Aggressive skin allograft rejection in CD28-/- mice independent of the CD40/CD40L costimulatory pathway.

CD28-/- mice have been utilized to study the role of B7/CD28 and B7-CTLA4 interactions. There is evidence that CTLA4 ligation may be critical for tolerance induction. The aim of the current study is to further investigate rejection responses of CD28-/- mice and to define the role of B7-CTLA4 interactions in the absence of the CD40 and CD28 pathways. Balb/c skin allografts were transplanted onto C57BL/6 (B6) wild type or CD28-/- mice treated with anti-CD40L, CTLA4-Ig, or combination blockade. To investigate the cellular mechanism of rejection in CD28-/- recipients, mice were treated with anti-CD4 or anti-CD8 antibodies prior to treatment with costimulation blockade. The fluoroscein dye CFSE was utilized to study T cell expansion in vivo. Surprisingly, treatment of B6 CD28-/- mice with CTLA4-Ig alone (MST 12d), anti-CD40L alone (MST 13d), or combined blockade (MST 13d) had no effect on allograft survival compared to untreated B6 CD28 mice (MST 11d). CD28-/- recipients depleted of CD4+ cells and treated with CTLA4-Ig, anti-CD40L, or combination blockade also did not have prolonged survival compared with untreated mice (MST 10d). In contrast, CD28-/- recipients depleted of CD8+ cells had markedly prolonged allograft survival when treated with either anti-CD40L alone (MST 49d) or with combination blockade (MST 57d). Studies utilizing CFSE demonstrated that CD28-/- CD8+ T cells are not defective in in vivo proliferation responses compared with wild type CD8 cells. Thus, CD28-/- CD8+ T cells are responsible for aggressive rejection responses of CD28-/- mice independent of the CD40 pathway. In addition, CD40L blockade does not result in CD4+ T cell tolerance in CD28 recipients, despite an intact B7-CTLA4 pathway.     

Pathways of helper CD4 T cell allorecognition in generating alloantibody and CD8 T cell alloimmunity

BACKGROUND: The relative contributions of the "direct" and "indirect" pathways of CD4 T cell allorecognition in providing help for generating effective humoral and CD8 T cell alloimmunity remain unclear. Here, the generation of alloantibody and cytotoxic CD8 T cell responses to a vascularized allograft were examined in a murine adoptive-transfer model in which help could only be provided by transferred CD4 T cells recognizing alloantigen exclusively through the direct pathway. METHODS: Rejection kinetics and the development of alloantibody and cytotoxic CD8 T cell responses to MHC-mismatched H-2d heart grafts were compared when CD4 T cell help was present (wild-type H-2d recipients), or absent (CD4 T cell deficient, MHC class II-/- H-2b recipients [B6CII-/-]), or available only through the direct pathway (B6CII-/- mice reconstituted with wild-type CD4 T cells). RESULTS: BALB/c allografts were rejected by B6 mice rapidly (median survival time [MST] 7 days) with strong CD8 T cell effector and alloantibody responses, but were rejected by B6CII-/- mice more slowly (MST 23 days), with markedly reduced CD8 T cell responses and no detectable alloantibody. CD4 T cell reconstitution of B6CII-/- recipients accelerated heart graft rejection to near that of wild-type recipients (MST 13 days), with complete restoration of cytotoxic CD8 T cell responses but without detectable IgM or IgG alloantibody. CONCLUSIONS: Different pathways of helper T cell allorecognition are responsible for generating humoral and CD8 T cell alloimmunity. CD4 T cell help provided exclusively through the direct pathway generates strong cytotoxic CD8 T cell responses that effect rapid heart graft rejection.     

