Primed allospecific T cells prevent the effects of costimulatory blockade on prolonged cardiac allograft survival in mice.

Costimulatory blockade can induce long‐term allograft survival in naïve animals, but may not be as effective in animals with previously primed immune repertoires. We attempted to induce long‐term graft survival in B10.D2 recipients of B10.A cardiac allografts using donor‐specific transfusion (DST) plus anti‐CD40 ligand antibody (αCD40L). Recipients were either naïve mice, or mice previously primed to B10.A or third party alloantigens through engraftment and rejection of skin transplants. Untreated naïve mice rejected cardiac transplants by day 15 and contained a high frequency of primed, donor‐reactive T cells. Donor‐specific transfusion/αCD40L treatment of naïve animals induced long‐term graft survival associated with low frequencies of donor‐reactive T cells. Previous priming of donor‐specific T cells through rejection of B10.A, but not third party, skin grafts prevented the effects of DST/αCD40L on prolonging survival of B10.A hearts. Moreover, adoptive transfer of CD3+, CD4+ or CD8+ T cells from B10.A skin‐graft‐primed animals prevented the effects of DST/αCD40L. The data demonstrate that animals with immune repertoires containing previously primed, donor‐reactive T cells are resistant to the effects of costimulatory blockade. The findings have important implications for ongoing, costimulatory blockade‐based trials in humans, whose T‐cell repertoires are known to contain memory alloreactive T cells.     

The high‐risk corneal regraft model: a justification for tissue matching in humans

Models of high‐risk corneal graft rejection involve neovascularization induced via innate immune responses, e.g., suture‐mediated trauma. We describe a model of high‐risk corneal graft rejection using corneal graft donor‐recipient pairing based on a single‐antigen disparity. Donor corneas from transgenic mice on B10.BR (H‐2^k) background, in which hen‐egg lysozyme (HEL) as a membrane‐bound antigen (mHEL) was expressed under the major histocompatibility complex (MHC) Class I promoter (KLK‐mHEL, H‐2^k), were transplanted into wild type B10.BR recipient mice. Unmanipulated wild type recipient mice rejected KLK‐mHEL grafts (39%) slowly over 50–60 days. Graft rejection incidence was maximized (100%) and tempo accelerated (27 days) by priming with HEL‐pulsed syngeneic dendritic cells and less so by increasing T‐cell precursor frequency. Rejection also reached maximum levels (100%) and tempo (3–8 days) when mice which had rejected a first graft (‘rejectors’) were regrafted, and was associated with induction of HEL‐specific memory T cells. In contrast, ‘acceptors’ rejected a second graft at rates and tempo similar to naïve mice. These data reveal the importance of (i) donor MHC antigens as alloantigens for indirect recognition, (ii) alloantigen‐specific memory in high‐risk graft rejection involving regrafts, and (iii) suggest a role for tissue matching in human corneal graft to avoid sensitization to donor MHC antigens.     

Differential Role of Naïve and Memory CD4+ T‐Cell Subsets in Primary Alloresponses

The T cell response to major histocompatibility complex (MHC) alloantigens occurs via two main pathways. The direct pathway involves the recognition of intact allogeneic MHC:peptide complexes on donor cells and provokes uniquely high frequencies of responsive T cells. The indirect response results from alloantigens being processed like any other protein antigen and presented as peptide by autologous antigen‐presenting cells. The frequencies of T cells with indirect allospecificity are orders of magnitude lower and comparable to other peptide‐specific responses. In this study, we explored the contributions of naïve and memory CD4⁺ T cells to these two pathways. Using an adoptive transfer and skin transplantation model we found that naive and memory CD4⁺ T cells, both naturally occurring and induced by sensitization with multiple third‐party alloantigens, contributed equally to graft rejection when only the direct pathway was operative. In contrast, the indirect response was predominantly mediated by the naïve subset. Elimination of regulatory CD4⁺CD25⁺ T cells enabled memory cells to reject grafts through the indirect pathway, but at a much slower tempo than for naïve cells. These findings have implications for better targeting of immunosuppression to inhibit immediate and later forms of alloimmunity.     

