Comparative studies of specific acquired systemic tolerance induced by intrathymic inoculation of a single synthetic Wistar-Furth (RT1U) allo-MHC class I (RT1.AU) peptide or WAG (RT1U)-derived class I peptide.

BACKGROUND: Because T cell receptor-MHC class I/self-peptide interactions regulate T-cell development, the presence of MHC allopeptides in the thymus may influence T-cell tolerance to alloantigens. This hypothesis is supported by our most recent finding that intrathymic (IT) inoculation of nonimmunogenic synthetic peptides derived from "WAG" RT1.A induces tolerance to cardiac allografts in the Wistar-Furth (WF)-to-ACI model. To evaluate whether in vivo immunogenicity of MHC peptides is relevant to tolerance induction and to examine the effect of peptide specificity, we compared the effects on graft survival of well-defined, strain-specific immunogenic WF MHC class I peptides (RT1.AU) with closely related but non-strain-specific class I peptides derived from WAG (RT1U). METHODS: In vivo immunization of seven MHC class I peptides synthesized from RT1.AU sequences showed that two (u-5 and u-7) were immunogenic, whereas five others were not immunogenic in ACI recipients. We then examined the effects on cardiac allograft survival in the WF-to-ACI model of the two immunogenic RT1.AU peptides (u-5 and u-7) and three immunogenic WAG-derived peptides (peptides 1, 2, and 5). RESULTS: A combination of equal amounts (150 microg or 300 microg) of u-5 or u-7 each with 0.5 ml of antilymphocyte serum (ALS) on day -7 led to 60% and 100% permanent graft survival (>150 days), respectively. IT injection of the individual peptides on day -7 showed that only 300 microg of u-5 significantly prolonged graft survival to a median survival time of 17.3 days from 10.5 days in naive recipients. IT injection of 150, 300, and 600 microg of u-5 combined with 0.5 ml of ALS on day -7 led to permanent graft survival (> 150 days) in four of six, nine of nine, and six of six ACI recipients, respectively, compared with a median survival time of 15.4 days in ALS alone-treated controls. In contrast, similar treatments with peptide u-7 with or without 0.5 ml of ALS did not prolong graft survival, thus demonstrating that peptide u-5 alone mediates the observed effects on graft prolongation. A total of 300 microg of u-5 injected IT combined with ALS led to acute rejection of third-party (Lewis) grafts. Intravenous injection of 300 microg of u-5 with ALS also did not prolong WF graft survival in ACI recipients. The long-term unresponsive ACI recipients accepted permanently donor-type (WF) but not third-party (Lewis) second-set cardiac and islet allografts. Similarly, we showed that although IT injection of 600 and 1200 microg of a mixture of immunogenic WAG-derived peptides 1, 2, and 5 combined with 0.5 ml of ALS on day -7 led to permanent WF graft survival in ACI, only IT injection of 300 microg of peptide 2 combined with ALS led to permanent graft survival (>150 days) in four of five animals. To define the underlying mechanisms of tolerance, we examined in vitro the mixed lymphocyte reaction (MLR), cell-mediated lymphocytotoxicity, and cytokine profile of unresponsive recipients. Although the results showed nonspecific T-cell suppression in the MLR at 25 days after transplantation, which correlated with the persistence of ALS immunosuppression, long-term unresponsive animals showed normal MLR to donor and third-party antigens. In contrast, the donor-specific reactive cytotoxic T lymphocytes remained suppressed in short-term and long-term unresponsive rats. CONCLUSION: Of interest is our finding that IT injection of a short segment of WAG-derived MHC class I peptide induces active acquired tolerance similar to results obtained with the use of pure WF-derived peptide u-5 in the WF-to-ACI rat combination. It is noteworthy that we could not confirm the T helper (Th)1/Th2 paradigm in this model by initial cytokine analysis. Whether induction of tolerance by IT injection of allo-MHC peptides will have clinical usefulness must await results of similar studies in large animals. However, of major interest is the finding that a short segment of RT1.AU represents the tolerogenic

Use of donor serum to prevent passive transfer of hyperacute rejection.

