Both CD4(+)CD25(+) and CD4(+)CD25(-) regulatory cells mediate dominant transplantation tolerance

CD4(+)CD25(+) T cells have been proposed as the principal regulators of both self-tolerance and transplantation tolerance. Although CD4(+)CD25(+) T cells do have a suppressive role in transplantation tolerance, so do CD4(+)CD25(-) T cells, although 10-fold less potent. Abs to CTLA-4, CD25, IL-10, and IL-4 were unable to abrogate suppression mediated by tolerant spleen cells so excluding any of these molecules as critical agents of suppression. CD4(+)CD25(+) T cells from naive mice can also prevent rejection despite the lack of any previous experience of donor alloantigens. However, this requires many more naive than tolerized cells to provide the same degree of suppression. This suggests that a capacity to regulate transplant rejection pre-exists in naive mice, and may be amplified in "tolerized" mice. Serial analysis of gene expression confirmed that cells sorted into CD4(+)CD25(+) and CD4(+)CD25(-) populations were distinct in that they responded to TCR ligation with very different programs of gene expression. Further characterization of the differentially expressed genes may lead to the development of diagnostic tests to monitor the tolerant state.     

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.     

Targeted CTLA-4 engagement induces CD4+CD25+CTLA-4high T regulatory cells with target (allo)antigen specificity

CTLA-4 (CD152) is actively involved in down-regulating T cell activation and maintaining lymphocyte homeostasis. Our earlier studies showed that targeted engagement of CTLA-4 can down-modulate T cell response and suppress allo- and autoimmune responses. In this study, we report that targeted CTLA-4 engagement can induce immune tolerance to a specific target through selective induction of an Ag-specific CD4(+)CD25(+)CTLA-4(high) regulatory T cell (Treg cell) population. Allogeneic cells coated with anti-CTLA-4 Ab induced immune hyporesponsiveness through suppression of proinflammatory cytokines IFN-gamma and IL-2, and up-regulation of the regulatory cytokines IL-10, TGF-beta1, and IL-4, presumably through the engagement of CTLA-4 on activated T cells. Although rechallenge with alloantigen failed to break the unresponsiveness, a transient recovery from tolerance was observed in the presence of high concentrations of exogenous IL-2, saturating concentrations of neutralizing anti-TGF-beta1 and anti-IL-10 Abs, and blocking anti-CTLA-4 Ab, and upon depletion of CD4(+)CD25(+) Treg cells. The CD4(+)CD25(+)CTLA-4(high) Treg cells from tolerant mice suppressed the effector function of CD25(-) T cells from Ag-primed mice. Adoptive transfer of these Treg cells into Ag-primed mice resulted in a significantly reduced alloantigen-specific response. Further characterization demonstrated that the Treg cells with memory phenotype (CD62L(-)) were more potent in suppressing the alloantigen-specific T cell response. These results strongly support that the targeted engagement of CTLA-4 has therapeutic potential for the prevention of transplant rejection.     

A role for TGF-beta in the generation and expansion of CD4+CD25+ regulatory T cells from human peripheral blood

An elusive goal in transplanting organs across histocompatibility barriers has been the induction of specific tolerance to avoid graft rejection. A considerable body of evidence exists that the thymus produces regulatory T cells that suppress the response of other T cells to antigenic stimulation. We report that TGF-beta can induce certain CD4+ T cells in the naive (CD45RA+RO-) fraction in human peripheral blood to develop powerful, contact-dependent suppressive activity that is not antagonized by anti-TGF-beta or anti-IL-10 mAbs. The costimulatory effects of TGF-beta on naive CD4+ T cells up-regulated CD25 and CTLA-4 expression, increased their transition to the activated phenotype, but decreased activation-induced apoptosis. Suppressive activity was concentrated in the CD25+ fraction. These CD4+CD25+ regulatory cells prevented CD8+ T cells from proliferating in response to alloantigens and from becoming cytotoxic effector cells. Moreover, these regulatory cells exerted their suppressive activities in remarkably low numbers and maintained these effects even after they are expanded. Once activated, their suppressive properties were Ag nonspecific. Although <1% of naive CD4+ T cells expressed CD25, depletion of this subset before priming with TGF-beta markedly decreased the generation of suppressive activity. This finding suggests that CD4+CD25+ regulatory T cells induced ex vivo are the progeny of thymus-derived regulatory T cells bearing a similar phenotype. The adoptive transfer of these regulatory T cells generated and expanded ex vivo has the potential to prevent rejection of allogeneic organ grafts.     

