CD40 expression on graft infiltrates and parenchymal CD154 (CD40L) induction in human chronic renal allograft rejection

BACKGROUND: CD40-CD154 (CD40L) costimulatory signaling plays a pivotal role in the effector mechanisms of transplant graft rejection. In animal models, CD40-CD154 blockade induces long-term graft acceptance concurrent with an absence of chronic rejection (CR) lesions. Given the critical importance of CD40-CD154 interactions in the development of chronic transplant allograft rejection, the relevance of in situ CD40 and CD154 expression was assessed in human chronic renal allograft rejection. METHODS: The expression of CD40, CD154, CD68, and T-cell receptor (TCR)alpha/beta was analyzed by immunohistochemistry. Serial cryostat sections of snap-frozen core renal allograft biopsies were obtained from 30 renal transplant patients. Biopsy specimens received diagnoses of CR (N = 23) according to the Banff classification and were compared with controls (N = 7) consisting of stable allografts and normal kidney tissue. RESULTS: Striking CD40 staining of graft cellular infiltrates (P = 0.016) was observed in renal allografts with CR compared with controls. The CD40+ cellular infiltrates in CR were predominantly TCR alpha/beta + T cells and some CD68+ macrophages. These findings were contrasted by the low-level CD40 expression detected in glomeruli and tubules of CR and controls. However, glomerular induction of CD154 was observed in CR allografts (P = 0.028) as compared with controls. CD154 immunoreactivity was demonstrated on glomerular endothelial, epithelial, and mesangial cells. Moderate CD154 expression was detected on tubular epithelial cells, and only weak CD154 immunoreactivity was observed on the infiltrates in isolated CR cases. CONCLUSION: In human chronic renal allograft rejection, CD40 is expressed on graft-infiltrating cells of the T cell and macrophage compartments. CD154 expression is induced on glomerular and tubular epithelial cells during CR, demonstrating another novel source of CD154 expression. The data substantiate the potential contributory role of an interaction between CD40+ graft-destructive effector T cells and macrophages with CD154+ renal allograft parenchymal cells in the development of chronic renal allograft rejection.     

OX40 controls islet allograft tolerance in CD154 deficient mice by regulating FOXP3+ Tregs

The role of OX40 in the islet allograft tolerance, especially in the absence of CD154 costimulation, remains poorly defined. In the present study, we used CD154 deficient mice to critically examine the role of OX40 in the activation of T effector cells and Foxp3+ Tregs and the effect of blocking OX40 on the induction of islet allograft tolerance. We found that blocking OX40 costimulation in CD154 deficient mice induced donor specific tolerance but stimulating OX40 resulted in prompt islet allograft rejection. We also found that OX40 differentially regulates T effector cells and Foxp3+ Tregs, OX40 signaling mediates proliferation of CD154 deficient T effector cells but blocks the induction and suppressor functions of Foxp3+ Tregs. Our data suggest that the role of OX40 in the induction of islet allograft tolerance involves modifying not only the T effector cells but also the Foxp3+ Tregs in CD154 deficient mice.     

CD154 Deficiency Uncouples Allograft CD8+ T‐Cell Effector Function from Proliferation and Inhibits Murine Airway Obliteration

Obliterative bronchiolitis (OB) limits the long‐term success of lung transplantation, while T‐cell effector mechanisms in this process remain incompletely understood. Using the murine heterotopic tracheal transplant model of obliterative airway disease (OAD) to characterize airway allograft rejection, we previously reported an important role for CD8⁺ T cells in OAD. Herein, we studied the role of CD154/CD40 costimulation in the regulation of allospecific CD8⁺ T cells, as airway rejection has been reported to be CD154‐dependent. Airway allografts from CD154^−/− recipients had significantly lower day 28 OAD scores compared to wild‐type (WT) recipients, and adoptive transfer of CD8⁺ T cells from WT recipients, but not CD154^−/− recipients, were capable of airway rejection in fresh CD154^−/− allograft recipients. Intragraft CD8⁺ T cells from CD154^−/− mice showed similar expression of the surface markers CD69, CD62L^low CD44^high and PD‐1, but markedly impaired IFN‐γ and TNF‐α secretion and granzyme B expression versus WT controls. Unexpectedly, intragraft and systemic CD8⁺ T cells from CD154^−/− recipients demonstrated robust in vivo expansion similar to WT recipients, consistent with an uncoupling of proliferation from effector function. Together, these data suggest that a lack of CD154/CD40 costimulation results in ineffective allospecific priming of CD8⁺ T cells required for murine OAD.     

