Rat parathyroid allotransplantation: Influence of MHC antigen expression on graft survival

MHC antigen expression in parathyroid tissue and its influence on graft survival after allogeneic transplantation were investigated using a heterotopic rat transplantation model. MHC class I and II expression in parathyroid tissue of Lewis (LEW), Dark Agouti (DA), and Wistar-Furth (WF) rats was analysed semi-quantitatively by using immunohistochemistry. MHC class I expression was strong in DA, moderate in WF, and weak in LEW rats parenchyma, whereas MHC class II expression was negative. In the interstitium of all investigated tissue specimens, the proportion of MHC class II-expressing cells was low. Additionally, four groups were transplanted: 1) LEW to LEW, 2) DA to DA, 3) LEW to DA, and 4) WF to LEW. After syngeneic transplantation, graft survival could be documented over the whole observation period. A median graft survival of 20 (+/-2) days was observed after transplantation from LEW to DA, whereas grafts in the group WF to LEW were rejected after 13 (+/-1) days. The number of intra-graft leucocytes expressing MHC class II molecules was the same in all groups, whereas increased levels of MHC class I in parathyroid tissue before transplantation resulted in a more rapid rejection. These results indicate that immunogenicity of rat parathyroid tissue might be determined by the amount of MHC class I expressed in donor parenchymal cells. Further experiments are necessary to validate this observation.     

Short-term immunosuppression after rat parathyroid allotransplantation

The aim of this study was to evaluate whether short-term postoperative immunosuppression is able to sufficiently prolong graft survival after experimental allogeneic parathyroid transplantation. Heterotopic parathyroid transplantation was performed in 6 groups: 1) syngeneic control Lewis (LEW) to LEW; 2) allogeneic control Wistar-Furth (WF) to LEW; 3-5) WF to LEW plus short-term immunosuppression, postoperative days 1-13 (cyclosporine 5/10/20 mg/kg); and 6) WF to LEW plus 10 mg/kg CyA from preoperative day 7 to postoperative day 7. Graft function was examined up to 60 days; histological and immunohistological examination was performed on all grafts with impaired function. Graft function after syngeneic transplantation was indefinite, while recipients of allogeneic grafts turned hypocalcemic after 13 +/- 2 days. With immunosuppression, graft function was 21 +/- 2 days (groups 5 and 6) and 28 +/- 3 days (groups 3 and 4). Histologically, a cellular infiltrate responsible for graft destruction was found. The results show that indefinite parathyroid allograft survival cannot be achieved by short-term immunosuppression alone. Whether the combination of an additional graft pretreatment and immunosuppression has an impact on graft function will be further examined.     

Intrathymic immunomodulation in sensitized rat recipients of cardiac allografts: requirements for allorecognition pathways.

BACKGROUND: Intrathymic injection of alloantigen in the form of donor cells, soluble major histocompatibility complex (MHC) molecules, or MHC allopeptides induces donor-specific tolerance in a variety of acute allograft rejection models. We have previously shown that a single intrathymic injection of donor spleen cells into pre-sensitized rats abrogates accelerated (circa 24-hour) rejection and prolongs the survival of cardiac allografts to about 7 days. The present study was designed to investigate the mechanisms by which intrathymic administration of donor cells modifies the course of accelerated rejection. METHODS: Lewis RT1(1) (LEW) rats sensitized by transplantation with Wistar-Furth RT1(u) (WF) skin grafts received WF cardiac allografts 7 days later-a classic model of accelerated rejection. At the time of skin challenge, however, certain animals received intrathymic cell suspensions (either allogeneic or syngeneic) or donor-derived class I and/or class II MHC peptides. RESULTS: Control animals (sensitized by skin grafts but receiving no other treatment) rejected cardiac allografts within 24 hours. Intrathymic injection of WF splenocytes at the time of skin transplantation abrogated rejection at 24 hours and prolonged cardiac allograft survival to 6.6+/-0.6 days (p<0.001), whereas intrathymic administration of syngeneic (LEW) or allogeneic third party Brown Norway RT1(n) cells was ineffective in this regard. Intrathymic injection of gamma-irradiated donor cells marginally extended cardiac allograft survival to 3.0+/-0.9 days (p< 0.001), but the grafts were still rejected in an accelerated fashion. Intrathymic injection of donor-derived class I and/or class II MHC allopeptides at the same time period also failed to prolong cardiac allograft survival beyond 3 days. In the group receiving unmodified donor cells, elevated immunoglobulin M (IgM) and immunoglobulin G (IgG) allo-antibodies were found at the time of cardiac transplantation; this pattern was not observed with any other treatment. CONCLUSION: The superiority of non-modified donor spleen cells over gamma-irradiated donor cells or donor specific allopeptides in modifying the course of accelerated cardiac rejection suggests that direct allorecognition is the dominant pathway initiating rejection in sensitized transplant recipients. Marked alterations in the antidonor IgM and IgG responses are associated with successful abrogation of accelerated rejection by thymic immunomodulatory mechanisms.     

