Different mechanisms control peripheral and central tolerance in hematopoietic chimeric mice.

Regulatory T cells (Treg) are important in peripheral tolerance, but their role in establishing and maintaining hematopoietic mixed chimerism and generating central tolerance is unclear. We now show that costimulation blockade using a donor-specific transfusion and anti-CD154 antibody applied to mice given bone marrow and simultaneously transplanted with skin allografts leads to hematopoietic chimerism and permanent skin allograft survival. Chimeric mice bearing intact skin allografts fail to generate effector/memory T cells against allogeneic targets as shown by the absence of IFNgamma-producing CD44(high)CD8+ T cells and in vivo cytotoxicity. Depletion of Tregs by injection of anti-CD4 or anti-CD25 antibody prior to costimulation blockade prevents chimerism, shortens skin allograft survival and leads to generation of effector/memory cytotoxic T cells. Depletion of Tregs by injection of anti-CD4 or anti-CD25 antibody two months after transplantation leads to loss of skin allografts even though mice remain chimeric and exhibit little in vivo cytotoxicity. In contrast, chimerism is lost, but skin allografts survive following naïve T-cell injection. We conclude that hematopoietic chimerism and peripheral tolerance may be maintained by different mechanisms in mixed hematopoietic chimeras.

Skin allograft maintenance in a new synchimeric model system of tolerance.

Treatment of mice with a single donor-specific transfusion plus a brief course of anti-CD154 mAb uniformly induces donor-specific transplantation tolerance characterized by the deletion of alloreactive CD8+ T cells. Survival of islet allografts in treated mice is permanent, but skin grafts eventually fail unless recipients are thymectomized. To analyze the mechanisms underlying tolerance induction, maintenance, and failure in euthymic mice we created a new analytical system based on allo-TCR-transgenic hemopoietic chimeric graft recipients. Chimeras were CBA (H-2(k)) mice engrafted with small numbers of syngeneic TCR-transgenic KB5 bone marrow cells. These mice subsequently circulated a self-renewing trace population of anti-H-2(b)-alloreactive CD8+ T cells maturing in a normal microenvironment. With this system, we studied the maintenance of H-2(b) allografts in tolerized mice. We documented that alloreactive CD8+ T cells deleted during tolerance induction slowly returned toward pretreatment levels. Skin allograft rejection in this system occurred in the context of 1) increasing numbers of alloreactive CD8+ cells; 2) a decline in anti-CD154 mAb concentration to levels too low to inhibit costimulatory functions; and 3) activation of the alloreactive CD8+ T cells during graft rejection following deliberate depletion of regulatory CD4+ T cells. Rejection of healed-in allografts in tolerized mice appears to be a dynamic process dependent on the level of residual costimulation blockade, CD4+ regulatory cells, and activated alloreactive CD8+ thymic emigrants that have repopulated the periphery after tolerization.

Allogeneic hematopoietic chimerism in mice treated with sublethal myeloablation and anti-CD154 antibody: absence of graft-versus-host disease, induction of skin allograft tolerance, and prevention of recurrent autoimmunity in islet-allografted NOD/Lt mice.

We describe a tolerance-based stem cell transplantation protocol that combines sublethal radiation with transient blockade of the CD40-CD154 costimulatory pathway using an anti-CD154 antibody. With this protocol, we established hematopoietic chimerism in BALB/c mice transplanted with fully allogeneic C57BL/6 bone marrow. The percentage of donor-origin mononuclear cells in recipients was more than 99%. In addition, all chimeric mice treated with anti-CD154 antibody remained free of graft-versus-host disease (GVHD) and accepted donor-origin but not third-party skin allografts. It was similarly possible to create allogeneic hematopoietic chimerism in NOD/Lt mice with spontaneous autoimmune diabetes. Pancreatic islet allografts transplanted into chimeric NOD/Lt mice were resistant not only to allorejection but also to recurrence of autoimmunity. We conclude that it is possible to establish robust allogeneic hematopoietic chimerism in sublethally irradiated mice without subsequent GVHD by blocking the CD40-CD154 costimulatory pathway using as few as 2 injections of anti-CD154 antibody. We also conclude that chimerism created in this way generates donor-specific allograft tolerance and reverses the predisposition to recurrent autoimmune diabetes in NOD/Lt mice, enabling them to accept curative islet allografts. (Blood. 2000;95:2175-2182)

Virus-induced abrogation of transplantation tolerance induced by donor-specific transfusion and anti-CD154 antibody.

