The FOXP3+ subset of human CD4+CD8+ thymocytes is immature and subject to intrathymic selection

FOXP3, believed to be the regulatory T (Treg)-cell determining factor, is already expressed at the CD4+CD8+ thymocyte stage, but there is disagreement whether these cells are the precursors of mature CD4+CD8(-) Treg cells. Here, we provide a quantitative analysis of FOXP3 expression in the human thymus. We show that a subset of CD4+CD8+ cells already expressed as much FOXP3 as the FOXP3+ CD4+CD8(-) cells, and like mature Treg cells were CD127 low. In contrast to earlier data, CD8+CD4(-) thymocytes expressed significantly lower levels of FOXP3 than either the CD4+CD8+ or CD4+CD8(-) subsets. The CD4+CD8+ double-positive cells also expressed recombination-activating gene-2, suggesting that they were still immature. Although the FOXP3+ double-positive cells are thus putatively the precursors of the mature CD4+CD8(-)FOXP3+ subset, their frequency did not predict the frequency of more mature Treg cells, and analysis of T-cell antigen receptor repertoire showed clear differences between the two subsets. Although these data do not rule out an independent CD4+CD8+ Treg cell subset, they are consistent with a model of human Treg cell development in which the upregulation of FOXP3 is an early event, but the first FOXP3+ population is still immature and subject to further selection. The upregulation of FOXP3 may thus not be the final determining factor in the commitment of human thymocytes to the Treg cell lineage.     

CD4dimCD25bright Treg cell frequencies above a standardized gating threshold are similar in asthmatics and controls.

BACKGROUND: Thymus selected CD4(+)CD25(bright) natural regulatory Treg cells expressing FOXP3 may contribute to control of immune responses. No unique markers have been available to identify and characterize Treg. We present a gating strategy that allows enumeration of Treg on the basis of CD4 and CD25 and investigate whether asthmatics have fewer Treg than controls. METHODS: Asthmatics and controls were selected from responses to a mailed questionnaire. CD25, CD4, HLA DR, and appropriate isotypes were recorded by flow cytometry. RESULTS: The CD4 T cells expressing most CD25 are a separate population expressing FOXP3 and lower levels of CD4 and CD127. On a CD4 CD25 dot-plot, the CD4 MFI of Treg for 152 participants was calculated to be 0.83 +/- 0.043*MFI of CD25(bright) T-cells. CD4(dim)CD25(bright) T cells in a rectangular gate with a CD4 MFI 相似文献   

Functional regulatory T cells are collected in stem cell autografts by mobilization with high-dose cyclophosphamide and granulocyte colony-stimulating factor

High-dose cyclophosphamide (Cy) and G-CSF are widely used to mobilize hemopoietic stem cells for treating patients with high-dose chemotherapy and autologous stem cell transplantation (ASCT). Because lymphocyte count in the graft collected after Cy-G-CSF treatment is an independent survival factor after ASCT for patients with multiple myeloma, our purpose was to study how Cy-G-CSF treatment affects the phenotype and function of T cells in patients with multiple myeloma. Cy induced a 3-fold decrease of T cell counts with a slow and partial T cell recovery of one-third at the time of hemopoietic stem cell collection. Cy-G-CSF treatment did not affect the relative ratios of central memory, effector memory, and late effector CD4+ or CD8+ T cells, but a decrease in the percentage of naive CD4+ cells was observed. The percentages of CD25+ cells increased 2- to 3-fold in CD4+ and CD8+ T cells, the former including both activated CD25low and CD25high cells. CD4+CD25high cells were regulatory T cells (Treg) that expressed high levels of FOXP3, CTLA-4, and GITR and displayed in vitro suppressive properties. The recovery of Treg absolute counts after Cy-G-CSF treatment was higher than the recovery of other lymphocyte subpopulations. In conclusion, Cy-G-CSF treatment induces a severe T cell count decrease without deleting Treg, which are potent inhibitors of antitumor response. The present data encourage novel therapeutic strategies to improve T cell recovery following ASCT while limiting Treg expansion.     

