Indefinite Graft Survival Research Articles

The role of immunosuppression in the long-term survival of tracheal allografts.

To assess the functional aspects of vascularized tracheal allograft transplant models and the long-term fate of these allografts. To examine the effects of cyclosporin A (CsA), 10 mg/kg per day, on the long-term survival of vascularized tracheal allografts and the presence and significance of host immune tolerance after cessation of immunosuppression. In a rabbit model, tracheal allografts were orthotopically transplanted after an initial period of heterotopic fascia revascularization. A full-thickness skin allograft from the same donor was used as an external monitor of the rejection process. Treatment with CsA was discontinued (group 2) or given intermittently (group 3) after an initial course of continuous CsA administration varying from 2 to 10 weeks. The first group received a continuous regimen of CsA for 10 weeks. Tracheal and skin allograft deterioration, microcirculation of tracheal transplant, mucociliary clearance, and histopathologic examination. Tracheal allografts under continuous immunosuppression with CsA, 10 mg/kg per day, showed no rejection and remained functional with preservation of mucociliary activity. After an initial course of CsA to achieve solid tracheal union, subsequent cessation and intermittent pulsing of the immunosuppressant were insufficient in maintaining indefinite graft survival. Chronic allograft rejection with simultaneous endothelial graft repopulation could be induced by intermittent pulsing of CsA, which led to prolonged allograft survival in some group 3 animals. Continuous immunosuppression is necessary to preserve an optimal morphological and functional condition of tracheal allografts.

Late adjunctive therapy with single doses of rapamycin in skin-allografted mice treated with antilymphocyte serum and donor bone marrow cells

Temporary donor-specific unresponsiveness induced by treatment of skin allografted mice with antilymphocyte serum (ALS) and donor bone marrow cells (BMC) can be converted to long-term graft survival and tolerance by adding adjunctive immunosuppressive agents, including rapamycin (Rapa). Rapa was given in a suboptimal dosing schedule to probe several aspects of its use in the promotion of tolerization. Single doses were given at 2 weeks post-transplantation to recipients prepared with ALS and donor BMC. Graft survival was markedly prolonged in a dose-dependent fashion by day +14, Rapa, doses ranging from 0.75 to 6.0 mg/kg. Indefinite (> 300 day) graft survival was observed in 26% of recipeints given the highest Rapa done. Short-term treatment with cyclosporin A (CsA) or deoxyspergualin (DSG) was ineffective when injected at this time. Rapa augmented the tolerizing effect of grafting with skin from newborn mice but had no significant additive or synergistic effects with the short course of CsA or with DSG given on days +1 to +3, even though the latter prolonged graft survival when added to the ALS/BMC protocol. Single doses of Rapa on day +1 also prolonged graft survival, but without any of the grafts surviving indefinitely. Later dosing on day +28 resulted in > 70% of grafts surviving > 300 days. Challenge grafting of these mice after day +300 resulted in delayed rejection of donor strain, but not third-party skin grafts. Rapa was very effective when given as widely spaced doses on days +14 and +49. Also, grafts showing the earliest signs of rejection could be rescued with a single Rapa dose in recipients treated with ALS and BMC but not ALS alone. Transfer of prolonged graft survival with spleen cells from ALS plus BMC treated recipients was not adversely affected by Rapa given to the suppressor-like spleen cell donors approximately 1 week before cell harvest. We conclude that the use of Rapa as an adjunctive agent in allograft recipients treated with ALS plus donor BMC is very flexible in terms of timing of administration, and that the drug can be effectively given as widely spaced doses or as a rescue agent after ALS/BMC treatment. Additionally, an active immunoregulatory mechanism induced by ALS/BMC treatment appears to be spared by Rapa.

