Intrathymic Research Articles

REGULATORY T CELLS MAINTAIN PERIPHERAL TOLERANCE TO ISLET ALLOGRAFTS INDUCED BY INTRATHYMIC INJECTION OF MHC CLASS I ALLOPEPTIDES

123 While transplantation remains the only potentially useful treatment for end-stage organ failure, immunological rejection of allografts continues to be a major problem. We have investigated if the fate of T cell precursors developing in the thymus may be determined by the presence of allo MHC class I peptides, since T cell receptor-MHC class I / self peptide interaction regulates thymocyte development. This study examined the effects and underlying mechanisms of intrathymic (IT) injection of a short segment of a synthetic immunogenic MHC class I peptide [P-2; Residues 67 - 85] of the hypervariable domain of RT1.A derived from WAG rat [RT1u] on islet graft survival in the WF [RT1u]-to-ACI [RT1a]. The results showed that while IT injection of 150 µg P-2 on day -7 did not prolong graft survival in naive recipients [MST of 14.0 days vs 9.6 days in controls], IT injection of 300 µg or 600 µg P-2 led to normoglycemia and permanent islet survival [>200 days] in 4/6 and 3/5 STZ induced diabetic ACI recipients, respectively. IT injection of 150 µg, 300 µg, or 600 µg P-2 combined with 0.5 ml ALS immunosuppression on day -7 led to 100% permanent islet graft survival [>200 days] compared to MST of 15.0 ± 2.3 days in ALS alone treated controls. Similarly prepared animals rejected 3rd party [BN] islets in an acute fashion, thus demonstrating donor specificity. Intravenous injection of 300 µg P-2 combined with 0.5 ml ALS did not prolong islet allografts. The long-term unresponsive islet allograft recipients challenged with 2nd set grafts accepted permanently 4/4 donor-type cardiac allografts while rejecting 3rd party [Lewis] hearts without rejecting the primary [WF] islets. In analyzing the underlying mechanisms of acquired systemic tolerance, we found no suppresser / regulatory cells in the adoptive transfer studies in tolerant animals at 30 days after IT injection of allopeptides. In contrast, adoptive transfer of 5 × 107 unseparated spleen cells or 2 × 107 purified T-cells obtained from tolerant animals 60 and 120 days after islet transplantation into lightly irradiated [200 rad TBI] ACI led to donor-specific permanent islet graft survival in 75 and 100% animals respectively compared to an MST of 14.5 days in lightly irradiated ACI recipients of unmodified syngenic spleen cells. Our findings suggest that maintenance of acquired thymic tolerance induced by class I MHC allopeptides is dependent on generation of regulatory cells and that this strategy may have potential clinical application in the induction of acquired tolerance.

Intrathymic administration of B cells induces prolonged survival of fully allogeneic cardiac grafts without prolonged deletion of donor-specific thymocytes

Intrathymic (IT) injection of alloantigen has been shown to induce unresponsiveness to allografts although the exact mechanisms of tolerance induction remains unclear, C57BL/10 (H2 b) cardiac allografts were accepted in C3H/He (H2 k) mice pretreated with IT inoculation of donor splenocytes (1 × 10 6) in combination with a depleting anti-CD4 monoclonal antibody 27 days before cardiac transplantation. To investigate which cell types were responsible for tolerance induction by IT injection of alloantigen, resting B (rB) cells or dendritic cells were used as the thymic inoculum instead of whole splenocytes. IT injection of rB cells induced indefinite graft prolongation in all recipients while only 20%of mice that had received IT injection of dendritic cells accepted grafts for over 100 days. In contrast, IT injection of dendritic cells resulted in significant deletion of donor-specific thymocytes whereas rB cells were relatively ineffective. IT deletion is not essential for the induction of tolerance by IT injection of rB cells; nondeletional mechanisms can be involved.

