Expression of ICOS on human melanoma-infiltrating CD4+CD25highFoxp3+ T regulatory cells: implications and impact on tumor-mediated immune suppression

OBJECTIVE: Interaction of ICOS with its ligand (ICOSL, B7-H2) promotes T cell responses. As CD4+CD25highFoxp3+ naturally occurring T regulatory cells in melanoma patients express ICOS, we investigated the impact of ICOS on naturally occurring T regulatory cell function. METHODS: Expression of ICOS and T regulatory (Treg) cell markers was determined on CD4+CD25high T cells in PBMC and tumor-infiltrating lymphocytes from melanoma patients (n=10) and PBMC of normal controls (n=10) by multicolor flow cytometry. Suppression mediated by sorted ICOShigh and ICOSlow Treg was assessed in CFSE-based suppression assays with autologous CD4+CD25- responder cells (RC). Transwell inserts separating Treg from RC were used to evaluate suppression mechanisms used by Treg. ICOShigh or ICOSlow Treg were coincubated with RC+/-TCR and IL-2 stimulation. ICOShigh and ICOS- Treg were also expanded under conditions previously shown to induce Tr1 from RC. RESULTS: Treg in tumor-infiltrating lymphocytes expressed ICOS (mean fluorescence intensity=70+/-10), while Treg in PBMC had low ICOS expression (mean fluorescence intensity=3.5+/-2.5, p相似文献   

CD18 is required for optimal development and function of CD4+CD25+ T regulatory cells

CD4+CD25+ T regulatory (Treg) cells inhibit immunopathology and autoimmune disease in vivo. CD4+CD25+ Treg cells' capacity to inhibit conventional T cells in vitro is dependent upon cell-cell contact; however, the cell surface molecules mediating this cell:cell contact have not yet been identified. LFA-1 (CD11a/CD18) is an adhesion molecule that plays an established role in T cell-mediated cell contact and in T cell activation. Although expressed at high levels on murine CD4+CD25+ Treg cells, the role of LFA-1 in these cells has not been defined previously. We hypothesized that LFA-1 may play a role in murine CD4+CD25+ Treg function. To evaluate this, we analyzed LFA-1-deficient (CD18-/-) CD4+CD25+ T cells. We show that CD18-/- mice demonstrate a propensity to autoimmunity. Absence of CD18 led to diminished CD4+CD25+ T cell numbers and affected both thymic and peripheral development of these cells. LFA-1-deficient CD4+CD25+ T cells were deficient in mediating suppression in vitro and in mediating protection from colitis induced by the transfer of CD4+CD25- T cells into lymphopenic hosts. Therefore, we define a crucial role for CD18 in optimal CD4+CD25+ Treg development and function.     

IL-4 modulation of CD4+CD25+ T regulatory cell-mediated suppression

Murine CD4(+)CD25(+) T regulatory (Treg) cells were cocultured with CD4(+)CD25(-) Th cells and APCs or purified B cells and stimulated by anti-CD3 mAb. Replacement of APCs by B cells did not significantly affect the suppression of CD4(+)CD25(-) Th cells. When IL-4 was added to separate cell populations, this cytokine promoted CD4(+)CD25(-) Th and CD4(+)CD25(+) Treg cell proliferation, whereas the suppressive competence of CD4(+)CD25(+) Treg cells was preserved. Conversely, IL-4 added to coculture of APCs, CD4(+)CD25(-) Th cells, and CD4(+)CD25(+) Treg cells inhibited the suppression of CD4(+)CD25(-) Th cells by favoring their survival through the induction of Bcl-2 expression. At variance, suppression was not affected by addition of IL-13, although this cytokine shares with IL-4 a receptor chain. When naive CD4(+)CD25(-) Th cells were replaced by Th1 and Th2 cells, cell proliferation of both subsets was equally suppressed, but suppression was less pronounced compared with that of CD4(+)CD25(-) Th cells. IL-4 production by Th2 cells was also inhibited. These results indicate that although CD4(+)CD25(+) Treg cells inhibit IL-4 production, the addition of IL-4 counteracts CD4(+)CD25(+) Treg cell-mediated suppression by promoting CD4(+)CD25(-) Th cell survival and proliferation.     

