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.     

G protein-coupled receptor 83 overexpression in naive CD4+CD25- T cells leads to the induction of Foxp3+ regulatory T cells in vivo

Foxp3 functions as a lineage specification factor for the development of naturally occurring thymus-derived CD4+CD25+ regulatory T (Treg) cells. Recent evidence suggests that naive Foxp3-CD4+CD25- T cells can be converted in the periphery into Foxp3+ Treg cells. In this study, we have identified the G protein-coupled receptor (GPR)83 to be selectively up-regulated by CD4+CD25+ Treg cells of both murine and human origin in contrast to naive CD4+CD25- or recently activated T cells. Furthermore, GPR83 was induced upon overexpression of Foxp3 in naive CD4+CD25- T cells. Transduction of naive CD4+CD25- T cells with GPR83-encoding retroviruses did not confer in vitro suppressive activity. Nevertheless, GPR83-transduced T cells were able to inhibit the effector phase of a severe contact hypersensitivity reaction of the skin, indicating that GPR83 itself or GPR83-mediated signals conferred suppressive activity to conventional CD4+ T cells in vivo. Most strikingly, this in vivo acquisition of suppressive activity was associated with the induction of Foxp3 expression in GPR83-transduced CD4+ T cells under inflammatory conditions. Our results suggest that GPR83 might be critically involved in the peripheral generation of Foxp3+ Treg cells in vivo.     

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.     

CD8+ CD205+ splenic dendritic cells are specialized to induce Foxp3+ regulatory T cells

Foxp3(+)CD25(+)CD4(+) regulatory T cells (Treg) mediate immunological self-tolerance and suppress immune responses. A subset of dendritic cells (DCs) in the intestine is specialized to induce Treg in a TGF-beta- and retinoic acid-dependent manner to allow for oral tolerance. In this study we compare two major DC subsets from mouse spleen. We find that CD8(+) DEC-205/CD205(+) DCs, but not the major fraction of CD8(-) DC inhibitory receptor-2 (DCIR2)(+) DCs, induce functional Foxp3(+) Treg from Foxp3(-) precursors in the presence of low doses of Ag but without added TGF-beta. CD8(+)CD205(+) DCs preferentially express TGF-beta, and the induction of Treg by these DCs in vitro is blocked by neutralizing Ab to TGF-beta. In contrast, CD8(-)DCIR2(+) DCs better induce Foxp3(+) Treg when exogenous TGF-beta is supplied. In vivo, CD8(+)CD205(+) DCs likewise preferentially induce Treg from adoptively transferred, Ag-specific DO11.10 RAG(-/-) Foxp3(-)CD4(+) T cells, whereas the CD8(-)DCIR2(+) DCs better stimulate natural Foxp3(+) Treg. These results indicate that a subset of DCs in spleen, a systemic lymphoid organ, is specialized to differentiate peripheral Foxp3(+) Treg, in part through the endogenous formation of TGF-beta. Targeting of Ag to these DCs might be useful for inducing Ag-specific Foxp3(+) Treg for treatment of autoimmune diseases, transplant rejection, and allergy.     

Experience-driven development: effector/memory-like alphaE+Foxp3+ regulatory T cells originate from both naive T cells and naturally occurring naive-like regulatory T cells

Naturally occurring Foxp3+CD25+CD4+ regulatory T cells (Treg) have initially been described as anergic cells; however, more recent in vivo studies suggest that Tregs vigorously proliferate under both homeostatic as well as inflammatory conditions. We have previously identified a subset of murine CD4+ Tregs, which is characterized by expression of the integrin alphaEbeta7 and which displays an effector/memory-like phenotype indicative of Ag-specific expansion and differentiation. In the present study, the alphaE+ Treg subset was found to contain a large fraction of cycling cells under homeostatic conditions in healthy mice. Using an adoptive transfer system of Ag-specific T cells, we could demonstrate that the vast majority of transferred natural, naive-like CD25+CD4+ Tregs acquired expression of the integrin alphaEbeta7 upon tolerogenic application of Ag via the oral route. In addition, using the same system, Foxp3+ Tregs could be de novo induced from conventional naive CD25-CD4+ T cells, and this conversion was associated with concomitant expression of alphaE. These findings suggest that Tregs expressing the integrin alphaE are effector/memory Tregs with a high turnover rate that can develop in the periphery upon Ag contact under tolerogenic conditions, both from thymic-derived CD25+CD4+ Tregs with a naive-like phenotype as well as from conventional naive T cells.     

