Unaltered Graft Survival and Intragraft Lymphocytes Infiltration in the Cardiac Allograft of Cxcr3−/- Mouse Recipients

Previous studies showed that absence of chemokine receptor Cxcr3 or its blockade prolong mouse cardiac allograft survival. We evaluated the effect of the CXCR3 receptor antagonist MRL-957 on cardiac allograft survival, and also examined the impact of anti-CXCR3 mAb in human CXCR3 knock-in mice. We found only a moderate increase in graft survival (10.5 and 16.6 days, p < 0.05) using either the antagonist or the antibody, respectively, compared to control (8.7 days). We re-evaluated cardiac allograft survival with two different lines of Cxcr3^−/- mice. Interestingly, in our hands, neither of the independently derived Cxcr3^−/- lines showed remarkable prolongation, with mean graft survival of 9.5 and 10.8 days, respectively. There was no difference in the number of infiltrating mononuclear cells, expansion of splenic T cells or IFN-γ production of alloreactive T cells. Mechanistically, an increased other chemokine receptor fraction in the graft infiltrating CD8 T cells in Cxcr3^−/- recipients compared to wild-type recipients suggested compensatory T-cell trafficking in the absence of Cxcr3. We conclude Cxcr3 may contribute to, but does not govern, leukocyte trafficking in this transplant model.     

Role of CXCR3 and CCR5 in allograft rejection

Chemokines are known to participate in allograft rejection by mediating leukocyte trafficking. Despite redundancy in chemokine family, several chemokine-chemokine receptor interactions have proven critical in alloimmune responses. We sought to determine the effect of combined blockade of CXCR3 and CCR5, two critical chemokine receptors, in acute rejection. METHODS: Heterotopic heart transplantation was performed using BALB/c to B6/129 mice deficient in CCR5. Following transplantation these mice were treated with goat anti-CXCR3 serum every other day. In the control group, BALB/c hearts were transplanted in wild type B6/129 recipients and treated with goat serum alone. No immunosuppression was given to either group. Recipient mice were then assessed daily for allograft function by abdominal palpation, and graft survival was confirmed by laparotomy. RESULTS: The donor hearts in the control group were rejected at 6 +/- 1 days posttransplantation. Combined blockade of CXCR3 and CCR5 prolonged allograft survival versus control; all allografts survived to 24 days. In addition, there was a decrease in graft infiltrating CD4 and CD8 lymphocytes in the experimental group at 24 days. CONCLUSION: Combined CXCR3 and CCR5 blockade is effective in prolonging allograft survival in a fully MHC mismatched murine model. Combined chemokine blockade holds promise in control of acute rejection in organ transplantation.     

A novel small-molecule compound targeting CCR5 and CXCR3 prevents acute and chronic allograft rejection

BACKGROUND: Chemokines and chemokine receptors are critical in leukocyte recruitment, activation, and differentiation. Among them, CC chemokine receptor 5 (CCR5) and CXC chemokine receptor 3 (CXCR3) have been reported to play important roles in alloimmune responses and may be potential targets for posttransplant immunosuppression. METHODS: Fully major histocompatibility complex (MHC)-mismatched murine cardiac and islet transplant models were used to test the effect in vivo of a novel, small-molecule compound TAK-779 by targeting CCR5 and CXCR3 in acute allograft rejection. An MHC class II mismatched cardiac transplant model was used to evaluate its efficacy in chronic allograft rejection. Intragraft expression of cytokines, chemokines, and chemokine receptors was measured by quantitative real-time polymerase chain reaction and by histological analysis. RESULTS: Treatment of TAK-779 significantly prolonged allograft survival across the MHC barrier in two distinct transplant models. The treatment downregulated local immune activation as observed by the reduced expression of several chemokines, cytokines, and chemokine receptors. Thereby, the recruitment of CD4, CD8, and CD11c cells into transplanted allografts were inhibited. Furthermore, TAK-779 treatment significantly attenuated the development of chronic vasculopathy, fibrosis, and cellular infiltration. CONCLUSIONS: Antagonism of CCR5 and CXCR3 has a substantial therapeutic effect on inhibiting both acute and chronic allograft rejection. CCR5 and CXCR3 are functional in the process of allograft rejection and may be potential targets in clinical transplantation in the future.     

