Allergic Conjunctivitis Renders CD4+ T Cells Resistant to T Regulatory Cells and Exacerbates Corneal Allograft Rejection

Allergic diseases rob corneal allografts of immune privilege and increase immune rejection. Corneal allograft rejection in BALB/c allergic hosts was analyzed using a short ragweed (SWR) pollen model of allergic conjunctivitis. Allergic conjunctivitis did not induce exaggerated T‐cell responses to donor C57BL/6 (B6) alloantigens or stimulate cytotoxic T lymphocyte (CTL) responses. Allergic conjunctivitis did affect T regulatory cells (Tregs) that support graft survival. Exogenous IL‐4, but not IL‐5 or IL‐13, prevented Treg suppression of CD4⁺ effector T cells isolated from naïve mice. However, mice with allergic conjunctivitis developed Tregs that suppressed CD4⁺ effector T‐cell proliferation. In addition, IL‐4 did not inhibit Treg suppression of IL‐4Rα^?/? CD4⁺ T‐cell responses, suggesting that IL‐4 rendered effector T cells resistant to Tregs. SRW‐sensitized IL‐4Rα^?/? mice displayed the same 50% graft survival as nonallergic WT mice, that was significantly less than the 100% rejection that occurred in allergic WT hosts, supporting the role of IL‐4 in the abrogation of immune privilege. Moreover, exacerbation of corneal allograft rejection in allergic mice was reversed by administering anti‐IL‐4 antibody. Thus, allergy‐induced exacerbation of corneal graft rejection is due to the production of IL‐4, which renders effector T cells resistant to Treg suppression of alloimmune responses.     

Interleukin‐33 prolongs allograft survival during chronic cardiac rejection

Interleukin‐33 (IL‐33) stimulates the generation of cells and cytokines characteristic of a Th2 immune response. We examined the effects of IL‐33 on allografted heart tissue in a chronic cardiac rejection model, including analysis of the peripheral myeloid and lymphoid compartments. B6.C‐H2bm12/KhEg hearts were transplanted into MHC class II‐mismatched C57Bl/6J mice; IL‐33 was administered daily. Cells from allografts and spleens were isolated for flow cytometry and cultured for cytokine production; some tissues were used for immunohistochemistry. Animals treated with IL‐33 showed significantly longer allograft survival, which was associated with a distinct cytokine profile produced by graft‐infiltrating cells. Proinflammatory IL‐17A production was decreased with IL‐33 treatment, while increased levels of IL‐5, IL‐10, and IL‐13 were observed. After IL‐33 therapy, flow cytometry showed a direct induction of CD4⁺ Foxp3⁺ Treg, whereas the number of B220⁺ CD19⁺ B cells, and circulating, as well as allograft deposited, alloantibodies was reduced. Following IL‐33 treatment, a significant decrease in graft‐infiltrating CD11b^highGr1^high granulocytes coincided with a significant increase in CD11b^highGr1^intermediate myeloid‐derived suppressor cells (MDSC). In conclusion, IL‐33 treatment in the setting of chronic rejection promotes the development of a Th2‐type immune response that favors MDSC and Treg expansion, reduces antibody‐mediated rejection (AMR), and ultimately, prolongs allograft survival.     

B cell–derived IL‐1β and IL‐6 drive T cell reconstitution following lymphoablation

Understanding the mechanisms of T cell homeostatic expansion is crucial for clinical applications of lymphoablative therapies. We previously established that T cell recovery in mouse heart allograft recipients treated with anti‐thymocyte globulin (mATG) critically depends on B cells and is mediated by B cell–derived soluble factors. B cell production of interleukin (IL)‐1β and IL‐6 is markedly upregulated after heart allotransplantation and lymphoablation. Neutralizing IL‐1β or IL‐6 with mAb or the use of recipients lacking mature IL‐1β, IL‐6, IL‐1R, MyD88, or IL‐6R impair CD4⁺ and CD8⁺ T cell recovery and significantly enhance the graft‐prolonging efficacy of lymphoablation. Adoptive co‐transfer experiments demonstrate a direct effect of IL‐6 but not IL‐1β on T lymphocytes. Furthermore, B cells incapable of IL‐1β or IL‐6 production have diminished capacity to mediate T cell reconstitution and initiate heart allograft rejection upon adoptive transfer into mATG treated B cell deficient recipients. These findings reveal the essential role of B cell–derived IL‐1β and IL‐6 during homeostatic T cell expansion in a clinically relevant model of lymphoablation.     

