Differential expression of Smad7 transcripts identifies the CD4+CD45RChigh regulatory T cells that mediate type V collagen-induced tolerance to lung allografts

Regulatory T cells (Tregs) induced by oral tolerance may suppress immunity by production of TGF-beta that could also enhance Treg activity. However, all cells that are phenotypically Tregs in rats (CD4(+)CD45RC(high)-RC(high)) may not have regulatory function. Because Smad7 expression in T cells is associated with inflammation and autoimmunity, then lack of Smad7 may identify those cells that function as Tregs. We reported that feeding type V collagen (col(V)) to WKY rats (RT1(l)) induces oral tolerance to lung allografts (F344-RT1(lvl)) by T cells that produce TGF-beta. The purpose of the current study was to identify the Tregs that mediate col(V)-induced tolerance, and determine Smad7 expression in these cells. RC(high) cells from tolerant rats were unresponsive to allogeneic stimulation and abrogated rejection after adoptive transfer. In contrast, CD4(+)CD45RC(low) (RC(low)) cells from tolerant rats and RC(high) or RC(low) cells from normal rats or untreated allograft recipients proliferated vigorously in response to donor Ags, and did not suppress rejection after adoptive transfer. TGF-beta enhanced proliferation in response to col(V) presented to tolerant RC(high), but not other cells. In contrast to other cells, only RC(high) cells from tolerant rats did not express Smad7. Collectively, these data show that the Tregs that mediate col(V)-induced tolerance to lung allografts do not express SMAD7 and, therefore, are permissive to TGF-beta-mediated signaling.     

CD4+ T lymphocytes are not necessary for the acute rejection of vascularized mouse lung transplants

Acute rejection continues to present a major obstacle to successful lung transplantation. Although CD4(+) T lymphocytes are critical for the rejection of some solid organ grafts, the role of CD4(+) T cells in the rejection of lung allografts is largely unknown. In this study, we demonstrate in a novel model of orthotopic vascularized mouse lung transplantation that acute rejection of lung allografts is independent of CD4(+) T cell-mediated allorecognition pathways. CD4(+) T cell-independent rejection occurs in the absence of donor-derived graft-resident hematopoietic APCs. Furthermore, blockade of the CD28/B7 costimulatory pathways attenuates acute lung allograft rejection in the absence of CD4(+) T cells, but does not delay acute rejection when CD4(+) T cells are present. Our results provide new mechanistic insight into the acute rejection of lung allografts and highlight the importance of identifying differences in pathways that regulate the rejection of various organs.     

Induction of IL-10 suppressors in lung transplant patients by CD4+25+ regulatory T cells through CTLA-4 signaling

T cell-mediated autoimmunity to collagen V (col-V), a sequestered yet immunogenic self-protein, can induce chronic lung allograft rejection in rodent models. In this study we characterized the role of CD4+ CD25+ regulatory T cells (Tregs) in regulating col-V autoimmunity in human lung transplant (LT) recipients. LT recipients revealed a high frequency of col-V-reactive, IL-10-producing CD4+ T cells (T IL-10 cells) with low IL-2-, IFN-gamma-, IL-5-, and no IL-4-producing T cells. These T(IL-10) cells were distinct from Tregs because they lacked constitutive expression of both CD25 and Foxp3. Expansion of T IL-10 cells during col-V stimulation in vitro involved CTLA-4 on Tregs, because both depleting and blocking Tregs with anti-CTLA4 F(ab')2 mAbs resulted in loss of T IL-10 cells with a concomitant increase in IFN-gamma producing Th1 cells (TIFN-gamma cells). A Transwell culture of col-V-specific T IL-10 cells with Th1 cells (those generated in absence of Tregs) from the same patient resulted in marked inhibition of IFN-gamma and proliferation of T(IFN-gamma) cells, which was reversed by neutralizing IL-10. Furthermore, the T IL-10 cells were HLA class II restricted because blocking HLA class II on APCs resulted in the loss of IL-10 production. Chronic lung allograft rejection was associated with the loss of Tregs with a concomitant decrease in T IL-10 cells and an increase in T IFN-gamma cells. We conclude that LT patients have col-V-specific T cells that can be detected in the peripheral blood. The predominant col-V-specific T cells produce IL-10 that suppresses autoreactive Th1 cells independently of direct cellular contact. Tregs are pivotal for the induction of these "suppressor" T IL-10 cells.     