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   

TLR2 stimulation drives human naive and effector regulatory T cells into a Th17-like phenotype with reduced suppressive function

Naturally occurring CD4(+)CD25(+)FOXP3(+) regulatory T cells suppress the activity of pathogenic T cells and prevent development of autoimmune responses. There is growing evidence that TLRs are involved in modulating regulatory T cell (Treg) functions both directly and indirectly. Specifically, TLR2 stimulation has been shown to reduce the suppressive function of Tregs by mechanisms that are incompletely understood. The developmental pathways of Tregs and Th17 cells are considered divergent and mutually inhibitory, and IL-17 secretion has been reported to be associated with reduced Treg function. We hypothesized that TLR2 stimulation may reduce the suppressive function of Tregs by regulating the balance between Treg and Th17 phenotype and function. We examined the effect of different TLR2 ligands on the suppressive functions of Tregs and found that activation of TLR1/2 heterodimers reduces the suppressive activity of CD4(+)CD25(hi)FOXP3(low)CD45RA(+) (naive) and CD4(+)CD25(hi)FOXP3(hi)CD45RA(-) (memory or effector) Treg subpopulations on CD4(+)CD25(-)FOXP3(-)CD45RA(+) responder T cell proliferation while at the same time enhancing the secretion of IL-6 and IL-17, increasing RORC, and decreasing FOXP3 expression. Neutralization of IL-6 or IL-17 abrogated Pam3Cys-mediated reduction of Treg suppressive function. We also found that, in agreement with recent observations in mouse T cells, TLR2 stimulation can promote Th17 differentiation of human T helper precursors. We conclude that TLR2 stimulation, in combination with TCR activation and costimulation, promotes the differentiation of distinct subsets of human naive and memory/effector Tregs into a Th17-like phenotype and their expansion. Such TLR-induced mechanism of regulation of Treg function could enhance microbial clearance and increase the risk of autoimmune reactions.     

Natural regulatory (CD4+CD25+FOXP+) T cells control the production of pro-inflammatory cytokines during Plasmodium chabaudi adami infection and do not contribute to immune evasion

Different functions have been attributed to natural regulatory CD4+CD25+FOXP+ (Treg) cells during malaria infection. Herein, we assessed the role for Treg cells during infections with lethal (DS) and non-lethal (DK) Plasmodium chabaudi adami parasites, comparing the levels of parasitemia, inflammation and anaemia. Independent of parasite virulence, the population of splenic Treg cells expanded during infection, and the absolute numbers of activated CD69+ Treg cells were higher in DS-infected mice. In vivo depletion of CD25+ T cells, which eliminated 80% of CD4+FOXP3+CD25+ T cells and 60-70% of CD4+FOXP3+ T cells, significantly decreased the number of CD69+ Treg cells in mice with lethal malaria. As a result, higher parasite burden and morbidity were measured in the latter, whereas the kinetics of infection with non-lethal parasites remained unaffected. In the absence of Treg cells, parasite-specific IFN-gamma responses by CD4+ T cells increased significantly, both in mice with lethal and non-lethal infections, whereas IL-2 production was only stimulated in mice with non-lethal malaria. Following the depletion of CD25+ T cells, the production of IL-10 by CD90(-) cells was also enhanced in infected mice. Interestingly, a potent induction of TNF-alpha and IFN-gamma production by CD4+ and CD90(-) lymphocytes was measured in DS-infected mice, which also suffered severe anaemia earlier than non-depleted infected controls. Taken together, our data suggest that the expansion and activation of natural Treg cells represent a counter-regulatory response to the overwhelming inflammation associated with lethal P.c. adami. This response to infection involves TH1 lymphocytes as well as cells from the innate immune system.     

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.     

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.     

IL-2 immunotoxin therapy modulates tumor-associated regulatory T cells and leads to lasting immune-mediated rejection of breast cancers in neu-transgenic mice

