Attenuation of Donor‐Reactive T Cells Allows Effective Control of Allograft Rejection Using Regulatory T Cell Therapy

Regulatory T cells (Tregs) are essential for the establishment and maintenance of immune tolerance, suggesting a potential therapeutic role for Tregs in transplantation. However, Treg administration alone is insufficient in inducing long‐term allograft survival in normal hosts, likely due to the high frequency of alloreactive T cells. We hypothesized that a targeted reduction of alloreactive T effector cells would allow a therapeutic window for Treg efficacy. Here we show that preconditioning recipient mice with donor‐specific transfusion followed by cyclophosphamide treatment deleted 70–80% donor‐reactive T cells, but failed to prolong islet allograft survival. However, infusion of either 5 × 10⁶ Tregs with direct donor reactivity or 25 × 10⁶ polyclonal Tregs led to indefinite survival of BALB/c islets in more than 70% of preconditioned C57BL/6 recipients. Notably, protection of C3H islets in autoimmune nonobese diabetic mice required islet autoantigen‐specific Tregs together with polyclonal Tregs. Treg therapy led to significant reduction of CD8⁺ T cells and concomitant increase in endogenous Tregs among graft‐infiltrating cells early after transplantation. Together, these results demonstrate that reduction of the donor‐reactive T cells will be an important component of Treg‐based therapies in transplantation.     

Further Analysis of the T-Cell Subsets and Pathways of Murine Cardiac Allograft Rejection

The present study examined the role of CD4+ and CD8+ T cells in cardiac allograft rejection when either the direct or indirect pathway was eliminated for the CD4+ portion of the response. To study the pathways in vivo, we used genetically altered mouse strains that lack class II antigens as either the donors or recipients for cardiac transplantation. In contrast to earlier published studies, which used different strain combinations, we found that either CD4- or CD8-depletion prolonged cardiac allograft survival moderately, but not indefinitely, in an MHC-mismatched, minor-matched combination. When the CD4+ indirect pathway was eliminated, rapid graft rejection occurred when both T-cell subsets were present and when either CD4+ or CD8+ T cells were depleted. When the CD4+ direct pathway was eliminated, rapid graft rejection occurred when both T-cell subsets were present, there was slow rejection when CD4+ T cells were eliminated, and no rejection was seen for more than 100 days when CD8+ T cells were eliminated. However, the long-surviving allografts on the recipients with only CD4+ cells and an indirect pathway did show evidence of chronic vasculopathy. Thus, either CD4+ or CD8+ T cells can mediate acute cardiac allograft rejection in these experiments when both pathways are available. In addition, CD4+ T cells can provide help for acute rejection through either the direct or indirect pathway. Finally, recipients who have only CD4+ cells and an indirect pathway do not demonstrate acute rejection, but do show evidence of chronic rejection.     

Non‐Classical Pathways of Cell‐Mediated Allograft Rejection: New Challenges for Tolerance Induction?

Allograft rejection results from separate pathways primarily controlled by CD4+ T cells. Refinement of transplantation models together with investigations on rejection occurring despite co-stimulation blockade revealed unexpected pathways involving CD8+ T cells, NK cells and Th2 cytokines. In this minireview, we discuss these non-classical pathways of allograft rejection and their relevance for the induction of tolerance in the clinics.     

The Role of TNF Receptor and TNF Superfamily Molecules in Organ Transplantation

The rapid increase in the number of molecules demonstrated to regulate immune responses has provided new opportunities for manipulation of the recipient immune response to transplanted organs. Molecules belonging to the TNF receptors and TNF superfamily are increasingly recognized as playing a major role in the regulation of immune responses to tumor, viral, and autoantigens. The mechanisms by which these molecules regulate immune responses are diverse. TNF receptor-related molecules have been shown to control the development of secondary lymphoid organs, affect the activation and survival of T cells and antigen presenting cells, and alter cytokine and chemokine production. An increasing amount of data suggest that some TNFR superfamily members are particularly important for the function of CD8+ T cells. Based on our current understanding of these molecules it seems highly likely that strategies that target selected TNFR/TNF superfamily molecules will be useful for controlling or preventing the rejection of transplanted organs and tissues.     