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.     

Allografts Stimulate Cross‐Reactive Virus‐Specific Memory CD8 T Cells with Private Specificity

Viral infections have been associated with the rejection of transplanted allografts in humans and mice, and the induction of tolerance to allogeneic tissues in mice is abrogated by an ongoing viral infection and inhibited in virus‐immune mice. One proposed mechanism for this ‘heterologous immunity’ is the induction of alloreactive T cell responses that cross‐react with virus‐derived antigens. These cross‐reactive CD8 T cells are generated during acute viral infection and survive into memory, but their ability to partake in the immune response to allografts in vivo is not known. We show here that cross‐reactive, virus‐specific memory CD8 T cells from mice infected with LCMV proliferated in response to allografts. CD8 T cells specific to several LCMV epitopes proliferated in response to alloantigens, with the magnitude and hierarchy of epitope‐specific responses varying with the private specificities of the host memory T cell repertoire, as shown by adoptive transfer studies. Last, we show that purified LCMV‐specific CD8 T cells rejected skin allografts in SCID mice. These findings therefore implicate a potential role for heterologous immunity in virus‐induced allograft rejection.     

Induction of Allograft Tolerance by Monoclonal CD3 Antibodies: A Matter of Timing

Despite remarkable progress in organ transplantation through the development of a wealth of immunosuppressive drugs highly effective at controlling acute rejection, two major problems still remain, the loss of transplants due to chronic rejection and the growing number of sensitized recipients due to previous transplants, transfusions or pregnancies. Induction of immune tolerance appears to be the only way to curb this complex situation. Here we describe that a therapy, already successfully used to restore immune tolerance to self‐antigens in overt autoimmunity, is effective at promoting transplant tolerance. We demonstrate that a short low‐dose course with CD3 antibodies started after transplantation, at the time of effector T cell priming to alloantigens, induces permanent acceptance of fully mismatched islet allografts. Mechanistic studies revealed that antigen‐specific regulatory and effector T cells are differentially affected by the treatment. CD3 antibody treatment preferentially induces apoptosis of activated alloreactive T cells which is mandatory for tolerance induction. In contrast, regulatory T cells are relatively spared from CD3 antibody‐induced depletion and can transfer antigen‐specific tolerance thus arguing for their prominent role in sustaining long‐term graft survival.     

B Cells Help Alloreactive T Cells Differentiate Into Memory T Cells

B cells are recognized as effector cells in allograft rejection that are dependent upon T cell help to produce alloantibodies causing graft injury. It is not known if B cells can also help T cells differentiate into memory cells in the alloimmune response. We found that in B‐cell‐deficient hosts, differentiation of alloreactive T cells into effectors was intact whereas their development into memory T cells was impaired. To test if B cell help for T cells was required for their continued differentiation into memory T cells, activated T cells were sorted from alloimmunized mice and transferred either with or without B cells into naïve adoptive hosts. Activated T cells cotransferred with B cells gave rise to more memory T cells than those transferred without B cells and upon recall, mediated accelerated rejection of skin allografts. Cotransfer of B cells led to increased memory T cells by enhancing activated CD4 T‐cell proliferation and activated CD8 T‐cell survival. These results indicate that B cells help alloreactive T‐cell differentiation, proliferation and survival to generate optimal numbers of functional memory T cells.     

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.     

