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.     

Imaging Tolerance Induction in the Classic Medawar Neonatal Mouse Model: Active Roles of Multiple F1‐Donor Cell Types

The immature immune system is uniquely susceptible to tolerance induction and thus an attractive target for immunomodulation strategies for organ transplantation. Newborn mice injected with adult semi‐allogeneic lymphohematopoietic cells accept transplants without immunosuppressive drugs. Early in vivo/in situ events leading to neonatal tolerance remain poorly understood. Here, we show by whole body/organ imaging that injected cells home to lymphoid organs and liver where various F1‐donor cell types selectively alter neonatal immunity. In host thymus, F1‐donor dendritic cells (DC) interact with developing thymocytes and regulatory T cells suggesting a role in negative selection. In spleen and lymph nodes, F1‐donor regulatory T/B cells associate with host alloreactive cells and by themselves prolong cardiac allograft survival. In liver, F1‐donor cells give rise to albumin‐containing hepatocyte‐like cells. The neonatal immune system is lymphopenic, Th‐2 immunodeviated and contains immature DC, suggesting susceptibility to regulation by adult F1‐donor cells. CD8a T cell inactivation greatly enhances chimerism, suggesting that variable emerging neonatal alloreactivity becomes a barrier to tolerance induction. This comprehensive qualitative imaging study systematically shows contribution of multiple in vivo processes leading simultaneously to robust tolerance. These insights into robust tolerance induction have important implications for development of strategies for clinical application.     

Lymphodepletion and Homeostatic Proliferation: Implications for Transplantation

Control of the alloimmune response requires elimination and/or suppression of alloreactive immune cells. Lymphodepleting induction therapies are increasingly used to accomplish this goal, both as part of tolerance induction protocols or to reduce the requirements for maintenance immunosuppression in the peritransplant setting. However, it is well recognized that lymphopenia induces compensatory proliferation of immune cells, generally termed ``homeostatic proliferation,'' which favors the emergence of memory T cells. Paradoxically therefore, the result may be a situation that favors graft rejection and/or makes tolerance difficult to achieve or sustain. Yet all depletion is not alike, particularly with respect to the timing of reconstitution and the types of cells that repopulate the host. Thus, to design more effective induction strategies it is important to understand the homeostatic mechanisms, which exist to maintain a balanced repertoire of naïve and memory T and B cells in the periphery and how they respond to lymphodepletion. Here we will review the biology of homeostatic proliferation stimulated by lymphopenia, the effects of specific depleting agents on reconstitution of the T‐ and B‐cell immune repertoire, drawing from both from animal models and human experience, and potential strategies to enhance allodepletion while minimizing the adverse effects of homeostatic proliferation.     

Persistence of Indirect but Not Direct T Cell Xenoresponses in Baboon Recipients of Pig Cell and Organ Transplants

We investigated the contributions of direct and indirect T cell antigen recognition pathways to the immune response to porcine antigens in naïve baboons and baboon recipients of pig xenografts. In naïve baboons, in vitro culture of peripheral blood T cells with intact pig cells (direct xenorecognition pathway) or pig cell sonicates and baboon antigen‐presenting cells (indirect xenorecognition pathway) induced the activation and expansion of xenoreactive T cells producing proinflammatory cytokines, interleukin‐2 and interferon‐γ. Primary indirect xenoresponses were mediated by preexisting memory T cells, whose presence is not typically observed in primary alloresponses. Next, baboons were conditioned with a nonmyeloablative regimen before short‐term immunosuppression and transplantation of xenogeneic peripheral blood progenitor cells and a kidney, heart, or pancreatic islets from a miniature swine. All transplants were rejected acutely within 30 days after their placement. Posttransplantation, we observed an inhibition of the direct xenoresponse but a significant expansion of indirectly activated proinflammatory T cells. These results suggest that additional treatment to suppress indirect T cell immunity in primates may be required to achieve tolerance of pig xenografts through hematopoietic chimerism.     

