Inhibitory feedback loop between tolerogenic dendritic cells and regulatory T cells in transplant tolerance

An active role of T regulatory cells (Treg) and tolerogenic dendritic cells (Tol-DC) is believed important for the induction and maintenance of transplantation tolerance. However, interactions between these cells remain unclear. We induced donor-specific tolerance in a fully MHC-mismatched murine model of cardiac transplantation by simultaneously targeting T cell and DC function using anti-CD45RB mAb and LF 15-0195, a novel analog of the antirejection drug 15-deoxyspergualin, respectively. Increases in splenic Treg and Tol-DC were observed in tolerant recipients as assessed by an increase in CD4(+)CD25(+) T cells and DC with immature phenotype. Both these cell types exerted suppressive effects in MLR. Tol-DC purified from tolerant recipients incubated with naive T cells induced the generation/expansion of CD4(+)CD25(+) Treg. Furthermore, incubation of Treg isolated from tolerant recipients with DC progenitors resulted in the generation of DC with Tol-DC phenotype. Treg and Tol-DC generated in vitro were functional based on their suppressive activity in vitro. These results are consistent with the notion that tolerance induction is associated with a self-maintaining regulatory loop in which Tol-DC induce the generation of Treg from naive T cells and Treg programs the generation of Tol-DC from DC progenitors.     

Elevated T regulatory cells in long-term stable transplant tolerance in rhesus macaques induced by anti-CD3 immunotoxin and deoxyspergualin

Regulatory T cells (Tregs) are implicated in immune tolerance and are variably dependent on IL-10 for in vivo function. Brief peritransplant treatment of multiple nonhuman primates (NHP) with anti-CD3 immunotoxin and deoxyspergualin has induced stable (5-10 years) rejection-free tolerance to MHC-mismatched allografts, which associated with sustained elevations in serum IL-10. In this study, we demonstrate that resting and activated PBMC from long-term tolerant NHP recipients are biased to secrete high levels of IL-10, compared with normal NHP PBMC. Although IL-10-producing CD4+ Tregs (type 1 regulatory cells (TR1)/IL-10 Tregs) were undetectable (<0.5%) in normal rhesus monkeys, 7.5 +/- 1.7% of circulating CD4+ T cells of tolerant rhesus recipients expressed IL-10. In addition to this >15-fold increase in Tr1/IL-10 Tregs, the tolerant monkeys exhibited a nearly 3-fold increase in CD4+CD25+ Tregs, 8.1 +/- 3.0% of CD4 T cells vs 2.8 +/- 1.4% in normal cohorts (p < 0.02). The frequency of CD4+CD25+IL-10+ cells was elevated 5-fold in tolerant vs normal NHP (1.8 +/- 0.9% vs 0.4 +/- 0.2%). Rhesus CD4+CD25+ Tregs exhibited a memory phenotype, and expressed high levels of Foxp3 and CTLA-4 compared with CD4+CD25- T cells. Also, NHP CD4+CD25+ Tregs proliferated poorly after activation and suppressed proliferation of CD4+CD25- effector T cells, exhibiting regulatory properties similar to rodent and human CD4+CD25+ Tregs. Of note, depletion of CD4+CD25+ Tregs restored indirect pathway antidonor responses in tolerant NHP. Our study demonstrates an expanded presence of Treg populations in tolerant NHP recipients, suggesting that these adaptations may be involved in maintenance of stable tolerance.     

