A major role for host MHC class I antigen presentation for promoting islet allograft survival

The purpose of this study was to determine the role for CD8 T cells versus generalized MHC class I-restricted antigen presentation in islet allograft rejection and tolerance. Diabetic C57BI/6 (B6, H-2(b)) controls, C57BI/6 CD8-deficient (CD8 KO), or MHC class I-deficient C57BI/6 (beta 2m KO) recipients were grafted with allogeneic BALB/c (H-2(d)) islets. Islet allografts were acutely rejected in untreated B6, CD8 KO, and in beta 2m KO mice, indicating that neither CD8 T cells nor host MHC class I is required for allograft rejection. We then determined the efficacy of costimulation blockade in these same strains. Costimulation blockade with anti-CD154 therapy facilitated long-term islet allograft survival in both B6 and in CD8 KO recipients. However, anti-CD154 treated beta 2m KO recipients were completely refractory to anti-CD154 therapy; all treated animals acutely rejected islet allografts with or without therapy. Also, anti-NK1.1 treatment of wild-type B6 mice abrogated graft prolongation following anti-CD154 therapy. Taken together, results show a dramatic distinction between two forms of MHC class I-restricted pathways in allograft prolongation. Although anti-CD154-induced allograft survival was CD8 T-cell independent, an intact host MHC class I-restricted (beta 2m-dependent) pathway is nevertheless necessary for allograft survival. This pathway required NK1.1+ cells, implicating NK and/or NKT cells in promoting allograft prolongation in vivo.     

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.     

CD4+ T cells are critical for corneal, but not skin, allograft rejection

BACKGROUND: The relative contribution of CD4+ or CD8+ T cells in allograft rejection remains to be fully characterized. Some reports indicate that there is an absolute requirement for CD4+ T cells in allogeneic rejection, whereas others report that CD4-depleted mice are capable of rejecting certain types of allografts. METHODS: We compared the ability of CD4- knockout (KO), CD8- KO, and normal CD4+/CD8+ mice to reject allogeneic corneal or skin grafts. We also examined delayed-type hypersensitivity and CTL responses to donor alloantigens. RESULTS: Engraftment of C57BL/6 corneas to C.B6-(n5-7) CD4-KO mice resulted in significantly higher rates of acceptance (>85%) than either C.B6-(n5-7) CD8- KO (30%) or normal BALB/c mice (40%). Likewise, mean survival times for B6 skin grafts placed on C.B6-(n5-7) CD4- KO mice (29.2 +/- 3.5 days) were significantly increased over those of normal BALB/c mice (13.2 +/- 1 days), although most CD4- KO mice (70%) eventually reject their grafts. C.B6-(n5-7) CD4- KO mice that reject allogeneic grafts fail to develop a delayed-type hypersensitivity response, but they did demonstrate significantly greater cytotoxic T lymphocyte precursor (CTLp) frequencies than did CD4- KO mice that accepted such grafts or that were not grafted. CONCLUSIONS: This study indicates that mice lacking CD4+ T cells have a significantly impaired ability to reject corneal allografts, but are able, in most cases, to reject allogeneic skin grafts. Thus, in the absence of CD4+ T cells, the likely mechanism for rejection appears to involve the generation of CD8+ CTLs.     

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.     

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.     

Decay-accelerating factor prevents acute humoral rejection induced by low levels of anti-alphaGal natural antibodies

