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.     

Enhanced engagement of CTLA-4 induces antigen-specific CD4+CD25+Foxp3+ and CD4+CD25- TGF-beta 1+ adaptive regulatory T cells

CTLA-4 is a critical negative regulator of T cell response and is instrumental in maintaining immunological tolerance. In this article, we report that enhanced selective engagement of CTLA-4 on T cells by Ag-presenting dendritic cells resulted in the induction of Ag-specific CD4(+)CD25(+)Foxp3(+) and CD4(+)CD25(-)TGF-beta1(+) adaptive Tregs. These cells were CD62L(low) and hyporesponsive to stimulation with cognate Ag but demonstrated a superior ability to suppress Ag-specific effector T cell response compared with their CD62L(high) counterparts. Importantly, treatment of mice with autoimmune thyroiditis using mouse thyroglobulin (mTg)-pulsed anti-CTLA-4 agonistic Ab-coated DCs, which results in a dominant engagement of CTLA-4 upon self-Ag presentation, not only suppressed thyroiditis but also prevented reemergence of the disease upon rechallenge with mTg. Further, the disease suppression was associated with significantly reduced mTg-specific T cell and Ab responses. Collectively, our results showed an important role for selective CTLA-4 signaling in the induction of adaptive Tregs and suggested that approaches that allow dominant CTLA-4 engagement concomitant with Ag-specific TCR ligation can be used for targeted therapy.     

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.     

Blockade of T cell activation using a surface-linked single-chain antibody to CTLA-4 (CD152)

CTLA-4 (CD152) engagement can down-regulate T cell activation and promote the induction of immune tolerance. However, the strategy of attenuating T cell activation by engaging CTLA-4 has been limited by sharing of its natural ligands with the costimulatory protein CD28. In the present study, a CTLA-4-specific single-chain Ab (scFv) was developed and expressed on the cell surface to promote selective engagement of this regulatory molecule. Transfectants expressing anti-CTLA-4 scFv at their surface bound soluble CTLA-4 but not soluble CD28. Coexpression of anti-CTLA-4 scFv with anti-CD3epsilon and anti-CD28 scFvs on artificial APCs reduced the proliferation and IL-2 production by resting and preactivated bulk T cells as well as CD4+ and CD8+ T cell subsets. Importantly, expression of anti-CTLA-4 scFv on the same cell surface as the TCR ligand was essential for the inhibitory effects of CTLA-4-specific ligation. CTLA-4-mediated inhibition of tyrosine phosphorylation of components of the proximal TCR signaling apparatus was similarly dependent on coexpression of TCR and CTLA-4 ligands on the same surface. These findings support a predominant role for CTLA-4 function in the modification of the proximal TCR signal. Using T cells from DO11.10 and 2C TCR transgenic mice, negative regulatory effects of selective CTLA-4 ligation were also demonstrated during the stimulation of Ag-specific CD4+ and CD8+ T cells by MHC/peptide complexes. Together these studies demonstrate that selective ligation of CTLA-4 using a membrane-bound scFv results in attenuated T cell responses only when coengaged with the TCR during T cell/APC interaction and define an approach to harnessing the immunomodulatory potential of CTLA-4-specific ligation.     

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.     

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.     

Targeting CD28, CTLA-4 and PD-L1 Costimulation Differentially Controls Immune Synapses and Function of Human Regulatory and Conventional T-Cells

CD28, CTLA-4 and PD-L1, the three identified ligands for CD80/86, are pivotal positive and negative costimulatory molecules that, among other functions, control T cell motility and formation of immune synapse between T cells and antigen-presenting cells (APCs). What remains incompletely understood is how CD28 leads to the activation of effector T cells (Teff) but inhibition of suppression by regulatory T cells (Tregs), while CTLA-4 and PD-L1 inhibit Teff function but are crucial for the suppressive function of Tregs. Using alloreactive human T cells and blocking antibodies, we show here by live cell dynamic microscopy that CD28, CTLA-4, and PD-L1 differentially control velocity, motility and immune synapse formation in activated Teff versus Tregs. Selectively antagonizing CD28 costimulation increased Treg dwell time with APCs and induced calcium mobilization which translated in increased Treg suppressive activity, in contrast with the dampening effect on Teff responses. The increase in Treg suppressive activity after CD28 blockade was also confirmed with polyclonal Tregs. Whereas CTLA-4 played a critical role in Teff by reversing TCR-induced STOP signals, it failed to affect motility in Tregs but was essential for formation of the Treg immune synapse. Furthermore, we identified a novel role for PD-L1-CD80 interactions in suppressing motility specifically in Tregs. Thus, our findings reveal that the three identified ligands of CD80/86, CD28, CTLA-4 and PD-L1, differentially control immune synapse formation and function of the human Teff and Treg cells analyzed here. Individually targeting CD28, CTLA-4 and PD-L1 might therefore represent a valuable therapeutic strategy to treat immune disorders where effector and regulatory T cell functions need to be differentially targeted.     

