Donor-specific Unresponsiveness Research Articles

Transplantation tolerance to rat cardiac and islet allografts by posttransplant intrathymic inoculation of soluble alloantigens.

The search for strategies for induction of specific tolerance in adult animals that will avoid long-term host immunosuppression with its complications has led to the deliberate introduction of alloantigens (Ag) into the adult thymus. However, pretransplant intrathymic (IT) inoculation of alloantigens (Ag), which has consistently induced tolerance to vascularized and neovascularized allografts in adult rodents, has limited future clinical application. To overcome the practical limitations of pretreatment, we have examined in the Lewis-to-WF combination the effect on graft survival of either simultaneous or posttransplant IT inoculation of soluble Ag obtained from 3M KCl extracts of donor T cells in transiently rabbit antirat lymphocyte serum (ALS) immunosuppressed recipients. While IT injection of 2.0 mg soluble Ag alone on day of cardiac transplantation caused acute graft rejection, IT inoculation of 2.0 mg Ag combined with 1 ml ALS transient immunosuppression of the recipient on day 0 led to long-term graft survival (> 250 days) in 5/6 recipients. Similarly, IT injection of soluble Ag on posttransplant day 3 or day 7 combined with 1 ml ALS on day 0 relative to allografting resulted in permanent graft survival in all recipients. In contrast, intravenous injection of soluble Ag combined with ALS immunosuppression on day 0 led to acute graft rejection that paralleled the rejection seen in ALS treated controls. Third-party Brown Norway (BN) hearts were acutely rejected in similarly prepared recipients of IT-Ag, thus confirming donor specificity. The long-term unresponsive Wistar-Furth (WF) recipients challenged 100 days after cardiac transplantation with a second-set graft specifically and permanently (> 100 days) accepted the second-set donor cardiac allografts, thus demonstrating donor-specific tolerance. In similar experiments, IT inoculation of 2 mg soluble Ag combined with transient ALS immunosuppression resulted in donor-specific unresponsiveness to islets in the same rat combination of Lewis-to-WF. Our findings suggest that this new strategy of immunologic manipulation of the adult thymus offers a safe, effective, and reproducible method of inducing tolerance that may have therapeutic application in cadaveric organ transplantation.

Transplantation tolerance induced by antigen pretreatment and depleting anti-CD4 antibody depends on CD4+ T cell regulation during the induction phase of the response.

Adult mice pretreated with donor-specific transfusion and depleting anti-CD4 antibody 28 days before transplant accept fully allogeneic heart grafts and become specifically tolerant without further treatment. The induction of tolerance in this model is not simply a function of CD4+ T cell ablation, but appears to depend on residual CD4+ T cells which escape depletion and engage donor alloantigen during a transient period of antibody blockade. To test the hypothesis that these CD4+ T cells might be responsible for regulating immune responses toward the graft, mice were reconstituted with naive recipient leukocytes at various times after pretreatment. Reconstitution either shortly after pretreatment or shortly after transplant had little effect on graft survival. However, when pretreated mice were given an additional dose of depleting anti-CD4 antibody at the time of transplant to target putative regulatory cells, naive leukocytes were able to cause acute graft rejection. These data suggest that in clinical transplantation specific T cell regulation might develop following pretreatment with antigen and non-depleting anti-CD4 antibodies. Such an approach could provide donor-specific unresponsiveness prior to transplant without the risks associated with sustained CD4+ T cell depletion.

Donor-Specific Unresponsiveness to Murine Cardiac Allografts Induced by Intrathymic-Soluble Alloantigens Is Dependent on Alternate Pathway of Antigen Presentation