LF 15-0195 treatment protects against central nervous system autoimmunity by favoring the development of Foxp3-expressing regulatory CD4 T cells

Experimental autoimmune encephalomyelitis (EAE) is an instructive model for the human demyelinating disease multiple sclerosis. Lewis (LEW) rats immunized with myelin-basic protein (MBP) develop EAE characterized by a single episode of paralysis, from which they recover spontaneously and become refractory to a second induction of disease. LF 15-0195 is a novel molecule that has potent immunosuppressive effects in several immune-mediated pathological manifestations, including EAE. In the present study, we show that a 30-day course of LF 15-0195 treatment not only prevents MBP-immunized LEW rats from developing EAE but also preserves their refractory phase to reinduction of disease. This effect is Ag driven since it requires priming by the autoantigen during the drug administration. In contrast to other immunosuppressive drugs, short-term treatment with this drug induces a persistent tolerance with no rebound of EAE up to 4 mo after treatment withdrawal. This beneficial effect of LF 15-0195 on EAE does not result from the deletion of MBP-specific Vbeta8.2 encephalitogenic T cells. In contrast, this drug favors the differentiation of MBP-specific CD4 T cells into Foxp3-expressing regulatory T cells that, upon adoptive transfer in syngeneic recipients, prevent the development of actively induced EAE. Finally, we demonstrate that the tolerance induced by LF 15-0195 treatment is not dependent on the presence of TGF-beta. Together, these data demonstrate that short-term treatment with LF 15-0195 prevents MBP-immunized LEW rats from EAE by favoring the development of Foxp-3-expressing regulatory CD4 T cells.     

Regulatory T cells induced by 1 alpha,25-dihydroxyvitamin D3 and mycophenolate mofetil treatment mediate transplantation tolerance

1alpha,25-dihydroxyvitamin D3, the active form of vitamin D3, and mycophenolate mofetil, a selective inhibitor of T and B cell proliferation, modulate APC function and induce dendritic cells (DCs) with a tolerogenic phenotype. Here we show that a short treatment with these agents induces tolerance to fully mismatched mouse islet allografts that is stable to challenge with donor-type spleen cells and allows acceptance of donor-type vascularized heart grafts. Peritransplant macrophages and DCs from tolerant mice express down-regulated CD40, CD80, and CD86 costimulatory molecules. In addition, DCs from the graft area of tolerant mice secrete, upon stimulation with CD4+ cells, 10-fold lower levels of IL-12 compared with DCs from acutely rejecting mice, and induce a CD4+ T cell response characterized by selective abrogation of IFN-gamma production. CD4+ but not CD8+ or class II+ cells from tolerant mice, transferred into naive syngeneic recipients, prevent rejection of donor-type islet grafts. Graft acceptance is associated with impaired development of IFN-gamma-producing type 1 CD4+ and CD8+ cells and an increased percentage of CD4+CD25+ regulatory cells expressing CD152 in the spleen and in the transplant-draining lymph node. Transfer of CD4+CD25+ cells from tolerant but not naive mice protects 100% of the syngeneic recipients from islet allograft rejection. These results demonstrate that a short treatment with immunosuppressive agents, such as 1alpha,25-dihydroxyvitamin D3/mycophenolate mofetil, induces tolerance to islet allografts associated with an increased frequency of CD4+CD25+ regulatory cells that can adoptively transfer transplantation tolerance.     

Tolerance to antigen-presenting cell-depleted islet allografts is CD4 T cell dependent

Pretreatment of pancreatic islets in 95% oxygen culture depletes graft-associated APCs and leads to indefinite allograft acceptance in immunocompetent recipients. As such, the APC-depleted allograft represents a model of peripheral alloantigen presentation in the absence of donor-derived costimulation. Over time, a state of donor-specific tolerance develops in which recipients are resistant to donor APC-induced graft rejection. Thus, persistence of the graft is sufficient to induce tolerance independent of other immune interventions. Donor-specific tolerance could be adoptively transferred to immune-deficient SCID recipient mice transplanted with fresh immunogenic islet allografts, indicating that the original recipient was not simply "ignorant" of donor antigens. Interestingly, despite the fact that the original islet allograft presented only MHC class I alloantigens, CD8+ T cells obtained from tolerant animals readily collaborated with naive CD4+ T cells to reject donor-type islet grafts. Conversely, tolerant CD4+ T cells failed to collaborate effectively with naive CD8+ T cells for the rejection of donor-type grafts. In conclusion, the MHC class I+, II- islet allograft paradoxically leads to a change in the donor-reactive CD4 T cell subset and not in the CD8 subset. We hypothesize that the tolerant state is not due to direct class I alloantigen presentation to CD8 T cells but, rather, occurs via the indirect pathway of donor Ag presentation to CD4 T cells in the context of host MHC class II molecules.     

