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Tissue-specific and tissue-restricted histocompatibility antigens David Steinmuller Strictly defined, tissue-specific antigens are antigens characteristic of one particular tissue or cell. They are usually associated with autoimmunity and are remarkably homologous between species. In contrast, histocompatibility (H) antigens reflect polymorphbm within species - they are alloantigens - and class-I major H complex ( M H C ) antigens - - at least mouse H - 2 D and H-2 K and human H L A - A and-B, the commonest targets of acute allograft rejection are widely distributed in the body; class-H M H C antigens - mouse Ia and human D R - have a much more limited distribution, being expressed primarily on B lymphocytes and on macrophages and other cells involved in antigen presentation and immune activation. This review is devoted to H antigens other than class-H M H C antigens with limited if not highly specific, tissue distribution. Some of these antigens are classic tissue-specific antigens," others are alloantigens with limited tissue expression. Much of the evidence that they evoke immune responses that damage or destroy transplanted tissue is incomplete or circumstantial, but some is convincing and includes the immunogenetic characterization of new antigen systems that may have to be reckoned with clinically, especially when dealing with HLA-matched transplants.
Kidney-specific antigens, serologicaUy defined Biopsies or fragments of rejected human kidney allografts can be trypsinized to bring cells into suspension that can be typed like lymphocytes for H L A antigens. The excessive cytotoxicity sometimes detected with kidney cells v. lymphocytes from the same donor suggests that some H L A typing sera contain antibodies to kidneyspecific antigens. For example, Perkins et al. 1 described a patient who rejected a kidney hyperacutely at a time when the only detectable cytotoxic antibodies in his serum were kidney-specific; they reacted with and were absorbed by kidney cells but not lymphocytes. Mohanakumar et al. ,2 eluted antibodies from rejected human kidney allografts that reacted specifically with kidney cells. The antibodies could be completely absorbed with the latter but not with platelets or T or B cells. Ende et al. 3 compared the sera of cadaver kidney allograft recipients who rejected their Histocompatibility Laboratory, Transplantation Society of Michigan Ann Arbor, MI 48104, USA
© 1984, Elsevier Science Publishers B,V., Amsterdam 0167 - 4919/84/$02.00
grafts within one year with that of those who had maintained them at least five years. Much higher levels of what appeared to be kidney-specific antibodies - they did not react with spleen cells, platelets or lung, skin and m u s c l e were found in the sera of the short-lived transplant group. The antibodies were not correlated with the presence or absence of lymphocytotoxic antibodies, and they did not appear to be H L A antibodies. However, except for one case of a donor-specific contralateral kidney, the target cells came from random donors; hence it is not clear whether the antibodies were allo- or organ-specific. However, allospecific n o n - M H C kidney antigens have been detected in rats. If allograft survival is maintained for a month or more by immunosuppression in certain strain combinations, antibodies that react specifically with donor but not host tubular basement membrane (TBM) appear in host serum, and the antibody response is associated with tubular injury in the allograft but not in the host's own non-grafted kidney 4. Similarly distributed lesions have been detected in human kidney allograft
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recipients 4, and Kashiwabara et al. 5 reported that antibody titers to a purified T B M antigen correlated with the onset and grade of acute kidney allograft rejection. Thoenes et al. 6 studied an immune-complex glomerulonephritis that develops five weeks or more after kidney allografting in certain MHC-compatible rat strain combinations. Antibody binds to the brush border of the proximal tubules of the allograft, but not to the host's own kidney, and proteinuria subsides when the allograft is removed. Although the antigen involved is a tissuespecific alloantigen, the disease depends for its induction on reactions to conventional H antigens. It does not occur in kidney allografts transplanted to tolerant hosts but does occur when tolerance is broken by the adoptive transfer of syngeneic lymphoid cells sensitized to donor H antigensT. In contrast, Hart and Fabre ~ studied a n o n - M H C kidney-specific alloantigen that induces a strong antiT B M antibody response in rats but has no role in allograft rejection. Kidney allografts mismatched for the antigen are not rejected in accelerated fashion, even in the face of an ongoing response, and no histological abnormalities are observed after grafting.
Kidney-specific antigens, cellularly defined Mashimo et al. 9 reported that rat kidney cells stimulate lymphocyte blastogenesis in one of four MHC-compatible rat strain combinations where mixed lymphocyte reactions (MLRs) are negative. Pawelec et aL 1o made a similar observation in 18 of 20 MHC-compatible, M L R negative combinations of pigs. In the latter study, at least, the stimulation by kidney cells cannot be attributed to 'contaminating' dendritic cells - powerful, bonemarrow-derived lymphocyte activators 'l _ because the investigators used established kidney cell lines as stimulator cells and dendritic cells do not proliferate and survive only for a few days in vitro 1~. Roth et aI. 12found that autologous as well as allogeneic human kidney cortical cells stimulate lymphocyte blastogenesis. They speculated that the autologous response might reflect events leading to progressive kidney cell damage in vivo secondary to tissue alterations induced by allograft rejection. Williams et al. ,3 tested human kidney allograft recipients daily for the first month after transplantation with a leukocyte migrationinhibition assay and found that the tests were more predictive of rejection when antigen was prepared from kidneys than from donor leukocytes. They confirmed this in a subsequent study with dogs 1~ where, as in Roth's study 12, the hosts responded to their own as well as to donor kidney antigen. Histological damage, accompanied by increasing proteinuria, also was noted in the hosts' own kidneys as well as in the allografts, indicative of an autoimmune nephropathy triggered by allograft rejection. Vegt et aL 15 generated cytotoxic T cells (CTLs) in dog MLRs that lysed both PHA-induced lymphoblasts and kidney cells from the same 1-haplotype mismatched donors. Monolayer absorption tests revealed a population of C T L reactive with kidney cells that had little or no affinity for leukocytes. Even a second absorption on a leukocyte monolayer did not further reduce the antikidney cell cytotoxicity, so the results were not explicable in terms of a lower density of shared antigens on leuko-
cytes. PHA blasts also did not inhibit the lysis of kidney cells very well in cold-target inhibition tests. Since the kidney-specific C T L were generated in MLRs, Vegt et al. suggested that the target-cell determinants either belonged to the endothelial cell-monocyte system (see below) or were class-II M H C antigens found on kidney cells but on only a limited number of P H A blasts (they were not class-I antigens because the C T L had no affinity for a class I-enriched monolayer). However, it is not clear why the investigators did not test the second alternative by trying to adsorb the C T L on a class II-enriched monolayer, and the first alternative is inconsistent with their claim that endothelial cells were not present in their kidney cell cultures. Manca et al. 16 reported that lymphocytes extracted from a rejected human kidney allograft and expanded in vitro with interleukin 2 lysed donor kidney fibroblasts but not resting leukocytes, suggesting the presence of fibroblast-specific determinants. However, the graftinfiltrating cells should have been assayed with lymphoblasts, not just resting leukocytes, to be sure that they were not reacting with an antigen shared by fibroblasts and lymphoblasts.
Heart-specific antigens Judd and Trentin 17 compared the ability of cell-free extracts of heart, spleen and liver to induce tolerance of newborn heart allografts in mice treated with antithymocyte globulin. The heart extract produced longer graft survival than the spleen or liver extracts when injected at the time of or four days after grafting, and only the heart extract produced prolonged graft survival when injected eight days after grafting. Although the results suggest heart-specific alloantigens, they also could be explained in terms of trival differences in the tolerogenicity of the three different extracts, which apparently were not standardized in terms of protein content, expression of serologically-defined antigens, etc. Hart and FabrC s detected a n o n - M H C heart alloantibody in the sera of LEW rats immunized with a DA heart homogenate or heart allografts. However, the use of backcross donors revealed that expression of the antigen did not influence heart allograft rejection time. Harkiss et aL 19 detected antibodies that bound to rat cardiac and skeletal muscle in 12 of 21 human heart allograft recipients. However, antibody kinetics and graft rejection were not well correlated, though heart-specific antibodies were present in four of six patients who died with acute rejection. Like kidney cells 9, enzymatically dissociated heart cells stimulate allogeneic lymphocytes, but in rats, at least, the reactions are poorly correlated with the fate of heart allografts. For example, BN heart cells strongly stimulate M A X X lymphocytes but M A X X rats accept BN hearts 2°.