This study extends the finding that intrathymic (IT) inoculation of uv-B irradiated donor spleen cells (SC) or soluble alloantigens (Ag) induces peripheral tolerance to organ allografts in the rat to the murine cardiac allograft model. In our initial experiment, we showed that IT inoculation of uv-B irradiated SC combined with transient immunosuppression of the recipient with either sublethal TBI or ALS on Day-7 led to donor-specific, long-term cardiac allograft survival (>300 days) in the completely mismatched A/J-to-C57BL/6 mice combination. To test our hypothesis that peripheral tolerance induced by IT injection of donor Ag is dependent on presentation of the foreign MHC molecule by thymic APC to T cell precursors, we examined the effect of IT injection of donor APC-free-soluble Ag inoculum obtained from 3 M KCl extracts of purified MHC class I resting T cells on cardiac allograft survival in the A/J-to-C3H mice combination. The results showed that IT injection of the optimal dose of 500 μg soluble Ag combined with 0.5 ml ALS on Day-7 led to donor-specific permanent graft survival (>200 days). This finding could not be reproduced by intravenous administration of soluble Ag, thus confirming the privileged position of the thymus in tolerance induction. To further define the role of host APC in allorecognition, we studied the presentation of soluble Ag by responder APC in MLR. The results showed that primed T cells obtained from A/J skin graft-sensitized C3H T cells specifically developed alloreactivity to A/J-soluble Ag in MLR. Addition of anti-Ia mAb blocked the proliferative response, thus confirming that primed T cells can respond to soluble Ag presented by responder-type APC. This study suggests that induction of peripheral tolerance by IT inoculation of soluble Ag is reproducible in the murine cardiac allograft model, where tolerance is dependent on the Ag dose and the indirect pathway of Ag presentation. This approach of immunologic manipulation of the adult thymus may have clinical therapeutic applications.

Intrathymic tolerance in the Lewis-to-F344 chronic cardiac allograft rejection model.

Successful induction of donor-specific unresponsiveness by intrathymic inoculation of alloantigen in several experimental acute rejection models has led us to hypothesize that similar immune manipulations can prevent chronic rejection and development of graft arteriosclerosis in the Lewis-to-F344 rat chronic cardiac allograft rejection model. Recipient F344 rats were treated with donor (Lewis) splenocytes by intrathymic injection (i.t.) alone (10 x 10(6) cells/lobe); with donor splenocytes i.t. plus a one-time dose of ALS (1 mg) by intraperitoneal injection (i.p.); or with ALS i.p. (1 mg) alone 2 and 6 weeks prior to heterotopic Lewis heart transplantation. Control F344 recipients received saline i.t. Allografts were monitored by daily palpation, and long-term surviving grafts were harvested on day 90 for histopathologic analysis. Control allografts had 28.6% long-term survival (> 90 days) with mean graft survival of 46.7 +/- 12.2 days. At day 90 the surviving control allografts were enlarged and fibrotic with barely palpable heartbeat (mean heartbeat grade 0.29 +/- 0.18), and histologically showed diffuse moderate mononuclear cell infiltrates and advanced graft arteriosclerosis (mean vessel score 3.57 +/- 0.10 and 89 +/- 1% vessels diseased). Recipient treatment with intrathymic donor splenocytes alone significantly prolonged graft survival (89% long-term survival; mean 83.8 +/- 6.2 days, P < 0.04), but did not significantly inhibit the development of graft arteriosclerosis (score 2.98 +/- 0.53 and 79 +/- 8% diseased, P = NS). By contrast, treatment with i.t. donor splenocytes plus ALS 2 weeks prior to transplantation prolonged graft survival (100% long-term; mean 90.0 +/- 0.0 days, P < 0.04), and markedly inhibited graft arteriosclerosis (score 0.80 +/- 0.14, P < 0.05; 27 +/- 4% diseased, P < 0.05). ALS alone given two weeks prior to transplantation also prolonged graft survival (100% long-term; mean 90.0 +/- 0.0 days, P < 0.04), and inhibited graft arteriosclerosis (score 0.89 +/- 0.31, P < 0.05; 25 +/- 7% diseased, P < 0.05). However, when ALS was given 6 weeks prior to heart transplantation the beneficial effect of ALS alone was abolished, suggesting that lymphocyte depletion may have been responsible for the observed effects when ALS was administered at 2 weeks. Interestingly, intrathymic donor splenocytes plus ALS 6 weeks prior to transplantation, on the other hand, showed significant prolongation of allograft survival (100% long-term, mean 90.0 +/- 0.0 days, P < 0.04), and inhibited graft arteriosclerosis (score 0.41 +/- 0.02, P < 0.05; 16 +/- 2% diseased, P < 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)

Failure of intrathymic inoculation of donor-specific splenocytes to prolong cardiac or renal allograft survival in dogs

