Acute Xenograft Rejection Mediated by Antibodies Produced Independently of TH1/TH2 Cytokine Profiles

There is substantial support for the hypothesis that T(H)1 cytokine responses are critical for the normal elaboration of allograft rejection. Recent studies by Wang et al. (1) underscore the importance of T(H)2 responses in xenograft rejection and revealed that T(H)1 cytokines, IL-12 and interferon-gamma (IFN-gamma), can negatively regulate the development of humoral responses necessary for xenograft rejection. Their exceptional studies prompted us to test whether the ability of allografts to elicit cellular rejection and xenografts to induce humoral rejection also result from the differential ability to induce T(H)1 and T(H)2 responses. We compared the kinetics of antibody and cytokine (IFN-gamma and IL-4) production in C57BL/6 mice following allograft transplantation with BALB/c hearts and in C57BL/6 and BALB/c mice following transplantation with Lewis rat hearts. We also compared the ability of BALB/c mice, deficient in the ability to produce IL-4 or IFN-gamma, to reject xenografts and produce xenoantibodies. We observed that T(H)1/T(H)2 cytokine production minimally affected the kinetics of graft rejection but regulated the magnitude of IgG subclass production. Anti-graft IgM played a critical role in initiating acute antibody-mediated xenograft rejection, and the production antigraft IgM was unaffected by IL-4 or IFN-gamma deficiency. In contrast to the report by Wang et al. (1), we conclude that antibody-mediated xenograft rejection in the concordant Lewis rat heart-to-C57BL/6 mouse xenotransplantation model is dependent on anti-IgM production but independent of T(H) cytokine profiles.     

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.     

Non-depleting anti-CD4, but not anti-CD8, antibody induces long-term survival of xenogeneic and allogeneic hearts in alpha1,3-galactosyltransferase knockout (GT-Ko) mice

The anti-galactose-alpha1,3-galactose (Gal) antibody (Ab) response following pig-to-human transplantation is vigorous and largely resistant to currently available immunosuppression. The recent generation of GT-Ko mice provides a unique opportunity to study the immunological basis of xenograft-elicited anti-Gal Ab response in vivo, and to test the efficacy of various strategies at controlling this Ab response [1]. In this study, we compared the ability of non-depleting anti-CD4 and anti-CD8 to control rejection and antibody production in GT-Ko mice following xenograft and allograft transplantation. Hearts from baby Lewis rat or C3H mice were transplanted heterotopically into GT-Ko. Non-depleting anti-CD4 (YTS177) and anti-CD8 (YTS105) Abs were used at 1 mg/mouse, and given as four doses daily from day -2 to 1 then q.o.d. till day 21. Xenograft rejection occurred at 3 to 5 days post-transplantation in untreated GT-Ko recipients, and was histologically characterized as vascular rejection. Anti-CD4, but not anti-CD8, Ab treatment prolonged xenograft survival to 68 to 74 days and inhibited anti-Gal Ab as well as xeno-Ab production. In four of the five hearts from anti-CD4 mAbs-treated GT-Ko mice, we observed classic signs of chronic rejection, namely, thickened intima in the lumen of vessels, significant IgM deposition, fibrosis and modest mononuclear cell infiltrate of Mac-1+ macrophages and scattered T cells (CD8>CD4). Xenograft rejection in untreated, as well as anti-CD4- and anti-CD8-treated, recipients was associated with increased intragraft IL-6, IFN-gamma and IL-10 mRNA. C3H allografts were rejected in 7 to 9 days by untreated GT-Ko mice and were histologically characterized as cellular rejection. Treatment with anti-CD4 and anti-CD8 mAb resulted in graft survivals of >94.8 and 11.8 days, respectively. Anti-CD4 mAb treatment resulted in a transient inhibition of alloreactive and anti-Gal Ab production. The presence of circulating alloreactive and anti-Gal Abs at >50 days post-transplant was associated with significant IgM and IgG deposition in the graft. Yet, in the anti-CD4 mAb-treated group, the allografts showed no signs of rejection at the time of sacrifice (>100 days post-transplantation). All rejected allografts had elevated levels of intragraft IL-6, IFN-gamma and IL-10 mRNA, while the long-surviving anti-CD4-treated allografts had reduced mRNA levels of these cytokines. Collectively, our studies suggest that the elicited xeno-antibody production and anti-Gal Ab production in GT-Ko mice are CD4+ T-cell dependent. The majority of xenografts succumbed to chronic rejection, while allografts survived with minimal histological change, despite elevated levels of circulating alloAbs. Thus, immunosuppression with anti-CD4 mAb therapy induces long-term survival of allografts more effectively than to xenografts.     