Induction of suppressive allogeneic regulatory T cells via rabbit antithymocyte polyclonal globulin during homeostatic proliferation in rat kidney transplantation

Experimental studies have shown that rabbit antithymocyte polyclonal globulin (ATG) can expand human CD4+CD25++Foxp3+ cells (Tregs). We investigated the major biological effects of a self‐manufactured rabbit polyclonal anti‐rat thymoglobulin (rATG) in vitro, as well as its effects on different peripheral T‐cell subsets. Moreover, we evaluated the allogeneic suppressive capacity of rATG‐induced Tregs in an experimental rat renal transplant model. Our results show that rATG has the capacity to induce apoptosis in T lymphocyte lymphocytes as a primary mechanism of T‐cell depletion. Our in vivo studies demonstrated a rapid but transient cellular depletion of the main T cell subsets, directly proportional to the rATG dose used, but not of the effector memory T cells, which required significantly higher rATG doses. After rATG administration, we observed a significant proliferation of Tregs in the peripheral blood of transplanted rats, leading to an increase in the Treg/T effector ratio. Importantly, rATG‐induced Tregs displayed a strong donor‐specific suppressive capacity when assessed in an antigen‐specific allogeneic co‐culture. All of these results were associated with better renal graft function in rats that received rATG. Our study shows that rATG has the biological capacity immunomodulatory to promote a regulatory alloimmune milieu during post‐transplant homeostatic proliferation.     

ATG‐Induced Accelerated Immune Senescence: Clinical Implications in Renal Transplant Recipients

Persistent ATG‐induced CD4⁺ T cell lymphopenia is associated with serious clinical complications. We tested the hypothesis that ATG induces accelerated immune senescence in renal transplant recipients (RTR). Immune senescence biomarkers were analyzed at transplant and one‐year later in 97 incident RTR ?62 patients receiving ATG and 35 receiving anti‐CD25 mAb (α‐CD25). This consisted in: (i) thymic output; (ii) bone marrow renewal of CD34⁺ hematopoietic progenitor cells (CD34⁺HPC) and lymphoid (l‐HPC) and myeloid (m‐HPC) progenitor ratio; (iii) T cell phenotype; and (iv) measurement of T cell relative telomere length (RTL) and telomerase activity (RTA). Clinical correlates were analyzed with a 3 year follow‐up. Thymic output significantly decreased one‐year posttransplant in ATG‐treated patients. ATG was associated with a significant decrease in l‐HPC/m‐HPC ratio. Late stage differentiated CD57⁺/CD28^? T cells increased in ATG‐treated patients. One‐year posttransplant T cell RTL and RTA were consequently lower in ATG‐treated patients. ATG is associated with accelerated immune senescence. Increased frequency of late differentiated CD4⁺ T cell frequency at transplantation tended to be predictive of a higher risk of subsequent opportunistic infections and of acute rejection only in ATG‐treated patients but this needs confirmation. Considering pretransplant immune profile may help to select those patients who may benefit from ATG to prevent severe infections and acute rejection.

     

Biologic mechanisms and clinical consequences of pregnancy alloimmunization

Alloimmunization occurs during pregnancy when tissue antigens derived from the fetus and/or placenta prime maternal immune cells to divide and differentiate. For many women, the result of pregnancy alloimmunization is the formation of anti‐HLA antibody that can endure for decades and preclude transplantation by limiting donor compatibility. Pregnancy alloimmunization may also generate memory B cells that can rapidly produce anti‐HLA antibody after transplantation as well as pathogenic memory T cells, which pose a threat to graft survival. However, emerging data suggest that pregnancy also programs the differentiation of anergic, dysfunctional, and regulatory T cell populations, which may not mediate accelerated graft rejection. Hence, some of the immune mechanisms responsible for maternal immunologic tolerance of the fetus may persist into postpartum life and affect the response to an allograft. This review discusses these emerging data as well as the persistent knowledge gaps that affect women at multiple stages of their transplant care.     

Depletion of CD8 Memory T Cells for Induction of Tolerance of a Previously Transplanted Kidney Allograft

Heterologous immunologic memory has been considered a potent barrier to tolerance induction in primates. Induction of such tolerance for a previously transplanted organ may be more difficult, because specific memory cells can be induced and activated by a transplanted organ. In the current study, we attempted to induce tolerance to a previously transplanted kidney allograft in nonhuman primates. The conditioning regimen consisted of low dose total body irradiation, thymic irradiation, antithymocyte globulin, and anti‐CD154 antibody followed by a brief course of a calcineurin inhibitor. This regimen had been shown to induce mixed chimerism and allograft tolerance when kidney transplantation (KTx) and donor bone marrow transplantation (DBMT) were simultaneously performed. However, the same regimen failed to induce mixed chimerism when delayed DBMT was performed after KTx. We found that significant levels of memory T cells remained after conditioning, despite effective depletion of naïve T cells. By adding humanized anti‐CD8 monoclonal antibody (cM‐T807), CD8 memory T cells were effectively depleted and these recipients successfully achieved mixed chimerism and tolerance. The current studies provide ‘proof of principle’ that the mixed chimerism approach can induce renal allograft tolerance, even late after organ transplantation if memory T‐cell function is adequately controlled.     