CD4+CD25+ regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation

Mature donor T cells cause graft-versus-host disease (GVHD), but they are also the main mediators of the beneficial graft-versus-tumor (GVT) activity of allogeneic bone marrow transplantation. Suppression of GVHD with maintenance of GVT activity is a desirable outcome for clinical transplantation. We have previously shown that donor-derived CD4+CD25+ regulatory T cells inhibit lethal GVHD after allogeneic bone marrow transplantation across major histocompatibility complex (MHC) class I and II barriers in mice. Here we demonstrate that in host mice with leukemia and lymphoma, CD4+CD25+ regulatory T cells suppress the early expansion of alloreactive donor T cells, their interleukin-2-receptor (IL-2R) alpha-chain expression and their capacity to induce GVHD without abrogating their GVT effector function, mediated primarily by the perforin lysis pathway. Thus, CD4+CD25+ T cells are potent regulatory cells that can separate GVHD from GVT activity mediated by conventional donor T cells.     

Role of CD4+ and CD8+ T cells in allorecognition: lessons from corneal transplantation

Corneal transplantation represents an interesting model to investigate the contribution of direct vs indirect Ag recognition pathways to the alloresponse. Corneal allografts are naturally devoid of MHC class II+ APCs. In addition, minor Ag-mismatched corneal grafts are more readily rejected than their MHC-mismatched counterparts. Accordingly, it has been hypothesized that these transplants do not trigger direct T cell alloresponse, but that donor Ags are presented by host APCs, i.e., in an indirect fashion. Here, we have determined the Ag specificity, frequency, and phenotype of T cells activated through direct and indirect pathways in BALB/c mice transplanted orthotopically with fully allogeneic C57BL/6 corneas. In this combination, only 60% of the corneas are rejected, while the remainder enjoy indefinite graft survival. In rejecting mice the T cell response was mediated by two T cell subsets: 1) CD4+ T cells that recognize alloantigens exclusively through indirect pathway and secrete IL-2, and 2) IFN-gamma-producing CD8+ T cells recognizing donor MHC in a direct fashion. Surprisingly, CD8+ T cells activated directly were not required for graft rejection. In nonrejecting mice, no T cell responses were detected. Strikingly, peripheral sensitization to allogeneic MHC molecules in these mice induced acute rejection of corneal grafts. We conclude that only CD4+ T cells activated via indirect allorecognition have the ability to reject allogeneic corneal grafts. Although alloreactive CD8+ T cells are activated via the direct pathway, they are not fully competent and cannot contribute to the rejection unless they receive an additional signal provided by professional APCs in the periphery.     

Prevention of acute and chronic allograft rejection with CD4+CD25+Foxp3+ regulatory T lymphocytes

A major challenge in transplantation medicine is controlling the very strong immune responses to foreign antigens that are responsible for graft rejection. Although immunosuppressive drugs efficiently inhibit acute graft rejection, a substantial proportion of patients suffer chronic rejection that ultimately leads to functional loss of the graft. Induction of immunological tolerance to transplants would avoid rejection and the need for lifelong treatment with immunosuppressive drugs. Tolerance to self-antigens is ensured naturally by several mechanisms; one major mechanism depends on the activity of regulatory T lymphocytes. Here we show that in mice treated with clinically acceptable levels of irradiation, regulatory CD4+CD25+Foxp3+ T cells stimulated in vitro with alloantigens induced long-term tolerance to bone marrow and subsequent skin and cardiac allografts. Regulatory T cells specific for directly presented donor antigens prevented only acute rejection, despite hematopoietic chimerism. By contrast, regulatory T cells specific for both directly and indirectly presented alloantigens prevented both acute and chronic rejection. Our findings demonstrate the potential of appropriately stimulated regulatory T cells for future cell-based therapeutic approaches to induce lifelong immunological tolerance to allogeneic transplants.     

IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo

We present evidence that donor-reactive CD4(+) T cells present in mice tolerant to donor alloantigens are phenotypically and functionally heterogeneous. CD4(+) T cells contained within the CD45RB(high) fraction remained capable of mediating graft rejection when transferred to donor alloantigen-grafted T cell-depleted mice. In contrast, the CD45RB(low) CD4(+) and CD25(+)CD4(+) populations failed to induce rejection, but rather, were able to inhibit rejection initiated by naive CD45RB(high) CD4(+) T cells. Analysis of the mechanism of immunoregulation transferred by CD45RB(low) CD4(+) T cells in vivo revealed that it was donor Ag specific and could be inhibited by neutralizing Abs reactive with IL-10, but not IL-4. CD45RB(low) CD4(+) T cells from tolerant mice were also immune suppressive in vitro, as coculture of these cells with naive CD45RB(high) CD4(+) T cells inhibited proliferation and Th1 cytokine production in response to donor alloantigens presented via the indirect pathway. These results demonstrate that alloantigen-specific regulatory T cells contained within the CD45RB(low) CD4(+) T cell population are responsible for the maintenance of tolerance to donor alloantigens in vivo and require IL-10 for functional activity.     

Regulation of murine inflammatory bowel disease by CD25+ and CD25- CD4+ glucocorticoid-induced TNF receptor family-related gene+ regulatory T cells

CD4(+)CD25(+) regulatory T cells in normal animals are engaged in the maintenance of immunological self-tolerance and prevention of autoimmune disease. However, accumulating evidence suggests that a fraction of the peripheral CD4(+)CD25(-) T cell population also possesses regulatory activity in vivo. Recently, it has been shown glucocorticoid-induced TNFR family-related gene (GITR) is predominantly expressed on CD4(+)CD25(+) regulatory T cells. In this study, we show evidence that CD4(+)GITR(+) T cells, regardless of the CD25 expression, regulate the mucosal immune responses and intestinal inflammation. SCID mice restored with the CD4(+)GITR(-) T cell population developed wasting disease and severe chronic colitis. Cotransfer of CD4(+)GITR(+) population prevented the development of CD4(+)CD45RB(high) T cell-transferred colitis. Administration of anti-GITR mAb-induced chronic colitis in mice restored both CD45RB(high) and CD45RB(low) CD4(+) T cells. Interestingly, both CD4(+)CD25(+) and CD4(+)CD25(-) GITR(+) T cells prevented wasting disease and colitis. Furthermore, in vitro studies revealed that CD4(+)CD25(-)GITR(+) T cells as well as CD4(+)CD25(+)GITR(+) T cells expressed CTLA-4 intracellularly, showed anergic, suppressed T cell proliferation, and produced IL-10 and TGF-beta. These data suggest that GITR can be used as a specific marker for regulatory T cells controlling mucosal inflammation and also as a target for treatment of inflammatory bowel disease.     

Abrogation of antibody-mediated allograft rejection by regulatory CD4 T cells with indirect allospecificity

Alloantibody is an important effector mechanism for allograft rejection. In this study, we tested the hypothesis that regulatory T cells with indirect allospecificity can prevent humoral rejection by using a rat transplant model in which acute rejection of MHC class I-disparate PVG.R8 heart grafts by PVG.RT1(u) recipients is mediated by alloantibody and is dependent upon help from CD4 T cells that can recognize the disparate MHC alloantigen only via the indirect pathway. Pretransplant treatment of PVG.RT1(u) recipients with anti-CD4 mAb plus donor-specific transfusion abrogated alloantibody production and prolonged PVG.R8 graft survival indefinitely. Naive syngeneic splenocytes injected into tolerant animals did not effect heart graft rejection, suggesting the presence of regulatory mechanisms. Adoptive transfer experiments into CD4 T cell-reconstituted, congenitally athymic recipients confirmed that regulation was mediated by CD4 T cells and was alloantigen-specific. CD4 T cell regulation could be broken in tolerant animals either by immunizing with an immunodominant linear allopeptide or by depleting tolerant CD4 T cells, but surprisingly this resulted in neither alloantibody generation nor graft rejection. These findings demonstrate that anti-CD4 plus donor-specific transfusion treatment results in the development of CD4 regulatory T cells that recognize alloantigens via the indirect pathway and act in an Ag-specific manner to prevent alloantibody-mediated rejection. Their development is associated with intrinsic tolerance within the alloantigen-specific B cell compartment that persists after T cell help is made available.     