B7-deficient autoreactive T cells are highly susceptible to suppression by CD4(+)CD25(+) regulatory T cells

CD4(+)CD25(+) regulatory T cells (Tregs) suppress immunity to infections and tumors as well as autoimmunity and graft-vs-host disease. Since Tregs constitutively express CTLA-4 and activated T cells express B7-1 and B7-2, it has been suggested that the interaction between CTLA-4 on Tregs and B7-1/2 on the effector T cells may be required for immune suppression. In this study, we report that autopathogenic T cells from B7-deficient mice cause multiorgan inflammation when adoptively transferred into syngeneic RAG-1-deficient hosts. More importantly, this inflammation is suppressed by adoptive transfer of purified wild-type (WT) CD4(+)CD25(+) T cells. WT Tregs also inhibited lymphoproliferation and acquisition of activation markers by the B7-deficient T cells. An in vitro suppressor assay revealed that WT and B7-deficient T cells are equally susceptible to WT Treg regulation. These results demonstrate that B7-deficient T cells are highly susceptible to immune suppression by WT Tregs and refute the hypothesis that B7-CTLA-4 interaction between effector T cells and Tregs plays an essential role in Treg function.     

Follicular lymphoma intratumoral CD4+CD25+GITR+ regulatory T cells potently suppress CD3/CD28-costimulated autologous and allogeneic CD8+CD25- and CD4+CD25- T cells

Regulatory T cells (T(R)) play a critical role in the inhibition of self-reactive immune responses and as such have been implicated in the suppression of tumor-reactive effector T cells. In this study, we demonstrate that follicular lymphoma (FL)-infiltrating CD8+ and CD4+ T cells are hyporesponsive to CD3/CD28 costimulation. We further identify a population of FL-infiltrating CD4+CD25+GITR+ T(R) that are significantly overrepresented within FL nodes (FLN) compared with that seen in normal (nonmalignant, nonlymphoid hyperplastic) or reactive (nonmalignant, lymphoid hyperplastic) nodes. These T(R) actively suppress both the proliferation of autologous nodal CD8+CD25- and CD4+CD25- T cells, as well as cytokine production (IFN-gamma, TNF-alpha and IL-2), after CD3/CD28 costimulation. Removal of these cells in vitro by CD25+ magnetic bead depletion restores both the proliferation and cytokine production of the remaining T cells, demonstrating that FLN T cell hyporesponsiveness is reversible. In addition to suppressing autologous nodal T cells, these T(R) are also capable of suppressing the proliferation of allogeneic CD8+CD25- and CD4+CD25- T cells from normal lymph nodes as well as normal donor PBL, regardless of very robust stimulation of the target cells with plate-bound anti-CD3 and anti-CD28 Abs. The allogeneic suppression is not reciprocal, as equivalent numbers of CD25+FOXP3+ cells derived from either normal lymph nodes or PBL are not capable of suppressing allogeneic CD8+CD25- and CD4+CD25- T cells, suggesting that FLN T(R) are more suppressive than those derived from nonmalignant sources. Lastly, we demonstrate that inhibition of TGF-beta signaling partially restores FLN T cell proliferation suggesting a mechanistic role for TGF-beta in FLN T(R)-mediated suppression.     

An important role of CD80/CD86-CTLA-4 signaling during photocarcinogenesis in mice

Although previous studies have shown that altered B7 costimulation plays a critical role in UV irradiation-induced regulation of immunity, the individual roles of the B7 receptors (CD28 and CTLA-4) or the B7 family members (CD80 and CD86) have not been explored. Thus, we investigated CTLA-4 signaling during photocarcinogenesis of chronically UV-B-exposed mice using an antagonistic anti-CTLA-4 Ab. Anti-CTLA-4-treated mice developed significantly fewer UV-induced tumors. Moreover, anti-CTLA-4 treatment induced long-lasting protective immunity because progressively growing UV tumors inoculated into anti-CTLA-4- and UV-treated mice that had not developed tumors were rejected. Next, we used mice deficient for CD80, CD86, or both in photocarcinogenesis studies to assess the relative contributions of these CTLA-4 ligands. Double-deficient mice showed significantly reduced UV-induced skin tumor development, whereas CD86(-/-) mice produced skin cancer earlier compared with CD80(-/-) and control mice. The growth of UV-induced tumors appears to be controlled by UV-induced suppressor T cells, because CD80(-/-)/CD86(-/-) mice had strongly reduced numbers of UV-induced CD4(+)CD25(+) suppressor T cells. In vitro, CTLA-4 blockade inhibited the suppressor activity of UV-induced CD4(+)CD25(+) T cells, suggesting that reduced photocarcinogenesis might be due to decreased numbers or function of suppressor T cells. Together, these data indicate that blocking CD80/86-CTLA-4 signaling induced immune protection against the development of UV-induced skin tumors. Furthermore, CD86-mediated costimulation appears to play a more critical role in the protection against photocarcinogenesis than CD80.     