Mechanism of allorecognition and skin graft rejection in CD28 and CD40 ligand double-deficient mice

BACKGROUND: It has been shown that simultaneous blockade of CD28- and CD40-mediated costimulatory signals significantly prolongs allograft survival. Although these results led to an expectation of the establishment of specific immunotolerant therapy for organ transplantation, it became evident that these treatments rarely resulted in indefinite allograft survival. To uncover the mechanisms underlying these costimulation blockade-resistant allograft rejections, we studied the process of allogenic skin graft rejection in CD28 and CD40 ligand (L) double-deficient (double-knockout [dKO]) mice. METHODS: Skin grafts from BALB/c or BALB.B mice were transplanted to C57BL/6 background dKO mice. The frequency of CD4+ and CD8+ T cells responding to alloantigens presented by direct or indirect pathways were defined by the use of a cytostaining assay. RESULTS: BALB/c skin grafts were rapidly rejected by dKO mice. This CD28 and CD40L independent allograft rejection was inhibited by the depletion of CD8+ T cells. In vitro studies indicated that CD8+ T cells from BALB/c skin-grafted dKO mice responded to donor antigen presented only by the direct pathway. Unlike major histocompatibility complex (MHC)-mismatched donors, allogenic skin grafts from MHC-matched donors were accepted by dKO mice. CONCLUSION: In the absence of CD28 and CD40 costimulatory signals, CD8+ T cells recognize MHC antigens by the direct pathway, resulting in the rejection of skin grafts from MHC-mismatched donors. In contrast, MHC-matched and non-MHC-mismatched donor skin grafts indefinitely survive in dKO mice. These results indicated that donor-host MHC matching may still be critical to costimulation blockade therapy for organ transplantation.     

The Immunobiology of Inductive Anti-CD40L Therapy in Transplantation: Allograft Acceptance is Not Dependent Upon the Deletion of Graft-Reactive T Cells

CD40–CD40L costimulatory interactions are crucial for allograft rejection, in that treatment with anti‐CD40L mAb markedly prolongs allograft survival in several systems. Recent reports indicate that costimulatory blockade results in deletion of graft‐reactive cells, which leads to allograft tolerance. To assess immunologic parameters that were influenced by inductive CD40–CD40L blockade, cardiac allograft recipients were treated with multiple doses of the anti‐CD40L mAb MR1, which was remarkably effective at prolonging allograft survival. Acute allograft rejection responses such as IL‐2 producing helper cell priming, Th1 priming, and alloantibody production were abrogated by anti‐CD40L treatment. Interestingly, the spleens of mice bearing long‐term cardiac allografts following inductive anti‐CD40L treatment retained precursor donor alloantigen‐reactive CTL, IL‐2 producing helper cells, and Th1 in numbers comparable to those observed in naïve mice. These mice retained the ability to reject donor‐strain skin allografts, but were incapable of rejecting the original cardiac allograft, or a second donor‐strain cardiac allograft. Further, differentiated effector cells were incapable of mediating rejection following adoptive transfer into mice bearing long‐term allografts, suggesting that regulatory cell function, rather than effector cell deletion was responsible for long‐term graft acceptance. Collectively, these data demonstrate that inductive CD40–CD40L blockade does not result in the deletion of graft‐reactive T cells, but induces the maintenance of these cells in a quiescent precursor state. They further point to a tissue specificity of this hyporesponsiveness, suggesting that not all donor alloantigen‐reactive cells are subject to this regulation.     

CD8 T cell-mediated rejection of intestinal allografts is resistant to inhibition of the CD40/CD154 costimulatory pathway

BACKGROUND: Disruption of the CD40/CD154 pathway inhibits rejection in numerous models. The importance of this pathway on intestinal allograft rejection was examined in this study. METHODS: Intestinal grafts from B6C3F1 mice transplanted into C57BL/6 recipients were assessed histologically for rejection. RESULTS: The monoclonal antibody to CD154, MR1, failed to inhibit rejection in wild-type mice. Similarly, CD154-/- recipient mice rejected intestinal allografts. MR1 did inhibit early rejection in CD8-/- mice, but had no effect in CD4-/- recipients. All MR1-treated CD8-/- recipients eventually developed rejection. No benefit was observed when blockade of the CD40/CD154 pathway by MR1 was combined with blockade of the CD28/B7 pathway by mCTLA4Ig. CONCLUSIONS: These data suggest that CD4+ T cells mediating intestinal allograft rejection may be more dependent upon the CD40/CD154 pathway than CD8+ T cells. This finding highlights the importance of identifying agents that suppress CD8+ T cell-mediated rejection.     

