Detection of minor alloantigen-specific cytotoxic T cells after rejection of murine orthotopic corneal allografts: evidence that graft antigens are recognized exclusively via the "indirect pathway".

BACKGROUND: The immune mechanisms by which corneal allografts are rejected in normal ocular graft beds have not been identified. Both acceptors and rejectors of these types of grafts display donor-specific delayed hypersensitivity and in vitro proliferating primed T cells, yet neither develop conventional, donor-specific cytotoxic T cells. We wished to determine whether unconventional donor-specific cytotoxic T cells are generated in rejector mice that recognize donor minor alloantigens presented by recipient major histocompatibility complex (MHC) molecules. METHODS: BALB/c mice received orthotopic corneal allografts from C57BL/10 donors in normal eyes. At 4 weeks (when 50% of grafts can be designated as rejected), primed cytotoxic T lymphocyte (CTL) activity in draining lymph nodes and spleen was assayed on targets selected to present donor-type minor H antigens on recipient MHC molecules. Control mice received heterotopic corneal allografts and were similarly examined. RESULTS: Lymphoid organs of recipients that rejected orthotopic or heterotopic corneal allografts contained CTL that lysed targets expressing donor-type minor H antigens presented by recipient MHC molecules. By contrast, no CTL activity was detected from lymphoid cells of recipients that accepted orthotopic corneal allografts. Rejection of orthotopic corneal allografts placed into normal mouse eyes correlates directly with the generation of donor-specific CTL that recognize minor H antigens in the context of recipients MHC molecules. CONCLUSIONS: These results indicate the indirect pathway of alloantigen presentation is the only pathway operative in the process by which orthotopic corneal allografts are rejected. The roles of emigrant Langerhans cells and corneal lymphatics in the indirect pathway are discussed.     

The failure of skin grafting to break tolerance to class I-disparate renal allografts in miniature swine despite inducing marked antidonor cellular immunity.

Long-term specific tolerance to one haplotype class I plus minor antigen disparate renal allografts develops without exogenous immunosuppression in approximately 35% of miniature swine (n = 128). Previous studies have suggested that this phenomenon is related to limited class I-specific helper T cell activity as evidenced by the failure of antibody class switching in vivo and the ability of exogenous interleukin 2 to elicit antidonor responses in vitro. To determine whether tolerance could be broken by inducing antidonor reactivity with donor antigen and a source of T cell help, multiple skin grafts bearing donor class I plus third-party class II antigens were placed on tolerant animals. Skin grafts were placed at least 3 months after the kidney transplant, at which time all recipients had normal renal function as measured by blood urea nitrogen and serum creatinine. First-set rejection of skin grafts by SLAad and SLAdd hosts occurred in 11.8 +/- 1.1 days (mean +/- SEM, n = 6) and in 9.3 +/- 0.9 days (n = 4), respectively. Coincident with skin rejection, most animals developed a transient rise in BUN to 62 +/- 11 mg/dl (n = 10) and a similar rise in Cr to 4.9 +/- 1.2 mg/dl (n = 10), with normal levels returning in all animals within two weeks. Subsequent skin grafts with the same disparity did not undergo second-set rejection and did not induce BUN or Cr elevations. Prior to skin grafting, animals showed no antidonor activity in mixed lymphocyte reaction or cell-mediated lymphocytotoxicity assays. After two skin grafts, all animals developed donor-specific CML and secondary MLR responses, and additional skin grafts amplified this cellular immunity. Development of marked antidonor immunity without a break in tolerance suggested that either graft adaptation or local suppression might be involved in maintaining tolerance to class I MHC antigens. In preliminary studies, an immunized SLAad animal and an immunized SLAdd animal were retransplanted with kidneys MHC matched to their first allografts. In both cases, the second graft was accepted permanently without immunosuppression, suggesting that graft adaptation is not necessary for the maintenance of tolerance to renal allografts in miniature swine.     

Tolerance to non-H-2 histocompatibility antigens. Transplantation tolerance to the H-4 and H-7 histocompatibility antigens.

