Guanyi Lu

CD8+ Th17 Mediate Costimulation Blockade-Resistant Allograft Rejection in T-bet-Deficient Mice

While studying Th responses induced by cardiac transplantation, we observed that mice deficient in the Th1 transcription factor T-bet (T-bet−/−) mount both Th1 and Th17 responses, whereas wild-type recipients mount only Th1 responses. Cells producing both IFN-γ and IL-17 were readily detectable within the rejecting graft of T-bet−/− recipients, but were absent from the spleen, indicating that the in vivo microenvironment influences Th function. In addition, disrupting CD40-CD40L costimulatory interactions was highly effective at prolonging allograft survival in WT mice, but ineffective in T-bet−/− recipients. In this study, we report that CD8+ Th17 mediate costimulation blockade-resistant rejection in T-bet−/− allograft recipients. Depleting CD8+ cells or neutralizing IL-17 or the Th17-inducing cytokine IL-6 ablated the Th17 response and reversed costimulation blockade-resistant graft rejection. Neutralizing IL-4 in IFN-γ−/− allograft recipients did not induce Th17, suggesting that T-bet, rather than IL-4 and IFN-γ (known inhibitors of Th17), plays a critical role in negatively regulating Th17 in the transplant setting.

Role of T Cell TGFβ Signaling and IL-17 in Allograft Acceptance and Fibrosis Associated with Chronic Rejection

Chronic allograft rejection (CR) is the main barrier to long-term transplant survival. CR is a progressive disease defined by interstitial fibrosis, vascular neointimal development, and graft dysfunction. The underlying mechanisms responsible for CR remain poorly defined. TGFβ has been implicated in promoting fibrotic diseases including CR, but is beneficial in the transplant setting due to its immunosuppressive activity. To assess the requirement for T cell TGFβ signaling in allograft acceptance and the progression of CR, we used mice with abrogated T cell TGFβ signaling as allograft recipients. We compared responses from recipients that were transiently depleted of CD4+ cells (that develop CR and express intragraft TGFβ) with responses from mice that received anti-CD40L mAb therapy (that do not develop CR and do not express intragraft TGFβ). Allograft acceptance and suppression of graft-reactive T and B cells were independent of T cell TGFβ signaling in mice treated with anti-CD40L mAb. In recipients transiently depleted of CD4+ T cells, T cell TGFβ signaling was required for the development of fibrosis associated with CR, long-term graft acceptance, and suppression of graft-reactive T and B cell responses. Furthermore, IL-17 was identified as a critical element in TGFβ-driven allograft fibrosis. Thus, IL-17 may provide a therapeutic target for preventing graft fibrosis, a measure of CR, while sparing the immunosuppressive activity of TGFβ.

Connective Tissue Growth Factor Promotes Fibrosis Downstream of TGFβ and IL-6 in Chronic Cardiac Allograft Rejection

Cardiac transplantation is an effective treatment for multiple types of heart failure refractive to therapy. Although immunosuppressive therapeutics have increased survival rates within the first year posttransplant, chronic rejection (CR) remains a significant barrier to long‐term graft survival. Indicators of CR include patchy interstitial fibrosis, vascular occlusion and progressive loss of graft function. Multiple factors have been implicated in the onset and progression of CR, including TGFβ, IL‐6 and connective tissue growth factor (CTGF). While associated with CR, the role of CTGF in CR and the factors necessary for CTGF induction in vivo are not understood. To this end, we utilized forced expression and neutralizing antibody approaches. Transduction of allografts with CTGF significantly increased fibrotic tissue development, though not to levels observed with TGFβ transduction. Further, intragraft CTGF expression was inhibited by IL‐6 neutralization whereas TGFβ expression remained unchanged, indicating that IL‐6 effects may potentiate TGFβ‐mediated induction of CTGF. Finally, neutralizing CTGF significantly reduced graft fibrosis without reducing TGFβ and IL‐6 expression levels. These findings indicate that CTGF functions as a downstream mediator of fibrosis in CR, and that CTGF neutralization may ameliorate fibrosis and hypertrophy associated with CR.