LF 15-0195 inhibits the development of rat central nervous system autoimmunity by inducing long-lasting tolerance in autoreactive CD4 T cells

Experimental autoimmune encephalomyelitis (EAE) is a T cell-dependent autoimmune disease induced in susceptible animals by a single immunization with myelin basic protein (MBP). LF 15-0195 is a novel immunosuppressor that has been shown to have a potent immunosuppressive effect in several pathological manifestations. The purpose of this study was to investigate the effect of this drug on the induction and progression of established rat EAE and to dissect the mechanisms involved. We show that LF 15-0195 administration at the time of MBP immunization reduces the incidence and severity of EAE in Lewis rats. This drug also inhibits ongoing and passively induced EAE, indicating that LF 15-0195 affects already differentiated pathogenic lymphocytes. Compared with lymph node cells from untreated rats, lymphocytes from MBP-immunized rats treated with LF 15-0195 proliferated equally well in response to MBP in vitro, while their ability to produce effector cytokines and to transfer EAE into syngeneic recipients was significantly reduced. This phenomenon is stable and long-lasting. Indeed, neither IL-12 nor repeated stimulation with naive APC and MBP in vitro rendered MBP-specific CD4 T cells from protected rats encephalitogenic. In conclusion, LF 15-0195 treatment suppresses EAE by interfering with both the differentiation and effector functions of autoantigen-specific CD4 T cells.     

Oral tolerance to nickel requires CD4+ invariant NKT cells for the infectious spread of tolerance and the induction of specific regulatory T cells

Previously, oral administration of nickel to C57BL/6 wild-type (WT) mice was shown to render both their splenic T cells and APCs (i.e., T cell-depleted spleen cells) capable of transferring nickel tolerance to naive syngeneic recipients. Moreover, sequential adoptive transfer experiments revealed that on transfer of tolerogenic APCs and immunization, the naive T cells of the recipients differentiated into regulatory T (Treg) cells. Here, we demonstrate that after oral nickel treatment Jalpha18(-/-) mice, which lack invariant NKT (iNKT) cells, were not tolerized and failed to generate Treg cells. However, transfer of APCs from those Jalpha18(-/-) mice did tolerize WT recipients. Hence, during oral nickel administration, tolerogenic APCs are generated that require iNKT cell help for the induction of Treg cells. To obtain this help, the tolerogenic APCs must address the iNKT cells in a CD1-restricted manner. When Jalpha18(-/-) mice were used as recipients of cells from orally tolerized WT donors, the WT Treg cells transferred the tolerance, whereas WT APCs failed to do so, although they proved tolerogenic on transfer to WT recipients. However, Jalpha18(-/-) recipients did become susceptible to the tolerogenicity of transferred WT APCs when they were reconstituted with IL-4- and IL-10-producing CD4(+) iNKT cells. We conclude that CD4(+) iNKT cells are required for the induction of oral nickel tolerance and, in particular, for the infectious spread of tolerance from APCs to T cells. Once induced, these Treg cells, however, can act independently of iNKT cells.     

