64: 259-261, 1947. Starzl, T. E. and Kaupp, H. A., Jr.: Mass homotransplantation of abdominal organs in dogs. S. Forum 11: 28-30, 1960. Stetson, C. A.: Role of humoral antibodies in homograft reaction. In Advances in Immunology 3: 97-130, 1963. Stetson, C. A. and Demopoulos, R.: Reactions of skin homografts .with specific immune sera. Ann. New York Acad. Sc. 73: 687-697,1958. Sturim, H. S. and Newton, W. T.: Renal homotransplantation after thoracic duct drainage. Clin. Res. (in press). Taylor, A. C. and Lehrfeld, J. W.: Definition of survival time of homografts. Ann. New York Acad. Sc. 59: 351-360, 1955. Teilum, G., Engbach, H. C. and Simonsen, M.: Effects of cortisone on plasma cells and reticulo-endothelial system in hyperimmunized rabbits. Acta endocrinol, 5: 181-193, 1950. Toolan, H. W.: Possible role of cortisone in overcoming resistance to the growth of human tissues in heterologous hosts. Ann. New York Acad. Sc. 59: 394-400, 1955. Triplett, E. L.: On the mechanism of immunologic self recognition. J. Immunol, 89: 505-510, 1962. Uhr, J. W. and Finkelstein, M. S.: Antibody formation. IV. Formation of rapidly and slowly sedimenting antibodies and immunological memory to bacteriophage X 174. J. Exper. Med. 117: 457-477,1963. Uhr, J. W., Salvin, S. B. and Pappenheimer, A. M., Jr.: Delayed hypersensitivity. II. Induction of delayed hypersensitivity in guinea pigs by means of antigenantibody complexes. J. Exper. Med. 105: 11-24, 1957. Wolstenholme, G. E. W. and O'Connor, M. (Eds.): Ciba Foundation Symposium on Cellular Aspects of Immunity. Boston, Little, Brown, & Co., 1960, 495 pp. Woodruff, M. F. A.: Transplantation of tissues and organs. Springfield, Ill., Charles C Thomas, 1960, 816 pp. Zukoski, C. F., Calloway, J. M. and Rhea, W. G.: Tolerance to canine renal homograft induced by prednisolone. S. Forum 14: 208-210, 1963. Zukoski, C. F., Lee, H. M. and Hume, D. M.: The prolongation of functional survival of canine renal homografts by 6-mercaptopurine. S. Forum 11: 470-472, 1960.
4960 Audubon Avenue St. Louis, Missouri 63110
This is a short summary of modern advances in transplantation ' biology. It is written for that busy practitioner who, in a reasonable economy of time, wishes to become intelligently conversant in this field. And, to this end, literary citations of reviews and summations of work are preferred over documentation of original sources. The interested student will wish to consult the volume by Woodruff 81 and the current scholarly review by Russell and Monaco. 63 It will shortly become clear that the failure of one individual to accept and maintain a tissue graft from another is an immune response on the part of the host. Consequently, this author chooses to deviate from the pattern of other published reviews and to examine the phenomena of homograft sensitivity against a background review of the general nature of immune responses. INTRODUCTION
Progress in organ homotransplantation required that three basic questions be answered. How mayan excised organ be joined to a new host? By what process does a temporarily successful union of an individual's organ to another host almost invariably fail? Can the processes that have directed the transplantation failure be governed? The first question was answered almost in entirety in the decade from 1902 to 1912 by the studies of Carrel and Guthrie." Satisfactory surgical union of major blood vessels allowed the transplantation of almost every organ of the body to new sites in new hosts. Yet the transplant, after functioning quite well for a few days, invariably failed. It remained for Medawar" to provide experimental From the Department of Surgery, Washington University School of Medicine and the Veterans Administration Hospital, St. Louis, Missouri * Associate Professor of Surgery, Washington University School of Medicine; Chief of Surgery, John Cochran Veterans Administration Hospital; Staff Physician, Barnes Hospital
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evidence in answer to the second question, the means by which destruction of the graft is mediated. He observed that a second graft of skin from one rabbit to another, after a first graft had been rejected, was now rejected in a (more rapid and intense fashion. He concluded that the host had been immunized by the first graft, and that the rejection of grafts was an immune response on the part of the host. The third question, regarding the control of immune processes, had already been demonstrated in some immune systems. Transplantation research provided the impetus to rapid development of immunosuppressive techniques. The answer to the first question made it obvious that any organ could be transplanted. Certain technical features of transplantation of the liver" and heart" required refinement, but success was almost guaranteed after successful union of blood vessels had been achieved. The major contributions of the surgeon as a technician came to an end in the first decade of this century.