The intrathymic inoculation (ITI) of donor splenocytes into potential organ transplant recipients has been demonstrated to result in donor-specific unresponsiveness and greatly prolonged survival of subsequent organ allografts in rodents without the need for long-term pharmacological immunosuppressive therapy. We have studied the effect of the ITI of saline (controls) (groups 1 ( n = 6) and 3 ( n = 6)) or donor splenocytes (groups 2 ( n = 10) and 4 ( n = 8)) in dogs that received either pharmacological immunosuppression (with cyclosporine and prednisone, ±azathioprine/cyclophosphamide) (groups 1 and 2) or rabbit anti-dog antithymocyte globulin (groups 3 and 4) at the time of ITI. Kidney or heart allografting (from the donor of the splenocytes) was carried out 16–74 days after ITI; all but four transplants were performed within 16–22 days after ITI. Mean kidney allograft survival was 6, 10, 9, and 9 days, respectively, in groups 1–4. Mean cardiac allograft survival was 7, 14, 8, and 7 days, respectively. There was no statistical difference in allograft survival between those dogs that received ITI of saline and those that received donor splenocytes. These results would suggest that the protocols developed to date using ITI in rodent species may not be successful in dogs.

Effects of FK 506 on chimerism and the induction of donor-specific unresponsiveness following fully allogeneic bone marrow transplantation in mice

Effects of FK 506 on chimerism and the induction of donor-specific unresponsiveness following fully allogeneic bone marrow transplantation in mice

TOLERANCE INDUCTION IN A FULLY ALLOGENEIC COMBINATION USING ANTI-T CELL RECEPTOR-αβ MONOCLONAL ANTIBODY, LOW DOSE IRRADIATION, AND DONOR BONE MARROW TRANSFUSION

In a murine strain combination disparate in both H-2 antigens and minor histocompatibility antigens consisting of C57BL/6 (B6; H-2b, Mls-1b) mice as recipients and AKR/J (AKR; H-2b, Mls-1a) mice as donors, we reported that the administration of anti-TCR-αβ mAb nonspecifically suppresses the ability to reject allografts. However, such an effect was only temporary and all grafts were eventually rejected within 40 days in the anti-TCR-αβ mAb-treated mice. In this study, to induce donor-specific tolerance, the transfer of donor bone marrow cells was added to the administration of anti-TCR-αβ mAb but donor bone marrow cells were rejected and failed to cause donor-specific unresponsiveness. After donor bone marrow cell transfer in the anti-TCR-αβ mAb-treated mice, the B cells of the recipients were observed along with the production of antidonor antibody. To abolish the residual lymphocyte populations, low dose irradiation was also added. A long-lasting skin allograft tolerance can be achieved by the tolerance system, in which low dose irradiation was added to the combined treatment with anti-TCR-αβ mAb and transfer of donor bone marrow cells. Such a protocol also established central and peripheral chimerism, which suggests that hematopoietic chimerism is necessary to maintain the tolerance. B cells were completely abolished in recipients given this combined treatment and their antibody production against donor antigens after the transfer of donor bone marrow cells was also completely suppressed. A possible role of B cells in the rejection of donor bone marrow cells before the establishment of chimerism is discussed.

TOLERANCE INDUCTION IN A FULLY ALLOGENEIC COMBINATION USING ANTI-T CELL RECEPTOR-αβ MONOCLONAL ANTIBODY, LOW DOSE IRRADIATION, AND DONOR BONE MARROW TRANSFUSION

In a murine strain combination disparate in both H-2 antigens and minor histocompatibility antigens consisting of C57BL/6 (B6; H-2b, Mls-1b) mice as recipients and AKR/J (AKR; H-2k, Mls-1a) mice as donors, we reported that the administration of anti-TCR-alpha beta mAb nonspecifically suppresses the ability to reject allografts. However, such an effect was only temporary and all grafts were eventually rejected within 40 days in the anti-TCR-alpha beta mAb-treated mice. In this study, to induce donor-specific tolerance, the transfer of donor bone marrow cells was added to the administration of anti-TCR-alpha beta mAb but donor bone marrow cells were rejected and failed to cause donor-specific unresponsiveness. After donor bone marrow cell transfer in the anti-TCR-alpha beta mAb-treated mice, the B cells of the recipients were observed along with the production of antidonor antibody. To abolish the residual lymphocyte populations, low dose irradiation was also added. A long-lasting skin allograft tolerance can be achieved by the tolerance system, in which low dose irradiation was added to the combined treatment with anti-TCR-alpha beta mAb and transfer of donor bone marrow cells. Such a protocol also established central and peripheral chimerism, which suggests that hematopoietic chimerism is necessary to maintain the tolerance. B cells were completely abolished in recipients given this combined treatment and their antibody production against donor antigens after the transfer of donor bone marrow cells was also completely suppressed. A possible role of B cells in the rejection of donor bone marrow cells before the establishment of chimerism is discussed.