An instructive role of donor macrophages in mixed chimeras in the induction of recipient CD4(+)Foxp3(+) Treg cells

The immune regulatory function of macrophages (M?s) in mixed chimeras has not been determined. In the present study, with a multi-lineage B6-to-BALB/c mixed chimeric model, we examined the ability of donor-derived splenic M?s in the induction of regulatory T cells (Treg). B6 splenic M?s from mixed chimeras induced significantly less cell proliferation, more IL-10 and TGF-β, and less IL-2 and IFN-γ productions of CD4(+) T cells from BALB/c mice than naive B6 M?s did, whereas they showed similar stimulatory activity to the third part C3H CD4(+) T cells. Importantly, highly purified donor F4/80(+)CD11c(-) M?s efficiently induced recipient CD4(+)Foxp3(+) Treg cells from CD4(+)CD25(-)Foxp3(-) T cells. Furthermore, donor M?s of mixed chimeras produced more IL-10 and less IFN-γ than those of naive mice when cultured with BALB/c but not the third party C3H CD4(+) T cells. Induction of recipient CD4(+) Treg cells by donor M?s was significantly blocked by anti-IL-10, but not by anti-TGF-β mAb. Therefore, donor M?s have the ability to induce recipient CD4(+)Foxp3(+) Treg cells in a donor antigen-specific manner, at least partially, via an IL-10-dependent pathway. This study for the first time showed that, in mixed allogeneic chimeras, donor M?s could be specifically tolerant to recipients and gained the ability to induce recipient but not the third party Foxp3(+) Treg cells. Whether this approach is involved in transplant immune tolerance needs to be determined.     

Alloantigen-induced CD25+CD4+ regulatory T cells can develop in vivo from CD25-CD4+ precursors in a thymus-independent process

The capacity of naturally occurring autoreactive CD25+CD4+ regulatory T cells (Treg) to control immune responses both in vivo and in vitro is now well established. It has been demonstrated that these cells undergo positive selection within the thymus and appear to enter the periphery as committed CD25+CD4+ Treg. We have shown previously that CD25+CD4+ Treg with the capacity to prevent skin allograft rejection can be generated by pretreatment with donor alloantigen under the cover of anti-CD4 therapy. Here we demonstrate that this process does not require an intact thymus. Furthermore, generation of these Treg is not dependent on the expansion of CD25+CD4+ thymic emigrants, because depletion of CD25+ cells before pretreatment does not prevent Treg development, and Treg can be generated from CD25-CD4+ precursors. Taken together, these results clearly demonstrate that CD25+CD4+ Treg can be generated in the periphery from CD25-CD4+ precursors in a pathway distinct to that by which naturally occurring autoreactive CD25+CD4+ Treg develop. These observations may have important implications for the design of protocols, both experimental and clinical, for the induction of tolerance to autoantigens or alloantigens in adults with limited thymic function.     

Induction of IL-10 suppressors in lung transplant patients by CD4+25+ regulatory T cells through CTLA-4 signaling

T cell-mediated autoimmunity to collagen V (col-V), a sequestered yet immunogenic self-protein, can induce chronic lung allograft rejection in rodent models. In this study we characterized the role of CD4+ CD25+ regulatory T cells (Tregs) in regulating col-V autoimmunity in human lung transplant (LT) recipients. LT recipients revealed a high frequency of col-V-reactive, IL-10-producing CD4+ T cells (T IL-10 cells) with low IL-2-, IFN-gamma-, IL-5-, and no IL-4-producing T cells. These T(IL-10) cells were distinct from Tregs because they lacked constitutive expression of both CD25 and Foxp3. Expansion of T IL-10 cells during col-V stimulation in vitro involved CTLA-4 on Tregs, because both depleting and blocking Tregs with anti-CTLA4 F(ab')2 mAbs resulted in loss of T IL-10 cells with a concomitant increase in IFN-gamma producing Th1 cells (TIFN-gamma cells). A Transwell culture of col-V-specific T IL-10 cells with Th1 cells (those generated in absence of Tregs) from the same patient resulted in marked inhibition of IFN-gamma and proliferation of T(IFN-gamma) cells, which was reversed by neutralizing IL-10. Furthermore, the T IL-10 cells were HLA class II restricted because blocking HLA class II on APCs resulted in the loss of IL-10 production. Chronic lung allograft rejection was associated with the loss of Tregs with a concomitant decrease in T IL-10 cells and an increase in T IFN-gamma cells. We conclude that LT patients have col-V-specific T cells that can be detected in the peripheral blood. The predominant col-V-specific T cells produce IL-10 that suppresses autoreactive Th1 cells independently of direct cellular contact. Tregs are pivotal for the induction of these "suppressor" T IL-10 cells.     