Blockade of CTLA-4 signals inhibits Th2-mediated murine chronic graft-versus-host disease by an enhanced expansion of regulatory CD8+ T cells

CTLA-4 (CD152) is thought to be a negative regulator of T cell activation. Little is known about the function of CTLA-4 in Th2-type immune responses. We have investigated the effect of initial treatment with anti-CTLA-4 mAb on murine chronic graft-vs-host disease. Transfer of parental BALB/c splenocytes into C57BL/6 x BALB/c F1 mice induced serum IgE production, IL-4 expression by donor CD4+ T cells, and host allo-Ag-specific IgG1 production at 6-9 wk after transfer. Treatment with anti-CTLA-4 mAb for the initial 2 wk significantly reduced IgE and IgG1 production and IL-4 expression. Analysis of the splenic phenotype revealed the enhancement of donor T cell expansion, especially within the CD8 subset, and the elimination of host cells early after anti-CTLA-4 mAb treatment. This treatment did not affect early IFN-gamma expression by CD4+ and CD8+ T cells and anti-host cytolytic activity. Thus, blockade of CTLA-4 greatly enhanced CD8+ T cell expansion, and this may result in the regulation of consequent Th2-mediated humoral immune responses. These findings suggest a new approach for regulating IgE-mediated allergic immune responses by blockade of CTLA-4 during a critical period of Ag sensitization.     

Functional analysis of B and T lymphocyte attenuator engagement on CD4+ and CD8+ T cells

T cell activation can be profoundly altered by coinhibitory and costimulatory molecules. B and T lymphocyte attenuator (BTLA) is a recently identified inhibitory Ig superfamily cell surface protein found on lymphocytes and APC. In this study we analyze the effects of an agonistic anti-BTLA mAb, PK18, on TCR-mediated T cell activation. Unlike many other allele-specific anti-BTLA mAb we have generated, PK18 inhibits anti-CD3-mediated CD4+ T cell proliferation. This inhibition is not dependent on regulatory T cells, nor does the Ab induce apoptosis. Inhibition of T cell proliferation correlates with a profound reduction in IL-2 secretion, although this is not the sole cause of the block of cell proliferation. In contrast, PK18 has no effect on induction of the early activation marker CD69. PK18 also significantly inhibits, but does not ablate, IL-2 secretion in the presence of costimulation as well as reduces T cell proliferation under limiting conditions of activation in the presence of costimulation. Similarly, PK18 inhibits Ag-specific T cell responses in culture. Interestingly, PK18 is capable of delivering an inhibitory signal as late as 16 h after the initiation of T cell activation. CD8+ T cells are significantly less sensitive to the inhibitory effects of PK18. Overall, BTLA adds to the growing list of cell surface proteins that are potential targets to down-modulate T cell function.     

Established T cell-driven germinal center B cell proliferation is independent of CD28 signaling but is tightly regulated through CTLA-4

CD4 T cell activation is positively (CD28) and negatively (CTLA-4) regulated by the costimulatory ligands CD80 and CD86. A central question is how the balance between these two opposing forces is controlled as T cells differentiate. We have previously shown that CD28 signaling is absolutely required to prime naive CD4 T cells to differentiate into effectors that provide help for germinal centers and class-switched Ab responses. In this study, we show that the requirement for CD28 signaling is transient and effector CD4 T cells do not require CD28 signals to sustain their function. The CD28 independence of effector T cells within germinal centers suggested that a key function for CD80/CD86 under these circumstances might be to provide negative regulatory signals via the CD28 homologue CTLA-4. By examining germinal center responses in mice where the ability to signal through T cell CTLA-4 was compromised, we provide data that supports a critical role for CTLA-4 in down-regulating T cell help for germinal center B cells.     

