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.     

The frequency of CD25+CD4+ and FOXP3+ regulatory T cells in ectopic endometrium and ectopic decidua

Background  

The presence of regulatory T (Treg) cells in human endometrium is crucial for maintaining immunological homeostasis within the uterus. For this study we decided to evaluate the subpopulations of Treg cells in conditions where a disturbance in the immunological equilibrium in ectopic endometrium and decidua has been observed, such as in cases of ovarian endometriosis (involving local immune cell suppression) and ectopic pregnancy (involving an increase in local immune system activity). We then compared these findings to what we observed in the normal eutopic endometrium of women during the secretory phase of the menstrual cycle (with immune cells under individual control).     

CD25- T cells generate CD25+Foxp3+ regulatory T cells by peripheral expansion

Naturally occurring CD4(+) regulatory T cells are generally identified through their expression of CD25. However, in several experimental systems considerable T(reg) activity has been observed in the CD4(+)CD25(-) fraction. Upon adoptive transfer, the expression of CD25 in donor-derived cells is not stable, with CD4(+)CD25(+) cells appearing in CD4(+)CD25(-) T cell-injected animals and vice versa. We show in this study that CD25(+) cells arising from donor CD25(-) cells upon homeostatic proliferation in recipient mice express markers of freshly isolated T(reg) cells, display an anergic state, and suppress the proliferation of other cells in vitro. The maintenance of CD25 expression by CD4(+)CD25(+) cells depends on IL-2 secreted by cotransferred CD4(+)CD25(-) or by Ag-stimulated T cells in peripheral lymphoid organs.     

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.     

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.     

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.     

The 4-1BB costimulation augments the proliferation of CD4+CD25+ regulatory T cells

The thymus-derived CD4(+)CD25(+) T cells belong to a subset of regulatory T cells potentially capable of suppressing the proliferation of pathogenic effector T cells. Intriguingly, these suppressor cells are themselves anergic, proliferating poorly to mitogenic stimulation in culture. In this study, we find that the 4-1BB costimulator receptor, best known for promoting the proliferation and survival of CD8(+) T cells, also induces the proliferation of the CD4(+)CD25(+) regulatory T cells both in culture and in vivo. The proliferating CD4(+)CD25(+) T cells produce no detectable IL-2, suggesting that 4-1BB costimulation of these cells does not involve IL-2 production. The 4-1BB-expanded CD4(+)CD25(+) T cells are functional, as they remain suppressive to other T cells in coculture. These results support the notion that the peripheral expansion of the CD4(+)CD25(+) T cells is controlled in part by costimulation.     

Identification of circulating human antigen-reactive CD4+ FOXP3+ natural regulatory T cells

Circulating human CD4(+)CD25(++)CD127(-)FOXP3(+) T cells with a persistent demethylated regulatory T cell (Treg)-specific demethylated region Foxp3 gene are considered natural Tregs (nTregs). We have shown that it is possible to identify functional Ag-reactive nTregs cells for a range of different common viral and vaccination Ags. The frequency of these Ag-reactive nTregs within the nTreg population is strikingly similar to the frequency of Ag-reactive T effector cells within the CD4(+) T cell population. The Ag-reactive nTregs could be recognized with great specificity by induction of CD154 expression. These CD154(+) Ag-reactive nTregs showed a memory phenotype and shared all phenotypical and functional characteristics of nTregs. The isolated CD154(+) nTregs could be most efficiently expanded by specific antigenic stimulation, while their Ag-reactive suppressive activity was maintained. After an in vivo booster Ag challenge, the ratio of Ag-reactive T cells to Ag-reactive Tregs increased substantially, which could be attributed to the rise in effector T cells but not Tregs. In conclusion, the nTreg population mirrors the effector T cell population in the frequency of Ag-reactive T cells. Isolation and expansion of functional Ag-reactive nTregs is possible and of potential benefit for specific therapeutic goals.     

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.     

Intracerebral human regulatory T cells: analysis of CD4+ CD25+ FOXP3+ T cells in brain lesions and cerebrospinal fluid of multiple sclerosis patients

Impaired suppressive capacity of CD4⁺CD25⁺FOXP3⁺ regulatory T cells (Treg) from peripheral blood of patients with multiple sclerosis (MS) has been reported by multiple laboratories. It is, however, currently unresolved whether Treg dysfunction in MS patients is limited to reduced control of peripheral T cell activation since most studies analyzed peripheral blood samples only. Here, we assessed early active MS lesions in brain biopsies obtained from 16 patients with MS by FOXP3 immunohistochemistry. In addition, we used six-color flow cytometry to determine numbers of Treg by analysis of FOXP3/CD127 expression in peripheral blood and cerebrospinal fluid (CSF) of 17 treatment-naïve MS patients as well as quantities of apoptosis sensitive CD45RO^hiCD95^hi cells in circulating and CSF Treg subsets. Absolute numbers of FOXP3⁺ and CD4⁺ cells were rather low in MS brain lesions and Treg were not detectable in 30% of MS biopsies despite the presence of CD4⁺ cell infiltrates. In contrast, Treg were detectable in all CSF samples and Treg with a CD45RO^hiCD95^hi phenotype previously shown to be highly apoptosis sensitive were found to be enriched in the CSF compared to peripheral blood of MS patients. We suggest a hypothetical model of intracerebral elimination of Treg by CD95L-mediated apoptosis within the MS lesion.     