Organ transplantation in presensitized recipients continues to be contraindicated for heart and kidney recipients due to the risk of hyperacute rejection, which has no known treatment at this time. We tested whether donor serum, which contains soluble MHC class I antigen, is able to neutralize the effect of anti-donor antibody in the recipient and prevent hyperacute or accelerated rejection. A rat model of passive immunization was used to test the role of anti-donor antibody in hyperacute rejection. Seven of 10 recipients of hyperimmune serum (HyS), derived from Lewis rats (RT1l) following 3 ACI (RT1a) skin grafts, developed hyperacute or accelerated rejection. Intravenous injection of ACI serum prior to the HyS administration prevented hyperacute rejection in all recipients tested. When third-party (Wistar-Furth, RT1u) serum was given to Lewis rats injected with HyS, hyperacute rejection was not abrogated. When examining the mechanism of this effect, a simple antibody blocking phenomenon was found to be unlikely since flow cytometry analysis showed that ACI serum needed to be present at > or = 256-fold excess compared to HyS to block anti-ACI antibody binding to RT1.Aa+cells by 50%. We tested whether the RT1.Aa class I antigen in ACI serum had other biologic properties that resulted in the prolonged graft survival. However, removal of RT1.Aa antigen from ACI serum prior to use in the passive transfer model did not abrogate the graft prolongation observed previously. These data suggest that components of donor serum other than MHC class I antigen may be useful for preventing the antibody-mediated component of hyperacute rejection.

The azaspirane SKF 105685 ameliorates renal allograft rejection in rats.

The azaspirane SKF 105685 (N,N-dimethyl-8, 8-dipropyl-2-azaspiro (4.5) decane-2-propanamine dihydrochloride) has been shown to attenuate or reverse the course of immunologic disease in several animal models, possibly through the induction of nonspecific suppressor activity. To investigate its effects on immune-mediated renal disease, SKF 105685 was administered by gavage to rats with kidney allografts. Six days after transplantation, GFR (inulin clearance, 1.46 +/- 0.27 versus 0.41 +/- 0.15 mL/min per kg; P < 0.005) and RPF (p-aminohippurate clearance, 5.48 +/- 0.98 versus 1.99 +/- 0.72 mL/min per kg; P < 0.01) were significantly higher in SKF 105685-treated rats compared with vehicle-treated control rats. In addition, mononuclear inflammatory cell infiltrates were significantly reduced in SKF 105685-treated animals compared with controls. Treatment also reduced renal production of thromboxane B2 (81 +/- 22 versus 424 +/- 76 pg/min per mg of protein; P < 0.0005), prostaglandin E2 (612 +/- 165 versus 2,059 +/- 351 pg/min per mg of protein; P < 0.005), and 6-keto prostaglandin F1 alpha (217 +/- 56 versus 943 +/- 186 pg/min per mg of protein; P < 0.005), but interleukin-1 beta mRNA levels within kidney allografts were not affected by treatment. Thus, the azaspirane SKF 105685 is a novel immunosuppressive agent that substantially ameliorates renal allograft rejection in the rat. Although the mechanism of action is unknown, the beneficial effects of SKF 105685 in rejection may relate to its ability to induce suppressor activity and/or its effects on eicosanoid production.

Rat thymic epithelium positively selects mouse T cells with specificity for rat MHC class II antigens but fails to induce detectable tolerance in the mouse T cells to the rat MHC antigens.

BALB/c (H-2d) nude mice were grafted with allogeneic AKR/J (H-2k) or xenogeneic (ACI-N rat, RT1av1) fetal thymuses which were depleted of hemopoietic cells by incubating with 2'-deoxyguanosine (2'dGuo) in vitro prior to grafting. The nylon-wool-passed LN T cells from nude mice grafted with 2'dGuo-treated AKR/J thymus showed a poor proliferative response to B10BR (H-2k) stimulator cells, confirming that mouse thymic epithelium has the capacity to induce tolerance against the mouse MHC antigens on the thymic epithelium. On the other hand, the nylon-wool-passed LN T cells from nude mice grafted with untreated or 2'dGuo-treated ACI/N rat thymus showed significant proliferative responses to ACI/N, which can be blocked by anti-rat MHC class II mAb, whereas the nylon-wool-passed LN T cells from nude mice grafted with syngeneic thymus hardly responded to the xenogeneic stimulator cells. These results suggest that rat thymic stromal cells including thymic epithelium can not induce detectable tolerance in mouse T cells to rat MHC antigens; but rat thymic epithelium may positively select mouse T cells with specificity for rat MHC class II antigens, resulting in a mouse T cell repertoire with strong xeno-reactivity.

Suppression of hepatic allograft rejection in the rat by mitomycin C-treated donor splenocytes: analysis of the immune status.