There have been several reports of observations which suggest that transplantation tolerance may be a result of positive immunoregulation rather than simply unresponsiveness attributable to a lack of competent effector cells. In particular, several investigators have reported that tolerance of the H-Y and H-1 histocompatibility antigens is mediated by a population of thymus-derived lymphocytes. In a companion report, we have presented evidence that supports the existence of a suppressor cell to the H-Y antigen. Furthermore, we have observed that female mice rendered tolerant of the H-Y antigens by neonatal exposure to male lymphoid cells or by multiparity accept male skin grafts indefinitely, but inactivate male peritoneal exudate cells (PEC) in a second-set fashion. This observation has led us to investigate whether tolerance of other non-H-2 antigens is controlled by a similar mechanism. Using mice congenic with C57BL/10 at the H-4 and H-7 loci, we have shown that mice rendered tolerant of the H-7a and H-4b antigens by neonatal exposure to histoincompatibe lymphoid cells are incapable of rejecting either skin or peritoneal cell allografts, suggesting that identical histocompatibility antigens are present on skin and peritoneal cells. Tolerance induced in neonatal mice to the H-4b and H-7a antigens could not be adoptively transferred to syngeneic recipients. These results suggest that tolerance involving the H-4 and H-7 antigens is most likely because of a clonal inactivation of alloantigen-reactive cells as a consequence of neonatal exposure to antigen.     

Role of the thymus in transplantation tolerance in miniature swine: V. Deficiency of the graft-to-thymus pathway of tolerance induction in recipients of cardiac transplants

BACKGROUND: We have previously shown that both thymic immigrants (graft to thymus pathway) and thymic emigrants (thymus to graft pathway) are involved in tolerance to renal allografts in miniature swine treated with a short course of calcineurin inhibitors. This study investigates the role of these pathways in cardiac transplant survival in recipients treated with a short course of tacrolimus. METHODS: Eleven animals received two-haplotype fully MHC-mismatched cardiac grafts with a 12-day course of tacrolimus. Recipients were thymectomized on day -21 (n=5) or day 0 (n=3), or were left euthymic (n=3). Two of the day -21 thymectomized animals received a day 0 host-MHC matched thymocyte infusion. RESULTS: Euthymic recipients of cardiac grafts treated with an immunosuppressive regimen identical to that previously shown to induce tolerance in euthymic recipients of renal allografts all rejected their grafts. Although no animal became tolerant, animals that were euthymic or thymectomized on day 0, as well as recipients of day 0 host-type thymocyte infusions following thymectomy on day -21, developed donor-specific hyporesponsiveness and maintained their cardiac grafts for markedly prolonged periods. In contrast, all animals thymectomized on day -21 that did not receive thymocyte infusions developed strong antidonor CTL responses and rejected their grafts by day 35. CONCLUSIONS: The graft-to-thymus pathway that plays an important role in tolerance induction to renal allografts appears to be relatively deficient in recipients of cardiac grafts. Strategies to increase donor antigen migration to the host thymus might therefore assist in tolerance induction to cardiac allografts.     

Allograft rejection and immune responses against allogeneic antigens in neonatally thymectomized mice

Skin allograft survival and immune responses against allogeneic antigens homologous to skin grafts were observed in BALB/c Cr Slc (BALB) mice (H-2d) thymectomized at 1 day after birth and grafted with skin from major histocompatibility complex (MHC)-incompatible, fully allogeneic C3H/HeN (C3H) (H-2k) or MHC-compatible allogeneic DBA/2 Cr Slc (DBA) mice (H-2d), at 14 weeks of age. In neonatally thymectomized (NTx) BALB mice, survival of C3H skin grafts was not prolonged at all, but survival of DBA skin grafts was prolonged significantly, although the survival periods of DBA skin grafts were very different among individual recipients. In NTx recipients grafted with C3H skin, delayed foot-pad reaction (DFR) was not reduced, but cytotoxic lymphocyte (CTL) activity and cytotoxic antibody (CTAb) production were appreciably depressed. CTL and CTAb were reduced profoundly and consistently in all NTx mice grafted with DBA skin, while DFR was reduced to various degrees in each. The degrees of depression of DFR in these NTx mice correlated well with the prolongation of DBA skin survival, although the sample number was small. The rejection of skin allografts appears to be attributable largely to a T cell subset, the function of which can be expressed as DFR. Thymus dependency in the ontogenic development is low as compared with other T cell subsets.     

The immediately vascularized skin allograft.