Cutting edge: IL-4-induced protection of CD4+CD25- Th cells from CD4+CD25+ regulatory T cell-mediated suppression

CD4(+)CD25(+) T regulatory (Treg) cells are a CD4(+) T cell subset involved in the control of the immune response. In vitro, murine CD4(+)CD25(+) Treg cells inhibit CD4(+)CD25(-) Th cell proliferation induced by anti-CD3 mAb in the presence of APCs. The addition of IL-4 to cocultured cells inhibits CD4(+)CD25(+) Treg cell-mediated suppression. Since all cell types used in the coculture express the IL-4Ralpha chain, we used different combinations of CD4(+)CD25(-) Th cells, CD4(+)CD25(+) Treg cells, and APCs from wild-type IL-4Ralpha(+/+) or knockout IL-4Ralpha(-/-) mice. Results show that the engagement of the IL-4Ralpha chain on CD4(+)CD25(-) Th cells renders these cells resistant to suppression. Moreover, the addition of IL-4 promotes proliferation of IL-4Ralpha(+/+)CD4(+)CD25(+) Treg cells, which preserve full suppressive competence. These findings support an essential role of IL-4 signaling for CD4(+)CD25(-) Th cell activation and indicate that IL-4-induced proliferation of CD4(+)CD25(+) Treg cells is compatible with their suppressive activity.     

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.     

Lipopolysaccharide-activated CD4+CD25+ T regulatory cells inhibit neutrophil function and promote their apoptosis and death

CD4+CD25+ T regulatory (Treg) cells play a central role in the suppression of immune response and prevention of autoimmune reactions. Pathogen recognition receptors expressed by immune cells, such as TLRs, may provide a critical link between the innate and adaptive immune systems. There is also evidence that TLR ligands can directly modulate the suppressive capacity of Treg cells. Here, we showed that CD4+CD25+ Treg cells affect neutrophil function and survival and that the TLR4 ligand is involved in the regulation of the cell interactions. We found that LPS-activated Treg cells inhibit reactive oxygen intermediates and cytokine production by neutrophils. Moreover, Treg cells reverse LPS-induced survival of neutrophils and promote their apoptosis and death. We also found that TCR-activated Treg cells induce the same effects on polymorphonuclear neutrophils as those achieved by TLR4 stimulation. Importantly, the suppressive potential of CD4+CD25+ Treg cells induced by LPS seems to be partially IL-10 and TGF-beta dependent, whereas anti-CD3/CD28 stimulation is rather contact dependent. Together, these observations suggest that Treg cells have the ability to directly regulate neutrophil function and life span when both types of the cells are exposed to LPS.     

4-1BB Signaling in Conventional T Cells Drives IL-2 Production That Overcomes CD4+CD25+FoxP3+ T Regulatory Cell Suppression

Costimulation with the recombinant SA-4-1BBL agonist of 4-1BB receptor on conventional CD4⁺ T cells (Tconvs) overcomes the suppression mediated by naturally occurring CD4⁺CD25⁺FoxP3⁺ T regulatory cells (Tregs). The mechanistic basis of this observation has remained largely unknown. Herein we show that Tconvs, but not Tregs, are the direct target of SA-4-1BBL-mediated evasion of Treg suppression. IL-2 produced by Tconvs in response to 4-1BB signaling is both necessary and sufficient for overcoming Treg suppression. Supernatant from Tconvs stimulated with SA-4-1BBL contains high levels of IL-2 and overcomes Treg suppression in ex vivo Tconv:Treg cocultures. Removal of IL-2 from such supernatant restores Treg suppression and repletion of Tconv:Treg cocultures with exogenous recombinant IL-2 overcomes suppression. This study establishes 4-1BB signaling as a key circuit that regulates physical and functional equilibrium between Tregs and Tconvs with important implications for immunotherapy for indications where a fine balance between Tregs and Teffs plays a decisive role.     