Lack of correlation between the induction of donor class I and class II major histocompatibility complex antigens and graft rejection

The induction of donor major histocompatibility complex (MHC) antigens on nonrejected and rejected rat renal allografts was compared at various times after transplantation in two strain combinations, DA-to-PVG and LEW-to-DA. Graft rejection was prevented by preoperative donor-specific blood transfusion (DST). Quantitative absorption analysis and immunohistology were performed using monoclonal antibodies specific for donor class I and class II MHC antigens. A significant increase in the expression of donor MHC antigens, both class I and class II, was demonstrated on nonrejected as well as rejected kidneys after transplantation. A kinetic analysis showed that induction of donor class I antigens was accelerated on the nonrejected grafts, and by day 5 the nonrejected kidneys showed increased expression of class I antigen when compared with the rejected grafts (a 37- vs. a 25-fold increase in expression). Increased expression of donor class I antigens persisted on the nonrejected grafts and was still detectable on long-term-surviving kidneys, 50 days after transplantation. The magnitude of class II antigen induction was similar on both rejected and nonrejected grafts (8-fold by 5 days after transplantation). Immunohistology demonstrated that class I and class II antigens were induced on identical structures in the kidney in both situations. In particular the vessel endothelia, which do not express class II antigens in normal kidney, become strongly positive in both rejected and nonrejected grafts 5 days after transplantation. Although renal allograft rejection is completely suppressed in rats given a single donor-specific blood transfusion before transplantation, graft survival cannot be explained by the lack of induction of donor MHC antigens. Donor MHC antigens are induced on these nonrejected kidney grafts, and therefore they could act as target molecules for the effector cells that mediate graft destruction. Thus the induction of donor MHC antigens on tissue allografts should not be considered as indicative of a rejection response resulting in graft destruction.     

The role of passenger leukocyte genotype in rejection and acceptance of rat liver allografts

BACKGROUND: Although graft-resident passenger leukocytes are known to mediate acute rejection by triggering direct allorecognition, they may also act in an immunomodulatory fashion and play an important role in tolerance induction. Our purpose in the current study was to utilize rat bone marrow chimeras to evaluate the role of the genotype of passenger leukocytes in both acute rejection and tolerance of liver allografts. METHODS: The fate of livers bearing donor-type, recipient-type, and third-party passenger leukocytes was evaluated in the MHC class I and II mismatched rejector combination ACI-->LEW and the acceptor combination PVG-->DA. RESULTS: We report that although treatment of ACI liver donors with lethal irradiation does not lead to prolongation of graft survival in the ACI-->LEW strain combination, ACI livers bearing recipient-type (LEW) or third-party passenger leukocytes (BN) are rejected at a significantly slower rate. We confirm that lethal irradiation of PVG donor animals leads to abrogation of tolerance induction with acute rejection of their livers by DA recipients. However, the majority of PVG livers carrying donor-type (PVG), recipient-type (DA), or third-party (LEW) passenger leukocytes are accepted for >100 days. These DA recipients develop immune tolerance to the donor parenchyma (PVG). CONCLUSIONS: Our findings demonstrate that long-term acceptance of liver allografts and tolerance induction is not dependent on the presence of donor-type passenger leukocytes and can be achieved with organs carrying donor-type, recipient-type, or third-party passenger leukocytes. The importance of the MHC framework on the surface of passenger leukocytes as a critical regulator of the immune response after transplantation of chimeric organs is substantiated by the delayed tempo of rejection of ACI livers bearing recipient-type or third-party passenger leukocytes in the ACI-->LEW strain combination.     