Survival Of Mhc-Deficient Mouse Heterotopic Cardiac Allografts

The immunologic mechanisms involved in the destruction of murine cardiac allografts were evaluated using MHC-deficient mice. Specifically, we examined the survival of immediately vascularized heterotopic adult cardiac grafts deficient in MHC class I, MHC class II, or both MHC class I and II antigens following transplantation to allogeneic hosts. We observed indefinite cardiac graft survival when donors lacked MHC class II or both MHC I and II antigens. In parallel experiments, we studied the survival of cardiac grafts harvested from normal donors in recipients severely depleted of either CD4 (class II-deficient mice) or CD8 (class I-deficient mice) T cells. Graft survival was dramatically prolonged in the absence of CD4 but not CD8 T cells. Collectively, our results demonstrate that the interaction of host CD4 T cells with donor class II antigens is critical to the rejection of murine cardiac grafts.

Role of CD4+ T cells in the rat to mouse cardiac xenotransplantation.

T cell subsets involved in rejection of xenografts were analyzed using a rat to mouse cardiac xenotransplant model. Proliferating response and interleulin-2 (IL-2) production in recipients' spleen cells were almost completely abrogated by elimination of L3T4+ T cells, but not by elimination of Lyt2.1+ T cells. Cytotoxic T lymphocyte (CTL) activities were mediated by both L3T4+ and Lyt2.1+ T cells with the help of IL-2-producing L3T4+ T cells. Administration of anti-L3T4 monoclonal antibody (mAb) into recipient mice resulted in a significant prolongation of graft survival (mean graft survival was 29.2 days). Moreover, anti-L3T4 mAb treatment plus thymectomy led to indefinite graft survival. Anti-rat endothelial cell (EC) antibody production in the grafted mice was remarkably suppressed by anti-L3T4 mAb treatment. In contrast, Lyt2.1 mAb treatment did not prolong the graft survival and did not suppress anti-EC antibody production. These results indicated the absolute requirement of L3T4+ T cells in the rejection of rat to mouse cardiac xenografts.

TRANSPLANTATION TOLERANCE INDUCED BY CTLA4-Ig1

The rejection of the transplanted allograft is dependent on T cell activation, which requires T cell receptor engagement by antigen and costimulatory signals delivered by T cell surface molecules such as CD28. CTLA4-Ig is a fusion protein that has previously been shown to block the CD28-mediated costimulatory signal and inhibit immune responses in vitro and in vivo. In this report we show that treatment of the C3H/He recipient of a BALB/c vascularized cardiac allograft with a 12-day course of CTLA4-Ig produced indefinite graft survival (> 100 days) in the majority of recipients. In addition, these recipients demonstrated donor-specific transplantation tolerance when tested with donor-specific (BALB/c) and third-party (C57BL/10) skin grafts. These results demonstrate that CTLA4-Ig can induce transplantation tolerance in the adult murine cardiac allograft model.

LEFLUNOMIDE IN EXPERIMENTAL TRANSPLANTATION

Leflunomide is a compound recently shown to reduce T and B cell-mediated responses in a number of experimental rat, mouse, and human systems. To explore its potential as an immunosuppressant, we studied leflunomide in 128 Brown-Norway/Lewis cardiac transplants and in 48 unoperated Lewis rats. At doses ranging from 0.63 mg/kg to 10 mg/kg given for 7 days, leflunomide significantly prolonged graft survival compared with controls. When cyclosporine or leflunomide was given for 21 days at a dose of 5 mg/kg, indefinite graft survival occurred in 3/6 animals receiving leflunomide but in none of the 21-day cyclosporine-treated animals. When acute rejection was allowed to develop for four days in untreated rats, leflunomide but not cyclosporine reversed the rejection, returning histology to a normal appearance by seven days. Alloantibody responses measured in microcytoxicity assays as well as total allospecific IgG and IgM in the rejecting animals also were returned to baseline levels by leflunomide but not cyclosporine. When both drugs were used together, a synergistic effect was observed at low doses of both drugs. Pharmacokinetics studies showed that their combined use for up to 28 days did not affect the trough levels of cyclosporine or cyclosporine elimination, suggesting that the synergistic effect was not caused by reduced elimination. The toxicity of each drug was negligible in a group of 32 rats receiving the drugs alone or in combination as measured by serial observation of general appearance, testing of serum ALT, AST, bilirubin, creatinine, white blood cell counts, hemoglobin, and gross necropsy appearance. Weight gain was slightly reduced by both drugs but combined drug use did not alter the pattern. The results of these experiments show leflunomide to be a potent, well-tolerated immunosuppressant, synergistic in its activity with cyclosporine, and would seem to encourage a closer look at this drug for potential use in man.

