Regulatory function of human CD4+ cytolytic T lymphocytes.

Allograft rejection is mediated by both CD4+ and CD8+ T cells. The lytic function of the classic CD8+ cytolytic T lymphocytes (CTL) occurs through recognition of allogeneic major histocompatibility complex (MHC) class I on the surface of the graft. CD4+ CTL recognize MHC class II through a direct recognition pathway or an indirect pathway where MHC peptides are presented in the context of self MHC class II. Lytic CD4+ cells may destroy graft tissue or, we hypothesize, the indirect CD4+ T cell may down regulate CD8+ CTL by recognition of donor MHC peptides presented by self MHC class II expressed on CD8+ T cells. To define the role of CD4+ CTL in allograft outcome we used a CD4+ CTL that is MHC class II restricted, recognizing human leucocyte antigen (HLA)-A1 and HLA-B8 peptides in the context of HLA-DR4. This line (MDSxA1/B8) will lyse DR4+ B lymphoblastoid cells (LCL) pulsed with HLA-A1/B8 peptides (amino acids 60-84 of the alpha1 domain of the MHC class I molecule). These T cells will also lyse peptide-pulsed antigen-specific T cell clones, both CD4+ and CD8+, that express HLA-DR4. These clones must process and present the MHC class I peptides for recognition and lysis to occur. These results suggest a possible mechanism to explain allograft tolerance. Lytic CD4+ T cells, that recognize donor HLA peptides through an indirect antigen presentation pathway, down-regulate donor-specific CTL through peptide-specific lysis resulting in graft tolerance.     

EBV-B cells as antigen presenting cells in characterization of the self/donor context of allorecognition by T lymphocytes

Alloreactivity is caused by T cell recognition of foreign histocompatibility antigens according to two models: (i) indirect recognition, in which processed allogeneic antigens are presented by self-major histocompatibility complexes like any other foreign antigen, and (ii) direct recognition, where the foreign MHC itself is recognized breaking the T cell recognition rule of self-restriction. This paper uses these two cases of alloantigen presentation as illustrative examples to investigate (i) the capacity of Epstein-Barr virus-transformed B cells (EBV-B cells) to process alloantigens, and (ii) in vitro assays with EBV-B cell lysate as a source of alloantigen, in order to characterize alloreactive T cell populations. A microculture system was established using donor EBV-B cell lysate as a source of the allogeneic antigen and donor or recipient EBV-B cells as antigen presenting cells to investigate whether alloantigen is recognized by effector T cells from the recipient. T lymphocytes produced after expansion in the presence of interleukin-2 from four samples of liver biopsies (three patients) and four samples of bronchoalveolar lavages (four patients) were used as effector cells. Upon human leucocyte antigen class II typing, these expressed the patient phenotype. When the T lymphocytes were from liver grafts, the recognition involved donor antigens presented by donor EBV-B cells (direct recognition). On the other hand, when the T lymphocytes were cultured from lung grafts, they mainly recognized antigens of donor EBV-B cell lysates in a self-restricted context (indirect recognition). These data suggest that EBV-B cells can provide allogeneic determinants recognized by T cells in donor or self-contexts, i.e. through either direct or indirect recognition.     

Role of three quantitatively dominant endogenous peptides from HLA-DRB10401 molecules in class II specific alloreactivity