Liver-specific antigens Hart and Fabre 21 also studied a liver-specific alloantibody that is a minor component of the sera of L E W rats immunized with DA liver homogenate or liver allografts. However, like antibodies to the mouse liver-specific F antigen 22, there is no evidence that these antibodies affect
236 liver allografts. Pawelec et al. lo found that cultured liver cells stimulated lymphocyte blastogenesis in 7 of 20 MHC-compatible, MLR-negative combinations of pigs, and Sakai et aL 23 claimed that hepatocytes were more than three times more stimulatory than spleen cells in an MHC-compatible rat strain combination. Pawelec's liver cell lines were free of non-parenchymal cells, but Sakai's hepatocyte preparations contained 20% Kupfer cells, which could have accounted for their superior immunogenicity. Endothelial cell - monocyte antigens Sometimes patients who acutely reject kidney allografts never develop lymphocytoxic antibodies, yet immunoglobulin and complement still are deposited in and around the blood vessels of the rejected grafts 2.. Vascular lesions are prominent in rejected kidneys, and vascular endothelial cells (VEC) express unique specificities detectable with xenoantisera 24and stimulate allogeneic T cells in vitro 25. These considerations motivated Moraes and Stastny to develop a method for performing cytotoxicity tests with umbilical cord VEC, with which they showed that sera of kidney allograft recipients frequently react with VEC but not lymphocytes 24. The antibodies involved are not absorbed by platelets, a rich source of HLA-A and B antigens, and lysostripping proved the independence of class I and VEC antigens 24. In criss-cross absorption tests, VEC and lymphocytes remove cytotoxicity against the homogolous cells but usually there is no reduction in anti-VEC activity when sera are absorbed with lymphocytes. However, peripheral blood monocytes absorb and are themselves lysed by these antibodies24; hence, the target-cell determinants are known as endothelial-monocyte (E-M) antigens. E-M antigens are not D R antigens because treating monocytes with antiD R sera blocks anti-DR but not anti-E-M cytotoxicity24. Paul and his collaborators 26 have fully confirmed these results with immunotluorescence techniques, and have also shown that absorption ofE-M antisera with erythrocytes of various species or with plasma proteins does not reduce anti-E-M titers, thus excluding the possibility that the antibodies involved are directed against heterophile antigens or plasma proteins adherent to vascular endothelium. At least eight groups of human E-M antigens have been defined, many of which are determined by genes linked to the H L A complex 27. E-M antibodies frequently are detected in the sera of kidney allograft recipients with poor clinical courses and in eluates of rejected kidneys, and the correlation between positive E-M antigen crossmatches and rejection of H L A mismatched as well as matched kidney allografts is highly significant 24'26. For example, Cerilli et al. 2~ detected E-M antibodies in 19 of 25 patients who either rejected their HLA-identical grafts or experienced severe rejection crises. The median onset of rejection in 15 patients with antibodies before transplantation was 10 days, including four patients who rejected their grafts within three days; the other four patients rejected their grafts within two weeks after the appearance of E-M antibodies. Cerilli also has evidence that those few patients with positive monocyte crossmatches who do not reject H L A identical grafts in fact have antibodies directed against monocyte-specific
Immunology Today, vol. 5, No. 8, 1984
antigens, not antigens shared by monocytes and VEC 29. Paul et al. 30 described a case ofhyperacute kidney allograft rejection associated with pre-existing E-M but not lymphocytoxic antibodies. They suggested that E-M antibodies may help to explain the contradictory data on graft outcome in patients with positive B-cell crossmatches because B-cell preparations often contain variable numbers ofmonocytes. In a cogent article in this journal on the 'antigenic anatomy' of the human kidney, Baldwin et al. 3~ suggest that the strong immunogenicity of E-M antigens may be related to their co-expression on peritubular tubular capillaries with D R antigens, which provide strong helper signals. This, along with the strong evidence that microvascular endotheliam is the primary target in the destruction of skin, kidney and heart allografts 32, emphasizes the importance of the E-M system in transplantation immunity. Direct evidence comes from Paul et al. 33 who defined a rat VEC alloantigen. When VEC antigen-incompatible kidneys were transplanted into nonimmune hosts, most of the grafts showed the spontaneous long-term survival expected in the MHC-compatible strain combination involved. However, when the kidneys were transplanted into specifically preimmunized hosts, 75 % were rejected in accelerated fashion, and the VEC antibodies disappeared in the 25% of the recipients who became longterm survivors. The reactions were specific because thirdparty, VEC-compatible but MHC-incompatible kidneys were rejected like first-set grafts. However, the investigators were careful to point out that passive transfer experiments would be needed to establish whether the VEC antibodies actually caused graft rejection because there was no clear correlation between pre-transplant antibody titers and rejection rate, and cellular immunity could have been responsible. As for the latter, Hirschberg and his collaborators 25 repeatedly have shown that human VEC stimulate allogeneic T cells in vitro, though it is not clear whether the reactions are mediated by D R or E-M antigens. However, Groenewegen et al. 3~ evoked C T L that apparently could distinguish between VEC obtained from the dog external jugular vein and common carotid artery. Bulk C T L generated in M L R (monocytes presumably being the source of E-M antigen) lysed allogeneic PHA-indueed lymphoblasts as well as venous and arterial VEC. Although each cell type inhibited the lysis of its homogolous target in cold-target inhibition assays, arterial but not venous VEC inhibited the lyses of P H A blasts, suggesting that venous VEC express a unique antigen. In a subsequent study, Groenewegen et al. 3s used allogeneic VEC themselves as stimulator cells and found that C T L generated with venous VEC lysed venous and arterial VEC but C T L generated with arterial VEC only lysed the latter. Confirmation of these intriguing results awaits the establishment ofVEC-specific C T L clones. Pancreas-specific antigens Pancreas-specific alloantigens were first serologically defined in the rabbit, rhesus monkey and man in the early 1960s, but no histological changes resulted from the passive transfer of antisera or immune cells 36. However, more recent research indicates an important influence of
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pancreas-specific antigens on the rejection of islet allografts as well as the pathogenesis of insulin-dependent diabetes mellitus ~7. For example, the incidence of disease in the diabetes-susceptible BB rat is strikingly reduced by the neonatal inoculation of bone marrow cells from normal donors, by neonatal thymectomy or by treatment with whole-body radiation or antilymphocyte serum, and diabetes can be adoptively transferred to normal rats with lymphocytes of acutely diabetic ones 37. In addition, BB rats tolerant of skin allografts are not tolerant of and reject islet allografts from the skin donor strain. Miller's group reported that both dog ~8 and human ~2 islets stimulate autologous as well as allogeneic T cells in vitro. The reactions are tissue-specific because accelerated secondary responses to islets are not observed when the responding lymphocytes are primed with lymphocytes from the islet donors. Baekkeskov et al. have precipitated specific membrane glycoproteins from fl cells with antibodies from the sera of newly diagnosed diabetic children 39 and diabetic BB rats 4°. However, it is not known whether these antigens function as target-cell determinants in islet or pancreas graft rejection.
Tissue-restricted alloantigens in the skin, serologically defined Skin and pancreatic islets share the distinction of being the allografts most susceptible to rejection, both frequently being rejected acutely in donor-recipient combinations where other types of allografts (e.g. heart and kidney) are accepted 4I. In contrast to islet allografts, however, the immunological basis of this vulnerability is at least in part explicable in terms of two systems of tissue-restricted alloantigens preferentially expressed on epidermal cells (EC), the serologically defined Skn and cellularly defined Epa systems. Boyse and Old 42predicted the existence of Skn (originally known as Sk) antigens to explain a phenomenon that is not widely appreciated in transplantation immunology; the rejection of donor-type skin grafts by persistent bonemarrow chimeras. Although there are many examples of this 'split-tolerance '43, the model that has been most thoroughly analysed consists of chimeras produced by exposing strain B6 mice to a lethal dose of whole-body radiation and giving them an injection of B6AFI hybrid bone marrow or spleen cells. About two months later, virtually all the hosts reject B6AFj or strain A skin grafts within 20-30 days while remaining tolerant of B6AF1 myeloid and lymphoid cells. In fact, some hosts can reject several consecutive A skin grafts in increasingly accelerated fashion while remaining lifelong chimeras 44. On the dual assumption that, like lymphocytes, EC express differentiation alloantigens, and that self-tolerance of the body's own differentiated cells is antigen-driven, Boyse and Old 42reasoned that lymphocytes arising from transplanted stern cells would be denied the opportunity to become tolerant of donor-strain EC alloantigens because they never were exposed to them, and they eventually would recognize donor-strain skin grafts as foreign and reject them. These hypotheses were later verified when it was shown that A skin grafts are accepted by B6AF 1-to-B6 chimeras if the latter are exposed to strain A skin in the form of EC injections or skin grafts at the time of marrow
237 transplantation 44. Later, Scheid et al. 45 showed that chimeras that reject A skin grafts make antibodies that are cytotoxic to A EC but not to A myeloid or lymphoid cells and not to B6 EC. Thus, the antibodies are tissue-specific alloantibodies, not autoantibodies that lyse host as well as donor EC. The antibodies do react with donor strain neuroblastoma cells, however, indicating the sharing of Skn antigens by EC and brain cells, which have a common embryological origin. Scheid et al. 45 used antisera raised by skin grafting reciprocal radiation chimeras to define two Skn alleles, and by typing various inbred strains they proved that Skn antigens are determined by non-H-2 genes. The genetics of the Skn system is reviewed in detail elsewherC TM. There is good evidence of at least two independent Skn loci, and both the immunogenicity of Skn antigens and the host response to them are under H-2 control.