Anti-CD2 monoclonal antibodies synergize with FK506 but not with cyclosporine or rapamycin to induce tolerance.

CsA, FK506, and rapamycin prolong allograft survival; however, each has significant associated side effects at therapeutic doses. Anti-CD2 mAbs also prolong survival but without toxicity. We tested whether alpha CD2 mAbs in combination with subtherapeutic immunosuppression could prolong allograft survival in a synergistic fashion. C57BL/6 (H-2b) mouse hearts were transplanted to CBA (H-2k) mice in a heterotopic, non-vascularized cardiac allograft model. Recipients received immunosuppressants intraperitoneally for 14 days and/or alpha CD2 mAb intravenously for 2 days starting at the time of grafting. Survival was determined by electrocardiogram monitoring. Anti-CD2 alone prolonged survival to 22.4 +/- 1.0 days versus 13.4 +/- 0.5 days for untreated controls (P < 0.05), while low dose FK506 minimally prolonged survival to 16.7 +/- 0.7 days (P < 0.057). However, FK506 plus alpha CD2 resulted in synergistic prolongation of graft survival to 28.0 +/- 2.1 days. Several doses of CsA and rapamycin in combination with alpha CD2 did not prolong survival over alpha CD2 administered alone. A 60-day course of low dose FK506 plus alpha CD2 resulted in indefinite graft survival (> 165 days). These animals were tolerant since they accepted a second donor-specific graft. CTL and MLR activity in long-term recipients were normal to both donor-specific and third party alloantigen. The combination of alpha CD2 with low dose FK506 is synergistic in prolonging cardiac allograft survival, while combinations with CsA and rapamycin are not. Continuous administration of low dose FK506 plus alpha CD2 results in a state of tolerance. This suggests that FK506 acts at a different locus in allograft immunity compared with the other immunosuppressants and this may be related to the alternative CD2 T cell activation pathway.

A role for nonspecific (cyclosporin A) or specific (monoclonal antibodies to ICAM-1, LFA-1, and IL-10) immunomodulation in the prolongation of skin allografts after antigen-specific pretransplant immunization or transfusion.

C3H/HEJ mice given pretransplant immunization via the portal vein with irradiated B10.BR spleen cells or non-irradiated B10.BR peripheral blood leukocytes showed delayed rejection of B10.BR but not BALB.K skin grafts. No increased graft survival was seen if pretransplant immunization was given by the lateral tail vein. Addition of a single treatment with cyclosporin A after pretransplant immunization via the portal vein led to indefinite graft survival in over 70% of recipients. As reported earlier, increased graft survival in vivo was correlated with preferential production of IL-4 (from Th2 type cells) rather than IL-2 from lymphocytes stimulated in vitro. When mice were given pretransplant transfusion via the lateral tail vein and a combination of anti-LFA-1 and anti-ICAM-1 mAbs, increased graft survival was again seen. In addition, in vivo injection of anti-IL-10 mAbs (but not anti-IFN-gamma or anti-IL-4 mAbs) abolished the delayed rejection seen when mice received pretransplant immunization via the portal vein. In all cases, delayed graft rejection in vivo was correlated with preferential activation of Th2-type cells, as assessed by lymphokine production from cells harvested from treated mice and activated in vitro.

Effect of transplantation site and culture pretreatment on islet xenograft survival (rat to mouse) in experimental diabetes without immunosuppression of the host.