INDUCTION OF DONOR SPECIFIC ACQUIRED THYMIC TOLERANCE BY INTRATHYMIC INOCULATION OF A SINGLE SYNTHETIC ALLO-MHC CLASS I PEPTIDE[RT1.Au

499 This study extends the finding that intrathymic (IT) injection of closely related WAG (RT1u) class I peptides induces transplant tolerance to WF (RT1u) to the use of well defined, strain specific WF MHC class I peptides [RT1.Au]. It is reasoned that the effectiveness of TCR-MHC/peptide interaction which controls T cell ontogeny may be influenced by the presence of allopeptides in the thymus. Seven peptides were synthesized from RT1.Au sequences as published by Walter et al(1995) and Joly et al (1995), respectively. Following in-vivo immunization, we determined that while two peptides [U-5 & U-7] were immunogenic, 5 others [U-1, U-2, U-3, U-4, & U-6] were not immunogenic in ACI recipients. We then examined the effects of IT injection of a combination or single doses of the immunogenic RT1.Au peptides on cardiac allograft survival in the WF-to-ACI rat combination. The results showed that a combination of equal amounts [150 µg or 300 µg each] of the two peptides combined with 0.5 ml ALS on day -7 relative to cardiac transplantation led to 60 & 100% permanent WF graft survival (>100 days) respectively in ACI recipients. We then studied the effects of the individual peptides on graft survival. While 150 µg U-5 injected IT on day -7 did not prolong graft survival, 300µg U-5 significantly prolonged graft survival to MST of 17.3 ± 3.6 days from 9.8 days in naive recipients. In contrast, IT injection of 150, 300, or 600 µg U-7 with or without ALS on day -7 did not affect graft rejection. On the other hand, IT injection of 150, 300, and 600 µg U-5 combined with 0.5 ml ALS on day -7 led to permanent graft survival (> 100 days) in 4/6, 6/6, and 4/4 ACI recipients, respectively, compared to an MST of 15.4 days in ALS alone treated controls; thus demonstrating that peptide U-5 alone mediates the observed effects on graft prolongation. Similar treatment with 300 µg U-5 combined with ALS led to acute rejection of third party(Lewis) grafts. Intravenous injection of 300 µg U-5 with ALS also did not prolong WF graft survival in ACI recipients. The long-term unresponsive ACI recipients challenged with 2nd-set allografts accepted permanently donor-type (WF) but not 3rd party (Lewis) cardiac allografts. In conclusion, we have demonstrated that a short segment of RT1.Au represents the tolerogenic epitope that induces acquired systemic tolerance. These studies have relevant clinical implications since only a single peptide can be potentially used in future large animal and human studies.

INDUCTION OF DONOR SPECIFIC ACQUIRED THYMIC TOLERANCE BY INTRATHYMIC INOCULATION OF A SINGLE SYNTHETIC ALLO-MHC CLASS I PEPTIDE [RT1.Au

499 This study extends the finding that intrathymic (IT) injection of closely related WAG (RT1u) class I peptides induces transplant tolerance to WF(RT1u) to the use of well defined, strain specific WF MHC class I peptides [RT1.Au]. It is reasoned that the effectiveness of TCR-MHC / peptide interaction which controls T cell ontogeny may be influenced by the presence of allopeptides in the thymus. Seven peptides were synthesized from RT1.Au sequences as published by Walter et al (1995) and Joly et al(1995), respectively. Following in-vivo immunization, we determined that while two peptides [U-5 & U-7] were immunogenic, 5 others [U-1, U-2, U-3, U-4,& U-6] were not immunogenic in ACI recipients. We then examined the effects of IT injection of a combination or single doses of the immunogenic RT1.Au peptides on cardiac allograft survival in the WF-to-ACI rat combination. The results showed that a combination of equal amounts [150 μg or 300 μg each] of the two peptides combined with 0.5 ml ALS on day -7 relative to cardiac transplantation led to 60 & 100% permanent WF graft survival (>100 days) respectively in ACI recipients. We then studied the effects of the individual peptides on graft survival. While 150 μg U-5 injected IT on day -7 did not prolong graft survival, 300 μg U-5 significantly prolonged graft survival to MST of 17.3 ± 3.6 days from 9.8 days in naive recipients. In contrast, IT injection of 150, 300, or 600μg U-7 with or without ALS on day -7 did not affect graft rejection. On the other hand, IT injection of 150, 300, and 600 μg U-5 combined with 0.5 ml ALS on day -7 led to permanent graft survival (>100 days) in 4/6, 6/6, and 4/4 ACI recipients, respectively, compared to an MST of 15.4 days in ALS alone treated controls; thus demonstrating that peptide U-5 alone mediates the observed effects on graft prolongation. Similar treatment with 300 μg U-5 combined with ALS led to acute rejection of third party (Lewis) grafts. Intravenous injection of 300 μg U-5 with ALS also did not prolong WF graft survival in ACI recipients. The long-term unresponsive ACI recipients challenged with 2nd-set allografts accepted permanently donor-type (WF) but not 3rd party (Lewis) cardiac allografts. In conclusion, we have demonstrated that a short segment of RT1.Au represents the tolerogenic epitope that induces acquired systemic tolerance. These studies have relevant clinical implications since only a single peptide can be potentially used in future large animal and human studies.

