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.

Engendering allograft ignorance in a mouse model of allogeneic skin transplantation to the distal hind limb.

OBJECTIVE: The aim of this study was to demonstrate lymphatic isolation in a model of hind limb lymph node (LN) excision, consisting of ipsilateral popliteal and inguinal LN excision and to evaluate the immunologic response to allogeneic skin transplanted onto this region of lymphatic isolation. METHODS: To study lymphatic flow, C57BL/6 mice underwent lymphadenectomy (n = 5), sham lymphadenectomy (n = 5), or no intervention (n = 5), followed by methylene blue injection. Mice were dissected to determine whether methylene blue traveled to the iliac LN. To study host response to skin transplantation, C57BL/6 mice underwent allogeneic skin transplantation with LN excision (n = 6), allogeneic skin transplantation alone (n = 6), or syngeneic skin transplantation (n = 4). Skin grafts were placed distal to the popliteal fossa and mice were euthanized at day 10. Grafts were stained for endothelial cell and proliferation markers (CD31 and Ki67, respectively). Secondary lymphoid tissues (spleen, ipsilateral axillary LN, and contralateral inguinal LN) were removed and rechallenged with BALB/c alloantigen in vitro with subsequent assay of interferon-γ and interleukin 4 cell expression using ELISPOT technique. RESULTS: Mice that underwent LN excision had no evidence of methylene blue in the iliac nodes; mice without surgical intervention or with sham LN excision consistently had methylene blue visible in the ipsilateral iliac nodes. Mice treated with allogeneic skin transplantation and LN excision had lower expression of interferon-γ and interleukin 4 in the secondary lymphoid tissues. CONCLUSIONS: Lymph node excision completely interrupts lymphatic flow of the hind limb. This model of lymphatic isolation impairs the ability of the transplant recipient to acutely mount a Th1 or Th2 response to allogeneic skin transplants.

New insights on OX40 in the control of T cell immunity and immune tolerance in vivo.

OX40 is a T cell costimulatory molecule that belongs to the TNFR superfamily. In the absence of immune activation, OX40 is selectively expressed by Foxp3(+) regulatory T cells (Tregs), but not by resting conventional T cells. The exact role of OX40 in Treg homeostasis and function remains incompletely defined. In this study, we demonstrate that OX40 engagement in vivo in naive mice induces initial expansion of Foxp3(+) Tregs, but the expanded Tregs have poor suppressive function and exhibit features of exhaustion. We also show that OX40 enables the activation of the Akt and Stat5 pathways in Tregs, resulting in transient proliferation of Tregs and reduced levels of Foxp3 expression. This creates a state of relative IL-2 deficiency in naive mice that further impacts Tregs. This exhausted Treg phenotype can be prevented by exogenous IL-2, as both OX40 and IL-2 agonists drive further expansion of Tregs in vivo. Importantly, Tregs expanded by both OX40 and IL-2 agonists are potent suppressor cells, and in a heart transplant model, they promote long-term allograft survival. Our data reveal a novel role for OX40 in promoting immune tolerance and may have important clinical implications.

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.

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.

Glucocorticoid-induced TNFR-related protein stimulation reverses cardiac allograft acceptance induced by CD40-CD40L blockade.

CD40-CD40L blockade has potent immunosuppressive effects in cardiac allograft rejection but is less effective in the presence of inflammatory signals. To better understand the factors that mediate CD40-CD40L blockade-resistant rejection, we studied the effects of stimulation through glucocorticoid-induced TNFR-related protein (GITR), a costimulatory protein expressed by regulatory and effector T cells. Stimulation of CD40-/- or wild-type recipient mice treated with anti-CD40L mAb (WT+anti-CD40L) and with agonistic anti-GITR mAb resulted in cardiac allograft rejection. GITR stimulation did not induce rejection once long-term graft acceptance was established. In vitro, GITR stimulation increased proliferation of effector T cells and decreased regulatory T cell (Treg) differentiation in both treatment groups. GITR-stimulated CD40-/- recipients rejected their allografts more rapidly compared to GITR-stimulated WT+anti-CD40L recipients, and this rejection, characterized by a robust Th2 response and significant eosinophilic infiltrate, could be mediated by CD4+ T cells alone. In contrast, both CD4+ and CD8+ T cells were required to induce rejection in GITR-stimulated WT+anti-CD40L-treated recipients, and the pathology of rejection was less severe. Hence, early GITR stimulation could initiate graft rejection despite CD40 deficiency or anti-CD40L mAb treatment, though the recipient response was dependent on the mechanism of CD40-CD40L disruption.

