Pretransplant Immune‐Regulation Predicts Allograft Tolerance

CD4⁺ Tregs specific for noninherited maternal antigens (NIMA^d) are detectable in some but not all B6 × BDF₁ backcross, H‐2^b homozygous offspring, and their presence is strongly correlated with extent of maternal (BDF₁) microchimerism. We hypothesized that the level of pretransplant donor antigen‐specific Tregs could predict allograft tolerance. To test this idea, mice were screened for bystander suppression in a DTH assay, followed 1 week later by DBA/2 heterotopic heart transplantation. NIMA^d‐exposed, H‐2^b offspring that failed to suppress DTH uniformly rejected heart allografts (12/12) by d15. In contrast, 5/6 NIMA^d‐exposed DTH ‘regulators’ accepted their allografts >100 days. The defect in ‘nonregulator“ offspring could be corrected by transfer of CD4⁺CD25⁺, but not CD4⁺CD25^neg or CD8⁺T cells from transplant acceptor mice. In conclusion, donor‐specific T reg screening of F1 backcross offspring correctly predicted which recipients would accept a heart allograft. If translated to the clinic, similar pretransplant Treg screening could greatly enhance the effectiveness of tolerance as a clinical strategy in transplantation between family members.     

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.     

IL‐6 Induced by Staphylococcus aureus Infection Prevents the Induction of Skin Allograft Acceptance in Mice

Clinical correlations between bacterial infections and rejection suggest a hypothesis that innate immune stimulation by bacterial infections results in the production of inflammatory cytokine that facilitate bystander T‐cell activation, increased alloreactivity and inhibition of tolerance induction. Previous studies demonstrated that IFNβ produced during an infection with a model bacterium, Listeria monocytogenes, prevented the induction of transplantation tolerance in mice with anti‐CD154 and donor‐specific transfusion (DST) ( 1 ). We investigated the impact of two clinically relevant bacterial infections at the time of transplantation on the ability of anti‐CD154 and DST to induce skin allograft acceptance in mice. Staphylococcus aureus (SA) infection prevented skin allograft acceptance whereas maximally tolerated doses of Pseudomonas aeruginosa infection had no effect. SA induced an acute production of IL‐6, which was necessary and sufficient for the prevention of skin allograft acceptance. Furthermore, a single pulse of methylprednisolone modulated IL‐6 production during SA infection and facilitated skin allograft acceptance in SA‐infected recipients. Taken together, our results suggest that bacterial infections elicit specific proinflammatory cytokines signatures that can serve as barriers to tolerance induction, and that inhibiting the production of or neutralizing these inflammatory cytokines can synergize with costimulatory blockade‐based therapies to facilitate the development of transplantation tolerance.     

γδT Cells Are Involved in Liver Transplant Tolerance

The role of nonconventional T cells in innate and adaptive immunity is just emerging; γδT cells play important roles in anti-tumor and anti-infectious diseases. The involvement of γδT cells in immunologic responses to hematopoietic cell transplantation remains controversial; divergent results have been reported depending on the murine strains and model systems. Whether γδT cells are involved in solid organ transplantation is understudied. We have characterized the γδT cells in mouse livers and spleens to evaluate their contributions to liver transplant tolerance posttransplantation using a murine allogeneic liver transplant model which induces spontaneous T regulatory cell (Treg)-dependent tolerance. Our studies revealed that γδT cells comprised about 20% of the population of liver nonparenchymal cells (NPCs). In naïve C3H mice they were CD4, CD8, and NK1.1 negative. The percentage of γδT cells decreased in spontaneously tolerated liver grafts posttransplantation from 20% in naïve C3H livers to <10% in allografts throughout the time course. In contrast, they increased in liver grafts with rejection induced by anti-CTLA4 plus anti-CD25 mAb administration. CD4 and CD8 expression on γδT cells dramatically increased in the tolerated but not rejected livers posttransplantation to >20% of CD4⁺ and 30% of CD8⁺. Our results suggested that γδT cells are involved in allogeneic immune responses. Whether γδT cells function as the causal or the effector cells in allograft tolerance rejection warrants further investigation.     