Different mechanisms of cardiac allograft rejection in wildtype and CD28-deficient mice.

Although CD28 blockade results in long-term cardiac allograft survival in wildtype mice, CD28-deficient mice effectively reject heart allografts. This study compared the mechanisms of allogeneic responses in wildtype and CD28-deficient mice. Adoptive transfer of purified CD28-deficient T cells into transplanted nude mice resulted in graft rejection. However, this model demonstrated that the allogeneic T cell function was severely impaired when compared with wildtype T cells, despite similar survival kinetics. Cardiac allograft rejection depended on both CD4+ and CD8+ T cell subsets in CD28-deficient mice, whereas only CD4+ T cells were necessary in wildtype recipients. These results suggested that CD8+ T cells were more important in CD28-deficient than wildtype mice. In addition to the CD8+ T cell requirement, allograft rejection in CD28-deficient mice was dependent on a sustained presence of CD4+ T cells, whereas it only required the initial presence of CD4+ T cells in wildtype mice. Taken together, these data suggest that CD4+ T cells from CD28-deficient mice have impaired responses to alloantigen in vivo, thus requiring long-lasting cooperation with CD8+ T cell responses to facilitate graft rejection. These results may help to explain the failure to promote graft tolerance in some preclinical and clinical settings.     

Distinct requirements for host CD80/CD86 costimulatory molecules in cardiac versus islet rejection

The role of B7 family members CD80 and CD86 in providing costimulatory signals to T cells is well established. Interestingly, previous studies show that host CD80/CD86 expression is required for cardiac allograft rejection. However, the role for host costimulation by CD80/CD86 molecules for the rejection of neovascularized islet allografts and xenografts is unknown. The purpose of this study was to determine whether islet allografts and/or rat islet xenografts required host CD80/CD86 molecules for acute rejection. Streptozotocin-induced diabetic C57Bl/6 (B6, H-2(b)) or B6 CD80/CD86 double-deficient mice were grafted with allogeneic BALB/c (H-2(d)) islet allografts or with WF (RT1(u)) islet xenografts. Nondiabetic B6 mice were grafted with BALB/c heterotopic cardiac allografts. Consistent with previous reports, BALB/c islet allografts were acutely rejected in wild-type B6 mice could survive long-term (>100 days) in B6 CD80/CD86-deficient animals. In stark contrast, both islet allografts and WF rat islet xenografts demonstrated acute rejection in both control B6 and in B6 CD80/CD86 deficient hosts. In conclusion, varied studies imply that the inherent pathways for rejecting primarily vascularized versus cellular allografts or xenografts may be distinct. The present study illustrates this concept by showing a marked difference in the role of host-derived CD80/CD86 costimulatory molecules for cardiac allograft versus islet allograft/xenograft rejection in vivo. Although such costimulation is rate limiting for cardiac allograft rejection, these same molecules are not necessary for acute rejection of either islet allografts or xenografts.     

Prolongation of islet allograft survival by coexpression of CTLA4Ig and CD40LIg in mice

BACKGROUND: B7/CD28 and CD40/CD40L have been well established as important costimulatory pathways. Cytotoxic T lymphocyte-associated antigen-4 (CTLA4) delivers negative signals to antigen-presenting cells to down-regulate proinflammatory responses and competitively inhibits the binding of B7 and CD28. Signals from the CD40/CD40L costimulatory pathway also play an important role in acute rejection of organ grafts. METHODS: Recombinant adenoviruses Ad-sCD40LIg-IRES2-CTLA4Ig, Ad-CTLA4Ig, and Ad-sCD40LIg were constructed to express sCD40LIg and CTLA4Ig simultaneously or separately as described previously. Streptozocin-induced diabetic BALB/c mice were injected with recombinant adenovirus, receiving approximately 500 donor islets isolated from C57BL/6 mice under the left kidney capsule. Five groups were assigned according to the treatment: nontreated group, Ad-Shuttle-CMV-treated group, Ad-CTLA4Ig-treated group, Ad-sCD40LIg-treated group, and Ad-sCD40LIg-IRES2-CTLA4Ig-treated group. The islet graft mean survival time (MST) was evaluated in the present study. RESULTS: Compared to the islet graft MST of the nontreated group (7.3+/-0.82 days) or Ad-Shuttle-CMV-treated group (7.2+/-1.47 days), the Ad-CTLA4Ig-treated and Ad-CD40LIg-treated islet graft survivals in recipients were 56.3+/-13.71 days (P<.01) and 47.3+/-15.64 days (P<.05), respectively. The islet graft MST was dramatically prolonged to 116.3+/-20.32 days in the Ad-sCD40LIg-IRES2-CTLA4Ig-treated group (P<.01). CONCLUSION: Simultaneous blockade of the CD40/CD40L and B7/CD28 costimulatory pathways via coexpression of sCD40LIg and CTLA4Ig mediated by replication-defective adenovirus may be an acceptable method to induce immune tolerance.     