Terminology The terms used to designate various types of grafts first proposed by Medawar" have been widely used, although Gorer'" has pointed out etymological inconsistencies. A graft of tissue from one part of an individual to another part of the same individual is an autograft. A transplantation from one individual to another of identical genetic constitution, such as in identical twins or a member of the same inbred animal strain, is an isograft. Transplants between individual members of the same species, but of different genetic make-up, are homografts. Transplants from one species to a member of another species are heterografts. In the newer terminology proposed by Gorer, autografts and isografts remain unchanged and are adjectively referred to as autologous and isogeneic (or syngeneic) transplantations. The homograft becomes an allogeneic transplantation of an allograft. Heterotransplants become xenogeneic transplantations of xenografts.
THE NATURE OF THE HOMOGRAFT REACTION
Biologic Observations A transplant of skin from one rabbit to another heals in well and has the same appearance as a skin autograft. Capillaries from the host grow across the graft bed and link with those of the graft or invade the graft. There are no significant collections of cells associated with inflammation. There is no pre-existing incompaiibilitu of host and graft. The incompatibility develops after the graft has been performed. Between the fourth and fifteenth day after grafting, biopsies of the graft show collections of lymphocytes, plasma cells, and perhaps eosinophils in the graft-host junction. Shortly thereafter, observation through a dissecting microscope will show a slowing
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of circulation in some of the capillaries and formation of microthrombi. 74 Suddenly, the circulation ceases completely, and the graft becomes black. If, after rejection of such a skin graft, another graft is attempted between the same original donor and host, the rejection occurs after only three to six days and is associated with a more intense inflammatory process, the "second-set" reaction. In fact, the graft may not heal in at all-the "white graft" type of second-set reaction. Similar reactions have been found in almost all vertebrates studied, from fishes" to man. Aside from certain differences in tempo and pathology, the reaction has been noted in tissues or organs derived from all three germ layers. The reaction has been noted in such disparate tissues as kidney.P liver," lung," gut/o nerve" and bone." In all cases there is a brief period of apparent acceptance followed by rejection associated with inflammation and vascular occlusion. There may be reservations regarding this generality in the case of endocrine tissues. 39 Homografts of one tissue will so sensitize a recipient as to show an accelerated second-set reaction to a subsequent graft of an entirely different tissue. Thus, the sensitivity is individual specific, not organ specific. However, there is considerable variation in the duration of the sensitive state. Homografts of skin must follow grafts of lymphoid tissues by only a few days in order to show second-set skin graft rejections. On the other hand, renal homografts will show accelerated rejection if they are performed many weeks after a skin homograft rejection. Red blood cells are very poor sensitizers except in those species with nucleated red blood cells. The route of immunization has a significant role in these variations. For example, dissociated epidermal cells are more effective sensitizers injected intracutaneously than subcutaneously, and, if injected intravenously, a delay in second-set rejection may occur. 7 There exist certain peculiar sites where grafts may exist apparently invulnerable to the processes of rejection. Particularly the anterior chamber of the eye 28 and the cerebral hemispheres" are sites where tissue transplants may survive for long periods without evidence of rejection. These sites lack lymphatic drainage. The cheek pouch of the hamster" and the allantoic membrane of the hen's egg" are sites where even heterografts may be grown under favorable conditions. The Homograft Reaction as an Immune Response
Biologically, the homograft reaction appears to be but one other of a group of phenomena termed immune responses. Evidence for this statement is the lag period required for the development of the first-set reaction, the intensification of the reaction in hosts subjected to a prior exposure to donor tissue, the relative specificity of the reaction for one donor over others (there is some cross-reactivity of a sensitive host to the tissues of a population of donors"), and, finally, the demonstration by Mitchison'" that the sensitive state could be transferred from one host to another by isolated
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lymph node cells. It is appropriate, then, to examine the nature of immune responses in general and their application to allograft (homograft) sensitivity. Immune responses are characteristic of the vertebrate portion of the animal kingdom." The immune response is a result of reaction of an antigen with an immunologically competent cell.