Regulatory T Cells Are Critical to Tolerance Induction in Presensitized Mouse Transplant Recipients Through Targeting Memory T Cells

Memory T cells are a significant barrier to induction of transplant tolerance. However, reliable means to target alloreactive memory T cells have remained elusive. In this study, presensitization of BALB/c mice with C57BL/6 skin grafts generated a large number of OX40⁺CD44^hieffector/memory T cells and resulted in rapid rejection of donor heart allografts. Recognizing that anti‐OX40L monoclonal antibody (mAb) (α‐OX40L) monotherapy prolonged graft survival through inhibition and apoptosis of memory T cells in presensitized recipients, α‐OX40L was added to the combined treatment protocol of LF15–0195 (LF) and anti‐CD45RB (α‐CD45RB) mAb—a protocol that induced heart allograft tolerance in non‐presensitized recipients but failed to induce tolerance in presensitized recipients. Interestingly, this triple therapy restored donor‐specific heart allograft tolerance in our presensitized model that was associated with induction of tolerogenic dendritic cells and CD4⁺CD25⁺Foxp3⁺ T regulatory cells (Tregs). Of note, CD25⁺ T cell depletion in triple therapy recipients prevented establishment of allograft tolerance. In addition, adoptive transfer of donor‐primed effector/memory T cells into tolerant recipients markedly reduced levels of Tregs and broke tolerance. Our findings indicated that targeting memory T cells, by blocking OX40 costimulation in presensitized recipients was very important to expansion of Tregs, which proved critical to development of tolerance.     

Infection with the Intracellular Bacterium,Listeria monocytogenes,Overrides Established Tolerance in a Mouse Cardiac Allograft Model

Infections and TLR signals at the time of transplantation have been shown to prevent the induction of tolerance, but their effect on allografts after tolerance has been established is unclear. We here report that infection with Listeria monocytogenes precipitated the loss of tolerance and the MyD88‐ and T cell‐dependent rejection of accepted cardiac allografts in mice. This loss of tolerance was associated with increases in the numbers of graft‐infiltrating macrophages and dendritic cells, as well as CD4⁺FoxP3^? and CD8⁺ T cells. Rejection was also associated with increased numbers of graft‐infiltrating alloreactive as well as Listeria‐reactive IFNγ‐producing T cells. Rejection of the established grafts required both IL‐6 and IFNß, cytokines produced during acute Listeria infection. However, IL‐6 and IFNß alone, even when present at higher concentrations than during Listeria infection, were insufficient to break tolerance, while the combination of IL‐6 and IFNß was sufficient to break tolerance. These and in vitro observations that IL‐6 but not IFNß enhanced T cell proliferation while IFNß but not IL‐6 enhanced IFNγ production support a hypothesis that these cytokines play nonredundant roles. In conclusion, these studies demonstrate that the proinflammatory effects of infections can induce the loss of tolerance and acute rejection of accepted allografts.     

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.     

Treadmill exercise induces murine cardiac allograft survival and generates regulatory T cell

Exercise therapy has been associated with improvement in functional capacity and quality of life. The role of exercise therapy in heart transplant recipients is of great interest for the transplant society, although concerning the effect of exercise therapy, there is little knowledge at present. We analyzed the effects of exercise on alloimmune responses in murine cardiac allograft transplantation. CBA mice (H2^k) underwent transplantation of C57Bl/6 (H2^b) hearts and exercised on a treadmill. Untreated CBA recipients rejected C57Bl/6 cardiac grafts acutely (median survival time [MST], 7 days). CBA recipients treated with treadmill for 1 week after transplantation, and for 1 week both before and after transplantation prolonged allograft survivals (MSTs, 35 and 18 days, respectively). However, treadmill exercise recipients for 1 week before transplantation were not effective to allograft survival (MST, 8 days). Adoptive transfer of whole splenocytes and CD4⁺ cells from treadmill exercise recipients significantly prolonged allograft survival in naive secondary recipients (MSTs, 30 and 52 days, respectively), suggesting that regulatory cells was generated after treadmill exercise. Moreover, flow cytometry studies showed that CD4⁺CD25⁺Foxp3⁺ cell population increased in treadmill exercise recipients. Therefore, postoperative but not pre‐operative exercise could induce prolongation of survival of fully allogeneic cardiac allografts and generate CD4⁺CD25⁺Foxp3⁺ regulatory T cells.     

Combined donor specific transfusion and anti-CD154 therapy achieves airway allograft tolerance

BACKGROUND: The state of tolerance allows long term graft survival without immunosuppressants. Lung transplantation tolerance has not been consistently achieved in either small or large animal models. METHODS: The mechanisms and effectiveness of a tolerance induction protocol consisting of donor specific transfusion (DST; day 0) and a short course of co-stimulatory blockade (anti-CD154 antibody; days -7, -4, 0 and +4) were studied in the mouse heterotopic tracheal transplant model of chronic lung rejection. C57BL/6 mice received BALB/c tracheal grafts (day 0) and were treated with DST alone, anti-CD154 alone, the combination (DST/anti-CD154), or no treatment. No non-specific immunosuppressants were used. RESULTS: DST/anti-CD154 in combination, but neither treatment alone, markedly prolonged the lumen patency and survival (>100 days) of fully histo-incompatible allografts (p<0.05 versus control allografts at every time point studied up to 16 weeks) without immunosuppression. This protocol was donor antigen specific as third party grafts (C3H) were promptly rejected. In addition, DST/anti-CD154 did not result in mixed chimerism but induced transplantation tolerance via a peripheral mechanism(s), which included significantly reduced cytotoxic T cell activity (p<0.001) and a significantly increased percentage of CD4+CD25+ cells (p = 0.03). CONCLUSIONS: The DST/anti-CD154 protocol successfully induced and maintained long term, donor specific tolerance in the mouse heterotopic airway graft model of chronic lung rejection. This finding may lead us closer to successful tolerance induction in lung transplantation.     