口服卡介菌诱导免疫耐受的CD4+CD25+调节性T 细胞机制研究

目的:研究口服卡介菌诱导免疫耐受对CD4+CD25+调节性T细胞的影响。方法:采用口服MPB制备EAE大鼠模型,随机分为BCG组(0.5mg/kg)和EAE模型组(PBS),每组各15只,连续经口灌服给药14d,同时选取15只健康大鼠作为对照组。分别于免疫后15d、27d流式细胞术检测外周血、胸腺及脾脏中CD4+CD25+T淋巴细胞百分率,ELISA检测血清IL-6、TGF-β、IgE、IgG含量。结果:与EAE模型组相比,免疫后BCG组大鼠外周血、胸腺及脾脏中CD4+CD25+T淋巴细胞百分率增加,血清IL-6、TGF-β含量上升,血清IgE、IgG抗体水平下降。结论:口服BCG通过上调淋巴器官中CD4+CD25+T淋巴细胞比例,抑制效应性T细胞活性,发挥免疫耐受作用。     

Tracking the immunoregulatory mechanisms active during allograft tolerance

Immunoregulatory mechanisms dependent on regulatory CD4+ T cells are believed to be critical in the maintenance of peripheral tolerance to allografts. However, a detailed characterization of the effects of these regulatory T cells has been hampered by the absence of a simple means to track and study them. In this work we provide evidence that in a murine model of islet transplantation the interactions between alloaggressive and regulatory T cells can be studied in vitro and in vivo at the single-cell level. The observations made in both an in vitro coculture system and an in vivo CFSE-based adoptive transfer model indicate that lymphocytes from tolerant allograft recipients 1) proliferate weakly to donor strain allogeneic cells but vigorously to third-party strain cells; and 2) suppress the proliferation of naive syngeneic CD4+ and CD8+ T cells to donor tissue in a cell dose- and Ag-specific manner. These effects depend on the presence of CD4+CD25+ T cells and are neutralized by anti-CTLA4 mAb or rIL-2. The principal effect of anti-CTLA4 is directed against the naive, not regulatory, T cell population. These results can be replicated in vivo by transferring lymphocyte populations into transplant recipients, proving that the graft-protecting actions of regulatory T cells are blunted by a rise in the number of allodestructive T cells (pool size model) and depend on the presence of CD4+CD25+ T cells and the integrity of the CTLA4/B7 pathway.     

Regulatory cells potentiate the efficacy of IL-4 gene transfer by up-regulating Th2-dependent expression of protective molecules in the infectious tolerance pathway in transplant recipients