Lymphoid cell subclasses in rejecting renal allograft in the rat

We have quantitated the frequency of lymphoid cell subsets in rejecting renal allografts and in the spleen of the allograft recipient during drug-unmodified rejection in the rat. The number of inflammatory (white) cells in the graft was approximately similar to the number of white cells responding to the allograft in the recipient spleen. The inflammatory population of the graft consisted of lymphoid cells and mononuclear phagocytes, with increasing numbers of macrophages toward the end of rejection. Analysis of allograft cellular dispersates with monoclonal antibodies directed to the lymphoid cell subsets demonstrated that although the majority of allograft-infiltrating lymphocytes were T cells, a sizable B-cell proliferation and immunoglobulin synthesis was associated with the inflammatory response of rejection. Within the T-cell subset, the T suppressor/killer cells predominated in the graft whereas the predominant lymphoid cell subset responding to the allograft in the recipient spleen was the T helper cell.     

Role of autoimmunity in organ allograft rejection: a focus on immunity to type V collagen in the pathogenesis of lung transplant rejection

Lung transplantation is the only definitive treatment modality for many forms of end-stage lung disease. However, the lung is rejected more often than any other type of solid organ allograft due to chronic rejection known as bronchiolitis obliterans (BO). Indeed, BO is the primary reason why the 5- and 7-yr survival rates are worse for the lung than for any other transplanted organ. Alloimmunity to donor antigens is established as the primary mechanism that mediates rejection responses. However, newer immunosuppressive regimens designed to abrogate alloimmune activation have not improved survival. Therefore, these data suggest that other antigens, unrelated to donor transplantation antigens, are involved in rejection. Utilizing human and rodent studies of lung transplantation, our laboratory has documented that a native collagen, type V collagen [col(V)], is a target of the rejection response. Col(V) is highly conserved; therefore, these data indicate that transplant rejection involves both alloimmune and autoimmune responses. The role of col(V) in lung transplant rejection is described in this review article. In addition, the potential role of regulatory T cells that are crucial to modulating autoimmunity and alloimmunity is also discussed.     

Increased expression of Tim-3 and its ligand Galectin-9 in rat allografts during acute rejection episodes

The aim of the study is to elucidate the profiles of T-cell immunoglobulin and mucin domain-3 (Tim-3) and its ligand Galecin-9 in acute pulmonary rejection by using a rat model of lung transplantation. Left lung grafts retrieved from Lewis or Fisher 344 rats were orthotopically transplanted into Lewis recipients without any immunosuppressions; the grafts were harvested at day 3, 7 or 10 after transplantation. The grade of acute rejection was histopathologically evaluated. Tim-3, Galectin-9, immune antigen and related cytokines expression were assessed with immunological techniques and real-time polymerase chain reaction (RT-PCR), respectively. Then, our results showed that Tim-3 and its ligand Galectin-9 were markedly up-regulated at protein and mRNA levels in allografts compared with syngrafts. Meanwhile, the decreased CD4/CD8 ratio was associated with acute rejection occurring and Tim-3 expression on CD4⁺ and CD8⁺ T cells in allografts was increased. Therefore, our study firstly described that enhanced Tim-3 and its ligand Galectin-9 in allografts might play an important role in the pathogenesis of rat lung transplant rejection, implying new valuable markers for detecting acute allograft rejection.     

Donor IFN-gamma receptors are critical for acute CD4(+) T cell-mediated cardiac allograft rejection.