Tissue-restricted alloantigens in the skin, cellularly defined Sakai et al. 20 found that EC stimulated allogeneic lymphocytes in MHC-compatible, MLR-negative rat strain combinations and that the magnitude of the stimulation by EC and skin allograft survival times were well correlated. Hirschberg and Thorsby 46used a hot pulse of [3H]thymidine to specifically eliminate lymphocytes proliferating in one-way human MLRs. The remaining lymphocytes no longer responded upon restimulation with donor lymphocytes but they did respond to donor EC, indicating the presence of tissue-specific lymphocyteactivating alloantigens on human EC, whose murine counterparts may be the Epa antigens defined in my laboratory. In contrast with Skn antigens, Epa antigens so far have been defined only by cell-mediated cytotoxicity (CMC). We discovered the Epa system when we found that prim ° ing and boosting C3H/He mice with H-2 compatible strain A K R or CBA EC generate C T L that preferentially lyse donor EC as opposed to lymph node or spleen cells 44. Lysis of the EC targets represents allo-immunity not autoimmunity because there is little or no lysis of syngeneic EC, and the targets must come from a donor of the same H - 2 haplotype as the host to be lysed. Thns, the cytotoxicity is a classic example of MHC-restricted CMC, the novelty being the tissue specificity. Genemapping and backcross studies revealed that the restricting antigen is a product of the K region of the H - 2 complex and that the non-H-2 target-ceU determinant, which we designated Epidermal alloantigen-1 (Epa-1), is determined by a single Mendelian gene ". By using appropriate combinations o f I-~-2 congenic lines as donors and hosts, we also determined that the ability to generate Epa1-specific C T L is under H - 2 gene control, with K/D region rather than/-region gene products serving as Irgene products 47. Subsequently we cloned and subcloned Epa- 1 killer cells, which are classic Thy- 1 +, Lyt-2 ÷ CTL, whose antigenic and H-2-restriction specificities have remained remarkedly stable in vitro for over two years ~8, These clones are very cytotoxic to EC, only slightly cytotoxic to Con A and LPS blasts and not cytotoxic at all to resting lymphocytes from Epa-1 + strains. They also readily lyse cultured adherent EC and fibroblasts 49, which
238 demonstrates that the preferential lysis of these targets is not an artifact trypsinization. We also found that although fresh peritoneal microphages (M+) are susceptible to lysis by other CTL, they are resistant to Epa-1 C T L but become susceptible after 12-24 hours in vitro. However., fresh PE M+ are susceptible to Epa-1 C T L if they come from mice that have been treated with the M+ activating agents Con A and BCG as opposed to the sterile inflammatory agents peptone broth or thioglycoltate 49. The correlation between Epa-1 expression and M+ activation suggests that Epa-1 may be a strainspecific marker for activated M+ as well as an inducible H antigen in vivo, and the 'contamination' of lymphocyte cultures with activated M~ may explain the slight cytotoxicity of Epa-1 C T L towards lymphoblast targets. We also used radiation chimeras as a source of stimulator and target cells to test the influence of Langerhans cells (LC) on Epa-l-mediated CMC s°. (The importance ofLC, the small population of bone-marrow derived, Ia + dendritic cells in the epidermis for skin-associated immune responses has been previously reviewed in this journal 51.) By making reciprocal radiation chimeras and controls between Epa-1 ÷ and Epa-1 - strains, we had at our disposal EC suspensions in which the keratinocytes and LC came from strains of the same or different Epa- 1 phenotype. EC suspensions from Epa- 1 ÷ hosts with longestablished Epa-1 - bone marrow were indistinguishable from Epa-1 ÷ EC controls at the priming, boosting and effector phases of Epa-l-specific CMC. Thus Epa-1 is fully expressed on keratinocytes, whose immunogenicity and susceptibility to lysis is not dependent on Epa- 1 + LC, which is consistent with the evidence that LC normally are not critical target antigens in the rejection of skin allografts 52. Our evidence that Epa-1 functions in vivo is fivefold: (1) In some cases Epa- 1 ÷ skin allografts are rejeeted faster than Epa-1 - skin allografts 44. (2) Injections ofEpa-1 + EC are much more effective than Epa-1 - EC at priming hosts for the accelerated rejection of Epa- 1 ÷ skin allografts 44. (3) Merely matching backcross recipients of parent-strain skin allografts for Epa-1 and ignoring all other H antigen differences prolongs the survival time of first-set Epa-1 ~ skin allografts very significantly, strong evidence that Epa- 1, or the product of a very closely linked gene, is an H antigen 44. (4) Epa-l-specific C T L can be extracted from spongematrix allografts impregnated with Epa-I * EC as well as from lymph nodes draining the site of sponge implantation 5~. (5) Cloned Epa-1-specific C T L mediate gross necrotizing skin lesions when injected intradermally, and the ability of the C T L to induce these lesions in vivo is subject to the very same antigen and H-2 restriction specificities as their ability to lyse EC targets in vitro ~. Thus, reactions to Epa antigens may in part explain why the skin is such a preferred target in graft-versus-host disease. More definitive studies on the role of Epa-1 in skin allograft rejection and graft-versus-host reactions await the establishment of an Epa-l-disparate congenic line, which currently is at the 15th backcross generation in my mouse colony. Although we have identified only one Epa-1 allele to
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date, the wide range of target-cell susceptibility and priming ability of EC of the numerous mouse and rat strains we have tested 5~suggest that the Epa-1 immunogenicity of a given strain may be determined by the additive effects of several molecularly distinct antigens or by several distinct determinants on the same molecule. Thus, Epa antigens may influence transplantation immunity by their multiplicity or polymorphism. In fact, we already have provisional evidence of the antigenic products of three more Epa loci. For example, CBA EC evoke C T L in B10.BR hosts that preferentially lyse CBA EC. Since both strains are Epa-1 +, the C T L must be directed against the antigenic product of a second Epa locus, provisionally designated Epa-2. In addition, the ability of human EC but not human lymphocytes to cross-prime C3H/He mice for the subsequent generation of Epa- 1-specific C T L 55indicates that Epa-like antigens are expressed in human skin, something we hope to establish directly with EC-specific C T L of human origin.
The hemopoietic histocompatibility (Hh) system The recent death of Gustavo Cudkowicz tragically ended the career of the investigator most responsible for our knowledge of H h antigens, which represent a tissuespecific obstacle to bone-marrow transplantation in mice and other species. The antigens are expressed by stem ceils and tumor cells of the lymphomyeloid series but not ceils of other histological types 56. They are unusual in several respects, most notedly because of their noncodominant expression. Only homozygous mice express Hh gene products. Thus, F 1 hybrids reject parent-strain bone marrow cells by responding to parental Hh antigens. H h responsiveness is resistant to a single dose of whole-body radiation up to about 1 000 rads, but splitdose or chronic irradiation weakens Hh resistance. H h reactions also are thymus independent, and thymectomized or congenitally athymic (nude) mice are hyperreactive to Hh-disparate grafts. These and other features of the Hh system, such as its delayed maturation, bonemarrow dependence and exquisite genetic control are reviewed elsewhere 56. The threshold nature of anti-Hh immunity, which can be overcome by increasing the number of cells in a bone marrow transplant or increasing the dose of radiation, and the non-codominant nature of Hh genes makes it unlikely that H h antigens are a serious problem in clinical marrow transplantation. However, there is much renewed interest in the system because of the striking similarity of anti-Hh effector cells and N K (natural killer) cells 57, which may have an important role in immune surveillance against tumors, intracellular bacteria and viruses.
Methodological considerations and conclusions The investigation of H antigens found on the fixed cells of solid organs and tissues involves problems that simply are not present in comparable studies of the free ceils of the blood, bone marrow and lymphoid organs. Semiquantitative data on tissue distribution and antigen expression in situ can be obtained by immunolluorescence or immunoelectron microscopy if appropriate antibodies are available. However, it is notoriously difficult to raise workable titers of antibody to most non-MHC antigens, which must be defined in vitro in cellular assays that
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require essentially pure single-cell suspensions for use as stimulator or target cells. Proteases, usually typsin or collagenase, almost invariably are required to free cells from solid organs and tissues, and it is well known that these enzymes can destroy or distort normal cell-surface antigens. The solution to this dilemma is to culture enzymatically dispersed cells in vitro long enough to allow cell surfaces to regenerate and normalize but not so long as to lose the tissue-specific antigens under investigation. The purity of cell preparations is a major problem when dealing with complicated organs such as kidney, liver or pancreas that contain several different types of parenchymal cells in addition to stromal and vascular components, and many reports of tissue-specific H antigens suffer from the lack of definition of the cell types involved. The better characterization of epidermal 44 and endothelial 2~cell alloantigens in large part reflects the ease of obtaining essentially pure preparations of these cell types. Still another problem is the contamination of cell suspensions prepared from solid organs and tissues with small numbers of highly immunogenic marrow-derived cells, such as dendritic cells 11, whose presence even in small numbers can give a false indication of the immunogenicity of the cell type presumably under investigation. One way to avoid this problem is to culture the desired ceils under conditions that result in the selective loss of bone-marrow derivatives 10.3s;another is to use established chimeras with a bone marrow syngeneic to the effector cell strain as a source of stimulator or target cells. This was the strategy we used to determine that Langerhans cells are not a critical source of Epa antigens 5°. Finally, it is particularly important to distinguish true tissue-specificity from mere quantitative differences in the expression of H antigens shared by different cell types when dealing with apparent tissue-specific transplantation immunity. Quantitative absorption and blocking tests with antibodies of known specificities are critical procedures for the serological definition of new antigens; discriminative assays such as monolayer adsorption, antigen-driven suicide and cold-target inhibition are obligatory for cellular definition. In spite of these problems, significant and interesting new information has been obtained by studying antigens expressed by the fixed cells of transplanted tissue and organs. The common assumption that all significant graft antigens are shared with leukocytes clearly is incorrect. Thus, donor-recipient matching based on tissue-typing and crossmatching lymphocytes only may not be the best way to avoid rejection reactions and promote graft survival. However, many practical and logistic problems will have to be solved before non-lymphoid cells can be used routinely in tissue-typing laboratories. [:[']
Acknowledgements The work from my own laboratory was supported by U.S. Public Health Service Grants AI 21208 and CA 09127 and the Mayo Foundation. I am grateful to Lois A. Garmon for help in preparing the manuscript.