Antigens Systematic study begins with the antigen, that is, a substance which excites an immunologically competent cell to undergo an immune response. Antigens are large molecules of greater than, say, 10,000 molecular weight, typically proteins or polysaccharides. Normally, a given host appears to recognize its own proteins as "self" and does not develop an immune response to them. The reason for this failure to react to "self" proteins that are quite antigenic in another species is the subject of much speculation'"- 31, 77 but remains in question. The immune apparatus develops late in embryogenesis. It appears that there is suppression of the growth and differentiation of those immune cells with propensity to react to antigens in the environment at that time. In exceptional circumstances, a host may react to its own antigens to produce an autoimmune reaction. Perhaps, under these circumstances, there has been a mutation of an antigenic determinant or a hapten substitution (vide infra) to produce a new antigen. In spite of the requirement that an antigen be a large molecule, the area of antigenic specificity of the molecule may be rather small, of the order of only one or two amino acid residues. And there may be many of these areas or "antigenic determinants" in a single antigen molecule. Artificial antigens have been created." Copolymers of glutamic acid, alanine and lysine are particularly effective antigens." However, polymers of a single amino acid, polylysine or polyglutamic acids have not been found to be antigenic." Antigenic specificity may be created by the chemical substitution of a simple chemical on the protein. For example, substitution of the terminal epsilon amino group of lysine in a protein.P or even in polylysine.i" by 2,4-dinitrophenyl groups produces an antigen whose specificity seems directed toward dinitrophenyllysine. Other simple chemicals may act similarly; toluene sulfonic acid, arsanilic acid, and certain azo compounds are examples. The simple chemical is called a hapten when its conjugation with protein or polyamino acid produces an antigen with specificity directed toward the substituted group. There have been many attempts to isolate a single molecular species from tissues that might represent an allograft antigen. Cell free materials have been found that have antigenic properties.P: 32, 37 The antigenic material seems to be a lipoprotein, perhaps associated with cell membranes or the enveloping membrane of cell microsomes. The lipid portion of the
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antigen may have haptenic properties in the determination of specificity, but chemical analyses of the lipoproteins isolated so far has shown a wide variety of lipids in any given preparation. There is evidence that the allograft antigen, in vivo, remains closely associated with donor cells. Algire'' constructed small diffusion chambers of a ring of lucite with circles of millipore filters of O.45JL pore size glued to the two edges of the ring. Bits of tissue could be placed within these chambers, and the whole could be implanted in the peritoneal cavity of a host. The filter pore size allowed the exchange of fluids and proteins but excluded host cells from entry and graft cells from exit. The grafts survived for long periods under these circumstances, and, pertinent to the present discussion, the hosts did not seem to be sensitized to subsequent test grafts from the original tissue donor. Antigen apparently did not pass from the cells within the chamber through the pores and to the site of reaction , with the host immune apparatus. Questions have been raised recently as to the permeability of these chambers to antibody and complement, and perhaps to antigen also.
The Immunologically Competent Cell Antigen, allowed to pierce the skin barrier, may either make its way in solution to a regional lymph node through the lymphatic drainage, or it may be carried there by a cell. There is evidence that a preliminary processing of antigen by macrophages is a prerequisite to the induction of the immune response." Other evidence points to the small lymphocyte as the wandering cell that carries antigen to the lymph node. The cell on arrival at the lymph node either passes on the antigenic information to other cells, or itself becomes localized to the node and differentiates into cells productive of immune responses. Particularly, the development of large lymphocytes and, later, plasma cells" seems associated with immune responses. The stimulated lymph node shows changes perhaps related to an increase in protein synthesis. An increase in the mass of the node and a greater relative increase in ribonucleic acid than in deoxyribonucleic acid occurs. Microsections of the nodes show reticular hyperplasia with engorgement of the medullary sinuses with lymphocytes, histocytes, and plasma cells. All these changes are seen in lymph nodes regional to the injection site of a potent purified antigen and also in nodes regional to rejection sites of tissue allografts;" 'The cellular participation in immune responses is the subject of comprehensive reviews.": 80 Cells teased from stimulated lymph nodes have the capacity to continue to evoke immune responses when they are transferred to another host of the same species. These cells continue to produce antibodies" or participate in delayed hypersensitivity reactions, as the case may be. As noted earlier, allograft sensitivity can be transferred in this fashion. Evidence indicates that the lymphocyte and plasma cell of the lymph node