Recent studies using mouse models demonstrate that CD4(+) T cells are sufficient to mediate acute cardiac allograft rejection in the absence of CD8(+) T cells and B cells. However, the mechanistic basis of CD4-mediated rejection is unclear. One potential mechanism of CD4-mediated rejection is via elaboration of proinflammatory cytokines such as IFN-gamma. To determine whether IFN-gamma is a critical cytokine in CD4-mediated acute cardiac allograft rejection, we studied whether the expression of IFN-gamma receptors on the donor heart was required for CD4-mediated rejection. To investigate this possibility, purified CD4(+) T cells were transferred into immune-deficient mice bearing heterotopic cardiac allografts from IFN-gamma receptor-deficient (GRKO) donors. While CD4(+) T cells triggered acute rejection of wild-type heart allografts, they failed to trigger rejection of GRKO heart allografts. The impairment in CD4-mediated rejection of GRKO hearts appeared to primarily involve the efferent phase of the immune response. This conclusion was based on the findings that GRKO stimulator cells provoked normal CD4 proliferation in vitro and that intentional in vivo challenge of CD4 cells with wild-type donor APC or the adoptive transfer of in vitro primed CD4 T cells failed to provoke acute rejection of GRKO allografts. In contrast, unseparated lymph node cells acutely rejected both GRKO and wild-type hearts with similar time courses, illustrating the existence of both IFN-gamma-dependent and IFN-gamma-independent mechanisms of acute allograft rejection.     

Prolonged allograft survival in TNF receptor 1-deficient recipients is due to immunoregulatory effects, not to inhibition of direct antigraft cytotoxicity.

TNF-alpha and lymphotoxin (LT)alpha have been shown to be important mediators of allograft rejection. TNF-R1 is the principal receptor for both molecules. Mice with targeted genetic deletions of TNF-R1 demonstrate normal development of T and B lymphocytes but exhibit functional defects in immune responses. However, the role of TNF-R1-mediated signaling in solid organ transplant rejection has not been defined. To investigate this question, we performed vascularized heterotopic allogeneic cardiac transplants in TNF-R1-deficient (TNF-R1(-/-)) and wild-type mice. Because all allografts in our protocol expressed TNF-R1, direct antigraft effects of TNF-alpha and LTalpha were not prevented. However, immunoregulatory effects on recipient inflammatory cells by TNF-R1 engagement was eliminated in TNF-R1(-/-) recipients. In our study, cardiac allograft survival was significantly prolonged in TNF-R1(-/-) recipients. Despite this prolonged allograft survival, we detected increased levels of CD8 T cell markers in allografts from TNF-R1(-/-) recipients, suggesting that effector functions, but not T cell recruitment, were blocked. We also demonstrated the inhibition of multiple chemokines and cytokines in allografts from TNF-R1(-/-) recipients including RANTES, IFN-inducible protein-10, lymphotactin, and IL-1R antagonist, as well as altered levels of chemokine receptors. We correlated gene expression with the physiologic process of allograft rejection using self-organizing maps and identified distinct patterns of gene expression in allografts from TNF-R1(-/-) recipients. These findings indicate that in our experimental system TNF-alpha and LTalpha exert profound immunoregulatory effects through TNF-R1.     

The role of the CC chemokine, RANTES, in acute lung allograft rejection

Lung transplantation is a therapeutic option for patients with end-stage lung disease. Acute allograft rejection is a major complication of lung transplantation and is characterized by the infiltration of activated mononuclear cells. The specific mechanisms that recruit these leukocytes have not been fully elucidated. The CC chemokine, RANTES, is a potent mononuclear cell chemoattractant. In this study we investigated RANTES involvement during acute lung allograft rejection in humans and in a rat model system. Patients with allograft rejection had a 2.3-fold increase in RANTES in their bronchoalveolar lavages compared with healthy allograft recipients. Rat lung allografts demonstrated a marked time-dependent increase in levels of RANTES compared with syngeneic control lungs. RANTES levels correlated with the temporal recruitment of mononuclear cells and the expression of RANTES receptors CCR1 and CCR5. To determine RANTES involvement in lung allograft rejection, lung allograft recipients were passively immunized with either anti-RANTES or control Abs. In vivo neutralization of RANTES attenuated acute lung allograft rejection and reduced allospecific responsiveness by markedly decreasing mononuclear cell recruitment. These experiments support the idea that RANTES, and the expression of its receptors have an important role in the pathogenesis of acute lung allograft rejection.     

Monokine induced by IFN-gamma is a dominant factor directing T cells into murine cardiac allografts during acute rejection.