The Programmed Death (PD)‐1/PD‐Ligand 1 Pathway Regulates Graft‐Versus‐Host‐Reactive CD8 T Cells After Liver Transplantation

Acute graft‐versus‐host disease (aGVHD) is a life‐threatening complication after solid‐organ transplantation, which is mediated by host‐reactive donor T cells emigrating from the allograft. We report on two liver transplant recipients who developed an almost complete donor chimerism in peripheral blood and bone marrow‐infiltrating T cells during aGVHD. By analyzing these T cells directly ex vivo, we found that they died by apoptosis over time without evidence of rejection by host T cells. The host‐versus‐donor reactivity was selectively impaired, as anti‐third‐party and antiviral T cells were still detectable in the host repertoire. These findings support the acquired donor‐specific allotolerance concept previously established in animal transplantation studies. We also observed that the resolution of aGVHD was not accompanied by an expansion of circulating immunosuppressive CD4/CD25/FoxP3‐positive T cells. In fact, graft‐versus‐host‐reactive T cells were controlled by an alternative negative regulatory pathway, executed by the programmed death (PD)‐1 receptor and its ligand PD‐L1. We found high PD‐1 expression on donor CD4 and CD8 T cells. In addition, blocking PD‐L1 on host‐derived cells significantly enhanced alloreactivity by CD8 T cells in vitro. We suggest the interference with the PD‐1/PD‐L1 pathway as a therapeutic strategy to control graft‐versus‐host‐reactive T cells in allograft recipients.     

IL‐17 Deficiency Attenuates Allograft Injury and Prolongs Survival in a Murine Model of Fully MHC‐Mismatched Renal Allograft Transplantation

IL‐17 is a pro‐inflammatory cytokine implicated in the pathogenesis of inflammatory and autoimmune diseases. However the role of IL‐17 in renal allograft rejection has not been fully explored. Here, we investigate the impact of IL‐17 in a fully MHC‐mismatched, life‐sustaining, murine model of kidney allograft rejection using IL‐17 deficient donors and recipients (IL‐17^?/? allografts). IL‐17^?/? allografts exhibited prolonged survival which was associated with reduced expression of the Th1 cytokine IFN‐γ and histological attenuation of acute and chronic allograft rejection, as compared to wild‐type allograft recipients. Results were confirmed in WT allograft recipients treated with an IL‐17 blocking antibody. Subsequent experiments using either donors or recipients deficient in IL‐17 showed a trend towards prolongation of survival only when recipients were IL‐17^?/?. Administration of a depleting anti‐CD25 antibody to IL‐17^?/? recipients abrogated the survival advantage conferred by IL‐17 deficiency, suggesting the involvement of a CD4⁺CD25⁺ T cell regulatory mechanism. Therefore, IL‐17 deficiency or neutralization was protective against the development of kidney allograft rejection, which may be mediated by impairment of Th1 responses and/or enhanced protection by Tregs.

     

Neutralizing IL-7 Promotes Long-Term Allograft Survival Induced by CD40/CD40L Costimulatory Blockade

Memory T cells are somewhat resistant to immunosuppresion. They therefore pose a threat to inducing long‐term allograft survival. IL‐7 is essential for memory T‐cell generation. Here, we investigated whether neutralizing IL‐7 promotes allograft survival. We found that neutralizing IL‐7 alone did not significantly prolong allograft survival. However, blocking both IL‐7 and CD154 signaling synergistically prolonged allograft survival. In contrast, neutralizing IL‐2 failed to further prolong allograft survival induced by CD40/CD154 costimulatory blockade. Allospecific memory CD8+ T‐cell generation was severely impaired under the treatment of anti‐IL‐7 plus anti‐CD154 Ab while administering recombinant IL‐7 enhanced CD8+ memory generation even under donor‐specific transfusion plus anti‐CD154 Ab treatment. Neutralizing IL‐7, but not IL‐2, together with blocking CD154 synergistically suppressed the proliferation of naïve/effector CD8+ T cells infiltrating grafts. Nevertheless, neutralizing IL‐7 did not alter regulatory T‐cell generation while neutralizing IL‐2 suppressed their generation. Hence, targeting IL‐7 represents a new strategy to prolong allograft survival by acting on both naïve and memory T cells. Long‐term allograft survival may be achieved by neutralizing IL‐7 plus CD40/CD154 blockade, since CD40/CD154 costimulatory blockade prevents acute rejection while neutralizing IL‐7 suppresses the generation of memory T cells that persist and mediate late or chronic rejection.     