Recently, we reported on indefinite islet graft survival in allotransplantation (rat to rat). This was achievable without the use of any immunosuppression by performing transplantation of culture-pretreated (22 degrees C) islets of Langerhans under the renal capsule (r.c.) of chemically induced diabetic recipients. The aim of this study was to test this successful islet modulation technique in a xenogeneic animal model. Six groups of chemically induced diabetic, inbred, C57BL/6J mice received by transplantation either into the liver via the portal vein (i.po.) or under the renal capsule (r.c.) 300-350 either freshly or culture-pretreated (37 degrees C and 22 degrees C) Lewis rat islets without any immunosuppressive therapy. Histology was performed after rejection or post-transplant normoglycaemia (> 120 days) for evaluation of the graft. Transplantation of freshly isolated islets resulted in 75% graft rejection 17 days after transplantation. Using culture pretreatment at 37 degrees C, we noted 75% graft rejection 31 days after transplantation. In contrast, culture pretreatment at 22 degrees C resulted in a marked prolongation of xenograft survival, 75% graft rejection occurring 58 days after transplantation, and in two cases there was indefinite graft survival (> 120 days). Statistical analysis showed a significant prolongation of xenograft survival after culture pretreatment, with the most beneficial effect appearing after low-temperature culture at 22 degrees C (P < 0.05). Interestingly, xenograft survival was markedly prolonged only using the r.c. approach. Statistical comparison revealed a highly significant prolongation using the r.c. as transplantation site compared with i.po. (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-CD2 receptor and anti-CD2 ligand (CD48) antibodies synergize to prolong allograft survival.

Indefinite graft survival was obtained with murine cardiac allografts using the combined administration of monoclonal antibodies (mAbs) directed against the receptor ligand pair CD2-CD48. Although each antibody could prolong graft survival when given alone, neither resulted in the indefinite graft survival seen with the combination. Combined mAb administration is associated with inhibition of T cell priming and help and subsequent cytotoxic T lymphocyte generation. This indicates that the interaction between CD2 and its ligand is important for antigen priming and recognition, and combined mAbs may prove to be a useful therapeutic regimen for transplantation.

Combined anti-CD2 and anti-CD3 receptor monoclonal antibodies induce donor-specific tolerance in a cardiac transplant model.

Administration of mAb against either the CD2 or CD3 receptor prolongs graft survival in CBA recipients in a heterotopic, nonvascularized cardiac transplant model, whereas the combination of mAb produces indefinite survival. Combined alpha-CD2 plus alpha-CD3 mAb synergistically prolonged allograft survival indefinitely for C57BL/6 donor hearts (> 150 vs 13.4 +/- 0.5 days for controls, p < 0.001, Wilcoxon's sign rank). All second donor-specific C57BL/6 allografts survived > 100 days (p < 0.001) without any additional immunosuppression. Third-party BALB/c allografts were rejected in a first set fashion (14.2 +/- 0.5 days). Anti-CD2 mAb of other epitopic specificities and isotypes demonstrate equivalent immunosuppressive capacity. The combination of mAb resulted in indefinite graft survival in other strain combinations. Therefore, these results are not restricted to a particular alpha-CD2 mAb or MHC combination. Combinations of alpha-CD2 plus mAb with specificities other than to CD3 did not result in tolerance, showing that the CD2-CD3 interaction was critical for tolerance induction. CTL and MLR responses from tolerant animals were normal both to H-2b and H-2d stimulators, indicating that clonal deletion of effector T cells did not occur. Adoptive transfer of naive recipient type cells broke tolerance, showing that graft adaptation was not the major determinant of tolerance maintenance. Flow cytometric analysis demonstrated that tolerance was not associated with deletion of T cells. The results imply that the mechanism of tolerance induction is related to suppression and/or anergy of helper and effector cells at the time of allografting, whereas maintenance of tolerance is associated with anergy in the Th cell compartment.

Induction of specific unresponsiveness to rat islet allografts by intrathymic UVB donor spleen cells.