Heart allograft survival in rats following immunization with soluble peptide MHC class I donor antigens: evidence for the role of indirect recognition in rejection

Abstract The current series of experiments addressed the question of whether indirect priming with donor MHC antigens affects heart allograft survival. LEW (RT-1¹) rats were immunized with a mixture of two peptides corresponding to the variable region of MHC class I locus A^a antigen (α1 and α2 domain). The recipients were transplanted with a DA (RT1-1^a) heart 1 month after immunization, and graft survival was closely monitored by ECG. All peptide-treated recipients presented with anti-peptide antibodies at the time of transplantation and developed a strongly accelerated graft rejection. These findings indicated that indirect recognition of MHC I donor antigens promotes heart allograft rejection.     

Immunohistological observations of rat fetal pancreas allografts transplanted into unmodified and cyclosporine-treated recipients

Administration of CsA (15 mg/kg/day) prolonged the survival of DA (RT1a) rat fetal pancreas transplanted to the renal subcapular site of both PVG (RT1c) and Lewis (RT1(1] recipients. Sections of fetal pancreas examined 40 days after transplantation into allogeneic CsA-treated recipients showed growth and development of the fetal pancreas tissue, and the presence of numerous insulin-containing islets. CsA treatment prevented the induction of MHC antigen within allografts. Whereas at day 4, both rejecting and CsA treated grafts showed donor class I MHC expression on duct epithelium and islet cells, only rejecting grafts displayed class I MHC induction on acinar cells. Rejecting grafts showed strong induction of class II MHC antigen expression on duct epithelium from day 4 onward but this was completely prevented by CsA treatment. Islet cells in both rejecting and CsA treated allografts remained class II-negative throughout. CsA also resulted in a reduction in the day 6 cellular infiltrate of allografts (median area leukocyte infiltrate reduced from 43% to 10%) with a marked decrease in the number of MRC OX-8-positive cells. These results show a favorable effect of CsA on rat fetal pancreas allografts with a reduction in MHC antigen expression within the graft and prolonged survival of insulin-rich endocrine tissue.     

Analysis of cellular events in hepatic allografts: Donor progenitors induce intragraft chimerism

Abstract Long‐term graft acceptance and tolerance induction after allogeneic rat liver transplantation are well described. However, the underlying mechanisms remain unclear. In this study we investigated the cellular events within the liver graft during initial immunosuppression and long‐term acceptance. Orthotopic liver transplantation was performed in the Dark Agouti (DA)‐to‐Lewis (LEW) and LEW‐to‐DA rat strain combination. In order to achieve long‐term acceptance, LEW recipients of DA livers were treated with two different short‐term therapies. Non‐parenchymal cells (NPC) were isolated from liver allografts on days + 10 and + 100 after transplantation and donor‐specific leukocytes were immunophenotyped by flow cytometry. Both the monotherapy and triple therapy prolonged graft survival (> 100 days). Liver allografts from LEW donors into DA recipients were spontaneously accepted across a complete MHC mismatch without immunosuppression. Liver allograft rejection was induced by infiltrating alloreactive immunocompetent cells. But the intensities of cell infiltration in the early and late phases after transplantation did not correlate with eventual outcome. Donor‐specific NPC decreased to 18‐25% on day + 10 in both therapeutic groups, but had rebounded to up to 40% by day + 100. Recurrence of donor‐specific cells was caused almost exclusively by rising T cell counts. The persistence of dendritic cells in the late phase after transplantation could be clearly demonstrated. Repopulation by donor‐specific T lymphocytes was observed in long‐term accepted liver grafts. This recurrence may be based on the differentiation of liver‐derived progenitor cells. The persistent coexistence of donor and recipient cells within the liver allograft (intrahepatic chimerism) appears to be characteristic and may be important for long‐term acceptance.     

