Sherri C. Wood

Immunobiology of Allograft Rejection in the Absence of IFN-γ: CD8+ Effector Cells Develop Independently of CD4+ Cells and CD40-CD40 Ligand Interactions

Both wild-type (WT) and IFN-γ-deficient (IFN-γ−/−) C57BL/6 mice can rapidly reject BALB/c cardiac allografts. When depleted of CD8+ cells, both WT and IFN-γ−/− 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-γ−/− recipients rapidly rejected them. Hence, IFN-γ−/−, but not WT mice developed an unusual CD8-mediated, CD4-independent, mechanism of allograft rejection. Allograft rejection in IFN-γ−/− 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-γ−/− mice. However, anti-CD40L treatment did prevent CD4-mediated rejection in IFN-γ−/− 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-γ 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.

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.

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.

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.

Transient Blockade of Delta-like Notch Ligands Prevents Allograft Rejection Mediated by Cellular and Humoral Mechanisms in a Mouse Model of Heart Transplantation

Rejection remains a major clinical challenge limiting allograft survival after solid organ transplantation. Both cellular and humoral immunity contribute to this complication, with increased recognition of Ab-mediated damage during acute and chronic rejection. Using a mouse model of MHC-mismatched heart transplantation, we report markedly protective effects of Notch inhibition, dampening both T cell and Ab-driven rejection. T cell–specific pan-Notch blockade prolonged heart allograft survival and decreased IFN-γ and IL-4 production by alloreactive T cells, especially when combined with depletion of recipient CD8+ T cells. These effects were associated with decreased infiltration by conventional T cells and an increased proportion of regulatory T cells in the graft. Transient administration of neutralizing Abs specific for delta-like (Dll)1/4 Notch ligands in the peritransplant period led to prolonged acceptance of allogeneic hearts, with superior outcome over Notch inhibition only in T cells. Systemic Dll1/4 inhibition decreased T cell cytokines and graft infiltration, germinal center B cell and plasmablast numbers, as well as production of donor-specific alloantibodies and complement deposition in the transplanted hearts. Dll1 or Dll4 inhibition alone provided partial protection. Thus, pathogenic signals delivered by Dll1/4 Notch ligands early after transplantation promote organ rejection through several complementary mechanisms. Transient interruption of these signals represents an attractive new therapeutic strategy to enhance long-term allograft survival.

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.

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.