BACKGROUND: Hyperacute and delayed vascular rejection due to natural antibodies (NAb) present major obstacles in pig-to-primate xenotransplantation. Although "supraphysiologic" expression of human complement regulatory proteins (CRPs) can prevent hyperacute rejection in discordant xenogenic recipients, their physiologic role in the homologous setting is undefined. We have evaluated the effect of the absence of decay-accelerating factor (DAF) on cardiac allograft rejection in the presence of different levels of antidonor antibodies (Ab). METHODS: DAF1-deficient (DAF KO; B6129F2 H-2) mice were used as heart graft donors to alpha1,3-galactosyltransferase deficient (GalT KO; B6, H-2) recipients. Heterotopic heart grafting was performed with or without presensitization. Graft survival, histology, and anti-alphaGal Ab levels were monitored. RESULTS: DAF knockout (KO) but not wild-type (WT) grafts showed hyperacute or acute humoral rejection in nonsensitized GalT KO mice with low levels of anti-alphaGal IgM NAb. However, humoral rejection of both DAF KO and DAF WT donor grafts occurred in presensitized GalT KO recipients. CONCLUSIONS: The expression of DAF prevents hyperacute rejection in mice with low titers of anti-alphaGal antibody. These studies demonstrate the physiologic role of DAF in preventing humoral rejection in the presence of low levels of NAb and have implications for transplantation of discordant vascularized xenografts.     

Dependence of murine obstructive airway disease on CD40 ligand.

BACKGROUND: Human lung transplantation carries a poor prognosis because of chronic rejection in the form of obliterative bronchiolitis syndrome (OBS). Using the mouse model of heterotopic tracheal transplantation, we examined the role of costimulation in the allograft rejection that characterizes obstructive airway disease (OAD). METHODS: C57BL/6 or BALB/c tracheae were implanted into wild-type control, CD28-/-, muMT (B-cell deficient), or CD40L-/- recipient mice. Grafts were explanted from 7 to 42 days posttransplantation and evaluated. RESULTS: Thickening of the basement membrane and a decrease in patent luminal area were first noted at 2 weeks in wild-type allogeneic trachea recipients and to a slightly lesser degree in CD28-/- recipients. In contrast, CD40L-/- recipient mice showed no evidence of cellular infiltrates or fibrosis in transplanted tracheae. To determine whether CD40L interacted with host or donor CD40, CD40-deficient tracheae were transplanted into CD40L+/+, CD40+/+ wild-type mice. Wild-type mice rejected CD40-/- tracheae. Tracheae were transplanted into B-cell-deficient mice to determine the role of B-cell CD40 in chronic pulmonary allograft rejection. The OAD reaction was identical in wild-type and B-cell-deficient mice. CONCLUSIONS: Development of OAD in the mouse trachea transplant model is primarily dependent on CD40L and is relatively CD28 independent. The ability of mice to reject CD40-/- tracheae demonstrated that host, not donor, CD40 is required for rejection. Furthermore, the ability of B-cell-deficient mice to reject allogeneic tracheae demonstrated that B-cell CD40-mediated responses are not required for the development of OAD.     

Paradoxical Functions of B7: CD28 Costimulation in a MHC Class II‐Mismatched Cardiac Transplant Model

Blockade of the B7: CD28 costimulatory pathway has emerged as a promising therapy to prevent allograft rejection. However, this pathway has also been demonstrated to be important for the generation and maintenance of regulatory T cells. In this study, we investigated the role of the B7: CD28 pathway in the ‘bm12 into B6’ MHC class II‐mismatched vascularized cardiac transplant model of chronic rejection. Allograft rejection was remarkably accelerated in B6 background B7DKO and CD28KO recipients compared with B6 wild‐type (WT) recipients. Allograft rejection was associated with a significantly enhanced Th1/Th2 alloreactivity and marked reduction in the ratio of regulatory T cells to CD4⁺ effector/memory cells. We noted that administration of anti‐B7‐1 and anti‐B7‐2 mAb prior to transplantation also accelerated allograft rejection. Furthermore, depleting CD25⁺ cells in B6 WT recipients of bm12 hearts prior to transplant also precipitated rejection at a similar rate. Neither B7/CD28 deficiency nor CD25 depletion affected graft survival in single MHC class I‐mismatched (bm1 into B6) recipients. This study highlights the paradoxical functions of B7: CD28 costimulation in a MHC class II‐mismatched model, in which the B7: CD28 pathway is demonstrated to be important in preventing rejection through the generation and maintenance of Tregs.     