Intragraft CD11b+IDO+ Cells Mediate Cardiac Allograft Tolerance by ECDI‐Fixed Donor Splenocyte Infusions

We have previously shown that pre‐ and post‐transplant infusions of donor splenocytes treated with 1‐ethyl‐3‐(3’‐dimethylaminopropyl)‐carbodiimide (ECDI‐SPs) provide permanent donor‐specific protection of islet allografts. The efficacy of donor ECDI‐SPs in protecting vascularized cardiac allografts and mechanism(s) of protection are unknown. In this study, we show that infusions of ECDI‐SPs significantly prolong cardiac allograft survival concomitant with an impressive accumulation of CD11b⁺IDO⁺ cells in the cardiac allograft, and that the presence of this population is dependent on Gr1⁺ cells. Consequently, depletion of Gr1⁺ cells or inhibition of indoleamine 2,3 dioxygenase (IDO) activity abrogates graft protection by ECDI‐SPs infusions. In addition, T cells from ECDI‐SPs treated recipients secrete high levels of interleukin 10 and interleukin 13 upon in vitro restimulation, which are also dampened in recipients treated with the IDO inhibitor. Furthermore, combination of donor ECDI‐SPs with a short course of rapamycin provides indefinite cardiac allograft survival in 100% of the recipients. These findings reveal a novel mechanism of donor ECDI‐SPs in inducing cardiac transplant tolerance and provide several targets that are amenable to therapeutic manipulations for tolerance induction for cardiac transplantation.     

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.     

PD‐1 expression on CD8+ T cells regulates their differentiation within lung allografts and is critical for tolerance induction

Immunological requirements for rejection and tolerance induction differ between various organs. While memory CD8⁺ T cells are considered a barrier to immunosuppression‐mediated acceptance of most tissues and organs, tolerance induction after lung transplantation is critically dependent on central memory CD8⁺ T lymphocytes. Here we demonstrate that costimulation blockade‐mediated tolerance after lung transplantation is dependent on programmed cell death 1 (PD‐1) expression on CD8⁺ T cells. In the absence of PD‐1 expression, CD8⁺ T cells form prolonged interactions with graft‐infiltrating CD11c⁺ cells; their differentiation is skewed towards an effector memory phenotype and grafts are rejected acutely. These findings extend the notion that requirements for tolerance induction after lung transplantation differ from other organs. Thus, immunosuppressive strategies for lung transplant recipients need to be tailored based on the unique immunological properties of this organ.     

IL‐22 is required for the induction of bronchus‐associated lymphoid tissue in tolerant lung allografts

Long‐term survival after lung transplantation remains profoundly limited by graft rejection. Recent work has shown that bronchus‐associated lymphoid tissue (BALT), characterized by the development of peripheral nodal addressin (PNAd)‐expressing high endothelial venules and enriched in B and Foxp3⁺ T cells, is important for the maintenance of allograft tolerance. Mechanisms underlying BALT induction in tolerant pulmonary allografts, however, remain poorly understood. Here, we show that the development of PNAd‐expressing high endothelial venules within intragraft lymphoid follicles and the recruitment of B cells, but not Foxp3⁺ cells depends on IL‐22. We identify graft‐infiltrating gamma‐delta (γδ) T cells and Type 3 innate lymphoid cells (ILC3s) as important producers of IL‐22. Reconstitution of IL‐22 at late time points through retransplantation into wildtype hosts mediates B cell recruitment into lymphoid follicles within the allograft, resulting in a significant increase in their size, but does not induce PNAd expression. Our work has identified cellular and molecular requirements for the induction of BALT in pulmonary allografts during tolerance induction and may provide a platform for the development of new therapies for lung transplant patients.     