The use of chemokine antagonism as a strategy to inhibit leukocyte trafficking into inflammatory sites requires identification of the dominant chemokines mediating recruitment. The chemokine(s) directing T cells into cardiac allografts during acute rejection remain(s) unidentified. The role of the CXC chemokines IFN-gamma inducible protein 10 (IP-10) and monokine induced by IFN-gamma (Mig) in acute rejection of A/J (H-2(a)) cardiac grafts by C57BL/6 (H-2(b)) recipients was tested. Intra-allograft expression of Mig was observed at day 2 posttransplant and increased to the time of rejection at day 7 posttransplant. IP-10 mRNA and protein production were 2.5- to 8-fold lower than Mig. Whereas allografts were rejected at day 7-9 in control recipients, treatment with rabbit antiserum to Mig, but not to IP-10, prolonged allograft survival up to day 19 posttransplant. At day 7 posttransplant, allografts from Mig antiserum-treated recipients had marked reduction in T cell infiltration. At the time of rejection in Mig antiserum-treated recipients (i.e., days 17-19), intra-allograft expression of macrophage-inflammatory protein-1alpha, -1beta, and their ligand CCR5 was high, whereas expression of CXCR3, the Mig receptor, was virtually absent. Mig was produced by the allograft endothelium as well as by recipient allograft-infiltrating macrophages and neutrophils, indicating the synergistic interactions between innate and adaptive immune compartments during acute rejection. Collectively, these results indicate that Mig is a dominant recruiting factor for alloantigen-primed T cells into cardiac allografts during acute rejection. Although Mig antagonism delays acute heart allograft rejection, the results also suggest that the alloimmune response circumvents Mig antagonism through alternative mechanisms.     

Long-term cardiac allograft survival across an MHC mismatch after "pruning" of alloreactive CD4 T cells

Specific tolerance to allografts has been achieved by a variety of means. We have previously shown that ex vivo removal of dividing CD4(+) T cells from an MLR or "pruning" delays skin allograft rejection. We tested pruning of alloreactive T cells as a strategy for retaining a broad T cell repertoire while removing alloreactive T cells in a model of cardiac allograft transplant. Using CFSE staining of responder BALB/c cells with stimulator C57BL/6 cells in an MLR, SCID mice were reconstituted with either dividing (D) or nondividing (ND) CD4(+) T cells derived from an MLR and then challenged with heterotopic cardiac allografts. Mice reconstituted with D CD4(+) T cells rejected cardiac allografts from the stimulator strain with a median survival time (MST) of 29 days, while mice reconstituted with ND CD4(+) T cells maintained allografts from the stimulator strain (MST of >100 days) while rejecting third-party allografts (B10.BR) (MST = 11 days). ELISPOT assays demonstrate donor-specific hyporesponsiveness of the ND CD4(+) T cells. TCR beta-chain V region (TRBV) repertoire analysis demonstrates clonal expansion within both rejecting D cardiac allografts and ND cardiac allografts surviving for the long-term. Histology showed greater allograft infiltration by the D CD4(+) T cells. The surviving ND cardiac allografts demonstrated reduced cellular infiltration and reduced incidence of allograft vasculopathy, but with the development of chronic fibrosis. Thus, pruning of alloreactive T cells allows long-term-specific cardiac allograft survival while retaining the ability to reject third-party allografts.     

CD4 T cell-mediated rejection of cardiac allografts in B cell-deficient mice

CD4 T cell-dependent mechanisms promoting allograft rejection include expression of inflammatory functions within the graft and the provision of help for donor-reactive CD8 T cell and Ab responses. These studies tested CD4 T cell-mediated rejection of MHC-mismatched cardiac allografts in the absence of both CD8 T and B lymphocytes. Whereas wild-type C57BL/6 recipients depleted of CD8 T cells rejected A/J cardiac grafts within 10 days, allografts were not rejected in B cell-deficient B6.muMT(-/-) recipients depleted of CD8 T cells. Isolated wild-type C57BL/6 and B6.muMT(-/-) CD4 T cells had nearly equivalent in vivo alloreactive proliferative responses. CD4 T cell numbers in B6.muMT(-/-) spleens were 10% of that in wild-type mice but were only slightly decreased in peripheral lymph nodes. CD8 T cell depletion did not abrogate B6.muMT(-/-) mice rejection of A/J skin allografts and this rejection rendered these recipients able to reject A/J cardiac allografts. Redirection of the alloimmune response to the lymph nodes by splenectomy conferred the ability of B6.muMT(-/-) CD4 T cells to reject cardiac allografts. These results indicate that the low number of splenic CD4 T cells in B6.muMT(-/-) mice underlies the inability to reject cardiac allografts and this inability is overcome by diverting the CD4 T cell response to the peripheral lymph nodes.     