Treatment with a 2-week course of anti-CD154 antibody and a single transfusion of donor leukocytes (a donor-specific transfusion or DST) permits skin allografts to survive for >100 days in thymectomized mice. As clinical trials of this methodology in humans are contemplated, concern has been expressed that viral infection of graft recipients may disrupt tolerance to the allograft. We report that acute infection with lymphocytic choriomeningitis virus (LCMV) induced allograft rejection in mice treated with DST and anti-CD154 antibody if inoculated shortly after transplantation. Isografts resisted LCMV-induced rejection, and the interferon-inducing agent polyinosinic:polycytidylic acid did not induce allograft rejection, suggesting that the effect of LCMV is not simply a consequence of nonspecific inflammation. Administration of anti-CD8 antibody to engrafted mice delayed LCMV-induced allograft rejection. Pichinde virus also induced acute allograft rejection, but murine cytomegalovirus and vaccinia virus (VV) did not. Injection of LCMV approximately 50 days after tolerance induction and transplantation had minimal effect on subsequent allograft survival. Treatment with DST and anti-CD154 antibody did not interfere with clearance of LCMV, but a normally nonlethal high dose of VV during tolerance induction and transplantation killed graft recipients. We conclude that DST and anti-CD154 antibody induce a tolerant state that can be broken shortly after transplantation by certain viral infections. Clinical application of transplantation tolerance protocols may require patient isolation to facilitate the procedure and to protect recipients.

Treatment of allograft recipients with donor-specific transfusion and anti-CD154 antibody leads to deletion of alloreactive CD8+ T cells and prolonged graft survival in a CTLA4-dependent manner.

A two-element protocol consisting of one donor-specific transfusion (DST) plus a brief course of anti-CD154 mAb greatly prolongs the survival of murine islet, skin, and cardiac allografts. To study the mechanism of allograft survival, we determined the fate of tracer populations of alloreactive transgenic CD8+ T cells in a normal microenvironment. We observed that DST plus anti-CD154 mAb prolonged allograft survival and deleted alloreactive transgenic CD8+ T cells. Neither component alone did so. Skin allograft survival was also prolonged in normal recipients treated with anti-CD154 mAb plus a depleting anti-CD8 mAb and in C57BL/6-CD8 knockout mice treated with anti-CD154 mAb monotherapy. We conclude that, in the presence of anti-CD154 mAb, DST leads to an allotolerant state, in part by deleting alloreactive CD8+ T cells. Consistent with this conclusion, blockade of CTLA4, which is known to abrogate the effects of DST and anti-CD154 mAb, prevented the deletion of alloreactive transgenic CD8+ T cells. These results document for the first time that peripheral deletion of alloantigen-specific CD8+ T cells is an important mechanism through which allograft survival can be prolonged by costimulatory blockade. We propose a unifying mechanism to explain allograft prolongation by DST and blockade of costimulation.

NOD mice have a generalized defect in their response to transplantation tolerance induction.