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.     

Accelerated acute rejection of the intestinal graft in CD28-deficient mice

OBJECTIVE: Multiple in vivo studies have shown that the pace and severity of graft rejection is little or not at all changed by deleting CD28 molecules in the recipient. These findings contrast with the effects of monoclonal antibody therapy aimed the same costimulatory target. The objective of the present study was to evaluate how the acute rejection process is affected in CD28-deficient mice using a fully allogeneic, highly immunologically reactive transplant model. METHODS: Heterotopic vascularized small bowel transplants were performed in 24 recipient mice divided into 4 groups: 2 wild-type and 2 knockout groups. Each group consisted of 5 to 7 animals in which BalbC mice were used as intestinal donors to either wild-type C57BL6 or C57BL6 background CD28-deficient recipient mice. Selected endpoints were 3 and 6 postoperative days (POD). Intestinal rejection was evaluated by mucosal laser Doppler flowmetry (expressed in perfusion units) and histology (expressed in rejection grades). RESULTS: Acute rejection occurred in both wild-type and CD28-deficient groups. At POD 3, no significant difference was noted between groups in terms of mucosal perfusion and histology. At POD 6, significant differences in graft mucosal perfusion and histology revealed a more aggressive rejection in the CD28-deficient group compared to the wild-type group. CONCLUSIONS: The present study showed that the severity of intestinal graft rejection responses was amplified by deleting CD28 molecules. Together with data from other studies, these results suggest a different pattern of distribution and/or activation of CD28/B7 receptors in various organs.     

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.     

Early expression of interferon-gamma inducible protein 10 and monokine induced by interferon-gamma in cardiac allografts is mediated by CD8+ T cells

BACKGROUND: Our goal was to test the intragraft mRNA expression and production of two chemokines that are potent chemoattractants for antigen-primed T cells, interferon-gamma inducible protein 10 (IP-10) and monokine-induced by IFN-gamma, (Mig), in allogeneic heart grafts. METHODS: Syngeneic or allogeneic A/J (H-2a) hearts were heterotopically transplanted to wild-type, CD4-/-, CD8alpha-/-, or IFN-gamma-/- C57BL/6 (H-2b) recipients. To test expression of IP-10 and Mig, grafts were removed 1-8 days posttransplant for RNA isolation and Northern blot analysis. To test the potential recipient leukocyte populations mediating intraallograft expression of IP-10 and Mig, recipients were treated with anti-NK 1.1, anti-CD4, and/or anti-CD8 monoclonal antibodies before transplantation. RESULTS: Allogeneic heart grafts transplanted to wild-type, but not IFN-gamma-/-, recipients expressed IP-10 and Mig at day +2 posttransplant that increased thereafter until rejection was completed. Expression of IP-10 and Mig in isografts was low or undetectable. Cardiac allografts from CD8+ T cell depleted, but not NK cell or CD4+ T cell depleted, recipients had low to undetectable expression of IP-10 and Mig on day +2 posttransplant. Similarly, cardiac allografts from CD8-/-, but not CD4-/-, recipients had low to undetectable expression of IP-10 and Mig on day +2 posttransplant. CONCLUSIONS: Early intraallograft expression of Mig and IP-10 during primary rejection of cardiac allografts is dependent on the activities of recipient CD8+ T cells.     

CD4+ T-cell-dependent immune damage of liver parenchymal cells is mediated by alloantibody