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.     

Siplizumab selectively depletes effector memory T cells and promotes a relative expansion of alloreactive regulatory T cells in vitro

Siplizumab, a humanized anti‐CD2 monoclonal antibody, has been used in conditioning regimens for hematopoietic cell transplantation and tolerance induction with combined kidney‐bone marrow transplantation. Siplizumab‐based tolerance induction regimens deplete T cells globally while enriching regulatory T cells (Tregs) early posttransplantation. Siplizumab inhibits allogeneic mixed‐lymphocyte reactions (MLRs) in vitro. We compared the impact of siplizumab on Tregs versus other T cell subsets in HLA‐mismatched allogeneic MLRs using PBMCs. Siplizumab predominantly reduced the percentage of CD4⁺ and CD8⁺ effector memory T cells, which express higher CD2 levels than naïve T cells or resting Tregs. Conversely, siplizumab enriched proliferating CD45RA^? FoxP3^HI cells in MLRs. FoxP3 expression was stable over time in siplizumab‐containing cultures, consistent with enrichment for bona fide Tregs. Consistently, high‐throughput TCRβ CDR3 sequencing of sorted unstimulated and proliferating T cells in MLRs revealed selective expansion of donor‐reactive Tregs along with depletion of donor‐reactive CD4⁺ effector/memory T cells in siplizumab‐containing MLRs. These results indicate that siplizumab may have immunomodulatory functions that may contribute to its success in tolerance‐inducing regimens. Our studies also confirm that naïve in addition to effector/memory T cells contribute to the allogeneic MLR and mandate further investigation of the impact of siplizumab on alloreactive naïve T cells.     

Occurrence of specific humoral non‐responsiveness to swine antigens following administration of GalT‐KO bone marrow to baboons

Griesemer A, Liang F, Hirakata A, Hirsh E, Lo D, Okumi M, Sykes M, Yamada K, Huang CA, Sachs DH. Occurrence of specific humoral non‐responsiveness to swine antigens following administration of GalT‐KO bone marrow to baboons. Xenotransplantation 2010; 17: 300–312. © 2010 John Wiley & Sons A/S. Abstract: Background: Hematopoietic chimerism induces transplantation tolerance across allogeneic and xenogeneic barriers, but has been difficult to achieve in the pig‐to‐primate model. We have now utilized swine with knockout of the gene coding for α‐1,3‐galactosyltransferase (GalT‐KO pigs) as bone marrow donors in an attempt to achieve chimerism and tolerance by avoiding the effects of natural antibodies to Gal determinants on pig hematopoietic cells. Methods: Baboons (n = 4; Baboons 1 to 4 = B156, B158, B167, and B175, respectively) were splenectomized and conditioned with TBI (150 cGy), thymic irradiation (700 cGy), T cell depletion with rabbit anti‐thymocyte globulin (rATG) and rat anti‐primate CD2 (LoCD2b), and received FK506 and supportive therapy for 28 days. All animals received GalT‐KO bone marrow (1 to 2 × 10⁹ cells/kg) in two fractions on days 0 and 2, and were thereafter monitored for the presence of pig cells by flow cytometry, for porcine progenitor cells by PCR of BM colony‐forming units, and for cellular reactivity to pig cells by mixed lymphocyte reaction (MLR). In vitro antibody formation to LoCD2b and rATG was tested by ELISA; antibody reactivity to GalT‐KO pig cells was tested by flow cytometry and cytotoxicity assays. Additionally, Baboons 3 and 4 received orthotopic kidney transplants on days 17 and 2, respectively, to test the potential impact of the protocol on renal transplantation. Results: None of the animals showed detectable pig cells by flow cytometry for more than 12 h post‐BM infusion. However, porcine progenitor cell engraftment, as evidenced by pig‐derived colony forming units in the BM, as well as peripheral microchimerism in the thymus, lymph node, and peripheral blood was detected by PCR in baboons 1 and 2 for at least 28 days post‐transplant. ELISA results confirmed humoral immunocompetence at time of transplantation as antibody titers to rat (LoCD2b) and rabbit (ATG) increased within 2 weeks. However, no induced antibodies to GalT‐KO pig cells or increased donor specific cytotoxicity was detectable by flow cytometry. In contrast, baboons 3 and 4 developed serum antibodies to pig cells as well as to rat and rabbit immunoglobulin by day 14. Retrospective analysis revealed that although all four baboons possessed low levels of antibody‐mediated cytotoxicity to GalT‐KO cells prior to transplantation, the two baboons (3 and 4) that became sensitized to pig cells (and rejected pig kidneys) had relatively high pre‐transplantation titers of anti–non‐Gal IgG detectable by flow cytometry, whereas baboons 1 and 2 had undetectable titers. Conclusions: Engraftment and specific non‐responsiveness to pig cells has been achieved in two of four baboons following GalT‐KO pig‐to‐baboon BMT. Engraftment correlated with absence of preformed anti–non‐Gal IgG serum antibodies. These results are encouraging with regard to the possibility of achieving transplantation tolerance across this xenogeneic barrier.     