落新妇甙对肺移植术后急性排斥反应的作用

目的探讨落新妇甙对大鼠肺移植后机体急性排斥反应的影响和机制，以明确落新妇甙对大鼠肺移植急性排斥反应的作用。方法建立大鼠原位肺移植模型，术后将60只受体大鼠随机分为两组，对照组：术后用生理盐水1ml／d灌胃，实验组：术后用落新妇甙1ml／kg·d灌胃。观察肺移植后大鼠的存活时间、大鼠脾细胞T淋巴细胞转化率、脾淋巴细胞白细胞介素2（IL-2）的活性以及外周血中活化T淋巴细胞凋亡情况。在电子显微镜下观察肺血管超微结构变化。结果实验组大鼠肺移植后存活时间较对照组明显延长（25．4±2．1d vs．13．4±1．2d；t=2．042，P〈0．05）。实验组脾细胞T淋巴细胞转化率较对照组明显降低（23465．8±8783．4 cpm vs．74567．3±12874．6cpm；t=2．284，P〈0．05）；实验组移植大鼠脾淋巴细胞IL-2活性较对照组明显降低（4．25±2．65U／ml vs．23．46±1．82 U／ml；t=3．165，P〈0．01）。实验组能有效地诱导急性排斥反应中活化T淋巴细胞凋亡。实验组肺组织超微结构损伤较对照组减轻。结论落新妇甙通过下调IL-2产生，诱导活化T淋巴细胞凋亡，抑制T淋巴细胞增殖分化，广泛抑制了以T淋巴细胞为主的肺移植术后急性排斥反廊，从而延长肺移槽大鼠的存活时间．     

Belatacept and Sirolimus Prolong Nonhuman Primate Islet Allograft Survival: Adverse Consequences of Concomitant Alefacept Therapy

Calcineurin inhibitors (CNI) and steroids are known to promote insulin resistance, and their avoidance after islet transplantation is preferred from a metabolic standpoint. Belatacept, a B7‐specific mediator of costimulation blockade (CoB), is clinically indicated as a CNI alternative in renal transplantation, and we have endeavored to develop a clinically translatable, belatacept‐based regimen that could obviate the need for both CNIs and steroids. Based on the known synergy between CoB and mTOR inhibition, we studied rhesus monkeys undergoing MHC‐mismatched islet allotransplants treated with belatacept and the mTOR inhibitor, sirolimus. To extend prior work on CoB‐resistant rejection, some animals also received CD2 blockade with alefacept (LFA3‐Ig). Nine rhesus macaques were rendered diabetic with streptozotocin and underwent islet allotransplantation. All received belatacept and sirolimus; six also received alefacept. Belatacept and sirolimus significantly prolonged rejection‐free graft survival (median 225 days compared to 8 days in controls receiving basiliximab and sirolimus; p = 0.022). The addition of alefacept provided no additional survival benefit, but was associated with Cytomegalovirus reactivation in four of six animals. No recipients produced donor‐specific alloantibodies. The combination of belatacept and sirolimus successfully prevents islet allograft survival in rhesus monkeys, but induction with alefacept provides no survival benefit and increases the risk of viral reactivation.     

Alloreactive (CD4-Independent) CD8+ T Cells Jeopardize Long-Term Survival of Intrahepatic Islet Allografts

Despite success of early islet allograft engraftment and survival in humans, late islet allograft loss has emerged as an important clinical problem. CD8⁺ T cells that are independent of CD4⁺ T cell help can damage allograft tissues and are resistant to conventional immunosuppressive therapies. Previous work demonstrates that islet allografts do not primarily initiate rejection by the (CD4-independent) CD8-dependent pathway. This study was performed to determine if activation of alloreactive CD4-independent, CD8⁺ T cells, by exogenous stimuli, can precipitate late loss of islet allografts. Recipients were induced to accept intrahepatic islet allografts (islet 'acceptors') by short-term immunotherapy with donor-specific transfusion (DST) and anti-CD154 mAb. Following the establishment of stable long-term islet allograft function for 60–90 days, recipients were challenged with donor-matched hepatocellular allografts, which are known to activate (CD4-independent) CD8⁺ T cells. Allogeneic islets engrafted long-term were vulnerable to damage when challenged locally with donor-matched hepatocytes. Islet allograft loss was due to allo specific immune damage, which was CD8- but not CD4-dependent. Selection of specific immunotherapy to suppress both CD4- and CD8-dependent immune pathways at the time of transplant protects islet allografts from both early and late immune damage.     