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are able to produce antibodies and mediate delayed hypersensitivity reactions. In fact, nonsensitized lymph node cells or small lymphocytes from the thoracic duct may be sensitized in vitro by exposure to antigen, washed free of antigen, and produce an immune response when transferred to a host rendered incapable of an immune response of its own by virtue of total body irradiation." The course of events of these cells may be followed by a tritiated thymidine label. Thymidine is incorporated only into deoxyribonucleic acid of cells, and, since the turnover rate of deoxyribonucleic acid is very slow, the radioactive thymidine becomes a good label of the whole cell. Labeled cells from tuberculin sensitive hosts on transfer to a new host are found to congregate at the site of an injection of tuberculin in the new host." On the contrary, labeled cells from allograft sensitive donors do not appear at allograft rejection sites in new hosts in any great number." In summary, it can be said that the allograft reaction requires a cell for its genesis and its mediation. On the other hand, the reaction does not have all the characteristics of the known cell-associated, delayed hypersensitivity reactions. The Immune Response The biologic phenomena of immune responses are essentially of two varieties: (1) responses mediated by a humoral factor in the serum of an immunized host and capable of being transferred to another host by injecting serum of the immunized host into the second host, and (2) responses in which our imperfect techniques do not allow the demonstration of humoral antibodies and which can only be transferred to another host by living lymph node cells. The greater mass of immune responses are of the first type with demonstrable antibodies in the serum. The second types are restricted to a group of reactions termed delayed hypersentiv1."ty reactions. An individual with humoral antibody in his serum responds to an intracutaneous injection of antigen with the development of a wheal within a few minutes followed by surrounding erythema. This is an immediate reaction. The delayed type is characterized by the absence of immediate reaction and development over a period of twelve to 24 hours of an indurated erythematous reaction without a wheal. The classic example is the tuberculin reaction. The contact skin sensitivity associated with certain protein reactive chemicals is a delayed hypersensitivity reaction. 17, 40 Immunization with very small amounts of antigen or of antigen-antibody complex may be followed by a temporary state of delayed hypersensitivity to the antigen, superseded eventually by serum antibody formation and immediate skin reactivity." Delayed hypersensitivity reactions are associated only with protein antigenic stimulation; polysaccharide antigens have been ineffective. The antibodies that mediate the majority of immune responses are a
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subgroup of serum proteins, the "immune globulins," of which three or four types can be separated in mammals." The principal separatory process is related to the electrophoretic migration of proteins in an electric field. Ordinarily, all serum proteins are negatively charged at pH 8.6 and migrate toward the positively charged anode in an electric field. The most rapid migration is by the serum albumins followed by the alpha, beta and gamma globulins, in that order. Each of the globulin peaks can be shown to consist of subgroups on precise analysis. Antibody activity has been found to be associated with the {32A globulins, and with the 1'1 and 1'2 subpeaks of the gamma globulin fraction. Further, the 1'1 fraction can be separated by ultracentrifugation, or by molecular sieve action, into a heavy ('Ylm) fraction of over one million molecular weight with sedimentation velocity of over 18 Svedberg units, and a light (1'188) fraction of approximately 160,000 molecular weight and 7S sedimentation. The 1'2 antibody is also of the 7S , variety. Varying biologic activity has been found for the different antibody fractions, although all four antibody types ({32A, 'Y1m, 1'188 and 1'2) may be produced in response to a single antigenic stimulus. Allergic states, such as ragweed sensitivity, may be associated only with {32A antibody." Gamma-j, antibodies are apt to be produced early in an immune response4 • 78 with the 7S antibodies appearing later. Guinea pig 1'188 antibodies are incapable of binding complement" as opposed to the 1'2 type. The definition of chemical structure of these antibodies and its relationship to biologic activity is a fermentive field of research today, and these relationships may have significant impact on the allograft sensitivity problem. Since all known immunologic processes that injure cells appear to be mediated by a binding of complement to an antigen-antibody complex, it is feasible that the 1'1 antibodies or digested fragments of antibodies that are incapable of binding complement" might be protective rather than destructive when they join a tissue graft. A reasonable explanation of the phenomenon of tumor transplant enhancement," might be offered through this mechanism. In this phenomenon, immunization to certain tumors across strains in mice produces an ability to accept tumor transplants rather than increased resistance. The protection can be shown to be associated with serum l' globulin. Early failures to transfer allograft sensitivity by serum from animals that had rejected tissue allografts, and the ability to transfer the sensitivity with lymph node cells compelled many to consider the allograft reaction as a cell-bound antibody response of the delayed hypersensitivity type. Controversy was aroused when it was shown that allograft sensitivity could be transferred in some experiments by serum from hyperimmunized mice.?' Furthermore, regular transfer of sensitivity has recently been obtained with extracts of sensitive lymph node cells." The active material in these extracts seems to be a gamma globulin. This controversy is the subject of a comprehensive current review. 71