Interleukin‐7 receptor blockade by an anti‐CD127 monoclonal antibody in nonhuman primate kidney transplantation

IL‐7 is an important cytokine for T cell lymphopoiesis. Blockade of the IL‐7 signaling pathway has been shown to induce long‐term graft survival or graft tolerance in murine transplant models through inhibiting T cell homeostasis and favoring immunoregulation. In this study, we assessed for the first time the effects of a blocking anti‐human cluster of differentiation 127 (CD127) mAb administered in combination with low‐dose tacrolimus or thymoglobulin in a life‐sustaining kidney allograft model in baboons. Contrary to our expectation, the addition of an anti‐CD127 mAb to the treatment protocols did not prolong graft survival compared to low‐dose tacrolimus alone or thymoglobulin alone. Anti‐CD127 mAb administration led to full CD127 receptor occupancy during the follow‐up period. However, all treated animals lost their kidney graft between 1 week and 2 weeks after transplantation. Unlike in rodents, in nonhuman primates, anti‐CD127 mAb treatment does not decrease the absolute numbers of lymphocyte and lymphocyte subsets and does not effectively inhibit postdepletional T cell proliferation and homeostasis, suggesting that IL‐7 is not a limiting factor for T cell homeostasis in primates.     

Contrasting effects of B cell depletion on CD4+ and CD8+ memory T cell responses generated after transplantation

Alloreactive memory T cells play a key role in transplantation by accelerating allograft rejection and preventing tolerance induction. Some studies using µMT mice, which are constitutionally devoid of B cells, showed that B cells were required for the generation of memory T cells after allotransplantation. However, whether B cell depletion in normal adult mice has the same effect on memory responses by CD4⁺ and CD8⁺ T cells activated after transplantation has not been thoroughly investigated. In this study, we tested the effect of anti‐CD20 antibody‐mediated B cell depletion on CD4⁺ and CD8⁺ memory T cell alloresponses after skin transplantation in wild‐type mice. We found that B cell depletion prevented the development of memory alloresponses by CD4⁺ T cells but enhanced that of CD8⁺ memory T cells. Next, we tested the influence of B cell depletion on hematopoietic chimerism. In OT‐II CD4⁺ anti‐OVA TCR transgenic mice sensitized to ovalbumin antigen, B cell depletion also impaired allospecific memory T cell responses and thereby enhanced donor hematopoietic chimerism and T cell deletion after bone marrow transplantation. This study underscores the complexity of the relationships between B and T cells in the generation and reactivation of different memory T cell subsets after transplantation.     

The evolving role of mTOR inhibition in transplantation tolerance

The mammalian target of rapamycin (mTOR) plays a key role in the immune response. mTOR inhibitors suppress T cell activation and proliferation and are effective immunosuppressants. Today there is growing interest in their potential role in inducing tolerance after transplantation. mTOR inhibitors induce anergy in na?ve T cells, promote the expansion of regulatory T cells, and inhibit the maturation of dendritic cells, thus promoting immunologic tolerance. Here we review the mechanisms by which mTOR inhibitors promote tolerance. We discuss the clinical relevance of these mechanisms and suggest how they might be used in the design of future protocols to induce tolerance.     