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.

TGFbeta neutralization within cardiac allografts by decorin gene transfer attenuates chronic rejection.

Chronic allograft rejection (CR) is the leading cause of late graft failure following organ transplantation. CR is a progressive disease, characterized by deteriorating graft function, interstitial fibrosis, cardiac hypertrophy, and occlusive neointima development. TGFbeta, known for its immunosuppressive qualities, plays a beneficial role in the transplant setting by maintaining alloreactive T cells in a hyporesponsive state, but has also been implicated in promoting graft fibrosis and CR. In the mouse vascularized cardiac allograft model, transient depletion of CD4(+) cells promotes graft survival but leads to CR, which is associated with intragraft TGFbeta expression. Decorin, an extracellular matrix protein, inhibits both TGFbeta bioactivity and gene expression. In this study, gene transfer of decorin into cardiac allografts was used to assess the impact of intragraft TGFbeta neutralization on CR, systemic donor-reactive T cell responses, and allograft acceptance. Decorin gene transfer and neutralization of TGFbeta in cardiac allografts significantly attenuated interstitial fibrosis, cardiac hypertrophy, and improved graft function, but did not result in systemic donor-reactive T cell responses. Thus, donor-reactive T and B cells remained in a hyporesponsive state. These findings indicate that neutralizing intragraft TGFbeta inhibits the cytokine's fibrotic activities, but does not reverse its beneficial systemic immunosuppressive qualities.

Role of T cell TGFbeta 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. TGFbeta 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 TGFbeta signaling in allograft acceptance and the progression of CR, we used mice with abrogated T cell TGFbeta signaling as allograft recipients. We compared responses from recipients that were transiently depleted of CD4(+) cells (that develop CR and express intragraft TGFbeta) with responses from mice that received anti-CD40L mAb therapy (that do not develop CR and do not express intragraft TGFbeta). Allograft acceptance and suppression of graft-reactive T and B cells were independent of T cell TGFbeta signaling in mice treated with anti-CD40L mAb. In recipients transiently depleted of CD4(+) T cells, T cell TGFbeta 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 TGFbeta-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 TGFbeta.

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-gamma 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-gamma(-/-) allograft recipients did not induce Th17, suggesting that T-bet, rather than IL-4 and IFN-gamma (known inhibitors of Th17), plays a critical role in negatively regulating Th17 in the transplant setting.

Fibroblastic niches prime T cell alloimmunity through Delta-like Notch ligands.

Alloimmune T cell responses induce graft-versus-host disease (GVHD), a serious complication of allogeneic bone marrow transplantation (allo-BMT). Although Notch signaling mediated by Delta-like 1/4 (DLL1/4) Notch ligands has emerged as a major regulator of GVHD pathogenesis, little is known about the timing of essential Notch signals and the cellular source of Notch ligands after allo-BMT. Here, we have shown that critical DLL1/4-mediated Notch signals are delivered to donor T cells during a short 48-hour window after transplantation in a mouse allo-BMT model. Stromal, but not hematopoietic, cells were the essential source of Notch ligands during in vivo priming of alloreactive T cells. GVHD could be prevented by selective inactivation of Dll1 and Dll4 in subsets of fibroblastic stromal cells that were derived from chemokine Ccl19-expressing host cells, including fibroblastic reticular cells and follicular dendritic cells. However, neither T cell recruitment into secondary lymphoid organs nor initial T cell activation was affected by Dll1/4 loss. Thus, we have uncovered a pathogenic function for fibroblastic stromal cells in alloimmune reactivity that can be dissociated from their homeostatic functions. Our results reveal what we believe to be a previously unrecognized Notch-mediated immunopathogenic role for stromal cell niches in secondary lymphoid organs after allo-BMT and define a framework of early cellular and molecular interactions that regulate T cell alloimmunity.