A protocol consisting of a single donor-specific transfusion (DST) plus a brief course of anti-CD154 monoclonal antibody (anti-CD40 ligand mAb) induces permanent islet allograft survival in chemically diabetic mice, but its efficacy in mice with autoimmune diabetes is unknown. Confirming a previous report, we first observed that treatment of young female NOD mice with anti-CD154 mAb reduced the frequency of diabetes through 1 year of age to 43%, compared with 73% in untreated controls. We also confirmed that spontaneously diabetic NOD mice transplanted with syngeneic (NOD-Prkdc(scid)/Prkdc(scid)) or allogeneic (BALB/c) islets rapidly reject their grafts. Graft survival was not prolonged, however, by pretreatment with either anti-CD154 mAb alone or anti-CD154 mAb plus DST. In addition, allograft rejection in NOD mice was not restricted to islet grafts. Anti-CD154 mAb plus DST treatment failed to prolong skin allograft survival in nondiabetic male NOD mice. The inability to induce transplantation tolerance in NOD (H2g7) mice was associated with non-major histocompatibility complex (MHC) genes. Treatment with DST and anti-CD154 mAb prolonged skin allograft survival in both C57BL/6 (H2b) and C57BL/6.NOD-H2g7 mice, but it was ineffective in NOD, NOD.SWR-H2q, and NOR (H2g7) mice. Mitogen-stimulated interleukin-1beta production by antigen-presenting cells was greater in strains susceptible to tolerance induction than in the strains resistant to tolerance induction. The results suggest the existence of a general defect in tolerance mechanisms in NOD mice. This genetic defect involves defective antigen-presenting cell maturation, leads to spontaneous autoimmune diabetes in the presence of the H2g7 MHC, and precludes the induction of transplantation tolerance irrespective of MHC haplotype. Promising islet transplantation methods based on overcoming the alloimmune response by interference with costimulation may require modification or amplification for use in the setting of autoimmune diabetes.

CTLA4 signals are required to optimally induce allograft tolerance with combined donor-specific transfusion and anti-CD154 monoclonal antibody treatment.

Sensitization to donor Ags is an enormous problem in clinical transplantation. In an islet allograft model, presensitization of recipients through donor-specific transfusion (DST) 4 wk before transplantation results in accelerated rejection. We demonstrate that combined DST with anti-CD154 (CD40L) therapy not only prevents the deleterious presensitization produced by pretransplant DST in the islet allograft model, it also induces broad alloantigen-specific tolerance and permits subsequent engraftment of donor islet or cardiac grafts without further treatment. In addition, our data strongly indicate that CTLA4-negative T cell signals are required to achieve prolonged engraftment of skin allograft or tolerance to islet allograft in recipients treated with a combination of pretransplant DST and anti-CD154 mAb. We provide direct evidence that a CD28-independent CTLA4 signal delivers a strong negative signal to CD4+ T cells that can block alloimmune MLR responses. In this study immune deviation into a Th2 (IL-4) response was associated with, but did not insure, graft tolerance, as the inopportune timing of B7 blockade with CTLA4/Ig therapy prevented uniform tolerance but did not prevent Th2-type immune deviation. While CTLA4-negative signals are necessary for tolerance induction, Th1 to Th2 immune deviation cannot be sufficient for tolerance induction. Combined pretransplant DST with anti-CD154 mAb treatment may be attractive for clinical deployment, and strategies aimed to selectively block CD28 without interrupting CTLA4/B7 interaction might prove highly effective in the induction of tolerance.

Prolonged survival of rat islet and skin xenografts in mice treated with donor splenocytes and anti-CD154 monoclonal antibody.

Treatment of C57BL/6 mice with one transfusion of BALB/c spleen cells and a brief course of anti-CD154 (anti-CD40 ligand) antibody permits BALB/c islet grafts to survive indefinitely and BALB/c skin grafts to survive for approximately 50 days without further intervention. We now report adaptation of this protocol to the transplantation of islet and skin xenografts. We observed prolonged survival of rat islet xenografts in mice treated with donor-specific spleen cell transfusion and anti-CD154 monoclonal antibody (mAb). Challenge islet xenografts placed on these animals indicated that graft acceptance was species-specific but not strain specific. Spleen cells from recipients bearing intact grafts led to rejection of rat islet xenografts in scid mice, suggesting that graft acceptance was not due to complete clonal deletion of xenoreactive cells. We also observed prolonged survival of rat skin xenografts in mice treated with donor-specific transfusion and anti-CD154 mAb. Prolonged survival of skin xenografts was also species specific. We conclude that treatment with appropriately timed donor-specific transfusion and anti-CD154 mAb induces durable survival of both islet and skin xenografts in mice. Because this procedure is targeted directly at CD154, a co-activation molecule expressed predominantly by activated CD4+ T-cells, the results suggest that CD4+ cells have a major role in the cellular immune response to xenografts.

Long-term survival of skin allografts induced by donor splenocytes and anti-CD154 antibody in thymectomized mice requires CD4(+) T cells, interferon-gamma, and CTLA4.