Pretransplant Antithymocyte Globulin Has Increased Efficacy in Controlling Donor‐Reactive Memory T Cells in Mice

Antibody‐mediated lymphocyte depletion is frequently used as induction therapy in sensitized transplant patients. Although T cells with an effector/memory phenotype remain detectable after lymphoablative therapies in human transplant recipients, the role of preexisting donor‐reactive memory in reconstitution of the T cell repertoire and induction of alloimmune responses following lymphoablation is poorly understood. We show in a mouse cardiac transplantation model that antidonor immune responses following treatment with rabbit antimouse thymocyte globulin (mATG) were dominated by T cells derived from the preexisting memory compartment. Administration of mATG 1 week prior to transplantation (pre‐TP) was more efficient in targeting preexisting donor‐reactive memory T cells, inhibiting overall antidonor T cell responses, and prolonging heart allograft survival than the commonly used treatment at the time of transplantation (peri‐TP). The failure of peri‐TP mATG to control antidonor memory responses was due to faster recovery of preexisting memory T cells rather than their inefficient depletion. This rapid recovery did not depend on T cell specificity for donor alloantigens suggesting an important role for posttransplant inflammation in this process. Our findings provide insights into the components of the alloimmune response remaining after lymphoablation and may help guide the future use of ATG in sensitized transplant recipients.     

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.     

Memory T-Cell Predominance Following T-Cell Depletional Therapy Derives from Homeostatic Expansion of Naive T Cells

T-cell depletion reportedly leads to alterations in the T-cell compartment with predominant survival of memory phenotype CD4 T cells. Here, we asked whether the prevalence of memory T cells postdepletion results from their inherent resistance to depletion and/or to the homeostatic expansion of naive T cells and their phenotypic conversion to memory, which is known to occur in lymphopenic conditions. Using a 'mosaic memory' mouse model with trackable populations of alloreactive memory T cells, we found that treatment with murine antithymocyte globulin (mATG) or antilymphocyte serum (ALS) effectively depleted alloreactive memory CD4 T cells, followed by rapid homeostatic proliferation of endogenous CD4 T cells peaking at 4 days postdepletion, with no homeostatic advantage to the antigen-specific memory population. Interestingly, naive (CD44lo) CD4 T cells exhibited the greatest increase in homeostatic proliferation following mATG treatment, divided more extensively compared to memory (CD44hi) CD4 T cells and converted to a memory phenotype. Our results provide novel evidence that memory CD4 T cells are susceptible to lymphodepletion and that the postdepletional T-cell compartment is repopulated to a significant extent by homeostatically expanded naive T cells in a mouse model, with important important implications for immune alterations triggered by induction therapy.     