Lung resident mesenchymal stem cells isolated from human lung allografts inhibit T cell proliferation via a soluble mediator

Development of allograft rejection continues to be the major determinant of morbidity and mortality postlung transplantation. We have recently demonstrated that a population of donor-derived mesenchymal stem cells is present in human lung allografts and can be isolated and expanded ex vivo. In this study, we investigated the impact of lung resident mesenchymal stem cells (LR-MSCs), derived from allografts of human lung transplant recipients, on T cell activation in vitro. Similar to bone marrow-derived MSCs, LR-MSCs did not express MHC II or the costimulatory molecules CD80 or CD86. In vitro, LR-MSCs profoundly suppressed the proliferative capacity of T cells in response to a mitogenic or an allogeneic stimulus. The immunosuppressive function of LR-MSCs was also noted in the absence of direct cell contact, indicating that LR-MSCs mediated their effect predominantly via a soluble mediator. LR-MSCs isolated from lung transplant recipients demonstrated PGE(2) secretion at baseline (385 +/- 375 pg/ml), which increased in response to IL-1beta (1149 +/- 1081 pg/ml). The addition of PG synthesis inhibitors (indomethacin and NS-398) substantially abrogated LR-MSC-mediated immunosuppression, indicating that PGE(2) may be one of the major soluble mediators impacting T cell activity. This is the first report to demonstrate that human tissue-derived MSCs isolated from an allogeneic environment have the potential to mediate immunological responses in vitro.     

Cutting edge: blockade of the CD28/B7 costimulatory pathway inhibits intestinal allograft rejection mediated by CD4+ but not CD8+ T cells.

The effect of blocking the CD28/B7 costimulatory pathway on intestinal allograft rejection was examined in mice. Murine CTLA4Ig failed to prevent the rejection of allografts transplanted into wild-type or CD4 knockout (KO) mice but did inhibit allograft rejection by CD8 KO recipients. This effect was associated with decreased intragraft mRNA for IFN-gamma and TNF-alpha and increased mRNA for IL-4 and IL-5. This altered pattern of cytokine production was not observed in allografts from murine CTLA4Ig-treated CD4 KO mice. These data demonstrate that blockade of the CD28/B7 pathway has different effects on intestinal allograft rejection mediated by CD4+ and CD8+ T cells and suggest that these T cell subsets have different costimulatory requirements in vivo. The results also suggest that the inhibition of CD4+ T cell-mediated allograft rejection by CTLA4Ig may be related to down-regulation of Th1 cytokines and/or up-regulation of Th2 cytokines.     

Anti-type V collagen humoral immunity in lung transplant primary graft dysfunction

Primary graft dysfunction (PGD) is a major complication following lung transplantation. We reported that anti-type V collagen (col(V)) T cell immunity was strongly associated with PGD. However, the role of preformed anti-col(V) Abs and their potential target in PGD are unknown. Col(V) immune serum, purified IgG or B cells from col(V) immune rats were transferred to WKY rat lung isograft recipients followed by assessments of lung pathology, cytokines, and PaO(2)/FiO(2), an index of lung dysfunction in PGD. Immune serum, purified IgG, and B cells all induced pathology consistent with PGD within 4 days posttransfer; up-regulated IFN-gamma, TNF-alpha, and IL-1beta locally; and induced significant reductions in PaO(2)/FiO(2). Depleting anti-col(V) Abs before transfer demonstrated that IgG2c was a major subtype mediating injury. Confocal microscopy revealed strong apical col(V) expression on lung epithelial, but not endothelial cells; which was consistent with the ability of col(V) immune serum to induce complement-dependent cytotoxicity only in the epithelial cells. Examination of plasma from patients with or without PGD revealed that higher levels of preformed anti-col(V) Abs were strongly associated with PGD development. This study demonstrates a major role for anti-col(V) humoral immunity in PGD, and identifies the airway epithelium as a target in PGD.     