Tolerance induction by intrathymic inoculation prevents chronic renal allograft rejection.

These experiments investigated the ability of the donor-specific unresponsiveness created by the intrathymic inoculation of donor alloantigen to effectively prevent chronic rejection in an established rat model of chronic renal allograft rejection. Three study groups were examined: (1) Allograft controls--F-344 rats received a Lewis renal allograft plus 10 days of low-dose cyclosporine (CsA); (2) isograft controls--F-344 rats received an F-344 renal isograft and low-dose CsA; (3) experimental group--F-344 rats received a T-cell depleted syngeneic bone marrow transplant and intrathymic injection of Lewis bone marrow. Twenty-one days after bone marrow transplant, these animals received a Lewis renal allograft. Allograft controls demonstrated severe parenchymal fibrosis; isograft controls and intrathymic (IT) animals failed to develop this lesion. Immunohistochemical analysis revealed increased CD4+ T cells infiltrating the cortex of the allograft controls. Cytokine interferon-gamma and interleukin-2 transcripts were strongly positive in allograft controls and were absent from isograft controls and IT allografts as determined by reverse transcriptase-polymerase chain reaction. Analysis of tolerant grafts by flow microfluorimetry and genomic DNA amplification could not detect chimerism to a level of < 0.1%. IT inoculation of donor alloantigen can confer long-term unresponsiveness and prevent the development of the characteristic lesions of chronic rejection.

Evidence for clonal deletion and clonal anergy after intrathymic antigen injection in a transplantation model.

Intrathymic (IT) antigen injection has been shown to induce antigen-specific systemic tolerance in the rodent. To delineate the mechanisms responsible for the induction of tolerance, we used the 2C line of T cell receptor transgenic mice. The majority of T cells in 2C mice express an antigen receptor specific for the major histocompatibility complex class I alloantigen Ld and can be identified with the clonotypic monoclonal antibody 1B2. IT injection of lymphoid cells expressing Ld was found to induce a significant prolongation in BALB/c skin allograft survival. The allograft prolongation was associated with a marked reduction in the number of developing 1B2+ thymocytes (clonal deletion), which occurred primarily at the CD4+ CD8+ stage of thymocyte development, as well as a reduction in the number of mature CD8+ 1B2+ 2C T cells in peripheral lymphoid tissue. In addition, CD8+ 1B2+ 2C T cells that survive deletion have decreased CD8 expression levels and a significantly reduced in vitro proliferative response to specific alloantigen (clonal anergy). Exogenous recombinant interleukin 2 restores the capacity of 2C T cells to respond in vitro to alloantigen. Experiments involving separation of cells by fluorescence-activated cell sorter indicate that there is a precise correlation between the reduction in CD8 expression and anergy induction. Collectively, these data indicate that IT antigen injection can induce antigen-specific systemic tolerance by both clonal deletion and clonal anergy.