Alloreactivity remains an important barrier to organ transplantation and is caused by T cell recognition of foreign histocompatibility antigens (HAg) in two ways: (1) indirect recognition, in which processed HAg peptides are presented by self MHC like any other foreign antigen, and (2) direct recognition, where the foreign MHC itself is recognized in contravention of the T cell recognition rule of self restriction. Whereas the role of endogenous peptides in direct MHC class I specific recognition is now established, their role in class II specific direct alloreactivity remains controversial, since no defined endogenous peptide has been shown to be required for alloreactivity. That mutations resulting in defective antigen processing impair class II specific allostimulation, however, suggests that the endogenous pathway is important for class II as well as class I alloreactivity. We attempted to establish the importance of endogenous peptides for alloreactivity by identifying common sequences of peptides bound by DR molecules of an HLA-DRB10401 homozygous B cell line. Peptides corresponding to three of these (calreticulin, HLA class I and an unidentified molecule) were used to restimulate established allospecific HLA-Dw4 reactive T cell clones, as well as to sensitize allogeneic T cells de novo in vitro. Xenogeneic chinese hamster ovary (CHO) cells coexpressing the relevant DR allele together with CD80 were used as antigen presenting cells. The role of CD80 could be determined on these cells because (1) they are xenogeneic and (2) they do not express B7 family members bound by CTLA-4Ig. However, we were unable to obtain evidence for allorecognition of any of these three peptides, although alloreactive T cell clones could be obtained only following stimulation of naive T cells by CHO cells in the presence of synthetic peptide, and not after stimulation by DR/CD80-bearing transfectants alone. Therefore, it seems unlikely that the most common endogenous peptides bound to DR molecules of B cell lines are directly relevant to allorecognition and, consequently, that attempts to influence alloreactivity by peptide blockade are unlikely to succeed.     

Patterns of T cell-accessory cell interaction in the generation of primary alloresponses in the pig.

Partially inbred, MHC-homozygous miniature swine provide a unique model for the study of organ transplantation and the induction of tolerance in large animals. Models of both vascularized solid organ transplantation and bone marrow transplantation have previously been established. The availability of monoclonal antibodies reactive with porcine leukocyte subset antigens now makes possible studies of the cellular immunology in this species, affording the opportunity to examine mechanisms of transplant tolerance and graft rejection in increasing detail. Using such antibodies and peripheral blood leukocytes from pigs of recombinant MHC haplotypes, we have examined porcine T cell-accessory cell interactions in vitro with attention to T cell subsets and the class of MHC alloantigen stimulation. Primary allospecific MLR and CML cultures were studied after depletion of accessory cells from responder and/or stimulator populations. Although class II MHC antigens were expressed on the majority of porcine T cells before and after depletion, these cells were insufficient for antigen presentation, since there was an absolute requirement for ACs in the generation of primary alloresponses. Proliferative and CTL alloresponses could be generated provided that ACs of either stimulator or responder type were present. Selective depletion of CD4+ T cells from the responder population demonstrated: (a) that the interaction mediated by self ACs was CD4-dependent; (b) that two pathways exist for interaction involving allogeneic ACs; and (c) that the interaction involving allogeneic class II is CD4-dependent, while that with allogeneic class I is not.     

Antigenicity of passively acquired major histocompatibility antigens on T cells

T cell blasts and lines passively acquire MHC molecules in vitro. To determine the role of these molecules in immunoregulatory reactions, we examined whether T cell lines grown on irradiated F1 spleen cells were able to supply allogeneic MHC antigens for the stimulation of T cell proliferation. Immunofluorescence analysis demonstrates that autoreactive T cell lines grown with irradiated F1 spleen cells acquire allogeneic class II molecules and subsequently lose the MHC molecules within 4 days of coculture with syngeneic cells. The proliferative response of (H-2k x H-2d)F1T cells stimulated by a T cell line grown on (H-2k x H-2d)F1 cells is inhibited by the addition of hybridoma-culture supernatants containing anti-IAd as well as anti-IEk antibodies. The proliferation of the F1 T cells to the T cell line grown on H-2k spleen cells is only affected by supernatants containing anti-IEk antibodies. To investigate the role of acquired class I MHC antigens, we examined their ability to serve as antigens for cytotoxic cells. Anti-H-2k cytotoxic T cells are generated when H-2b T cells are cultured with an H-2b-derived T cell line, only if the line has been grown on (H-2k x H-2b)F1 cells. An H-2b-derived T cell line exposed to (H-2k x H-2b)F1 cells can be lysed by anti-H-2k cytotoxic T cells from a primary MLR. Similarly, an H-2k anti-H-2b cytotoxic T cell clone will kill an H-2k-derived T cell clone grown on (H-2k x H-2b)F1 spleen cells. These results demonstrate that passively acquired class I molecules can stimulate the generation of cytotoxic T cells that lyse cells expressing the class I antigens and that passively acquired class I molecules expressed on T cells serve as the target for cytotoxic T cells.     