Recipient–derived EDA fibronectin promotes cardiac allograft fibrosis

Advances in donor matching and immunosuppressive therapies have decreased the prevalence of acute rejection of cardiac grafts; however, chronic rejection remains a significant obstacle for long‐term allograft survival. While initiating elements of anti‐allograft immune responses have been identified, the linkage between these factors and the ultimate development of cardiac fibrosis is not well understood. Tissue fibrosis resembles an exaggerated wound healing response, in which extracellular matrix (ECM) molecules are central. One such ECM molecule is an alternatively spliced isoform of the ubiquitous glycoprotein fibronectin (FN), termed extra domain A‐containing cellular fibronectin (EDA cFN). EDA cFN is instrumental in fibrogenesis; thus, we hypothesized that it might also regulate fibrotic remodelling associated with chronic rejection. We compared the development of acute and chronic cardiac allograft rejection in EDA cFN‐deficient (EDA−/−) and wild‐type (WT) mice. While EDA−/− mice developed acute cardiac rejection in a manner indistinguishable from WT controls, cardiac allografts in EDA−/− mice were protected from fibrosis associated with chronic rejection. Decreased fibrosis was not associated with differences in cardiomyocyte hypertrophy or intra‐graft expression of pro‐fibrotic mediators. Further, we examined expression of EDA cFN and total FN by whole splenocytes under conditions promoting various T‐helper lineages. Conditions supporting regulatory T‐cell (Treg) development were characterized by greatest production of total FN and EDA cFN, though EDA cFN to total FN ratios were highest in Th1 cultures. These findings indicate that recipient‐derived EDA cFN is dispensable for acute allograft rejection responses but that it promotes the development of fibrosis associated with chronic rejection. Further, conditions favouring the development of regulatory T cells, widely considered graft‐protective, may drive production of ECM molecules which enhance deleterious remodelling responses. Thus, EDA cFN may be a therapeutic target for ameliorating fibrosis associated with chronic cardiac allograft rejection. Copyright

Transforming growth factor beta-induced connective tissue growth factor and chronic allograft rejection.

Late loss of allograft function is primarily attributed to chronic rejection (CR). There are no effective treatments for CR and the underlying cause of the disease is unknown. This study compared events that occurred within cardiac allografts placed in mice that received either anti‐CD4 therapy and develop CR or anti‐CD40L therapy and do not develop CR. Both TGFβ and connective tissue growth factor (CTGF), which is induced by TGFβ, were expressed in grafts with CR but were not expressed in grafts without CR. TGFβ transfection of allografts in anti‐CD40L‐treated recipients resulted in CTGF expression and CR. However, TGFβ transfection of syngeneic grafts did not result in CTGF expression or CR. These data indicate that TGFβ alone is insufficient to induce CR and that CTGF is required. Further, antigenic stimulation is required for TGFβ induction of CTGF. Thus, CTGF may serve as a therapeutic target for CR.

IL-6 Promotes Cardiac Graft Rejection Mediated by CD4+ Cells

IL-6 mediates numerous immunologic effects relevant to transplant rejection; however, its specific contributions to these processes are not fully understood. To this end, we neutralized IL-6 in settings of acute cardiac allograft rejection associated with either CD8+ or CD4+ cell-dominant responses. In a setting of CD8+ cell-dominant graft rejection, IL-6 neutralization delayed the onset of acute rejection while decreasing graft infiltrate and inverting anti-graft Th1/Th2 priming dominance in recipients. IL-6 neutralization markedly prolonged graft survival in the setting of CD4+ cell-mediated acute rejection and was associated with decreased graft infiltrate, altered Th1 responses, and reduced serum alloantibody. Furthermore, in CD4+ cell-dominated rejection, IL-6 neutralization was effective when anti–IL-6 administration was delayed by as many as 6 d posttransplant. Finally, IL-6–deficient graft recipients were protected from CD4+ cell-dominant responses, suggesting that IL-6 production by graft recipients, rather than grafts, is necessary for this type of rejection. Collectively, these observations define IL-6 as a critical promoter of graft infiltration and a shaper of T cell lineage development in cardiac graft rejection. In light of these findings, the utility of therapeutics targeting IL-6 should be considered for preventing cardiac allograft rejection.