Recently, we showed that intrathymic (i.t.) injection of UVB-irradiated (600 J/m2) spleen cells induces donor-specific unresponsiveness to cardiac allografts in the sublethally irradiated (200 rads, TBI) recipients in the Lewis-to-ACI rat combination. This study examined if i.t. injection of UVB donor SC could induce specific unresponsiveness to neovascularized (islet) allografts in the same rat combination of Lewis-to-ACI. Streptozotocin-induced diabetic ACI rats pretreated with sublethal TBI (200 rads) 7 days prior to intraportal transplantation of freshly isolated Lewis islets reject their grafts in 10.2 +/- 2.9 days compared with rejection of islets in 8.5 +/- 2.6 days in unmodified controls. Recipient pretreatment with i.t. injection of UVB donor SC combined with sublethal TBI (200 rads) 7 days prior to islet transplantation induced indefinite graft survival (> 200 days) in 3 of 7 animals. Similar treatment failed to prevent acute rejection of third-party (WF) islets, thus demonstrating donor-specificity. Treatment with sublethal TBI (200 rads) on the day of islet transplantation led to permanent graft survival in 2 of 5 animals that received i.t. inoculation of UVB donor SC 7 days prior to islet transplantation. Conditioning of the recipients with a sublethal TBI dose of 300 rads on the day of islet transplantation, which alone delayed graft rejection for only 19 days, led to indefinite graft survival (> 150 days) in all animals pretreated with i.t. injection of UVB donor SC. Extrathymic inoculation of donor UVB SC via subcutaneous, intraperitoneal, intratesticular, and intravenous routes, respectively in similarly prepared animals failed to prolong islet survival, thus confirming the privileged position of the thymus in the induction of tolerance. Our findings suggest that this new strategy of immunomodulation with donor UVB SC is potentially useful for induction of donor-specific unresponsiveness.

Tolerance Induction To Rat Islet Allografts By Intrathymic Inoculation Of Donor Spleen Cells

Intrathymic (i.t.) injection of UVB donor spleen cells induces donor-specific unresponsiveness to cardiac and islet allografts in sublethally irradiated recipients in the low-responder Lewis-to-ACI rat combination. This study examined whether unresponsiveness to islet allografts could be achieved following i.t. inoculation of untreated or UVB-irradiated donor SC under the cover of peritransplant immunosuppression with sublethal TBI or ALS in the high-responder combination of WF-to-Lewis rats. The results of this study show that i.t. injection of untreated SC combined with sublethal TBI or ALS led to permanent islet allograft survival in 50% of recipients, while i.t. injection of UVB donor SC combined with sublethal TBI or ALS resulted in indefinite graft survival in 80-100% of recipients. Third-party (BN) islets were rejected normally in this model, confirming donor-specificity of unresponsiveness. Extrathymic inoculation of UVB-treated donor SC by the subcutaneous, intratesticular, or intravenous routes in similarly immunosuppressed animals did not result in any prolongation of islet allograft survival, thus confirming the importance of the thymus in induction of tolerance in this model. The unresponsive recipients that were challenged with 2nd-set allografts 100 days after islet transplantation, permanently accepted donor-type but not third-party (ACI) cardiac allografts, thus proving that recipients are indeed tolerant to donor alloantigens and that such tolerance is donor- and not organ-specific. This study proposes a novel strategy of immunomodulation that may be useful in induction of specific unresponsiveness to organ allografts.

Induction of donor-specific unresponsiveness to rat cardiac allografts by pretreatment with intrathymic donor MHC class I antigens.

