Regulatory T cells maintain peripheral tolerance to islet allografts induced by intrathymic injection of MHC class I allopeptides.

Although transplantation remains the treatment of choice for diabetes mellitus, immunological rejection of allografts continues to be a major problem. The search for strategies to prevent graft rejection led us to examine if the fate of developing T cells may be influenced by the presence of allo MHC class I peptides in the thymus because T cell receptor-MHC class I/self-peptide interaction regulates thymocyte development. We studied the effects of intrathymic (IT) injection of a short segment of a synthetic immunogenic MHC class I peptide (peptide 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 combination. Adult diabetic male recipients were treated with IT injection of a single WAG-derived MHC class I peptide 7 days before intraportal islet transplantation. Long-term unresponsive islet recipients were examined for the development of alloantigen (Ag)-specific regulatory cells. The results showed that while IT injection of 150 microg peptide 2 on day -7 did not prolong graft survival in naive recipients [median survival time (MST) of 14.0 days vs. 9.6 in controls], IT injection of 300 or 600 microg peptide 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, 300, or 600 microg peptide 2 combined with 0.5 antilymphocyte serum (ALS) immunosuppression on day -7 led to 100% permanent islet allograft survival (> 200 days) compared to MST of 15.0 +/- 2.3 days in ALS alone-treated controls. Similarly prepared animals rejected third-party Brown Norway (BN) islets in an acute fashion, thus demonstrating donor specificity. Intravenous injection of 300 microg peptide 2 combined with 0.5 ml ALS did not prolong islet allograft survival. The long-term unresponsive islet allograft recipients challenged with second set grafts accepted permanently 100% donor-type cardiac allografts while rejecting third-party (BN) hearts without rejecting the primary Wistar Furth (WF) islets. In analyzing the underlying mechanisms of acquired systemic tolerance, we found no suppressor/regulatory cells in adoptive transfer studies in tolerant animals at 30 days after IT injection of allopeptides. In contrast, adoptive transfer of 5 x 10(7) unseparated spleen cells from tolerant animals at 60 and 100 days after islet transplantation into lightly irradiated [200 rad total body irradiation (TBI)] ACI recipients led to donor-specific permanent islet graft survival in 2/3 and 4/5 secondary recipients, respectively, compared to an MST of 13.8 days in lightly irradiated ACI given unmodified syngeneic spleen cells. In addition, adoptive transfer of 2 x 10(7) purified T cells obtained from long-term functioning islet recipients led to permanent donor-specific islet survival in secondary recipients. The finding that IT injection of a short segment of a synthetic immunodominant MHC class I peptide derived from WAG that shares the RT1.A(U) domain with the graft donor is capable of inducing acquired systemic tolerance to WF islets suggests that linked recognition or epitope suppression may be involved in the induction of unresponsiveness. Generation of peripheral Ag-specific regulatory cells that suppress Ag-specific alloreactive T cells is, in part, responsible for the maintenance of tolerance in this model.

Mechanism of acquired thymic tolerance induced by a single major histocompatibility complex class I peptide with the dominant epitope: differential analysis of regulatory cytokines in the lymphoid and intragraft compartments.

BACKGROUND: We have recently shown that intrathymic injection of a combination of immunogenic WAG-derived or Wistar-Furth (WF) (RT1.Au) major histocompatibility complex class I peptides induces acquired systemic tolerance to cardiac and islet allografts in the WF-to-ACI rat combination and therefore hypothesized that identification of the class I peptide dominance may play an important role in the induction of antigen (Ag)-specific tolerance. This study defined the peptide with the dominant epitope among the seven synthetic RT1.Au peptides and analyzed the immunoregulatory cytokines within the lymphoid and intragraft compartments associated with acquired thymic tolerance. METHODS: ACI recipients were pretreated with intrathymic (IT) injection of 300 microg of the individual seven RT1.Au peptides 7 days before WF or Lewis cardiac transplantation. Cytokine profile of mixed lymphocyte reaction supernatants of T cells obtained from the thymus, mesenteric lymph nodes, spleen, peripheral blood leukocytes, and graft infiltrating cells after donor (WF) or third-party (Lewis) Ag stimulation were measured by enzyme-linked immunosorbent assay, whereas cytokine gene expression was determined by reverse transcription-polymerase chain reaction. RESULTS: Only IT injection of peptide 5 (93-109) among the seven RT1.Au peptides induced donor-spe cific tolerance to cardiac allografts in the WF-to-ACI rat combination. In addition, intravenous injection of peptide 5 did not prolong WF graft survival in ACI recipients. Analysis of cytokine production by the tolerant recipients showed significant Ag-specific reduction in the production of interleukin (IL)-2 and interferon-gamma (IFN-gamma) in the thymus, mesenteric lymph nodes, spleen, and peripheral blood leukocytes, which was not associated with a concomitant Ag-specific increase in IL-4 and IL-10 production. Measurement of cytokine mRNA expression confirmed undetectable