Treatment of C57BL/6 mice with one transfusion of BALB/c spleen cells and anti-CD154 (anti-CD40-ligand) antibody permits BALB/c islet grafts to survive indefinitely and BALB/c skin grafts to survive for approximately 50 d without further intervention. The protocol induces long-term allograft survival, but the mechanism is unknown. We now report: (a) addition of thymectomy to the protocol permitted skin allografts to survive for > 100 d, suggesting that graft rejection in euthymic mice results from thymic export of alloreactive T cells. (b) Clonal deletion is not the mechanism of underlying long-term graft survival, as recipient thymectomized mice were immunocompetent and harbor alloreactive T cells. (c) Induction of skin allograft acceptance initially depended on the presence of IFN-gamma, CTLA4, and CD4(+) T cells. Addition of anti-CTLA4 or anti-IFN-gamma mAb to the protocol was associated with prompt graft rejection, whereas anti-IL-4 mAb had no effect. The role of IFN-gamma was confirmed using knockout mice. (d) Graft survival was associated with the absence of IFN-gamma in the graft. (e) Long-term graft maintenance required the continued presence of CD4(+) T cells. The results suggest that, with modification, our short-term protocol may yield a procedure for the induction of long-term graft survival without prolonged immunosuppression.

Prolonged survival of mouse skin allografts in recipients treated with donor splenocytes and antibody to CD40 ligand.

Combined treatment with antibody against CD40 ligand and one transfusion of donor splenocytes prolonged survival of fully mismatched BALB/c skin allografts on C57BL/6 recipients, with approximately 20% of grafts surviving > 100 days. In vitro alloresponsiveness in treated animals was reduced in the immediate post-transplantation period, but by day 100 was increased despite the presence of a successful allograft. The presence of alloreactivity on day 100 was confirmed in vivo by adoptive transfer, which suggests that our protocol had induced either a state of "split tolerance" or "graft accommodation." Mice with skin grafts that had survived for > or = 100 days revealed no evidence of lymphoid chimerism. Treatment with donor splenocytes and antibody against CD40 ligand permits long-term survival of highly antigenic donor skin allografts despite the presence of functionally intact alloreactive lymphocytes.

Association between epidermal interleukin-10 secretion and the ability of cotransplanted skin from neonatal donors to prolong adult allograft survival.

Cotransplantation of skin from neonatal donors prolongs the survival of adult skin allografts on rabbit anti-mouse lymphocyte serum-treated and donor bone marrow cell-treated mice relative to controls (the cotransplant effect). In B6AF1 (H2a/b) recipients, cotransplants of skin from C3HeB/FeJ (C3H; H2k) neonates up to 6-7 days old prolonged the survival of adult C3H skin grafts. Skin from 9- to 10-day-old neonates was inactive. The magnitude of the cotransplant effect declined with increasing cotransplant age. Although few class II+ cells are present in skin from < 24-hr-old mice, the numbers of these cells increase rapidly after birth. On days 3-4, when the cotransplant effect is strong, their numbers in neonatal skin are greater than in adult skin. Development of class II expression continues when neonatal skin is grafted, but with an apparent 2-day lag. Because class II+ cell numbers decline in grafted adult skin, we speculate that this apparent developmental lag may be due to Langerhans cells migrating from the graft as they mature. Epidermal cells from litters of neonates were cultured overnight and supernatants were tested for interleukin (IL)-10. All 11 samples from 0- to 4-day-old neonates and 4 of 9 samples from 4- to 7-day-old neonates were positive. IL-10 was found in 1 of 9 samples from donors 8-16 days old and 1 of 7 samples from individual adult mice. Thus, there is a temporal association between the ability of neonatal skin to produce a cotransplant effect and its ability to secrete IL-10.

Effect of rapamycin on renal allograft survival in canine recipients treated with antilymphocyte serum, donor bone marrow, and cyclosporine.