Viral IL-10 Gene Transfer Inhibits the Expression of Multiple Chemokine and Chemokine Receptor Genes Induced by Inflammatory or Adaptive Immune Stimuli

Previously we have shown that adenovirus-mediated gene transfer and expression of vIL-10 are able to prolong cardiac allograft survival, through the inhibition of the immune response to both alloantigen and adenoviral antigens. In the current study, we have defined further mechanisms of Ad.vIL-10-mediated prolongation of cardiac allograft survival. E1- and E3-deleted adenoviral vectors encoding beta-galactosidase or vIL-10 were transferred into grafts at the time of transplantation, chemokine and chemokine receptor expression were evaluated by a pathway-specific cDNA array, and the results were confirmed with real time RT-PCR on selected genes. Ischemic injury, alloantigen and adenovirus vector induced the expression of multiple pro-inflammatory chemokines in the grafts, which likely amplify allograft rejection. Most of these Th1-related chemokine genes were inhibited or down-regulated by Ad.vIL-10 administration, which may help to decrease leukocytic infiltration and improve graft survival. Among the potent Th1 type chemokines inhibited were the CXCR3 ligands CXCL9 and CXCL10, which could directly inhibit vector-mediated gene expression in myoblasts, although targeting CXCR3 or its ligands did not prolong allograft survival with vIL-10 gene transfer. Ad.vIL-10 administration also induced the expression of the Th2-associated chemokines eotaxin-2 and MIP-1 gamma, suggesting Th1 to Th2 immune deviation. These results demonstrated that the vIL-10 gene transfer inhibits chemokine expression, preventing stimulation of innate and adaptive immunity.     

Function of the vascular endothelial growth factor receptors Flt-1 and Flk-1/KDR in the alloimmune response in vivo

BACKGROUND: We have recently reported that vascular endothelial growth factor (VEGF) functions as a proinflammatory cytokine to regulate the trafficking of leukocytes into allografts in the early posttransplant period. VEGF binds two major VEGF receptors: VEGFR-1 (flt-1) and VEGFR-2 (flk-1/KDR). Here, we wished to investigate the expression and function of VEGF receptors in the process of acute allograft rejection in vivo. METHODS: We performed fully MHC-mismatched C57BL/6 (H-2b) into BALB/c (H-2d) vascularized heterotopic murine cardiac transplants and we examined the expression of VEGF and VEGF receptors by immunohistochemistry during acute allograft rejection. Next, we treated mice with specific neutralizing monoclonal antibodies against murine VEGFR-1 and VEGFR-2 and examined their effect on the development of acute allograft rejection by histology and by analysis of graft survival. The intragraft expression of cytokines and chemokines were also evaluated by quantitative real-time PCR analysis. RESULTS: The expression of VEGF, VEGFR-1 and VEGFR-2 were significantly up-regulated during allograft rejection as compared to isografts. Administration of either anti-VEGFR-1 or anti-VEGFR-2 alone failed to inhibit allograft rejection. However, coadministration of both antibodies together inhibited leukocyte infiltration of allografts and prolonged allograft survival. Furthermore, the effect of VEGFR blockade was associated with the downregulation of intragraft cytokine and chemokine expression. CONCLUSIONS: Our data suggest that VEGF-VEGFR interactions function in the alloimmune response in vivo. Targeting VEGFRs may represent a novel therapy to protect allografts following clinical transplantation.     

Absence of host B7 expression is sufficient for long-term murine vascularized heart allograft survival

CD28 antagonists have been shown to promote long-term graft survival and induce donor-specific tolerance. In this study, the role of CD28/B7 costimulation and the relative importance of host versus donor B7 expression in allograft rejection was assessed in a murine abdominal vascularized heterotopic heart transplant model. Wild-type, CD28-deficient, or B7-1/B7-2-deficient C57BL/6 (B6) mice were grafted with allogeneic wild type or B7-1/B7-2-deficient hearts. The results demonstrate allogeneic heart grafts survive long-term in mCTLA4Ig-treated B6 and untreated B7-1/B7-2-deficient B6 recipients but not CD28KO B6 mice. B7-1/B7-2KO B6 recipients treated with anti-CD28 (PV-1) or recombinant human IL-2 rejected the heart transplants indicating that these mice are immunologically competent to reject grafts if costimulatory signals are supplied or bypassed. Finally, there was no difference in rejection between normal animals transplanted with wild-type versus B7-1/B7-2-deficient hearts. These results support a critical role for B7-expressing host antigen presenting cells in the rejection of heart allografts in mice and differences among B7KO and CD28KO animals.     