References 1 Perkins, H. A., Gantan, Z., Siegel, S., Howell, E., Belzer, F. O. and Kountz, S. L. (1975) Tissue Antigens 5, 88-98 2 Mohanakumar, T., Phibbs, M., Haar, J., Mendez, G., Kaplan, A. M. and Lee, H. M. (1980) Transplant. Proe. 12, Suppl. 1, 65-68
239 3 Ende, N., Orsi, E. V., Baturay, N. Z. and Britten, T. L. (1979) Am. ,f Glin. Pathol. 71,543-548 4 Carpenter, C. B., d'Apice, J. F. and Abbas, A. K. (1976)Adv. lmmunol. 22, 1-65 5 Kashiwabara, H., Wakashin, Y, Benltani, A., Oomori, K., Hachisu, T., Shishido, H., Yokoyama, T., Wakashin, M. and Iwasaki, Y. (1979) Transplant. Proc. 11,434-437 6 Thoenes, G. H., Pielstieker, K. and Schubert, G. (1979) Lab. Invest. 41, 321-333 7 Thoenes, G. H., Krieger, A. and Pielsticker, K. (1979) Transplant. Proc. 11, 1604-1607 8 Hart, D. N.J. and Fabre, J. W. (1980)./[ Exp. Med. 151,651-666 9 Mashimo, S., Sakai, A., Ochiai, T. and Kountz, S. L. (1976) Tissue Antigens 7, 291-300 10 Pawelec, G., Davies, H. ft. S., Pearson; J. D., Steele, C. and Brons, G. (1979) Tissue Antigens 14, 367-378 11 Steinman, R. M., Kaplan, G., Witmer, M. D. and Cohn, Z. A. (1979) J. Exp. Meal 149, 1-16 12 Roth, D., Russell, T., Fuller, L., Kyriakides, G. K., Mintz, D. and Miller, J. (1984) Transplant. Proc. 16, 854-856 13 Williams, R.J., Mallick, N. F., Taylor, G., Orr, W. MeN. (1974), Glin. NephroL 2, 100-105 14 Williams, R.J., Eyres, K., Mallick, N. P., Orr, W. MEN., Taylor, G. and Williams, G. (1976) Transplantation 21, 188-194 15 Vegt, P. A., Buurman, W. A., van der Linden, C. J., Daemen, A. J. J. M., Greep, J. M. and Jeekel, J. (1982) Transplantation 33, 465-469 16 Manca, F., Barocci, S., Kunkl, A., Gurreri, G., Costantini, M. and Celada, F. (1983) Transplantation 36, 670-674 17 Judd, K. P. and Trentin, J . J . (1973) Transplantation 16, 351-358 18 Hart, D. N.J. and Fabre, J. W. (1981) Transplantation 31, 174-177 19 Harkiss, G. D., Cave, P., Brown, D. L. and English, T. A. H. (1984) Int. Arch. Allerg~AppL Immunol. (1973) 18-22 20 Sakai, A., Kashiwabara, H., Taha, M. and Kontz, S. L. (1977) Transplant. Proc. 9, 629-632 21 Hart, D. N.J. and Fabre, J. W. (1981) Transplantation31, 178-182 22 Lane, D. P. and Silver, D. M. (1976) Eur. J. Immunol. 6, 480-485 23 Sakai, A., Tanaka, S. and Kountz, S. L. (1978) Transplantation 25, 110-114 24 Stastny, P. (1980) Transplant. Proc. 12, Suppl. 1, 32-36 25 Hirsehberg, H., Bergh, O.J. and Thorsby, E. (1980),]: Exp. Med. 152, 249s-255s 26 Paul, L. C. and Carpenter, C. B. (1980) Transplant. Proc. 12, Suppl. 1, 43-48 27 Moraes, J. R. and Stastny, P. (1976) Tissue Antigens 8, 273-276 28 Cerilli, J., Brasile, L. and Clarke, J. (1983) in Non-HLA Antigens in Health, Aging and Malignancy (Cohen, E. and Singal, ~D. P., eds), pp. 251-256, Allan R. Liss, New York 29 Cerilli, J., Brasile, L. Clarke, J. and Simak, C. (1983) Abstracts ofthe Ninth Annual Meeting of theAmerican Associationfor Histocompatibility Testing, p. 23 30 Paul, L. C., Claas, F. H.J., van Es, L. A., Kalff, M. W. and de Graeff, J. (1979) N. Eng. J. Med. 300, 1258-1260 31 Balwin, W. M., Claas, F. H. J., van Es, L. A. and van Rood, J. J. (1980) Immunol. Today 1, 110-111 32 Forbes, R. D. C., Guttmann, R. D., Gomersall, M. and Hibberd, J. (1983)Am. J. Pathol. 111,184-196 33 Paul, L. C., Busch, G. J., Paradysz, J. M., Carpenter, C. B. (1983) Transplantation 36, 533-539 34 Groenewegen, G., Buurman, W. A., van der Linden, C.J., Jeunhomrae, J. M. A. A. and Kootstra, G. (1983) Tissue Antigens 21, 114-128 35 Groenewegen, G., Buurman, W. A.,Jeunhomme, J. M. A. A., van der Linden, C. J., Vegt, P. A. and Kootstra, G. (1984) Transplantation 37, 206-210 36 Metzgar, R. S. (1980) Transplant. Proc. 12, Suppl. 1, 123-128 37 Naji, A., Silvers, W. K. and Barker, C. F. (1980) Transplantation 36, 355-361 38 Rabinoviteh, A., Fuller, L., Mintz, D., Severyn, W., Noel, J., Flaa, C., Kyriakides, G. and Miller, J. (1981),]. Clin. Invest. 67, 1507-1516 39 Baekkeskov, S., Nielsen, J. H., Marner, B., Bilde, T., Ludvigsson, J. and Lernmark, A. (1982) Nature (London) 298, 167-169 40 Baekkeskov, S,, Dyrberg, T. and Lernmark, A. (1982) Diabetologia 25, 138 41 Nash, J. R., Peters, M. and Bell, P. R. F. (1977) Transplantation 70-73 42 Boyse, E. A. and Old, L.J. (1968) Transplantation 6, 19 43 Steinmuller, D. and Waehtel, S. S. (1980) Transplant. Proc. 12, Suppl. 1, 100-106 44 Steinmuller, D. (1983) in Traumatic lnjury: Infection and OtherImmunologic Sequellae (Ninnemann, J. L. ed.), pp. 181-196, University Park Press, Baltimore 45 Scheid, M., Boyse, E. A., Carswell, E. A. and Old, L.J. (1972)J. Exp. Med. 135, 238-255
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46 Hirschberg, H. and Thorsby, E. (1975) Tissue Antigens 183-194 47 Tyler, J. D., Burlingham, W. J., David, C. S. and Steinrnuller, D. (1982) Immunogenetics 16, 23-36 48 Tyler, J. D., Galli, S. J., Snider, M. E., Dvorak, A. M. and Steinmuller, D. (1984)0( Exp. Med. 159, 234-243 49 Burlingham, W. J., Snider, M. E., Tyler, J. D. and Steinmuller, D. Cell. Immunol. (in press) 50 Burlingham, W. J. and Steinmuller, D. (1983) Transplantation 35, 130-135
Friedmann, P. S. (1981) Immunol. Today 2, 124-128 Steinmulter, D. (1981) Immunol. Today 2 (No. 11), ii Snider, M. E. and SteinmuUer, D. Transplant. Proc. (in press) Steinmuller, D., Tyler, J. D. and Zinsmeister, A. R. (1984) Transplant. Proc. (in press) 55 Steinmuller, D. and Tyler, J. D. (1983) Transplant. Proc. 15, 238-241 56 Cudkowicz, G. and Nakamura, I. (1983) Transplant. Proc. 15, 2058-2063 57 Miller, S. C. (1983)J. ImmunoL 131, 92-97
51 52 53 54
Immunosuppressive activity of the retroviral envelope protein P15E and its possible relationship to neoplasia Ralph Snyderman and GeorgeJ. Cianciolo Type C retroviral infections can causeprofound immunosuppression as well as neoplasms. The retroviral envelopeprotein p l 5E has both immunosuppressive and anti-inflammatory activities which may contribute to the pathogenicity of retroviruses. Murine and human neoplastic cells, not infected with retroviruse~, have recently beenfound to contain p l 5Elike antigens. In this article Ralph Snyderman and George Cianciolo discuss the potential relationship between p l 5 E production, irnmunosuppression and neoplasia.
Immunological reactions can destroy neoplastic cells in vivo, and the accumulation of macrophages within a tumor can lead to its destruction 1'2. Moreover, cytotoxic T lymphocytes, natural killer (NK) cells, and activated macrophages can kill tumor cells in vitro 3-5. These observations suggest that the immune system provides some resistance against the development and spread of cancer, a contention strengthened by the increased incidence of spontaneous tumors in individuals with congenital or acquired itumunodeficiency diseases 6. In animals infected by tumor-producing retroviruses immunosuppression frequently precedes the development of tumors 7-9 and a causal relationship is now suspected between infection by the human retroviruses named T-cell lymphotropic virus III ( H T L V III) or lymphadenopathy-associated retrovirus (LAV) and the development of the acquired immunodeficiency syndrome (AIDS) and Kaposi' s sarcoma 1°-~2. Since immune mechanisms may limit the development or spread of cancer, clinically apparent tumors may develop when transformed cells acquire the means to escape immunological host defence mechanisms. In this article we discuss factors produced by tumor cells which i depress macrophage-mediated functions, and the recent observation that these substances may be related to the retroviral structural envelope protein p 15E. There is data to suggest that retroviruses may induce immunosuppresstun, m part, by initiating the production of pl5E. We suggest that this property of retroviruses enhances their ability to produce neoplastic cells capable of escaping immune surveillance. Spontaneous tumor cells may likewise resist immune destruction ifa cellular pl 5E-like gene is activated. Laboratory of Immune Effector Function, Howard Hughes Medical Institute Division of Rheumatology and Immunology, Department of Medicine Duke University Medical Center, Durham, NC, USA. © 1984,ElsevierSciencePublishersB.V.,Amsterdam 0167-4919/84/$02.00
Immunosuppression associated with retroviruses and their structural components Imtuunosuppression often accompanies retroviral infection 7-9'11'12.For example, avian, murine, feline and probably human leukemia viruses cause acquired immunodeficieney states in their respective species. getroviruses also diminish immune responses in vitro13-17. The pathological events accompanying retroviral infections are complex but viral structural components are important in the induction of immunosuppression in animals. Olsen et al. 18 and Schaller et al. 19 reported that immunization of cats with UV-inactivated feline leukemia virus (FeLV) abrogated tumor immunity and increased tumor incidence after challenge with infectious feline sarcoma virus (FeSV). Similarly, in-vitro blastogenic responses of feline lytuphocytes to concanavalin A (Con A) were suppressed by up to 65 % by UV-inactivated FeLV 2°. Freeze-thawed preparations of murine Rauscher leukemia virus (RLV) suppressed the in-vitro blastogenic response of mouse splenic lymphocytes to phytohemagglutinin (PHA) or to allogeneic cells in a two-way mixed leukocyte reaction (MLR), 21 perhaps because cell recognition sites were altered by a virion envelope component. Mathes et al. 22.23 reported the inhibition of in-vitro blastogenic responses of feline lymphocytes to Con A using the purified FeLV envelope protein p15E. P15E is the hydrophobic transmembrane protein (around 19 000 dahons) of the retroviral envelope which is synthesized as part of a precursor of mol. wt 80-90 000 (Ref. 24). This precursor is cleaved into p15E and gp70 during protein maturation (Fig. 1). Mathes also demonstrated that treatment of cats with purified FeLV pl5E suppressed their ability to resist a subsequent challenge with infectious FeSV and resulted in increased tumor incidence in the FeSV-challenged animals 23. Con A-induced blastogenesis of hutuan lymphocytes was inhibited by disrupted type
Immunology Today, vol. 5, No. 8, 1984
Tissue-specific and tissue-restricted histocompatibility antigens David Steinmuller Strictly defined, tissue-specific antigens are antigens characteristic of one particular tissue or cell. They are usually associated with autoimmunity and are remarkably homologous between species. In contrast, histocompatibility (H) antigens reflect polymorphbm within species - they are alloantigens - and class-I major H complex ( M H C ) antigens - - at least mouse H - 2 D and H-2 K and human H L A - A and-B, the commonest targets of acute allograft rejection are widely distributed in the body; class-H M H C antigens - mouse Ia and human D R - have a much more limited distribution, being expressed primarily on B lymphocytes and on macrophages and other cells involved in antigen presentation and immune activation. This review is devoted to H antigens other than class-H M H C antigens with limited if not highly specific, tissue distribution. Some of these antigens are classic tissue-specific antigens," others are alloantigens with limited tissue expression. Much of the evidence that they evoke immune responses that damage or destroy transplanted tissue is incomplete or circumstantial, but some is convincing and includes the immunogenetic characterization of new antigen systems that may have to be reckoned with clinically, especially when dealing with HLA-matched transplants.