DONOR-SPECIFIC TOLERANCE BY PERIOPERATIVE INTRATHYMIC INJECTION OF BONE MARROW CELLS IN THE RAT CARDIAC ALLOGRAFT MODEL

Many strategies of tolerance induction by intrathymic (IT) injection of donor alloantigens have been reported to date; however, the timing of IT injection is usually 1-3 weeks before transplantation. To apply IT injection to cadaveric organ transplantation, 1 x 10(8) fully allogeneic bone marrow cells (BMC) of Buffalo (BUF; RT1b) rats were intrathymically injected into Wistar Furth (WF; RT1u) rats at the time of BUF cardiac allografting with short-course therapy of antilymphocyte serum (ALS) and FK506 in our experimental model. Allogeneic IT injection of BUF BMC with ALS and FK506 indefinitely prolonged graft survival (mean survival time > 210 days) in all WF rats. On day 130 after grafting, tolerant WF rats accepted donor BUF skin grafts (> 120 days) but not third-party Lewis skin grafts. In control groups, syngeneic IT injection of WF BMC or intravenous injection of donor BUF BMC in combination with ALS/FK506 therapy failed to induce tolerance. In vivo testing was performed during induction (1 month) or during maintenance (6 months of tolerance. In the mixed lymphocyte reaction (MLR), spleen T cells of tolerant rats at 1 month after grafting displayed hyporesponsiveness after stimulation with donor cells. The addition of interleukin (IL)-2 to MLR culture did not restore T-cell responsiveness. Tolerant rats had a significantly decreased frequency of T cytotoxic cell precursors (fTcp) of 1:4,926, and frequency of IL-2-producing T helper cell precursors (fThp) of 1:23,925, compared with naive rats (1: 2,158 and 1:4,266, respectively). By 6 months after grafting, however, the anti-donor MLR proliferative responses of tolerant rats had been restored to the levels of naive splenic T cells. These tolerant rats displayed restoration of the (fTcp) of 1:2,842 and of the (fThp) of 1:5,630, which were comparable frequencies of naive rats. Suppressor T cells did not contribute in this model. In cardiac grafts of tolerant rats induced by IT injection, expression of both Th1 (interferon-gamma and IL-2) and Th2 (IL-4 and IL-10) cytokines was detected in the early phase; thereafter, expression was completely inhibited, except for interferon-gamma in the chronic phase. Perfect donor-specific tolerance was obtained by IT injection of donor BMC at the time of transplantation, while alloimmune responses were maintained at levels similar to those of naive rats.

Deletion of alloantigen-reactive thymocytes as a mechanism of adult tolerance induction following intrathymic antigen administration.

Direct injection of foreign antigen into the adult thymus is a potent route of antigen delivery for the induction of tolerance in vivo. In this report, we demonstrate that tolerance to C57BL/10 (H2b/BL10) alloantigens can be induced in CBA/Ca (H2k/CBA) mice by intrathymic (IT) administration of BL10 spleen leukocytes coincident with transient peripheral immunomodulation of CD4+ T cells using a depleting anti-CD4 monoclonal antibody. T cell receptor (TCR) transgenic mice (BM3.6; H2k) expressing a CD8-independent TCR specific for H2Kb were used as recipients to facilitate investigation of the mechanisms responsible for tolerance induction by allowing visualization of events in the thymus following IT injection. IT administration of 5 x 10(7) BL10 spleen leukocytes and concomitant transient peripheral T cell depletion in BM3.6 mice resulted in a substantial H2Kb-specific deletion of transgenic-TCR+ (tg-TCR) thymocytes which was dependent on the level of tg-TCR expression. IT deletion and the failure to export CD8+ T cells to the peripheral lymphoid organs correlated with the induction of tolerance to H2Kb; TCR transgenic mice that had received IT injection of BL10 splenocytes and peripheral T cell depletion accepted a H2Kb+ cardiac allograft indefinitely. Analysis of tolerant BM3.6 mice revealed that there were low numbers of CD8+ T cells in the periphery giving rise to a substantially reduced reactivity in vitro despite the fact that no donor cells or IT deletion were observed in the thymi of the majority of tolerant mice. These results demonstrate for the first time that IT injection of foreign alloantigen into an adult thymus results in the deletion of thymocytes expressing a TCR specific for the injected alloantigen and suggest that this is an important mechanism of tolerance induction following IT injection of alloantigen in vivo. Furthermore, analysis of tolerant TCR-transgenic mice suggests that IT deletion is not required for the maintenance of tolerance, and that peripheral mechanisms enforce continued hyporesponsiveness to H2Kb following transplantation.

Localization of tolerogenic epitopes in the α1 helical region of the rat class I major histocompatibility complex molecule

Induction of tolerance to organ allografts remains an elusive goal in transplant biology. Tolerance toward heart allografts was induced by preoperative intrathymic (IT) inoculation of extracted soluble histocompatibility antigens (HAg) in combination with a single dose of anti-T-cell antibodies. 1 In contrast, peritransplant intravenous (iv) or intraportal administration of HAg combined with a short course of cyclosporine (CyA) prolonged allograft survival but did not induce transplantation tolerance. 2 IV injection of peptides corresponding to amino acid (AA) residues 75–84 of the human HLA-B7 protein combined with a short course of CyA before and after grafting induced donor-specific tolerance to heart allograft in rats. 3 The present experiments examined the localization of tolerogenic epitopes in rat class I major histocompatibility complex RTI.A u and RT1.A 1 molecules.