Specific B cell tolerance is induced by cyclosporin A plus donor-specific blood transfusion pretreatment: prolonged survival of MHC class I disparate cardiac allografts

Donor-specific blood transfusion (DST), designed to prolong allograft survival, sensitized recipients of the high-responder PVG-RT1u strain, resulting in accelerated rejection of MHC-class I mismatched (PVG-R8) allografts. Rejection was found to be mediated by anti-MHC class I (Aa) alloantibody. By pretreating recipients 4 wk before grafting with cyclosporin A (CsA) daily (x7), combined with once weekly (x4) DST, rejection was prevented. The investigation explores the mechanism for this induced unresponsiveness. CD4 T cells purified from the thoracic duct of CsA/DST-pretreated RT1u rats induced rejection when transferred to R8 heart-grafted RT1u athymic nude recipients, indicating that CD4 T cells were not tolerized by the pretreatment. To determine whether B cells were affected, nude recipients were pretreated, in the absence of T cells, with CsA/DST (or CsA/third party blood) 4 wk before grafting. The subsequent transfer of normal CD4 T cells induced acute rejection of R8 cardiac allografts in third party- but not DST-pretreated recipients; prolonged allograft survival was reversed by the cotransfer of B cells with the CD4 T cells. Graft survival correlated with reduced production of anti-MHC class I (Aa) cytotoxic alloantibody. The results indicated that the combined pretransplant treatment of CsA and DST induced tolerance in allospecific B cells independently of T cells. The resulting suppression of allospecific cytotoxic Ab correlated with the survival of MHC class I mismatched allografts. The induction of B cell tolerance by CsA has important implications for clinical transplantation.     

Donor deficiency of decay-accelerating factor accelerates murine T cell-mediated cardiac allograft rejection

Decay-accelerating factor (DAF) is a cell surface regulator that accelerates the dissociation of C3/C5 convertases and thereby prevents the amplification of complement activation on self cells. In the context of transplantation, DAF has been thought to primarily regulate antibody-mediated allograft injury, which is in part serum complement-dependent. Based on our previously delineated link between DAF and CD4 T cell responses, we evaluated the effects of donor Daf1 (the murine homolog of human DAF) deficiency on CD8 T cell-mediated cardiac allograft rejection. MHC-disparate Daf1(-/-) allografts were rejected with accelerated kinetics compared with wild-type grafts. The accelerated rejection predominantly tracked with DAF's absence on bone marrow-derived cells in the graft and required allograft production of C3. Transplantation of Daf1(-/-) hearts into wild-type allogeneic hosts augmented the strength of the anti-donor (direct pathway) T cell response, in part through complement-dependent proliferative and pro-survival effects on alloreactive CD8 T cells. The accelerated allograft rejection of Daf1(-/-) hearts occurred in recipients lacking anti-donor Abs. The results reveal that donor DAF expression, by controlling local complement activation on interacting T cell APC partners, regulates the strength of the direct alloreactive CD8(+) T cell response. The findings provide new insights into links between innate and adaptive immunity that could be exploited to limit T cell-mediated injury to an allograft following transplantation.     

An essential contribution by IFN-gamma to CD8+ T cell-mediated rejection of pancreatic islet allografts

CD8+ T cells have long been considered to be the prototypical cytotoxic lymphocyte subpopulation. However, whether alloreactive CD8+ T cells require traditional cytolytic pathways such as perforin and Fas ligand (FasL) to mediate graft rejection has been a controversial issue. In the present studies, we examined the role of varied effector pathways in CD8+ T cell-mediated rejection of pancreatic islet allografts. Our goal was to systematically determine the relative requirements, if any, of perforin and FasL as well as the proinflammatory cytokine IFN-gamma in triggering graft destruction. To study CD8+ T cell effector pathways independently of other lymphocyte populations, purified alloreactive CD8+ T cells were adoptively transferred into severe combined immune-deficient (SCID) recipients bearing established islet allografts. Results indicate that to reject established islet allografts, primed CD8+ T cells do not require the individual action of the conventional cytotoxic effectors perforin and Fas ligand. In contrast, the ability to produce IFN-gamma is critical for efficient CD8+ T cell-mediated rejection of established islet allografts. Furthermore, alloreactive CD8+ TCR transgenic T cells (2C) also show IFN-gamma dependence for mediating islet allograft rejection in vivo. We speculate from these results that the production of IFN-gamma by alloreactive CD8+ T cells is a rate-limiting step in the process of islet allograft rejection.     