A model for immediate vascularization of skin was devised to examine one of the possible explanations for the differential survival rates of transplants of freely grafted skin and organs, i.e., the increased vulnerability of skin to early ischemic necrosis prior to revascularization. Female Fischer (F) rat skin was transplanted beneath the kidney capsule of tolerant female Lewis (LEW) recipients. This skin healed in and initially appeared to be normal grossly and microscopically. In 27 rats, after 30 days, the composite grafts were excised, and immediately transplanted by means of vascular anastomosis into normal LEW recipients (syngeneic to the kidney portion of the graft and allogeneic to the skin). For 5 days after transplantation of the composite graft, the skin appeared to be normal with minimal or no inflammation in a panel of 11 recipients. From the 6th to 11th day, an active rejection reaction developed at the skin-kidney interface in a panel of six rats. In 10 rats, in which the composite grafts remained in their secondary hosts for 12 to 21 days, rejection of the skin was complete. The renal portion of all composite grafts appeared to be normal histologically. All recipients of composite grafts rejected subsequent orthotopic F skin grafts in an accelerated manner, with median survival times of 8.2 +/- 0.3 days compared to 11.5 +/- 0.7 days in untreated F leads to LEW controls, demonstrating that the skin on the composite graft was fully immunogenic. These results demonstrate that immediately vascularized skin allografts between rats compatible at the major Ag-B1 locus will still be rejected within 2 weeks compared to survivals of up to 48 weeks in renal allografts in the same histocompatibility combinations, suggesting that anatomical factors are not sufficient to account for the differences in survival times between skin and solid organs.     

Thymic transplantation in miniature swine: III. Induction of tolerance by transplantation of composite thymokidneys across fully major histocompatibility complex-mismatched barriers

BACKGROUND: This study determines whether composite thymokidney (TK) grafts, created by implantation of autologous thymic tissue beneath the donor's renal capsule before transplantation, could induce allogeneic transplantation tolerance across two-haplotype fully major histocompatibility complex (MHC)- mismatched barriers in juvenile MGH-miniature swine. METHODS: TK grafts were prepared by implanting autologous thymic tissue under the renal capsule of donor animals 2 to 3 months before transplantation. Four recipients were treated with a T-cell-depleting immunotoxin and received fully MHC-mismatched TK grafts plus a 12-day course of cyclosporine A (CsA). Control animals were treated with CsA alone or both CsA and immunotoxin, but with a normal kidney or a kidney implanted with autologous lymph node rather than thymus. Renal graft function was assessed by plasma creatinine levels and histologic analyses. Immunologic status was monitored by cell-mediated lympholysis assays. RESULTS: All four recipients of fully MHC-mismatched TK transplants treated with immunotoxin and a 12-day course of CsA accepted their composite renal allografts long-term. All control recipients receiving a TK and CsA alone, a normal kidney or a composite kidney containing lymph node tissue acutely rejected their grafts. CONCLUSIONS: To our knowledge, this is the first demonstration that functional vascularized thymic grafts can induce transplantation tolerance across fully MHC-mismatched barriers in a large animal model.     

Effector mechanism in rejection of allografts expressing an isolated minor histocompatibility disparity. Importance of cytotoxic T lymphocytes in the rejection of H-43a allografts by H-43b mice

To explore effector mechanisms in allograft rejection, we transplanted skin grafts (SG) across a single minor histocompatibility locus (H-43) using mouse strains carrying the H-43b allele as SG recipients and those carrying the H-43a allele as SG donors. Recipients' spleen cells (SC) were assayed at various intervals for 200 days for anti-H-43a cytotoxic T lymphocyte (CTL) responsiveness, as well as delayed-type hypersensitivity (DTH) responsiveness. When H-43a SG from C3H.SW mice were transplanted to H-43b CWB mice, two thirds of the recipients rejected the SG, and recipients' SC showed marked self-H-2Kb-restricted anti-H-43a CTL responsiveness until the end of the observation period. In contrast,H-43aSG transplanted to H-43b (B10.BRxCWB)F1 (BWF1) mice survived in almost all of the BWF1 recipients. The anti-H-43a CTL responsiveness of the recipients' SC was evident until day 40 but thereafter started to wane and eventually disappeared. Notably, BWF1 mice whose self-H-2Kb-restricted anti-H-43a CTL precursors had been primed by prior injection with H-43a SC rejected H-2Kb-bearing H-43a CSW SG but not H-2k, H-43a C3H/HeN SG. In contrast, an anti-H-43a DTH response was not induced in any of the CWB and BWF1 recipients, including CWB recipients who rejected the H-43a SG. Since it has been well documented that anti-H-43a CTL are restricted solely by self-H-2Kb, the results in this study indicate that self-H-2Kb-restricted anti-H-43a CTL are responsible for rejection of H-43a allografts by H-43b recipient mice.     