Since intrathymic injection of UV-B-irradiated spleen cells induces donor-specific unresponsiveness in the sublethally irradiated (200 rads TBI) recipients, while intrathymic injection of naive SC leads to acute graft rejection, we hypothesized that presentation of MHC class I rather than MHC class II antigens to immature T cells in the thymus may convey a tolerogenic signal to the recipient. The present study was designed to examine if intrathymic injection of naive MHC class I-positive resting T lymphocytes can induce antigen-specific unresponsiveness to cardiac allografts in the Lewis-to-ACI rat combination. The results showed that intrathymic injection of resting Lewis T cells consistently induced indefinite graft survival (> 300 days) in sublethally irradiated (200 rads TBI) ACI recipients while similar treatment failed to prevent the rejection of third-party (Wistar-Furth) cardiac allografts, thus demonstrating the specificity of the immunologic unresponsiveness to donor alloantigens. Examination of the timing of intrathymic antigen presentation relative to cardiac transplantation that would achieve 100% permanent graft survival in the Lewis-to-ACI rat combination showed that the optimal time was 7 days before allografting, while peritransplant and immediate post-transplant intrathymic inoculation of donor T cells was relatively ineffective in the induction of unresponsiveness to donor grafts. We also showed that removal of the antigen-containing thymus in the sublethally irradiated recipients with functioning cardiac allografts consistently caused graft rejection if performed earlier than 21 days after heart transplantation; thymectomy after 21 days of organ transplantation did not affect indefinite survival of the grafts. Thus, it appears that the maintenance of peripheral tolerance to the grafts after 21 days of transplantation may be dependent on the presence of a new clone of antigen-specific tolerant host T cells. These results confirm the immunologic privileged position of the thymus in the induction of central and peripheral tolerance, and suggest that pretreatment with intrathymic MHC class I alloantigens is potentially useful in the induction of unresponsiveness to donor vascularized allografts in adult animals and in man.

Induction of Donor-Specific Tolerance to Rat Cardiac and Small Bowel Allografts by Intrathymic Inoculation of Donor T-Cells

We have shown that donor-specific unresponsiveness to cardiac and islet allografts can be induced by intrathymic (IT) inoculation of uv-B-irradiated spleen cells or resting T-cells in the Lewis-to-ACI rat combination. To examine if tolerance to cardiac and small intestinal allografts can be induced in adult animals, we employed intrathymic inoculation of resting donor T-cells combined with sublethal total body irradiation of 200 rads on Day -7 relative to organ transplant and consistently induced permanent donor-specific cardiac allograft survival (>300 days) and small bowel transplant (SBT) survival (>100 days) in the Lewis-to-ACI rat combination. Similarly, IT inoculation of T-cells on Day -7 combined with 1 ml ALS on Days -7 and 0 relative to SBT resulted in indefinite donor-specific graft survival (>100 days) in all recipients without any evidence of graft-versus-host disease (GVHD) in the high responder of Wistar-Furth (WF)-to-Lewis rat combination. In contrast, WF SBT was promptly rejected in recipients pretreated with intravenous administration of donor T-cells and ALS, thus confirming the privileged position of the thymus in the induction of tolerance. The long-term unresponsive ACI recipients challenged 150 days after cardiac transplant with a second-set graft specifically and permanently (>120 days) accepted the second-set SBT without rejecting the primary cardiac grafts and without developing GVHD. Third-party grafts were rejected in a normal fashion. In vitro cell-mediated lymphocytotoxicity assay showed that T-cells obtained from the long-term unresponsive animals had no detectable cytotoxic activity to donor target cells compared to cells obtained from naive rats. Clonal anergy or deletion of precursor cytotoxic T-cells may be the explanation of our findings. This new strategy of thymic reeducation in adult animals is potentially useful in the induction of specific tolerance and in the prevention of GVHD in SBT.

Intestinal allograft survival in the rat following pretreatment with donor-specific UV-B-irradiated leukocytes and peritransplant immunosuppression with cyclosporine.