Critical role of proinflammatory cytokine IL-6 in allograft rejection and tolerance

The proinflammatory cytokine IL-6 plays an important role in controlling T-cell differentiation, especially the development of Th17 and regulatory T cells. To determine the function of IL-6 in regulating allograft rejection and tolerance, BALB/c cardiac grafts were transplanted into wild-type or IL-6-deficient C57BL/6 mice. We observed that production of IL-6 and IFN-γ was upregulated during allograft rejection in untreated wild-type mice. In IL-6-deficient mice, IFN-γ production was greater than that observed in wild-type controls, suggesting that IL-6 production affects Th1/Th2 balance during allograft rejection. CD28-B7 blockade by CTLA4-Ig inhibited IFN-γ production in C57BL/6 recipients, but had no effect on the production of IL-6. Although wild-type C57BL/6 recipients treated with CTLA4-Ig rejected fully MHC-mismatched BALB/c heart transplants, treatment of IL-6-deficient mice with CTLA4-Ig resulted in graft acceptance. Allograft acceptance appeared to result from the combined effect of costimulatory molecule blockade and IL-6-deficiency, which limited the differentiation of effector cells and promoted the migration of regulatory T cells into the grafts. These data suggest that the blockade of IL-6, or its signaling pathway, when combined with strategies that inhibit Th1 responses, has a synergistic effect on the promotion of allograft acceptance. Thus, targeting the effects of IL-6 production may represent an important part of costimulation blockade-based strategies to promote allograft acceptance and tolerance.     

Prolongation of allograft survival in ccr7-deficient mice

BACKGROUND: Lymphocyte homing to secondary lymphoid organs is thought to be required for initiation of the alloreactive immune response. Because CCR7 is the essential chemokine receptor responsible for lymphocyte and dendritic cell homing to secondary lymphoid organs, allograft survival was analyzed in CCR7-deficient (CCR7) mice. METHODS: Heterotopic heart and skin allotransplantation was performed in CCR7 and wild-type (WT) recipients. Graft survival was monitored daily. Grafts and draining lymph nodes were analyzed by immunohistology and flow cytometry at different time points. Groups of mice were splenectomized at the day of allotransplantation. RESULTS: A significant though modest prolongation of allograft survival in CCR7 recipients was observed for heart grafts (WT, 7.3 +/- 0.5 days; CCR7, 10.7 +/- 2.8 days) and skin grafts (WT, 8.9 +/- 0.9 days; CCR7, 12.3 +/- 0.9 days). This was accompanied by a delay in the cellular infiltration of allografts. T-cell accumulation and expansion in the draining lymph nodes in CCR7 recipients was severely impaired. Splenectomy had only a moderate prolongation effect on allograft survival in CCR7 mice. CONCLUSIONS: These results suggest that CCR7-dependent processes support allograft rejection yet are dispensable for the rejection response.     

Anti‐Complement Component C5 mAb Synergizes with CTLA4Ig to Inhibit Alloreactive T cells and Prolong Cardiac Allograft Survival in Mice

While activation of serum complement mediates antibody‐initiated vascular allograft injury, increasing evidence indicates that complement also functions as a modulator of alloreactive T cells. We tested whether blockade of complement activation at the C5 convertase step affects T cell‐mediated cardiac allograft rejection in mice. The anti‐C5 mAb BB5.1, which prevents the formation of C5a and C5b, synergized with subtherapeutic doses of CTLA4Ig to significantly prolong the survival of C57BL/6 heart grafts that were transplanted into naive BALB/c recipients. Anti‐C5 mAb treatment limited the induction of donor‐specific IFNγ‐producing T cell alloimmunity without inducing Th2 or Th17 immunity in vivo and inhibited primed T cells from responding to donor antigens in secondary mixed lymphocyte responses. Additional administration of anti‐C5 mAb to the donor prior to graft recovery further prolonged graft survival and concomitantly reduced both the in vivo trafficking of primed T cells into the transplanted allograft and decreased expression of T cell chemoattractant chemokines within the graft. Together these results support the novel concept that C5 blockade can inhibit T cell‐mediated allograft rejection through multiple mechanisms, and suggest that C5 blockade may constitute a viable strategy to prevent and/or treat T cell‐mediated allograft rejection in humans.     