We have previously shown that the tolerant state in allograft recipients can be maintained and perpetuated by an "infectious" T cell-dependent regulatory mechanism. Hence, 1) treatment of LEW rats with RIB-5/2, a CD4 nondepleting mAb, produces indefinite survival of LBNF1 cardiac allografts; 2) donor-specific tolerance can be then transferred by spleen cells into new cohorts of test allograft recipients; and 3) putative regulatory CD4+ Th2-like cells are instrumental in this tolerance model. We now report on studies aimed at exposing mechanisms underlying the infectious tolerance pathway, with emphasis on the interactions between intragraft adenovirus-IL-4 gene transfer and systemic infusion of regulatory cells from tolerant hosts. Unlike individual treatment regimens, adjunctive therapy with adenovirus-IL-4 and suboptimal doses of regulatory spleen cells was strongly synergistic and extended donor-type test cardiac allograft survival to about 2 mo. RT-PCR-based expression of intragraft mRNA coding for IL-2 and IFN-gamma remained depressed, whereas that of IL-4 and IL-10 reciprocally increased selectively in the combined treatment group, data supported by ELISA studies. In parallel, only adjunctive treatment triggered intragraft induction of molecules with anti-oxidant (HO-1) and anti-apoptotic (Bcl-xL/Bag-1) but not with pro-apoptotic (CPP-32) functions, both in the early and late posttransplant phases. Hence, systemic infusion of regulatory cells potentiates the effects of local adenovirus-IL-4 gene transfer in transplant recipients. Th2-driven up-regulation of protective molecule programs at the graft site, such as of anti-oxidant HO-1 and/or anti-apoptotic Bcl-xL and Bag-1, may contribute, at least in part, to the maintenance of the infectious tolerance pathway in transplant recipients.     

CD25 is a marker for CD4+ thymocytes that prevent autoimmune diabetes in rats, but peripheral T cells with this function are found in both CD25+ and CD25- subpopulations

Previously we have shown that autoimmune diabetes, induced in rats by a protocol of adult thymectomy and split-dose gamma irradiation, can be prevented by the transfer of a subset of CD4+ T cells with a memory phenotype (CD45RC-), as well as by CD4+CD8- thymocytes, from syngeneic donors. Further studies now reveal that in the thymus the regulatory cells are observed in the CD25+ subset of CD4+CD8- cells, whereas transfer of the corresponding CD25- thymocyte subset leads to acceleration of disease onset in prediabetic recipients. However, in the periphery, not all regulatory T cells were found to be CD25+. In thoracic duct lymph, cells that could prevent diabetes were found in both CD25- and CD25+ subsets of CD4+CD45RC- cells. Further, CD25- regulatory T cells were also present within the CD4+CD45RC- cell subset from spleen and lymph nodes, but were effective in preventing diabetes only after the removal of CD25- recent thymic emigrants. Phenotypic analysis of human thymocytes showed the presence of CD25+ cells in the same proportions as in rat thymus. The possible developmental relationship between CD25+ and CD25- regulatory T cells is discussed.     

In vitro induction of CD8 expression on thymic pre-T cells. I. Transforming growth factor-beta and tumor necrosis factor-alpha induce CD8 expression on CD8- thymic subsets including the CD25+CD3-CD4-CD8- pre-T cell subset.

We previously reported that IL-7 maintains the viability and differentiation potential of CD25 (IL-2R p55) positive CD3-CD4-CD8- thymic pre-T cells in vitro. This culture system is suitable for studying signals that regulate differentiation of T cell precursors in the thymus. In this study, we screened cytokines for their capacity to induce CD4 or CD8 in murine thymic pre-T cells cultured with IL-7. Of 15 cytokines tested, only transforming growth factor (TGF-beta) and TNF-alpha induced CD8 (Lyt-2), while no cytokine was able to induce CD4 on CD25+CD3-CD4-CD8- thymocytes. The combination of TGF-beta and TNF-alpha was synergistic, and the majority of cells recovered after 2 to 3 days in culture expressed CD8 (but not CD3 or CD4). A similar effect of TGF-beta and TNF-alpha was observed using day-15 fetal thymocytes, CD3+CD4-CD8- or CD3+CD4+CD8- adult thymocytes, although the combination of these cytokines resulted in an additive rather than a synergistic effect in these subsets. In contrast, neither TGF-beta nor TNF-alpha induced CD8 expression on splenic CD4+CD8- T cells. These observations suggest a role for these cytokines in the induction of CD8 expression in CD8- thymocyte subsets including CD3-CD4-CD8- thymic pre-T cells.     