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.     

Interleukin‐17 positive cells accumulate in renal allografts during acute rejection and are independent predictors of worse graft outcome

Interleukin‐17 (IL‐17) plays an important role in the regulation of cellular and humoral immune responses. Recent studies suggest a role for IL‐17 in transplantation. Our study investigated whether quantifying IL‐17⁺ cells in renal transplant biopsies during acute rejection could have additional prognostic value for better stratifying patients at risk for nonresponsiveness to anti‐rejection therapy and future graft dysfunction. Forty‐nine renal biopsies with acute rejection were double immunostained and quantitatively analyzed for IL‐17 and CD3 (IL‐17⁺ T‐lymphocytes), tryptase (IL‐17⁺ mast cells) or CD15 (IL‐17⁺ neutrophils). Total IL‐17⁺ cell count correlated with total percentage of inflamed biopsy and estimated GFR during rejection. Most IL‐17⁺ cells were mast cells and neutrophils. We could hardly find any IL‐17⁺ T‐lymphocytes. IL‐17⁺ mast cells correlated with interstitial fibrosis/tubular atrophy (IF/TA). None of the IL‐17⁺ cell counts had an additional prognostic value for response to anti‐rejection treatment. Multivariate analysis correcting for C4d positivity and time from transplantation to biopsy showed that total IL‐17⁺ cell count independently predicts graft dysfunction at the last follow‐up, which was validated in an independent cohort of 48 renal biopsies with acute rejection. We conclude that intragraft IL‐17⁺ cell count during acute allograft rejection could have an additional value for predicting late graft dysfunction.     

Anti‐TCRβ mAb Induces Long‐Term Allograft Survival by Reducing Antigen‐Reactive T Cells and Sparing Regulatory T Cells

TCR specific antibodies may modulate the TCR engagement with antigen–MHC complexes, and in turn regulate in vivo T cell responses to alloantigens. Herein, we found that in vivo administration of mAbs specific for mouse TCRβ (H57–597), TCRα or CD3 promptly reduced the number of CD4⁺ and CD8⁺ T cells in normal mice, but H57–597 mAb most potently increased the frequency of CD4⁺Foxp3⁺ Treg cells. When mice were injected with staphylococcal enterotoxin B (SEB) superantigen and H57–597 mAb, the expansion of SEB‐reactive Vβ8⁺ T cells was completely abrogated while SEB‐nonreactive Vβ2⁺ T cells remained unaffected. More importantly, transient H57–597 mAb treatment exerted long‐lasting effect in preventing T cell responses to alloantigens, and produced long‐term cardiac allograft survival (>100 days) in 10 out of 11 recipients. While Treg cells were involved in maintaining donor‐specific long‐term graft survival, T cell homeostasis recovered over time and immunity was retained against third party allografts. Moreover, transient H57–597 mAb treatment significantly prolonged survival of skin allografts in naïve recipients as well as heart allografts in skin‐sensitized recipients. Thus, transient modulation of the TCRβ chain by H57–597 mAb exhibits potent, long‐lasting therapeutic effects to control alloimmune responses.     

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.

     

The Immunobiology of Inductive Anti-CD40L Therapy in Transplantation: Allograft Acceptance is Not Dependent Upon the Deletion of Graft-Reactive T Cells

CD40–CD40L costimulatory interactions are crucial for allograft rejection, in that treatment with anti‐CD40L mAb markedly prolongs allograft survival in several systems. Recent reports indicate that costimulatory blockade results in deletion of graft‐reactive cells, which leads to allograft tolerance. To assess immunologic parameters that were influenced by inductive CD40–CD40L blockade, cardiac allograft recipients were treated with multiple doses of the anti‐CD40L mAb MR1, which was remarkably effective at prolonging allograft survival. Acute allograft rejection responses such as IL‐2 producing helper cell priming, Th1 priming, and alloantibody production were abrogated by anti‐CD40L treatment. Interestingly, the spleens of mice bearing long‐term cardiac allografts following inductive anti‐CD40L treatment retained precursor donor alloantigen‐reactive CTL, IL‐2 producing helper cells, and Th1 in numbers comparable to those observed in naïve mice. These mice retained the ability to reject donor‐strain skin allografts, but were incapable of rejecting the original cardiac allograft, or a second donor‐strain cardiac allograft. Further, differentiated effector cells were incapable of mediating rejection following adoptive transfer into mice bearing long‐term allografts, suggesting that regulatory cell function, rather than effector cell deletion was responsible for long‐term graft acceptance. Collectively, these data demonstrate that inductive CD40–CD40L blockade does not result in the deletion of graft‐reactive T cells, but induces the maintenance of these cells in a quiescent precursor state. They further point to a tissue specificity of this hyporesponsiveness, suggesting that not all donor alloantigen‐reactive cells are subject to this regulation.     