Role of the ICOS-B7h costimulatory pathway in the pathophysiology of chronic allograft rejection

BACKGROUND: Inducible costimulator (ICOS) is the third member of the CD28 superfamily and has a unique role in T cell activation and function. Recent studies indicated that the ICOS-B7h pathway plays an important role in alloimmune responses. We further investigated the role of the ICOS pathway in the pathologic process of chronic rejection in vivo. METHODS: An established major histocompatibility complex class II disparate cardiac transplantation model was used. We treated mice with a blocking anti-B7h monoclonal antibody (mAb) either in the initiation phase (early blockade) or in the progression phase (delayed blockade) of disease. In addition, some mice received mAb in the entire period (whole blockade). At 6 weeks after transplantation, cardiac grafts were evaluated by histopathologic analysis in terms of vasculopathy, fibrosis, and cellular infiltration. The intragraft expressions of cytokines and chemokines were also examined by quantitative real-time polymerase chain reaction analysis. RESULTS: Early blockade of the ICOS-B7h pathway did not show any protective effect on chronic allograft rejection compared with untreated controls. In contrast, delayed blockade significantly inhibited the development of vasculopathy, fibrosis, and cellular infiltration (P=0.043, P=0.004, and P=0.03 vs. untreated control, respectively). Interestingly, whole blockade did not prevent the chronic rejection process. Furthermore, the inhibitory effect of delayed ICOS blockade on chronic rejection was associated with down-regulation of local intragraft expression of several cytokines and chemokines. CONCLUSIONS: These data suggest that the ICOS-B7h pathway is critical in the activation of effector/memory T cells that are necessary for the progression of chronic rejection and provide the rationale to develop novel and specific therapies to prevent this process.     

Prolonged survival of heart allografts from p53-deficient mice

BACKGROUND: Acute rejection of the heart allograft is the major cause of heart failure in the first month after transplantation. Most studies on the prevention of acute rejection have concentrated on immune suppression of the recipients, whereas little is known about the effects of genetically manipulated donor organs on heart allograft survival. Herein, we describe a mouse model of heart allografts donated by p53-/- mice that can prolong the survival time of the grafts. METHODS: Hearts of p53-/- or p53+/+ C57BL/6J mice were grafted to the neck carotid artery and jugular vein of BALB/c mice using a cuff technique. The graft survival was observed daily. The hearts were analyzed using several techniques, including histology, immunofluorescence, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), and Western blot analysis. RESULTS: p53+/+ allografts ceased beating at 7.6+/-0.5 days, whereas p53-/- hearts were beating at 10.5+/-1.1 days after transplantation (P<0.01). Mean histological rejection scores were significantly lower in allografts donated by p53-deficient mice. Furthermore, apoptotic cells, determined by TUNEL and a reagent kit for detection of cardiac apoptosis, were of high numbers in the allograft sections from wild-type hearts but rare in p53-/- allografts (4.2+/-1.3 vs. 0.7+/-0.5/250x field). Immunofluorescence staining and Western blot analysis revealed that high levels of p53 and proapoptotic protein Bax were expressed in wild-type grafts but not p53-/- allografts. Interestingly, Bcl-2, an antiapoptotic protein, was abundant in cardiac allografts from p53-/- mice and almost undetectable in grafts from wild-type mice. CONCLUSIONS: Thus, p53 is involved in cardiac apoptosis induced by alloimmune reaction, and prolonged survival of heart allografts can be achieved when p53 is lacking.     

Different mechanisms of cardiac allograft rejection in wildtype and CD28-deficient mice.

Although CD28 blockade results in long-term cardiac allograft survival in wildtype mice, CD28-deficient mice effectively reject heart allografts. This study compared the mechanisms of allogeneic responses in wildtype and CD28-deficient mice. Adoptive transfer of purified CD28-deficient T cells into transplanted nude mice resulted in graft rejection. However, this model demonstrated that the allogeneic T cell function was severely impaired when compared with wildtype T cells, despite similar survival kinetics. Cardiac allograft rejection depended on both CD4+ and CD8+ T cell subsets in CD28-deficient mice, whereas only CD4+ T cells were necessary in wildtype recipients. These results suggested that CD8+ T cells were more important in CD28-deficient than wildtype mice. In addition to the CD8+ T cell requirement, allograft rejection in CD28-deficient mice was dependent on a sustained presence of CD4+ T cells, whereas it only required the initial presence of CD4+ T cells in wildtype mice. Taken together, these data suggest that CD4+ T cells from CD28-deficient mice have impaired responses to alloantigen in vivo, thus requiring long-lasting cooperation with CD8+ T cell responses to facilitate graft rejection. These results may help to explain the failure to promote graft tolerance in some preclinical and clinical settings.     