The Classical Complement Pathway in Transplantation: Unanticipated Protective Effects of C1q and Role in Inductive Antibody Therapy

Though complement (C) deposition within the transplant is associated with allograft rejection, the pathways employed have not been established. In addition, evidence suggests that C-mediated cytolysis may be necessary for the tolerance-inducing activities of mAb therapies. Hence, we assessed the role of the classical C pathway in acute allograft rejection and its requirement for experimental mAb therapies. C1q-deficient (C1q-/-) recipients rejected allografts at a faster rate than wild-type (WT) recipients. This rejection was associated with exacerbated graft pathology but not with enhanced T-cell responses in C1q-/- recipients. However, the humoral response to donor alloantigens was accelerated in C1q-/- mice, as an early IgG response and IgG deposition within the graft were observed. Furthermore, deposition of C3d, but not C4d was observed in grafts isolated from C1q-/- recipients. To assess the role of the classical C pathway in inductive mAb therapies, C1q-/- recipients were treated with anti-CD4 or anti-CD40L mAb. The protective effects of anti-CD4 mAb were reduced in C1q-/- recipients, however, this effect did not correlate with ineffective depletion of CD4+ cells. In contrast, the protective effects of anti-CD40L mAb were less compromised in C1q-/- recipients. Hence, this study reveals unanticipated roles for C1q in the rejection process.     

Critical Role for IL-6 in Hypertrophy and Fibrosis in Chronic Cardiac Allograft Rejection

Chronic cardiac allograft rejection is the major barrier to long term graft survival. There is currently no effective treatment for chronic rejection except re-transplantation. Though neointimal development, fibrosis, and progressive deterioration of graft function are hallmarks of chronic rejection, the immunologic mechanisms driving this process are poorly understood. These experiments tested a functional role for IL-6 in chronic rejection by utilizing serial echocardiography to assess the progression of chronic rejection in vascularized mouse cardiac allografts. Cardiac allografts in mice transiently depleted of CD4+ cells that develop chronic rejection were compared with those receiving anti-CD40L therapy that do not develop chronic rejection. Echocardiography revealed the development of hypertrophy in grafts undergoing chronic rejection. Histologic analysis confirmed hypertrophy that coincided with graft fibrosis and elevated intragraft expression of IL-6. To elucidate the role of IL-6 in chronic rejection, cardiac allograft recipients depleted of CD4+ cells were treated with neutralizing anti-IL-6 mAb. IL-6 neutralization ameliorated cardiomyocyte hypertrophy, graft fibrosis, and prevented deterioration of graft contractility associated with chronic rejection. These observations reveal a new paradigm in which IL-6 drives development of pathologic hypertrophy and fibrosis in chronic cardiac allograft rejection and suggest that IL-6 could be a therapeutic target to prevent this disease.     

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.     