Cyclophosphamide-induced type-1 diabetes in the NOD mouse is associated with a reduction of CD4+CD25+Foxp3+ regulatory T cells

Regulatory T cells (Tregs) have been implicated as key players in immune tolerance as well as suppression of antitumor responses. The chemotherapeutic alkylating agent cyclophosphamide (CY) is widely used in the treatment of tumors and some autoimmune conditions. Although previous data has demonstrated that Tregs may be preferentially affected by CY, its relevance in promoting autoimmune conditions has not been addressed. The nonobese diabetic mouse spontaneously develops type-1 diabetes (T1D). We demonstrate in this study that CY targets CD4+CD25+Foxp3+ Tregs in vivo. CD4+CD25+ T cells isolated from CY-treated mice display reduced suppressive activity in vitro and increased expression of apoptotic markers. Although Treg numbers rapidly recovered to pretreatment levels in the peripheral lymphoid tissues, Tregs failed to recover proportionally within pancreatic infiltrates. T1D progression was effectively prevented by adoptive transfer of a small number of islet Ag-specific CD4+CD25+ Tregs to CY-treated recipients. Prevention of T1D was associated with reduced T cell activation and higher Treg proportions in the pancreas. We conclude that acceleration of T1D by CY is associated with a reduction in CD4+CD25+Foxp3+ Tregs and can be prevented by transfer of CD4+CD25+ Tregs.     

IFN-gamma controls the generation/activation of CD4+ CD25+ regulatory T cells in antitumor immune response

Immunization with serological identification of Ags by recombinant expression cloning (SEREX)-defined self-Ags leads to generation/activation of CD4+ CD25+ regulatory T cells with suppressive activities and enhanced expression of Foxp3. This is associated with increased susceptibility to pulmonary metastasis following challenge with syngeneic tumor cells and enhanced development of 3-methylcholanthrene-induced primary tumors. In contrast, coimmunization with the same SEREX-defined self-Ags mixed with a CTL epitope results in augmented CTL activity and heightened resistance to pulmonary metastasis, both of which depend on CD4+ Th cells. These active regulatory T cells and Th cells were derived from two distinct CD4+ T cell subsets, CD4+ CD25+ T cells and CD4+ CD25- T cells, respectively. In the present study, IFN-gamma was found to abrogate the generation/activation of CD4+ CD25+ regulatory T cells by immunization with SEREX-defined self-Ag. CD4+ CD25+ T cells from these IFN-gamma-treated mice failed to exhibit immunosuppressive activity as measured by 1) increased number of pulmonary metastasis, 2) enhanced development of 3-methylcholanthrene-induced primary tumors, 3) suppression of peptide-specific T cell proliferation, and 4) enhanced expression of Foxp3. The important role of IFN-gamma produced by CD8+ T cells was shown in experiments demonstrating that CD4+ CD25+ T cells cotransferred with CD8+ T cells from IFN-gamma(-/-) mice, but not from wild-type BALB/c mice, became immunosuppressive and enhanced pulmonary metastasis when recipient animals were subsequently immunized with a SEREX-defined self-Ag and a CTL epitope. These findings support the idea that IFN-gamma regulates the generation/activation of CD4+ CD25+ regulatory T cells.     

Foxp3+ regulatory T cells impede the priming of protective CD8+ T cells

T cell activation is controlled by incompletely defined opposing stimulation and suppression signals that together sustain the balance between optimal host defense against infection and peripheral tolerance. In this article, we explore the impacts of Foxp3(+) regulatory T cell (Treg) suppression in priming Ag-specific T cell activation under conditions of noninfection and infection. We find the transient ablation of Foxp3(+) Tregs unleashes the robust expansion and activation of peptide-stimulated CD8(+) T cells that provide protection against Listeria monocytogenes infection in an Ag-specific fashion. By contrast, Treg ablation had nonsignificant impacts on the CD8(+) T cell response primed by infection with recombinant L. monocytogenes. Similarly, nonrecombinant L. monocytogenes administered with peptide stimulated the expansion and activation of CD8(+) T cells that paralleled the response primed by Treg ablation. Interestingly, these adjuvant properties of L. monocytogenes did not require CD8(+) T cell stimulation by IL-12 produced in response to infection, but instead were associated with sharp reductions in Foxp3(+) Treg suppressive potency. Therefore, Foxp3(+) Tregs impose critical barriers that, when overcome naturally during infection or artificially with ablation, allow the priming of protective Ag-specific CD8(+) T cells.     