CD4+CD25+ and CD4+CD25- T cells act respectively as inducer and effector T suppressor cells in superantigen-induced tolerance

The repeated injection of low doses of bacterial superantigens (SAg) is known to induce specific T cell unresponsiveness. We show in this study that the spleen of BALB/c mice receiving chronically, staphylococcal enterotoxin B (SEB) contains SEB-specific CD4(+) TCRBV8(+) T cells exerting an immune regulatory function on SEB-specific primary T cell responses. Suppression affects IL-2 and IFN-gamma secretion as well as proliferation of T cells. However, the suppressor cells differ from the natural CD4(+) T regulatory cells, described recently in human and mouse, because they do not express cell surface CD25. They are CD152 (CTLA-4)-negative and their regulatory activity is not associated with expression of the NF Foxp3. By contrast, after repeated SEB injection, CD4(+)CD25(+) splenocytes were heterogenous and contained both effector as well as regulatory cells. In vivo, CD4(+)CD25(-) T regulatory cells prevented SEB-induced death independently of CD4(+)CD25(+) T cells. Nevertheless, SEB-induced tolerance could not be achieved in thymectomized CD25(+) cell-depleted mice because repeated injection of SEB did not avert lethal toxic shock in these animals. Collectively, these data demonstrate that, whereas CD4(+)CD25(+) T regulatory cells are required for the induction of SAg-induced tolerance, CD4(+)CD25(-) T cells exert their regulatory activity at the maintenance stage of SAg-specific unresponsiveness.     

Cutting edge: CD4+CD25+ alloantigen-specific immunoregulatory cells that can prevent CD8+ T cell-mediated graft rejection: implications for anti-CD154 immunotherapy

Blockade of CD40-CD154 interactions can facilitate long-term allograft acceptance in selected rodent and in primate models, but, due to the ability of CD154-independent CD8(+) T cells to initiate graft rejection, this strategy is not always effective. In this work we demonstrate that blockade of the CD40-CD154 pathway at the time of transplantation enables the generation of donor alloantigen-specific CD4(+)CD25(+) regulatory T cells, and that if the regulatory cells are present in sufficient numbers they can suppress allograft rejection mediated by CD154-independent CD8(+) T cells.     

Antigen exposure during enhanced CTLA-4 expression promotes allograft tolerance in vivo

The role of CTLA-4 in tolerance is primarily inferred from knockout and blocking studies. Anti-CD45RB mediates allograft tolerance in mice by inducing CTLA-4 expression on CD4 cells, providing a novel opportunity to determine how therapeutic enhancement of CTLA-4 promotes tolerance. We now show that induced CTLA-4 expression normally resolves by day 17. Although thymectomy prolongs enhanced CTLA-4 expression, long-term engraftment is unaffected. To address the temporal relationship between increased CTLA-4 expression and engraftment, transplantation was delayed for various times after anti-CD45RB treatment. Delaying transplantation for 7 days (when CTLA-4 expression had peaked but treatment mAb was no longer detectable), resulted in long-term engraftment comparable to transplantation with no delay (day 0). Delaying transplantation from 10 to 18 days led to a progressively poorer outcome as CTLA-4 expression returned to baseline. This suggested that Ag exposure while CTLA-4 expression is enhanced is sufficient to induce long-term engraftment. To substantiate this, on day 0, anti-CD45RB-treated mice received BALB/c vs unrelated alloantigen, followed by transplantation of BALB/c islets 10 days later. Whereas recipients exposed to unrelated Ag experienced acute rejection, recipients exposed to donor Ag achieved long-term engraftment. Anti-CD45RB-treated mice exposed to alloantigen exhibited anergic CD4(+)CD25(-) effector cells and regulatory CD4(+)CD25(+) cells. Moreover, CD25 depletion in the peritransplant period prevented anti-CD45RB-mediated engraftment. Thus, exposure of CD4 cells expressing CTLA-4 to donor Ag is necessary and sufficient to induce long-term engraftment which appears to be mediated by both regulation and anergy.     