A detailed analysis of the potential of water-soluble classical class I MHC molecules for the suppression of kidney allograft rejection and in vitro cytotoxic T cell responses

Water-soluble classical (RT1-A) class I MHC molecules were purified from aqueous extracts of DA strain liver. Following monoclonal antibody affinity, lentil lectin affinity, and gel filtration chromatography, 600 micrograms of soluble RT1-A class I molecules with antigen activity equivalent to 1.3 x 10(11) nucleated DA spleen cells (greater than 500 DA spleens) was obtained. Both PVG and LEW strain recipients of DA kidney allografts were pretreated with intravenous injections of the DA soluble class I molecules, in doses with antigen activity equivalent to 10(8) nucleated DA spleen cells. Three protocols of pretreatment were used: twice-weekly injections for 4-5 weeks, with grafting 3 or 4 days after the last injection; a single injection 7 days pregraft; or a single injection 1 day pregraft. The PVG and LEW rats received the soluble class I pretreatment either alone or in combination with suboptimal doses (2 mg/kg/day) of cyclosporine after grafting, making a total of 12 experimental groups treated with soluble class I antigen. In no case did treatment with soluble class I antigen elicit an antibody response in prospective graft recipients; influence kidney graft survival in any way; or enhance or suppress the antibody response to the kidney graft. The soluble DA class I MHC molecules were tested in vitro for their effect on the generation and effector function of allospecific PVG and LEW anti DA RT1-A class I cytotoxic T cells and TNP specific, self RT1-Aa restricted cytotoxic T cells. Concentrations up to 5 micrograms/ml (10(-7) M), equivalent to 10(9) nucleated DA spleen cells/ml, were without any effect. We conclude that monomeric forms of water-soluble classical class I molecules are poor immunogens--and, at doses conventionally used for active enhancement, do not influence cytotoxic T cell responses and have little potential for donor-specific immunosuppression.     

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.     

The effects of perioperative portal venous inoculation with donor lymphocytes on renal allograft survival in the rat. I. Specific prolongation of donor grafts and suppressor factor in the serum

In order to investigate the in vivo functional role of the liver in the immune responses in organ transplantation, effects of perioperative portal venous p.v. administration of donor lymphocytes on renal allograft survival were tested in the rat kidney transplant model. Donor lymphocytes were prepared from BN (BN, RT-1n) or third-party DA (RT1a) rat spleens and lymph nodes and injected p.v. or intravenously to Lewis (LEW, RT-1l) hosts on the day of transplantation (day 0). Untreated LEW hosts rejected BN renal grafts at 7.8 +/- 0.6 days (n = 10). Intravenous administration of 1 x 10(8) BN cells to LEW hosts on day 0 caused a slight, but not significant, prolongation of renal allograft survival (MST = 9.5 +/- 3.0 days, n = 13, NS), whereas portal venous inoculation of 1 x 10(8) BN cells on day 0 remarkably prolonged renal graft survival to 22.2 +/- 5.3 (n = 10, P less than 0.01). The prolongation of graft survival was antigen-specific; the administration of 1 x 10(8) DA cells p.v. to LEW hosts did not prolong the survival of BN renal grafts (MST = 7.4 +/- 0.8, n = 5). Spleen cells from p.v. treated LEW hosts 10 days after transplantation had no suppressor effect on the one-way MLC reaction of normal LEW responder cells toward donor BN or third-party DA stimulators. On the other hand, when serum from p.v.-treated LEW hosts was added to MLC at a concentration of 3 per cent of total volume, it suppressed the MLC reaction toward donor BN cells by 71.6 per cent, but not toward third-party DA stimulators (-8.5 per cent suppression, NS). Histological examination of p.v.-treated LEW hosts at 10 days after transplantation revealed that the liver had normal lobular architecture without expansion of portal tracts and infiltration of inflammatory cells. On the other hand, the transplanted kidney demonstrated a moderate mononuclear cell infiltration around the artery without an interstitial hemorrhage. Moreover, adoptive transfer of the serum from p.v.-treated LEW rats into the virgin secondary LEW hosts significantly prolonged the graft survival of BN kidneys from 7.8 days to 18.9 +/- 5.5 days (P less than 0.01), but not third-party DA graft survivals (MST = 7.5 +/- 0.6 days), indicating that an antigen-specific tolerogenic factor was released into the circulation through the process of allogeneic cells in the liver.     

Donor-derived soluble MHC antigens plus low-dose cyclosporine induce transplantation unresponsiveness independent of the thymus by down-regulating T cell-mediated alloresponses in a rat transplantation model.