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SUPPRESSION OF IMMUNE RESPONSE
The reaction of a population of animals to the same antigenic stimulation varies widely. In some cases one genetic strain of animals may react intensely to a given antigen, but another strain may react poorly if at all. Until the resurgence of interest in tissue transplantation, immunologists had been more interested in the intensification of immune responses than in their suppression. However, some indication of the possibility of immune suppression came from Felton's observations on "immune paralysis.i'" He observed that mice immunized with very large amounts of pneumococcal polysaccharide not only failed to develop serum antibody to the polysaccharide but were refractory for long periods thereafter to subsequent attempts to immunize the mice with the usual amounts. Subsequently, the suppressive effect on immune responses of large antigen excesses has been confirmed in a number of immune systems. Martinez et al. 43 have demonstrated that the phenomenon applies to tissue transplantation. Mice given large amounts of tissue brei from another strain show a significantly prolonged survival of test skin grafts from that strain. Application of these findings to organ transplantation in man is tempered by the observation that animals "paralyzed" by large antigen excesses may eventually develop immunity to the antigen after long periods. It was also known that total body irradiation'! and the alkylating nitrogen mustards" that destroyed lymphoid tissue preferentially could inhibit both the induction and expression of immune responses. It was known that cortisone administration caused an abrupt decline in circulating lymphocytes and inhibition of the intensity of immune responses. 75 Chronologically, the next major advance in immune suppression was the discovery of neonatal tolerance by Owens" and Billingham et al." after the predictions of Burnet.!" Owens' basic observation involved freemartin cattle. Frequently, in bovine twin births, the twins have shared placental vascular anastomoses, with the blood of each twin perfusing the other. Under these circumstances the twins may become chimeras with cells of both genotypes circulating in their blood in later life. These twins will accept grafts of skin from one another. It was reasoned that exposure to the cells of one twin before the immune apparatus had been developed and subsequent persistence of these cells had caused the immune apparatus of the second twin to recognize these antigens as "self" in later life. Billingham et al. showed that injection of cells from one animal strain into embryos or the newborn of another strain produced a state of acquired tolerance to tissue grafts from the donor strain. However, if the cells injected into the fetus or newborn were in themselves immunologically competent (lymph node cells or spleen cells) a high proportion of the recipients developed "runt disease."! This general wasting and inanition was later shown to be mediated by the transfer of small lymphocytes'"
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and the development of a "graft versus host" reaction, the development of immunity by the transferred cells to the tissues of the host. For maintenance of the tolerant state the antigen must replicate and persist. Acquired tolerance to noncellular antigens eventually disappears. The discovery of acquired tolerance led to intensified research in the development of immune capacity in neonatal life and the rediscovery that the thymus has a regulatory influence over the development of immune maturity in mammals." Thymectomy in newborn mice produces a state of immune unresponsiveness as the animal matures and the ability to accept test skin grafts from other strains. Thymectomy later in life is ineffective except when the skin donor and recipient bear only weak histocompatibility differences." Since thymus tissue implanted in cell-impenetrable diffusion chambers in thymectomized animals allows the development of immune , capacity, it is assumed that the regulation is humoral in nature rather than by population of the reticuloendothelial system by cells born in the thymus. In the rare state of congenital agammaglobulinemia, skin homografts have been observed to survive for long periods." These children do have the capacity to develop delayed hypersensitivity to tuberculin. Schwartz and Damashek'" reasoned that if the introduction of antigen to an inactive, immature lymphoid system in the newborn produced tolerance to the antigen, it might be possible to produce tolerance in the
Table 1. Immunosuppressive Techniques TECHNIQUE
Antigen excess
REFERENCE
.