Transient Lymphopenia Breaks Costimulatory Blockade‐Based Peripheral Tolerance and Initiates Cardiac Allograft Rejection

Lymphopenia is induced by lymphoablative therapies and chronic viral infections. We assessed the impact of lymphopenia on cardiac allograft survival in recipients conditioned with peritransplant costimulatory blockade (CB) to promote long‐term graft acceptance. After vascularized MHC‐mismatched heterotopic heart grafts were stably accepted through CB, lymphopenia was induced on day 60 posttransplant by 6.5 Gy irradiation or by administration of anti‐CD4 plus anti‐CD8 mAb. Long‐term surviving allografts were gradually rejected after lymphodepletion (MST = 74 ± 5 days postirradiation). Histological analyses indicated signs of severe rejection in allografts following lymphodepletion, including mononuclear cell infiltration and obliterative vasculopathy. Lymphodepletion of CB conditioned recipients induced increases in CD44^high effector/memory T cells in lymphatic organs and strong recovery of donor‐reactive T cell responses, indicating lymphopenia‐induced proliferation (LIP) and donor alloimmune responses occurring in the host. T regulatory (CD4⁺ Foxp³⁺) cell and B cell numbers as well as donor‐specific antibody titers also increased during allograft rejection in CB conditioned recipients given lymphodepletion. These observations suggest that allograft rejection following partial lymphocyte depletion is mediated by LIP of donor‐reactive memory T cells. As lymphopenia may cause unexpected rejection of stable allografts, adequate strategies must be developed to control T cell proliferation and differentiation during lymphopenia.     

Differential Requirement of CD27 Costimulatory Signaling for Naïve Versus Alloantigen‐Primed Effector/Memory CD8+ T Cells

CD8⁺ memory T cells endanger allograft survival by causing acute and chronic rejection and prevent tolerance induction. We explored the role of CD27:CD70 T‐cell costimulatory pathway in alloreactive CD8⁺/CD4⁺ T‐cell activation. CD27‐deficient (CD27^?/?) and wild‐type (WT) B6 mice rejected BALB/c cardiac allografts at similar tempo, with or without depletion of CD4⁺ or CD8⁺ T cells, suggesting that CD27 is not essential during primary T‐cell alloimmune responses. To dissect the role of CD27 in primed effector and memory alloreactive T cells, CD27^?/? or WT mice were challenged with BALB/c hearts either 10 or 40 days after sensitization with donor‐type skin grafts. Compared to WT controls, allograft survival was prolonged in day 40‐ but not day 10‐sensitized CD27^?/? recipients. Improved allograft survival was accompanied by diminished secondary responsiveness of memory CD8⁺ T cells, which resulted from deficiency in memory formation rather than their lack of secondary expansion. Chronic allograft vasculopathy and fibrosis were diminished in CD27^?/? recipients of class I‐ but not class II‐mismatched hearts as compared to WT controls. These data establish a novel role for CD27 as an important costimulatory molecule for alloreactive CD8⁺ memory T cells in acute and chronic allograft rejection.     

Role of Natural Killer Cell Subsets in Cardiac Allograft Rejection

To achieve donor-specific immune tolerance to allogeneic organ transplants, it is imperative to understand the cell types involved in acute allograft rejection. In wild-type mice, CD4(+) T cells are necessary and sufficient for acute rejection of cardiac allografts. However, when T-cell responses are suboptimal, such as in mice treated with costimulation-targeting agents or in CD28-deficient mice, and perhaps in transplanted patients taking immunosuppressive drugs, the participation of other lymphocytes such as CD8(+) T cells and NK1.1(+) cells becomes apparent. We found that host NK but not NKT cells were required for cardiac rejection. Ly49G2(+) NK cells suppressed rejection, whereas a subset of NK cells lacking inhibitory Ly49 receptors for donor MHC class I molecules was sufficient to promote rejection. Notably, rejection was independent of the activating receptors Ly49D and NKG2D. Finally, our experiments supported a mechanism by which NK cells promote expansion and effector function of alloreactive T cells. Thus, therapies aimed at specific subsets of NK cells may facilitate transplantation tolerance in settings of impaired T-cell function.     

An Anti‐CD154 Domain Antibody Prolongs Graft Survival and Induces Foxp3+ iTreg in the Absence and Presence of CTLA‐4 Ig

The use of monoclonal antibodies targeting the CD154 molecule remains one of the most effective means of promoting graft tolerance in animal models, but thromboembolic complications during early clinical trials have precluded their use in humans. Furthermore, the role of Fc‐mediated deletion of CD154‐expressing cells in the observed efficacy of these reagents remains controversial. Therefore, determining the requirements for anti‐CD154‐induced tolerance will instruct the development of safer but equally efficacious treatments. To investigate the mechanisms of action of anti‐CD154 therapy, two alternative means of targeting the CD40–CD154 pathway were used: a nonagonistic anti‐CD40 antibody and an Fc‐silent anti‐CD154 domain antibody. We compared these therapies to an Fc‐intact anti‐CD154 antibody in both a fully allogeneic model and a surrogate minor antigen model in which the fate of alloreactive cells could be tracked. Results indicated that anti‐CD40 mAbs as well as Fc‐silent anti‐CD154 domain antibodies were equivalent to Fc‐intact anti‐CD154 mAbs in their ability to inhibit alloreactive T cell expansion, attenuate cytokine production of antigen‐specific T cells and promote the conversion of Foxp3⁺ iTreg. Importantly, iTreg conversion observed with Fc‐silent anti‐CD154 domain antibodies was preserved in the presence of CTLA4‐Ig, suggesting that this therapy is a promising candidate for translation to clinical use.

