Cutting edge: CD28 controls peripheral homeostasis of CD4+CD25+ regulatory T cells

CD28/B7 blockade leads to exacerbated autoimmune disease in the nonobese diabetic mouse strain as a result of a marked reduction in the number of CD4(+)CD25(+) regulatory T cells (Tregs). Herein, we demonstrate that CD28 controls both thymic development and peripheral homeostasis of Tregs. CD28 maintains a stable pool of peripheral Tregs by both supporting their survival and promoting their self-renewal. CD28 engagement promotes survival by regulating IL-2 production by conventional T cells and CD25 expression on Tregs.     

CD4+CD25+ regulatory T cells in HIV infection

The immune system faces the difficult task of discerning between foreign, potentially pathogen-derived antigens and self-antigens. Several mechanisms, including deletion of self-reactive T cells in the thymus, have been shown to contribute to the acceptance of self-antigens and the reciprocal reactivity to foreign antigens. Over the last decade it has become increasingly clear that CD4(+)CD25(+) T(Reg) cells are crucial for maintenance of T cell tolerance to self-antigens in the periphery, and to avoid development of autoimmune disorders. Recently, evidence has also emerged that demonstrates that CD4(+)CD25(+) T(Reg) cells can also suppress T cell responses to foreign pathogens, including viruses such as HIV. In this article we review the current knowledge and potential role of CD4(+)CD25(+) T(Reg) cells in HIV infection.     

Distinct effects of STAT5 activation on CD4+ and CD8+ T cell homeostasis: development of CD4+CD25+ regulatory T cells versus CD8+ memory T cells

Using transgenic mice that express a constitutively active version of STAT5b, we demonstrate that STAT5 plays a key role in governing B cell development and T cell homeostasis. STAT5 activation leads to a 10-fold increase in pro-B, but not pro-T, cells. Conversely, STAT5 signaling promotes the expansion of mature alphabeta T cells (6-fold increase) and gammadelta and NK T cells (3- to 4-fold increase), but not of mature B cells. In addition, STAT5 activation has dramatically divergent effects on CD8(+) vs CD4(+) T cells, leading to the selective expansion of CD8(+) memory-like T cells and CD4(+)CD25(+) regulatory T cells. These results establish that activation of STAT5 is the primary mechanism underlying both IL-7/IL-15-dependent homeostatic proliferation of naive and memory CD8(+) T cells and IL-2-dependent development of CD4(+)CD25(+) regulatory T cells.     

Antigen-specific T cell repertoire modification of CD4+CD25+ regulatory T cells

T cell immune responses are regulated by the interplay between effector and suppressor T cells. Immunization with Ag leads to the selective expansion and survival of effector CD4(+) T cells with high affinity TCR against the Ag and MHC. However, it is not known if CD4(+)CD25(+) regulatory T cells (T(reg)) recognize the same Ag as effector T cells or whether Ag-specific TCR repertoire modification occurs in T(reg). In this study, we demonstrate that after a primary Ag challenge, T(reg) proliferate and TCR repertoire modification is observed although both of these responses were lower than those in conventional T cells. The repertoire modification of Ag-specific T(reg) after primary Ag challenge augmented the total suppressive function of T(reg) against TCR repertoire modification but not against the proliferation of memory CD4(+) T cells. These results reveal that T cell repertoire modification against a non-self Ag occurs in T(reg), which would be crucial for limiting excess primary and memory CD4(+) T cell responses. In addition, these studies provide evidence that manipulation of Ag-specific T(reg) is an ideal strategy for the clinical use of T(reg).     

Distinct IL-2 receptor signaling pattern in CD4+CD25+ regulatory T cells

Despite expression of the high-affinity IL-2R, CD4(+)CD25(+) regulatory T cells (Tregs) are hypoproliferative upon IL-2R stimulation in vitro. However the mechanisms by which CD4(+)CD25(+) T cells respond to IL-2 signals are undefined. In this report, we examine the cellular and molecular responses of CD4(+)CD25(+) Tregs to IL-2. IL-2R stimulation results in a G(1) cell cycle arrest, cellular enlargement and increased cellular survival of CD4(+)CD25(+) T cells. We find a distinct pattern of IL-2R signaling in which the Janus kinase/STAT pathway remains intact, whereas IL-2 does not activate downstream targets of phosphatidylinositol 3-kinase. Negative regulation of phosphatidylinositol 3-kinase signaling and IL-2-mediated proliferation of CD4(+)CD25(+) T cells is inversely associated with expression of the phosphatase and tensin homologue deleted on chromosome 10, PTEN.     