Induction of peripheral chimerism in an allogenic model of small bowel transplantation with intrathymic inoculation of donor-derived cells

A SELECTED population of patients with intestinal failure and unable to support the iatrogenic consequences of long-term parenteral nutrition would greatly benefit from intestinal transplantation. Despite preliminary trials to establish this technique, early graft loss and subsequent infections are very common, and the clinical result has been very deceptive.’ Better than aggressive, general immunosuppression therapies, the implementation of more selective, toleranceinducing, therapies also represents a current challenge in the field of clinical transplantation. Recently and in various models related to solid organ transplants and autoimmunity, the intrathymic (IT) injection of donor-derived cells/ Ags (or autologous targets in the case of autoimmunity) has been shown efficient in the induction of Ag-specific tolerance.2-4 The rational of this manipulation is based in the attempt to negatively select the newly emerging alloreactive T-cell specificities when confronted with their targets, as it happens normally inside the thymus.” We present some preliminary findings in a new experimental system of heterotopic intestinal grafts and IT injections of donor-derived cells (splenocytes + bone marrow cells) in inbred mouse strains. The experimental groups have been also submitted to several combinations of lethal X-radiation and syngenic bone marrow (BM) transplantation and to short courses of parenteral cyclosporine A (CyA). The evolution of grafted tissues, quantification of the degree of peripheral chimerism, and modification of allospecific T-cytotoxic lymphocytes have been evaluated during 3 to 4 month periods of follow-up. Very high percentages of donor MHC class I-positive cells are present in the periphery, but not inside the thymus, of the grafted mice for at least 2 to 3 months. Surprisingly, the presence of injected cells inside the thymus is undetectable after 1 week of the IT manipulation. Also, clonal deletion of recipient thymocytes reacting against donor-derived superantigens (Mls-la) is not observed. The vast majority of chimeric cells represent (Ag-presenting) dendritic cells, likely coming from both the grafted intestine and the IT inoculum. Allospecific T-cell cytotoxic reactivities are significantly reduced in the mice receiving IT injections. This suggests

Dependence of acquired systemic tolerance to rat islet allografts induced by intrathymic soluble alloantigens on host responsiveness, MHC differences, and transient immunosuppression in the high responder recipient.

Recent studies suggest that the adult immune system can be manipulated by intrathymic (IT) inoculation of donor Ag to accept cardiac and islet allografts in the low responder rat combination of Lewis-to-WF. We have now extended this study to examine the effect of IT inoculation of soluble protein Ag obtained from 3M KCl extracts of resting T cells combined with transient ALS immunosuppression on islet allograft survival in the high responder combination of WF-to-Lewis. We first confirmed the earlier observation that IT injection of 2 mg soluble Ag on day -7 led to permanent islet graft survival (>200 days) in the Lewis-to-WF rat combination without the use of recipient immunosuppression and found this to be true in the Lewis-to-ACI rat combination. In the high responder combination of WF-to-Lewis, unmodified Lewis rats pretreated with IT inoculation of 2 mg soluble Ag acutely rejected WF and BN islet allografts. IT inoculation of donor Ag combined with 1 ml ALS transient immunosuppression on day -7 led to a modest graft prolongation [24.8+/-10.1 days as compared with 15.2+/-3.6 days in ALS only treated controls]. Intrathymic injection of soluble Ag on day -7 combined with 1 ml ALS on days -7 and 0 relative to allografting resulted in 100% permanent islet graft survival (>200 days) compared with an MST of 20.6+/-2.3 days in ALS only-treated controls. Similar treatment led to acute rejection of 3rd party (BN) grafts, thus demonstrating donor-specificity. In addition, extrathymic inoculation of donor Ag in similarly immunosuppressed animals did not result in islet graft prolongation, once again confirming the importance of the thymus in tolerance induction. To examine them for donor-specific tolerance, long-term unresponsive (>120 days) Lewis recipients of renal subcapsular islets underwent nephrectomy of the islet bearing kidneys and were challenged with intraportal donor- or third party-type islets after becoming diabetic. All the nonimmunosuppressed recipients of donor-type (WF) islets became permanently normoglycemic (>100 days) while the third-party (BN) grafts were promptly rejected, with an MST of 10.6 days. These findings confirm that acquired thymic tolerance induced by IT inoculation of soluble protein Ag in the low to moderate responder rat combinations is reproducible in the high responder combination provided that adequate peritransplant immunosuppression is used. This study suggests that acquired thymic tolerance in the rat model is dependent on host responsiveness to alloantigens, MHC differences between the donor-recipient pair, and the use of transient immunosuppression in the high responder recipient. This model may have potential clinical application in the development of strategies for specific transplantation tolerance.