CD154 Blockade Abrogates Allospecific Responses and Enhances CD4+ Regulatory T‐Cells in Mouse Orthotopic Lung Transplant

Acute cellular rejection (ACR) is a common and important clinical complication following lung transplantation. While there is a clinical need for the development of novel therapies to prevent ACR, the regulation of allospecific effector T‐cells in this process remains incompletely understood. Using the MHC‐mismatched mouse orthotopic lung transplant model, we investigated the short‐term role of anti‐CD154 mAb therapy alone on allograft pathology and alloimmune T‐cell effector responses. Untreated C57BL/6 recipients of BALB/c left lung allografts had high‐grade rejection and diminished CD4⁺: CD8⁺ graft ratios, marked by predominantly CD8⁺>CD4⁺ IFN‐γ⁺ allospecific effector responses at day 10, compared to isograft controls. Anti‐CD154 mAb therapy strikingly abrogated both CD8⁺ and CD4⁺ alloeffector responses and significantly increased lung allograft CD4⁺: CD8⁺ ratios. Examination of graft CD4⁺ T‐cells revealed significantly increased frequencies of CD4⁺CD25⁺Foxp3⁺ regulatory T‐cells in the lung allografts of anti‐CD154‐treated mice and was associated with significant attenuation of ACR compared to untreated controls. Together, these data show that CD154/CD40 costimulation blockade alone is sufficient to abrogate allospecific effector T‐cell responses and significantly shifts the lung allograft toward an environment predominated by CD4⁺ T regulatory cells in association with an attenuation of ACR.     

Intrinsic renal cell expression of CD40 directs Th1 effectors inducing experimental crescentic glomerulonephritis

Evidence suggests that human and experimental crescentic GN results from Th1-predominant immunity to glomerular antigens. CD40/CD154 signaling plays a key role in initiating Th1 responses and may direct Th1 effector responses. The role of CD40 in the development of GN was assessed in murine experimental anti-glomerular basement membrane GN. In this model, C57BL/6 wild-type (WT) mice sensitized to sheep globulin develop crescentic GN resulting from Th1 effector responses when challenged with sheep globulin planted in glomeruli. CD40-/- mice do not develop immunity in response to sheep globulin and thus fail to develop effector responses or significant GN. CD40 is expressed in nephritic glomeruli, suggesting a potential role for intrarenal CD40-CD154 interactions in injurious effector responses. Immune neutralization of the CD40 ligand (CD154) at the time of challenge significantly reduced accumulation of Th1 effectors and injury. The role of CD40 expression by renal cells was assessed by comparing GN in WT-->CD40-/- chimeras (absent renal but intact bone marrow CD40) and sham chimeric mice (WT-->WT). Both groups developed strong antigen-specific immune responses (antibody and IFN-gamma production). However, WT-->CD40-/- chimeras demonstrated reduced renal monocyte chemotactic protein 1 and IFN-inducible protein 10 mRNA levels and minimal T cell and macrophage influx and were protected from renal injury. Sham chimeric mice developed reduced GFR, with prominent renal expression of monocyte chemotactic protein 1 and IFN-inducible protein 10 mRNA and effector cell accumulation. In conclusion, the expression of CD40 by nonimmune renal cells plays a major role in Th1 effector responses by inducing Th1 chemokine production. Therefore, CD40-CD154 interactions are a potential therapeutic target in GN.     