Th17 cells and transplant acceptance.

The discovery of Th17 cells has revealed a novel pathway of T-cell maturation. As with Th1 and Th2 lineages, Th17 cells promote graft pathology. However, a growing body of evidence indicates that Th17 cells may exhibit resistance to current methods of immunosuppression. Identification of this lineage provides an additional and challenging target for promoting graft acceptance.

TGF-beta, IL-6, IL-17 and CTGF direct multiple pathologies of chronic cardiac allograft rejection.

Cardiac transplantation is an effective treatment for heart failure refractive to therapy. Although immunosuppressive therapeutics have increased first year survival rates, chronic rejection remains a significant barrier to long-term graft survival. Chronic rejection manifests as patchy interstitial fibrosis, vascular occlusion and progressive loss of graft function. Recent evidence from experimental and patient studies suggests that the development of cardiomyocyte hypertrophy is another hallmark of chronic cardiac allograft rejection. This pathologic hypertrophy is tightly linked to the immune cytokine IL-6, which promotes facets of chronic rejection in concert with TGF-beta and IL-17. These factors potentiate downstream mediators, such as CTGF, which promote the fibrosis associated with the disease. In this article, we summarize contemporary findings that have revealed several elements involved in the induction and progression of chronic rejection of cardiac allografts. Further efforts to elucidate the interplay between these factors may direct the development of targeted therapies for this disease.

Regulation of alloimmune Th1 responses by the cyclin-dependent kinase inhibitor p21 following transplantation.

BACKGROUND: The cyclin-dependent kinase (cdk) inhibitor p21 inhibits cellular proliferation of many cell types, including T cells. Autoimmune models, however, have yielded conflicting results regarding the role of cdk inhibitors and T-cell function. The role of p21 in T-cell function after transplantation has not been investigated directly. We hypothesized that p21 plays an important role in alloantigen-driven responses in vitro in mixed lymphocyte cultures (MLC) and in vivo using the heterotopic murine cardiac allograft model. METHODS: Wild type (WT) and p21-deficient (p21-/-) mice were used as recipients, and the effects of p21 overexpression were assessed by transplanting p21 adenoviral-transfected cardiac allografts. Enzyme-linked immunospot (ELISPOT) and 3H-thymidine incorporation were used to evaluate for T-cell priming and proliferation in vitro, whereas graft histology was evaluated for rejection. RESULTS: When stimulated with alloantigens in vitro, splenocytes from p21-/- mice mounted enhanced proliferative responses and decreased Th2 responses relative to their WT counterparts. No differences in Th1 responses were noted when p21-/- cells were stimulated with alloantigens in vitro; however, after cardiac transplantation, Th1 responses were enhanced in p21-/- recipients relative to WT mice. This enhanced in vivo Th1 response was associated with exacerbated graft rejection in p21-/- recipients. Interestingly, p21 transfection of WT allografts inhibited graft rejection and Th1 priming. CONCLUSIONS: p21 controls the intensity of the immune response posttransplantation, with overexpression inhibiting allograft rejection. Our data demonstrate that p21 controls T-cell priming and suggest that p21 and other cdk inhibitors may serve as potential targets for therapeutic manipulation of alloimmune responses.

Anti-CD40L monoclonal antibody treatment induces long-term, tissue-specific, immunologic hyporesponsiveness to peripheral nerve allografts.

The CD40/CD40L costimulatory pathway plays a crucial role in allograft rejection. The purpose of this study was to determine the effectiveness of anti-CD40L monoclonal antibody (mAb) treatment as a method to induce long-term, tissue-specific, immunologic hyporesponsiveness to peripheral nerve allografts. Sciatic nerve allografts were performed from BALB/c donor mice into C57BL/6 recipients. Anti-CD40L mAb (1 mg) was administered intraperitoneally to recipient mice on postoperative days 0, 1, and 2. After a 14-, 28-, or 60-day recovery period, the mice were rechallenged with either a BALB/c cardiac or peripheral nerve allograft. Rejection was assessed by measuring the production of interferon gamma (IFN-gamma), interleukin (IL)-2, -4, and -5, and alloantibodies immunoglobulin (Ig) M and IgG. IFN-gamma, IL-2, IL-4, IL-5, IgM, and IgG responses were much lower in the anti-CD40L mAb group compared with controls. Nerve allograft and nerve isograft rechallenge 60 days following the original nerve allotransplantation produced low cytokine responses, whereas cardiac allograft rechallenge produced high cytokine production, indicative of acute rejection. Short-term anti-CD40L treatment may cause long-term, tissue-specific, immunologic hyporesponsiveness. This may allow time for native axons to traverse the transplanted nerve allograft and replace the graft with autogenous peripheral nerve tissue.