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.     

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.     

CTLA-4 and CD4+ CD25+ regulatory T cells inhibit protective immunity to filarial parasites in vivo

The T cell coinhibitory receptor CTLA-4 has been implicated in the down-regulation of T cell function that is a quintessential feature of chronic human filarial infections. In a laboratory model of filariasis, Litomosoides sigmodontis infection of susceptible BALB/c mice, we have previously shown that susceptibility is linked both to a CD4+ CD25+ regulatory T (Treg) cell response, and to the development of hyporesponsive CD4+ T cells at the infection site, the pleural cavity. We now provide evidence that L. sigmodontis infection drives the proliferation and activation of CD4+ Foxp3+ Treg cells in vivo, demonstrated by increased uptake of BrdU and increased expression of CTLA-4, Foxp3, GITR, and CD25 compared with naive controls. The greatest increases in CTLA-4 expression were, however, seen in the CD4+ Foxp3- effector T cell population which contained 78% of all CD4+ CTLA-4+ cells in the pleural cavity. Depletion of CD25+ cells from the pleural CD4+ T cell population did not increase their Ag-specific proliferative response in vitro, suggesting that their hyporesponsive phenotype is not directly mediated by CD4+ CD25+ Treg cells. Once infection had established, killing of adult parasites could be enhanced by neutralization of CTLA-4 in vivo, but only if performed in combination with the depletion of CD25+ Treg cells. This work suggests that during filarial infection CTLA-4 coinhibition and CD4+ CD25+ Treg cells form complementary components of immune regulation that inhibit protective immunity in vivo.     

CD4+ Primary T Cells Expressing HCV-Core Protein Upregulate Foxp3 and IL-10, Suppressing CD4 and CD8 T Cells

Adaptive T cell responses are critical for controlling HCV infection. While there is clinical evidence of a relevant role for regulatory T cells in chronic HCV-infected patients, based on their increased number and function; mechanisms underlying such a phenomena are still poorly understood. Accumulating evidence suggests that proteins from Hepatitis C virus can suppress host immune responses. We and others have shown that HCV is present in CD4+ lymphocytes from chronically infected patients and that HCV-core protein induces a state of unresponsiveness in the CD4+ tumor cell line Jurkat. Here we show that CD4+ primary T cells lentivirally transduced with HCV-core, not only acquire an anergic phenotype but also inhibit IL-2 production and proliferation of bystander CD4+ or CD8+ T cells in response to anti-CD3 plus anti-CD28 stimulation. Core-transduced CD4+ T cells show a phenotype characterized by an increased basal secretion of the regulatory cytokine IL-10, a decreased IFN-γ production upon stimulation, as well as expression of regulatory T cell markers, CTLA-4, and Foxp3. A significant induction of CD4+CD25+CD127^lowPD-1^highTIM-3^high regulatory T cells with an exhausted phenotype was also observed. Moreover, CCR7 expression decreased in HCV-core expressing CD4+ T cells explaining their sequestration in inflamed tissues such as the infected liver. This work provides a new perspective on de novo generation of regulatory CD4+ T cells in the periphery, induced by the expression of a single viral protein.     