Inhibition of IL-2 synthesis by donor-specific suppressor T cells in a renal transplant recipient.

A study was conducted to elucidate the mechanism of donor-specific Mixed Lymphocyte Reaction (MLR and Cell Mediated Lymphotoxicity (CML) unresponsiveness in a renal transplant recipient with a long-term well-functioning kidney. The peripheral blood lymphocytes (PBL) of the recipient, who had not shown rejection since his transplantation 5 years previously, and those of his mother (donor), his father and two healthy third parties were examined. MLR, CML, semimicro MLR in a double chamber, interleukin-2 (IL-2) synthesis assay and limiting dilution assay were performed. This recipient showed donor-specific MLR and CML unresponsiveness. IL-2 assay showed that the PBL of the recipient produced less IL-2 against the donor than against the father and the third parties. The addition of exogenous recombinant IL-2 (rIL-2; Takeda Co.) to the priming MLR caused a recovery of CML against the donor. A limiting dilution assay indicated that cytotoxic T cell precursor (CTLp) frequencies against the donor and father did not differ. The suppressor assay in a double chamber indicated that the PBL of the recipient stimulated by the donor PBL had a non-specific suppressive effect on MLR, CML and IL-2 synthesis of the PBL across the Major Histocompatibility Complex (MHC) barrier. This suppressive effect was abolished by OKT3 or OKT8 monoclonal antibody and complement. Thus, the recipient had donor-specific suppressor T cells that produced a humoral non-specific suppressive factor only when stimulated by the donor PBL, and this factor suppressed MLR and CML by inhibiting IL-2 synthesis of the PBL.

Thromboxane A2 receptor blocking abrogates donor-specific unresponsiveness to renal allografts induced by thymic recognition of major histocompatibility allopeptides.

Recent in vitro studies have documented that thromboxane (Tx)A2 induces thymocyte apoptosis by acting on specific receptors abundantly expressed on the surface of immature T lymphocytes. No information is available on the in vivo relevance of this observation in development of self- or acquired tolerance. We and others have previously documented that injection of donor cells into adult thymus of experimental animals induced specific systemic unresponsiveness to allografts in the rat and mouse models. More recently, we have shown that intrathymic injection of synthetic class II major histocompatibility complex (MHC) allopeptides resulted in donor-specific unresponsiveness to renal allografts. The induction of unresponsiveness was abrogated by recipient thymectomy within the first week. We now report the effect of TxA2 blockade on acquired thymic tolerance to renal allografts induced by intrathymic injection of synthetic class II MHC allopeptides in the Wistar-Furth (WF) to Lewis rat strain combination. Administration of the TxA2 receptor blocker prior to transplantation or 2 wk postengraftment completely abrogated the unresponsive state. In addition, inhibiting the TxA2-forming enzyme by aspirin or dexamethasone also abolished the induction of acquired thymic tolerance. Evidence is also provided for a critical "dose" of peptides to be injected into the thymus to induce systemic unresponsiveness to renal allografts. These data, coupled with observations that activated peripheral T cells can circulate through the thymus, provide evidence that TxA2/TxA2 receptor interaction in the thymic microenvironment, leading to anergy/programmed cell death of activated T cells, may play an important role in the development of acquired unresponsiveness in vivo.

EFFECTIVENESS OF INTRATHYMIC INOCULATION OF SOLUBLE ANTIGENS IN THE INDUCTION OF SPECIFIC UNRESPONSIVENESS TO RAT ISLET ALLOGRAFTS WITHOUT TRANSIENT RECIPIENT IMMUNOSUPPRESSION