Prevention of Chronic Rejection with Immunoregulatory Cells Induced by Intrathymic Immune Modulation with Class I Allopeptides

Intrathymic immune modulation with RT1.A^a allopeptides in the PVG.R8‐to‐PVG.1 U rat strain combination leads to long‐term survival of cardiac allografts. This regimen, however, does not induce transplantation tolerance, since most long‐surviving allografts undergo chronic rejection. We investigated recipients with chronic rejection for donor‐specific immune nonresponsiveness and immunoregulatory cells as possible mechanisms responsible for long‐term graft survival. There was a significant reduction in the proliferative response of T cells from long‐term allograft recipients to donor alloantigens as compared with that of naïve T cells. Adoptive transfer of splenocytes from intrathymically manipulated primary long‐term graft survivors into minimally irradiated secondary hosts resulted in indefinite survival of > 80% of allografts, providing evidence for immunoregulatory cells. Secondary recipients had total absence of donor‐reactive cellular and humoral responses. Immunoregulation was also transferable from secondary to tertiary graft recipients. More importantly, there was a significant reduction in the incidence of chronic rejection in secondary hosts (> 85%) and complete prevention of acute and chronic rejection in tertiary hosts. This study demonstrates that intrathymic immunomodulation with class I allopeptides results in the generation of immunoregulatory cells that do not block chronic rejection in primary hosts where they develop, but prevent both acute and chronic allograft rejection when adoptively transferred into secondary and tertiary recipients.     

Role of secondary lymphoid tissues in primary and memory T-cell responses to a transplanted organ

Secondary lymphoid tissues are the hub of adaptive immune responses wherein rare cognate lymphocytes encounter dendritic cells bearing antigen from peripheral tissues and differentiate into effector and memory cells that eliminate antigen. It is accepted that immune responses against microbial and tumor antigens are initiated within secondary lymphoid tissues. There is less agreement on whether the same principle applies to immune responses to a transplanted organ because an allograft expresses foreign major histocompatibility complex and contains donor antigen presenting cells that could activate T cells directly in situ leading to rejection. Recent studies confirm that although naïve T cells can be primed within the allograft, their differentiation to effect rejection is dependent on secondary lymphoid tissues. Antigen-experienced memory T cells, unlike Naïve T cells, function largely independent of secondary lymphoid tissues to cause allograft rejection. In an alloimmune response, secondary lymphoid tissues support not only immune activation but also immune regulation essential for allograft survival. Here, we will review recent findings and discuss the role of secondary lymphoid tissues in primary and memory alloimmune responses.     

Antigen‐specific regulatory T cells can induce tolerance to immunogenic grafts without the need for chronic immunosuppression

Regulatory CD4⁺CD25⁺Foxp3⁺ T cells (Tregs) play an important role in the induction of allospecific tolerance. However tolerance in solid organ transplantation by mere transfer of Tregs has been difficult. Besides this the stability of the differentiation phenotype of Tregs has recently been questioned. We therefore aimed in generating large numbers of stable allospecific Tregs from naïve T cells by retroviral transduction with Foxp3. These were tested in an immunogenic skin transplantation model (C57BL/6→BALB/c). We established a system of transduction of mouse T cells with ecotropic retroviruses expressing Foxp3 and Thy1.1 as a surface marker to follow up transduced T cells. Alloantigen‐specific Tregs were generated by stimulating naïve recipient CD4⁺ T cells with irradiated donor splenocytes. CD25⁺ and/or CD69⁺ allospecific recipient CD4⁺ T cells were isolated and transduced with Foxp3. Alloantigen‐specific Foxp3 T cells (iTregs) showed high expression for the Treg markers Foxp3, CTLA4 and GITR. They could suppress a MLR in an alloantigen‐specific manner. Furthermore, they could be expanded up to 18 fold in vitro while maintaining their Treg phenotype and expression of lymph node homing markers like CCR7 and CD62L. iTregs prevented skin graft rejection without the need for chronic immunosuppression and recipients showed systemic allospecific allotolerance. Alloantigen‐specific Tregs were far more potent than polyspecific Tregs. Mechanisms of tolerance were graft specific homing, expansion and long‐term persistence of Tregs within the graft (>100 days, 90% of intragraft Tregs were alloantigen‐specific). In fact, tolerance could be transferred with re‐transplantation of the tolerant graft onto secondary recipients. Third party grafts were readily rejected demonstrating specificity of tolerance. Due to the Foxp3 transduction, iTregs did not lose their Treg phenotype. The results prove that large numbers of stable alloantigen‐specific Tregs can be generated from a polyclonal repertoire of naïve T cells. This is the first time that allotolerance was achieved in a non‐lymphopenic transplant model using skin grafts in an immunogenic strain combination. Therefore, antigen‐specific Tregs might have a huge therapeutic potential after solid organ transplantation.     