Induced expression of murine gamma2a by CD40 ligation independently of IFN-gamma.

IgG2a, with gamma2a H chains, is important for protection against viruses and other intracellular pathogens. Although a large portion of IgG2a expression is dependent upon IFN-gamma, some germline transcription and switch recombination to the murine gamma2a H chain gene expression are independent of IFN-gamma. We found that agonistic anti-CD40 Abs injected into IFN-gamma-deficient mice induce a > 200-fold increase in the amount of serum Ig2a, while other Ig isotypes are increased by 16-fold or less. In vitro, ligation of CD40 on B cells, without the addition of other B cell activators or cytokines, results in germline transcription and switch recombination that are largely restricted to the gamma2a gene. These results suggest that some immune responses to infectious agents can result in large amounts of IgG2a expression through ligation of CD40, without the expression of IFN-gamma by Th1 or other cells.

Short-term anti-CD40 ligand costimulatory blockade induces tolerance to peripheral nerve allografts, resulting in improved skeletal muscle function.

BACKGROUND: Reconstruction of long or multiple peripheral nerve defects with peripheral nerve autografts may not be possible due to insufficient quantities of donor nerve. There are promising preliminary data that nerve allografting has the potential to improve functional outcome and quality of life after devastating nerve injuries or large tumor resections. The authors previously demonstrated that blockade of the CD40/CD40 ligand costimulatory pathway, via anti-CD40 ligand monoclonal antibody (MR1) therapy, induces tolerance to peripheral nerve allografts in mice. In this study, the authors sought to correlate the immunomodulatory effects of MR1 treatment with functional muscle recovery after peripheral nerve allografting in the murine model. METHODS: In the mouse hindlimb model, peroneal nerve isografts (C57BL/6 into C57BL/6) and allografts (BALB/c into C57BL/6) were utilized to reconstruct 0.8-cm peroneal nerve gaps. MR1 versus vehicle was administered on days 0, 1, and 2. At 60 days after transplantation, splenocyte production of interferon-gamma and interleukins 2, 4, and 5 were quantified using ELISPOT analysis, and in vitro maximum tetanic isometric force of the extensor digitorum longus muscle was measured. RESULTS: At 60 days after transplantation, immunomodulation persisted in MR1-treated, allografted animals, as evidenced by significantly muted interferon-gamma, interleukin 4, and interleukin 2 splenocyte production. Functional extensor digitorum longus muscle recovery after nerve allografting and MR1 administration was improved due to the tolerance induced by MR1 compared with untreated allograft recipients. CONCLUSIONS: Three-day inductive therapy with MR1 produces 60-day immunologic tolerance to peripheral nerve allografts, as evidenced by dramatic decreases in interferon-gamma, interleukin 4, and interleukin 2 production, and results in increased muscle force recovery. This work emphasizes the potential promise of CD40-CD40 ligand costimulatory blockade in reducing or eliminating peripheral nerve 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-beta1) prolongs transplant survival provided that recipients are initially depleted of CD8+ T cells. To test if graft survival was prolonged by persistent TGF-beta1 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-beta1 transgene expression did not suppress effector cell function. We next asked whether TGF-beta1 gene transfer was associated with the development of regulatory cells. When splenocytes obtained from mice bearing long-term TGF-beta1-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-beta1 transfected cardiac allografts. The effector cells failed to mediate rejection in mice bearing TGF-beta1-transfected allografts and we observed a significant increase in intra-graft Foxp3 expression. These findings indicate that TGF-beta1 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.