TGF-beta-mediated suppression by CD4+CD25+ T cells is facilitated by CTLA-4 signaling

CD4+CD25+ T cells play a pivotal role in immunological homeostasis by their capacity to exert immunosuppressive activity. However, the mechanism by which these cells function is still a subject for debate. We previously reported that surface (membrane) TGF-beta produced by CD4+CD25+ T cells was an effector molecule mediating suppressor function. We now support this finding by imaging surface TGF-beta on Foxp3+CD4+CD25+ T cells in confocal fluorescence microscopy. Then, using a TGF-beta-sensitive mink lung epithelial cell (luciferase) reporter system, we show that surface TGF-beta can be activated to signal upon cell-cell contact. Moreover, if such TGF-beta signaling is blocked in an in vitro assay of CD4+CD25+ T cell suppression by a specific inhibitor of TGF-betaRI, suppressor function is also blocked. Finally, we address the role of CTLA-4 in CD4+CD25+ T cell suppression, showing first that whereas anti-CTLA-4 does not block in vitro suppressor function, it does complement the blocking activity of anti-TGF-beta. We then show with confocal fluorescence microscopy that incubation of CD4+CD25+ T cells with anti-CTLA-4- and rB7-1/Fc-coated beads results in accumulation of TGF-beta at the cell-bead contact site. This suggests that CTLA-4 signaling facilitates TGF-beta-mediated suppression by intensifying the TGF-beta signal at the point of suppressor cell-target cell interaction.     

B7 interactions with CD28 and CTLA-4 control tolerance or induction of mucosal inflammation in chronic experimental colitis

CD28-B7 interaction plays a critical costimulatory role in inducing T cell activation, while CTLA-4-B7 interaction provides a negative signal that is essential in immune homeostasis. Transfer of CD45RB(high)CD4(+) T cells from syngeneic mice induces transmural colon inflammation in SCID recipients. This adoptive transfer model was used to investigate the contribution of B7-CD28/CTLA-4 interactions to the control of intestinal inflammation. CD45RB(high)CD4(+) cells from CD28(-/-) mice failed to induce mucosal inflammation in SCID recipients. Administration of anti-B7.1 (but not anti-B7.2) after transfer of wild-type CD45RB(high)CD4(+) cells also prevented wasting disease with colitis, abrogated leukocyte infiltration, and reduced production of proinflammatory cytokines IL-2 and IFN-gamma by lamina propria CD4(+) cells. In contrast, anti-CTLA-4 treatment led to deterioration of disease, to more severe inflammation, and to enhanced production of proinflammatory cytokines. Of note, CD25(+)CD4(+) cells from CD28(-/-) mice similar to those from the wild-type mice were efficient to prevent intestinal mucosal inflammation induced by the wild-type CD45RB(high) cells. The inhibitory functions of these regulatory T cells were effectively blocked by anti-CTLA-4. These data show that the B7-CD28 costimulatory pathway is required for induction of effector T cells and for intestinal mucosal inflammation, while the regulatory T cells function in a CD28-independent way. CTLA-4 signaling plays a key role in maintaining mucosal lymphocyte tolerance, most likely by activating the regulatory T cells.     

Induction of polyclonal CD8+ T cell activation and effector function by Pertussis toxin

Pertussis toxin (PTX) has pronounced adjuvant activity and strongly enhances innate and adaptive immune responses, including increased antibody production and Th1/Th2 cytokine production. Adjuvant effects of PTX on Th1 and Th2 cells are primarily mediated via CD80/86 costimulation via enhanced expression of these molecules by APCs. However, it has remained unresolved whether PTX modulates the expression of costimulatory and inhibitory molecules on CD4+ and CD8+ T cells. To address this question, we determined the expression kinetics of CD28, CTLA-4, and CD40L on spleen CD4+ and CD8+ T cells after incubation with PTX. The results show that PTX upregulated the expression of CD28 by CD8+ T cells, but not by CD4+ T cells. In contrast, the expression of CTLA-4 and CD40L was not substantially altered on CD4+ or CD8+ T cells. CD28 upregulation by CD8+ T cells was paralleled by upregulation of CD69 and the induction of IFN-γ, Granzyme B (GrB), and IL-17. CD8+ T cell activation and cytokine production could be substantially blocked with anti-CD80 and CD86 antibodies, consistent with CD28 mediated signaling. Treatment of highly purified CD8+ T cells with PTX resulted in upregulation of CD28 and CD69, and production of IFN-γ. Incubation with CD28 mAb further enhanced this effect, suggesting that PTX has direct effects on CD8+ T cells which are enhanced by CD80/86-mediated costimulation provided by APCs.     