     

Long‐Term Hepatic Allograft Acceptance Based on CD40 Blockade by ASKP1240 in Nonhuman Primates

Blockade of the CD40–CD154 costimulatory signal is an attractive strategy for immunosuppression and tolerance induction in organ transplantation. Treatment with anti‐CD154 monoclonal antibodies (mAbs) results in potent immunosuppression in nonhuman primates (NHPs). Despite plans for future clinical use, further development of these treatments was halted by complications. As an alternative approach, we have been focusing on the inhibition of the counter receptor, CD40 and have shown that a novel human anti‐CD40 mAb, ASKP1240, markedly prolongs renal allograft survival in NHPs, although allografts eventually underwent chronic allograft nephropathy. On the basis of our previous findings that a CD40–CD154 costimulation blockade induces tolerance to hepatic, but not cardiac, allografts in rodents, we tested here our hypothesis that a blockade of CD40 by ASKP1240 allows acceptance of hepatic allografts in NHPs. A 2‐week ASKP1240 induction treatment prolonged liver allograft survival in NHPs; however, the graft function deteriorated due to chronic rejection. In contrast, a 6‐month ASKP1240 maintenance monotherapy efficiently suppressed both cellular and humoral alloimmune responses and prevented rejection on the hepatic allograft. No serious side effects, including thromboembolic complications, were noted in the ASKP1240‐treated monkeys. We conclude that CD40 blockade by ASKP1240 would be a desirable immunosuppressant for clinical liver transplantation.     

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.     

Transplant Acceptance Following Anti‐CD4 Versus Anti‐CD40L Therapy: Evidence for Differential Maintenance of Graft‐Reactive T Cells

Inductive therapy with anti‐CD4 or anti‐CD40L monoclonal antibodies (mAb) leads to long‐term allograft acceptance but the immune parameters responsible for graft maintenance are not well understood. This study employed an adoptive transfer system in which cells from mice bearing long‐term cardiac allografts following inductive anti‐CD4 or anti‐CD40L therapy were transferred into severe combined immunodeficiency (SCID) allograft recipients. SCID recipients of cells from anti‐CD4‐treated mice (anti‐CD4 cells) did not reject allografts while those receiving cells from anti‐CD40L‐treated mice (anti‐CD40L cells) did reject allografts. Carboxyfluorescein succinimidyl ester (CFSE) labeling of transferred cells revealed that this difference was not associated with differential proliferative capacities of these cells in SCID recipients. Like cells from naïve mice, anti‐CD40L cells mounted a Th1 response following transfer while anti‐CD4 cells mounted a dominant Th2 response. Early (day 10) T‐cell priming was detectable in both groups of primary allograft recipients but persisted to day 30 only in recipients treated with anti‐CD4 mAb. Thus, anti‐CD40L therapy appears to result in graft‐reactive T cells with a naïve phenotype while anti‐CD4 therapy allows progression to an altered state of differentiation. Additional data herein support the notion that anti‐CD40L mAb targets activated, but not memory, cells for removal or functional silencing.     

Identification of Gene Markers for the Prediction of Allograft Rejection or Permanent Acceptance

The clinical success of new treatment strategies aiming on inducing permanent graft acceptance will rely on the ability to determine whether specific unresponsiveness to donor alloantigens has developed and for how long it is maintained. To identify markers for such posttransplant monitoring, genes differentially expressed by graft infiltrating leukocytes during tolerance induction or rejection after kidney transplantation in rats were compared. A subsequently performed full kinetic analysis in two different transplant models, kidney and heart, in two species, rat and mouse identified two markers (TOAG-1, alpha-1,2-mannosidase) with high specificity and reproducibility, which are highly expressed during induction and maintenance of acceptance, and downregulated during rejection. Expression level of these markers showed a strong positive correlation with graft function. In addition, expression of both genes was downregulated in the peripheral blood and the graft prior to rejection, suggesting that these markers may be useful for monitoring in clinical transplantation where peripheral blood is the most easily accessible patient sample. Interestingly, downregulation of TOAG-1 and alpha-1,2-mannosidase expression occurred in graft infiltrating cells and expression of both genes was also downregulated after T-cell activation in vitro.