Our finding that intrathymic inoculation of resting T cells but not dendritic cells induces donor-specific unresponsiveness to organ allografts led us to hypothesize that presentation of MHC class I alloantigens by thymic antigen-presenting cells to T cell precursors during their ontogeny may convey a tolerogenic signal to the recipient. In this study, we examined whether intrathymic inoculation of soluble antigen obtained from 3M KCl extracts of allogeneic T cells could induce donor-specific unresponsiveness to islet allografts in the Lewis-to-WF rat combination. Our results showed that while intrathymic injection of 0.5 mg soluble antigen on day −7 relative to islet transplantation caused acute graft rejection, intrathymic inoculation of 1.0 mg soluble antigen significantly prolonged the survival of Lewis islet allografts from 10.3±1.1 days in controls to 53.5±15.6 days (P< 0.001) in naive (nonimmunosuppressed) STZ (streptozotocin)-induced diabetic WF recipients. In contrast, intrathymic inoculation of 2.0 or 4.0 mg soluble Ag on day −7 led to indefinite Lewis islet survival (>150 days) in all naive diabetic WF recipients; a finding that suggests that 2.0 mg soluble Ag is the optimal effective dose of intrathymic inoculum required to induce donor-specific unresponsiveness in naive recipients in this model. This finding could not be reproduced by intravenous injection of 2.0 mg soluble Ag, thus confirming the privileged position of the thy- mus in the induction of Ag-specific unresponsiveness. Third-party (BN) islet allografts were rejected in an acute fashion in similarly prepared recipients. Our results suggest that (1) intrathymic inoculation of soluble Ag, unlike cellular Ag, induces donor-specific unresponsiveness to islet allografts without the use of transient recipient immunosuppression; (2) induction of specific unresponsiveness appears to be dose dependent; and (3) the tolerogenic effect of soluble Ag is dependent on the indirect pathway of Ag-presentation in the thymus in the absence of donor antigen-presenting cells in the inoculum. This novel approach.

EFFECTIVENESS OF INTRATHYMIC INOCULATION OF SOLUBLE ANTIGENS IN THE INDUCTION OF SPECIFIC UNRESPONSIVENESS TO RAT ISLET ALLOGRAFTS WITHOUT TRANSIENT RECIPIENT IMMUNOSUPPRESSION

Our finding that intrathymic inoculation of resting T cells but not dendritic cells induces donor-specific unresponsiveness to organ allografts led us to hypothesize that presentation of MHC class I alloantigens by thymic antigen-presenting cells to T cell precursors during their ontogeny may convey a tolerogenic signal to the recipient. In this study, we examined whether intrathymic inoculation of soluble antigen obtained from 3M KCl extracts of allogeneic T cells could induce donor-specific unresponsiveness to islet allografts in the Lewis-to-WF rat combination. Our results showed that while intrathymic injection of 0.5 mg soluble antigen on day -7 relative to islet transplantation caused acute graft rejection, intrathymic inoculation of 1.0 mg soluble antigen significantly prolonged the survival of Lewis islet allografts from 10.3 +/- 1.1 days in controls to 53.5 +/- 15.6 days (P < 0.001) in naive (nonimmunosuppressed) STZ (streptozotocin)-induced diabetic WF recipients. In contrast, intrathymic inoculation of 2.0 or 4.0 mg soluble Ag on day -7 led to indefinite Lewis islet survival (> 150 days) in all naive diabetic WF recipients; a finding that suggests that 2.0 mg soluble Ag is the optimal effective dose of intrathymic inoculum required to induce donor-specific unresponsiveness in naive recipients in this model. This finding could not be reproduced by intravenous injection of 2.0 mg soluble Ag, thus confirming the privileged position of the thymus in the induction of Ag-specific unresponsiveness. Third-party (BN) islet allografts were rejected in an acute fashion in similarly prepared recipients. Our results suggest that (1) intrathymic inoculation of soluble Ag, unlike cellular Ag, induces donor-specific unresponsiveness to islet allografts without the use of transient recipient immunosuppression; (2) induction of specific unresponsiveness appears to be dose dependent; and (3) the tolerogenic effect of soluble Ag is dependent on the indirect pathway of Ag-presentation in the thymus in the absence of donor antigen-presenting cells in the inoculum. This novel approach of thymic reeducation of adult animals by the deliberate intrathymic inoculation of soluble major histocompatibility complex Ag without the use of recipient immunosuppression may have therapeutic potential in the induction of transplantation tolerance.