Comparison of successful and unsuccessful islet/Sertoli cell cotransplant grafts in streptozotocin-induced diabetic mice

Sertoli cells (SC) protect islet allografts from immune destruction in diabetic rodents. In this study, we examined the difference between successful and rejected islet/SC cografts in order to further improve this procedure for optimal extension of islet allograft survival. We cotransplanted 500 BALB/c islets with 1-8 million BALB/c SC under the kidney capsule of diabetic BALB/c, C3H-HeJ, and C57BL/6 mice. Cotransplantation of islets with up to 8 million SC was not detrimental to long-term islet graft function in syngeneic mice. However, large numbers of SC were detrimental to islet graft survival in allogeneic mice with the optimal dose for cotransplantation of 4 or 1 million SC in C3H-HeJ or C57BL/6 mice, respectively. Examination of successful grafts, from euglycemic recipients, revealed the presence of SC arranged in tubule structures with islets surrounding these tubules. Cellular infiltrate in successful grafts revealed CD4 T cells and macrophages along the periphery and within the grafts, and very few CD8 T cells. Conversely, examination of unsuccessful grafts, harvested from hyperglycemic recipients at the time of rejection, revealed the presence of SC arranged randomly with islets adjacent to the Sertoli cells, when present, and massive CD4 and CD8 T cell as well as macrophage cell infiltration. Prolongation of islet allograft survival appeared to be a function of SC transplant mass and recipient genetic background. A consequence of long-term graft acceptance is the formation of SC tubule structures, which may be an additional requirement for optimal protection of islet allografts.     

Cyclophosphamide-induced tolerance in rat orthotopic liver transplantation

BACKGROUND: We previously established a cyclophosphamide (CP)-induced tolerance system in rodent skin graft models. In this study, we applied this system to rat liver transplantation. METHODS: Lewis recipients were inoculated on day -2 with spleen and bone marrow cells (SC+BMC) from Dark Agouti (DA) donors, followed by 100 mg/kg CP on day 0. On day 25, DA livers were orthotopically grafted. We assessed the alloresponses to the donors of the long-term surviving recipients, using the second skin grafting and in vitro assay. RESULTS: The recipients that had been treated with SC+BMC and CP survived for more than 165 days. None of control group that received SC+BMC alone (mean survival times [MST]=13.8 days), CP alone (MST=40.0), SC+BMC from third-party PVG rats and CP (MST=45.0), or no treatment (MST=13.8) survived over 50 days. The donor-specific tolerance was confirmed by second skin grafts onto recipients with permanent DA liver grafts, which accepted DA skins (MST>75) but not PVG (MST=8.3). However, the lymphocytes from the tolerant recipients showed alloresponse to DA in vitro. To investigate whether the T helper type 2 deviation contributed to this "split tolerance," we assessed the production of cytokines in mixed lymphocyte reaction. Interleukin 2 and interferon-gamma were detected but interleukin 4 was not. CONCLUSIONS: These data showed that this protocol induced split tolerance in rat liver transplantation and, furthermore, the mechanism of split tolerance was not due to T helper 2 deviation.     

Effect of organ culture on the survival of thyroid allografts in mice.

Mouse thyroid can be maintained in organ culture for 4 weeks. Uncultured BALB/c thyroid is rejected 10-15 days after transplantation under the kidney capsule of H-2 disparate recipients (C57BL, CBA). Organ culture of thyroid tissue prior to transplantation prolongs allograft survival. This prolongation of graft survival increases with increasing time in culture and 80-90% of BALB/c thyroids maintained in culture for 26 days survive in allogeneic CBA recipients for a 60- to 70-day test period. These allografts show normal function as measured by 125I uptake, and show no histological evidence of chronic rejection. Cultured allografts can be rejected if the host's immune system is stimulated with viable leukocytes of donor origin. Host animals carrying a functioning allograft are not tolerant of donor tissues and will reject a second uncultured allograft from the same donor strain.     