Urinary fractalkine is a marker of acute rejection

Chemokines and their receptors play an important role in the development of allograft rejection through directing mononuclear cell invasion of the graft. To study whether chemokine assays in the urine could prove to be predictive of acute rejection, we measured the urinary excretion of several chemokines, including fractalkine, chemokine monokine induced by interferon-gamma, interferon-gamma-inducible protein 10, macrophage inflammatory protein-3 alpha, granzyme B, and perforin in 215 allograft recipients and in 80 healthy control subjects. The 67 patients with acute rejection had significantly higher levels of all urinary chemokines compared to the healthy controls or patients having chronic allograft nephropathy but with stable renal function. Only changes in urinary fractalkine differentiated patients with acute rejection from those with acute tubular necrosis. The 7 patients who lost their grafts had greater urinary fractalkine, interferon-gamma, and macrophage inflammatory protein-3 alpha concentrations than those patients with reversible acute rejection. The area under the receiver operating characteristic curve for fractalkine was the best indicator among all of the markers differentiating 39 patients diagnosed with steroid-resistant from the 28 patients with steroid-sensitive acute rejection and in predicting graft loss. Our study shows that measuring urinary fractalkine levels is a noninvasive approach for detecting acute rejection where high levels were associated with steroid-resistance and poor outcome.     

From basic science to clinical practice: use of cytokines and chemokines as therapeutic targets in renal diseases

Cytokines are soluble factors that are critical for the pathophysiology of the immune system and exhibit other important functions. Recently, several cytokines have been suggested as possible therapeutic targets in renal diseases, and their possible clinical utility is now being tested in phase I-II clinical trials. Chemokines are a family of small, structurally related, cytokines that regulate cell trafficking of different subsets of leukocytes, thus critically regulating renal inflammation. The initial hope that targeting of chemokines may result in the attenuation of inflammation without inducing generalized immunosuppression is hampered by the redundancy in chemokine activity with the potential risk of severe side effects. However, a deep comprehension of the complex biology of chemokines has allowed the identification of some chemokine receptors as possible theraputic targets in the pathogenesis of selected renal diseases, in as much as they display non-redundant roles. More importantly the discovery of the critical involvement of CXCR3 in transplant tolerance, suggests that this chemokine receptor might represent a critical target for treatment of both acute rejection and chronic allograft nephropathy. Finally, we recently demonstrated that pretransplant serum levels of CXCL10/IP-10, a CXCR3-ligand, represent a clinically useful parameter for the identification of subjects exhibiting high risk of acute rejection, chronic allograft nephropathy and graft failure, a finding that might be used to individualize immunosuppressive therapies.     

Chemokines in transplantation: what can atypical receptors teach us about anti-inflammatory therapy?

More than 45 members of the family of chemotactic cytokines have been described. These chemokines control the migration of leukocytes throughout the whole alloimmune response from initial ischemic damage to acute inflammation and eventual resolution. Several chemokines have been strongly linked to allograft rejection. Recent studies have described powerful endogenous mechanisms that regulate chemokine biology. This review will describe a new class of chemokine receptor that bind ligands with high affinity but lack the capacity for signaling. Atypical receptors represent a new paradigm in chemokine biology and may hold the key to our eventual manipulation of chemokine-driven allograft inflammation.     

Chemokines and their receptors in islet allograft rejection and as targets for tolerance induction

Graft rejection is a major barrier to successful outcome of transplantation surgery. Islet transplantation introduces insulin secreting tissue into type 1 diabetes mellitus recipients, relieving patients from exogenous insulin injection. However, insulitis of grafted tissue and allograft rejection prevent long-term insulin independence. Leukocyte trafficking is necessary for the launch of successful immune responses to pathogen or allograft. Chemokines, small chemotactic cytokines, direct the migration of leukocytes through their interaction with chemokine receptors found on cell surfaces of immune cells. Unique receptor expression of leukocytes, and the specificity of chemokine secretion during various states of immune response, suggest that the extracellular chemokine milieu specifically homes certain leukocyte subsets. Thus, only those leukocytes required for the current immune task are attracted to the inflammatory site. Chemokine blockade, using antagonists and monoclonal antibodies directed against chemokine receptors, is an emerging and specific immunosuppressive strategy. Importantly, chemokine blockade may potentiate tolerance induction regimens to be used following transplantation surgery, and prevent the need for life-long immunosuppression of islet transplant recipients. Here, the role for chemokine blockade in islet transplant rejection and tolerance is reviewed.     