Hyperlipidemia Promotes Anti‐Donor Th17 Responses That Accelerate Allograft Rejection

Hyperlipidemia occurs in 95% of organ transplant recipients, however its effect on organ allograft rejection has not been investigated. We found that induction of hyperlipidemia in mice caused a significant acceleration of rejection of cardiac allografts. Accelerated rejection was associated with an aggressive T cell infiltrate that mediated significant tissue damage as well as increased serum levels of the proinflammatory cytokines IL‐2, IL‐6, and IL‐17. Hyperlipidemic mice had an increased number of Th17 cells in their periphery and rejecting allografts from hyperlipidemic mice contained significant numbers of IL‐17 producing T cells that were not detectable in transplants harvested from controls. Neutralization or genetic ablation of IL‐17 prolonged survival of cardiac allografts transplanted into hyperlipidemic recipients, suggesting that IL‐17 production promotes accelerated rejection. Analysis of alloreactive T cell frequencies directly ex vivo in naïve mice revealed that the frequency of donor reactive IL‐17 producing cells in hyperlipidemic was increased prior to antigen exposure, suggesting that hyperlipidemia was sufficient to alter T cell alloreactivity and promote anti‐donor Th17 responses on first exposure to antigen. Together, our data suggest that hyperlipidemia alters rejection by altering the types of T cell subsets that respond to donor antigen by promoting Th17 biased anti‐donor reactivity.     

CD8+ T‐Cell Depletion and Rapamycin Synergize with Combined Coreceptor/Stimulation Blockade to Induce Robust Limb Allograft Tolerance in Mice

The growing development of composite tissue allografts (CTA) highlights the need for tolerance induction protocols. Herein, we developed a mouse model of heterotopic limb allograft in a stringent strain combination in which potentially tolerogenic strategies were tested taking advantage of donor stem cells in the grafted limb. BALB/c allografts were transplanted into C57BL/6 mice treated with anti‐CD154 mAb, nondepleting anti‐CD4 combined to either depleting or nondepleting anti‐CD8 mAbs. Some groups received additional rapamycin. Both depleting and nondepleting mAb combinations without rapamycin only delayed limb allograft rejection, whereas the addition of rapamycin induced long‐term allograft survival in both combinations. Nevertheless, robust donor‐specific tolerance, defined by the acceptance of a fresh donor‐type skin allograft and simultaneous rejection of third‐party grafts, required initial CD8⁺ T‐cell depletion. Mixed donor‐recipient chimerism was observed in lymphoid organs and recipient bone marrow of tolerant but not rejecting animals. Tolerance specificity was confirmed by the inability to produce IL‐2, IFN‐γ and TNF‐α in MLC with donor antigen while significant alloreactivity persisted against third‐ party alloantigens. Collectively, these results show that robust CTA tolerance and mixed donor‐recipient chimerism can be achieved in response to the synergizing combination of rapamycin, transient CD8⁺ T‐cell depletion and costimulation/coreceptor blockade.     

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.     

LFA‐1 Antagonism Inhibits Early Infiltration of Endogenous Memory CD8 T Cells into Cardiac Allografts and Donor‐Reactive T Cell Priming