Malignant B cells induce the conversion of CD4+CD25- T cells to regulatory T cells in B-cell non-Hodgkin lymphoma

Recent evidence has demonstrated that regulatory T cells (Treg) were enriched in the tumor sites of patients with B-cell non-Hodgkin lymphoma (NHL). However, the causes of enrichment and suppressive mechanisms need to be further elucidated. Here we demonstrated that CD4(+)CD25(+)FoxP3(+)CD127(lo) Treg were markedly increased and their phenotypes were different in peripheral blood (PB) as well as bone marrow (BM) from newly diagnosed patients with B-cell NHL compared with those from healthy volunteers (HVs). Involved lymphatic tissues also showed higher frequencies of Treg than benign lymph nodes. Moreover, the frequencies of Treg were significantly higher in involved lymphatic tissues than those from PB as well as BM in the same patients. Suppression mediated by CD4(+)CD25(+) Treg co-cultured with allogeneic CFSE-labeled CD4(+)CD25(-) responder cells was also higher in involved lymphatic tissues from B-cell NHL than that mediated by Treg from HVs. In addition, we found that malignant B cells significantly induced FoxP3 expression and regulatory function in CD4(+)CD25(-) T cells in vitro. In contrast, normal B cells could not induce the conversion of CD4(+)CD25(-) T cells to Treg. We also showed that the PD-1/B7-H1 pathway might play an important role in Treg induction. Taken together, our results suggest that malignant B cells induce the conversion of CD4(+)CD25(-) T cells to Treg, which may play a role in the pathogenesis of B-cell NHL and represent a promising therapeutic target.     

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.     

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.     

Cutting edge: IL-12 induces CD4+CD25- T cell activation in the presence of T regulatory cells

IL-12p40 cytokines have been implicated in the development of organ-specific autoimmune diseases as well as pathogen-specific adaptive immunity. In addition to inducing IFN-gamma, IL-12 stimulates effector CD4(+) T cells to express adhesion molecules and homing receptors that facilitate their migration to sites of inflammation. In this study, we expand upon those observations by demonstrating an alternative pathway by which IL-12 could promote Th1 inflammatory responses in mice, namely, by restoring proliferation and cytokine expression by effector T cells in the presence of CD4(+)CD25(+) regulatory T cells (Treg). This effect of IL-12 was not replicated by IL-23 or IFN-gamma and was dependent on signaling through the IL-12R expressed on CD25(-) responder cells, but not on Treg. Our studies suggest that IL-12 could act in concert with other proinflammatory factors to stimulate CD4(+)CD25(-) T cell activation in the presence of Treg.     

The effector T cells of diabetic subjects are resistant to regulation via CD4+ FOXP3+ regulatory T cells

Defects in immune regulation have been implicated in the pathogenesis of diabetes in mouse and in man. In vitro assays using autologous regulatory (Treg) and responder effector (Teff) T cells have shown that suppression is impaired in diabetic subjects. In this study, we addressed whether the source of this defect is intrinsic to the Treg or Teff compartment of diabetic subjects. We first established that in type 1 diabetes (T1D) individuals, similar levels of impaired suppression were seen, irrespective of whether natural (nTreg) or adaptive Treg (aTreg) were present. Then using aTreg, we examined the ability of T1D aTreg to suppress Teff of healthy controls, as compared with the ability of control aTreg to suppress Teff of diabetic subjects. Taking this approach, we found that the aTregs from T1D subjects function normally in the presence of control Teff, and that the T1D Teff were resistant to suppression in the presence of control aTreg. This escape from regulation was seen with nTreg as well and was not transferred to control Teff coincubated with T1D Teff. Thus, the "defective regulation" in T1D is predominantly due to the resistance of responding T cells to Treg and is a characteristic intrinsic to the T1D Teff. This has implications with respect to pathogenic mechanisms, which underlie the development of disease and the target of therapies for T1D.     

Cutting edge: direct suppression of B cells by CD4+ CD25+ regulatory T cells

Regulatory T cells (Tregs) can potentially migrate to the B cell areas of secondary lymphoid tissues and suppress T cell-dependent B cell Ig response. T cell-dependent Ig response requires B cell stimulation by Th cells. It has been unknown whether Tregs can directly suppress B cells or whether they must suppress Th cells to suppress B cell response. We report here that Foxp3+ Tregs are found in T-B area borders and within germinal centers of human lymphoid tissues and can directly suppress B cell Ig response. Although Tregs can effectively suppress T cells, they can also directly suppress B cell response without the need to first suppress Th cells. The direct suppression of B cell Ig production by Tregs is accompanied by inhibition of Ig class switch recombination.