Immunobiology of allograft rejection in the absence of IFN-gamma: CD8+ effector cells develop independently of CD4+ cells and CD40-CD40 ligand interactions

Both wild-type (WT) and IFN-gamma-deficient (IFN-gamma(-/-)) C57BL/6 mice can rapidly reject BALB/c cardiac allografts. When depleted of CD8(+) cells, both WT and IFN-gamma(-/-) mice rejected their allografts, indicating that these mice share a common CD4-mediated, CD8-independent mechanism of rejection. However, when depleted of CD4(+) cells, WT mice accepted their allografts, while IFN-gamma(-/-) recipients rapidly rejected them. Hence, IFN-gamma(-/-), but not WT mice developed an unusual CD8-mediated, CD4-independent, mechanism of allograft rejection. Allograft rejection in IFN-gamma(-/-) mice was associated with intragraft accumulation of IL-4-producing cells, polymorphonuclear leukocytes, and eosinophils. Furthermore, this form of rejection was resistant to treatment with anti-CD40 ligand (CD40L) mAb, which markedly prolonged graft survival in WT mice. T cell depletion studies verified that anti-CD40L treatment failed to prevent CD8-mediated allograft rejection in IFN-gamma(-/-) mice. However, anti-CD40L treatment did prevent CD4-mediated rejection in IFN-gamma(-/-) mice, although grafts were eventually rejected when CD8(+) T cells repopulated the periphery. The IL-4 production and eosinophil influx into the graft that occurred during CD8-mediated rejection were apparently epiphenomenal, since treatment with anti-IL-4 mAb blocked intragraft accumulation of eosinophils, but did not interfere with allograft rejection. These studies demonstrate that a novel, CD8-mediated mechanism of allograft rejection, which is resistant to experimental immunosuppression, can develop when IFN-gamma is limiting. An understanding of this mechanism is confounded by its association with Th2-like immune events, which contribute unique histopathologic features to the graft but are apparently unnecessary for the process of allograft rejection.     

Requirement for donor and recipient CD40 expression in cardiac allograft rejection: induction of Th1 responses and influence of donor-derived dendritic cells

Costimulation through the CD40-CD40 ligand (CD40L) pathway is critical to allograft rejection, in that anti-CD40L mAb therapy prolongs allograft survival. However, the majority of studies exploring CD40-CD40L interactions have targeted CD40L. Less is known about the requirement for donor- and/or host-derived CD40 during rejection. This study assessed the relative contributions of donor and recipient CD40 expression to the rejection process. As the effectiveness of costimulatory blockade may be mouse strain dependent, this study explored the requirement for donor and recipient CD40 expression in BALB/c and C57BL/6 mice. Wild-type (WT) and CD40(-/-) BALB/c recipients readily rejected WT and CD40(-/-) C57BL/6 allografts, and rejection was associated with a prominent Th1 response. In contrast, CD40(-/-) C57BL/6 recipients failed to reject WT or CD40(-/-) BALB/c allografts and did not mount Th1 or Th2 responses. However, injection of donor CD40(-/-) dendritic cells induced both Th1 and Th2 responses and allograft rejection in CD40(-/-) C57BL/6 recipients. Finally, WT C57BL/6 mice rejected CD40(-/-) allografts, but this rejection response was associated with muted Th1 responses. These findings demonstrate that 1) CD40 expression by the recipient or the graft may impact on the immune response following transplantation; 2) the requirement for CD40 is influenced by the mouse strain; and 3) the requirement for CD40 in rejection may be bypassed by donor DC. Further, as CD40 is not required for rejection in BALB/c recipients, but anti-CD40L mAb prolongs graft survival in these mice, these results suggest that anti-CD40L therapy functions at a level beyond disruption of CD40-CD40L interactions.