Normal Donor Bone Marrow is Superior to Flt3 Ligand-Mobilized Bone Marrow in Prolonging Heart Allograft Survival when Combined with Anti-CD40L (CD154)

Flt3 ligand (FL) administration markedly increases bone marrow (BM) stem cells and immature dendritic cells. We investigated the influence of CD40-CD40Ligand (CD154) pathway blockade on antidonor immunity, cytokine production, microchimerism and heart graft survival in BALB/c (H2d) recipients of fully allogeneic C57BL/10 (H2b) FL-mobilized BM (FL-BM) or normal BM. Anti-CD40L mAb strongly suppressed anti-donor T-cell proliferative responses in recipients of either normal or FL-BM, but was less efficient in inhibiting antidonor cytolytic T-cell (CTL) activity, especially in recipients of FL-BM. Interestingly, CD40L blockade was more effective in recipients of multiple compared with single donor BM infusions. Anti-donor cytokine responses revealed complete impairment of IFN-gamma, IL-4 and IL-10 production in recipients of normal BM and CD40L mAb. By contrast, and in agreement with the CTL data, mice given FL-BM retained ability to produce IFN-gamma CD40-CD40L blockade did not promote microchimerism, as evidenced by immunohistology and real time polymerase chain reaction. Nevertheless, anti-CD40L mAb enhanced heart allograft survival in recipients of FL-BM, but the effect was inferior to that achieved with normal BM. These data provide insight into the influence of growth factor-expanded donor BM and costimulation blockade on antidonor immune reactivity and transplant outcome. The comparatively poor outcome obtained using FL-BM plus anti-CD40L mAb in this model may be ascribed to the failure of effectively interdicting antidonor CTL activity.     

Differences in suppressor of cytokine signaling-1 (SOCS-1) expressing islet allograft destruction in normal BALB/c and spontaneously-diabetic NOD recipient mice

BACKGROUND: The ability to block interferon signaling represents an important strategy in designing therapies to prevent beta-cell destruction during islet allograft rejection. METHODS: The SOCS proteins regulate cytokine signaling by blocking activation of JAK/STAT proteins. Using islets isolated from SOCS-1 transgenic mice (SOCS-1-Tg; these mice express SOCS-1 under the control of the human insulin promoter and are on the C57BL6/J background), we investigated whether SOCS proteins can prevent the destruction pancreatic islet cells transplanted beneath the kidney capsule of major histocompatibility complex mismatched normal BALB/c and spontaneously-diabetic NOD mouse recipients. RESULTS: Immunohistochemical staining for insulin confirmed the presence of donor SOCS-1-Tg islets in islet allografts harvested at 22 days posttransplant, whereas grafts of control non-Tg islets were destroyed by 14 days. In contrast, SOCS-1-Tg allogeneic islets were not protected from beta-cell destruction in clinically diabetic NOD mice. The islet allografts functioned for 1 week posttransplant; however, hyperglycemia returned after 2 weeks and the grafts were destroyed. Rejection of SOCS-1-Tg and non-Tg islets in autoimmune diabetic NOD mice was associated with an infiltrate of both CD4+ and CD8+ T cells and a T2-type cytokine response (IL-4) rather than the conventional T1-type cytokine response observed during islet allograft rejection. Self-antigen upregulation in response to IFN-gamma stimulation did not appear to be a factor in rejection of the islet allografts. CONCLUSIONS: These results demonstrate that expression of SOCS-1 in islets delays islet allograft rejection but cannot circumvent destruction of the islets by the recurrence of the tissue-specific autoimmune process of spontaneous diabetes.     

Long-Term Survival of Intestinal Allografts Induced by Costimulation Blockade, Busulfan and Donor Bone Marrow Infusion