Absence of allogeneic T cell response to human insulin-secreting cells following long-term culture

Lymphocyte proliferative responses (3H-Thymidine uptake) were studied in mixed culture combinations of peripheral blood lymphocytes as responder cells and human insulinoma cells as stimulator cells (MILR). Influence of culture time on the ability of insulinoma cells to stimulate allogeneic T cell proliferation was examined. Crude cell suspensions initiated a strong lymphoproliferative response with a stimulation index (SI) that ranged between 3 and 7, whereas insulinoma cells did not stimulate allogeneic T cells after one month of culture. Expression of cell surface determinants coded by the major histocompatibility complex (MHC) was evaluated simultaneously by indirect immunofluorescence using monoclonal antibodies to class I shared-determinant or class II molecules. Human insulinoma cells expressed class I but not class II molecules. Crude insulinoma cell suspensions were found to be contaminated by 2% of DR+ cells from nonislet components. It is postulated that loss of these DR+ lymphoreticular cells with culture time resulted in absence of immune recognition and lymphoproliferative response. These results emphasize the need of culturing human islet cells prior to transplantation in order to reduce cell immunogenicity.     

Antigen-specific tumor vaccine efficacy in vivo against prostate cancer with low class I MHC requires competent class II MHC

BACKGROUND: Cancers can escape immune recognition by means of evading class I major histocompatibility complex (MHC) -mediated recognition by cytotoxic T lymphocytes. However, immunization strategies targeting defined tumor-associated antigens have not been extensively characterized in murine prostate cancer models. Therefore, we evaluated antigen-specific, antitumor immunity after antigen-encoding vaccinia immunization against mouse prostate cancer cells expressing a model tumor-associated antigen (beta-galactosidase) and exhibiting partially deficient class I MHC. METHODS AND RESULTS: Low class I MHC expression in beta-galactosidase-expressing D7RM-1 prostate cancer cells was shown by fluorescence activated cell sorting, and deficient class I MHC-mediated antigen presentation was shown in resistance of D7RM-1 to cytolysis by beta-galactosidase-specific cytotoxic T lymphocytes (CTL). Despite partially deficient class I MHC presenting function, immunization with vaccinia encoding beta-galactosidase conferred antigen-specific protection against D7RM-1 cancer. Antigen-specific immunity was recapitulated in beta(2)m knockout mice (with deficient class I MHC and CTL function), confirming that class I MHC antigen presentation was not required for immunity against tumor partially deficient in class I MHC. Conversely, antigen-specific antitumor immunity was abrogated in A(b)beta knockout mice (with deficient class II MHC and helper T cell function), demonstrating a requirement for functional class II MHC. Resistant tumors from the otherwise effectively immunized beta(2)m knockout mice (among which tumor progression had been reduced or delayed) showed reduced target antigen expression, corroborating antigen-specificity (and showing an alternative immune escape mechanism), whereas antigen expression (like tumor growth) was unaffected among A(b)beta knockout mice. CONCLUSION: Our results demonstrate that class I MHC-restricted antigen presentation and CTL activity is neither necessary nor sufficient for antigen-encoding vaccinia immunization to induce protective immunity against class I MHC-low tumors, whereas host class II MHC-mediated antigen presentation facilitates antigen-specific immunity against prostate cancer in vivo. Reduced expression of the target antigen developed rapidly in vivo as an immune escape mechanism for such cancers.     