Spontaneous allograft tolerance in B7-deficient mice independent of preexisting endogenous CD4+CD25+ regulatory T-cells

BACKGROUND: Acute cardiac allograft rejection requires host, but not donor, expression of B7-1/B7-2 costimulatory molecules. However, acute cardiac rejection requires direct antigen presentation by donor-derived antigen presenting cells to CD4 T-cells and does not require indirect antigen presentation to CD4 T-cells. Given this discrepancy in the literature and that the consequence of allograft exposure in B7-deficient mice is unknown; the goal of the study was to examine the antidonor status of allografted B7-1/B7-2-deficient hosts. METHODS: C57Bl/6 B7-1/B7-2-/- mice were grafted with heterotopic BALB/c hearts. Recipients bearing long-term surviving allografts were used to examine the status of antidonor reactivity in vitro and in vivo. Tolerance was examined in vivo through adoptive transfer of splenocytes from graft-bearing animals to secondary immune-deficient Rag-1-/- hosts bearing donor-type or third-party cardiac allografts and by regulatory T-cell depletion with anti-CD25 antibody. RESULTS: When transferred to B7-replete Rag-1-/- recipients, cells from na?ve B7-1/B7-2-/- mice readily initiated cardiac allograft rejection. However, splenocytes transferred from long-term allograft acceptor B7-1/B7-2-/- hosts failed to reject donor-type hearts but acutely rejected third-party allografts. In addition, such cells did not reject (donorxthird-party) F1 allografts. Finally, in vivo depletion of regulatory T-cells did not prevent long-term acceptance. CONCLUSIONS: Results demonstrate that B7-deficient T-cells are capable of acute cardiac allograft rejection in a B7-replete environment. Importantly, results also show that B7-deficient hosts do not simply ignore cardiac allografts, but rather spontaneously develop transferable, donor-specific tolerance and linked suppression in vivo. Interestingly, this tolerant state does not require endogenous CD4+CD25+ regulatory T-cells.     

Effect of Inflammation on Costimulation Blockade-Resistant Allograft Rejection

Previously, we reported that allogeneic skin grafts were rapidly rejected by CD28 and CD40 ligand double deficient mice mediated by CD8⁺ T cells. These results indicated that some elements in addition to CD28- and CD40-mediated costimulation provide stimulatory signals for the activation of donor-specific CD8⁺ T cells. In this report, we investigated the role of inflammation associated with transplantation on costimulation-independent priming of CD8⁺ T cell during graft rejection. B6 RAG1 KO mice were transplanted with BALB/c-skin and adoptively transferred with syngeneic CD8⁺ T cells the same day or 50 days after transplantation. When blockade of CD28- and CD40-mediated costimulation failed to prevent acute rejection of freshly transplanted skin grafts, it efficiently delayed rejection of well-healed skin grafts. These results showed that factors associated with transplantation have essential roles in inducing costimulation blockade-resistant allograft rejection. Costimulation blockade failed to prevent acute graft-infiltration of NK cells and increasing expression of intragraft IL-12 and IL-15. These factors may trigger the graft-infiltration and priming of CD8⁺ T cells to induce costimulation blockade-resistant allograft rejection.     