Studies in cancer patients have suggested that breast tumors recruit regulatory T cells (Tregs) into the tumor microenvironment. The extent to which local Tregs suppress antitumor immunity in breast cancer is unknown. We questioned whether inhibiting systemic Tregs with an IL-2 immunotoxin in a model of neu-mediated breast cancer, the neu-transgenic mouse, could impact disease progression and survival. As in human breast cancer, cancers that develop in these mice attract Tregs into the tumor microenvironment to levels of approximately 10-25% of the total CD4+ T cells. To examine the role of Tregs in blocking immune-mediated rejection of tumor, we depleted CD4+CD25+ T cells with an IL-2 immunotoxin. The treatment depleted Tregs without concomitant lymphopenia and markedly inhibited tumor growth. Depletion of Tregs resulted in a persistent antitumor response that was maintained over a month after the last treatment. The clinical response was immune-mediated because adoptive transfer of Tregs led to a complete abrogation of the therapeutic effects of immunotoxin treatment. Further, Treg down-modulation was accompanied by increased Ag-specific immunity against the neu protein, a self Ag. These results suggest that Tregs play a major role in preventing an effective endogenous immune response against breast cancer and that depletion of Tregs, without any additional immunotherapy, may mediate a significant antitumor response.     

HLA-A*02:01-directed chimeric antigen receptor/forkhead box P3-engineered CD4+ T cells adopt a regulatory phenotype and suppress established graft-versus-host disease

Background aimsTo investigate the feasibility of using CD4 + T cells genetically modified to express an allo-HLA directed CAR and FOXP3 to suppress T cell proliferation and cytokine secretion in GvHD. Methods: Human CD4+ T cells from A*02:01 negative donors were transduced to express A*02 CAR and FOXP3 and co-cultured in mixed lymphocyte reaction assays to demonstrate T cell suppression. A*02- CAR/FOXP CD4+ T cells were then injected into mice engrafted with allogeneic T cells in a GvHD mouse model.ResultsCD4+ T cells genetically modified to express allo-HLA-directed CAR and FOXP3 proliferate rapidly, downregulate CD127 and interferon-γ, express high CD25 and Helios and convert to a stable antigen-dependent suppressive phenotype. In mixed lymphocyte reaction assays, these cells potently suppressed T-cell proliferation and secreted IL-10. In a graft-versus-host disease model, A*02-CAR/FOXP3 CD4+ T cells outperformed polyclonal Tregs by reducing liver and lung inflammation, inhibiting pro-inflammatory cytokine production and limiting grafted CD3+ T-cell expansion.ConclusionsCD4 + T cells expressing allo-antigen directed HLA-specific CAR and FOXP3 act as potent, specific and stable suppressors of inflammation that out-perform their Treg counterparts both in vitro and in vivo.     

Listeriolysin O expressed in a bacterial vaccine suppresses CD4+CD25high regulatory T cell function in vivo

CD4(+)CD25(high) regulatory T cells (Treg) protect the host from autoimmune diseases but are also obstacles against cancer therapies. An ideal cancer vaccine would stimulate specific cytotoxic responses and reduce/suppress Treg function. In this study, we showed that Escherichia coli expressing listeriolysin O and OVA (E. coli LLO/OVA) demonstrated remarkable levels of protection against OVA-expressing tumor cells. By contrast, E. coli expressing OVA only (E. coli OVA) showed poor protection. High-avidity OVA-specific CTL were induced in E. coli LLO/OVA-vaccinated mice, and CD8(+) depletion--but not NK cell depletion, abolished the antitumor activity of the E. coli LLO/OVA vaccine. Phenotypic analysis of T cells following vaccination with either vaccine revealed preferential generation of CD44(high)CD62L(low) CD8(+) effector memory T cells over CD44(high)CD62L(high) central memory T cells. Unexpectedly, CD4(+) depletion turned E. coli OVA into a vaccine as effective as E. coli LLO/OVA suggesting that a subset of CD4(+) cells suppressed the CD8(+) T cell-mediated antitumor response. Further depletion experiments demonstrated that these suppressive cells consisted of CD4(+)CD25(high) regulatory T cells. We therefore assessed these vaccines for Treg function and found that although CD4(+)CD25(high) expansion and Foxp3 expression within this population was similar in all groups of mice, Treg cells from E. coli LLO/OVA-vaccinated animals were unable to suppress conventional T cells proliferation. These findings provide the first evidence that LLO expression affects Treg cell function and may have important implications for enhancing antitumor vaccination strategies in humans.     