T-lymphocyte homeostasis and function in infant baboons: implications for transplantation

Laboratory mice are born lymphopenic and demonstrate lymphopenia‐induced proliferation that generates memory T cells, yet they are prone to immunologic tolerance. Here we tested whether these fundamental immunologic observations apply to higher animals by studying the immune system of infant baboons. Using flow cytometry of the peripheral blood cells, it was found that baboons are born relatively lymphopenic and subsequently expand their initially naïve T cell pool with increasing numbers of memory T cells. After transplantation of an artery patch allograft or xenograft, non‐immunosuppressed recipients readily mounted an immune response against donor‐type antigens, as evidenced by mixed lymphocyte reaction. Immunosuppression with anti‐thymocyte globulin (ATG), anti‐CD154 mAb, and mycophenolate mofetil prevented T cell‐mediated rejection. After lymphocyte depletion with ATG, homeostatic T cell proliferation was observed. In conclusion, the baboon proved a suitable model to investigate the infant immune system. In this study, neonatal lymphopenia and expansion of the memory T cell population were observed but, unlike mice, there were no indications that infant baboons are prone to T cell tolerance. The expansion of memory T cells during the neonatal period or after induction therapy may actually form an obstacle to tapering immunosuppressive therapy, or ultimately achieving immunologic tolerance.     

The Allo‐ and Viral‐Specific Immunosuppressive Effect of Belatacept,but Not Tacrolimus,Attenuates With Progressive T Cell Maturation

Tacrolimus impairs allo‐ and viral‐specific T cell responses. Belatacept, a costimulation‐based alternative to tacrolimus, has emerged with a paradoxical picture of less complete control of alloimmunity with concomitant impaired viral immunity limited to viral‐naïve patients. To reconcile these signatures, bulk population and purified memory and naïve lymphocytes from cytomegalovirus (CMV)‐seropositive (n = 10) and CMV‐seronegative (n = 10) volunteers were studied using flow cytometry, interrogating proliferation (carboxyfluorescein succinimidyl ester dilution) and function (intracellular cytokine staining) in response to alloantigens or CMV‐pp‐65 peptides. As anticipated, T cells from CMV‐experienced, but not naïve, individuals responded to pp‐65 with a small percentage of their repertoire (<2.5%) consisting predominantly of mature, polyfunctional (expressing interferon gamma, tumor necrosis factor alpha and IL‐2) T effector memory cells. Both CMV naïve and experienced individuals responded similarly to alloantigen with a substantially larger percentage of the repertoire (up to 48.2%) containing proportionately fewer polyfunctional cells. Tacrolimus completely inhibited responses of CMV‐ and allo‐specific T cells regardless of their maturation. However, belatacept's effects were decreasingly evident in increasingly matured cells, with minimal effect on viral‐specific triple cytokine producers and CD28‐negative allo‐specific cells. These data indicate that belatacept's immunosuppressive effect, unlike tacrolimus's, wanes on progressively developed effector responses, and may explain the observed clinical effects of belatacept.     