Tolerance-inducing strategies that infuse donor bone marrow cells in conjunction with costimulation blockade have not been applied to intestinal transplantation. Intestines from BALB/c mice were transplanted into C57BL/6 recipients treated with anti-CD40L mAb, CTLA4-Ig, donor bone marrow, and busulfan. The majority of mice transplanted after completion of this regimen developed hematopoietic macrochimerism, although the degree of chimerism varied widely between recipients, and experienced long-term allograft survival. T cells from these mice demonstrated donor-specific hyporesponsiveness in vitro. However, T cells from chimeric mice proliferated to donor alloantigen in vivo. Furthermore, chimeric mice bearing intestinal allografts were capable of rejecting subsequently placed donor-strain skin grafts. These data suggest that although long-term allograft survival occurs in the absence of acute or chronic rejection, recipient mice are not completely unresponsive to donor alloantigens. When intestinal transplantation was performed at the time of initial bone marrow infusion (initiation of the chimerism protocol), most recipients failed to develop chimerism and promptly rejected the intestinal allograft. Although this is the most effective protocol that we have tested using this stringent model of transplantation, our observations suggest that modifications will be necessary before it can be reliably applied to the transplantation of highly immunogeneic organs like the intestine.     

Peripheral nerve transplantation: the role of chemical acellularization in eliminating allograft antigenicity

This study tests the hypothesis that a chemically acellularized peripheral nerve allograft is as immunologically inactive as a peripheral nerve isograft. Cellular and acellular sciatic nerves were transplanted from BALB/c into C57BL/6 mice. C57BL/6 sciatic nerves were also transplanted into C57BL/6 recipients as isograft controls. Fourteen days post-transplantation, recipient splenocytes were isolated, stimulated with donor alloantigens, and IL-2, IL-4, IL-5, and gamma-IFN production was quantified using the ELISPOT technique. Cellular peripheral nerve allografts stimulated robust Th1 and Th2 systemic immune responses, whereas acellular peripheral nerve allografts elicited a response that is comparable to or lower than that quantified following peripheral nerve isograft transplantation. Chemical acellularization of peripheral nerve allografts dramatically reduces the cellular and humoral immunologic responses. These data indicate that chemically acellularized peripheral nerve constructs are relatively non-antigenic and may be a readily available source of nerve for peripheral nerve reconstruction.     

Use of anti-CD40 ligand monoclonal antibody as antirejection therapy in a murine peripheral nerve allograft model

Monoclonal antibody directed against CD40 ligand prevents acute allograft rejection in several models of solid-organ transplantation. This study describes the use of CD40 ligand as antirejection therapy in a mouse peripheral nerve allograft model. C3H mice received 8-mm nerve isografts (n = 2) or nerve allografts from C57BL donors. Treated animals (n = 11) received anti-CD40 ligand antibody applied to the graft and by intraperitoneal injections postoperatively. At 3 weeks, nerve histology from treated animals was comparable to isografts, whereas untreated allografts demonstrated virtually no signs of regeneration. Walking-track analysis demonstrated a trend toward improved functional recovery in treated animals. In conclusion, blockade of the CD40 pathway suppresses nerve allograft rejection in mice, and facilitates regeneration comparable to isografts.     

Critical Role for CD8+ T Cells in Allograft Acceptance Induced by DST and CD40/CD154 Costimulatory Blockade

Donor-specific transfusion (DST) and CD40/CD154 costimulation blockade is a powerful immunosuppressive strategy which prolongs survival of many allografts. The efficacy of DST and anti-CD154 mAb for prolongation of hepatocellular allograft survival was only realized in C57BL/6 mice that have both CD4- and CD8-dependent pathways available (median survival time, MST, 82 days). Hepatocyte rejection in CD8 KO mice which is CD4-dependent was not suppressed by DST and anti-CD154 mAb treatment (MST, 7 days); unexpectedly DST abrogated the beneficial effects of anti-CD154 mAb for suppression of hepatocyte rejection (MST, 42 days) and on donor-reactive alloantibody production. Hepatocyte rejection in CD4 KO mice which is CD8-dependent was suppressed by treatment with DST and anti-CD154 mAb therapy (MST, 35 days) but did not differ significantly from immunotherapy with anti-CD154 mAb alone (MST, 32 days). Induction of hepatocellular allograft acceptance by DST and anti-CD154 mAb immunotherapy was dependent on host CD8(+) T cells, as demonstrated by CD8 depletion studies in C57BL/6 mice (MST, 14 days) and CD8 reconstitution of CD8 KO mice (MST, 56 days). These studies demonstrate that both CD4(+) and CD8(+) T-cell subsets contribute to induction of hepatocellular allograft acceptance by this immunotherapeutic strategy.     