Therapeutic regulation of systemic inflammation in xenograft recipients

Inflammation is known to preclude tolerance after transplantation. We have previously shown that systemic inflammation in xenograft recipients (SIXR) precedes activation of coagulation in the absence of T cell responses. Accordingly, SIXR may amplify innate and adaptive immune responses against xenografts after pig‐to‐primate xenotransplantation, even with efficient immunosuppressive therapy. We evaluated the impact of anti‐inflammatory agents on pro‐inflammatory cytokines and chemokines in pig artery patch and heart xenograft recipients. Baboons received an artery patch (Group1, n=8) or heart (Group2, n=4) from genetically engineered pigs. All baboons received lymphodepletion with thymoglobulin (ATG) and costimulation blockade‐based immunosuppression (anti‐CD40 and/or CTLA4Ig). In Group1, baboons received either (i) no anti‐inflammatory agents (n=2), (ii) cobra venom factor (CVF, n=2), (iii) α1‐antitrypsin (AAT, n=2), or (iv) interleukin (IL)‐6 receptor antagonist (IL‐6RA, n=2). In Group2, all baboon received corticosteroids, either without (n=2) or with (n=2) IL‐6RA. Serum IFN‐γ, TNF‐α, IL‐1β, IL‐17, IL‐6, IL‐8, MCP‐1, and sCD40L levels were measured by Luminex. Fibrinogen, D‐dimers, and C‐reactive protein (C‐RP) were also measured. Recipient baboon T cell proliferation was evaluated by mixed lymphocyte reaction (MLR) before and after transplantation. Pig and baboon tissue factor (TF) mRNA levels in heart xenografts were measured by RT‐PCR. In no recipient was a marked increase in T cell response to pig cells observed after transplantation. In Groups 1 and 2, post‐transplantation levels of IFN‐γ, TNF‐α, IL‐1β, and IL‐17 remained comparable to or lower than pre‐transplant levels, except in one heart recipient that succumbed to CMV infection. In Group1, when no anti‐inflammatory agent was administered, post‐transplant levels of IL‐6, IL‐8, and MCP‐1 were elevated. After CVF, IL‐6, IL‐8, and MCP‐1 remained low. After IL‐6RA, IL‐6 and MCP‐1 were elevated. After AAT, IL‐8 was elevated. sCD40L became elevated intermittently in most recipients irrespective of the administered anti‐inflammatory agent. In Group2, IL‐6 was transiently elevated, particularly after IL‐6RA administration. MCP‐1 gradually increased by 2 months in Group2 recipients. sCD40L generally remained low except in one recipient. In Group1 and Group2 recipients, C‐RP levels were elevated except after IL‐6RA administration, while D‐dimers were elevated regardless of administration of anti‐inflammatory agent. In Group2, pig TF mRNA levels were increased in heart xenografts compared to naive pig hearts, irrespective of IL‐6 receptor antagonist administration. Additionally, baboon TF mRNA levels were detectable in heart xenografts, but not in naive pig hearts. Some pro‐inflammatory cytokines and chemokines are elevated in xenograft recipients, even with efficient T cell‐directed immunosuppressive therapy. Persistent elevation of D‐dimers, and individual cytokines and chemokines suggest a continuous inflammatory response, despite administration of anti‐inflammatory agents. Systemic administration of combined anti‐inflammatory agents as well as complement regulation may be essential to prevent SIXR after xenotransplantation.     

Antithymocyte globulins in renal transplantation—from lymphocyte depletion to lymphocyte activation: The doubled-edged sword

Compelling data suggest that lymphocyte depletion following T cell depleting therapy may induce prolonged CD4 T cell lymphopenia and trigger lymphocyte activation in some patients. These profound and non-reversible immune changes in T cell pool subsets are the consequence of both impaired thymic renewal and peripheral homeostatic proliferation. Chronic viral challenges by CMV play a major role in these immune alterations. Even when the consequences of CD4 T cell lymphopenia have been now well described, recent studies shed new light on the clinical consequences of immune activation. In this review, we will first focus on the mechanisms involved in T cell pool reconstitution after T cell depletion and further consider the clinical consequences of ATG-induced T cell activation and senescence in renal transplant recipients.     

Preclinical Efficacy and Immunological Safety of FR104, an Antagonist Anti‐CD28 Monovalent Fab′ Antibody

Antagonist anti‐CD28 antibodies prevent T cell costimulation and differentiate from CTLA4Ig since they cannot block CTLA‐4 and PDL‐1 coinhibitory signals. They demonstrated efficacy in suppressing effector T cells while enhancing regulatory T cells function and immune tolerance. However, anti‐CD28 antibodies devoid of immunotoxicity and with a good pharmacokinetic profile have not yet been developed. Here, we describe FR104, a novel humanized pegylated anti‐CD28 Fab′ antibody fragment presenting a long elimination half‐life in monkeys. In vitro, FR104 failed to induce human T cell proliferation and cytokines secretion, even in the presence of anti‐CD3 antibodies or when cross‐linked with secondary antibodies. Furthermore, in humanized NOD/SCID mice adoptively transferred with human PBMC, whereas superagonist and divalent antibodies elicited rapid cytokines secretion and human T cell activation, FR104 did not. These humanized mice developed a florid graft‐versus‐host disease, which was prevented by administration of FR104 in a CTLA4‐dependent manner. Interestingly, administration of high doses of CTLA4‐Ig was ineffective to prevent GVHD, whereas administration of low doses was partially effective. In conclusion, we demonstrated that FR104 is devoid of agonist activity on human T cells and thus compatible with a clinical development that might lead to higher therapeutic indexes, by sparing CTLA‐4, as compared to CD80/CD86 antagonists.