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

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.

cAMP-mediated vascular protection in an orthotopic rat lung transplant model. Insights into the mechanism of action of prostaglandin E1 to improve lung preservation.

Prostaglandin E1 (PGE1) is often added to the donor pulmonary flush solution to enhance clinical lung preservation for transplantation. Although PGE1 is thought to act as a pulmonary vasodilator during the harvest period, the precise mechanism(s) of action whereby PGE1 enhances lung preservation is unknown. Because cAMP levels decline in endothelial and vascular smooth muscle cells exposed to hypoxia, we hypothesized that a PGE1-mediated increase in cAMP levels within the preserved lungs might improve pulmonary vascular homeostasis following lung transplantation. Rat lungs demonstrated a time-dependent decline in cAMP levels during hypothermic storage, with cAMP levels significantly increased by PGE1 supplementation (approximately 2-fold by 6 hours, P < .0005). To test whether augmenting cAMP levels may enhance lung preservation, experiments were performed using an orthotopic rat left lung transplant model. Compared with controls, supplementing the preservation solution with the membrane-permeable cAMP analogue dibutyryl-cAMP resulted in dose-dependent preservation enhancement, marked by reduced pulmonary vascular resistance (6.0-fold, P < .01), improved arterial oxygenation (3.0-fold, P < .01), reduced graft neutrophil infiltration (1.5-fold, P < .05), and improved recipient survival (7.0-fold, P < .005). Similar preservation enhancement was observed with another cAMP analogue (8-bromo-cAMP) or the phosphodiesterase inhibitor indolidan. Stimulating the cAMP second messenger system by PGE1 supplementation resulted in marked hemodynamic benefits and improved recipient survival, in parallel with reduced graft neutrophil infiltration, vascular permeability, and platelet deposition. These beneficial effects of PGE1 were abrogated by simultaneous administration of the cAMP-dependent protein kinase antagonist Rp-cAMPS. Although an arterial vasodilator (minoxidil) resulted in significant pulmonary vasodilation during harvest, it lacked other nonvasodilating effects of PGE1 and resulted in poor preservation. These data show that harvest vasodilation by itself is insufficient to enhance lung preservation and that PGE1 enhances lung preservation by stimulating the cAMP-dependent protein kinase and promoting non-vasodilatory mechanisms of pulmonary protection.

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.

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 MKCl 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 micrograms 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced preservation of orthotopically transplanted rat lungs by nitroglycerin but not hydralazine. Requirement for graft vascular homeostasis beyond harvest vasodilation.

Nitric oxide (NO) produced within the lungs maintains pulmonary vascular homeostatic properties, modulating leukocyte traffic, platelet aggregation, and vasomotor tone. Because reactive oxygen intermediates generated during reperfusion react rapidly with available NO, we hypothesized that the NO donor nitroglycerin (NTG) would enhance lung preservation for transplantation by improving graft blood flow and reducing graft neutrophil and platelet sequestration. By use of an orthotopic rat left lung transplant model, with ligation of the native right pulmonary artery to ensure that recipient survival and physiological measurements depend entirely on the transplanted lung, transplants were performed in 70 male Lewis rats after 6-hour 4 degrees C preservation in Euro-Collins solution (EC) alone or EC with supplemental NTG. Compared with EC alone, supplemental NTG significantly increased pulmonary arterial flow (2.2 +/- 1.4 to 21.4 +/- 2.9 mL/min, P < .01), decreased pulmonary vascular resistance (7.4 +/- 2.0 to 1.4 +/- 0.1 x 10(3) Woods units, P < .05), improved arterial oxygenation (163 +/- 57 to 501 +/- 31 mm Hg, P < .01), and enhanced recipient survival (17% to 100%, P < .001). These beneficial effects of NTG were dose dependent over a range of 0.001 to 0.1 mg/mL. Although NTG caused significant pulmonary vasodilation during the harvest/flushing period, the direct-acting vasodilator hydralazine caused greater vasodilation than did NTG but was associated with poor graft function, elevated pulmonary vascular resistance, and poor recipient survival. To explore nonvasodilator protective mechanisms of NTG, graft neutrophil and platelet sequestration were studied; supplemental NTG significantly reduced both neutrophil and platelet accumulation compared with either hydralazine or EC alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of peripheral tolerance by intrathymic inoculation of soluble alloantigens: evidence for the role of host antigen-presenting cells and suppressor cell mechanism.