Acquired systemic tolerance to rat cardiac allografts induced by intrathymic inoculation of synthetic polymorphic MHC class I allopeptides.

This study extends the finding that intrathymic (IT) injection of 3M KC1 extracts of T cells induces transplant tolerance to the use of well defined polymorphic MHC class I allopeptides derived from the hypervariable domain of RT1.Au (WF MHC class I). While three of the six synthetic RT1.Au peptides were immunogenic, three others were nonimmunogenic when tested in ACI responders. In our initial studies, we examined the effects of IT injection of a mixture of equal concentrations of the three nonimmunogenic RT1.Au peptides on WF cardiac allograft survival in ACI recipients. The results showed that a single IT injection of 100 and 300 microg class I MHC allopeptides on day -7 relative to cardiac transplant did not significantly prolong graft survival in naive ACI recipients (MST of 9.8, and 12.3 days vs. 10.5 days in controls). In contrast, 600 microg allopeptides injected IT resulted in modest prolongation of graft to an MST of 19.5 days. However, IT injection of 600 microg allopeptides combined with 0.5 ml ALS on day -7 led to permanent acceptance (>200 days) of cardiac allografts in 7/9 ACI recipients compared with survival of 24.2 days in ALS alone treated controls. In contrast, similar treatment led to acute rejection of third party (Lewis) cardiac allografts. Intravenous injection of 600 microg allopeptides combined with ALS did not result in prolonged graft survival (26.8 days). The long-term unresponsive ACI recipients (>100 days) challenged with second-set cardiac grafts accepted permanently donor-type (WF) grafts while rejecting the third party (Lewis) grafts, a finding that confirms acquired systemic tolerance. These findings confirm the role of IT injection of synthetic polymorphic allopeptides in the induction of acquired thymic tolerance and provide the rationale for testing this strategy in large animals and eventually in man.

INDUCTION OF TOLERANCE TO SKIN ALLOGRAFTS BY INTRATHYMIC INJECTION OF DONOR SPLENOCYTES

The effect of donor-recipient strain combination and supplemental rapamycin (Rapa) on tolerance induction by intrathymic (IT) injection of donor splenocytes was examined in a mouse skin allograft model. In an MHC class I-mismatched C3H/He skin to (C57BL/6 x A)F1 (B6AF1) mouse combination, IT injection of 50 x 10(6) donor splenocytes with transient immunosuppression by rabbit anti-mouse lymphocyte serum (ALS) induced significant prolongation of skin allograft survival with a median survival time (MST) of 115 days versus an MST of 24.5 days in controls given ALS alone. With an additional short course of supplemental Rapa treatment at a dose of 1.5 mg/kg i.p. every other day from day 0 to 12, all C3H/He skin allografts survived indefinitely (> 350 days) in ALS-treated, donor splenocyte intrathymically injected B6AF1 recipient mice. Tolerance was antigen-specific, since the second donor-type skin allografts were accepted while third-party skin allografts were acutely rejected in these mice bearing long-term C3H/He skin allografts. In MHC class I- and II-disparate (DBA/2 to B6AF1) and fully MHC-incompatible (AKR to B6) strain combinations, IT injection of donor splenocytes and ALS treatment failed to prolong skin allograft survival over ALS controls. When supplemental Rapa was used, long-term skin allograft acceptance was observed with an MST of 127 days for the DBA/2 to B6AF1 combination and 70 days for the AKR to B6 combination. In contrast, supplemental treatment with cyclosporine was not effective in these combinations, which suggests that supplemental Rapa may have a unique effect in augmenting IT tolerance induction. Thymectomy within 7 days after IT injection significantly shortened the allograft survival, which suggests that interaction of the host thymus and the injected donor splenocytes, which takes place early after IT injection, plays an important role in the induction of allograft tolerance in this model.

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.