Previous studies from our laboratory showed that pretreatment with ultraviolet-B-irradiated donor leukocytes (UV-B DL) combined with brief peritransplant cyclosporine (CyA) resulted in indefinite survival of Wistar/Furth rat cardiac allografts in Lewis recipients. This study was designed to examine the effect of pretransplant UV-B DL with or without peritransplant CyA on orthotopic intestinal allografts in the same rat strain combination. The results showed that while low-dose CyA treatment alone (10 mg/kg i.m. on days 0, +1, and +2) had no effect on intestinal allograft rejection, 20 mg/kg (on days 0, +1, and +2) CyA significantly (P less than or equal to 0.001) prolonged graft survival, with 33% of the hosts surviving indefinitely. The highest dose of CyA (30 mg on days 0, +1, and +2) abrogated rejection, but most transplant recipients succumbed to infection and functional ileus due to a toxic side effect of CyA. Pretreatment with UV-B DL on days -14 and -7 alone did not prolong intestinal allograft survival. Combination of a subtherapeutic CyA dose (20 or 10 mg/kg) given on days 0, +1, and +2 with pretransplant UV-B DL on days -14 and -7 did not alter the survival of intestinal allografts compared to treatment with CyA alone. This suggests that pretreatment with UV-B DL with or without peritransplant administration of CyA has no effect on intestinal allograft survival, in contrast to the effect of such combined treatment on cardiac allograft survival, where indefinite graft survival is observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Intestinal allograft survival in the rat following pretreatment with donor-specific UV-B-irradiated leukocytes and peritransplant immunosuppression with cyclosporine

Abstract. Previous studies from our laboratory showed that pretreatment with ultraviolet-B-irradiated donor leukocytes (UV-B DL) combined with brief peritransplant cyclosporine (CyA) resulted in indefinite survival of Wistar/Furth rat cardiac allografts in Lewis recipients, This study was designed to examine the effect of pre-transplant UV-B DL with or without peritransplant CyA on orthotopic intestinal allografts in the same rat strain combination. The results showed that while low-dose CyA treatment alone (10 mg/kg i.m. on days 0, +1, and + 2) had no effect on intestinal allograft rejection, 20 mg/kg (on days 0, + 1, and + 2) CyA significantly (P< 0.001) prolonged graft survival, with 33% of the hosts surviving indefinitely. The highest dose of CyA (30 mg on days 0, + 1, and + 2) abrogated rejection, but most transplant recipients succumbed to infection and functional ileus due to a toxic side effect of CyA. Pretreatment with UV-B DL on days-14 and-7 alone did not prolong intestinal allograft survival. Combination of a subtherapeutic CyA dose (20 or 10 mg/kg) given on days 0, + 1, and + 2 with pretransplant UV-B DL on days-14 and-7 did not alter the survival of intestinal allografts compared to treatment with CyA alone. This suggests that pretreatment with UV-B DL with or without peritransplant administration of CyA has no effect on intestinal allograft survival, in contrast to the effect of such combined treatm̂ent on cardiac allograft survival, where indefinite graft survival is observed. This difference in the effect of pretransplant UV-B DL on intestinal and cardiac allograft survival is most likely due to organ-specific immunogenicity, particularly to the relative density of class I and II histocompatibility antigens present in heart or intestine.

Thymus-mediated immune tolerance to renal allograft is donor but not tissue specific.

Studies were conducted in Lewis (RT1l) rats to determine whether the process of unresponsiveness to kidney graft induced by the intrathymic glomerular transplantation were donor-strain specific as suggested by previous studies (Remuzzi et al., Lancet 1991;337:750-752). When glomeruli from Sprague-Dawley rats were injected in the thymus of Lewis rats, the subsequent kidney graft from a "third party" Brown-Norway (RT1n) rejected within 9 to 14 days. Moreover, an alternative site for glomerular antigen inoculation, such as i.p. administration, failed to induce a state of unresponsiveness to renal allograft. Whether tolerance was tissue specific was investigated by intrathymic injection of a preparation of donor blood cells that only included white cells. Such a maneuver, followed 10 days later by a kidney transplant, allowed indefinite renal graft survival in all animals, whereas all rats injected intrathymically with blood cell medium alone rejected the kidney graft in 8 to 11 days. Shortening the time interval between intrathymic injection of blood cells and kidney transplantation still allowed the graft to survive indefinitely. Finally, Lewis (RT1l) rats with chronic renal failure injected intrathymically with blood cells from Brown-Norway (RT1n) rats tolerated indefinitely a subsequent kidney graft from the same donor. These findings indicate that (1) the induction of immune tolerance to renal allograft induced by intrathymic injection of antigens is donor but not tissue specific; (2) the time interval between intrathymic injection of donor cells and the subsequent kidney transplantation can be reduced to 24 h; and (3) uremia does not preclude the possibility of renal allograft tolerance after the thymus procedure.