CD4+ CD25+ regulatory T cells impair HIV-1-specific CD4 T cell responses by upregulating interleukin-10 production in monocytes

T cell dysfunction in the presence of ongoing antigen exposure is a cardinal feature of chronic viral infections with persistent high viremia, including HIV-1. Although interleukin-10 (IL-10) has been implicated as an important mediator of this T cell dysfunction, the regulation of IL-10 production in chronic HIV-1 infection remains poorly understood. We demonstrated that IL-10 is elevated in the plasma of individuals with chronic HIV-1 infection and that blockade of IL-10 signaling results in a restoration of HIV-1-specific CD4 T cell proliferation, gamma interferon (IFN-γ) secretion, and, to a lesser extent, IL-2 production. Whereas IL-10 blockade leads to restoration of IFN-γ secretion by HIV-1-specific CD4 T cells in all categories of subjects investigated, significant enhancement of IL-2 production and improved proliferation of CD4 T helper cells are restricted to viremic individuals. In peripheral blood mononuclear cells (PBMCs), this IL-10 is produced primarily by CD14(+) monocytes, but its production is tightly controlled by regulatory T cells (Tregs), which produce little IL-10 directly. When Tregs are depleted from PBMCs of viremic individuals, the effect of the IL-10 signaling blockade is abolished and IL-10 production by monocytes decreases, while the production of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), increases. The regulation of IL-10 by Tregs appears to be mediated primarily by contact or paracrine-dependent mechanisms which involve IL-27. This work describes a novel mechanism by which regulatory T cells control IL-10 production and contribute to dysfunctional HIV-1-specific CD4 T cell help in chronic HIV-1 infection and provides a unique mechanistic insight into the role of regulatory T cells in immune exhaustion.     

Influence of CD4+CD25+ regulatory T cells on low/high-avidity CD4+ T cells following peptide vaccination

We have recently reported that NY-ESO-1-specific naive CD4+ T cell precursors exist in most individuals but are suppressed by CD4+CD25+ regulatory T cells (Tregs), while memory CD4+ T cell effectors against NY-ESO-1 are found only in cancer patients with spontaneous Ab responses to NY-ESO-1. In this study, we have analyzed mechanisms of CD4+ T cell induction following peptide vaccination in relation to susceptibility to Tregs. Specific HLA-DP4-restricted CD4+ T cell responses were elicited after vaccination with NY-ESO-1(157-170) peptide (emulsified in IFA) in patients with NY-ESO-1-expressing epithelial ovarian cancer. These vaccine-induced CD4+ T cells were detectable from effector/memory populations without requirement for in vitro CD4+CD25+ T cell depletion. However, they were only able to recognize NY-ESO-1(157-170) peptide but not naturally processed NY-ESO-1 protein and had much lower avidity compared with NY-ESO-1-specific pre-existing naive CD4+CD25- T cell precursors or spontaneously induced CD4+ T cell effectors of cancer patients with NY-ESO-1 Ab. We propose that vaccination with NY-ESO-1(157-170) peptide recruits low-avidity T cells with low sensitivity to Tregs and fails to modulate the suppressive effect of Tregs on high-avidity NY-ESO-1-specific T cell precursors.     

Therapeutic potential of CD4+ CD25+ regulatory T cells in allogeneic transplantation

The subpopulation of CD4+ CD25+ immunoregulatory T cells constitutes less than of the entire CD4+ T-cell pool in mice and 2% in humans. These cells play a crucial role in the control of autoimmune processes. More recently, in vitro and in vivo data also indicate that CD4+ CD25+ immunoregulatory T cells can regulate alloreactivity. This renders them good candidates for innovative strategies in the field of transplantation. Inducing a state of immune tolerance with immunoregulatory T cells would alleviate the need for immunosuppression, and the occurrence of late allograft failure represents a major goal of transplantation immunology. Here we discuss how these naturally occurring CD4+ CD25+ immunoregulatory T cells can be used to modulate alloreactivity in hematopoietic stem cell and solid organ transplantation.     

CD4+CD25+ T regulatory cells suppress NK cell-mediated immunotherapy of cancer

CD4+CD25+ regulatory T cells (Treg) that suppress T cell-mediated immune responses may also regulate other arms of an effective immune response. In particular, in this study we show that Treg directly inhibit NKG2D-mediated NK cell cytotoxicity in vitro and in vivo, effectively suppressing NK cell-mediated tumor rejection. In vitro, Treg were shown to inhibit NKG2D-mediated cytolysis largely by a TGF-beta-dependent mechanism and independently of IL-10. Adoptively transferred Treg suppressed NK cell antimetastatic function in RAG-1-deficient mice. Depletion of Treg before NK cell activation via NKG2D and the activating IL-12 cytokine, dramatically enhanced NK cell-mediated suppression of tumor growth and metastases. Our data illustrate at least one mechanism by which Treg can suppress NK cell antitumor activity and highlight the effectiveness of combining Treg inhibition with subsequent NK cell activation to promote strong innate antitumor immunity.     