Murine CD4 T cells selected in a highly disparate xenogeneic porcine thymus graft do not show rapid decay in the absence of selecting MHC in the periphery

CD4 repopulation can be achieved in T cell-depleted, thymectomized mice grafted with xenogeneic porcine thymus tissue. These CD4 T cells are specifically tolerant of the xenogeneic porcine thymus donor and the recipient, but are positively selected only by porcine MHC. Recent studies suggest that optimal peripheral survival of naive CD4 T cells requires the presence of the same class II MHC in the periphery as that of the thymus in which they were selected. These observations would suggest that T cells selected on porcine thymic MHC would die rapidly in the periphery, where porcine MHC is absent. Persistent CD4 reconstitution achieved in mice grafted with fetal porcine thymus might be due to increased thymic output to compensate for rapid death of T cells in the periphery. Comparison of CD4 T cell decay after removal of porcine or murine thymic grafts ruled out this possibility. No measurable role for peripheral murine class II MHC in maintaining the naive CD4 pool originating in thymic grafts was demonstrable. However, mouse class II MHC supported the conversion to, survival, and/or proliferation of memory-type CD4 cells selected in fetal porcine thymus. Thus, the same MHC as that mediating positive selection in the thymus is not critical for maintenance of the memory CD4 cell pool in the periphery. Our results support the interpretation that xenogeneic thymic transplantation is a feasible strategy to reconstitute CD4 T cells and render recipients tolerant of a xenogeneic donor.     

A tolerogenic semimature dendritic cells induce effector T-cell hyporesponsiveness by activation of antigen-specific CD4+CD25+ T regulatory cells that promotes skin allograft survival in mice

Semimature dendritic cells (smDCs) can induce autoimmune tolerance by activation of host antigen-specific CD4⁺CD25⁺ regulatory T (Treg) cells. We hypothesized that donor smDCs injected into recipients would induce effector T-cell hyporesponsiveness by activating CD4⁺CD25⁺Treg cells, and promote skin allograft survival. Myeloid smDCs were derived from C57BL/6J mice (donors) in vitro. BALB/c mice (recipients) were injected with smDCs to generate antigen-specific CD4⁺CD25⁺Treg cells in vivo. Allograft survival was prolonged when BALB/c recipients received either C57BL/6J smDCs prior to grafting or C57BL/6J smDC-derived CD4⁺CD25⁺Treg cells post-grafting, and skin flaps from these grafts showed the highest IL-10 production regardless of rapamycin treatments. Our findings confirm that smDCs constitute an independent subgroup of DCs that play a key role for inducing CD4⁺CD25⁺Treg cells to express high IL-10 levels, which induce hyporesponsiveness of effector T cells. Pre-treating recipients with donor smDCs may have potential for transplant tolerance induction.     

The generation of CD25+ CD4+ regulatory T cells that prevent allograft rejection does not compromise immunity to a viral pathogen

In all but a small minority of cases, continued survival of solid organ grafts after transplantation depends on lifelong, nonselective immunosuppression that, although effective, results in increased rates of infection, cancer, and vascular disease. Therapeutic strategies that engage or mimic self-tolerance may allow prolonged allograft survival without the disadvantages of nonspecific immunotherapy. Pretreatment of recipient mice with donor alloantigen combined with transient modulation of the peripheral T cell pool with anti-CD4 Ab leads to the indefinite survival of MHC-incompatible cardiac allografts without further therapy. Tolerance is dependent on CD25+ CD4+ regulatory T cells that arise from naive CD25- precursors and regulate rejection via both IL-10 and CTLA-4. Although these cells are clearly effective at controlling rejection, the proven ability of recently activated CD25+ cells to mediate bystander regulation raises the possibility that tolerized individuals might also have a reduced capacity to respond to environmental pathogens. We have examined anti-influenza responses in tolerized primary heart recipients, secondary recipients following adoptive transfer of regulatory populations, and tolerized mice in which bystander regulation has been deliberately induced. Neither virus-specific CTL activity in vitro nor the clearance of virus in vivo was significantly diminished in any of these treatment groups compared with infected unmanipulated controls. The data suggest that the induction of dominant allograft tolerance dependent on regulatory T cells does not necessarily result in attenuated responses to pathogens providing further support for the development of tolerance induction protocols in clinical transplantation.     