Allograft Acceptance Despite Differential Strain-Specific Induction of TGF-β/IL-10-Mediated Immunoregulation

We examined the immune approaches that C57BI/6 and BALB/c mice take when treated to accept cardiac allografts. C57BI/6 mice accept DBA/2 cardiac allografts when treated with gallium nitrate (GN) or anti-CD40L mAb (MR1). These allograft acceptor mice fail to mount donor-reactive delayed type hypersensitivity (DTH) responses, and develop a donor-induced immunoregulatory mechanism that inhibits DTH responses. In contrast, BALB/c mice accept C57BI/6 cardiac allografts when treated with MR1 but not with GN. These allograft acceptor mice display modest donor-reactive DTH responses, and do not develop donor-induced immune regulation of DTH responses. Real-time PCR analysis of rejecting graft tissues demonstrated no strain-related skewing in the production of cytokines mRNAs. In related studies, C57BI/6 recipients of cytokine and alloantigen educated syngeneic peritoneal exudate cells (PECs) failed to mount DTH responses to the alloantigens unless neutralizing antibodies to transforming growth factor-beta (TGF-p were present at the DTH site demonstrating regulation of cell-mediated alloimmune responses. In contrast, BALB/c recipients of cytokine-and alloantigen-educated PECs expressed strong DTH responses to alloantigens demonstrating a lack of regulated alloimmunity. In conclusion, C57BI/6 mice respond to immunosuppression by accepting cardiac allografts and generating TGF-beta-related regulation of donor-reactive T cell responses, unlike BALB/c mice that do not generate these regulatory responses yet still can accept cardiac allografts.     

Cellular rejection of fetal pancreas grafts: Differences between alio- and xenograft rejection

Abstract: Hyperacute rejection (HAR) is the major immunologic problem with vascularized xenografts between discordant donor/recipient combinations but does not occur in neovascularized grafts of organ-cultured fetal pig pancreas in either mice or cynomolgus monkeys. However, a form of cell-mediated acute rejection with quite different histopathologic features does occur with kinetics that are similar to acute cellular rejection of fetal pancreas allografts in non-immunosuppressed MHC-mismatched mice. Xenograft rejection is dominated by non-lymphoid cells, mostly eosinophils, that appear some days after transplantation. In contrast, in mouse allografts, mononuclear cells are the dominant population throughout the rejection process^. The rejected allograft site rapidly resolves to form a mature non-irifiltrated scar whereas the infiltrate in the xenograft site remains for weeks and forms a large granuloma before its eventual resolution. There are also differences in the intra-graft cytokine profile in the graft site between alio- and xenografts during acute rejection with an early predominance of IL-5 and TNF-α and an absence of TNF-γ in the xenografts. Immunosuppression with a depleting anti-CD4 mAb shows that xenograft rejection is more dependent on CD4+ve T cells but xenografts are more difficult to maintain with conventional immunosuppression that is often effective for allografts. Limited studies in primates have shown that the histopathology of fetal pig pancreas rejection is similar to that seen in mice but occurs at a faster tempo. Thus, although HAR may not be a problem in rejection of neovascularised xenografts, a vigorous form of cellular rejection is present that may require different immunosuppression than is usually used for the I control of allograft rejection.     