Preferential development of CD4 and CD8 T regulatory cells in RasGRP1-deficient mice

RasGRP1 and Sos are two Ras-guanyl-nucleotide exchange factors that link TCR signal transduction to Ras and MAPK activation. Recent studies demonstrate positive selection of developing thymocytes is crucially dependent on RasGRP1, whereas negative selection of autoreactive thymocytes appears to be RasGRP1 independent. However, the role of RasGRP1 in T regulatory (Treg) cell development and function is unknown. In this study, we characterized the development and function of CD4(+)CD25(+)Foxp3(+) and CD8(+)CD44(high)CD122(+) Treg lineages in RasGRP1(-/-) mice. Despite impaired CD4 Treg cell development in the thymus, the periphery of RasGRP1(-/-) mice contained significantly increased frequencies of CD4(+)Foxp3(+) Treg cells that possessed a more activated cell surface phenotype. Furthermore, on a per cell basis, CD4(+)Foxp3(+) Treg cells from mutant mice are more suppressive than their wild-type counterparts. Our data also suggest that the lymphopenic environment in the mutant mice plays a dominant role of favored peripheral development of CD4 Treg cells. These studies suggest that whereas RasGRP1 is crucial for the intrathymic development of CD4 Treg cells, it is not required for their peripheral expansion and function. By contrast to CD4(+)CD25(+)Foxp3(+) T cells, intrathymic development of CD8(+)CD44(high)CD122(+) Treg cells is unaffected by the RasGRP1(-/-) mutation. Moreover, RasGRP1(-/-) mice contained greater numbers of CD8(+)CD44(high)CD122(+) T cells in the spleen, relative to wild-type mice. Activated CD8 Treg cells from RasGRP1(-/-) mice retained their ability to synthesize IL-10 and suppress the proliferation of wild-type CD8(+)CD122(-) T cells, albeit at a much lower efficiency than wild-type CD8 Treg cells.     

Cutting edge: CD47 controls the in vivo proliferation and homeostasis of peripheral CD4+ CD25+ Foxp3+ regulatory T cells that express CD103

Peripheral CD103(+)Foxp3(+) regulatory T cells (Tregs) can develop both from conventional naive T cells upon cognate Ag delivery under tolerogenic conditions and from thymic-derived, expanded/differentiated natural Tregs. We here show that CD47 expression, a marker of self on hematopoietic cells, selectively regulated CD103(+)Foxp3(+) Treg homeostasis at the steady state. First, the proportion of effector/memory-like (CD44(high)CD62L(low)) CD103(+)Foxp3(+) Tregs rapidly augmented with age in CD47-deficient mice (CD47(-/-)) as compared with age-matched control littermates. Yet, the percentage of quiescent (CD44(low)CD62L(high)) CD103(-)Foxp3(+) Tregs remained stable. Second, the increased proliferation rate (BrdU incorporation) observed within the CD47(-/-)Foxp3(+) Treg subpopulation was restricted to those Tregs expressing CD103. Third, CD47(-/-) Tregs maintained a normal suppressive function in vitro and in vivo and their increased proportion in old mice led to a decline of Ag-specific T cell responses. Thus, sustained CD47 expression throughout life is critical to avoid an excessive expansion of CD103(+) Tregs that may overwhelmingly inhibit Ag-specific T cell responses.     

CD45RA−Foxp3 non-regulatory T cells in the CCR7−CD45RA−CD27+CD28+ effector memory subset are increased in synovial fluid from patients with rheumatoid arthritis

Increased numbers of regulatory T (Treg) cells are found in synovial fluid from patients with rheumatoid arthritis (RASF) compared with peripheral blood. However, Treg cells in RASF have been shown to have a decreased capacity to suppress T cells. Here we phenotypically classified CD4+ T cells in RASF into six subsets based on the expression of CD45RA, CCR7, CD27 and CD28, and demonstrated that the CCR7−CD45RA−CD27+CD28+ T_EM subset was significantly increased in synovial fluid compared with peripheral blood. In addition, the proportion of Foxp3+ Treg cells in the CCR7−CD45RA−CD27+CD28+ T_EM subset was significantly increased in RASF. Furthermore, most of the Foxp3+ Treg cells in RASF were non-suppressive CD45RA−Foxp3^low non-Treg cells, and the frequency of the non-Treg cells in the CCR7−CD45RA−CD27+CD28+ T_EM subset was significantly increased in RASF. Our findings suggest that the pro-inflammatory environment in RA joints may induce the increase of CD45RA−Foxp3^low non-Treg cells in synovial fluid.     