Allorecognition in T-cell receptor (beta-chain) transgenic mice.

Recent advances in knowledge of the structure of the T-cell receptor and of major histocompatibility complex (MHC) molecules have increased our understanding of the nature of their interaction in the immune response. Nevertheless, it remains unclear how the T-cell receptor recognizes foreign MHC molecules in the process of graft rejection. One approach to this problem is to characterize the alloreactivity of a given T-cell receptor. We have chosen to take this approach in vivo by examining patterns of rejection of vascularized heart allografts in transgenic mice carrying a rearranged T-cell receptor-beta-chain gene, in which essentially all alpha beta T cells bear the rearranged gene product. Heterotopic heart allografts were performed in transgene-positive and transgene-negative recipients. The data show that transgene-positive mice will reject fully allogeneic grafts of three different haplotypes after a modest delay, but will not reject grafts from F1 mice that bear H-2 antigens from these same haplotypes and from the recipient strain. Transgene-negative animals reject all grafts promptly. These results suggest that the restricted T-cell receptor repertoire expressed by transgene-positive recipients affects their ability to respond to an alloantigen as expressed on a vascularized graft and that this response is influenced by the presence of self-MHC molecules on the graft.     

Orthotopic corneal transplantation in mice--evidence that the immunogenetic rules of rejection do not apply.

The fate of orthotopic corneal transplants has been studied in inbred strains of mice. Using a surgical technique that achieves > 95% success of syngeneic cornea grafts, it was determined that a high proportion of orthotopic cornea allografts were accepted indefinitely, irrespective of the degree of immunogenetic disparity between graft donor and recipient. Grafts that succumbed to irreversible rejection developed extensive corneal edema and intrastromal neovascularization as harbingers of corneal opacity and endothelial cell failure. The highest rate of rejection occurred among grafts that confronted their hosts with multiple minor histocompatibility antigens, with or without major histocompatibility antigens. Much lower rates of rejection (< 35%) were observed when the donors of the grafts differed from recipients at class I and/or class II major histocompatibility loci. Corneal grafts that confronted their hosts with class II MHC alloantigens alone experienced early, acute inflammation, and eventually developed stomal neovascularization, but only a small minority of these grafts were eventually destroyed. Allogeneic corneas that were transplanted orthotopically into eyes of presensitized mice were uniformly subjected to an acute rejection process that produced opacity within three weeks; however, in a minority of instances, the inflammation and opacity subside, and after eight weeks the grafts displayed a clear, nonvascularized appearance. The high rate of success of even grossly histoincompatible orthotopic corneal allografts in mice resembles the extraordinary success of unmatched allogeneic corneas transplanted into human eyes. The results are discussed in terms of the possible mechanisms that permit orthotopic corneal allografts to enjoy significantly better survival than orthotopic grafts of other types of solid tissues.     

Immunological unresponsiveness induced by ultraviolet-B-irradiated and nonirradiated skin grafts

We previously reported that ultraviolet-B-irradiated B10.AQR tail skin grafts were permanently accepted by B10.T6R recipients in about half the cases. Such a beneficial effect on graft survival could only be demonstrated in this particular combination. We have now investigated whether these animals had become tolerant to the donor strain antigens. Nonirradiated B10.AQR tail skin grafted 50 days after acceptance of a UVB-irradiated B10.AQR graft was accepted in 9/9 cases, indicating that these animals had become tolerant to the B10.AQR alloantigens. However, secondary grafts on animals that had rejected the first graft also showed a prolonged or definite survival. This tolerance was specific; B10.T6R mice tolerant to B10.AQR grafts rejected B10 skin grafts, while F1(B10A X B10.AQR) and F1(B10 X B10A) grafts, sharing class II antigens with B10.AQR, had a slightly prolonged graft survival. Cells of tolerant animals showed normal proliferative responses against B10.AQR antigens. However, when autologous serum was added, proliferation was specifically suppressed. Likewise, this specific tolerance could be transferred with serum or serum and cells but not with cells only. Analysis of the sera of these animals showed long-lasting and donor-specific high-titered cytotoxic antibody titers, which are likely to play a pivotal role in the observed suppression.     