BACKGROUND: Allogeneic hepatocytes initiate both CD4- and CD8-dependent rejection responses. The current studies address the hypothesis that acute damage of allogeneic liver parenchymal cells by the CD4-dependent pathway is alloantibody-mediated and examines immune conditions which promote activation of this pathway. METHODS: The role of alloantibody in CD4-dependent hepatocyte rejection was evaluated by assessing hepatocyte (FVB/N, H-2q) survival in CD8-depleted B-cell knockout (KO) (H-2b) recipients and by monitoring hepatocyte survival in C57BL/6.SCID (H-2b) recipients transfused with donor-reactive alloantibody. The development of donor-reactive alloantibody in C57BL/6 (H-2b), CD8-depleted C57BL/6, CD8 KO (H-2b), IFN-gamma KO (H-2b), perforin KO (H-2b), and FasL mutant gld/gld (H-2b) hepatocyte recipients was assessed. RESULTS: Hepatocyte rejection in B-cell KO mice was significantly delayed by CD8+ T-cell depletion (median survival time [MST], 35 days) when compared to untreated (MST, 8 days) and CD4-depleted (MST, 10 days) recipient mice. Transfusion of donor-reactive alloantibody into SCID recipients with functional hepatocellular allografts was sufficient to precipitate rejection in a dose-dependent fashion. Donor-reactive alloantibody was minimal in the serum of C57BL/6 hepatocyte recipients, but was produced in significant quantities in hepatocyte recipients genetically deficient in or depleted of CD8+ T cells and in recipients with impaired cytotoxic effector mechanisms. In addition, recipients with defects in Th1 immunity, such as IFN-gamma KO recipients, also produced readily detectable alloantibody. CONCLUSIONS: Collectively, these data support the hypothesis that acute immune damage of allogeneic hepatocytes by the CD4-dependent pathway is mediated by alloantibody and that this pathway is favored when Th1- or cell-mediated cytotoxic effector immune mechanisms are impaired.     

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.     

The role of leucyl-leucine methyl ester-sensitive cytotoxic cells in skin allograft rejection.

Treatment of murine spleen cells (SpC) with L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) depletes L3T4(+) and Lyt2(+) cytotoxic T lymphocyte precursors and the capacity to generate lethal graft-versus-host disease in semiallogeneic class I + II MHC and multiple non-MHC-disparate recipient mice, whereas T helper cell function is preserved. In the present studies the role of Leu-Leu-OMe-sensitive CTL in skin graft rejection was examined. C57BL/6J (B6) mice were serially thymectomized, lethally irradiated, reconstituted with T cell-depleted bone marrow, and treated with intraperitoneal injections of anti-L3T4 and anti-Lyt2 monoclonal antibodies. These adult thymectomized, bone marrow-reconstituted, T cell-depleted (ATXBM, TCD) mice were unable to reject B6xDBA/2F1 (B6D2F1) skin grafts. When such ATXBM, TCD mice were reconstituted with 7 x 10(7) control B6 SpC, acute rejection of B6D2F1 skin was observed. When B6 donor SpC were Leu-Leu-OMe-treated prior to transfer to ATXBM, TCD mice, uniform rejection of B6D2F1 skin grafts was still observed, although a significant delay in the time to rejection was observed. More rigorous T cell depletion of ATXBM, TCD host mice by infusion of antithymocyte globulin did not prevent delayed rejection of B6D2F1 skin initiated by transfer of Leu-Leu-OMe-treated B6 SpC. Despite the lack of complete prevention of skin allograft rejection, Leu-Leu-OMe treatment of B6 donor cells prevented lethal GVHD even in thymectomized B6D2F1 recipients. Precursors of anti-B6D2F1-specific CTL were greatly reduced or undetectable in unreconstituted ATXBM, TCD mice or in irradiated B6D2F1 recipients of Leu-Leu-OMe-treated B6 SpC. By contrast, ATXBM, TCD recipients of Leu-Leu-OMe-treated B6 SpC were found to contain a population of anti-class I MHC-specific CTL precursors of host origin within 28 days of reconstitution. These findings have indicated a number of features of the cells involved in skin graft rejection. First, Leu-Leu-OMe-sensitive CTL play a major role in acute rejection of class I + II MHC and multiple non-MHC antigen-disparate skin grafts. Moreover, the thymus-independent expansion of host-derived CTL precursors in ATXBM, TCD mice reconstituted with syngeneic Leu-Leu-OMe-resistant T helper cells also appears to play a role in mediating rejection of allogeneic skin grafts.     