Role of STAT3 in CD4+CD25+FOXP3+ regulatory lymphocyte generation: implications in graft-versus-host disease and antitumor immunity

Immunological tolerance is maintained by specialized subsets of T cells including CD4(+)CD25(+)FOXP3(+) regulatory cells (Treg). Previous studies established that Treg thymic differentiation or peripheral conversion depend on CD28 and Lck signaling. Moreover, foxp3 gene transfer in murine CD4(+)CD25(-) T lymphocytes results in the acquisition of suppressive functions. However, molecular pathways leading to FOXP3 expression remain to be described. In this study, we investigated the molecular events driving FOXP3 expression. We demonstrated that CD28 activation in CD4(+)CD25(-) T lymphocytes leads to STAT3 Tyr(705) phosphorylation in an Lck-dependent manner. STAT3 neutralization during naive peripheral CD4(+)CD25(-) T cell conversion into Treg through costimulation with TCR/CD28 and TGF-beta1, decreased FOXP3 expression, prevented the acquisition of suppressive functions and restored the ability of the converted lymphocytes to produce IL-2 and IFN-gamma. Furthermore, we observed that STAT3 ablation using small interfering RNA strategies inhibited FOXP3 expression and suppressive functions among naturally differentiated CD4(+)CD25(+) T lymphocytes, suggesting a direct role of STAT3 in Treg phenotype and function maintenance. CD4(+)CD25(+) T lymphocytes transduced with specific STAT3 small interfering RNA were devoid of suppressive functions and failed to control the occurrence of acute graft-vs-host disease. Finally, STAT3 inhibition in CD4(+) lymphocytes enhanced the anti-tumor immunity conferred by a lymphocyte adoptive transfer. In summary, our findings determine that STAT3 is critical in the molecular pathway required for FOXP3 expression. STAT3 modulation should be taken into account when assessing how regulatory T cells contribute to inflammatory diseases and tumor immunosurveillance.     

Generation of antigen-specific, Foxp3-expressing CD4+ regulatory T cells by inhibition of APC proteosome function

We tested the hypothesis that immature APC, whose NF-kappaB-signaling pathway and thus maturation was blocked by the proteosome inhibitor benzyloxycarbonyl-isoleucyl-glutamyl(O-tert-butyl)-alanyl-leucinal (PSI), could be a source of Ag-specific regulatory T (Treg) cells. DO11.10 CD4(+) T cells that were incubated with Ag- and PSI-pulsed APC proliferated poorly, produced less IL-2, IFN-gamma, and IL-10 in secondary cultures, and inhibited the response of both naive and memory CD4(+) T cells stimulated by Ag-pulsed APC. The generation of PSI-APC Treg cells required IL-10 production by APC. PSI-APC Treg cell inhibition required cell-cell contact but not IL-10 or TGF-beta. Addition of IL-2 did not reverse, but Ab to CTLA-4 did reverse partially the inhibitory effect. Depletion of CD25(+) T cells before initial culture with PSI-APC did not affect Treg generation. PSI-APC Treg cells expressed high levels of Foxp3, inhibited proliferation of naive DO11.10 T cells in vivo, and abrogated colitis driven by a memory Th1 response to bacterial-associated Ag. We conclude that NF-kappaB-blocked, immature APC are able to induce the differentiation of Treg cells that can function in vitro and in vivo in an Ag-specific manner.     

P38 MAP kinase signaling is required for the conversion of CD4+CD25- T cells into iTreg

CD4+CD25+ regulatory T cells (Treg) are important mediators of immune tolerance. A subset of Treg can be generated in the periphery by TGF-beta dependent conversion of conventional CD4+CD25- T cells into induced Treg (iTreg). In chronic viral infection or malignancy, such induced iTreg, which limit the depletion of aberrant or infected cells, may be of pathogenic relevance. To identify potential targets for therapeutic intervention, we investigated the TGF-beta signaling in Treg. In contrast to conventional CD4+ T cells, Treg exhibited marked activation of the p38 MAP kinase pathway. Inhibition of p38 MAP kinase activity prevented the TGF-beta-dependent conversion of CD4+CD25- T cells into Foxp3+ iTreg in vitro. Of note, the suppressive capacity of nTreg was not affected by inhibiting p38 MAP kinase. Our findings indicate that signaling via p38 MAP kinase seems to be important for the peripheral generation of iTreg; p38 MAP kinase could thus be a therapeutic target to enhance immunity to chronic viral infection or cancer.     

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.     

Guarding the immune system: suppression of autoimmunity by CD4+CD25+ immunoregulatory T cells

CD4+CD25+Foxp3+ T cells (CD25+ T regulatory [Treg] cells) are a naturally occurring suppressor T-cell population that regulates a wide variety of immune responses. A major function of CD25+ Treg cells is to inhibit the activity of self-reactive T cells that can potentially cause autoimmune disease. This review examines the recent advances in CD25+ Treg cell biology, with particular focus on the thymic and peripheral development of CD25+ Treg cells, the signals that promote their expansion and maintenance in the periphery and the mechanism by which they mediate their suppressor activity in peripheral lymphoid tissues. An understanding of these issues is likely to facilitate the development of CD25+ Treg-cell-based therapies for the treatment of autoimmune disease.