A new T-cell receptor transgenic model of the CD4+ direct pathway: level of priming determines acute versus chronic rejection

BACKGROUND: T-cell receptor transgenic (TCR-tg) mouse models with direct CD4 alloreactivity will help elucidate mechanisms of transplant rejection and tolerance in vivo. Although such models exist, they are limited by unusual strain combinations or are based on model antigens. METHODS: A TCR-tg mouse with direct CD4 specificity in the widely used BALB/c donor --> C57BL/6 host strain combination was created. This TCR-tg mouse, named 4C, was selected for reactivity against BALB/c dendritic cells in order to model early priming events after transplantation. The response of 4C T cells to skin and heart transplants were characterized. RESULTS: The alloantigen is restricted by I-A and appears to be widely distributed in mouse tissues. 4C T cells are able to acutely reject skin but not heart allografts. Paradoxically, heart grafts elicited a stronger proliferation and effector function of TCR-tg T cells than skin grafts. 4C T cells caused cardiac allograft vasculopathy in the absence of other T cells and alloantibodies, suggesting a role for the direct pathway in chronic rejection. Augmentation of priming with an infusion of donor-derived dendritic cells resulted in acute heart allograft rejection by 4C T cells, demonstrating that the level of priming can play a role in determining acute versus chronic rejection by the CD4 direct pathway. CONCLUSIONS: Rejection of a graft by the direct CD4 pathway is determined by graft susceptibility to rejection, as well as the degree of T-cell priming caused by the graft. Grafts that are not acutely rejected can develop transplant vasculopathy mediated by the direct CD4 T cells.     

Reepithelialized orthotopic tracheal allografts expand memory cytotoxic T lymphocytes but show no evidence of chronic rejection

BACKGROUND: Acute rejection of mouse tracheal allografts is characterized by infiltration of the lamina propria with CD4+/CD8+ T cells that leads to the destruction of the epithelium and luminal obliteration. The donor epithelium is progressively replaced by recipient-derived epithelium. Once allograft reepithelialization has occurred, immunosuppression can be withdrawn without inciting acute rejection. We hypothesize that reepithelialization will also prevent chronic rejection of the trachea after withdrawal of immunosuppression. METHODS: BALB/c tracheal grafts were transplanted orthotopically into allogeneic C57BL/6 recipients. Allografted mice were nonimmunosuppressed for 10 or 100 days or immunosuppressed with cyclosporine A continuously for 50 days and then withdrawn from immunosuppression for an additional 50 days. In addition, grafts from this group were then heterotopically retransplanted into isogenic C57BL/6 or allogeneic BALB/c recipients to assess their immunogenicity. RESULTS: Cyclosporine A-treated mice showed no signs of chronic rejection or priming of cellular immunity as measured by proliferation and cytokine secretion in a mixed leukocyte reaction. However, there was a notable expansion of memory CD8+ T cells specific for donor major histocompatibility complex. When these tracheal allografts were retransplanted heterotopically into C57BL/6 or BALB/c, they demonstrated reduced responses toward BALB/c and primed responses toward C57BL/6, respectively. These results suggest that the grafts express a chimeric phenotype consisting of both BALB/c and C57BL/6 antigens. CONCLUSION: These observations suggest that long-term withdrawal of immunosuppression does not lead to chronic tracheal rejection even in the presence of alloantigen specific cytotoxic T-lymphocyte responses and that the reepithelialized grafts may contain donor elements that impact the generation of immunity.     

Contribution of CD40-CD154-mediated costimulation to an alloresponse in vivo.

BACKGROUND: Costimulation through CD40-CD154 plays an important role in T-cell activation. Although systemic administration of anti-CD154 antibody prevents or delays rejection of organ allografts in animal models, the molecular mechanisms responsible for this effect are not well defined. METHODS: We have previously demonstrated that priming of mice (H2d) with CD40-/- but not with wildtype naive B cells (H2b) leads to alloantigen-specific T-cell hyporesponsiveness in vitro. In the present study, we investigated whether such priming modifies allograft rejection in a major histocompatibility complex-mismatched murine cardiac transplantation model. RESULTS: Priming of hosts with donor-specific CD40-/- B cells delayed rejection of subsequently transplanted wild-type cardiac allografts by 8.0 days (P<0.001). The lack of CD40 on the cardiac graft delayed rejection in unprimed or primed hosts by 3-5 days. Prolongation of graft survival correlated with the failure of infused CD40-/- B cells to express B7.2 and ICAM-1 in vivo. CONCLUSIONS: Our data suggest that CD40-CD154 costimulation contributes to T cell priming to alloantigens in vivo and to a second set rejection phase in which donor antigens are presented to primed T cells.     