Rejection responses to allogeneic hepatocytes by reconstituted SCID mice, CD4, KO, and CD8 KO mice

INTRODUCTION: The purpose of the current study was to investigate the capacity of CD4+, CD8+, or non-T cells to independently initiate acute rejection of allogeneic hepatocytes using reconstituted SCID, CD4 or CD8 knockout (KO) recipient mice. METHODS: Allogeneic hepatocytes (FVB/N, H-2q) were transplanted into C57BL/6.SCID (H-2b), CD4 KO (H-2b), CD8 KO (H-2b), or beige/beige (H-2b) mice. SCID mice with functioning hepatocellular allografts subsequently received purified non-T cells (NTC), CD4+, or CD8+ splenocytes. Some mice were treated with anti-CD4, anti-CD8, and/or anti-nkl.1 mAb. Recipient mice were also assessed for donor-reactive delayed-type hypersensitivity (DTH) responses and donor-reactive alloantibody production. RESULTS: Median hepatocellular allograft survival time (MST) was 28 days in CD4+ reconstituted SCID mice and 14 days in CD8+ reconstituted SCID mice. SCID hosts reconstituted with NTC demonstrated indefinite hepatocellular allograft survival (>120 days). MST was 10 days in untreated beige/beige (NK cell deficient) mice. MST was 14 days in untreated, 35 days in anti-CD4 mAb treated, and 10 days in anti-nkl.1 mAb treated CD8 KO mice. MST was 10 days in untreated, 35 days in anti-CD8 mAb treated, and 7 days in anti-nk1.1 mAb treated CD4 KO mice. Donor-reactive DTH responses were not detected in reconstituted SCID mice, were minimal in CD4 KO mice, and were prominent in CD8 KO mice after rejection of allogeneic hepatocytes. Similarly, donor-reactive alloantibody, was not detected in CD4 KO hosts, but was readily detected in CD8 KO hosts. CONCLUSIONS: These studies show that both CD4+ and CD8+ T cells (but not host NTC) can independently initiate the rejection of allogeneic hepatocytes. While hepatocyte rejection by isolated CD4+ T cells is not surprising, rejection by CD8+ T cells (in the absence of CD4+ T cells) was unusual, and may explain the failure of "standard" immunosuppressive regimens to suppress acute rejection of allogeneic hepatocytes, as noted in prior studies. Furthermore, NK cells do not appear to be required for either CD4+ T cell or CD8+ T cell initiated hepatocyte rejection.     

Role of CD4+ and CD8+ T cells in murine skin and heart allograft rejection across different antigenic desparities

The factors that influence the relative contribution of the T cell subsets to allograft rejection remain unclear. We compared skin and heart rejection in CD4 Knockout (KO), and CD8 KO mice across full-, minor-, and class II histocompatibility antigen (HA) mismatches. Skin allografts were rejected by either CD4+ or CD8+ T cells alone at any degree of antigenic mismatch. However, either the absence of CD4+ cells or a lesser degree of HA mismatch resulted in prolongation of graft survival. In contrast, fully allogeneic heart grafts were accepted in CD4 KO recipients, and minor HA mismatched heart grafts were accepted by both CD4 KO and CD8 KO mice. Thus, the T cell subsets required for allograft rejection are determined by the immunogenicity of the tissue transplanted. In the absence of CD8+ T cells, perforin and Fas ligand (FasL) but not granzyme B mRNA were detected in rejecting grafts. Thus, granzyme B is a CD8+ cytotoxic T lymphocyte (CTL)-specific effector molecule.     