A single intraperitoneal injection of 3 x 10(6) donor splenocytes treated with mitomycin c (MMC) seven days before hepatic transplantation prolongs survival of hepatic allografts in the ACI (RT1a) to LEW (RT1l) rat combination. This effect is donor specific. An intravenous injection of the same dose of splenocytes treated with MMC seven days before transplantation also tends to prolong hepatic allograft survival. Furthermore, lymphocytotoxic antibody can be detected in rats 30 days after transplantation. Adoptive transfer of 5 x 10(7) splenocytes taken from long-term surviving hepatic allograft recipients pretreated with MMC-treated donor ACI splenocytes into irradiated (750 rads) LEW rats prolongs the survival of donor-type skin grafts, whereas third-party strain (BN) grafts are rejected. Similarly, prolonged survival of ACI cardiac allografts in irradiated (450 rads) LEW recipients is achieved following the transfer of spleen cells taken from longterm surviving hepatic allograft recipients pretreated with MMC-treated donor ACI splenocytes, whereas third-party (BN) cardiac allografts show rejection. These findings suggest the presence of donor-specific suppressor cells and indicate that a single injection of donor splenocytes treated with MMC to the recipient seven days before transplantation can induce specific suppression of rejection in a rat hepatic allograft model.

Induction of a suppressive serum factor, prevention of sensitization, and prolongation of kidney graft survival in rats by transfusion with antibody-coated blood cells.

Transfusion with antibody-coated allogeneic blood cells suppresses the cytotoxic antidonor antibody response in a strongly incompatible rat combination (BN----LEW). Cell coating with homologous recipient antidonor antiserum, rat monoclonal antibodies against MHC class I donor antigens, or rabbit antirat lymphocyte serum all were effective. The suppression was not abrogated by repeated booster transfusions with untreated donor blood. Moreover, the suppression extended to antibody-uncoated antigens present on the same donor cell. Not only the antibody response but also the Graft-versus-host reaction against donor antigens was suppressed. The serum of pretreated animals contained suppressive activity. It suppressed the cytotoxic antibody response as well as the cellular immune response (GVH) when transferred into syngeneic recipients. A weaker suppression of antibody response was obtained by transferring spleen cells of pretreated animals into syngeneic recipients. The transfer data suggest that broadly reactive serum factor(s) were mainly responsible for the suppressive effect. Transfusion with LEW-anti-BN-coated donor cells before transplantation induced markedly prolonged kidney graft survival in the BN----LEW combination without additional immunosuppression (untreated controls: 8.4 +/- 0.4, pretreated recipients: 124 +/- 36 days, P less than 10(-4)).

Effect of 15-deoxyspergualin on experimental organ transplantation.

DSPG had a definite but relatively feeble immunosuppressive effect in rats undergoing heterotopic heart transplantation and in dogs after renal transplantation. The drug was toxic in both species, although less so in rats. In dogs, synergistic interactions with cyclosporine and steroids were not evident.

Effect of FK506 in experimental organ transplantation.

FK506 is the most potent immunosuppressive agent known. Its toxicity is substantial in dogs, minor in rats, and unknown in subhuman primates. In small doses that are nontoxic even in dogs, it can be used in synergistic combination with cyclosporine, steroids, and presumably in other drugs.

Evidence that pretransplant donor blood transfusion prevents rat renal allograft dysfunction but not the in situ cellular alloimmune or morphologic manifestations of rejection.

The effects of preoperative donor-specific blood transfusion (DSBT) on the physiologic, morphologic, and immunologic aspects of allograft responsiveness were evaluated in a rat renal transplant model, using the ACI (RT1a) into PVG (RT1c) high-responder strain combination. Indefinite graft survival (mean greater than 63 days) could be induced by DSBT administration alone. In comparison, animals receiving autologous blood transfusion (ABT) all died within 7 days posttransplantation. As assessed by clearance of inulin and paraaminohippurate, renal allograft function in DSBT-pretreated recipients at 6 days was equivalent to that of isograft recipients, and in contrast to the significant reduction seen in ABT treated rats. Likewise, thromboxane B2 (TXB2) production by ex-vivo-perfused allografts from DSBT-treated recipients was comparable to that of isografts, and significantly lower than that of allografts from ABT-treated rats. A significant inverse correlation was found between renal TXB2 production and inulin clearance. Despite these substantial differences in renal function and eicosanoid metabolism, morphologic evaluation of renal allografts from DSBT-enhanced and ABT-rejecting recipients at comparable time points showed equivalent histologic manifestations of rejection. In addition, immunohistologic labeling of renal allograft sections and fluorescence-activated cell sorter analysis of cells eluted from allografts showed the same phenotype and pattern of infiltrating T cell subsets in both groups. Specific antidonor cytotoxic T lymphocyte precursor (pCTL) frequencies of cells eluted from kidney grafts were equivalent in DSBT and ABT-pretreated animals, and both groups expressed significantly higher (but equivalent) pCTL frequencies in the kidneys than spleens. Comparisons of the lysis of PVG.R1 (RT1.Aa on a PVG background) and ACI targets indicated that cytotoxic responses from effector cells freshly eluted from DSBT and ABT kidneys were primarily directed against allogeneic class I major histocompatibility complex (MHC) specificities, whereas several long term T cell lines generated from 6-day kidney transplants of both groups expressed a predominant W3/25+ (T helper) phenotype and cytotoxic activity against donor specificities other than RT1.Aa class I MHC. Specific antidonor proliferative T lymphocyte (pPTL) precursor frequencies of cells eluted from renal allografts were also equivalent for both DSBT- and ABT-treated recipients, and the range of pPTL frequencies for allograft cell eluates was similar to that in spleens, regardless of the source of the transfusion.(ABSTRACT TRUNCATED AT 400 WORDS)