A short course of FK506 can induce limited donor-specific graft acceptance

To examine the hypothesis that a short course of FK506 would induce permanent graft acceptance after lung transplantation, left lung allotransplantation was performed in 14 mongrel dogs. In group 1 (control; n = 3), no immunosuppressive agent was given. In group 2 (n = 7), FK506 (1.2 mg/kg intramuscularly) was given on posttransplantation days 0, 1, and 2. In group 3 (n = 4), FK506 was given at the same dose on posttransplantation days 0, 1, and 2 as well as on days 29 and 30. Allograft function was evaluated by temporarily occluding the right pulmonary artery. A mixed lymphocyte reaction study was performed preoperatively and monthly thereafter. Control lungs were all rejected within 8 days. Group 2 dogs showed improved survival, with a median survival of 49.5 days. One dog in group 2 lived more than 400 days after transplantation. The mixed lymphocyte reaction data suggests that some donor-specific unresponsiveness occurs, which lasts for only a limited time. Supplemental doses of FK506 did not significantly improve survival (median, 74 days). The whole blood level of FK506 was 17.7 ± 3.98 ng/mL on day 15; however, on day 29 the FK506 level was almost undetectable. We conclude that a 3-day course of 1.2 mg/kg of FK506 can induce donorspecific graft acceptance, but this acceptance is not permanent.

MECHANISMS OF ACQUIRED THYMIC UNRESPONSIVENESS TO RENAL ALLOGRAFTS

We have recently shown that a single intrathymic injection of synthetic 25mer peptides, representing full sequences of the hypervariable domain of RT1.Buβ (4 peptides) and RT1.Duβ (4 peptides) WF class II MHC molecules, 48 hr before transplantation induces donor-specific unresponsiveness to WF rat renal al-lografts in adult LEW recipients. The induction of unresponsiveness was abrogated by the recipient's thymectomy within the first week after intrathymic injection. Peripheral T cells of long-term survivors exhibited antigen-specific hyporesponsiveness in the LEWxWF MLR. Studies on the mechanisms of induction of acquired thymic unresponsiveness to alloantigen in vivo and in vitro are now reported. First, since we have previously demonstrated in LEW responders that only 4 of the 8 synthetic 25mer peptides, 2 RT1.Duβ and 2 RT1.Buβ sequences, were immunogenic in vitro and in vivo, we compared the tolerogenicity of the immunogenic versus the nonimmunogenic peptides. While LEW rats intrathymically injected with the nonimmunogenic peptides acutely rejected their renal allografts within 6–10 days, animals injected with the immunogenic peptides did not reject their grafts and are surviving >100 days with normal allograft function. In vitro studies established that peripheral T cells from intrathymically tolerized animals exhibited antigen-specific hyporesponsiveness in the LEWxWF MLR starting as early as 1 week posttransplant. Immunohistological evaluation of renal allografts from intrathymically tolerized animals 1 week postengraftment showed marked reduction in mononuclear cell infiltrates with no evidence of tubulitis, and marked reduction in intragraft staining for activation and inflammatory cytokines and alloantibodies, as compared with acutely rejecting controls. Systemic administration of 1000 U of rIL-2 daily for 5 days starting on the day of transplantation abrogated the tolerogenic effect of intrathymic MHC allopeptides. Injection of 100 μUg of a single immunogenic peptide, RT1.Duβ2 (residues 20–44), into the thymus of responder LEW rats 48 hrs before immunization with RT1.Duβ2 effected significant reduction of in vitro proliferation of primed lymphocytes to RT1.Duβ2, an effect that was abrogated by addition of rIL-2 in vitro. In contrast, thymectomy beyond 2 weeks and administration of rIL-2 at 4–6 weeks after transplantation failed to cause rejection. These observations indicate that thymic recognition of immunodominant class II MHC allopeptides leads to peripheral T cell anergy that mediates the induction phase of systemic unresponsiveness to renal allografts. The maintenance phase appears to be mediated by dense anergy or clonal deletion. These findings also confirm the role of indirect T cell recognition of alloantigen in vascularized allograft rejection, since functional inactivation/ deletion of T cells in this pathway is sufficient to prevent rejection.