Hyperexpression of Foxp3 and IDO during acute rejection of islet allografts

BACKGROUND: We investigated the hypothesis that Foxp3+ cells are an integral component of antiallograft immunity but are dominated by pathogenic effectors. METHODS: Wild-type H-2b C57BL/6 (B6) mice or B6 mice with a targeted disruption of c-Rel gene (c-Rel-/-) were used as recipients of islet grafts from allogeneic DBA/2 (H-2d) mice or syngeneic B6 mice. We developed kinetic quantitative polymerase chain reaction assays and measured intragraft expression of mRNA for Foxp3, IDO, cytolytic molecules, proinflammatory cytokines, and chemokines/receptors. RESULTS: Intraislet levels of mRNA for Foxp3, IDO, CD3, CD25, tumor necrosis factor-alpha, RANTES, IP-10, and CXCR3 were highest in DBA/2 islet allografts from WT B6 recipients compared to DBA/2 islet allografts from c-Rel-/- B6 recipients or syngeneic B6 islet grafts from WT B6 mice. The ratio of granzyme B or IFN-gamma to Foxp3 was higher with the DBA/2 islet allografts from the WT B6 recipients compared to DBA/2 islet allografts from c-Rel-/- B6 recipients or B6 islet grafts from WT B6 recipients. CONCLUSIONS: Foxp3+ cells are an integral component of acute rejection of allografts but may be dominated by pathogenic effectors.     

Allograft Rejection in a New Allospecific CD4+ TCR Transgenic Mouse

The application of TCR transgenic mice to transplantation immunology is hampered by the limited lines available. Recently, we reported CD4+ T cell receptor (TCR) transgenic mice specific for I-Abm12 expressed on B6.C.H-2bm12 mice. Here, we characterized rejection of skin and vascularized cardiac allografts in these mice, which we term ABM (for anti-bm12). In vivo proliferative experiments reveal that all CD4 T cells in ABM mice react to bm12 antigens. Surprisingly, while ABM mice have accelerated (compared to B6 recipients) rejection of bm12 skin allografts, they, like B6 recipients, fail to acutely reject bm12 cardiac allografts. This is not due to lack of immunogenicity of bm12 hearts, as these grafts are acutely rejected by primed ABM recipients, although not by primed B6 recipients. Lastly, long-term surviving bm12 grafts in ABM recipients are relatively free from chronic rejection (compared with B6 recipients), which may be due to skewing of the CD4 repertoire towards direct alloreactivity, and consequent lack of CD4 mediated indirect allorecognition as evidenced by the lack of IgG deposition in those grafts. The results indicate that a complex interplay between responder frequency, priming and repertoire dictates the occurrence, or lack thereof, of acute and chronic rejection.     

Interleukin-9 promotes eosinophilic rejection of mouse heart allografts

BACKGROUND: Eosinophils participate in allograft rejection when donor-reactive helper T lymphocytes are T-helper type 2 (Th2)-biased. Whereas the involvement of interleukin (IL)-4 and IL-5 in these forms of rejection is well established, the role of IL-9, another Th2-type cytokine promoting eosinophilia, has not been determined. METHODS: We first used real-time polymerase chain reaction to quantify IL-9 mRNA in rejected allografts in a mouse model of fully mismatched heart transplantation in which recipients were devoid of CD8 T cells and developed a Th2 alloimmune response. We then compared allograft survival in wild-type versus IL-9-deficient mice depleted of CD8 T cells. Finally, we compared the fate of major histocompatibility complex class II-mismatched cardiac transplants from wild-type versus IL-9 transgenic donors to determine the influence of IL-9 overexpression within the graft. RESULTS: The Th2 alloimmune response in CD8-deficient mice was associated with the accumulation of IL-9 mRNA in the rejected graft. In IL-9-deficient recipients depleted of CD8 T cells, eosinophil infiltration of heart allografts did not develop, but rejection still occurred. In the major histocompatibility complex class II disparate model, heart allografts from IL-9 transgenic donors were acutely rejected, whereas grafts from wild-type donors did not develop rejection. Acute rejection of IL-9 transgenic hearts was associated with massive eosinophil infiltration and prevented by neutralization of either IL-4 or IL-5. CONCLUSION: IL-9 is critically involved in heart transplant eosinophilia in conjunction with IL-4 and IL-5.