Human CD4+CD25+ regulatory T cells share equally complex and comparable repertoires with CD4+CD25- counterparts

CD4(+)CD25(+) T cells are critical mediators of peripheral immune tolerance. However, many developmental and functional characteristics of these cells are unknown, and knowledge of human regulatory T cells is particularly limited. To better understand how human CD4(+)CD25(+) T cells develop and function, we examined the diversity of CD4(+)CD25(+) and CD4(+)CD25(-) T cell repertoires in both thymus and peripheral blood. Levels of T receptor excision circles (TREC) were comparable in purified CD4(+)CD25(+) and CD4(+)CD25(-) thymic populations, but were significantly higher than those in samples derived from peripheral blood, consistent with murine studies demonstrating thymic development of CD4(+)CD25(+) regulatory T cells. Surprisingly, CD4(+)CD25(-) T cells isolated from peripheral blood had greater TREC quantities than their CD4(+)CD25(+) counterparts, supporting the possibility of extrathymic expansion as well. CD4(+)CD25(+) and CD4(+)CD25(-) T cells from a given individual showed overlapping profiles with respect to diversity by Vbeta staining and spectratyping. Interestingly, CD4(+)CD25(+) T cells have lower quantities of CD3 than CD4(+)CD25(-) T cells. Collectively, these data suggest that human CD4(+)CD25(+) T cells recognize a similar array of Ags as CD4(+)CD25(-) T cells. However, reduced levels of TCR on regulatory T cells suggest different requirements for activation and may contribute to how the immune system regulates whether a particular response is suppressed or augmented.     

IL-12R beta 2 promotes the development of CD4+CD25+ regulatory T cells

We have previously shown that mice lacking the IL-12-specific receptor subunit beta2 (IL-12Rbeta2) develop more severe experimental autoimmune encephalomyelitis than wild-type (WT) mice. The mechanism underlying this phenomenon is not known; nor is it known whether deficiency of IL-12Rbeta2 impacts other autoimmune disorders similarly. In the present study we demonstrate that IL-12Rbeta2(-/-) mice develop earlier onset and more severe disease in the streptozotocin-induced model of diabetes, indicating predisposition of IL-12Rbeta2-deficient mice to autoimmune diseases. T cells from IL-12Rbeta2(-/-) mice exhibited significantly higher proliferative responses upon TCR stimulation. The numbers of naturally occurring CD25(+)CD4(+) regulatory T cells (Tregs) in the thymus and spleen of IL-12Rbeta2(-/-) mice were comparable to those of WT mice. However, IL-12Rbeta2(-/-) mice exhibited a significantly reduced capacity to develop Tregs upon stimulation with TGF-beta, as shown by significantly lower numbers of CD25(+)CD4(+) T cells that expressed Foxp3. Functionally, CD25(+)CD4(+) Tregs derived from IL-12Rbeta2(-/-) mice were less efficient than those from WT mice in suppressing effector T cells. The role of IL-12Rbeta2 in the induction of Tregs was confirmed using small interfering RNA. These findings suggest that signaling via IL-12Rbeta2 regulates both the number and functional maturity of Treg cells, which indicates a novel mechanism underlying the regulation of autoimmune diseases by the IL-12 pathway.     