Marked inhibition of transplant vascular sclerosis by in vivo-mobilized donor dendritic cells and anti-CD154 mAb

BACKGROUND: Combination of donor dendritic cells (DC) and anti-CD40 Ligand (L) (CD154) monoclonal antibody (mAb) markedly prolongs heart or skin allograft survival, but the influence of this strategy in models of chronic rejection is unknown. Our aim was to ascertain the influence of in vivo-mobilized immature donor DC plus anti-CD40L mAb on vascular sclerosis in functional murine aortic allografts. METHODS: C3H He/J (C3H;H2k) mice received 2 x 106 freshly isolated, immunobead-purified (>90%) fms-like tyrosine kinase 3 ligand-mobilized C57BL/10 (B10;H2b) CD11c+ DC intravenously (IV), together with 500 microg of anti-CD40L mAb (MR1) intraperitoneally (IP) on days -7, 0, 4, and 10. Controls received either no donor cells, no mAb, or were untreated. B10 aortic grafts were transplanted in the abdominal aorta on day 0. At day 30, antidonor T-cell proliferative and cytotoxic responses and both complement fixing and immunoglobulin (Ig)G alloantibody levels were determined. Grafts were harvested on days 30 and 60 and examined by histology and immunohistochemistry. RESULTS: DC infusion alone enhanced ex vivo antidonor proliferative and cytotoxic T-cell activity. By contrast, complement-fixing alloantibody levels were reduced. Anti-CD40L mAb alone strongly suppressed each of these responses. Graft inflammatory cell infiltration, intimal smooth muscle cell proliferation, fibrosis, and elastic lamina disruption observed in untreated animals were reduced in response to anti-CD40L mAb or donor DC alone. Antidonor immune reactivity, including IgG levels, and intimal proliferation were all markedly suppressed to an overall greater extent in mice given both treatments. CONCLUSION: Whereas blockade of the CD40-CD40L pathway ameliorated transplant vasculopathy, preservation of near-normal vessel architecture was achieved by simultaneous administration of donor DC. This strategy represents a novel application of DC for suppression of chronic rejection.     

T-cell response to cardiac myosin persists in the absence of an alloimmune response in recipients with chronic cardiac allograft rejection

BACKGROUND: Immune-mediated injury to the graft has been implicated in the pathogenesis of chronic rejection. However, little is known regarding the nature of the antigen(s) involved in this immune process. We demonstrated that cardiac transplantation in mice induces an autoimmune T-cell response to a heart tissue-specific protein, cardiac myosin (CM). This response contributes to transplant rejection in that its modulation affects cardiac graft survival. This study investigates whether anti-CM T cells undergo activation and expansion in mice with chronic cardiac allograft rejection. METHODS: The frequency of CM- and donor major histocompatibility complex (MHC)-specific interferon (IFN)-gamma-producing T cells were assessed by ELISPOT in BALB/c mice, which were injected with anti-CD40L (MR1) mAb (chronic rejection group) or CTLA4Ig fusion protein (tolerant group) and transplanted with C57BL/6 cardiac allografts. RESULTS AND CONCLUSIONS: MR1-treated BALB/c recipients of C57BL/6 hearts with chronic rejection displayed a high frequency of activated CM-specific T cells, whereas the frequency of activated alloreactive T cells were similar to na?ve, nontransplanted mice. In contrast, no activation of CM-reactive T cells was detected in tolerant recipients after CTLA4Ig treatment. Therefore, in the absence of alloimmunity, chronic rejection is associated with persistence of a T-cell response against CM. Our data indicate that anti-CM autoimmunity may be involved in the immune mechanisms of chronic rejection and suggest that tolerance strategies should target both allo- and autoimmune responses to prevent this process.     