TOLERANCE INDUCTION TO CARDIAC ALLOGRAFTS BY SIMULTANEOUS OR SEQUENTIAL INTRATHYMIC INOCULATION OF DISPARATE ALLOANTIGENS

Recent studies show that the adult immune system can be manipulated to accept allografts by the intrathymic (IT) inoculation of donor alloantigens (Ag). To make this model clinically applicable to cadaveric transplants, we have examined the effect of simultaneous or sequential IT injection of two disparate Ags on graft survival. In initial experiments, Wistar Furth (WF) rats received IT injections of a single or an admixture of 2 x 10(7) Lewis T cells and 2 x 10(7) BN T cells 7 days prior to cardiac transplantation. Untreated WF recipients acutely rejected single Lewis, BN, and ACI heart allografts, respectively, at times equivalent to rejection of double hearts while IT injection of single or double 2 x 10(7) Lewis and 2 x 10(7) BN T cells on day -7 also led to acute graft rejection. As expected, IT injection of donor-type resting T cells combined with 1 ml ALS recipient immunosuppression on day -7 led to donor-specific Lewis or BN permanent graft survival (> 200 days). The long-term unresponsive WF recipients challenged 100 days after cardiac transplantation with 2nd-set grafts specifically and permanently accepted 2nd-set donor cardiac allografts. IT injection of an admixture of 2 x 10(7) Lewis and 2 x 10(7) BN T-cells combined with 1 ml ALS on day -7 resulted in 100% permanent double (Lewis and BN) allograft survivals (> 150 days). Similar treatment led to acute rejection of third-party (ACI) grafts while the simultaneously transplanted Lewis hearts survived indefinitely (> 150 days). In the second group of experiments, unresponsive recipients of single (Lewis or BN) cardiac allografts were given IT inoculation of a second Ag in a sequential fashion 30 or 100 days after primary heart transplant. Unresponsive WF recipients of Lewis grafts accepted permanently (> 100 days) second BN grafts that were transplanted 30 or 100 days after primary Lewis graft and 7 days after IT injection of BN T-cells and 1 ml ALS. Similarly, unresponsive WF recipients of BN grafts accepted second Lewis grafts after IT injection of Lewis T cells combined with ALS, 30 or 100 days after primary BN grafts. To define the mechanism of peripheral tolerance in this model, we have shown that adoptive transfer of unseparated spleen cells from unresponsive recipients induces prolonged double (Lewis and BN) graft survivals (> 60 days) but not ACI grafts in sublethally irradiated (400 rads) syngeneic (WF) recipients, thus suggesting that maintenance of tolerance is, in part, dependent on suppressor/regulatory cell network.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Induction of Peripheral Tolerance by Intrathymic Inoculation of Soluble Alloantigens: Evidence for the Role of Host Antigen-Presenting Cells and Suppresser Cell Mechanism

Intrathymic (IT) inoculation of soluble alloantigens (Ag) obtained from 3 M KCI extracts of resting T-cells induces donor-specific tolerance to cardiac allografts and islet allografts. This study examined the cellular basis of induction of transplantation tolerance by IT injection of soluble Ag. Our results show that while IT inoculation of 2 mg soluble donor Ag on Day -7 relative to Lewis islet transplantation induced specific unresponsiveness to islet allografts (>200 days) in naive diabetic recipients, IT inoculation of 2 mg soluble Ag on the same day as islet transplantation did not prolong islet allograft survival in the same Lewis-to-WF rat combination. To define the role of donor APCs in intrathymic tolerance, we showed that IT injection of an admixture of 1 × 10 4 donor DC and 2 mg soluble Ag caused acute islet graft rejection. In contrast, addition of 1 × 10 4 recipient-type DC to the IT inoculum did not prevent long-term graft survival. This finding suggests that while the presence of donor APCs in the inoculum does not appear neccessary for IT-alloantigen to induce peripheral tolerance, presentation of the soluble Ag in the thymus is dependent on host APCs. This conclusion is supported by our in vitro MLR experiments which showed that in vivo WF-Ag-primed Lewis T-cells proliferated specifically to WF-soluble Ag and that the response was enhanced 14-fold by the addition of responder-type DC. Addition of anti-Lewis MHC class II mAb specifically blocked the alloresponse, thus suggesting that in vivo Ag-primed T-cells are capable of recognizing and proliferating in response to allopeptides presented by responder APCs. We also showed that adoptive transfer of syngeneic naive T-cells into unresponsive recipients failed to break tolerance to long-term surviving islet allografts. This finding suggests that tolerance in this model is not due to a lack of T help. On the other hand, the adoptive transfer of spleen cells, but not sera, from the unresponsive WF recipients bearing long-term (> 120 days) functioning Lewis islets resulted in prolonged survival of donor-type but not third-party islet allografts in secondary syngeneic hosts. Our data suggest that the tolerogenic effect of IT inoculation of soluble Ag is dependent on the indirect pathway of Ag presentation and clonal deletion of alloreactive T-cells in the thymus, while suppresser/regulatory mechanism may be involved in the maintenance of peripheral tolerance.