The majority of human peripheral blood CD4+CD25highFoxp3+ regulatory T cells bear functional skin-homing receptors

CD4+CD25+ T regulatory cells (Treg) are thought to be important in the peripheral tolerance. Recent evidence suggests that human peripheral blood CD4+CD25+ T cells are heterogeneous and contain both CD4+CD25(high) T cells with potent regulatory activity and many more CD4+CD25(low/med) nonregulatory T cells. In this study, we found that virtually all peripheral blood CD4+CD25(high)Foxp3+ Treg expressed high levels of the chemokine receptor CCR4. In addition, 80% of Treg expressed cutaneous lymphocyte Ag (CLA) and 73% expressed CCR6. These molecules were functional, as CLA+ Treg showed CD62E ligand activity and demonstrable chemotactic responses to the CCR4 ligands CCL22 and CCL17 and to the CCR6 ligand CCL20. The phenotype and chemotactic response of these Treg were significantly different from those of CD4+CD25(med) nonregulatory T cells. We further demonstrated that blood CLA+ Treg inhibited CD4+CD25- T cell proliferation induced by anti-CD3. Based on homing receptor profile, CLA+ Treg should enter normal skin. We next isolated CD4+CD25(high) T cells directly from normal human skin; these cells suppressed proliferation of skin CD4+CD25- T cells. Therefore, the majority of true circulating Treg express functional skin-homing receptors, and human Treg may regulate local immune responses in normal human skin.     

CD4+ CD25+ regulatory T cells inhibit the maturation but not the initiation of an autoantibody response

To investigate the mechanism by which T regulatory (Treg) cells may control the early onset of autoimmunity, we have used an adoptive transfer model to track Treg, Th, and anti-chromatin B cell interactions in vivo. We show that anti-chromatin B cells secrete Abs by day 8 in vivo upon provision of undeviated, Th1- or Th2-type CD4+ T cell help, but this secretion is blocked by the coinjection of CD4+ CD25+ Treg cells. Although Treg cells do not interfere with the initial follicular entry or activation of Th or B cells at day 3, ICOS levels on Th cells are decreased. Furthermore, Treg cells must be administered during the initial phases of the Ab response to exert full suppression of autoantibody production. These studies indicate that CD25+ Treg cells act to inhibit the maturation, rather than the initiation, of autoantibody responses.     

CD39 is highly involved in mediating the suppression activity of tumor-infiltrating CD8+ T regulatory lymphocytes

CD39 is an ectoenzyme, present on different immune cell subsets, which mediates immunosuppressive functions catalyzing ATP degradation. It is not known whether CD39 is expressed and implicated in the activity of CD8+ regulatory T lymphocytes (Treg). In this study, CD39 expression and function was analyzed in both CD8+ and CD4+CD25^hi Treg from the peripheral blood of healthy donors as well as from tumor specimens. CD39 was found expressed by both CD8+ (from the majority of healthy donors and tumor patients) and CD4+CD25^hi Treg, and CD39 expression correlated with suppression activity mediated by CD8+ Treg. Importantly, CD39 counteraction remarkably inhibited the suppression activity of CD8+ Treg (both from peripheral blood and tumor microenvironment) suggesting that CD39-mediated inhibition constitutes a prevalent hallmark of their function. Collectively, these findings, unveiling a new mechanism of action for CD8+ Treg, provide new knowledge on intratumoral molecular pathways related to tumor immune escape, which could be exploited in the future for designing new biological tools for anticancer immune intervention.     

CD4+CD25+调节性T细胞

调节性T细胞(regulatory T cells,Treg)是机体维持自身耐受的重要组成部分。CD4^ CD25^ Treg细胞来源于胸腺,其主要功能是抑制自身反应性T细胞,并且其作用是通过直接的Treg-T效应细胞之间的相互接触方式来实现的。CD4^ CD25^ Treg细胞可分泌多种抑制性细胞因子,但与其抑制功能关系并不明确,目前有证据表明GITR和Foxp3与CD4^ CD25^ Treg细胞的抑制功能有关,并且Foxp3已作为CD4^ CD25^ Treg细胞的特异性标志。通过IL-10、TGF-β等抑制性细胞因子、imDC以及转基因技术可以产生具有免疫抑制功能的调节性T细胞。调节性T细胞在免疫相关性疾病、肿瘤免疫和抗感染免疫等方面具有重要意义。     