MECHANISMS OF ACQUIRED THYMIC UNRESPONSIVENESS TO RENAL ALLOGRAFTS

We have recently shown that a single intrathymic injection of synthetic 25mer peptides, representing full sequences of the hypervariable domain of RT1.BuB (4 peptides) and RT1.Du beta (4 peptides) WF class II MHC molecules, 48 hr before transplantation induces donor-specific unresponsiveness to WF rat renal allografts in adult LEW recipients. The induction of unresponsiveness was abrogated by the recipient's thymectomy within the first week after intrathymic injection. Peripheral T cells of long-term survivors exhibited antigen-specific hyporesponsiveness in the LEW x WF MLR. Studies on the mechanisms of induction of acquired thymic unresponsiveness to alloantigen in vivo and in vitro are now reported. First, since we have previously demonstrated in LEW responders that only 4 of the 8 synthetic 25mer peptides, 2 RT1.Du beta and 2 RT1.Bu beta sequences, were immunogenic in vitro and in vivo, we compared the tolerogenicity of the immunogenic versus the nonimmunogenic peptides. While LEW rats intrathymically injected with the nonimmunogenic peptides acutely rejected their renal allografts within 6-10 days, animals injected with the immunogenic peptides did not reject their grafts and are surviving > 100 days with normal allograft function. In vitro studies established that peripheral T cells from intrathymically tolerized animals exhibited antigen-specific hyporesponsiveness in the LEW x WF MLR starting as early as 1 week posttransplant. Immunohistological evaluation of renal allografts from intrathymically tolerized animals 1 week postengraftment showed marked reduction in mononuclear cell infiltrates with no evidence of tubulitis, and marked reduction in intragraft staining for activation and inflammatory cytokines and alloantibodies, as compared with acutely rejecting controls. Systemic administration of 1000 U of rIL-2 daily for 5 days starting on the day of transplantation abrogated the tolerogenic effect of intrathymic MHC allopeptides. Injection of 100 micrograms of a single immunogenic peptide, RT1.Du beta 2 (residues 20-44), into the thymus of responder LEW rats 48 hrs before immunization with RT1.Du beta 2 effected significant reduction of in vitro proliferation of primed lymphocytes to RT1.Du beta 2, an effect that was abrogated by addition of rIL-2 in vitro. In contrast, thymectomy beyond 2 weeks and administration of rIL-2 at 4-6 weeks after transplantation failed to cause rejection. These observations indicate that thymic recognition of immunodominant class II MHC allopeptides leads to peripheral T cell anergy that mediates the induction phase of systemic unresponsiveness to renal allografts. The maintenance phase appears to be mediated by dense anergy or clonal deletion.(ABSTRACT TRUNCATED AT 400 WORDS)

INDUCTION OF SPECIFIC TOLERANCE BY INTRATHYMIC INJECTION OF RECIPIENT MUSCLE CELLS TRANSFECTED WITH DONOR CLASS I MAJOR HISTOCOMPATIBILITY COMPLEX

Induction of tolerance to allogeneic MHC antigens has been a goal in the field of transplantation because it would reduce or eliminate the need for generalized immunosuppression. Although encouraging results have been obtained in experimental models by exposing recipient thymus to donor cells before transplantation, donor cells are not typically available at that time, and the donor antigens responsible for the effect are poorly defined. In the present study, thymic tolerance was demonstrated without using donor cells. Recipient thymus was injected before transplantation with autologous myoblasts and myotubes that were genetically modified to express allogeneic donor-type MHC class I antigen. Donor-specific unresponsiveness was induced to a completely MHC-disparate liver transplant and to a subsequent donor-type cardiac allograft, but not a third-party allograft. In vitro, recipient CTL demonstrated a 10-fold reduction in killing of donor cells, but not of third-party cells. Our results demonstrate: (1) that recipient muscle cells can be genetically engineered to induce donor-specific unresponsiveness when given intrathymically, and (2) transfected recipient cells expressing only donor MHC class I antigen can induce tolerance to a fully allogeneic donor.

Successful Engraftment of Autologous and Allogeneic Islets into the Porcine Thymus

Work in rodents has shown that injection of pancreatic islets of Langerhans into the thymus can induce donor-specific unresponsiveness to islets subsequently transplanted to extrathymic sites. The overall objective of our investigation is to test this in a large animal (pig) model. In the current study we examined whether autologous and allogeneic adult or fetal islets survive in the porcine thymus. Collagenase-digested adult and fetal porcine islets were injected into the thymic lobes of 5- to 13-month-old, normal pigs. Pigs receiving allografts were given either a standard triple regimen of oral cyclosporine, azathioprine, and prednisone or intravenous anti-lymphocyte globulin (ALG) for immunosuppression. Two pigs of each group that received an allograft or autograft were given no immunosuppression. Biopsies of the grafts were taken at 4, 6, or 12 weeks for examination. Islet survival was assessed by histology with hematoxylin and eosin and insulin staining and by measurement of tissue insulin content using acid alcohol extraction and radioimmunoassay. The insulin content of control thymus was found to be 0.71 ± 0.29 μU/mg ( n = 5). The insulin content of the islet-grafted thymic tissues ranged from 1.57 to 10.65 μU/mg. Since the amounts of injected islets were not equal and not distributed evenly, and only a part of the graft site was used for determination of tissue insulin, thymic insulin contents higher than 1.4 μU/mg (twice that of the control value) were considered to demonstrate the presence of viable islets. With this criterion, we concluded that islets were found to be viable in seven of nine pigs and two pigs which were treated with ALG were shown to be marginally positive. On histological examination, islets were found in the thymus as well as under the thymic capsule, except for the above two pigs. These results demonstrate that the pig thymus supports fetal and adult islet transplants.