OX40 Costimulation Prevents Allograft Acceptance Induced by CD40-CD40L Blockade

Disrupting the CD40-CD40L costimulation pathway promotes allograft acceptance in many settings. Herein, we demonstrate that stimulating OX40 overrides cardiac allograft acceptance induced by disrupting CD40-CD40L interactions. This effect of OX40 stimulation was dependent on CD4+ T cells, which in turn provided help for CD8+ T cells and B cells. Allograft rejection was associated with donor-reactive Th1 and Th2 responses and an unconventional granulocytic infiltrate and thrombosis of the arteries. Interestingly, OX40 stimulation induced a donor-reactive IgG class switch in the absence of CD40-CD40L interactions, and the timing of OX40 stimulation relative to transplantation affected the isotype of donor-reactive Ab produced. Inductive OX40 stimulation induced acute graft rejection, which correlated with both IgG1 and IgG2a deposition within the graft. Once graft acceptance was established following CD40-CD40L blockade, delayed OX40 stimulation did not induce acute allograft rejection despite priming of graft-reactive Th1 and Th2. Rather, chronic rejection was induced, which was characterized by IgG1 but not IgG2a deposition within the graft. These studies reveal both redundancy and key differences in function among costimulatory molecules that manifest in distinct pathologies of allograft rejection. These findings may help guide development of therapeutics aimed at promoting graft acceptance in transplant recipients.

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.

Transforming Growth Factor-Beta1 Gene Transfer is Associated with the Development of Regulatory Cells

Adenovirus‐mediated transfection of mouse cardiac allografts with active human transforming growth factor‐beta 1 (TGF‐β1) prolongs transplant survival provided that recipients are initially depleted of CD8+ T cells. To test if graft survival was prolonged by persistent TGF‐β1 transgene expression, long‐term transfected allografts were re‐transplanted into naïve mice that were transiently depleted of CD8+ T cells. Re‐transplanted allografts were acutely rejected, indicating that TGF‐β1 transgene expression did not suppress effector cell function. We next asked whether TGF‐β1 gene transfer was associated with the development of regulatory cells. When splenocytes obtained from mice bearing long‐term TGF‐β1‐transfected allografts were adoptively transferred into recipients of non‐transfected cardiac allografts, prolonged allograft survival was observed, and increased levels of the regulatory T cell transcription factor Foxp3 were present. To further test for regulation, differentiated effector cells were obtained from mice that had rejected cardiac allografts and were adoptively transferred into mice bearing long‐term TGF‐β1 transfected cardiac allografts. The effector cells failed to mediate rejection in mice bearing TGF‐β1‐transfected allografts and we observed a significant increase in intra‐graft Foxp3 expression. These findings indicate that TGF‐β1 gene transfer allows for the development of regulatory cells that control graft‐reactive T cell responses once therapeutic levels of the transgene product are no longer produced.

Graft rejection mediated by CD4+ T cells via indirect recognition of alloantigen is associated with a dominant Th2 response

CD4+ T cells that respond to indirectly presented alloantigen have been shown to mediate chronic rejection, however, the role of the indirect pathway in acute rejection has yet to be completely elucidated. To this end, BALB/c or C57BL/6 mice were depleted of CD8+ T cells and transplanted with class II transactivator (CIITA)‐deficient cardiac allografts, which cannot directly present class II alloantigens to CD4+ T cells. In this manner, the rejection response by CD4+ cells was forced to rely upon the indirect recognition pathway. When not depleted of CD8+ cells, both BALB/c and C57BL/6 mice rejected CIITA–/– allografts and a polarized Th1 response was observed. In contrast, when BALB/c recipients of CIITA–/– allografts were depleted of CD8+ T cells, the grafts were acutely rejected and a strong Th2 response characterized by eosinophil influx into the graft was observed. Interestingly, CD8‐depleted C57BL/6 recipients of CIITA–/– allografts did not acutely reject their transplants and a Th2 response was not mounted. These findings indicate that CD4+ T cells responding to indirectly presented alloantigens mediate graft rejection in a Th2‐dominant manner, and provide further evidence for the role of Th2 responses in acute graft rejection.