Cutting edge: TNFR-shedding by CD4+CD25+ regulatory T cells inhibits the induction of inflammatory mediators

CD4+CD25+ regulatory T (Treg) cells play an essential role in maintaining tolerance to self and nonself. In several models of T cell-mediated (auto) immunity, Treg cells exert protective effects by the inhibition of pathogenic T cell responses. In addition, Treg cells can modulate T cell-independent inflammation. We now show that CD4+CD25+ Treg cells are able to shed large amounts of TNFRII. This is paralleled by their ability to inhibit the action of TNF-alpha both in vitro and in vivo. In vivo, Treg cells suppressed IL-6 production in response to LPS injection in mice. In contrast, Treg cells from TNFRII-deficient mice were unable to do so despite their unhampered capacity to suppress T cell proliferation in a conventional in vitro suppression assay. Thus, shedding of TNFRII represents a novel mechanism by which Treg cells can inhibit the action of TNF, a pivotal cytokine driving inflammation.     

Cutting edge: size and diversity of CD4+CD25high Foxp3+ regulatory T cell repertoire in humans: evidence for similarities and partial overlapping with CD4+CD25- T cells

Both differentiation and function of CD4+CD25(high) naturally arising regulatory T cells (Treg), which play a key role in the control of autoimmunity, are thought to depend on TCR specificity. In the present study, we comparatively measured the alphabetaTCR repertoire sizes of human peripheral blood Treg and CD4+CD25- T cells by using a methodology based on PCR amplification and sequencing analysis. We show that Treg use a large unrestricted alphabeta TCR repertoire, the size and diversity of which are closely similar to those of CD4+CD25- T cells, with a mean estimated size of 3.5 x 10(6) distinct alphabeta TCR vs 4.7 x 10(6) distinct alphabetaTCR for CD4+CD25- T cells. In addition, a 24% overlap between the repertoires of these two CD4+ subsets in the periphery is found. These data emphasize the intersection between naturally occurring Treg and effector T cell peripheral repertoires and provide new insights into the ontogeny of Treg in humans.     

Protection from type 1 diabetes by invariant NK T cells requires the activity of CD4+CD25+ regulatory T cells

Invariant NK T (iNKT) cells regulate immune responses, express NK cell markers and an invariant TCR, and recognize lipid Ags in a CD1d-restricted manner. Previously, we reported that activation of iNKT cells by alpha-galactosylceramide (alpha-GalCer) protects against type 1 diabetes (T1D) in NOD mice via an IL-4-dependent mechanism. To further investigate how iNKT cells protect from T1D, we analyzed whether iNKT cells require the presence of another subset(s) of regulatory T cells (Treg), such as CD4+ CD25+ Treg, for this protection. We found that CD4+ CD25+ T cells from NOD.CD1d(-/-) mice deficient in iNKT cell function similarly in vitro to CD4+ CD25+ T cells from wild-type NOD mice and suppress the proliferation of NOD T responder cells upon alpha-GalCer stimulation. Cotransfer of NOD diabetogenic T cells with CD4+ CD25+ Tregs from NOD mice pretreated with alpha-GalCer demonstrated that activated iNKT cells do not influence the ability of T(regs) to inhibit the transfer of T1D. In contrast, protection from T1D mediated by transfer of activated iNKT cells requires the activity of CD4+ CD25+ T cells, because splenocytes pretreated with alpha-GalCer and then inactivated by anti-CD25 of CD25+ cells did not protect from T1D. Similarly, mice inactivated of CD4+ CD25+ T cells before alpha-GalCer treatment were also not protected from T1D. Our data suggest that CD4+ CD25+ T cells retain their function during iNKT cell activation, and that the activity of CD4+ CD25+ Tregs is required for iNKT cells to transfer protection from T1D.     