BACKGROUND: In vitro, soluble MHC (sMHC) antigens modulate and induce apoptosis in alloreactive and antigen-specific T cells, demonstrating their potency to regulate T cell-mediated immune responses. However, their efficacy to regulate immunological responses in vivo remains unclear. Here, we report that repetitive intraperitoneal injection of recombinant Lewis rat-derived MHC class I antigens in Dark Agouti (DA) rats modulates alloreactivity. METHODS: RT1.A1 (Lewis derived) genes were cloned into mammalian expression vectors, and RT1.Aa (DA derived) genes were used to transfect a rat myeloma cell line. RT1.A1 molecules were injected intraperitoneally in DA recipients that subsequently underwent transplantation with Lewis-derived cardiac allografts. RESULTS: Soluble class I antigens were secreted by the transfected cells and were shown to be heterodimeric, peptide-loaded, and conformationally folded. Injection of donor-derived soluble MHC significantly reduced the ability of recipient animals to mount a cytotoxic T-cell response to donor-derived tissue. More interestingly, this treatment significantly prolonged donor-graft survival and allowed 60% of treated animals to develop graft tolerance (>120 days), when donor sMHC were combined with a single subtherapeutic dosage of cyclosporine. Thymectomy of recipient animals before transplantation did not interfere with induction of peripheral tolerance. CONCLUSIONS: Donor-derived sMHC are potential tolerogens for down-regulating the cytotoxic T-cell response of animals that undergo transplantation. Thus, these data provide for the first time a rationale for the application of directly injected sMHC in vivo to down-regulate immunological responses and aid the induction of graft tolerance.     

Presentation of donor major histocompatibility complex class II antigens by dna vaccination prolongs heart allograft survival

BACKGROUND: Donor major histocompatibility complex (MHC) antigens play an important role in both allograft rejection and tolerance. With the use of several animal models, it has been shown that presentation of donor antigens before transplantation can lead to allograft tolerance. Vaccination of animals with a DNA plasmid encoding an antigen enables highly efficient expression of the protein in vivo. METHODS: In this study, we used DNA vaccination delivered through intramuscular, intraperitoneal, or intravenous routes to indirectly present donor antigens and to determine the effect in the modulation of the allograft response. LEW.1A recipients of a LEW.1W heart allograft were treated before grafting by vaccination with a plasmid encoding the donor RT1.D MHC class II or RT1.A class I molecules. RESULTS: Only anti-MHC II vaccination significantly prolonged allograft survival compared with untreated rats. We observed a significant prolongation of heart allograft survival with the intramuscular route of injection, but surprisingly we found the intravenous and intraperitoneal routes to be the best. CONCLUSION: After transplantation the anti-donor cellular response was significantly decreased in vaccinated rats. This was accompanied by a significant reduction in interferon-gamma mRNA expression in the grafted hearts and T helper 1-type alloantibody production, indicating that the vaccination modifies the alloresponse against the grafts.     

肝移植后供肝和宿主免疫学状态的变化

目的观察供肝免疫原性和宿主对供体抗原反应能力的动态改变。方法利用近交系LEW到WF的大鼠肝移植自发免疫耐受模型,取出不同时期的供肝,分别刺激长期生存的WF宿主,观察能否诱导出肝损害；另外对LEW→WF肝移植后不同时期的宿主,再次给予供体抗原刺激。结果(1)移植后第1、2天的同种移植肝,可激起长期生存的宿主出现暂时性肝损害(121±33、83±21),但第3天以后的则不能(28±9)。(2)给予供体同源的脾细胞刺激后,移植后7、14、28 d的宿主均未能诱导肝损害(56±17、66±11、61±35),但第56、84或112天的宿主均可被诱导出暂时性肝损害(98±25、158±43、330±82)。结论(1)肝移植后供肝的免疫原性在术后3 d基本消失。(2)移植术后1个月内宿主对供体抗原的刺激是处于低(无)反应状态的。     