Kidney-specific antigens, serologicaUy defined Biopsies or fragments of rejected human kidney allografts can be trypsinized to bring cells into suspension that can be typed like lymphocytes for H L A antigens. The excessive cytotoxicity sometimes detected with kidney cells v. lymphocytes from the same donor suggests that some H L A typing sera contain antibodies to kidneyspecific antigens. For example, Perkins et al. 1 described a patient who rejected a kidney hyperacutely at a time when the only detectable cytotoxic antibodies in his serum were kidney-specific; they reacted with and were absorbed by kidney cells but not lymphocytes. Mohanakumar et al. ,2 eluted antibodies from rejected human kidney allografts that reacted specifically with kidney cells. The antibodies could be completely absorbed with the latter but not with platelets or T or B cells. Ende et al. 3 compared the sera of cadaver kidney allograft recipients who rejected their Histocompatibility Laboratory, Transplantation Society of Michigan Ann Arbor, MI 48104, USA
© 1984, Elsevier Science Publishers B,V., Amsterdam 0167 - 4919/84/$02.00
grafts within one year with that of those who had maintained them at least five years. Much higher levels of what appeared to be kidney-specific antibodies - they did not react with spleen cells, platelets or lung, skin and m u s c l e were found in the sera of the short-lived transplant group. The antibodies were not correlated with the presence or absence of lymphocytotoxic antibodies, and they did not appear to be H L A antibodies. However, except for one case of a donor-specific contralateral kidney, the target cells came from random donors; hence it is not clear whether the antibodies were allo- or organ-specific. However, allospecific n o n - M H C kidney antigens have been detected in rats. If allograft survival is maintained for a month or more by immunosuppression in certain strain combinations, antibodies that react specifically with donor but not host tubular basement membrane (TBM) appear in host serum, and the antibody response is associated with tubular injury in the allograft but not in the host's own non-grafted kidney 4. Similarly distributed lesions have been detected in human kidney allograft
235
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recipients 4, and Kashiwabara et al. 5 reported that antibody titers to a purified T B M antigen correlated with the onset and grade of acute kidney allograft rejection. Thoenes et al. 6 studied an immune-complex glomerulonephritis that develops five weeks or more after kidney allografting in certain MHC-compatible rat strain combinations. Antibody binds to the brush border of the proximal tubules of the allograft, but not to the host's own kidney, and proteinuria subsides when the allograft is removed. Although the antigen involved is a tissuespecific alloantigen, the disease depends for its induction on reactions to conventional H antigens. It does not occur in kidney allografts transplanted to tolerant hosts but does occur when tolerance is broken by the adoptive transfer of syngeneic lymphoid cells sensitized to donor H antigensT. In contrast, Hart and Fabre ~ studied a n o n - M H C kidney-specific alloantigen that induces a strong antiT B M antibody response in rats but has no role in allograft rejection. Kidney allografts mismatched for the antigen are not rejected in accelerated fashion, even in the face of an ongoing response, and no histological abnormalities are observed after grafting.
Kidney-specific antigens, cellularly defined Mashimo et al. 9 reported that rat kidney cells stimulate lymphocyte blastogenesis in one of four MHC-compatible rat strain combinations where mixed lymphocyte reactions (MLRs) are negative. Pawelec et aL 1o made a similar observation in 18 of 20 MHC-compatible, M L R negative combinations of pigs. In the latter study, at least, the stimulation by kidney cells cannot be attributed to 'contaminating' dendritic cells - powerful, bonemarrow-derived lymphocyte activators 'l _ because the investigators used established kidney cell lines as stimulator cells and dendritic cells do not proliferate and survive only for a few days in vitro 1~. Roth et aI. 12found that autologous as well as allogeneic human kidney cortical cells stimulate lymphocyte blastogenesis. They speculated that the autologous response might reflect events leading to progressive kidney cell damage in vivo secondary to tissue alterations induced by allograft rejection. Williams et al. ,3 tested human kidney allograft recipients daily for the first month after transplantation with a leukocyte migrationinhibition assay and found that the tests were more predictive of rejection when antigen was prepared from kidneys than from donor leukocytes. They confirmed this in a subsequent study with dogs 1~ where, as in Roth's study 12, the hosts responded to their own as well as to donor kidney antigen. Histological damage, accompanied by increasing proteinuria, also was noted in the hosts' own kidneys as well as in the allografts, indicative of an autoimmune nephropathy triggered by allograft rejection. Vegt et aL 15 generated cytotoxic T cells (CTLs) in dog MLRs that lysed both PHA-induced lymphoblasts and kidney cells from the same 1-haplotype mismatched donors. Monolayer absorption tests revealed a population of C T L reactive with kidney cells that had little or no affinity for leukocytes. Even a second absorption on a leukocyte monolayer did not further reduce the antikidney cell cytotoxicity, so the results were not explicable in terms of a lower density of shared antigens on leuko-
cytes. PHA blasts also did not inhibit the lysis of kidney cells very well in cold-target inhibition tests. Since the kidney-specific C T L were generated in MLRs, Vegt et al. suggested that the target-cell determinants either belonged to the endothelial cell-monocyte system (see below) or were class-II M H C antigens found on kidney cells but on only a limited number of P H A blasts (they were not class-I antigens because the C T L had no affinity for a class I-enriched monolayer). However, it is not clear why the investigators did not test the second alternative by trying to adsorb the C T L on a class II-enriched monolayer, and the first alternative is inconsistent with their claim that endothelial cells were not present in their kidney cell cultures. Manca et al. 16 reported that lymphocytes extracted from a rejected human kidney allograft and expanded in vitro with interleukin 2 lysed donor kidney fibroblasts but not resting leukocytes, suggesting the presence of fibroblast-specific determinants. However, the graftinfiltrating cells should have been assayed with lymphoblasts, not just resting leukocytes, to be sure that they were not reacting with an antigen shared by fibroblasts and lymphoblasts.
Heart-specific antigens Judd and Trentin 17 compared the ability of cell-free extracts of heart, spleen and liver to induce tolerance of newborn heart allografts in mice treated with antithymocyte globulin. The heart extract produced longer graft survival than the spleen or liver extracts when injected at the time of or four days after grafting, and only the heart extract produced prolonged graft survival when injected eight days after grafting. Although the results suggest heart-specific alloantigens, they also could be explained in terms of trival differences in the tolerogenicity of the three different extracts, which apparently were not standardized in terms of protein content, expression of serologically-defined antigens, etc. Hart and FabrC s detected a n o n - M H C heart alloantibody in the sera of LEW rats immunized with a DA heart homogenate or heart allografts. However, the use of backcross donors revealed that expression of the antigen did not influence heart allograft rejection time. Harkiss et aL 19 detected antibodies that bound to rat cardiac and skeletal muscle in 12 of 21 human heart allograft recipients. However, antibody kinetics and graft rejection were not well correlated, though heart-specific antibodies were present in four of six patients who died with acute rejection. Like kidney cells 9, enzymatically dissociated heart cells stimulate allogeneic lymphocytes, but in rats, at least, the reactions are poorly correlated with the fate of heart allografts. For example, BN heart cells strongly stimulate M A X X lymphocytes but M A X X rats accept BN hearts 2°.