Peripheral tolerance via the anterior chamber of the eye: role of B cells in MHC class I and II antigen presentation

Ags introduced into the anterior chamber (AC) of the eye induce a form of peripheral immune tolerance termed AC-associated immune deviation (ACAID). ACAID mitigates ocular autoimmune diseases and promotes corneal allograft survival. Ags injected into the AC are processed by F4/80(+) APCs, which migrate to the thymus and spleen. In the spleen, ocular APCs induce the development of Ag-specific B cells that act as ancillary APCs and are required for ACAID induction. In this study, we show that ocular-like APCs elicit the generation of Ag-specific splenic B cells that induce ACAID. However, direct cell contact between ocular-like APCs and splenic B cells is not necessary for the induction of ACAID B cells. Peripheral tolerance produced by ACAID requires the participation of ACAID B cells, which induce the generation of both CD4(+) regulatory T cells (Tregs) and CD8(+) Tregs. Using in vitro and in vivo models of ACAID, we demonstrate that ACAID B cells must express both MHC class I and II molecules for the generation of Tregs. These results suggest that peripheral tolerance induced through the eye requires Ag-presenting B cells that simultaneously present Ags on both MHC class I and II molecules.     

Induction of high level IL-2 production in CD4+8- T helper lymphocytes requires post-thymic development.

Triggering of the CD3:TCR complex by optimal concentrations of anti-CD3, anti-TCR beta-chain, and allogeneic stimulator cells induced dramatically higher levels (fivefold for anti-CD3, greater than 10-fold for anti-TCR beta-chain, 84-fold for alloantigen) of IL-2 production in spleen CD4+8- T cells than their thymic counterparts, despite comparable levels of CD3 and TCR beta-chain expression. The nature of the reduced IL-2 production was examined by analysis of anti-CD3-induced IL-2 production at the single cell level. The frequency of IL-2-producing cells in spleen CD4+8- T cells (40.0%) was approximately threefold that of thymus CD4+8- T cells (14.5%). Furthermore, the average IL-2 levels among positive IL-2 producers was also approximately threefold higher in spleen CD4+8- T cells than their thymic counterparts. Adoptive transfer of purified Thy-1.2+ CD4+8- T cells into Thy-1.1-congenic hosts provided a physiologic and histocompatible system that enabled identification of transferred donor (Thy-1.2+) among a sea of host (Thy-1.2-) CD4+ T cells, whose immune function with respect to IL-2 inducibility was examined after isolation by electronic cell sorting. Donor CD4+ T cells thus isolated from host spleen shortly (1 day) after i.v. transfer of thymus CD4+8- T cells were similar to freshly isolated thymus CD4+8- T cells in that they both produced little IL-2 in response to anti-CD3. However, by day 3 post-transfer, IL-2 production by donor CD4+8- T cells had more than doubled and by day 8, they produced IL-2 levels comparable to those of host spleen CD4+8- T cells. A similar acquisition of high level IL-2 inducibility in thymus CD4+8- T cells upon i.v. transfer into Thy-1.1-congenic hosts was also observed using allogeneic cells as the stimulus of IL-2 production. When thymus CD4+8- T cells were intra-thymically transferred into Thy-1.1-congenic hosts, those donor cells that emigrated to the periphery became high IL-2 producers in a time-dependent manner, whereas those that remained inside the thymus showed no signs of up-regulation in IL-2 inducibility. Intrathymic transfer of CD4-8- thymocytes revealed that the most recent thymic emigrant CD4+8- T cells contained few IL-2-producing cells and were not functionally mature with respect to high level IL-2 inducibility.