Major histocompatibility complex antigen expression of parenchymal cells of thyroid allografts is not by itself sufficient to induce rejection.

H-2d thyroids cultured in oxygen at normal atmospheric pressure (suboptimal culture) were grafted into H-2b mice. Some of these tissues were cultured with recombinant mouse gamma-interferon (rIFN), and they expressed high levels of major histocompatibility complex antigens before grafting. Three weeks later, no difference in the rate of rejection of MHC-induced grafts was observed as compared with uninduced tissues (50% of each group). Fresh (uncultured) grafts were uniformly rejected in less than 2 weeks. Also, H-2d thyroids, freed of donor leukocytes by preculture in hyperbaric oxygen and more than 1 year parking in normal H-2b recipients, were incubated with and without rIFN, and then grafted into normal H-2b mice; 100% acceptance was observed in both groups regardless of the expression of allo-MHC molecules on thyroid cells. In another set of experiments, using grafts with a single antigenic difference at the MHC locus (bm1 into B6), graft rejection was observed only when the recipients were immunized with donor spleen cells and fresh tissues were implanted. In the same immune recipients, cultured and MHC-induced thyroids grafted in the opposite kidney were, in general, not rejected. These results demonstrated that the expression of allo-MHC molecules on graft cells was not by itself sufficient to engender tissue immunogenicity. This supports our previous hypothesis that the main effect of tissue culture is the inactivation of passenger leukocytes. MHC antigens appear to be immunogenic only when properly presented by these cells.     

Assessment of peripheral tolerance in anti-CD4 treated C57BL/6 mouse heart transplants recipients.

The study was designed to compare second heart and skin grafts and in vitro assays as a means of assessing peripheral tolerance in C57BL/6 mice. Vascularized heterotopic BALB/c hearts were placed in C57BL/6 recipients treated with anti-CD4, GK1.5 (1 mg total per 20 g mouse i.p. on days 0, 1, 2, 3). Those mice in which hearts survived for >60 days were challenged with donor and third-party (CBA) skin grafts or with second heart grafts, of donor or third-party origin, attached to the carotid artery and jugular vein. In vitro alloreactivity was assessed by mixed lymphocyte reactions (MLR) and cell mediated lympholysis (CML) using recipient spleen cells. Parenchymal damage, cellular infiltration and vascular disease were assessed from the histology of long-term allografts and isografts. Allografts in untreated recipients were rapidly rejected while isografts survived > 100 days. Primary allografts in anti-CD4 treated recipients also survived > 100 days, as did donor strain secondary heart transplants given at >60 days after the first graft. Third-party hearts were rapidly rejected, as were donor and third-party skin grafts placed on recipients with long-term allografts. These recipients showed low MLR response to both donor and third-party stimulators and donor-specific suppression of CML at 60 days post graft. Long-surviving heart allografts all showed evidence of parenchymal damage and vascular intimal thickening. Thus in the BALB/c to C57BL/6 donor-recipient strain combination, hearts, but not skin grafts, could be used to demonstrate peripheral tolerance, which seemed to be both organ and major histocompatibility complex (MHC) specific. Despite long survival, BALB/c hearts all showed evidence of parenchymal damage and vascular intimal thickening, a sign of chronic rejection.     

Dominant tolerance and linked suppression induced by therapeutic antibodies do not depend on Fas-FasL interactions

BACKGROUND: Nonlytic anti-CD4 monoclonal antibody therapy can be used to induce transplantation tolerance in rodent models. Such tolerance is often associated with dominant regulation, mediated by CD4+ cells, and characterized by infectious tolerance and linked suppression. Understanding the mechanisms by which CD4+ regulatory cells function may improve the manner in which current immunosuppressants are applied and may lead to the development of new tolerance-inducing therapeutics. Fas-mediated apoptosis has been characterized as an important mechanism of peripheral self-tolerance and we here examine whether it has any role in anti-CD4 monoclonal antibody-induced dominant tolerance. METHODS: Tolerance to transplanted skin and bone marrow, mismatched for multiple minor histocompatibility antigens, was induced in Fas mutant and control mice using anti-CD4 and anti-CD8 monoclonal antibodies. To test for linked suppression, animals were transplanted with a second graft-bearing tolerated and third party antigens. The ability of splenocytes from tolerant animals to suppress graft rejection was assessed by transfer into partially immunocompromised recipients. RESULTS: Monoclonal antibody therapy rendered Fas mutant mice tolerant of minor disparate skin and bone marrow. Splenocytes from these and control tolerant animals when transferred into partially immunocompromised Fas mutant or control recipients, induced antigen-specific suppression of graft rejection. Additionally, tolerant Fas mutant mice accepted grafts bearing tolerated and third party antigens. CONCLUSIONS: Signal transduction through the Fas receptor plays no essential role in the induction of tolerance using anti-CD4 and anti-CD8 monoclonal antibodies or its maintenance by active regulation.     