Inducible nitric oxide synthase inhibitors prolonged the survival of skin xenografts through selective down-regulation of pro-inflammatory cytokine and CC-chemokine expressions

To elucidate the possible immunoregulatory role of nitric oxide (NO) in cellular xenograft rejection we performed rat-to-mouse skin xenotransplantation. The rat skin engrafted mice were treated with the inducible NO synthase (iNOS) inhibitors, aminoguanidine (AMG, 200 mg/kg) and NG-nitro-L-arginine methyl ester (L-NAME, 60 mg/kg) every other day until rejection. Skin xenograft survival was monitored and immune cell infiltration and intragraft cytokine and chemokine mRNA expressions were analyzed 7 days after grafting. Compared with the control mice, the AMG- and L-NAME treated mice showed delayed xenograft rejection by approximately 3 days (8.9 +/- 0.7 days vs. 11.7 +/- 1.2 and 12.0 +/- 0.9 days, respectively). Infiltrations of CD11b+, MOMA-2+ cells and neutrophils were significantly reduced in both AMG- and L-NAME treated graft but CD4+ and CD8+ cells were not. The expression of cytokines such as IL-1beta, IL-2, IL-6, IL-12 and IFN-gamma in AMG- and L-NAME treated grafts were significantly decreased (P<0.01), whereas IL-10, TNF-alpha and TGF-beta1 were unchanged or enhanced. Additionally, the expressions of CC-chemokines, such as RANTES and MIP-1alpha, were significantly reduced (P<0.01) whereas the expressions of CXC-chemokines, such as IP-10 and MIG, were unchanged. These results imply that prolonged rat-to-mouse skin xenograft survival by iNOS inhibitors may be due to the selective inhibition of pro-inflammatory cytokines and chemokines and suggest the possible regulatory role of NO in cytokine and chemokine expressions during xenotransplant rejection.     

Multiple pathways to allograft rejection

Allograft rejection results from a complex process involving both the innate and acquired immune systems. The innate immune system predominates in the early phase of the allogeneic response, during which chemokines and cell adhesion play essential roles, not only for leukocyte migration into the graft but also for facilitating dendritic and T-cell trafficking between lymph nodes and the transplant. This results in a specific and acquired alloimmune response mediated by T cells. Subsequently, T cells and cells from innate immune system function synergistically to reject the allograft through nonexclusive pathways, including contact-dependent T cell cytotoxicity, granulocyte activation by either Th1 or Th2 derived cytokines, NK cell activation, alloantibody production, and complement activation. Blockade of individual pathways generally does not prevent allograft rejection, and long-term allograft survival is achieved only after simultaneous blockade of several of them. In this review, we explore each of these pathways and discuss the experimental evidence highlighting their roles in allograft rejection.     

Mechanism of allorecognition and skin graft rejection in CD28 and CD40 ligand double-deficient mice

BACKGROUND: It has been shown that simultaneous blockade of CD28- and CD40-mediated costimulatory signals significantly prolongs allograft survival. Although these results led to an expectation of the establishment of specific immunotolerant therapy for organ transplantation, it became evident that these treatments rarely resulted in indefinite allograft survival. To uncover the mechanisms underlying these costimulation blockade-resistant allograft rejections, we studied the process of allogenic skin graft rejection in CD28 and CD40 ligand (L) double-deficient (double-knockout [dKO]) mice. METHODS: Skin grafts from BALB/c or BALB.B mice were transplanted to C57BL/6 background dKO mice. The frequency of CD4+ and CD8+ T cells responding to alloantigens presented by direct or indirect pathways were defined by the use of a cytostaining assay. RESULTS: BALB/c skin grafts were rapidly rejected by dKO mice. This CD28 and CD40L independent allograft rejection was inhibited by the depletion of CD8+ T cells. In vitro studies indicated that CD8+ T cells from BALB/c skin-grafted dKO mice responded to donor antigen presented only by the direct pathway. Unlike major histocompatibility complex (MHC)-mismatched donors, allogenic skin grafts from MHC-matched donors were accepted by dKO mice. CONCLUSION: In the absence of CD28 and CD40 costimulatory signals, CD8+ T cells recognize MHC antigens by the direct pathway, resulting in the rejection of skin grafts from MHC-mismatched donors. In contrast, MHC-matched and non-MHC-mismatched donor skin grafts indefinitely survive in dKO mice. These results indicated that donor-host MHC matching may still be critical to costimulation blockade therapy for organ transplantation.