Long-term culture of islets abrogates cytokine-induced or lymphocyte-induced increase of antigen expression on beta cells

BACKGROUND: Immunogenicity of a graft depends on its expression of major histocompatability complex (MHC) antigens and adhesion molecules and on the amount of intragraft leukocytes, the so-called passenger leukocytes. Although long-term culture reduces passenger leukocytes, permanent acceptance is not necessarily observed after allogeneic transplantation. Because little is known about antigen expression on the surface of islet cells after long-term culture of islets, we investigated whether antigen expression of pancreatic beta cells is influenced by long-term culture and whether long-term culture can counteract the increase of antigen expression induced by cytokines or by allogeneic lymphocytes. We also investigated whether long-term cultured islets were able to stimulate allogeneic lymphocytes to produce cytokines. METHODS: Isolated LEW.1A (RT1a) rat islets of Langerhans were cultured for 14 and 28 days. Precultured and freshly-isolated islets were then incubated for 2 days with rat recombinant interferon (rIFN)-gamma (1,000 IU/mL), or were co-cultured for 4 days with LEW.1W (RT1u) splenic lymphocytes. RESULTS: Long-term culture significantly reduced CD45 leukocytes within the islets and decreased the amount of beta cells expressing intercellular adhesion molecule (ICAM)-1, whereas MHC antigen expression remained unchanged. After incubation of freshly isolated islets with IFN-gamma induction of MHC class II antigens on beta cells, an increase of MHC class I antigen density and an enhancement of ICAM-1+ beta cells were observed. Similar results were found after co-culturing with allogeneic lymphocytes. Using precultured islets, the induction of MHC class II on beta cells by IFN-gamma was still present but significantly lower and was absent after co-culture with allogeneic lymphocytes. Enhancement of ICAM-1+ beta cells by IFN-gamma or by allogeneic lymphocytes was markedly lowered because of preculturing. The proportion of MHC class I beta cells remained unchanged; however, antigen density of long-term cultured islets (28 days) could not be enhanced by allogeneic lymphocytes. Precultured islets were not able to stimulate allogeneic lymphocytes to produce and release normal amounts of cytokines (IFN-gamma or interleukin [IL]-2). CONCLUSIONS: In conclusion, in addition to reduction-depletion of passenger leukocytes, long-term culturing of islets also is able to counteract the IFN-gamma-induced or allogeneic lymphocyte-induced increase of antigen expression. Therefore, initiation of rejection and generation of cytotoxic cells might be altered or timely delayed when long-term cultured islets are transplanted. The variable and conflicting in vivo results after transplantation of long-term cultured islets might be explained by the possible indirect antigen presentation, which is not influenced by islet preculture.     

Analysis of immune response to tumor specific antigen restricted to HLA class II on renal cell carcinoma and its recognition mechanism

PURPOSE: In this study, we studied the immune response against to human renal cell carcinoma and its antigensity. METHODS: Mixed lymphocyte tumor culture test was performed using tumor cells as stimulator cells, peripheral blood lymphocytes from tumor patient (autologous) or healthy volunteer (allogeneic) as responder cells, and tumor cells or peripheral blood lymphocytes from tumor patient as target cells. The cytotoxic activity of mixed lymphocyte tumor culture test was assayed by 51Cr-relase test, and cell surface antigens presented on tumor cells or peripheral blood lymphocytes were assayed by antibody block test. RESULTS: The cytotoxic activity against to tumor cells was induced from allogeneic peripheral blood lymphocytes by mixed lymphocyte tumor culture test. Its cytotoxic activity was inhibited by anti-CD8 antibody treatment of peripheral blood lymphocytes and anti-HLA class II antibody treatment of tumor cells. Furthermore, allogeneic peripheral blood lymphocytes induced to tumor cells did not damage peripheral blood lymphocyte of the tumor patient derivation. CONCLUSION: Renal cell carcinoma may express tumor specific antigen restricted to HLA class II antigens that could be recognized by allogeneic CD8 positive T lymphocytes.     