WOFIE augments the immunosuppressive potency of FK-506

Bidirectional recognition of donor- and recipient-derived immunocompetent cells has been proven to play a pivotal role for the induction of long-term unresponsiveness to allogeneic grafts. This study investigated the fate of heterotopic heart grafts with respect to the timing of subtherapeutic doses of FK-506 and with respect to the time point and type of donor antigen application, leaving space for mutual adaptation of alloreactive lymphocytes, designated as the 'WOFIE-concept' (window of opportunity for immunological engagement), originally described by R Calne. METHODS: Heterotropic heart transplantation was performed using male DA (RT1.a) donor and LEW (RT1.1) recipient rats in the following groups (n = 6). FK-506 was applied intramuscularly (i.m.) using doses of 2 mg/kg x body weight per day. Donor antigen application was performed either by DA blood transfusion, 2 ml intravenously (i.v.), or by i.v. transfusion of 5 x 10(7) DA splenocytes. (i) LEW --> LEW, untreated; (ii) DA --> LEW, untreated; (iii) DA --> LEW, FK-506 days 0, 4-7; (iv) DA --> LEW, FK-506 as group (iii) plus 2 ml of DA blood 6 h post-Tx; (v) same as group (iv) but DA blood transfusion 24 h post-Tx; (vi) DA --> LEW, FK-506 as group (iii) plus DA splenocytes 6 h post-Tx; (vii) same as group (vi) but DA splenocyte transfusion 24 h post-Tx; (viii) DA --> LEW, FK-506 days 0-4 and (ix) DA --> LEW, FK-506 as group (viii) plus DA blood 6 h post-Tx. Immunohistochemical stainings (APAAP-method) of the allografts and flow cytometric analysis of recipient spleens were performed electively 3, 7 and 14 days after organ reperfusion. RESULTS: The mean graft survival differed significantly between groups and comprised (mean +/- SD days): (i) >100, (ii) 6.5 +/- 1.0, (iii) 31.6 +/- 12.1, (iv) 44.8 +/- 10.1, (v) 29.8 +/- 14.2, (vi) 27.2 +/- 4.7, (vii) 14.6 +/- 4.2, 17.5 +/- 4.2, (viii) 17.5 +/- 4.2 and (ix) 18.8 +/- 2.8 days. Prolongation of graft survival and long-term unresponsiveness (group iv) revealed a substantially different pattern of graft infiltration. CONCLUSIONS: Effective treatment with unspecific immunosuppressants like FK-506 can be substantially improved if (i) mutual antigen recognition between donor and recipient immunocompetent cells is warranted, (ii) donor-derived blood-borne antigens are given immediately after graft reperfusion, and (iii) the type of inoculated donor antigen has a strong impact on graft survival as splenocytes which contain a large population of professional antigen-presenting cells failed to prolong graft survival after interrupted FK-506 treatment.     

Different patterns of donor MHC antigen induction in rat kidney allografts following active and passive enhancement

We compare the expression of donor class I and class II major histocompatibility complex antigens in DA kidney grafts transplanted to PVG recipients treated by different protocols of donor-specific immunosuppression. MHC expression was evaluated using donor-specific antibodies and assays by immunohistology and quantitative absorption analysis. PVG recipients were either untreated or treated by (A) twice-weekly intravenous injections of 0.5 ml DA blood for 12 weeks; (B) 0.5 ml DA blood intravenously at 7 days pregraft; (C) as for (B), but with the addition or oral cyclosporine at 10 mg/kg/day from the day of grafting; and (D) passive enhancement with DA anti-PVG serum. Grafts were assessed at 3, 5, and 7 days after transplantation. In untreated controls at day 3, there is a periarteriolar leukocyte infiltrate, weak or absent class II induction, but strong class I induction. Class II induction in untreated controls is maximal at day 5. We confirm that active enhancement by blood transfusion, even using the intensive protocol of twice-weekly transfusions for 3 months, results in accelerated leukocyte infiltration and accelerated donor class I and class II MHC induction. At day 3, there is an intense, diffuse leukocyte infiltration and maximal class II induction. Cyclosporine treatment of blood-transfused recipients reduced the leukocyte infiltration and MHC induction to levels seen in untreated controls--i.e., the accelerated MHC induction caused by the transfusion was partially reversible by cyclosporine. In passively enhanced recipients, leukocyte infiltration and class I MHC induction were similar to untreated controls. However, class II induction was much delayed, not being evident until day 7.     

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.     

Cytotoxic T-cell elimination during anti-CD4-induced rat liver acceptance and rapid replacement of functional graft antigen-presenting cells.