New Insights in CD28-Independent Allograft Rejection

CD28 costimulatory blockade induces tolerance in most murine transplant models but fails to do so in stringent transplant models, such as skin transplantation. The precise immunological mechanisms of CD28-independent rejection remain to be fully defined. Using two novel mouse strains in which both CD28 and either CD4 or CD8 are knocked out (CD4(-/-)CD28(-/-) or CD8(-/-)CD28(-/-) mice), we examined mechanisms of CD28-independent CD4(+) or CD8(+) T-cell-mediated allograft rejection. CD4(-/-)CD28(-/-) and CD8(-/-)CD28(-/) deficient mice rejected fully allogeneic skin allografts at a tempo comparable with that in wild-type mice. Rejection proceeded despite significant reduction in alloreactive T-cell clone sizes suggesting the presence of a subset of T cells harnessing alternate CD28-independent costimulatory pathways. Blockade of CD40-CD154 and CD134-CD134L, but not ICOS-B7h pathways in combination significantly prolonged allograft survival in CD8(-/-)CD28(-/-) recipients and to a lesser extent in CD4(-/-)CD28(-/-) recipients. Prolongation in allograft survival was associated with reduced effector-memory T-cell generation, decreased allospecific Th1 cytokine generation and diminished alloreactive T-cell proliferation in vivo. In aggregate, the data identify these two pathways as critical mediators of CD28-independent rejection by CD4(+) and to a lesser extent CD8(+) T cells, and provide novel mechanistic insights into functions of novel T-cell co-stimulatory pathways in vivo.     

Role of the CTLA4 pathway in hyporesponsiveness induced by intratracheal delivery of alloantigen

BACKGROUND: The authors previously reported that intratracheal delivery (ITD) of donor alloantigen induced donor-specific hyporesponsiveness to C57BL/10 cardiac allografts in CBA recipients and that blockade of the B7 pathways abrogated that hyporesponsiveness. In this study, the authors used a CD28-deficient model to evaluate which signal, either through CD28 or cytotoxic T-lymphocyte-associated antigen (CTLA4), is involved in the induction of hyporesponsiveness. METHODS: Seven days before transplantation of hearts from C3H/HeJ (H2k) mice into C57BL/6 (H2b) or CD28-deficient (C57BL/6 background) mice, the transplant recipients were given ITD of donor splenocytes (1 x 10(7)), alone or in combination with human CTLA4-immunoglobulin (Ig) (200 microg). RESULTS: ITD of C3H splenocytes induced donor-specific hyporesponsiveness to C3H cardiac grafts in C57BL/6 recipients (graft median survival time [MST], 40 days). Administration of CTLA4-Ig concurrently with ITD abrogated the prolonged allograft survival (MST, 12 days). Interestingly, ITD of C3H splenocytes induced prolonged survival of C3H allografts in CD28-deficient recipients (MST, 55 days). Furthermore, administration of CTLA4-Ig combined with ITD of C3H splenocytes abrogated the prolonged survival of C3H allografts in CD28-deficient recipients (MST, 7 days), whereas recipients given isotype-control antibody in combination with ITD of splenocytes had prolonged survival of C3H allografts (MST, 58 days). CONCLUSIONS: Taken together, the authors' findings indicate that a signal through CTLA4, rather than through CD28, plays an important role in the induction of hyporesponsiveness by ITD of alloantigen in this model.     