Liver-specific antigens Hart and Fabre 21 also studied a liver-specific alloantibody that is a minor component of the sera of L E W rats immunized with DA liver homogenate or liver allografts. However, like antibodies to the mouse liver-specific F antigen 22, there is no evidence that these antibodies affect
236 liver allografts. Pawelec et al. lo found that cultured liver cells stimulated lymphocyte blastogenesis in 7 of 20 MHC-compatible, MLR-negative combinations of pigs, and Sakai et aL 23 claimed that hepatocytes were more than three times more stimulatory than spleen cells in an MHC-compatible rat strain combination. Pawelec's liver cell lines were free of non-parenchymal cells, but Sakai's hepatocyte preparations contained 20% Kupfer cells, which could have accounted for their superior immunogenicity. Endothelial cell - monocyte antigens Sometimes patients who acutely reject kidney allografts never develop lymphocytoxic antibodies, yet immunoglobulin and complement still are deposited in and around the blood vessels of the rejected grafts 2.. Vascular lesions are prominent in rejected kidneys, and vascular endothelial cells (VEC) express unique specificities detectable with xenoantisera 24and stimulate allogeneic T cells in vitro 25. These considerations motivated Moraes and Stastny to develop a method for performing cytotoxicity tests with umbilical cord VEC, with which they showed that sera of kidney allograft recipients frequently react with VEC but not lymphocytes 24. The antibodies involved are not absorbed by platelets, a rich source of HLA-A and B antigens, and lysostripping proved the independence of class I and VEC antigens 24. In criss-cross absorption tests, VEC and lymphocytes remove cytotoxicity against the homogolous cells but usually there is no reduction in anti-VEC activity when sera are absorbed with lymphocytes. However, peripheral blood monocytes absorb and are themselves lysed by these antibodies24; hence, the target-cell determinants are known as endothelial-monocyte (E-M) antigens. E-M antigens are not D R antigens because treating monocytes with antiD R sera blocks anti-DR but not anti-E-M cytotoxicity24. Paul and his collaborators 26 have fully confirmed these results with immunotluorescence techniques, and have also shown that absorption ofE-M antisera with erythrocytes of various species or with plasma proteins does not reduce anti-E-M titers, thus excluding the possibility that the antibodies involved are directed against heterophile antigens or plasma proteins adherent to vascular endothelium. At least eight groups of human E-M antigens have been defined, many of which are determined by genes linked to the H L A complex 27. E-M antibodies frequently are detected in the sera of kidney allograft recipients with poor clinical courses and in eluates of rejected kidneys, and the correlation between positive E-M antigen crossmatches and rejection of H L A mismatched as well as matched kidney allografts is highly significant 24'26. For example, Cerilli et al. 2~ detected E-M antibodies in 19 of 25 patients who either rejected their HLA-identical grafts or experienced severe rejection crises. The median onset of rejection in 15 patients with antibodies before transplantation was 10 days, including four patients who rejected their grafts within three days; the other four patients rejected their grafts within two weeks after the appearance of E-M antibodies. Cerilli also has evidence that those few patients with positive monocyte crossmatches who do not reject H L A identical grafts in fact have antibodies directed against monocyte-specific
Immunology Today, vol. 5, No. 8, 1984
antigens, not antigens shared by monocytes and VEC 29. Paul et al. 30 described a case ofhyperacute kidney allograft rejection associated with pre-existing E-M but not lymphocytoxic antibodies. They suggested that E-M antibodies may help to explain the contradictory data on graft outcome in patients with positive B-cell crossmatches because B-cell preparations often contain variable numbers ofmonocytes. In a cogent article in this journal on the 'antigenic anatomy' of the human kidney, Baldwin et al. 3~ suggest that the strong immunogenicity of E-M antigens may be related to their co-expression on peritubular tubular capillaries with D R antigens, which provide strong helper signals. This, along with the strong evidence that microvascular endotheliam is the primary target in the destruction of skin, kidney and heart allografts 32, emphasizes the importance of the E-M system in transplantation immunity. Direct evidence comes from Paul et al. 33 who defined a rat VEC alloantigen. When VEC antigen-incompatible kidneys were transplanted into nonimmune hosts, most of the grafts showed the spontaneous long-term survival expected in the MHC-compatible strain combination involved. However, when the kidneys were transplanted into specifically preimmunized hosts, 75 % were rejected in accelerated fashion, and the VEC antibodies disappeared in the 25% of the recipients who became longterm survivors. The reactions were specific because thirdparty, VEC-compatible but MHC-incompatible kidneys were rejected like first-set grafts. However, the investigators were careful to point out that passive transfer experiments would be needed to establish whether the VEC antibodies actually caused graft rejection because there was no clear correlation between pre-transplant antibody titers and rejection rate, and cellular immunity could have been responsible. As for the latter, Hirschberg and his collaborators 25 repeatedly have shown that human VEC stimulate allogeneic T cells in vitro, though it is not clear whether the reactions are mediated by D R or E-M antigens. However, Groenewegen et al. 3~ evoked C T L that apparently could distinguish between VEC obtained from the dog external jugular vein and common carotid artery. Bulk C T L generated in M L R (monocytes presumably being the source of E-M antigen) lysed allogeneic PHA-indueed lymphoblasts as well as venous and arterial VEC. Although each cell type inhibited the lysis of its homogolous target in cold-target inhibition assays, arterial but not venous VEC inhibited the lyses of P H A blasts, suggesting that venous VEC express a unique antigen. In a subsequent study, Groenewegen et al. 3s used allogeneic VEC themselves as stimulator cells and found that C T L generated with venous VEC lysed venous and arterial VEC but C T L generated with arterial VEC only lysed the latter. Confirmation of these intriguing results awaits the establishment ofVEC-specific C T L clones. Pancreas-specific antigens Pancreas-specific alloantigens were first serologically defined in the rabbit, rhesus monkey and man in the early 1960s, but no histological changes resulted from the passive transfer of antisera or immune cells 36. However, more recent research indicates an important influence of
Immunology Today, vol. 5, No. 8, 1984
pancreas-specific antigens on the rejection of islet allografts as well as the pathogenesis of insulin-dependent diabetes mellitus ~7. For example, the incidence of disease in the diabetes-susceptible BB rat is strikingly reduced by the neonatal inoculation of bone marrow cells from normal donors, by neonatal thymectomy or by treatment with whole-body radiation or antilymphocyte serum, and diabetes can be adoptively transferred to normal rats with lymphocytes of acutely diabetic ones 37. In addition, BB rats tolerant of skin allografts are not tolerant of and reject islet allografts from the skin donor strain. Miller's group reported that both dog ~8 and human ~2 islets stimulate autologous as well as allogeneic T cells in vitro. The reactions are tissue-specific because accelerated secondary responses to islets are not observed when the responding lymphocytes are primed with lymphocytes from the islet donors. Baekkeskov et al. have precipitated specific membrane glycoproteins from fl cells with antibodies from the sera of newly diagnosed diabetic children 39 and diabetic BB rats 4°. However, it is not known whether these antigens function as target-cell determinants in islet or pancreas graft rejection.
Tissue-restricted alloantigens in the skin, serologically defined Skin and pancreatic islets share the distinction of being the allografts most susceptible to rejection, both frequently being rejected acutely in donor-recipient combinations where other types of allografts (e.g. heart and kidney) are accepted 4I. In contrast to islet allografts, however, the immunological basis of this vulnerability is at least in part explicable in terms of two systems of tissue-restricted alloantigens preferentially expressed on epidermal cells (EC), the serologically defined Skn and cellularly defined Epa systems. Boyse and Old 42predicted the existence of Skn (originally known as Sk) antigens to explain a phenomenon that is not widely appreciated in transplantation immunology; the rejection of donor-type skin grafts by persistent bonemarrow chimeras. Although there are many examples of this 'split-tolerance '43, the model that has been most thoroughly analysed consists of chimeras produced by exposing strain B6 mice to a lethal dose of whole-body radiation and giving them an injection of B6AFI hybrid bone marrow or spleen cells. About two months later, virtually all the hosts reject B6AFj or strain A skin grafts within 20-30 days while remaining tolerant of B6AF1 myeloid and lymphoid cells. In fact, some hosts can reject several consecutive A skin grafts in increasingly accelerated fashion while remaining lifelong chimeras 44. On the dual assumption that, like lymphocytes, EC express differentiation alloantigens, and that self-tolerance of the body's own differentiated cells is antigen-driven, Boyse and Old 42reasoned that lymphocytes arising from transplanted stern cells would be denied the opportunity to become tolerant of donor-strain EC alloantigens because they never were exposed to them, and they eventually would recognize donor-strain skin grafts as foreign and reject them. These hypotheses were later verified when it was shown that A skin grafts are accepted by B6AF 1-to-B6 chimeras if the latter are exposed to strain A skin in the form of EC injections or skin grafts at the time of marrow
237 transplantation 44. Later, Scheid et al. 45 showed that chimeras that reject A skin grafts make antibodies that are cytotoxic to A EC but not to A myeloid or lymphoid cells and not to B6 EC. Thus, the antibodies are tissue-specific alloantibodies, not autoantibodies that lyse host as well as donor EC. The antibodies do react with donor strain neuroblastoma cells, however, indicating the sharing of Skn antigens by EC and brain cells, which have a common embryological origin. Scheid et al. 45 used antisera raised by skin grafting reciprocal radiation chimeras to define two Skn alleles, and by typing various inbred strains they proved that Skn antigens are determined by non-H-2 genes. The genetics of the Skn system is reviewed in detail elsewherC TM. There is good evidence of at least two independent Skn loci, and both the immunogenicity of Skn antigens and the host response to them are under H-2 control.