Real-time polymerase chain reaction analysis reveals an evolution of cytokine mRNA production in allograft acceptor mice

BACKGROUND: The relative contribution of pro-inflammatory and anti-inflammatory cytokines in promoting the rejection or acceptance of experimental cardiac allografts remains controversial. We hypothesized that the posttransplantation induction of a new immune response to graft alloantigens at a distant delayed-type hypersensitivity (DTH) site would force the immune system to reveal its current disposition toward graft alloantigen as it initiates the new immune response. Thus, we should be able to monitor the evolution of the immunologic relationship between allograft recipients and their grafts at any time posttransplantation by challenging the recipient for DTH responses to donor alloantigen and evaluating the cytokine profiles displayed at the DTH site. METHODS: We have used the sensitive and quantitative technique of real-time polymerase chain reaction to evaluate the patterns of donor alloantigen-induced cytokine mRNA production for interleukin (IL)-2, interferon (IFN)-gamma, IL-4, IL-10, and transforming growth factor (TGF)-beta. We evaluated cytokine mRNA expression in cardiac allografts and in donor alloantigen-challenged DTH sites in mice that have either accepted or rejected cardiac allografts. RESULTS: We observed the following. (1) Normal hearts and pinnae exhibited detectable baseline production of cytokine mRNAs: TGF-beta>IFN-gamma=IL-10>IL2->IL-4. (2) Both the accepted and rejecting cardiac allografts produced increased amounts of all cytokine mRNAs tested and displayed few quantitative differences in cytokine mRNA production. Notably, accepted allografts displayed enhanced IL-10 mRNA production on day 7 posttransplantation, but not on day 60 posttransplantation and reduced IFN-gamma mRNA production on day 60, but not day 7. (3) There was a high degree of variability in production levels among the various cytokine mRNAs, both for background levels and for allograft-stimulated levels. (4) Donor-reactive DTH sites of allograft rejector mice displayed a broad array of cytokine mRNAs, whereas the DTH sites of allograft acceptor mice displayed only IL-4 mRNA production. (5) Provision of exogenous TGF-beta or IL-10 at a DTH challenge site of allograft rejector mice caused a shift in the cytokine mRNA profile that minimized IFN-gamma and IL-2 mRNA production but spared IL-4, IL-10, and TGF-beta mRNA production. CONCLUSIONS: A broad array of cytokine mRNAs may be stockpiled for future use in cardiac allografts, regardless of whether the grafts will be accepted or rejected. This stockpile is continuously replenished for as long as the graft survives, thereby obscuring any changes in immune disposition of the graft recipient toward graft alloantigens. However, such changes can be revealed by challenge with donor alloantigens at a distant site (DTH challenge). In allograft acceptor mice, this disposition evolves from pro-inflammatory to anti-inflammatory.     

Costimulatory blockade induces hyporesponsiveness in T cells that recognize alloantigen via indirect antigen presentation

BACKGROUND: Blockade of T cell costimulation by treatment with donor-specific transfusion (DST) and anti-CD154 monoclonal antibody (mAb) induces prolonged allograft survival in mice. This effect is due in part to deletion of host CD8 and CD4 T cells that recognize alloantigen by direct presentation. The fate of host CD4 T cells that recognize alloantigen by indirect presentation, however, is unclear. METHODS: We studied Tg361 TCR transgenic CD4 T cells that recognize alloantigen by indirect presentation. Carboxyfluorescein diacetate, succinimidyl ester-labeled Tg361 cells were adoptively transferred into syngeneic nontransgenic recipients and their fate in the peripheral blood, spleen, and lymph nodes following treatment with DST and anti-CD154 was analyzed. RESULTS: Treatment of mice with DST plus anti-CD154 mAb does not delete Tg361 CD4 T cells, but instead renders them hyporesponsive to rechallenge with alloantigen. Mice circulating hyporesponsive CD4 T cells also fail to reject skin allografts. The hyporesponsive state of the T cells is not reversed by the addition of interleukin-2, anti-CD28 mAb, or an agonistic anti-CD134 mAb in the presence of antigen. These T cells are capable of activation, however, as evidenced by in vitro proliferation in response to anti-CD3 mAb. CONCLUSIONS: These results demonstrate that costimulation blockade can induce hyporesponsiveness of host CD4 T cells recognizing alloantigens by indirect presentation, thus prolonging graft survival by a mechanism that does not involve deletion of alloreactive T cells.