Failure of nonimmunogenic islet allografts to induce donor-specific immunological unresponsiveness [corrected

Highly purified perinatal rat islets, isolated by a nonenzymic in vitro culture technique, have been successfully transplanted across complete MHC barriers without immunosuppression. Acceptance of these allogeneically transplanted islets is hypothesized to result from an absence of antigen presenting cells (APCs) within the islets. This study was designed to examine the effects of organ transplantation and cyclosporine (CsA) therapy on the development of immunological unresponsiveness in recipients receiving a graft of culture-isolated islets. Kidneys were successfully allotransplanted into unilaterally nephrectomized rats, across a complete MHC barrier (Rt1lv1 to Rt1u) using CsA therapy initiated on the day of transplantation (7.5 mg/kg, orally for 14 days). Remaining native kidneys were removed 14 days following renal allotransplantation. Limited mononuclear cell (MNC) infiltrates were observed in biopsies of renal allografts, taken 30 days posttransplant, but failure of the renal allograft was not observed. Animals bearing established renal allografts (n = 10) received allografts of approximately 200 highly purified perinatal islets (ACl, n = 5; F-344, n = 5), transplanted to the kidney subcapsule of the established renal allograft at least 30 days following renal allotransplantation (at least 16 days following termination of CsA). Islet allografts were not rejected, and, as expected, did not initiate rejection of the renal allograft. Similar results were observed in renal allograft recipients rendered diabetic by a single injection of streptozotocin (STZ, 65 mg/kg, n = 5) and receiving islet allografts of sufficient mass (approximately 1200-1400 islets) to reverse STZ-induced hyperglycemia. Further, neither islet nor renal allografts were rejected following challenge by 1 x 10(7) donor-strain dendritic cells (DCs).(ABSTRACT TRUNCATED AT 250 WORDS)

The Relative Contribution of Intrathyrmic Inoculation of Donor Leukocyte Subpopulations in the Induction of Specific Tolerance

We have recently demonstrated that intrathymic (IT) inoculation of ultraviolet-B irradiation-modified donor spleen cells induces specific tolerance to organ allografts in transiently immunosuppressed adult rats. This study examines the splenic leukocyte component that plays a role in the induction of organ-specific tolerance when injected into the thymus of an adult animal. Our results showed that in the low responder combination of Lewis-to-ACI, IT inoculation of resting Lewis T-cells consistently induced specific unresponsiveness to cardiac allografts in sublethally irradiated (200 rad total body irradiation) ACI recipients while the same was true in the high responder rat combination of WF-to-Lewis, where recipient rats were transiently immunomodulated with antilymphocyte serum. In contrast, IT inoculation of donor B cells, macrophages, or dendritic cells in similarly prepared animals failed to prolong graft survival in both the low responder (Lewis-to ACI) and the high responder (WF-to-Lewis) rat strain combinations. This observation suggests that donor-specific unresponsiveness can be readily achieved by IT inoculation of major histocompatability (MHC) class I expressing donor-type resting T-cells rather than by any donor-type antigen presenting cells that express MHC class I and II molecules. Extrathymic inoculation of T-cells in transiently immunosuppressed recipients failed to prevent graft rejection, thus demonstrating the privileged position of the thymus in the induction of tolerance. The unresponsive recipients of cardiac allografts specifically and permanently accepted donor-type second-set islet allografts, thus confirming antigen-specific tolerance in this model. These results suggest that manipulation of the immune system through thymic reeducation of the developing T-cells by the deliberate introduction of foreign MHC class I cellular alloantigens has therapeutic potential in the induction of transplantation tolerance in adult animals.

Induction of donor specific unresponsiveness in rat islet allografts by intraportal grafting and FK506.

Induction of donor specific unresponsiveness in rat islet allografts by intraportal grafting and FK506.