Comparative study of the role of professional versus semiprofessional or nonprofessional antigen presenting cells in the rejection of vascularized organ allografts

The immune systems of transplant recipients are progressively challenged with exposure to the multiple lineages of donor cells that comprise the vascularized organ allograft. Each lineage of such donor tissue constitutively expresses or can be induced to express varying densities of MHC antigens ranging from no expression of MHC to MHC class I only to both MHC class I and class II. In addition, the cell surface expression of a diverse assortment of costimulatory and cell adhesion molecules also varies in density in a tissue specific fashion within the allograft. The MHC class I/II molecules displayed on the donor cells contain within their clefts a constellation of processed protein antigens in the form of peptides derived from intracellular and to some extent extracellular sources. Therefore, the potential for each cell lineage to induce alloactivation and serve as a target for allospecific immune responses is dependent on the diversity and density of peptide-bearing MHC molecules, constimulatory molecules, and cell adhesion molecules. In addition, the T cell receptor repertoire of the recipient also contributes to the magnitude of the allogeneic response. Consequently, the variety of clinical outcomes following organ transplantation even with the institution of potent immunosuppressive (drug) therapies is not suprising, as it appears reasonable for such therapies to influence the allogeneic response against distinct lineages differentially. Our failure to prevent chronic human allograft rejection may therefore be due to our limited appreciation of the full spectrum of alloactivating experiences encountered by host T cells as they interact with donor cells of diverse tissue lineages.Investigations by our laboratory of the immunopathogenesis of chronic cardiac allograft rejection have revealed an intrinsic inability of human cardiac myocytes to process and present antigens, not only for primary but also for secondary alloimmune responses. One obvious explanation for this phenomenon is the fact that cardiac myocytes do not constitutively express MHC class II molecules and express only low levels of class I molecules. However, this immunological unresponsiveness is maintained even after the induction of MHC class II and upregulation of MHC class I on these cells by interferon-gamma (IFN-γ). Similar results have also been reported for cells of different tissue lineages (e.g. chondrocytes, keratinocytes, neural cells). Until now, cells have been defined as professional or nonprofessional for the purposes of defining their potential for antigen presentation to T cells. Professional antigen presenting cells have been identified as cells that are of haematopoietic origin, that constitutively express MHC class I and class II molecules as well as potent costimulatory molecules, and that are able to induce both primary and secondary immune responses, whereas nonprofessional antigen presenting cells are not bone marrow derived, do not constitutively express MHC class II, but may in some cases initiate primary and secondary immune responses after induction of MHC class II antigen by proinflammatory cytokines (e.g. IFN-γ). The findings of our laboratory and others suggest that cells of certain lineages be considered in the separate class of ‘nonantigen presenting cells’. Indeed, nonprofessional antigen presenting cells can be reclassified into three categories: semiprofessional-, nonprofessional-, or nonantigen presenting cells that are able to present antigen to and activate naive T cells, activated T cells, or no T Cells, respectively.The aim of this review is to identify and (re)examine the antigen presentation characteristics of cells of different tissue lineages in terms of their ability to activate different subsets of T cells. This approach is taken in an attempt to synthesize these concepts into a unified picture of T cell activation in the context of antigen processing and presentation by different cell types.     

Immunogenicity of MHC class I alloantigens expressed on parenchymal cells in the human kidney.

The goal of the present study was to examine the capacity of human kidney cell lines (KCL) to elicit T cell responses to MHC class I alloantigens. KCL exhibited the phenotypic characteristics of tubular epithelial cells and were devoid of detectable contamination with leukocytes. Coculture of normal peripheral blood leukocytes (PBL) with allogeneic KCL elicited cytolytic T lymphocytes (CTL) that lysed the stimulating KCL but failed to lyse third-party KCL. Cell panel and antibody blocking studies demonstrated that the CTL were directed to the HLA class I alloantigens expressed on the KCL stimulators. Purified T cells completely failed to mount a CTL response to KCL, but the response could be reconstituted by supplementing the cultures with either autologous non-T cells or supernatant from a mixed lymphocyte culture (MLC). IL-2, but not IL-1, IL-4, IL-6, or gamma-interferon, restored the anti-KCL response, suggesting that IL-2 is the active factor in the MLC supernatant. Induction of class II antigens on the KCL stimulators with gamma-IFN failed to restore a CTL response, suggesting that KCL are deficient in a costimulatory factor important for class II restricted T helper responses. Nonetheless, our data demonstrate that parenchymal cells in the kidney are capable of presenting class I antigens to alloreactive T cells and, therefore, may contribute to the immunogenicity of renal allografts.     