Relative Resistance of Human CD4+ Memory T Cells to Suppression by CD4+CD25+ Regulatory T Cells

Successful expansion of functional CD4⁺CD25⁺ regulatory T cells (T_reg) ex vivo under good manufacturing practice conditions has made T_reg‐cell therapy in clinical transplant tolerance induction a feasible possibility. In animals, T_reg cells home to both transplanted tissues and local lymph nodes and are optimally suppressive if active at both sites. Therefore, they have the opportunity to suppress both naïve and memory CD4⁺CD25^? T cells (Tresp). Clinical transplantation commonly involves depleting therapy at induction (e.g. anti‐CD25), which favors homeostatic expansion of memory T cells. Animal models suggest that T_reg cells are less suppressive on memory, compared with naïve Tresp that mediate allograft rejection. As a result, in the context of human T_reg‐cell therapy, it is important to define the effectiveness of T_reg cells in regulating naïve and memory Tresp. Therefore, we compared suppression of peripheral blood naïve and memory Tresp by fresh and ex vivo expanded T_reg cells using proliferation, cytokine production and activation marker expression (CD154) as readouts. With all readouts, naïve human Tresp were more suppressible by approximately 30% than their memory counterparts. This suggests that T_reg cells may be more efficacious if administered before or at the time of transplantation and that depleting therapy should be avoided in clinical trials of T_reg cells.     

Cytokines affecting CD4+ T regulatory cells in transplant tolerance. Interleukin-4 does not maintain alloantigen specific CD4+CD25+ Treg

IL-4 is thought to promote induction of transplantation tolerance and alloantigen-specific CD4⁺CD25⁺ T regulatory cells (Treg). This study examined the effect of IL-4 on the induction and maintenance of the CD4⁺ T regulatory cells (Treg) that mediate transplantation tolerance. Tolerance was induced in DA rats with PVG heterotopic cardiac allografts by a short course of cyclosporine. Naïve and tolerant lymphocytes, including the CD4⁺ and CD4⁺CD25⁺ T cell subsets, were assayed in mixed lymphocyte cultures with or without recombinant (r)IL-4 or other cytokines. The proliferation, cell surface and cytokine phenotype of these cells was examined, as was their capacity to adoptively transfer tolerance. rIL-4 enhanced the proliferation of naïve and tolerant lymphoid cells, including CD4⁺ and CD4⁺CD25⁺ T cells, but this was not alloantigen specific. Naïve or tolerant CD4⁺ T cells cultured with rIL-4 and donor PVG antigen effected rapid graft rejection, even though before culture tolerant CD4⁺ T cells transferred antigen-specific tolerance. These rIL-4 cultured CD4⁺ T cells had a phenotype consistent with activated CD4⁺CD25⁺FoxP3⁻ Th2 cells. While naïve natural CD4⁺CD25⁺ T cells (nTreg) cultured with alloantigen and rIL-4 had enhanced proliferation and capacity to suppress rejection in vivo, the culture of tolerant CD4⁺CD25⁺ T cells with alloantigen and rIL-4 could not sustain their proliferation against specific donor, nor their capacity to transfer tolerance to specific donor allograft. Thus, IL-4 promotes both regulatory and effector T cells early in the immune response, but once alloimmune tolerance is established, IL-4 promoted the activation of effector cells to mediate rejection and did not support alloantigen-specific Treg that could transfer specific tolerance.     

Induction of regulatory T cells from mature T cells by allogeneic thymic epithelial cells in vitro

The ability of thymic epithelial cells (TEC) to re‐educate mature T cells to be regulatory T cells has not been addressed. In the present study, this issue was directly investigated by co‐culturing of mature T cells and allo‐TECs. B6 macrophage cell line 1C21‐cultured BALB/c splenocytes responded to B6 antigens in vitro. However, BALB/c splenocytes precultured with B6‐derived TECs 1‐4C18 or 1C6 did not proliferate to B6 antigens, but responded to rat antigens. Exogenous interleukin‐2 (IL‐2) failed to revise the unresponsiveness of these T cells. Allo‐TEC‐cultured T cells predominantly expressed Th2 cytokines (IL‐4 and IL‐10). B6 TEC‐cultured BALB/c splenocytes markedly inhibited the immune responses of naïve BALB/c splenocytes to B6 antigens, but not to rat or the third‐party mouse antigens. BALB/c nude mice that received naïve syngeneic splenocytes rejected B6 or rat skin grafts by 17 days postskin grafting; however, co‐injection of B6 TEC‐cultured BALB/c splenocytes significantly delayed B6 skin graft rejection (P < 0.01), with the unchanged rejection of rat skin grafts. These studies demonstrate that allo‐TECs are able to ‘educate’ mature T cells to be regulatory cells, and suggest that regulatory cells derived from mature T cells by TECs may play an important role in T cell tolerance to allo‐ and auto‐antigens.     