CD40‐Independent Help by Memory CD4 T Cells Induces Pathogenic Alloantibody But Does Not Lead to Long‐Lasting Humoral Immunity

CD40/CD154 interactions are essential for productive antibody responses to T‐dependent antigens. Memory CD4 T cells express accelerated helper functions and are less dependent on costimulation when compared with naïve T cells. Here, we report that donor‐reactive memory CD4 T cells can deliver help to CD40‐deficient B cells and induce high titers of IgG alloantibodies that contribute to heart allograft rejection in CD40?/? heart recipients. While cognate interactions between memory helper T and B cells are crucial for CD40‐independent help, this process is not accompanied by germinal center formation and occurs despite inducible costimulatory blockade. Consistent with the extrafollicular nature of T/B cell interactions, CD40‐independent help fails to maintain stable levels of serum alloantibody and induce differentiation of long‐lived plasma cells and memory B cells. In summary, our data suggest that while CD40‐independent help by memory CD4 T cells is sufficient to induce high levels of pathogenic alloantibody, it does not sustain long‐lasting anti‐donor humoral immunity and B cell memory responses. This information may guide the future use of CD40/CD154 targeting therapies in transplant recipients containing donor‐reactive memory T cells.

     

Tubulitis in renal allograft rejection: role of transforming growth factor-beta and interleukin-15 in development and maintenance of CD103+ intraepithelial T cells

BACKGROUND: Renal tubules normally show no lymphocyte infiltration, but tubulitis is a feature of renal allograft rejection with many intratubular T cells expressing CD8 and CD103 (the alphaEbeta7 integrin). We investigated the development and maintenance of allospecific CD103 T cells within the tubular microenvironment. METHODS: Mixed lymphocyte cultures were supplemented with transforming growth factor (TGF)-beta1 to model the expression and function of CD103 observed in situ on intratubular lymphocytes. Immunocytochemical techniques were used to identify cells coexpressing CD8 and interleukin (IL)-15Ralpha, to enumerate proliferating intratubular T cells, and to quantify IL-15 expression within the tubules of control and rejection-graded transplant biopsy specimens. These results were compared with a parallel analysis of the phenotype and proliferation of allospecific T cells expanded in vitro in the presence of TGF-beta1 and IL-15. RESULTS: TGF-beta1 only induced the expression of adhesive CD103 after at least one cycle of alloantigen-specific cell division in vitro. In the renal allograft, a similar proportion of intratubular T cells was observed to proliferate during and after acute rejection. Tubular epithelial cells expressed IL-15 constitutively, whereas intratubular CD8 T cells expressed IL-15 receptor alpha. IL-15 and TGF-beta1 synergized to promote expansion and survival of allospecific CD8 CD103 T cells in vitro, but IL-15 down-regulated perforin expression. CONCLUSIONS: These results suggest that activated, allospecific CD8 T cells are recruited to tubules during acute rejection where they encounter TGF-beta, up-regulate CD103 expression, and bind E-cadherin. A proportion of these cells proliferates and is maintained in a state of low perforin expression by the combined action of TGF-beta and IL-15.     

Tubulitis after renal transplantation: demonstration of an association between CD103+ T cells, transforming growth factor beta1 expression and rejection grade

BACKGROUND: Tubulitis is a defining feature for the diagnosis and management of acute renal allograft rejection. Lymphocytes extracted from rejecting renal tissue are known to express the alphaEbeta7-integrin (CD103), a receptor for E-cadherin expressed on epithelial cells. In this study, expression of CD103 was examined in situ in tubulitis associated with acute rejection. METHODS: Immuno-labeling detected CD8+ and CD103+ lymphocytes and E-cadherin on epithelial cells in cryostat sections from 34 diagnostic biopsy specimens and a limited number of transplant nephrectomies. CD8+ and CD103+ intratubular cells were enumerated as mean numbers per tubular crosssection and median values were compared between rejection grades as were median ratios of CD103+ to CD8+ cells. Active transforming growth factor (TGF) beta1 was quantified in paraffin sections by immunofluorescence and confocal microscopical analysis. A parallel in vitro study quantified CD103+ T cells after allospecific activation with and without exogenous TGFbeta1. RESULTS: CD8+ T cells were present in tubules and tubular interstitium in acute rejection. CD103+ T cells were restricted exclusively to the tubules. The numbers of intratubular CD8+ and CD103+ cells and the ratio of intratubular CD103+ to CD8+ cells increased significantly with tubulitis score (P values 0.005, 0.009, and 0.02, respectively). TGFbeta1 expression was wide-spread in tubules also increasing significantly with tubulitis score (P=0.034). In chronic rejection, CD103+ T cells and TGFbeta1 were present within both tubules and interstitial cell populations. The in vitro study demonstrated that addition of TGFbeta1 to activated, alloantigen-specific T cells increased the proportion of CD8+ cells that also expressed CD103. CONCLUSIONS: These data indicate that specific upregulation of the alphaEbeta7-integrin by activated, intratubular T cells in acute renal allograft rejection could be a consequence of exposure to high local concentrations of TGFbeta1. The capacity of CD103+ T cells to bind E-cadherin on tubular epithelial cells may be an important factor in the pathogenesis of specific tissue damage observed in acute renal allograft rejection.     