Expression of MHC class II antigens on human glioma cells modulated by transfection with genes encoding these antigens.

Four glioma cell lines not expressing human leukocyte antigen (HLA)-DR or DQ did so after transfection with genes encoding HLA-DR and DQ. The glioma cells had enhanced ability to stimulate allogeneic and autologous responding lymphocytes in the mixed lymphocyte response (MLR). Glioma cells expressing only HLA-DR had weaker MLR enhancement than those expressing both HLA-DR and DQ or only HLA-DQ. Stimulation by transformed glioma cells increased the killing activity of responding lymphocytes against autologous glioma cells 2.0 times. Both HLA-DR and DQ are important in MLR, and the immunogenicity of glioma cells might be increased by transfection with genes encoding these antigens.     

Simultaneous ligation of CD5 and CD28 with monoclonal antibodies restores impaired immunostimulatory function in human renal cell carcinoma

Tumor cells, including renal cell carcinoma (RCC) cells, do not effectively stimulate T lymphocyte responses against specific antigens presented on their surface. Reasons for this low immunogenicity may include low or absent expression of MHC class I and/or class II molecules, as well as accessory and costimulatory molecules. We used tumor cell pretreatment with cytokines, together with monoclonal antibodies (mAbs) directed at receptors for costimulatory molecules, to render RCC cells immunostimulatory. Interferon-gamma or tumor necrosis factor-alpha pretreatment enhanced expression of MHC class I and class II molecules, as well as CD54, but had only minimal effects on T cell activation. A CD28 mAb, or an even more effective combination of CD28 and CD5 mAb, induced strong primary proliferative responses of allogeneic resting T lymphocytes. Cytokine pretreatment further augmented this T cell response in vitro and allowed T cell expansion and establishment of T cell lines. Stimulation of T cells with autologous RCC cells resulted in a similar T cell activation but with the expansion of cytolytic T cells directed at autologous MHC class II molecules. These experiments demonstrate that cytokines combined with costimulatory mAbs are useful for increasing the immunogenicity of tumor cells. They also indicate. however, that autologous MHC class II expression on tumor cells, together with strong costimulation, may lead to the activation of autoreactive T cells.     

Immunosuppressive activity of class I antigens in the absence of antigen-presenting cells that are MHC-compatible with the host

Pretransplant transfusions of heat-treated spleen and lymph node cells were shown to prolong the survival of DA strain heart grafts in 3 allogeneic host strains: BS, HS, and AS2. To examine whether MHC incompatibility was necessary for immunosuppression mediated by heat-treated cells, AS strain skin-graft recipients were pretreated with fresh or heated inocula from either MHC compatible or incompatible congenic donor strains, AS2.1L(AS) and AS.1F(AS2) prior to transplanting donor strain skin. Prolonged survival was observed only in the MHC-incompatible strain combination, and in this MHC-incompatible strain combination, and in this instance heated cells were conspicuously more immunosuppressive than fresh cells. To determine the effect of intra-MHC differences between donor and host on graft survival, cells from a recombinant donor strain (r22), which shared class II antigens with the graft donor strain and class I antigens with the host, were transfused prior to heart transplantation. Neither fresh nor heated r22 cells prolonged graft survival. Our data accord with the suggestion that in the absence of MHC-compatible antigen-presenting cells, foreign class I antigen is immunosuppressive.     