Alloreactive memory T cells are present in virtually all transplant recipients due to prior sensitization or heterologous immunity and mediate injury undermining graft outcome. In mouse models, endogenous memory CD8 T cells infiltrate MHC‐mismatched cardiac allografts and produce IFN‐γ in response to donor class I MHC within 24 h posttransplant. The current studies analyzed the efficacy of anti‐LFA‐1 mAb to inhibit early CD8 T cell cardiac allograft infiltration and activation. Anti‐LFA‐1 mAb given to C57BL/6 6 (H‐2^b) recipients of A/J (H‐2^a) heart grafts on days –1 and 0 completely inhibited CD8 T cell allograft infiltration, markedly decreased neutrophil infiltration and significantly reduced intragraft expression levels of IFN‐γ‐induced genes. Donor‐specific T cells producing IFN‐γ were at low/undetectable numbers in spleens of anti‐LFA‐1 mAb treated recipients until day 21. These effects combined to promote substantial prolongation (from day 8 to 27) in allograft survival. Delaying anti‐LFA‐1 mAb treatment until days 3 and 4 posttransplant did not inhibit early memory CD8 T cell infiltration and proliferation within the allograft. These data indicate that peritransplant anti‐LFA‐1 mAb inhibits early donor‐reactive memory CD8 T cell allograft infiltration and inflammation suggesting an effective strategy to attenuate the negative effects of heterologous immunity in transplant recipients.     

Adoptive transfer of double negative T regulatory cells induces B-cell death in vivo and alters rejection pattern of rat-to-mouse heart transplantation

Abstract: Background: Antibody‐mediated hyperacute and acute graft rejection are major obstacles in achieving long‐term graft survival in xenotransplantation. It is well documented that regulatory T (Treg) cells play a very important role in regulating immune responses to self and non‐self antigens. Our previous studies have shown that TCRαβ⁺CD3⁺CD4^?CD8^? (double negative, DN)‐Treg cells can suppress anti‐donor T‐cell responses and prolong graft survival in allo‐ and xenotransplantation models. We have demonstrated that DN‐Treg cells can induce B‐cell apoptosis in vitro through a perforin‐dependent pathway. Methods: B6 mice received rat heart grafts, followed by 14 days of LF15‐0195 treatment. Some mice received Lewis rat cell activated DN‐Treg cells after LF treatment. DN‐Treg cells, purified from perforin^?/? mice and from B6 mice pre‐immunized with third party rat cells, were used as controls. Results: In this study, we investigated the possibility that adoptive transfer of xenoreactive DN‐Treg cells could suppress B cells in vivo, thus prolonging xenograft survival. We found that apoptotic death of B cells significantly increased after adoptive transfer of DN‐Treg cells. In addition, anti‐donor IgG subtypes were significantly inhibited in the DN‐Treg cell‐treated group, in which the rejection pattern was altered towards cellular‐mediated rejection rather than antibody‐mediated acute vascular rejection. However, perforin‐deficient DN‐Treg cells failed to induce B‐cell death and to prolong heart graft survival, indicating a perforin‐dependent mechanism contributes to B‐cell death in vivo. Conclusions: This study suggests that adoptive transfer of xenoreactive DN‐Treg cells can inhibit B‐cell responses in vivo. DN‐Treg cells may be valuable in controlling B‐cell responses in xenotransplantation.     

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.     

Suppressing memory T cell activation induces islet allograft tolerance in alloantigen‐primed mice

Memory T cells are known to play a key role in prevention of allograft tolerance in alloantigen‐primed mice. Here, we used an adoptively transferred memory T cell model and an alloantigen‐primed model to evaluate the abilities of different combinations of monoclonal antibodies (mAb) to block key signaling pathways involved in activation of effector and memory T cells. In the adoptively transferred model, the use of anti‐CD134L mAb effectively prevented activation of CD4⁺ memory T cells and significantly prolonged islet survival, similar to the action of anti‐CD122 mAb to CD8⁺ memory T cells. In the alloantigen‐primed model, use of anti‐CD134L and anti‐CD122 mAbs in addition to co‐stimulatory blockade with anti‐CD154 and anti‐LFA‐1 prolonged secondary allograft survival and significantly reduced the proportion of memory T cells; meanwhile, this combination therapy increased the proportion of regulatory T cells (Tregs) in the spleen, inhibited lymphocyte infiltration in the graft, and suppressed alloresponse of recipient splenic T cells. However, we also detected high levels of alloantibodies in the serum which caused high levels of damage to the allogeneic spleen cells. Our results suggest that combination of four mAbs can significantly suppress the function of memory T cells and prolong allograft survival in alloantigen primed animals.     