Effects of proteasome inhibitors on rat renal fibrosis in vitro and in vivo

Aim: Transforming growth factor‐β (TGF‐β) is involved in renal tubulointerstitial fibrosis. Recently, the ubiquitin proteasome system was shown to participate in the TGF‐β signalling pathway. The aim of this study was to examine the effects of proteasome inhibitors on TGF‐β‐induced transformation of renal fibroblasts and tubular epithelial cells in vitro and on unilateral ureteral obstruction (UUO) in vivo. Methods: Rat renal fibroblasts NRK‐49F cells and tubular epithelial cells, NRK‐52E, were treated with TGF‐β in the presence or absence of a proteasome inhibitor, MG132 or lactacystin. Rats were subjected to UUO and received MG132 i.p. for 7 days. Results: In cultured renal cells, both MG132 and lactacystin inhibited TGF‐β‐induced α‐smooth muscle actin (α‐SMA) protein expression according to both western blotting and immunofluorescent study results. MG132 also suppressed TGF‐β‐induced mRNA expression of α‐SMA and upregulation of Smad‐response element reporter activity. However, MG132 did not inhibit TGF‐β‐induced phosphorylation and nuclear translocation of Smad2. In contrast, MG132 increased the protein level of Smad co‐repressor SnoN, demonstrating that SnoN is one of the target molecules by which MG132 blocks the TGF‐β signalling pathway. Although the proteasome inhibitor suppressed TGF‐β‐induced transformation of cultured fibroblasts and tubular epithelial cells, MG132 treatment did not ameliorate tubulointerstitial fibrosis in the rat UUO model. Conclusion: Proteasome inhibitors attenuate TGF‐β signalling by blocking Smad signal transduction in vitro, but do not inhibit renal interstitial fibrosis in vivo.     

Back signaling of HLA class I molecules and T/NK cell receptor ligands in epithelial cells reflects the rejection‐specific microenvironment in renal allograft biopsies

The role of endothelial cells in the pathophysiology of antibody‐mediated rejection after renal transplantation has been widely investigated. We expand this scenario to the impact of epithelial cells on the microenvironment during rejection. Primary proximal tubular epithelial cells were stimulated via HLA class I, CD155 and CD166 based on their potential signal‐transducing capacity to mediate back signaling after encounter with either T/NK cells or donor‐specific antibodies. Upon crosslinking of these ligands with mAbs, PTEC secreted IL‐6, CXCL1,8,10, CCL2, and sICAM‐1. These proteins were also released by PTEC as consequence of a direct interaction with T/NK cells. Downmodulation of the receptor CD226 on effector cells confirmed the involvement of this receptor/ligand pair in back signaling. In vivo, CD155 and CD166 expression was detectable in proximal and distal tubuli of renal transplant biopsies, respectively. The composition of the protein microenvironment in these biopsies showed a substantial overlap with the PTEC response. Cluster and principal component analyses of the microenvironment separated unsuspicious from rejection biopsies and, furthermore, ABMR, TCMR, and borderline rejection. In conclusion, our results provide evidence that epithelial cells may contribute to the rejection process and pave the way to a better understanding of the pathomechanisms of kidney allograft rejection.     