IgM AND IgG ALLOANTIBODY PRODUCTION BY SPLENOCYTES AND DEPOSITION IN RAT RENAL ALLOGRAFTS ARE DECREASED BY DONOR-SPECIFIC BLOOD TRANSFUSION

Untreated PVG (RT1c) rats reject ACI (RT1a) renal grafts in 6-8 days. Autologous (PVG) blood transfusions (ABT) do not alter ACI allograft rejection, but donor-specific blood transfusions (DSBT) 7 or 11 days prior to transplantation usually results in indefinite graft survival. We have reported previously that DSBT is associated with the development of antiidiotypic antibody and reduced circulating cytotoxic alloantibodies in this model. To further define the effects of DSBT on the alloantibody responses to renal allografts, we examined PVG rats that received DSBT or ABT prior to ACI renal transplantation. Antibody production by cells in the spleen was investigated by tissue culture techniques; circulating antibody titers were measured by antibody binding to target ACI lymphoblasts with flow cytometry; and antibodies bound to the ACI allograft were recovered by hypertonic acid elution and quantitated by flow cytometry and ELISA. Seven days after DSBT alone, circulating IgM alloantibodies to ACI reached peak titers. After renal allografting, serum IgM alloantibody titers decreased in DSBT-pretreated rats and little IgG could be detected. In contrast, renal allografts in ABT-pretreated rats elicited high titers of IgM and moderate titers of IgG in the circulation by 5-7 days posttransplantation. Spleens harvested one week posttransplantation from ABT-pretreated rats produced high titers (16-32) of IgM and IgG antibodies to ACI antigens, but no such antibody production was detected in spleens cultured from DSBT-pretreated rats. In addition, 4-32-fold more IgM and IgG was eluted from kidneys removed 6-7 days after grafting to ABT-treated rats than from allografts in DSBT-treated rats. IgG2a was the predominant subclass of IgG that bound to target ACI cells. No IgA was detected in graft eluates from any rat. Polyacrylamide gel electrophoresis demonstrated that the eluates contained predominantly IgM and IgG without significant contamination by other serum proteins. These data suggest that DSBT decreases the levels of IgM and IgG normally produced in the spleen and deposited in the graft following renal transplantation. Because IgM fixes complement and IgG (especially IgG2a) triggers ADCC, the reduced deposition of IgM and IgG in the graft may be of particular importance in DSBT enhancement.

RO 23–6457 prolongs survival of vascularized allografts in rodents and primates

The ability of RO 23–6457, a retinoid compound with marked in vitro immunosuppressive properties, to prolong vascularized allografts was examined in several in vivo transplantation models. In the murine heterotopic heart model, efficacy was shown in two H-2 incompatible strain combinations, with indefinite graft survival at some doses. In the rat heterotopic heart model, oral administration prolonged Wistar—Furth grafts in Lewis hosts an average of 1 week, with no long-term survivors at a variety of doses. Given subcutaneously, grafts were further prolonged, but the compound proved toxic. In a bilaterally nephrectomized renal transplant model in cynomolgus monkeys treated intravenously at a dose of 2 mg/kg/day, host survival was prolonged to 18, 32, 33, and 74 days, compared with 11, 11, 12, 13, and 26 days in untreated controls ( P < 0.05 by rank-sum testing). The three shorter surviving recipients died from anorexia and weight loss with normal renal function, while the longest survivor rejected its kidney when exhaustion of iv sites precluded further treatment. The toxic effects of the compound resemble the syndrome of hypervitaminosis A. RO 23–6457 will prolong graft survival as a single agent, justifying further preclinical testing and efforts to reduce toxicity.