Rapamycin (Rapa) monotherapy can promote renal allograft survival in dogs, but it is very toxic. To attempt to augment the effectiveness of Rapa and reduce its toxicity in a tolerance induction protocol, canine renal allograft recipients were treated briefly with antilymphocyte serum (ALS), donor bone marrow cells (BMC), and a limited course of cyclosporine (CsA). Rapa had little effect when CsA-treated recipients were given ALS on days -5 to -1 and BMC on day +1. When combined with CsA given days +13 to +42, ALS on days -5 to +7, and BMC on day +10, Rapa at 0.3 mg/kg on day +8 plus alternate days +15 to +39 significantly increased overall survival and was compatible with long-term survival after immunosuppression (6 grafts, 1 graft > 212 days, 1 graft > 470 days). Rapa appeared to prevent early rejections that can occur during treatment with these ALS/BMC/CsA protocols. Little toxicity of Rapa was observed with any treatment.

Tolerogenic behavior of skin allografts from neonatal mice.

Neonatal skin allografts can be tolerogenic when transplanted to appropriately immunosuppressed hosts. Single grafts of neonatal skin survive longer than adult skin grafts when recipients are treated with antilymphocyte serum (ALS) and donor bone marrow cells (BMC). Neonatal skin grafts can also prolong the survival of adult grafts of the same donor strain simultaneously cotransplanted with the neonatal grafts. To probe the mechanisms involved in this cotransplantation phenomenon, we delayed placement of the neonatal cotransplants relative to grafting with adult skin. Neonatal allografts placed either 7-9 days or 14 days after grafting with adult skin significantly prolonged adult graft survival in mice treated with ALS and BMC. However, day 0-placed neonatal cotransplants must remain on the recipient for > 2 weeks to prolong adult graft survival. Removal of cotransplants from ALS- and BMC-treated recipients after 7 or 14 days abrogated the cotransplantation effect. If left in place until day 21, neonatal cotransplants could significantly prolong adult graft survival, but did not induce the long-term graft survival observed in approximately 50% of the recipients whose cotransplants were not removed. Cotransplant removal after 1 year did not affect subsequent adult graft survival. Additionally, cotransplants were removed from recipients either on day 14 or from long-term graft-bearing mice and retransplanted to other ALS/BMC-treated recipients. These retransplanted grafts were unable to prolong survival of adult grafts on the new recipients. After transplant, but not before transplant, cyclophosphamide treatment of recipients prevented expression of the cotransplant effect in ALS-treated mice. However, recipient splenectomy > or = 1 week before grafting did not interfere with the effect. These results reflect on the contributions of the donor tissue, and the recipients' response, to the tolerogenic signals that permit a neonatal cotransplant to prolong adult graft survival.

Prolonged adult skin allograft survival as a result of cotransplantation with neonatal tissue. The requirement for antigen sharing between graft and cotransplant.

The survival of an adult skin allograft can be prolonged by a cotransplant of neonatal, but not adult, skin on the same recipient. We demonstrated this phenomenon using a C3HeB/FeJ (H-2k; C3H) adult graft and neonatal cotransplant donors. The median survival time (MST) for adult graft survival on B6AF1 (H-2a/b) recipients was 59 days on recipients treated with antilymphocyte serum and donor bone marrow cells. With adult or neonatal cotransplants, the MSTs for adult graft survival were 55 and > 100 days, respectively. Our current experiments explore the specificity of this phenomenon by substituting neonatal cotransplants of several allogeneic and partially allogeneic strains. Cotransplants that do not share the antigens presented by the adult graft to the recipient as foreign do not produce any prolongation of adult graft survival. Thus, cotransplants of adult or neonatal C57BL/6J (H-2b) or A/J (H-2a) strain skins had no significant effect on adult C3H graft survival. In contrast to these results, neonatal (but not adult) cotransplants that share presented antigens produce a significant cotransplant effect. The presence, on a recipient, of a neonatal cotransplant of CBA/J (H-2k) resulted in significant prolongation of adult skin grafts (MST > 150 days; P < 0.05). As well, on a different recipient strain (CAF1; H-2a/d), neonatal C3H-H-2o2/SfSn (H-2o2) skin cotransplants, sharing only background antigens and H-2Dk with the adult graft donor, caused a significant prolongation of adult graft survival relative to that seen on recipients bearing only a single adult graft (MSTs = 53 and 31 days; P < 0.05). Our results suggest that this graft-prolonging effect of neonatal cotransplantation requires that the cotransplant shares antigens with the adult graft that are presented as foreign to the recipient.