Galectin‐1 Prolongs Survival of Mouse Liver Allografts From Flt3L‐Pretreated Donors

Liver allografts are spontaneously accepted across MHC barriers in mice. The mechanisms underlying this phenomenon remain poorly understood. Galectin‐1, an endogenous lectin expressed in lymphoid organs, plays a vital role in maintaining central and peripheral tolerance. This study was to investigate the role of galectin‐1 in spontaneous tolerance of liver allografts in mice, and to evaluate the therapeutic effects of galectin‐1 on liver allograft rejection induced by donor Flt3L pretreatment. Blockade of the galectin‐1 pathway via neutralizing antigalectin‐1 mAb did not affect survival of the liver allografts from B6 donors into C3H recipients. Administration of rGal‐1 significantly prolonged survival of liver allografts from Flt3L‐pretreated donors and ameliorated Flt3L‐triggered liver allograft rejection. This effect was associated with increased apoptosis of T cells in both allografts and spleens, decreased frequencies of Th1 and Th17 cells, decreased expression of Th1‐associated cytokines (IL‐12, IL‐2 and IFN‐γ), Th17‐associated cytokines (IL‐23 and IL‐17) and granzyme B, in parallel with selectively increased IL‐10 expression in liver allografts. In vitro, galectin‐1 inhibited Flt3L‐differentiated DC‐mediated proliferation of allo‐CD4⁺ T cells and production of IFN‐γ and IL‐17. These data provide new evidence of the potential regulatory effects of galectin‐1 in alloimmune responses in a murine model of liver transplantation.     

Complement Mediated Renal Inflammation Induced by Donor Brain Death: Role of Renal C5a‐C5aR Interaction

Kidneys retrieved from brain‐dead donors have impaired allograft function after transplantation compared to kidneys from living donors. Donor brain death (BD) triggers inflammatory responses, including both systemic and local complement activation. The mechanism by which systemic activated complement contributes to allograft injury remains to be elucidated. The aim of this study was to investigate systemic C5a release after BD in human donors and direct effects of C5a on human renal tissue. C5a levels were measured in plasma from living and brain‐dead donors. Renal C5aR gene and protein expression in living and brain‐dead donors was investigated in renal pretransplantation biopsies. The direct effect of C5a on human renal tissue was investigated by stimulating human kidney slices with C5a using a newly developed precision‐cut method. Elevated C5a levels were found in plasma from brain‐dead donors in concert with induced C5aR expression in donor kidney biopsies. Exposure of precision‐cut human kidney slices to C5a induced gene expression of pro‐inflammatory cytokines IL‐1 beta, IL‐6 and IL‐8. In conclusion, these findings suggest that systemic generation of C5a mediates renal inflammation in brain‐dead donor grafts via tubular C5a‐C5aR interaction. This study also introduces a novel in vitro technique to analyze renal cells in their biological environment.     

Interleukin-17 and CD40-ligand synergistically enhance cytokine and chemokine production by renal epithelial cells

Renal allograft rejection is characterized by an influx of inflammatory cells. Interaction between infiltrating T cells and resident parenchymal cells might play an important role in the ongoing inflammatory response. The present study demonstrates that CD40L, a product of activated T cells, is locally expressed in kidneys undergoing rejection. Furthermore, during rejection, CD40 expression not only is present on most graft infiltrating cells but also is increased on resident tubular epithelial cells (TEC). To obtain more detailed insight in the consequences of T cell/TEC interaction, we analyzed the production of chemokines, including interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1) and regulated upon activation, normal T cell expressed and secreted (RANTES), and the production of IL-6 by cultured human primary TEC in response to activation with CD40L in vitro. In addition, we studied the interaction with IL-17, a T-cell-specific cytokine previously demonstrated to be present during renal allograft rejection. The results, obtained by enzyme-linked immunosorbent assay, indicate that simultaneous activation of TEC with IL-17 and CD40L synergistically enhances production of IL-6 (2.1-fold higher than sum of single stimulations) and the chemokines IL-8 (15-fold) and RANTES (5.8-fold) as demonstrated by statistical analysis (P: < 0.05), whereas effects on MCP-1 (1.4-fold) are additive. Part of the synergy can be explained by increased CD40 expression on TEC upon IL-17 stimulation. The synergy is not unique for TEC, because similar responses were found with human synoviocytes and a foreskin fibroblast cell line (FS4). Stimulation of TEC with CD40L results in activation of NF-kappaB and induction of cytokine production by IL-17 and CD40L is prevented by addition of the NF-kappaB inhibitor pyrrolidine dithiocarbamate. These data suggest an important role for T cells in renal allograft rejection by acting on parenchymal cells via both soluble mediators and direct cellular contact.     