Intrathymic (IT) inoculation of soluble alloantigens (Ag) obtained from 3 M KCl 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 x 10(4) donor DC and 2 mg soluble Ag caused acute islet graft rejection. In contrast, addition of 1 x 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 necessary 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 suppressor/regulatory mechanism may be involved in the maintenance of peripheral tolerance.

Nitroglycerin maintains graft vascular homeostasis and enhances preservation in an orthotopic rat lung transplant model.

Transplanted lungs often fail during the peritransplantation period for poorly understood reasons. Because the nitric oxide pathway regulates pulmonary vascular tone, helps to maintain the integrity of the endothelial barrier, and modulates neutrophil adhesivity and activation, we hypothesized that perturbation of this pathway during the preservation and reperfusion of transplanted lungs might play a critical role in mediating early graft failure. To evaluate whether supplementing the preservation solution with the nitric oxide donor nitroglycerin enhances lung preservation for transplantation, we obtained hemodynamic measurements in a model of orthotopic left lung transplantation in the rat after ligation of the native right pulmonary artery. In these experiments, recipient survival and hemodynamics depended solely on the transplanted lung. The left lung was harvested from 22 rats, flushed with either lactated Ringer's solution alone (control, n = 11) or Ringer's solution supplemented with nitroglycerin (0.1 mg/ml, n = 11), preserved for 4 hours at 4 degrees C, and then transplanted using a rapid cuff technique for bronchial and vascular anastomoses. Nitroglycerin significantly improved arterial blood oxygenation (339 +/- 66 versus 130 +/- 12 mm Hg, p < 0.05), increased pulmonary arterial flow (7.6 +/- 1.9 versus 0.9 +/- 0.2 ml/min, p < 0.005), decreased pulmonary vascular resistance (1.7 +/- 0.4 versus 6.6 +/- 1.9 x 10(3) Wood units, p < 0.05), and enhanced recipient survival (64% versus 0%, p < 0.05). Control grafts had significantly greater neutrophil accumulation (50% greater as quantified by myeloperoxidase activity, p < 0.05) than grafts preserved in the presence of nitroglycerin. These studies show that supplementation of the preservation solution with the nitric oxide donor nitroglycerin maintains graft vascular homeostasis and significantly improves pulmonary function and recipient survival after transplantation.

The nitric oxide/cyclic GMP pathway in organ transplantation: critical role in successful lung preservation.

Reestablishment of vascular homeostasis following ex vivo preservation is a critical determinant of successful organ transplantation. Because the nitric oxide (NO) pathway modulates pulmonary vascular tone and leukocyte/endothelial interactions, we hypothesized that reactive oxygen intermediates would lead to decreased NO (and hence cGMP) levels following pulmonary reperfusion, leading to increased pulmonary vascular resistance and leukostasis. Using an orthotopic rat model of lung transplantation, a porphyrinic microsensor was used to make direct in vivo measurements of pulmonary NO. NO levels measured at the surface of the transplanted lung plummeted immediately upon reperfusion, with levels moderately increased by topical application of superoxide dismutase. Because cGMP levels declined in preserved lungs after reperfusion, this led us to buttress the NO pathway by adding a membrane-permeant cGMP analog to the preservation solution. Compared with grafts stored in its absence, grafts stored with supplemental 8-Br-cGMP and evaluated 30 min after reperfusion demonstrated lower pulmonary vascular resistances with increased graft blood flow, improved arterial oxygenation, decreased neutrophil infiltration, and improved recipient survival. These beneficial effects were dose dependent, mimicked by the type V phosphodiesterase inhibitor 2-o-propoxyphenyl-8-azapurin-6-one, and inhibited by a cGMP-dependent protein kinase antagonist, the R isomer of 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphorothioate. Augmenting the NO pathway at the level of cGMP improves graft function and recipient survival following lung transplantation.