Tissue-restricted alloantigens in the skin, cellularly defined Sakai et al. 20 found that EC stimulated allogeneic lymphocytes in MHC-compatible, MLR-negative rat strain combinations and that the magnitude of the stimulation by EC and skin allograft survival times were well correlated. Hirschberg and Thorsby 46used a hot pulse of [3H]thymidine to specifically eliminate lymphocytes proliferating in one-way human MLRs. The remaining lymphocytes no longer responded upon restimulation with donor lymphocytes but they did respond to donor EC, indicating the presence of tissue-specific lymphocyteactivating alloantigens on human EC, whose murine counterparts may be the Epa antigens defined in my laboratory. In contrast with Skn antigens, Epa antigens so far have been defined only by cell-mediated cytotoxicity (CMC). We discovered the Epa system when we found that prim ° ing and boosting C3H/He mice with H-2 compatible strain A K R or CBA EC generate C T L that preferentially lyse donor EC as opposed to lymph node or spleen cells 44. Lysis of the EC targets represents allo-immunity not autoimmunity because there is little or no lysis of syngeneic EC, and the targets must come from a donor of the same H - 2 haplotype as the host to be lysed. Thns, the cytotoxicity is a classic example of MHC-restricted CMC, the novelty being the tissue specificity. Genemapping and backcross studies revealed that the restricting antigen is a product of the K region of the H - 2 complex and that the non-H-2 target-ceU determinant, which we designated Epidermal alloantigen-1 (Epa-1), is determined by a single Mendelian gene ". By using appropriate combinations o f I-~-2 congenic lines as donors and hosts, we also determined that the ability to generate Epa1-specific C T L is under H - 2 gene control, with K/D region rather than/-region gene products serving as Irgene products 47. Subsequently we cloned and subcloned Epa- 1 killer cells, which are classic Thy- 1 +, Lyt-2 ÷ CTL, whose antigenic and H-2-restriction specificities have remained remarkedly stable in vitro for over two years ~8, These clones are very cytotoxic to EC, only slightly cytotoxic to Con A and LPS blasts and not cytotoxic at all to resting lymphocytes from Epa-1 + strains. They also readily lyse cultured adherent EC and fibroblasts 49, which
238 demonstrates that the preferential lysis of these targets is not an artifact trypsinization. We also found that although fresh peritoneal microphages (M+) are susceptible to lysis by other CTL, they are resistant to Epa-1 C T L but become susceptible after 12-24 hours in vitro. However., fresh PE M+ are susceptible to Epa-1 C T L if they come from mice that have been treated with the M+ activating agents Con A and BCG as opposed to the sterile inflammatory agents peptone broth or thioglycoltate 49. The correlation between Epa-1 expression and M+ activation suggests that Epa-1 may be a strainspecific marker for activated M+ as well as an inducible H antigen in vivo, and the 'contamination' of lymphocyte cultures with activated M~ may explain the slight cytotoxicity of Epa-1 C T L towards lymphoblast targets. We also used radiation chimeras as a source of stimulator and target cells to test the influence of Langerhans cells (LC) on Epa-l-mediated CMC s°. (The importance ofLC, the small population of bone-marrow derived, Ia + dendritic cells in the epidermis for skin-associated immune responses has been previously reviewed in this journal 51.) By making reciprocal radiation chimeras and controls between Epa-1 ÷ and Epa-1 - strains, we had at our disposal EC suspensions in which the keratinocytes and LC came from strains of the same or different Epa- 1 phenotype. EC suspensions from Epa- 1 ÷ hosts with longestablished Epa-1 - bone marrow were indistinguishable from Epa-1 ÷ EC controls at the priming, boosting and effector phases of Epa-l-specific CMC. Thus Epa-1 is fully expressed on keratinocytes, whose immunogenicity and susceptibility to lysis is not dependent on Epa- 1 + LC, which is consistent with the evidence that LC normally are not critical target antigens in the rejection of skin allografts 52. Our evidence that Epa-1 functions in vivo is fivefold: (1) In some cases Epa- 1 ÷ skin allografts are rejeeted faster than Epa-1 - skin allografts 44. (2) Injections ofEpa-1 + EC are much more effective than Epa-1 - EC at priming hosts for the accelerated rejection of Epa- 1 ÷ skin allografts 44. (3) Merely matching backcross recipients of parent-strain skin allografts for Epa-1 and ignoring all other H antigen differences prolongs the survival time of first-set Epa-1 ~ skin allografts very significantly, strong evidence that Epa- 1, or the product of a very closely linked gene, is an H antigen 44. (4) Epa-l-specific C T L can be extracted from spongematrix allografts impregnated with Epa-I * EC as well as from lymph nodes draining the site of sponge implantation 5~. (5) Cloned Epa-1-specific C T L mediate gross necrotizing skin lesions when injected intradermally, and the ability of the C T L to induce these lesions in vivo is subject to the very same antigen and H-2 restriction specificities as their ability to lyse EC targets in vitro ~. Thus, reactions to Epa antigens may in part explain why the skin is such a preferred target in graft-versus-host disease. More definitive studies on the role of Epa-1 in skin allograft rejection and graft-versus-host reactions await the establishment of an Epa-l-disparate congenic line, which currently is at the 15th backcross generation in my mouse colony. Although we have identified only one Epa-1 allele to
Immunology Today, voL 5, No. 8, 1984
date, the wide range of target-cell susceptibility and priming ability of EC of the numerous mouse and rat strains we have tested 5~suggest that the Epa-1 immunogenicity of a given strain may be determined by the additive effects of several molecularly distinct antigens or by several distinct determinants on the same molecule. Thus, Epa antigens may influence transplantation immunity by their multiplicity or polymorphism. In fact, we already have provisional evidence of the antigenic products of three more Epa loci. For example, CBA EC evoke C T L in B10.BR hosts that preferentially lyse CBA EC. Since both strains are Epa-1 +, the C T L must be directed against the antigenic product of a second Epa locus, provisionally designated Epa-2. In addition, the ability of human EC but not human lymphocytes to cross-prime C3H/He mice for the subsequent generation of Epa- 1-specific C T L 55indicates that Epa-like antigens are expressed in human skin, something we hope to establish directly with EC-specific C T L of human origin.
The hemopoietic histocompatibility (Hh) system The recent death of Gustavo Cudkowicz tragically ended the career of the investigator most responsible for our knowledge of H h antigens, which represent a tissuespecific obstacle to bone-marrow transplantation in mice and other species. The antigens are expressed by stem ceils and tumor cells of the lymphomyeloid series but not ceils of other histological types 56. They are unusual in several respects, most notedly because of their noncodominant expression. Only homozygous mice express Hh gene products. Thus, F 1 hybrids reject parent-strain bone marrow cells by responding to parental Hh antigens. H h responsiveness is resistant to a single dose of whole-body radiation up to about 1 000 rads, but splitdose or chronic irradiation weakens Hh resistance. H h reactions also are thymus independent, and thymectomized or congenitally athymic (nude) mice are hyperreactive to Hh-disparate grafts. These and other features of the Hh system, such as its delayed maturation, bonemarrow dependence and exquisite genetic control are reviewed elsewhere 56. The threshold nature of anti-Hh immunity, which can be overcome by increasing the number of cells in a bone marrow transplant or increasing the dose of radiation, and the non-codominant nature of Hh genes makes it unlikely that H h antigens are a serious problem in clinical marrow transplantation. However, there is much renewed interest in the system because of the striking similarity of anti-Hh effector cells and N K (natural killer) cells 57, which may have an important role in immune surveillance against tumors, intracellular bacteria and viruses.
Methodological considerations and conclusions The investigation of H antigens found on the fixed cells of solid organs and tissues involves problems that simply are not present in comparable studies of the free ceils of the blood, bone marrow and lymphoid organs. Semiquantitative data on tissue distribution and antigen expression in situ can be obtained by immunolluorescence or immunoelectron microscopy if appropriate antibodies are available. However, it is notoriously difficult to raise workable titers of antibody to most non-MHC antigens, which must be defined in vitro in cellular assays that
Immunology Today, vol. 5, No. 8, 1984
require essentially pure single-cell suspensions for use as stimulator or target cells. Proteases, usually typsin or collagenase, almost invariably are required to free cells from solid organs and tissues, and it is well known that these enzymes can destroy or distort normal cell-surface antigens. The solution to this dilemma is to culture enzymatically dispersed cells in vitro long enough to allow cell surfaces to regenerate and normalize but not so long as to lose the tissue-specific antigens under investigation. The purity of cell preparations is a major problem when dealing with complicated organs such as kidney, liver or pancreas that contain several different types of parenchymal cells in addition to stromal and vascular components, and many reports of tissue-specific H antigens suffer from the lack of definition of the cell types involved. The better characterization of epidermal 44 and endothelial 2~cell alloantigens in large part reflects the ease of obtaining essentially pure preparations of these cell types. Still another problem is the contamination of cell suspensions prepared from solid organs and tissues with small numbers of highly immunogenic marrow-derived cells, such as dendritic cells 11, whose presence even in small numbers can give a false indication of the immunogenicity of the cell type presumably under investigation. One way to avoid this problem is to culture the desired ceils under conditions that result in the selective loss of bone-marrow derivatives 10.3s;another is to use established chimeras with a bone marrow syngeneic to the effector cell strain as a source of stimulator or target cells. This was the strategy we used to determine that Langerhans cells are not a critical source of Epa antigens 5°. Finally, it is particularly important to distinguish true tissue-specificity from mere quantitative differences in the expression of H antigens shared by different cell types when dealing with apparent tissue-specific transplantation immunity. Quantitative absorption and blocking tests with antibodies of known specificities are critical procedures for the serological definition of new antigens; discriminative assays such as monolayer adsorption, antigen-driven suicide and cold-target inhibition are obligatory for cellular definition. In spite of these problems, significant and interesting new information has been obtained by studying antigens expressed by the fixed cells of transplanted tissue and organs. The common assumption that all significant graft antigens are shared with leukocytes clearly is incorrect. Thus, donor-recipient matching based on tissue-typing and crossmatching lymphocytes only may not be the best way to avoid rejection reactions and promote graft survival. However, many practical and logistic problems will have to be solved before non-lymphoid cells can be used routinely in tissue-typing laboratories. [:[']
Acknowledgements The work from my own laboratory was supported by U.S. Public Health Service Grants AI 21208 and CA 09127 and the Mayo Foundation. I am grateful to Lois A. Garmon for help in preparing the manuscript.