CD4+CD25+ 调节性T 细胞与冠状动脉粥样硬化病变的研究进展

CD4+CD25+调节性T细胞是一个具有独特免疫调节功能的T细胞亚群,人体主要通过CD4+CD25+调节性T细胞以免疫负向调节的方式来抑制自身反应性T细胞的作用,减少免疫性疾病的发生,从而维持机体内环境的稳定,维持免疫耐受。CD4+CD25+Treg已被证实其与肿瘤、感染、自身免疫病、移植免疫等多种疾病的发生、发展及转归均相关。随着社会的进步和人民生活水平的提高冠状动脉粥样硬化性病变作为一种慢性病变,其发病率越来越高,已经成为严重危害人类健康的常见病,近年来越来越多的证据表明炎症及免疫反应机制在冠状动脉粥样硬化性心脏病的发生、发展及预后过程中具有重要的作用。而CD4+CD25+调节性T细胞在冠状动脉粥样硬化性病变中所起的作用也受到越来越多的关注。本文就CD4+CD25+调节性T细胞与冠状动脉粥样硬化病变之间的关联做一综述。     

OX40-mediated differentiation to effector function requires IL-2 receptor signaling but not CD28, CD40, IL-12Rbeta2, or T-bet

Ag-specific CD4 T cells transferred into unirradiated Ag-bearing recipients proliferate, but survival and accumulation of proliferating cells is not extensive and the donor cells do not acquire effector functions. We previously showed that a single costimulatory signal delivered by an agonist Ab to OX40 (CD134) promotes accumulation of proliferating cells and promotes differentiation to effector CD4 T cells capable of secreting IFN-gamma. In this study, we determined whether OX40 costimulation requires supporting costimulatory or differentiation signals to drive acquisition of effector T cell function. We report that OX40 engagement drives effector T cell differentiation in the absence of CD28 and CD40 signals. Two important regulators of Th1 differentiation, IL-12R and T-bet, also are not required for acquisition of effector function in CD4 T cells responsive to OX40 stimulation. Finally, we show that CD25-deficient CD4 T cells produce little IFN-gamma in the presence of OX40 costimulation compared with wild type, suggesting that IL-2R signaling is required for efficient OX40-mediated differentiation to IFN-gamma secretion.     

Natural recovery and protection from autoimmune encephalomyelitis: contribution of CD4+CD25+ regulatory cells within the central nervous system

Immune regulation of autoimmune disease can function at two sites: at the secondary lymphoid organs or in the target organ itself. In this study, we investigated the natural resolution of autoimmune pathology within the CNS using murine experimental autoimmune encephalomyelitis (EAE). Recovery correlates with the accumulation of IL-10-producing CD4+CD25+ T cells within the CNS. These CD4+CD25+ cells represent as many as one in three of CD4+ cells in the CNS during recovery, they are FoxP3+ and express other markers associated with regulatory cells (CTLA-4, GITR, and alpha(E)beta7), and they have regulatory function ex vivo. Depletion of CD25+ cells inhibits the natural recovery from EAE. Also, depletion of CD25+ cells after recovery removes the resistance to reinduction of EAE observed in this model. Furthermore, passive transfer of CNS-derived CD4+CD25+ cells in low numbers provides protection from EAE in recipient mice. These are the first data demonstrating the direct involvement of CD4+CD25+ regulatory T cells in the natural resolution of autoimmune disease within the target organ.     

Increased natural CD4+CD25+ regulatory T cells and their suppressor activity do not contribute to mortality in murine polymicrobial sepsis

Regulatory T cells (Tregs), including natural CD4+CD25+ Tregs and inducible IL-10 producing T regulatory type 1 (T(R)1) cells, maintain tolerance and inhibit autoimmunity. Recently, increased percentages of Tregs have been observed in the blood of septic patients, and ex vivo-activated Tregs were shown to prevent polymicrobial sepsis mortality. Whether endogenous Tregs contribute to sepsis outcome remains unclear. Polymicrobial sepsis, induced by cecal ligation and puncture, caused an increased number of splenic Tregs compared with sham-treated mice. Splenic CD4+CD25+ T cells from septic mice expressed higher levels of Foxp3 mRNA and were more efficient suppressors of CD4+CD25- T effector cell proliferation. Isolated CD4+ T cells from septic mice displayed increased intracellular IL-10 staining following stimulation, indicating that T(R)1 cells may also be elevated in sepsis. Surprisingly, Ab depletion of total CD4+ or CD4+CD25+ populations did not affect mortality. Furthermore, no difference in survival outcome was found between CD25 or IL-10 null mice and wild-type littermates, indicating that Treg or T(R)1-generated IL-10 are not required for survival. These results demonstrate that, although sepsis causes a relative increase in Treg number and increases their suppressive function, their presence does not contribute significantly to overall survival in this model.