UV-B modulation of haplo-identical bone marrow cells in the prevention of GVHD and induction of specific transplantation tolerance to intestinal allografts.

Ultraviolet B (UV-B) irradiation of BM cells (BMC) prior to transplantation into lethally gamma irradiated allogeneic rats prevents graft-versus-host disease (GVHD), induces stable allogeneic chimerism and specific tolerance to donor-type allografts. This study evaluated the role of UV-B modulation of bone marrow transplant (BMT) in the prevention of GVHD in the parent (P)-to-F1 hybrid rat combination of Lewis-to-Lewis x Brown Norway F1 (LBNF1) and of subsequent tolerance to small bowel transplantation (SBT). Lethally gamma irradiated (10.5 Gy) LBNF1 recipients of unmodified Lewis BMT (admixture of 10(8) BMC and 5 x 10(6) spleen cells) developed lethal GVHD and died within 55 days. Similarly, groups of lethally irradiated LBNF1 recipients of Lewis BMT treated with 100-300 J/m2 UV-B developed GVHD and died within 47 days. All F1 hybrid recipients of Lewis BMT treated with 700 or 900 J/m2 of UV-B permanently accepted their BM grafts, gained weight, showed no clinical evidence of GVHD and survived for > 300 days. These stable chimeras expressed 94-98% donor T-cells in their lymph nodes and became tolerant to BM donor-type (Lewis) SBT when grafted 180 days after BMT. In contrast, similarly prepared F1 recipients rejected BN SBT, thus demonstrating donor specificity. The chimeric rats were specifically unresponsive to donor (Lewis) Ag in MLR while they maintained normal alloreactivity to BN stimulators. These results suggest that UV-B irradiation of BMT offers a promising approach to overcoming the limitations of T-cell depletion of BMT and may offer a useful approach for recipient conditioning for induction of transplantation tolerance.

Diastolic function in the heterotopic rat heart transplant model. Effects of edema, ischemia, and rejection.

Decreased systolic ventricular function and compliance and increased left ventricular edema and mass have been demonstrated in cardiac allograft rejection. Whether decreased left ventricular compliance in rejection is caused by myocardial edema has not been examined, and compliance in the Ono-Lindsey model has not been reported. Heterotopic rat abdominal cardiac transplantation was performed in ACI isografts (n = 24) and in ACI to Lewis allografts (n = 24). Subgroups were studied on posttransplantation days 0, 1, 3, and 5 (each n = 6). Both transplanted hearts and native hearts were arrested with potassium for the assessment of myocardial water content, heart weight, and the left ventricular pressure-volume relation. In transplanted hearts, myocardial water content did not change in isografts but increased on posttransplantation day 5 in allografts (81.1% on posttransplantation day 5 versus 76.1% on day 0, 77.2% on day 1, and 77.5% on day 3, p < 0.05). Wet and dry heart weight also increased on posttransplantation day 5 in allografts (p < 0.05). The left ventricular pressure-volume relation in transplanted hearts shifted to the left when compared with that in native hearts in all subgroups; these volume differences were statistically significant (p < 0.01) for all pressures above 7.5 mm Hg. This pattern was similar in isografts and allografts on posttransplantation days 0, 1, and 3, and no significant differences between isografts and allografts were demonstrated. On posttransplantation day 5, however, the pressure after a 0.05 ml injection in allografts was greater in transplanted hearts than in native hearts (24 +/- 3 versus 3 +/- 1 mm Hg, p < 0.01). The pressure difference between transplanted and native hearts was also significantly greater in allografts than in isografts (22 +/- 2 versus 6 +/- 1 mm Hg, p < 0.01), indicating an increase in stiffness of allografts. Thus edema and impaired diastolic properties occur concurrently with allograft rejection. Left ventricular volume is abnormal from posttransplantation days 0 to 5 in transplanted hearts but not native hearts in the Ono-Lindsey model with current methods, apparently because of ischemic injury during transplantation.

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.

The relative contribution of intrathymic 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 histocompatibility (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.