A function for IL-7R for CD4+CD25+Foxp3+ T regulatory cells

The IL-2/IL-2R interaction is important for development and peripheral homeostasis of T regulatory (Treg) cells. IL-2- and IL-2R-deficient mice are not completely devoid of Foxp3+ cells, but rather lack population of mature CD4+CD25+Foxp3high Treg cells and contain few immature CD4+CD25-Foxp3low T cells. Interestingly, common gamma chain (gammac) knockout mice have been shown to have a near complete absence of Foxp3+ Treg cells, including the immature CD25-Foxp3low subset. Therefore, other gammac-cytokine(s) must be critically important during thymic development of CD4+CD25+Foxp3+ Treg cells apart from the IL-2. The present study was undertaken to determine whether the gammac-cytokines IL-7 or IL-15 normally contribute to expression of Foxp3 and Treg cell production. These studies revealed that mice double deficient in IL-2Rbeta and IL-7Ralpha contained a striking lack in the CD4+Foxp3+ population and the Treg cell defect recapitulated the gammac knockout mice. In the absence of IL-7R signaling, IL-15/IL-15R interaction is dispensable for the production of CD4+CD25+Foxp3+ Treg cells, indicating that normal thymic Treg cell production likely depends on signaling through both IL-2 and IL-7 receptors. Selective thymic reconstitution of IL-2Rbeta in mice double deficient in IL-2Rbeta and IL-7Ralpha established that IL-2Rbeta is dominant and sufficient to restore production of Treg cells. Furthermore, the survival of peripheral CD4+Foxp3low cells in IL-2Rbeta-/- mice appears to depend upon IL-7R signaling. Collectively, these data indicate that IL-7R signaling contributes to Treg cell development and peripheral homeostasis.     

Human CD4+ T cells express TLR5 and its ligand flagellin enhances the suppressive capacity and expression of FOXP3 in CD4+CD25+ T regulatory cells

Germline encoded pattern recognition receptors, such as TLRs, provide a critical link between the innate and adaptive immune systems. There is also evidence to suggest that pathogen-associated molecular patterns may have the capacity to modulate immune responses via direct effects on CD4+ T cells. Given the key role of both CD4+CD25+ T regulatory (Treg) cells and the TLR5 ligand flagellin in regulating mucosal immune responses, we investigated whether TLR5 may directly influence T cell function. We found that both human CD4+CD25+ Treg and CD4+CD25- T cells express TLR5 at levels comparable to those on monocytes and dendritic cells. Costimulation of effector T cells with anti-CD3 and flagellin resulted in enhanced proliferation and production of IL-2, at levels equivalent to those achieved by costimulation with CD28. In contrast, costimulation with flagellin did not break the hyporesponsiveness of CD4+CD25+ Treg cells, but rather, potently increased their suppressive capacity and enhanced expression of FOXP3. These observations suggest that, in addition to their APC-mediated indirect effects, TLR ligands have the capacity to directly regulate T cell responses and modulate the suppressive activity of Treg cells.     

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.     

Ex vivo expansion of CD4+CD25+FoxP3+ T regulatory cells based on synergy between IL-2 and 4-1BB signaling

Naturally occurring CD4(+)CD25(+)FoxP3(+) T regulatory (Treg) cells require three distinct signals transduced via TCR, CD28, and IL-2R for their development and maintenance. These requirements served as the basis for several recently developed ex vivo expansion protocols that relied on the use of solid support-bound Abs to CD3 and CD28 in the presence of high dose IL-2. We report in this study that Treg cells up-regulate the expression of inducible costimulatory receptor 4-1BB in response to IL-2, and stimulation using this receptor via a novel form of 4-1BB ligand (4-1BBL) fused to a modified form of core streptavidin (SA-4-1BBL) was effective in expanding these cells up to 110-fold within 3 wk. Expanded cells up-regulated CD25, 4-1BB, and membranous TGF-beta, suppressed T cell proliferation, and prevented the rejection of allogeneic islets upon adoptive transfer into graft recipients. Importantly, SA-4-1BBL rendered CD4(+)CD25(-) T effector cells refractive to suppression by Treg cells. This dual function of signaling via 4-1BB, vis-à-vis Treg cell expansion and licensing T effector cells resistant to Treg cell suppression, as well as the up-regulation of 4-1BB by IL-2 may serve as important regulatory mechanisms for immune homeostasis following antigenic challenge. Stimulation using a soluble form of SA-4-1BBL represents a novel approach to expand Treg cells with potential therapeutic applications in autoimmunity and transplantation.