Common gamma chain cytokines promote rapid in vitro expansion of allo-specific human CD8+ suppressor T cells

Human CD8(+) regulatory T cells, particularly the CD8(+)CD28(-) T suppressor cells, have emerged as an important modulator of alloimmunity. Understanding the conditions under which these cells are induced and/or expanded would greatly facilitate their application in future clinical trials. In the current study, we develop a novel strategy that combines common gamma chain (γc) cytokines IL-2, IL-7 and IL-15 and donor antigen presenting cells (APCs) to stimulate full HLA-mismatched allogeneic human CD8(+) T cells which results in significant expansions of donor-specific CD8(+)CD28(-) T suppressor cells in vitro. The expanded CD8(+)CD28(-) T cells exhibit increased expressions of CTLA-4, FoxP3, and CD25, while down-regulate expressions of CD56, CD57, CD127, and perforin. Furthermore, these cells suppress proliferation of CD4(+) T cells in a contact-dependent and cytokine-independent manner. Interestingly, the specificity of suppression is restricted by the donor HLA class I antigens but promiscuous to HLA class II antigens, providing a potential mechanism for linked suppression. Taken together, our results demonstrate a novel role for common γc cytokines in combination with donor APCs in the expansion of donor-specific CD8(+)CD28(-) T suppressor cells, and represent a robust strategy for in vitro generation of such cells for adoptive cellular immunotherapy in transplantation.     

CTLA-4 engagement and regulatory CD4+CD25+ T cells independently control CD8+-mediated responses under costimulation blockade

Blockade of costimulatory signals is a promising therapeutic target to prevent allograft rejection. In this study, we sought to characterize to what extent CTLA-4 engagement contributes to the development of transplantation tolerance under the cover of CD40/CD40L and CD28/CD86 blockade. In vitro, we found that inhibition of the primary alloresponse and induction of alloantigen hyporesponsiveness by costimulation blockade was abrogated by anti-CTLA-4 mAb. In addition, regulatory CD4(+)CD25(+) T cells (T(REG)) were confirmed to play a critical role in the induction of hyporesponsiveness by anti-CD40L and anti-CD86 mAb. Our data indicated that CTLA-4 engagement is not required for activation or suppressor function of T(REG). Instead, in the absence of either CTLA-4 signaling or T(REG), CD8(+) T cell division was enhanced, whereas the inhibition of CD4(+) T cell division by costimulation blockade remained largely unaffected. In vivo, the administration of additional anti-CTLA-4 mAb abrogated anti-CD40L- and anti-CD86 mAb-induced cardiac allograft survival. Correspondingly, rejection was accompanied by enhanced allograft infiltration of CD8(+) cells. We conclude that CTLA-4 signaling and T(REG) independently cooperate in the inhibition of CD8(+) T cell expansion under costimulation blockade.     

CD25+ CD4+ T cells regulate the expansion of peripheral CD4 T cells through the production of IL-10

The mechanisms by which the immune system achieves constant T cell numbers throughout life, thereby controlling autoaggressive cell expansions, are to date not completely understood. Here, we show that the CD25(+) subpopulation of naturally activated (CD45RB(low)) CD4 T cells, but not CD25(-) CD45RB(low) CD4 T cells, inhibits the accumulation of cotransferred CD45RB(high) CD4 T cells in lymphocyte-deficient mice. However, both CD25(+) and CD25(-) CD45RB(low) CD4 T cell subpopulations contain regulatory cells, since they can prevent naive CD4 T cell-induced wasting disease. In the absence of a correlation between disease and the number of recovered CD4(+) cells, we conclude that expansion control and disease prevention are largely independent processes. CD25(+) CD45RB(low) CD4 T cells from IL-10-deficient mice do not protect from disease. They accumulate to a higher cell number and cannot prevent the expansion of CD45RB(high) CD4 T cells upon transfer compared with their wild-type counterparts. Although CD25(+) CD45RB(low) CD4 T cells are capable of expanding when transferred in vivo, they reach a homeostatic equilibrium at lower cell numbers than CD25(-) CD45RB(low) or CD45RB(high) CD4 T cells. We conclude that CD25(+) CD45RB(low) CD4 T cells from nonmanipulated mice control the number of peripheral CD4 T cells through a mechanism involving the production of IL-10 by regulatory T cells.