Artemisiae capillaris herba induces prolonged survival of fully cardiac allografts and generates regulatory cells in mice

Inchingorei-san (TJ-117), a 6-component herbal medicine, is used in Japan for the treatment of vomiting, urticaria, and liver and kidney disorders with few side effects. In this study we investigated the effect of TJ-117 on alloimmune responses in murine cardiac allograft transplantation. CBA (H2^k) mice underwent transplantation of a C57BL/6 (B6, H2^b) heart with oral administration of TJ-117 (or 1 component of TJ-117) from the day of transplantation for 7 days. CBA recipients given 1 g/kg/d of TJ-117 showed prolonged B6 allograft survival (median survival time [MST], 23 days). Naive CBA mice rejected B6 cardiac grafts acutely (MST, 7 days). Moreover, Artemisiae capillaris herba (ACH; 1g/kg/d) 1 component of TJ-117, significantly prolonged B6 allograft survival (MST, > 100 days). However, the other 5 components of TJ-117 were individually less effective than TJ-117 or ACH. ACH also suppressed splenocyte proliferation and interferon-γ production. Secondary CBA recipient showed prolonged survival of B6 hearts after treatments with whole splenocytes from primary ACH-treated CBA recipients carrying B6 cardiac allografts for 30 days (MST, >50 days). Flow cytometry studies showed increased CD4⁺CD25⁺Foxp3⁺ regulatory cells in transplant recipients given ACH. In conclusion, ACH, 1 component of TJ-117, as well as TJ-117 induced hyporesponsiveness to fully allogeneic cardiac allografts with generation of CD4⁺CD25⁺Foxp3⁺ regulatory cells.     

Vascularized Osteomyocutaneous Allografts Are Permissive to Tolerance by Induction‐Based Immunomodulatory Therapy

Vascularized composite allografts (VCAs) are unique among transplanted organs in that they are composed of multiple tissues with disparate antigenic and immunologic properties. As the predominant indications for VCAs are non‐life‐threatening conditions, there is an immediate need to develop tolerance induction strategies and to elucidate the mechanisms of VCA rejection and tolerance using VCA‐specific animal models. In this study, we explore the effects of in vitro induced donor antigen‐specific CD4^?CD8^? double negative (DN) Treg‐based therapy, in a fully MHC mismatched mouse VCA such as a vascularized osteomyocutaneous as compared to a non‐VCA such as a full thickness skin (FTS) transplantation model to elucidate the unique features of VCA rejection and tolerance. We demonstrate that combined therapy with antigen‐induced CD4 derived DN Tregs and a short course of anti‐lymphocyte serum, rapamycin and IL‐2/Fc fusion protein results in donor‐specific tolerance to VCA, but not FTS allografts. Macrochimerism was detected in VCA but not FTS allograft recipients up to >60 days after transplantation. Moreover, a significant increase of CD4⁺Foxp3⁺ Tregs was found in the peripheral blood of tolerant VCA recipients. These data suggest that VCA are permissive to tolerance induced by DN Treg‐based induction therapy.

     

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.     

Music exposure induced prolongation of cardiac allograft survival and generated regulatory CD4⁺ cells in mice

In clinical practice, music has been used to decrease stress, heart rate, and blood pressure and to provide a distraction from disease symptoms. We investigated sound effects on alloimmune responses in murine heart transplantation. Naïve and eardrum-ruptured CBA/N (CBA, H2^K) underwent transplantation of a C57BL/6 (B6, H2^b) heart and were exposed to 1 of 3 types of music-opera (La Traviata), classical (Mozart), and New Age (Enya)-or 1 of 6 different single sound frequencies for 7 days. An adoptive transfer study was performed to determine whether regulatory cells were generated in allograft recipients. Cell-proliferation, cytokine, and flow cytometry assessments were also performed. CBA recipients of a B6 graft exposed to opera and classical music had significantly prolonged allograft survival (median survival times [MSTs], 26.5 and 20 days, respectively), whereas those exposed to 6 single sound frequencies and New Age did not (MSTs, 7, 8, 9, 8, 8, 8, and 11 days, respectively). Untreated and eardrum-ruptured CBA rejected B6 grafts acutely (MSTs, 7 and 8.5 days, respectively). Adoptive transfer of whole splenocytes, CD4⁺ cells, and CD4⁺CD25⁺ cells from opera-exposed primary recipients resulted in significantly prolonged allograft survival in naive secondary recipients (MSTs, 36, 68, and >50 days, respectively). Cell-proliferation, interleukin (IL)-2 and interferon-γ were suppressed in opera-exposed mice, whereas IL-4 and IL-10 from opera-exposed recipients were up-regulated. Flow cytometry studies showed an increased CD4⁺CD25⁺Foxp3⁺ cell population in splenocytes from opera-exposed mice. In conclusion, exposure to some types of music may induce prolonged survival of fully allogeneic cardiac allografts and generate CD4⁺CD25⁺Foxp3⁺ regulatory cells.