Cytomegalovirus-induced regulation of major histocompatibility complex class I antigen expression in human aortic smooth muscle cells.

Cardiac allograft vasculopathy (accelerated transplant atherosclerosis) is considered by most to involve a chronic allogeneic immune response to one or more constituents in the coronary vascular wall. Recent evidence suggests that there is an association between cytomegalovirus infection and the development of cardiac allograft vasculopathy (CAV). To determine whether CMV directly infects and/or potentially influences immunogenicity of vascular tissue, human umbilical vein (HU-VECs) or human aortic (HAECs) endothelial cells and human aortic smooth muscle cells (HASMCs) were isolated, cultured, and infected with CMV strain AD 169. Infection was detected using an immunoperoxidase-labeled monoclonal antibody to CMV immediate-early antigen (L-14). The presence and relative quantity of MHC class I and II antigens were determined flow cytometrically using monoclonal antibodies to monomorphic class I and class II HLA determinants. Gamma interferon was used as a positive control stimulant for the upregulation of MHC determinants. Both pooled HUVECs as well as 2 cell lines of HAECs served as targets for CMV infection though less than 10% of the cells were infected despite inocula of 10 pfu/cell. Infection of the pooled HUVECs resulted in no significant changes in the cell surface density of either MHC class I or II determinants. In contrast, HASMCs were excellent targets for CMV infection with virtually 100% of cells infected. CMV infection of 2 distinct HASMC cultures resulted in an increase of 254 +/- 158 relative fluorescence units (RFUs) in MHC class I antigen expression, as assessed by fluorescence intensity, in a variable portion of the HASMCs. A second population of cells exhibited a decrease of 73 +/- 16 RFUs in MHC class I antigen expression. No significant change in MHC class II antigen expression was noted. These results demonstrate that while HUVECs and HAECs are targets of CMV infection, human aortic smooth muscle cells can more readily be infected by CMV. Furthermore, CMV can regulate smooth muscle cell MHC class I expression, hence potentially altering immunogenicity. A pathophysiologic link between cardiac allograft vasculopathy and CMV disease can therefore be hypothesized.     

Effect of cyclosporine on MHC class II antigen expression on arterial and venous endothelium in vitro

MHC class II antigens play a crucial role in immunological responses. The expression of MHC class II antigens on monocytes and endothelial cells is reported to be variable and able to be induced by gamma-interferon. In this study we report on MHC class II antigen expression in vitro by arterial and venous canine endothelial cells, as detected with FACS analysis and indirect immunofluorescence with a monoclonal antibody against canine MHC class II antigens. It appears that cultured endothelial cells do not express MHC class II antigens. Their expression could be induced during a three-day incubation period in lymphokine-containing supernatant produced in mixed leukocyte culture (MLC). Cyclosporine (CsA) added to allogeneically stimulated or unstimulated canine lymphocytes in MLC inhibited the induction of expression by the MLC supernatant. The addition of CsA to MLC supernatant did not have an inhibitory effect. It is concluded that CsA inhibits the production of an MHC class-II-antigen-inducing lymphokine produced by lymphocytes in mixed cultures; allogeneic stimulation is not necessary for production of the lymphokine. It is postulated that a possible mode of action of CsA in prolongation of allograft survival is based on prevention of the induction of MHC class II antigen expression by endothelial cells.     

MHC matching and mechanisms of alloactivation in corneal transplantation.