22
PROPOSED MECHANISM OF ACTION
Inhibition of both development and manifestation of immune response in presence of excess antigen Total body irradiation . 14 Destruction of lymphoid tissues Reticuloendothelial blockade. Engorgement of phagocytic capacity of reticuloendothelial system by Trypan Blue or Thorotrast Thymectomy . 25,49 Removal of regulatory center for development and differentiation of immune apparatus Antipurine chemotherapy .... 1,59,66,83 Competitive inhibition of ribonucleic acid synthesis in lymphoid tissues Corticoids . Inhibition of development of primary im82 mune reactions and inhibition of inflammatory effector cells Local irradiation of graft . 41 Destruction of effector cells Thoracic duct drainage . 16,42,46 Depletion of lymphoid cells Alpha globulin administration Coating of reactive sites on target cells 52 Amino acid administration ... Competition with other amino acids for 64 entry into antibody forming cells Agammaglobulinemia . 26 Failure to make antibody Nutritional deficiency, uremia Nonspecific inhibition of antibody synthesis
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adult if the antigen were introduced after the immune system had been drastically suppressed by chemotherapy. They found that adult rabbits given large doses of 6-mercaptopurine could accept skin homografts for long periods. Graft acceptance continued only so long as the .drug was administered. The effect of a variety of drugs on the survival time of kidney homografts was investigated.' Work of this type laid the basis for current protocols in human renal homotransplantation. These investigations together with those of Zukoski et al. 83 and of Pierce and Varc0 59 indicated that renal homotransplantation in dogs might be successful for long periods if antipurines were given continuously. Subsequently, the administration of corticoids" and local irradiation" of a graft have allowed long-term survival of a few dogs. These modalities have become widely used techniques in suppression of renal homograft rejection crises in man. The current techniques for immune suppression for organ transplantation in man are dangerous; large number of organ recipients continue to die of the toxicity of antimetabolite administration, both directly and through the development of uncontrollable infections. Examination of other immunosuppressive techniques is needed. Fortunately, a number of promising leads are open. McGregor and Gowans" have shown that depletion of small lymphocytes from the bodies of rats can be achieved by a few day's drainage of the thoracic duct. These animals do not develop primary immune responses but appear to be fully competent with regard to anamnestic booster responses. Prolongation of skin and renal homograft survival time in dogs and rats occurs when thoracic duct drainage is instituted at the same time the graft is performed." but there appears to be little prolongation if the drainage is done before the graft is placed." It is more difficult to deplete man of lymphoid tissue." In one personal case, drainage of about 20 billion lymphocytes from the recipient prior to a cadaver renal transplantation failed to alter the nature and severity of the rejection crisis 16 days later. Other immunosuppressive techniques are currently in laboratory stages of investigation. Administration of large amounts of an a globulin of serum seems to suppress primary immune responses and to increase the survival time of homografts.P Administration of large doses of the amino acid, phenylalanine;" seems to delay immune responses. The Relation of Host and the "Accepted" Allograft The greatest immediate threat to the individual who has undergone an organ homotransplantation is the forthcoming rejection crisis. Control with antipurines, prednisone, local graft irradiation or other immunosuppressive measures leads to a state of apparent tolerance, perhaps interspersed with milder rejection crises. The nature of this state is not clear at present. Is there complete immune suppression in the host maintained on antipurines? Has the graft taken on the antigenic identity of the host? Is there a state of tolerance on the part of the host to the tissues of the
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donor? The group at the Peter Bent Brigham Hospital have considered these questions in a series of incisive experiments." They found, as have others, that an occasional dog recipient of a kidney homograft can be taken off antipurines without damage to the graft. Both those animals taken off drugs and others still receiving drugs are immunologically competent; they have the capacity to reject skin grafts from indifferent donors. Further, these conditioned animals may reject a second kidney transplant from the original donor while the first transplant continues to function undisturbed! The "conditioned" graft may be returned to its original host with normal function, indicating that it has not taken on the antigenic features of the host in which it was accepted. And, if this "conditioned" homograft is transplanted to a third indifferent dog, it undergoes the usual homograft rejection indicating that it has not lost antigenic specificity. Thus, an allograft that is antigenic exists and functions in an immunologically competent animal. Clearly, under these circumstances, the transplanted tissue has undergone some type of adaptation to its new environment. The nature of this adaptation defies precise explanation in terms of known biologic mechanisms. It is appropriate to end this review on this note of uncertainty with its implication of the experimental character of tissue transplantation between separate individuals. The evidence cited here has provided the basis for clinical experimentation in man.
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