The role of B7 ligands (CD80 and CD86) in CD152-mediated allograft tolerance: a crosscheck hypothesis

BACKGROUND: The regulatory mechanism by which the B7 ligands (CD80 and CD86) direct the CD28/CD152 costimulatory pathways is unclear. This study investigated the role of CD80 and CD86 in a CD152-mediated allograft tolerance model. METHODS: A low-responding cardiac transplant model (BALB/c-->B10.A) with possible long-term acceptance was used. Immunocytochemical and flow cytometric analyses of the graft-infiltrating cells were conducted to characterize this transplant model. The influence of anti-CD80 and anti-CD86 treatments on the proliferation and interleukin (IL)-2 productions of the tolerated splenocytes (SC) was analyzed. The role of CD80 and CD86 in the induction and maintenance of the graft acceptance in this transplant model were also tested. RESULTS: B10.A mice could accept the BALA/c cardiac allografts (11/22), and an anti-CD152 antibody blocked the graft acceptance (10/10). Immunocytochemical and flow cytometric analyses showed that CD152+ cells were predominant among the CD4+ cells infiltrating the 100-day grafts of the B10.A recipients (B10.A-100). Either anti-CD80 or anti-CD86 treatment significantly enhanced polyclonal proliferation and IL-2 production of the B10.A-100 SC. Blockade of either CD80 or CD86 prohibited the tolerance transmitted by adoptive transfer, and anti-CD80 or anti-CD86 plus skin grafting undermined the established allograft tolerance. CONCLUSIONS: Both CD80 and CD86 were essential for the induction and maintenance of the CD152-mediated allograft tolerance.     

Blockade of LIGHT/HVEM and B7/CD28 signaling facilitates long-term islet graft survival with development of allospecific tolerance

BACKGROUND: Previous studies have shown that blockade of LIGHT, a T-cell costimulatory molecule belonging to the tumor necrosis factor (TNF) superfamily, by soluble lymphotoxin beta receptor-Ig (LTbetaR-Ig) inhibited the development of graft-versus-host disease. The cardiac allografts were significantly prolonged in LIGHT deficient mice. No data are yet available regarding the role of the LIGHT/HVEM pathway in more stringent fully allogeneic models such as skin and islet transplantation models. METHODS: Streptozotocin-induced chemical diabetic BALB/C mice underwent transplantation with allogeneic C57BL/6 islets and were treated with LTbetaR-Ig, CTLA4-Ig or a combination of both in the early peritransplant period. RESULTS: Administration of CTLA4-Ig or LTbeta R-Ig alone only increased graft survival to 55 days and 27 days respectively, whereas simultaneous blockade of both pathways significantly prolonged the islet allograft survival for more than 100 days. Long-term survivors were retransplanted with donor-specific (C57BL/6) islets and the grafted islets remained functional for more than 100 days. All of islet allografts were protected against rejection when the mixtures of 1x10(6) CD4+ T cells from tolerant mice and islet allografts were cotransplanted under the renal capsule of the na?ve BALB/c recipients. CONCLUSIONS: These data indicate that: 1) a synergistic effect for prolonged graft survival can be obtained by simultaneously blocking LIGHT and CD28 signaling in the stringent model of islet allotransplantation; 2) development of donor-specific immunological tolerance is associated with the presence of regulatory T-cell activity; and 3) local cotransplantation of the allografts with the regulatory T cells can effectively prevent allograft rejection and induce donor-specific tolerance in lymphocytes-sufficient recipients.     

CD4+ T-cell receptor transgenic T cells alone can reject vascularized heart transplants through the indirect pathway of alloantigen recognition

The vast array of epitopes presented by allografts and the diversity of T cells responding to them complicates mechanistic studies of rejection. To minimize these problems, we developed a transgenic (Tg) model system limited to a single T-cell receptor (TCR)/peptide/major histocompatibility complex molecule. Two alloantigen-specific CD4 T-cell clones were used to isolate cDNA encoding the TCRalpha and TCRbeta chains that recognize the Kd54-68/I-Ab epitope. Two different TCR Tg lines were produced in C57BL/6 (B6) mice and crossed onto the B6.Rag1-/- background. B6.Rag1-/- recipients of T cells from TCR Tg Rag1-/-mice promptly rejected B10.D2, but not irrelevant B10.BR, cardiac grafts. Thus, a single allogeneic epitope presented by self-major histocompatibility complex class II is sufficient to activate TCR Tg T cells and serve as a target for rejection.     