BACKGROUND: In human corneal transplantation the value of matching, particularly for MHC class II, is unclear and controversial. The contribution of the direct pathway to T cell activation is also uncertain. We have determined the relative contribution of class I, II and non-MHC antigens to graft rejection and of the direct and indirect pathways to T cell activation in a rat model mimicking human incompatibilities. METHODS: DA (RT1a) strain recipients received fully mismatched PVG (RT1c) strain grafts or grafts from one of three recombinant strains bearing DA MHC genes on a PVG background. Graft survival was assessed and the specificity of T cells generated in the draining lymph nodes was determined in mixed lymphocyte (MLR) proliferation assays. To assess the contribution of the direct pathway, fully mismatched graft were performed and allospecific proliferation was measured after depletion of recipient APC from the MLR reaction. RESULTS: There was no significant difference in survival of grafts between the four grades of mismatch, which ranged from a full mismatch to non-MHC mismatches alone (median survival 12.5, 11, 13 and 12.5 days respectively). In conformity with clinical results, strong secondary responses were generated against targets matched for MHC with the recipient. Depletion of recipient APC from a fully allogeneic secondary MLR did not fully abrogate donor-specific proliferation. CONCLUSIONS: Class II matching is of no benefit in this model. Strong indirect responses to non-MHC mismatches are sufficient to induce the rapid rejection, but the small numbers of class II+ cells in the donor appear sufficient to generate a direct response.     

An immunosuppressive effect by synthetic sulfonolipids deduced from sulfonoquinovosyl diacylglycerols of sea urchin

BACKGROUND: It is important to develop new immunosuppressive agents without clinical drawbacks. In this article, we reveal the possibility of a chemically synthetic sulfonolipid that acts as a novel immunosuppressive drug. METHODS: We evaluated the immunosuppressive effect of 3-O-(6-deoxy-6-sulfono-beta-D-glucopyranosyl)-1,2-di-O-acylglycerol (beta-SQDG) that contains a saturated C18 fatty acid, which is designated as beta-SQDG(18:0) by mixed lymphocyte reaction (MLR) and rat allogeneic skin graft. Then, we investigated the mechanism of immunosuppressive effect of beta-SQDG(18:0). RESULTS: beta-SQDG(18:0) inhibited human MLR in a dose-dependent manner without overt cytotoxic effect and prolonged rat skin allograft rejection in vivo. beta-SQDG(18:0) did not inhibit the direct activation of responder T. This reagent could not affect the expression of either major histocompatibility antigen complex (MHC) class I or class II molecules on the cell surface of the stimulator cells, antigen-presenting cells. In contrast, beta-SQDG(18:0) was demonstrated to inhibit the binding among allogeneic lymphocytes. However, the expression of known cell surface accessory and adhesion molecules, such as CD4, CD28, leukocyte function-associated antigen 1, intercellular adhesion molecule 1, and CTLA-4, was not affected by beta-SQDG(18:0) treatment. CONCLUSIONS: beta-SQDG(18:0) might be a new class of the immunosuppressive reagent, and the inhibition of responder T-lymphocyte activation in MLR by beta-SQDG(18:0) may be responsible for certain three-dimensional structures of this reagent or its quinovose binding to sulfonic acid.     

Prolonged survival of cardiac allografts in rats following the administration of heat-treated donor lymphocytes. A possible immunosuppressive role for class I major histocompatibility complex antigens

Pretransplant transfusions of spleen and lymph node cells heated to 45 degrees C or 50 degrees C for 1 hr prolong the survival of subsequent donor-specific heart grafts in the fully allogeneic donor-host combination DA (RT1a)----AS (RT1l). The results are comparable to survival times recorded following pretransplant transfusions of purified donor specific red blood cells (RBC) in the same strain combination. Both class I and class II major histocompatibility complex (MHC) antigens are serologically detectable on heat-treated cells; by contrast only class I antigens are expressed on red blood cells. Although heat-treated cells stimulate alloantibody formation, they fail to provoke a proliferative response in an in vivo host-versus-graft assay. Both red blood cells and heat-treated inocula persist in the host for long periods, possibly an important consideration in relation to their capacity to prolong the survival of subsequent donor strain allografts. The experimental data support the contention that class I MHC antigens can be immunosuppressive in the context of allografting. The present results recall the experiments carried out early in the century, which used heat-treated tumor cells to prolong the survival of subsequent viable tumor allografts, and which are sometimes cited as the first example of active enhancement.