Paradoxical Functions of B7: CD28 Costimulation in a MHC Class II‐Mismatched Cardiac Transplant Model

Blockade of the B7: CD28 costimulatory pathway has emerged as a promising therapy to prevent allograft rejection. However, this pathway has also been demonstrated to be important for the generation and maintenance of regulatory T cells. In this study, we investigated the role of the B7: CD28 pathway in the ‘bm12 into B6’ MHC class II‐mismatched vascularized cardiac transplant model of chronic rejection. Allograft rejection was remarkably accelerated in B6 background B7DKO and CD28KO recipients compared with B6 wild‐type (WT) recipients. Allograft rejection was associated with a significantly enhanced Th1/Th2 alloreactivity and marked reduction in the ratio of regulatory T cells to CD4⁺ effector/memory cells. We noted that administration of anti‐B7‐1 and anti‐B7‐2 mAb prior to transplantation also accelerated allograft rejection. Furthermore, depleting CD25⁺ cells in B6 WT recipients of bm12 hearts prior to transplant also precipitated rejection at a similar rate. Neither B7/CD28 deficiency nor CD25 depletion affected graft survival in single MHC class I‐mismatched (bm1 into B6) recipients. This study highlights the paradoxical functions of B7: CD28 costimulation in a MHC class II‐mismatched model, in which the B7: CD28 pathway is demonstrated to be important in preventing rejection through the generation and maintenance of Tregs.     

Peritransplant VLA‐4 blockade inhibits endogenous memory CD8 T cell infiltration into high‐risk cardiac allografts and CTLA‐4Ig resistant rejection

Recipient endogenous memory CD8 T cells expressing reactivity to donor class I MHC infiltrate MHC‐mismatched cardiac allografts within 24 hours after reperfusion and express effector functions mediating graft injury. The current study tested the efficacy of Very Late Antigen‐4 (VLA‐4) blockade to inhibit endogenous memory CD8 T cell infiltration into cardiac allografts and attenuate early posttransplant inflammation. Peritransplant anti‐VLA‐4 mAb given to C57BL6 (H‐2^b) recipients of AJ (H‐2^a) heart allografts completely inhibited endogenous memory CD4 and CD8 T cell infiltration with significant decrease in macrophage, but not neutrophil, infiltration into allografts subjected to either minimal or prolonged cold ischemic storage (CIS) prior to transplant, reduced intra‐allograft IFN‐γ‐induced gene expression and prolonged survival of allografts subjected to prolonged CIS in CTLA‐4Ig treated recipients. Anti‐VLA‐4 mAb also inhibited priming of donor‐specific T cells producing IFN‐γ until at least day 7 posttransplant. Peritransplant anti‐VLA plus anti‐CD154 mAb treatment similarly prolonged survival of allografts subjected to minimal or increased CIS prior to transplant. Overall, these data indicate that peritransplant anti‐VLA‐4 mAb inhibits early infiltration memory CD8 T cell infiltration into allografts with a marked reduction in early graft inflammation suggesting an effective strategy to attenuate negative effects of heterologous alloimmunity in recipients of higher risk grafts.     

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.     

Anti‐huCD20 Antibody Therapy for Antibody‐Mediated Rejection of Renal Allografts in a Mouse Model

We have reported that B6.CCR5^?/? mice reject renal allografts with high serum donor‐specific antibody (DSA) titers and marked C4d deposition in grafts, features consistent with antibody‐mediated rejection (AMR). B6.huCD20/CCR5^?/? mice, where human CD20 expression is restricted to B cells, rejected A/J renal allografts by day 26 posttransplant with DSA first detected in serum on day 5 posttransplant and increased thereafter. Recipient treatment with anti‐huCD20 mAb prior to the transplant and weekly up to 7 weeks posttransplant promoted long‐term allograft survival (>100 days) with low DSA titers. To investigate the effect of B cell depletion at the time serum DSA was first detected, recipients were treated with anti‐huCD20 mAb on days 5, 8, and 12 posttransplant. This regimen significantly reduced DSA titers and graft inflammation on day 15 posttransplant and prolonged allograft survival >60 days. However, DSA returned to the titers observed in control treated recipients by day 30 posttransplant and histological analyses on day 60 posttransplant indicated severe interstitial fibrosis. These results indicate that anti‐huCD20 mAb had the greatest effect as a prophylactic treatment and that the distinct kinetics of DSA responses accounts for acute renal allograft failure versus the development of fibrosis.