Marked prolongation of rat skin xenografts induced by intrathymic injection of xenogeneic splenocytes and a short course of rapamycin in antilymphocyte serum-treated mice.

The effect of intrathymic (IT) injection of donor splenocytes and a short course of rapamycin (Rapa) treatment on rat to mouse skin xenograft survival was investigated. ACI rat skin xenografts were transplanted to (C57BL/6 x A)F1 mice treated with rabbit anti-mouse lymphocyte serum (ALS) on days -1, +2, and +4 relative to skin grafting on day 0. Fifty million donor-type splenocytes were injected intrathymically on day 7 after transplantation. Rapa was given intraperitoneally every other day from day 0 to day 12 at a dose of 3.0 mg/kg. Prolonged skin xenograft survival was observed in ALS- and Rapa-treated recipients (no IT injection) with a median survival time of 47 days. However, skin graft survival was markedly more prolonged in the group treated with ALS, Rapa, and IT injection of donor splenocytes did not have a beneficial effect on skin xenograft survival in ALS-treated recipients. An increased presence of donor-type cells was observed in the thymus of the ALS- and Rapa-treated recipients for 7 days after IT injection of donor splenocytes. In conclusion, a short course of Rapa markedly augments rat skin xenograft survival in ALS-treated mice injected intrathymically with donor-type splenocytes.

Immunological tolerance to a defined myelin basic protein antigen administered intrathymically.

To explore the mechanisms responsible for the development of tolerance to allografts after intrathymic (IT) injection of alloantigen, the well-defined model of experimental autoimmune encephalomyelitis (EAE), which mimics the human autoimmune disease multiple sclerosis, was used. This inflammatory neurologic syndrome is initiated by myelin basic protein (MBP)-reactive CD4+ T lymphocytes restricted to self-MHC class II molecules. Naive adult, EAE-susceptible Lewis (RT1(1) rats were treated IT, i.v., or i.p. with a single dose (100 micrograms) of guinea pig-myelin basic protein (GP-MBP 1-176) in PBS plus 1 ml rabbit anti-rat lymphocyte serum i.p. Twenty-one days later, all rats were challenged by intradermal hind footpad injections of 50 micrograms GP-MBP in PBS emulsified in CFA. Only IT, but not i.p. or i.v., administration of GP-MBP plus anti-lymphocyte serum conferred marked resistance to a subsequent systemic challenge of GP-MBP, as demonstrated by the prevention of weight loss and paralysis characteristic of EAE. The IT administration dramatically decreased the size and number of histologic perivascular infiltrates observed per visual field in spinal cord of the tolerant animals and decreased GP-MBP-specific T lymphocyte in vitro proliferation (p < 0.01), whereas proliferation to a nonspecific mitogen (Con A) was not altered. With the addition of rIL-2, the decreased Ag-specific proliferative responses of IT-treated animals increased to control levels. Adoptive transfer of 100 x 10(6) splenocytes from tolerant hosts i.v. to naive syngeneic Lewis rats challenge with 100 micrograms GP-MBP in CFA had no effect on clinical or histologic EAE. Exposure of MBP to maturing thymocytes results in functionally immunounresponsive lymphocytes and prevention of autoimmune EAE.

A minimally invasive technique for intrathymic cell transplantation in the dog.

As an alternative to drug immunosuppression, attempts at inducing donor-specific tolerance by intrathymic (IT) inoculations to transplant recipient of donor origin alloantigenic products has proven very promising. Using fiber optic thoracoscopy, a technique for the study of this phenomena was developed for the dog. We show an approach to the dog thymus using fiber optics for injection of bone marrow (BM) cells as the tolerogen. Bone marrow was retrieved from the donor beagles and purified using an automated Ficoll-Paque gradient technique. The purified cellular suspension was injected into the thymus through a small intercostal incision with the use of an injection needle port guided by the use of a rigid fiberoptic scope. To demonstrate engraftment, supravital staining with Fluorescein Diacetate of the BM cells was performed prior to inoculation. Immunofluorescence of cryostat sections obtained at necropsy confirmed the presence of viable BM cells up to several days after transplantation. Results of this study show that the thoracoscopic approach to the thymus can be safely and effectively used for IT inoculation studies in dogs.