Relationship between antithymocyte globulin,T cell phenotypes,and clinical outcomes in pediatric kidney transplantation

Depletional induction using antithymocyte globulin (ATG) reduces rates of acute rejection in adult kidney transplant recipients, yet little is known about its effects in children. Using a longitudinal cohort of 103 patients in the Immune Development in Pediatric Transplant (IMPACT) study, we compared T cell phenotypes after ATG or non-ATG induction. We examined the effects of ATG on the early clinical outcomes of alloimmune events (development of de novo donor specific antibody and/or biopsy proven rejection) and infection events (viremia/viral infections). Long-term patient and graft outcomes were examined using the Scientific Registry of Transplant Recipients. After ATG induction, although absolute counts of CD4 and CD8 T cells were lower, patients had higher percentages of CD4 and CD8 memory T cells with a concomitant decrease in frequency of naïve T cells compared to non-ATG induction. In adjusted and unadjusted models, ATG induction was associated with increased early event-free survival, with no difference in long-term patient or allograft survival. Decreased CD4⁺ naïve and increased CD4⁺ effector memory T cell frequencies were associated with improved clinical outcomes. Though immunologic parameters are drastically altered with ATG induction, long-term clinical benefits remain unclear in pediatric patients.     

Phenotype,Distribution and Alloreactive Properties of Memory T Cells from Cynomolgus Monkeys

The high frequency of memory T cells present in primates is thought to represent a major barrier to tolerance induction in transplantation. Therefore, it is crucial to characterize these memory T cells and determine their functional properties. High numbers of memory T cells were detected in peripheral blood and all lymphoid tissues except lymph nodes, which were essentially the site of naïve T cells. The majority of CD8⁺ memory T cells were effector memory cells located in the blood and bone marrow while most CD4⁺ memory T cells were central memory cells present in the spleen. Next, memory T cells from over 100 monkeys were tested for their response to alloantigens by ELISPOT. Memory alloreactivity mediated via direct but not indirect allorecognition was detected in all animals. The frequency of allospecific memory T cells varied dramatically depending upon the nature of the responder/stimulator monkey combination tested. MHC gene matching was generally associated with a low‐memory alloreactivity. Nevertheless, low anamnestic alloresponses were also found in a significant number of fully MHC‐mismatched monkey combinations. These results show that selected donor/recipient combinations displaying a low memory alloresponsiveness can be found. These combinations may be more favorable for transplant tolerance induction.     

The Changed Balance of Regulatory and Naive T Cells Promotes Tolerance after TLI and Anti‐T‐Cell Antibody Conditioning

The goal of the study was to determine how the changed balance of host naïve and regulatory T cells observed after conditioning with total lymphoid irradiation (TLI) and antithymocyte serum (ATS) promotes tolerance to combined organ and bone marrow transplants. Although previous studies showed that tolerance was dependent on host natural killer T (NKT) cells, this study shows that there is an additional dependence on host CD4⁺CD25⁺ Treg cells. Depletion of the latter cells before conditioning resulted in rapid rejection of bone marrow and organ allografts. The balance of T‐cell subsets changed after TLI and ATS with TLI favoring mainly NKT cells and ATS favoring mainly Treg cells. Combined modalities reduced the conventional naïve CD4⁺ T cells 2800‐fold. The host type Treg cells that persisted in the stable chimeras had the capacity to suppress alloreactivity to both donor and third party cells in the mixed leukocyte reaction. In conclusion, tolerance induction after conditioning in this model depends upon the ability of naturally occurring regulatory NKT and Treg cells to suppress the residual alloreactive T cells that are capable of rejecting grafts.     

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.