Radiosensitivity of suppressor cells in neonatally tolerant rats.

Specific suppressor cells were demonstrated in rats that had carried tolerated skin allografts for long periods of time after being rendered tolerant at birth. These suppressor cells were able to transfer tolerance to sublethally irradiated syngeneic recipients and to inhibit cytotoxic antibody production in normal syngeneic recipients. Suppressive activity of these cells was shown to be radiosensitive. The presence of suppressor cells in tolerant animals was attributable to neonatal tolerance induction and not to skin grafting of neonatally treated animals. In some cases spleen cells from tolerant animals transferred adoptively or induced permenent tolerance to skin grafts, which suggests a long-lasting active mechanism of tolerance.     

Indefinite survival of rat islet allografts following infusion of donor bone marrow without cytoablation

We have tested the effect of donor bone marrow cell (DBMC) infusion on the survival of pancreatic islet allografts in the rat, without the use of cytoablative recipient conditioning. Lewis and diabetic Brown Norway rats were used as donors and recipients, respectively. Donor islets were placed beneath the left renal capsule. Infusion of DBMC and temporary immunosuppression followed by delayed islet transplantation resulted in indefinite survival of all islet grafts (MST >180 days). Control animals demonstrated recurrent hyperglycemia (islet allografts rejection). Donor bone marrow derived cells were detected in the spleen and cervical lymph nodes of BN recipients of LEW bone marrow but not in the recipients of islet transplants alone. Second set full thickness skin grafts were performed in normal BN and in recipients of a previously successful ITX. Donor specific skin grafts were accepted in the animals that had received DBMC 40 days before the islet allograft, while animals receiving DBMC at the time of the islet allograft rejected the donor specific skin graft similarly to the controls. However, these animals did not reject a second set donor-specific islet transplant. The results indicate that radiation conditioning of the recipients was not necessary to induce microchimerism and graft acceptance in this rodent model of islet allotransplantation.     

Role of persistence of antigen and indirect recognition in the maintenance of tolerance to renal allografts

BACKGROUND: We have previously shown that a 12-day treatment with cyclosporine A (CyA) facilitates induction of tolerance to class-I disparate kidneys, as demonstrated by acceptance of second, donor-matched kidneys without immunosuppression. In the present study, we have examined 1) the duration of tolerance in the absence of donor antigen and 2) the pathway of antigen recognition determining maintenance or loss of tolerance. METHODS: Seventeen miniature swine received class-I mismatched kidneys with 12 days of CyA, and received second donor-matched kidneys without immunosuppression at 0, 1, 3, or 4 months after nephrectomy of the primary graft. Five were sensitized 6 weeks after nephrectomy of the primary graft, three with donor-matched skin grafts, and two with donor class-I peptides to eliminate direct pathway involvement. In addition, two long-term tolerant animals received class-I peptides. RESULTS: Rejection of second grafts required at least a 3 month absence of donor antigen. Although donor-matched skin grafts in animals tolerant to kidneys induced antidonor cytotoxic T lymphocyte responses, second renal transplants revealed no evidence of sensitization. In contrast, immunization of recipients with donor class-I peptides after nephrectomy of the primary graft led to loss of tolerance at both T-cell and B-cell levels, as evidenced by rejection of the second graft in 5 days and development of antidonor immunoglobulin G. Peptide immunization of long-term tolerant in recipients bearing long-term renal grafts did not break tolerance. CONCLUSIONS: These data indicate that the renal allograft is required for the indefinite maintenance of tolerance, that indirect antigen presentation is capable of breaking tolerance, and that in tolerant animals, direct antigen presentation may suppress rejection, allowing tolerance to persist.