CD11b is a novel alternate receptor for CD154 during alloimmunity

Antagonism of the CD154/CD40 pathway is a highly effective means of inducing long‐term graft survival in preclinical models. Using a fully allogeneic murine transplant model, we found that CD154 blockade was more effective in prolonging graft survival than was CD40 blockade, raising the possibility that CD154 binds a second receptor. To test this, we queried the impact of CD154 antagonism in the absence of CD40. Data indicated that anti‐CD154 functioned to reduce graft‐infiltrating CD8⁺ T cells in both WT and CD40^?/? hosts. Because it has recently been reported that CD154 can ligate CD11b, we addressed the impact of blocking CD154‐CD11b interactions during transplantation. We utilized a specific peptide antagonist that prevents CD154 binding of CD11b but has no effect on CD154‐CD40 interactions. CD154:CD11b antagonism significantly increased the efficacy of anti‐CD40 in prolonging allograft survival as compared to anti‐CD40 plus control peptide. Mechanistically, CD154:CD11b antagonism functioned to reduce the frequency of graft‐infiltrating CD8⁺ T cells and innate immune cells. These data therefore demonstrate that blocking CD154 interactions with both CD40 and CD11b is required for optimal inhibition of alloimmunity and provide an explanation for why CD40 blockers may be less efficacious than anti‐CD154 reagents for the inhibition of allograft rejection.     

Evidence for Kidney Rejection After Combined Bone Marrow and Renal Transplantation Despite Ongoing Whole‐Blood Chimerism in Rhesus Macaques

Although there is evidence linking hematopoietic chimerism induction and solid organ transplant tolerance, the mechanistic requirements for chimerism‐induced tolerance are not clearly elucidated. To address this, we used an MHC‐defined primate model to determine the impact of impermanent, T cell‐poor, mixed‐chimerism on renal allograft survival. We compared two cohorts: one receiving a bone marrow and renal transplant (“BMT/renal”) and one receiving only a renal transplant. Both cohorts received maintenance immunosuppression with CD28/CD40‐directed costimulation blockade and sirolimus. As previously demonstrated, this transplant strategy consistently induced compartmentalized donor chimerism, (significant whole‐blood chimerism, lacking T cell chimerism). This chimerism was not sufficient to prolong renal allograft acceptance: the BMT/renal mean survival time (MST, 76 days) was not significantly different than the renal transplant alone MST (85 days, p = 0.46), with histopathology documenting T cell mediated rejection. Flow cytometric analysis revealed significant enrichment for CD28–/CD95+ CD4+ and CD8+ Tem cells in the rejected kidney, suggesting a link between CD28‐negative Tem and costimulation blockade‐resistant rejection. These results suggest that in some settings, transient T cell‐poor chimerism is not sufficient to induce tolerance to a concurrently placed renal allograft and that the presence of this chimerism per se is not an independent biomarker to identify tolerance.     

Mechanisms of Donor‐Specific Tolerance in Recipients of Haploidentical Combined Bone Marrow/Kidney Transplantation

We recently reported long‐term organ allograft survival without ongoing immunosuppression in four of five patients receiving combined kidney and bone marrow transplantation from haploidentical donors following nonmyeloablative conditioning. In vitro assays up to 18 months revealed donor‐specific unresponsiveness. We now demonstrate that T cell recovery is gradual and is characterized by memory‐type cell predominance and an increased proportion of CD4⁺CD25⁺CD127^?FOXP3⁺ Treg during the lymphopenic period. Complete donor‐specific unresponsiveness in proliferative and cytotoxic assays, and in limiting dilution analyses of IL‐2‐producing and cytotoxic cells, developed and persisted for the 3‐year follow‐up in all patients, and extended to donor renal tubular epithelial cells. Assays in two of four patients were consistent with a role for a suppressive tolerance mechanism at 6 months to 1 year, but later (≥18 months) studies on all four patients provided no evidence for a suppressive mechanism. Our studies demonstrate, for the first time, long‐term, systemic donor‐specific unresponsiveness in patients with HLA‐mismatched allograft tolerance. While regulatory cells may play an early role, long‐term tolerance appears to be maintained by a deletion or anergy mechanism.