CD4+CD25+ regulatory T cells control innate immune reactivity after injury

Major injury initiates a systemic inflammatory response that can be detrimental to the host. We have recently reported that burn injury primes innate immune cells for a progressive increase in TLR4 and TLR2 agonist-induced proinflammatory cytokine production and that this inflammatory phenotype is exaggerated in adaptive immune system-deficient (Rag1(-/-)) mice. The present study uses a series of adoptive transfer experiments to determine which adaptive immune cell type(s) has the capacity to control innate inflammatory responses after injury. We first compared the relative changes in TLR4- and TLR2-induced TNF-alpha, IL-1beta, and IL-6 production by spleen cell populations prepared from wild-type (WT), Rag1(-/-), CD4(-/-), or CD8(-/-) mice 7 days after sham or burn injury. Our findings indicated that splenocytes prepared from burn-injured CD8(-/-) mice displayed TLR-induced cytokine production levels similar to those in WT mice. In contrast, spleen cells from burn-injured CD4(-/-) mice produced cytokines at significantly higher levels, equivalent to those in Rag1(-/-) mice. Moreover, reconstitution of Rag1(-/-) or CD4(-/-) mice with WT CD4(+) T cells reduced postinjury cytokine production to WT levels. Additional separation of CD4(+) T cells into CD4(+)CD25(+) and CD4(+)CD25(-) subpopulations before their adoptive transfer into Rag1(-/-) mice showed that CD4(+)CD25(+) T cells were capable of reducing TLR-stimulated cytokine production levels to WT levels, whereas CD4(+)CD25(-) T cells had no regulatory effect. These findings suggest a previously unsuspected role for CD4(+)CD25(+) T regulatory cells in controlling host inflammatory responses after injury.     

Interleukin-7 influences FOXP3+CD4+ regulatory T cells peripheral homeostasis

Mechanisms governing peripheral CD4+ FOXP3+ regulatory T cells (Treg) survival and homeostasis are multiple suggesting tight and complex regulation of regulatory T cells homeostasis. Some specific factors, such as TGF-β, interleukin-2 (IL-2) and B7 costimulatory molecules have been identified as essentials for maintenance of the peripheral Treg compartment. Conversely, Treg dependency upon classical T cell homeostatic factors such as IL-7 is still unclear. In this work, we formally investigated the role of IL-7 in Treg homeostasis in vivo in murine models. We demonstrated that IL-7 availability regulated the size of peripheral Treg cell pool and thus paralleled the impact of IL-7 on conventional T cell pool. Moreover, we showed that IL-7 administration increased Treg cell numbers by inducing thymic-independent Treg peripheral expansion. Importantly the impact of IL-7 on Treg expansion was detected whether conventional T cells were present or absent as IL-7 directly participates to the peripheral expansion of Treg after adoptive transfer into lymphopenic hosts. Our results definitively identify IL-7 as a central factor contributing to Treg peripheral homeostasis, thus reassembling Treg to other T cell subsets in respect of their need for IL-7 for their peripheral maintenance.     

Natural regulatory CD4 T cells expressing CD25

In normal mice, a subpopulation of CD4 T cells constitutively express CD25. These cells behave as regulatory T cells in autoimmune and inflammatory reactions, in tolerance to superantigens, and in peripheral T-cell homeostasis. They are unable to produce interleukin (IL)-2, and are dependent on IL-2 for growth in vitro and in vivo. CD4 CD25(+) T cells spontaneously secrete IL-10, which is involved in some of their regulatory functions. They are resistant to apoptosis, but can be tolerized by anergy.     

CD4+CD25+ regulatory T cells inhibit the antigen-dependent expansion of self-reactive T cells in vivo

A deficiency of CD4+CD25+ regulatory T cells (CD25+ Tregs) in lymphopenic mice can result in the onset of autoimmune gastritis. The gastric H/K ATPase alpha (H/Kalpha) and beta (H/Kbeta) subunits are the immunodominant autoantigens recognized by effector CD4+ T cells in autoimmune gastritis. The mechanism by which CD25+ Tregs suppress autoimmune gastritis in lymphopenic mice is poorly understood. To investigate the antigenic requirements for the genesis and survival of gastritis-protecting CD25+ Tregs, we analyzed mice deficient in H/Kbeta and H/Kalpha, as well as a transgenic mouse line (H/Kbeta-tsA58 Tg line 224) that lacks differentiated gastric epithelial cells. By adoptive transfer of purified T cell populations to athymic mice, we show that the CD25+ Treg population from mice deficient in either one or both of H/Kalpha and H/Kbeta, or from the H/Kbeta-tsA58 Tg line 224 mice, is equally effective in suppressing the ability of polyclonal populations of effector CD4+ T cells to induce autoimmune gastritis. Furthermore, CD25+ Tregs, from either wild-type or H/Kalpha-deficient mice, dramatically reduced the expansion of pathogenic H/Kalpha-specific TCR transgenic T cells and the induction of autoimmune gastritis in athymic recipient mice. Proliferation of H/Kalpha-specific T cells in lymphopenic hosts occurs predominantly in the paragastric lymph node and was dependent on the presence of the cognate H/Kalpha Ag. Collectively, these studies demonstrate that the gastritis-protecting CD25+ Tregs do not depend on the major gastric Ags for their thymic development or their survival in the periphery, and that CD25+ Tregs inhibit the Ag-specific expansion of pathogenic T cells in vivo.     

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.