CD25+CD4+ regulatory T cells develop in mice not only during spontaneous acceptance of liver allografts but also after acute allograft rejection

BACKGROUND: Liver grafts transplanted across a major histocompatibility barrier are accepted spontaneously and induce donor specific tolerance in some species. Here, we investigated whether liver allograft acceptance is characterized by, and depends upon, the presence of donor reactive CD25CD4 regulatory T cells. METHODS: CD25 and CD25CD4 T cells, isolated from CBA. Ca (H2) recipients of C57BL/10 (B10; H2) liver and heart allografts 10 days after transplantation, were transferred into CBA. Rag1 mice to investigate their influence on skin allograft rejection mediated by CD45RBCD4 effector T Cells. RESULTS: Fully allogeneic B10 liver allografts were spontaneously accepted by naive CBA.Ca recipient mice, whereas B10 cardiac allografts were acutely rejected (mean survival time=7 days). Strikingly, however, CD25CD4 T cells isolated from both liver and cardiac allograft recipients were able to prevent skin allograft rejection in this adoptive transfer model. Interestingly, CD25CD4 T cells isolated from liver graft recipients also showed suppressive potency upon adoptive transfer. Furthermore, depletion of CD25CD4 T cells in primary liver allograft recipients did not prevent the acceptance of a secondary donor-specific skin graft. CONCLUSIONS: Our data provide evidence that the presence of CD25CD4 regulatory T cells is not a unique feature of allograft acceptance and is more likely the result of sustained exposure to donor alloantigens in vivo.     

Prolonged allograft survival in anti-CD4 antibody transgenic mice: lack of residual helper T cells compared with other CD4-deficient mice

BACKGROUND: Investigations of the role of CD4 T lymphocytes in allograft rejection and tolerance have relied on the use of mouse models with a deficiency in CD4 cells. However, in mice treated with depleting monoclonal antibody (mAb) and in MHC class II knockout (KO) mice, there are residual populations of CD4 cells. CD4 KO mice had increased CD4- CD8-TCRalphabeta+ helper T cells, and both strains of KO mice could reject skin allografts at the normal rate. In this study, transgenic mice with no peripheral CD4 cells were the recipients of skin and heart allografts. Results were compared with allograft survival in CD4 and MHC class II KO mice. METHODS: GK5 (C57BL/6 bml mice transgenic for a chimeric anti-CD4 antibody) had no peripheral CD4 cells. These mice, and CD4 and class II KO mice, received BALB/c or CBA skin or cardiac allografts. Some GK5 mice were treated with anti-CD8 mAb to investigate the role of CD8 cells in rejection. CD4 and CD8 cells were assessed by FACS and immunohistochemistry. RESULTS: BALB/c skin on GK5 mice had a mean survival time +/- SD of 24+/-6 days, compared with 9+/-2 days in wild-type mice. Anti-CD8 mAb prolonged this to 66+/-7 days. BALB/c skin survived 10+/-2 days on class II KO and 14+/-2 days on CD4 KO, both significantly less than the survival seen on GK5 recipients (P<0.001). BALB/c hearts survived >100 days in GK5 recipients and in wild-type recipients treated with anti-CD4 mAb at the time of grafting, in contrast to a mean survival time of 10+/-2 days in untreated wild-type mice. Immunohistochemistry revealed that long-term surviving heart allografts from the GK5 recipients had CD8 but no CD4 cellular infiltrate. These hearts showed evidence of transplant vasculopathy. CONCLUSIONS: The GK5 mice, with a complete absence of peripheral CD4 cells, provide the cleanest available model for investigating the role of CD4 lymphocytes in allograft rejection. Prolonged skin allograft survival in these mice compared with CD4 and MHC class II KO recipients was clearly the result of improved CD4 depletion. Nevertheless, skin allograft rejection, heart allograft infiltration, and vascular disease, mediated by CD8 cells, developed in the absence of peripheral CD4 T cells.