(Nva2)-cyclosporine--less potent than cyclosporine A in rats with lung and heart transplants.

The ability of a new cyclosporine (Cs) derivative, (Nva2)-Cs (CsG), to suppress rejection of lung and heart allografts in rats was determined and compared with that of CsA. Left lungs were transplanted orthotopically; hearts were transplanted heterotopically into the abdomen. (Nva2)-Cs was used in three experimental protocols: (1) single or three (Nva2)-Cs injections given to lung-transplanted rats, (2) daily oral (Nva2)-Cs treatment at different doses compared with similar CsA treatments in heart allografted rats, and (3) An 11-day (Nva2)-Cs treatment starting at increasing intervals after transplantation of hearts. (Nva2)-Cs was found to be immunosuppressive, and effective even when the treatment started as late as four days after transplantation. However, (Nva2)-Cs was less effective than CsA in suppressing rejection of lung and heart allografts at low doses. Because (Nva2)-Cs is possibly not nephrotoxic, it might be a useful drug if used in higher doses than CsA or in combination with other immunosuppressive agents.

Humoral presensitization in rat heart allotransplantation.

In these experiments an attempt was made to create a rat model of the past-positive, current-negative lymphocyte crossmatch (PPCNCx) phenomenon currently of concern in clinical renal transplantation. Lewis rats were sensitized with three to four serial ACI heart fragment (HF) or skin grafts. Subsequent ACI heart graft survival in the presence of high titer LEW-anti-ACI antibody was markedly shortened with 4 of 10 surviving for 24 hr or less in HF-sensitized rats and 11 of 11 surviving less than 24 hr following skin graft sensitization. Thirteen sensitized LEW rats were transplanted with ACI hearts 18 months later when their anti-ACI antibody was nil. Control rats (N = 5) had graft survival of 4.4 +/- 0.6 days; cyclosporine (CsA) therapy prolonged this to 8.0 +/- 3.6 days (N = 4), while combined cyclosporine and cyclophosphamide (Cy) resulted in an MST of 4.3 +/- 0.4 (N = 3). LEW-anti-ACI antibody was present on Day 3 or 4 in the control rats but was absent at the time of rejection in the CsA-Cy treated rats. Adoptive transfer of splenocytes from sensitized LEW rats into naive LEW hosts produced animals with humoral immune memory but no anti-ACI antibody at the time of transplantation. Nonimmunosuppressed adoptively transferred LEW recipients of ACI hearts rejected their grafts in an accelerated fashion (MST of 4.5 +/- 0.5 days) and displayed an anamnestic antibody production with first appearance on Day 3 or 4. Immunosuppression with CsA or Cy prolonged graft survival (greater than 30 days) in all cases and Cy prevented an anamnestic humoral response.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural studies of the class II histocompatibility antigens of the ACI rat.

The class II antigens of the ACI rat were studied using both conventional alloantisera and monoclonal antibodies. By sequential immunoprecipitation experiments and cell binding studies, alloantisera were shown to contain antibodies to both the RT1.B and the RT1.D gene products. Using one- and two-dimensional gel electrophoresis, the structures of these gene products were shown to be distinguishable. The importance of these differences for the immune response and antigen presentation is discussed.

Alloantibody to a sex-influenced esterase in rats (Rattus norvegicus).

An antiserum to a serum protein from female ACI rats was produced in DONRYU rats by alloimmunization. The serum protein reacting with this antiserum was shown to be a sex-influenced esterase which is identified by zymogram techniques. Evidence for this is as follows: this protein migrated in the albumin region on agarose gel immunoelectrophoresis and was not present in sera from mature males; all rats possessing the protein were sex-influenced esterase in sera specifically disappeared after treatment with this antiserum. The attempt in ACI rats to produce antiserum to the esterases from DONRYU rats was unsuccessful.