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239 3 Ende, N., Orsi, E. V., Baturay, N. Z. and Britten, T. L. (1979) Am. ,f Glin. Pathol. 71,543-548 4 Carpenter, C. B., d'Apice, J. F. and Abbas, A. K. (1976)Adv. lmmunol. 22, 1-65 5 Kashiwabara, H., Wakashin, Y, Benltani, A., Oomori, K., Hachisu, T., Shishido, H., Yokoyama, T., Wakashin, M. and Iwasaki, Y. (1979) Transplant. Proc. 11,434-437 6 Thoenes, G. H., Pielstieker, K. and Schubert, G. (1979) Lab. Invest. 41, 321-333 7 Thoenes, G. H., Krieger, A. and Pielsticker, K. (1979) Transplant. Proc. 11, 1604-1607 8 Hart, D. N.J. and Fabre, J. W. (1980)./[ Exp. Med. 151,651-666 9 Mashimo, S., Sakai, A., Ochiai, T. and Kountz, S. L. (1976) Tissue Antigens 7, 291-300 10 Pawelec, G., Davies, H. ft. S., Pearson; J. D., Steele, C. and Brons, G. (1979) Tissue Antigens 14, 367-378 11 Steinman, R. M., Kaplan, G., Witmer, M. D. and Cohn, Z. A. (1979) J. Exp. Meal 149, 1-16 12 Roth, D., Russell, T., Fuller, L., Kyriakides, G. K., Mintz, D. and Miller, J. (1984) Transplant. Proc. 16, 854-856 13 Williams, R.J., Mallick, N. F., Taylor, G., Orr, W. MeN. (1974), Glin. NephroL 2, 100-105 14 Williams, R.J., Eyres, K., Mallick, N. P., Orr, W. MEN., Taylor, G. and Williams, G. (1976) Transplantation 21, 188-194 15 Vegt, P. A., Buurman, W. A., van der Linden, C. J., Daemen, A. J. J. M., Greep, J. M. and Jeekel, J. (1982) Transplantation 33, 465-469 16 Manca, F., Barocci, S., Kunkl, A., Gurreri, G., Costantini, M. and Celada, F. (1983) Transplantation 36, 670-674 17 Judd, K. P. and Trentin, J . J . (1973) Transplantation 16, 351-358 18 Hart, D. N.J. and Fabre, J. W. (1981) Transplantation 31, 174-177 19 Harkiss, G. D., Cave, P., Brown, D. L. and English, T. A. H. (1984) Int. Arch. Allerg~AppL Immunol. (1973) 18-22 20 Sakai, A., Kashiwabara, H., Taha, M. and Kontz, S. L. (1977) Transplant. Proc. 9, 629-632 21 Hart, D. N.J. and Fabre, J. W. (1981) Transplantation31, 178-182 22 Lane, D. P. and Silver, D. M. (1976) Eur. J. Immunol. 6, 480-485 23 Sakai, A., Tanaka, S. and Kountz, S. L. (1978) Transplantation 25, 110-114 24 Stastny, P. (1980) Transplant. Proc. 12, Suppl. 1, 32-36 25 Hirsehberg, H., Bergh, O.J. and Thorsby, E. (1980),]: Exp. Med. 152, 249s-255s 26 Paul, L. C. and Carpenter, C. B. (1980) Transplant. Proc. 12, Suppl. 1, 43-48 27 Moraes, J. R. and Stastny, P. (1976) Tissue Antigens 8, 273-276 28 Cerilli, J., Brasile, L. and Clarke, J. (1983) in Non-HLA Antigens in Health, Aging and Malignancy (Cohen, E. and Singal, ~D. P., eds), pp. 251-256, Allan R. Liss, New York 29 Cerilli, J., Brasile, L. Clarke, J. and Simak, C. (1983) Abstracts ofthe Ninth Annual Meeting of theAmerican Associationfor Histocompatibility Testing, p. 23 30 Paul, L. C., Claas, F. H.J., van Es, L. A., Kalff, M. W. and de Graeff, J. (1979) N. Eng. J. Med. 300, 1258-1260 31 Balwin, W. M., Claas, F. H. J., van Es, L. A. and van Rood, J. J. (1980) Immunol. Today 1, 110-111 32 Forbes, R. D. C., Guttmann, R. D., Gomersall, M. and Hibberd, J. (1983)Am. J. Pathol. 111,184-196 33 Paul, L. C., Busch, G. J., Paradysz, J. M., Carpenter, C. B. (1983) Transplantation 36, 533-539 34 Groenewegen, G., Buurman, W. A., van der Linden, C.J., Jeunhomrae, J. M. A. A. and Kootstra, G. (1983) Tissue Antigens 21, 114-128 35 Groenewegen, G., Buurman, W. A.,Jeunhomme, J. M. A. A., van der Linden, C. J., Vegt, P. A. and Kootstra, G. (1984) Transplantation 37, 206-210 36 Metzgar, R. S. (1980) Transplant. Proc. 12, Suppl. 1, 123-128 37 Naji, A., Silvers, W. K. and Barker, C. F. (1980) Transplantation 36, 355-361 38 Rabinoviteh, A., Fuller, L., Mintz, D., Severyn, W., Noel, J., Flaa, C., Kyriakides, G. and Miller, J. (1981),]. Clin. Invest. 67, 1507-1516 39 Baekkeskov, S., Nielsen, J. H., Marner, B., Bilde, T., Ludvigsson, J. and Lernmark, A. (1982) Nature (London) 298, 167-169 40 Baekkeskov, S,, Dyrberg, T. and Lernmark, A. (1982) Diabetologia 25, 138 41 Nash, J. R., Peters, M. and Bell, P. R. F. (1977) Transplantation 70-73 42 Boyse, E. A. and Old, L.J. (1968) Transplantation 6, 19 43 Steinmuller, D. and Waehtel, S. S. (1980) Transplant. Proc. 12, Suppl. 1, 100-106 44 Steinmuller, D. (1983) in Traumatic lnjury: Infection and OtherImmunologic Sequellae (Ninnemann, J. L. ed.), pp. 181-196, University Park Press, Baltimore 45 Scheid, M., Boyse, E. A., Carswell, E. A. and Old, L.J. (1972)J. Exp. Med. 135, 238-255
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46 Hirschberg, H. and Thorsby, E. (1975) Tissue Antigens 183-194 47 Tyler, J. D., Burlingham, W. J., David, C. S. and Steinrnuller, D. (1982) Immunogenetics 16, 23-36 48 Tyler, J. D., Galli, S. J., Snider, M. E., Dvorak, A. M. and Steinmuller, D. (1984)0( Exp. Med. 159, 234-243 49 Burlingham, W. J., Snider, M. E., Tyler, J. D. and Steinmuller, D. Cell. Immunol. (in press) 50 Burlingham, W. J. and Steinmuller, D. (1983) Transplantation 35, 130-135
Friedmann, P. S. (1981) Immunol. Today 2, 124-128 Steinmulter, D. (1981) Immunol. Today 2 (No. 11), ii Snider, M. E. and SteinmuUer, D. Transplant. Proc. (in press) Steinmuller, D., Tyler, J. D. and Zinsmeister, A. R. (1984) Transplant. Proc. (in press) 55 Steinmuller, D. and Tyler, J. D. (1983) Transplant. Proc. 15, 238-241 56 Cudkowicz, G. and Nakamura, I. (1983) Transplant. Proc. 15, 2058-2063 57 Miller, S. C. (1983)J. ImmunoL 131, 92-97
51 52 53 54
Immunosuppressive activity of the retroviral envelope protein P15E and its possible relationship to neoplasia Ralph Snyderman and GeorgeJ. Cianciolo Type C retroviral infections can causeprofound immunosuppression as well as neoplasms. The retroviral envelopeprotein p l 5E has both immunosuppressive and anti-inflammatory activities which may contribute to the pathogenicity of retroviruses. Murine and human neoplastic cells, not infected with retroviruse~, have recently beenfound to contain p l 5Elike antigens. In this article Ralph Snyderman and George Cianciolo discuss the potential relationship between p l 5 E production, irnmunosuppression and neoplasia.
Immunological reactions can destroy neoplastic cells in vivo, and the accumulation of macrophages within a tumor can lead to its destruction 1'2. Moreover, cytotoxic T lymphocytes, natural killer (NK) cells, and activated macrophages can kill tumor cells in vitro 3-5. These observations suggest that the immune system provides some resistance against the development and spread of cancer, a contention strengthened by the increased incidence of spontaneous tumors in individuals with congenital or acquired itumunodeficiency diseases 6. In animals infected by tumor-producing retroviruses immunosuppression frequently precedes the development of tumors 7-9 and a causal relationship is now suspected between infection by the human retroviruses named T-cell lymphotropic virus III ( H T L V III) or lymphadenopathy-associated retrovirus (LAV) and the development of the acquired immunodeficiency syndrome (AIDS) and Kaposi' s sarcoma 1°-~2. Since immune mechanisms may limit the development or spread of cancer, clinically apparent tumors may develop when transformed cells acquire the means to escape immunological host defence mechanisms. In this article we discuss factors produced by tumor cells which i depress macrophage-mediated functions, and the recent observation that these substances may be related to the retroviral structural envelope protein p 15E. There is data to suggest that retroviruses may induce immunosuppresstun, m part, by initiating the production of pl5E. We suggest that this property of retroviruses enhances their ability to produce neoplastic cells capable of escaping immune surveillance. Spontaneous tumor cells may likewise resist immune destruction ifa cellular pl 5E-like gene is activated. Laboratory of Immune Effector Function, Howard Hughes Medical Institute Division of Rheumatology and Immunology, Department of Medicine Duke University Medical Center, Durham, NC, USA. © 1984,ElsevierSciencePublishersB.V.,Amsterdam 0167-4919/84/$02.00
Immunosuppression associated with retroviruses and their structural components Imtuunosuppression often accompanies retroviral infection 7-9'11'12.For example, avian, murine, feline and probably human leukemia viruses cause acquired immunodeficieney states in their respective species. getroviruses also diminish immune responses in vitro13-17. The pathological events accompanying retroviral infections are complex but viral structural components are important in the induction of immunosuppression in animals. Olsen et al. 18 and Schaller et al. 19 reported that immunization of cats with UV-inactivated feline leukemia virus (FeLV) abrogated tumor immunity and increased tumor incidence after challenge with infectious feline sarcoma virus (FeSV). Similarly, in-vitro blastogenic responses of feline lytuphocytes to concanavalin A (Con A) were suppressed by up to 65 % by UV-inactivated FeLV 2°. Freeze-thawed preparations of murine Rauscher leukemia virus (RLV) suppressed the in-vitro blastogenic response of mouse splenic lymphocytes to phytohemagglutinin (PHA) or to allogeneic cells in a two-way mixed leukocyte reaction (MLR), 21 perhaps because cell recognition sites were altered by a virion envelope component. Mathes et al. 22.23 reported the inhibition of in-vitro blastogenic responses of feline lymphocytes to Con A using the purified FeLV envelope protein p15E. P15E is the hydrophobic transmembrane protein (around 19 000 dahons) of the retroviral envelope which is synthesized as part of a precursor of mol. wt 80-90 000 (Ref. 24). This precursor is cleaved into p15E and gp70 during protein maturation (Fig. 1). Mathes also demonstrated that treatment of cats with purified FeLV pl5E suppressed their ability to resist a subsequent challenge with infectious FeSV and resulted in increased tumor incidence in the FeSV-challenged animals 23. Con A-induced blastogenesis of hutuan lymphocytes was inhibited by disrupted type