In previous studies, we showed that primed T cells were eliminated in long-term survival Wistar Furth (WF) recipient rats with spontaneously accepted Lewis (LEW) liver graft and that the grafted liver lost the ability to elicit rejection reaction early after liver transplantation. We hypothesized that the same phenomenon may be observed in tolerant animals after immunosuppression in a rejector rat strain combination (WF-->LEW). Furthermore, we proposed the repopulation of liver allograft with host antigen-presenting cells rapidly after transplantation. Recipient LEW rats that underwent anti-CD4 therapy accepted the WF liver allografts after a transient rejection reaction. In tolerant animals, alloreactive CD8 T cell precursors were present, but primed T cells were absent. Intraperitoneal challenge with grafted WF liver homogenates obtained from recipient LEW rats on day 4 after transplantation did not induce transient rejection responses in long-term survival recipient LEW rats, a finding that differed from the results of experiments using normal WF liver homogenates. However, challenge with grafted WF liver homogenates, similar to those of normal LEW liver homogenates, induced rejection responses in long-term survival recipient WF rats with LEW liver allograft. Flow cytometric analysis confirmed that most of nonparenchymal cells in the grafted WF liver were recipient (LEW) genotype. These observations showed that the deletional mechanism of effector T cells also is observed in this setting, and professional donor antigen-presenting cells are replaced by those of recipient genotype within the graft during the early phase of transplantation.     

Induction of indirect donor-specific hyporesponsiveness by transportal RT1-peptide pulse in rat skin transplantation

In the present study, we examined whether transportal pulse of class I major histocompatibility complex (MHC) allopeptides can induce indirect (non-chimeric) donor-specific hyporesponsiveness, using a high-responder rat skin transplantation model. Two donor-specific 8-amino acid peptides corresponding to residues 58-65 and 70-77 in the alpha(1) helical region of RT1.A(a) were synthesized. In order to test immunogenicity of these peptides, mixed lymphocyte reaction (MLR) was performed. Then, 100-microg portions of peptides were injected into recipient Lewis (LEW, RT1.A(l)) rats via the portal vein 14 days before skin transplantation. Skin allografts from August Copenhagen Irish (ACI, RT1(a)) or Wistar King A (WKA, RT1(k), third-party) donors were transplanted to LEW (RT1(l)) recipients. Transportal pulse of residues 58-65 and 70-77 prolonged graft survival significantly in ACI-to-LEW skin transplantation (17.6+/-0.40 and 18.0+/-0.45 days) compared with control (14.2+/-0.37 days). However, pulse of residues 106-113, a non-donor-specific control, did not prolong graft survival time (14.6+/-0.40 days) in the same combination. Regarding the third-party donor, residues 58-65 injected into LEW recipients had no effect on survival time of skin grafts (19.0+/-0.84 days) derived from WKA donors compared with the untreated WKA-to-LEW control (19.4+/-0.93 days). Transportal pulse of RT1.A(a) peptides induced donor-specific hyporesponsiveness even in a high-responder rat skin transplantation model. Our results suggest that graft enhancement by transportal exposure to donor cells may not be induced by a chimeric process but, instead, by an indirect mechanism not involving intervention of viable donor cells.     

The effectiveness of pretreatment with soluble or membrane-bound donor class I major histocompatibility complex antigens in the induction of unresponsiveness to a subsequent rat renal allograft.

We have examined the ability of two physical forms of RT1.A class I molecules to induce immunologic unresponsiveness to renal allografts in the rat. Both preparations of class I MHC antigen were derived from rat liver. Class I MHC antigen was presented either as purified membrane-bound molecules incorporated into protein micelles or as a water-soluble preparation containing soluble RT1.A class I molecules. The amount of RT1.A class I contained in each preparation was compared with the amount of class I antigen expressed by whole viable liver cells by quantitative absorption analysis using F16.4.4.11 mAb. The results demonstrated that DA recipients pretreated with a single dose of 1.75 x 10(10) cellular equivalents or multiple doses of 5 x 10(9) cellular equivalents of purified LEW membrane-bound class I molecules, delivered in aggregated micelle form, accepted their LEW renal allografts indefinitely (MST greater than 100 days). In contrast, no prolongation of graft survival was observed using the liver cell cytosol preparation containing soluble RT1.A class I molecules (MST 10 days) at the concentrations tested (10(8) -3 x 10(8) cell equivalents). However, when preoperative treatment with single (greater than or equal to 5 x 10(7) cellular equivalents of soluble class I MHC antigen) or multiple doses (greater than or equal to 10(7) cellular equivalents per dose) of the liver cell cytosol preparation was combined with a subtherapeutic dose of CsA given postoperatively (day +2, 10 mg/kg), suppression of renal allograft rejection was achieved with long-term survival (MST greater than 100 days). The immunologic unresponsiveness observed in both cases was donor specific.