Elevated T regulatory cells in long-term stable transplant tolerance in rhesus macaques induced by anti-CD3 immunotoxin and deoxyspergualin

Regulatory T cells (Tregs) are implicated in immune tolerance and are variably dependent on IL-10 for in vivo function. Brief peritransplant treatment of multiple nonhuman primates (NHP) with anti-CD3 immunotoxin and deoxyspergualin has induced stable (5-10 years) rejection-free tolerance to MHC-mismatched allografts, which associated with sustained elevations in serum IL-10. In this study, we demonstrate that resting and activated PBMC from long-term tolerant NHP recipients are biased to secrete high levels of IL-10, compared with normal NHP PBMC. Although IL-10-producing CD4+ Tregs (type 1 regulatory cells (TR1)/IL-10 Tregs) were undetectable (<0.5%) in normal rhesus monkeys, 7.5 +/- 1.7% of circulating CD4+ T cells of tolerant rhesus recipients expressed IL-10. In addition to this >15-fold increase in Tr1/IL-10 Tregs, the tolerant monkeys exhibited a nearly 3-fold increase in CD4+CD25+ Tregs, 8.1 +/- 3.0% of CD4 T cells vs 2.8 +/- 1.4% in normal cohorts (p < 0.02). The frequency of CD4+CD25+IL-10+ cells was elevated 5-fold in tolerant vs normal NHP (1.8 +/- 0.9% vs 0.4 +/- 0.2%). Rhesus CD4+CD25+ Tregs exhibited a memory phenotype, and expressed high levels of Foxp3 and CTLA-4 compared with CD4+CD25- T cells. Also, NHP CD4+CD25+ Tregs proliferated poorly after activation and suppressed proliferation of CD4+CD25- effector T cells, exhibiting regulatory properties similar to rodent and human CD4+CD25+ Tregs. Of note, depletion of CD4+CD25+ Tregs restored indirect pathway antidonor responses in tolerant NHP. Our study demonstrates an expanded presence of Treg populations in tolerant NHP recipients, suggesting that these adaptations may be involved in maintenance of stable tolerance.     

Analysis of Suppressor and Non-Suppressor FOXP3+ T Cells in HIV-1-Infected Patients

Recently, it was shown that peripheral blood FOXP3+CD4+ T cells are composed of three phenotypic and functionally distinct subpopulations. Two of them having in vitro suppressive effects were characterized as resting Treg cells (rTregs) and activated Treg cells (aTregs). A third subset, identified as FOXP3+ non-Tregs, does not display any suppressor activity and produce high levels of Th1 and Th17 cytokines upon stimulation. In the present study we focus on the characteristics of these three subsets of FOXP3+CD4+ T cells in untreated HIV-1-infected patients. We found that the absolute counts of rTregs, aTregs and FOXP3+ non-Tregs were reduced in HIV-1 patients compared with healthy donors. The relative frequency of rTregs and aTregs was similar in HIV-1 patients and healthy donors, while the frequency of FOXP3+ non-Tregs was significantly higher in HIV-1 patients, reaching a maximum in those patients with the lower values of CD4 counts. Contrasting with the observations made in FOXP3- CD4+ T cells, we did not find a negative correlation between the number of rTregs, aTregs or FOXP3+ non-Tregs and virus load. Studies performed with either whole PBMCs or sorted aTregs and FOXP3+ non-Tregs cells showed that these two populations of FOXP3+ T cells were highly permissive to HIV-1 infection. Upon infection, FOXP3+ non-Tregs markedly down-regulates its capacity to produce Th1 and Th17 cytokines, however, they retain the ability to produce substantial amounts of Th2 cytokines. This suggests that FOXP3+ non-Tregs might contribute to the polarization of CD4+ T cells into a Th2 profile, predictive of a poor outcome of HIV-1-infected patients.     

Functional Improvement of Regulatory T Cells from Rheumatoid Arthritis Subjects Induced by Capsular Polysaccharide Glucuronoxylomannogalactan

Objective

Regulatory T cells (Treg) play a critical role in the prevention of autoimmunity, and the suppressive activity of these cells is impaired in rheumatoid arthritis (RA). The aim of the present study was to investigate function and properties of Treg of RA patients in response to purified polysaccharide glucuronoxylomannogalactan (GXMGal).

Methods

Flow cytometry and western blot analysis were used to investigate the frequency, function and properties of Treg cells.

Results

GXMGal was able to: i) induce strong increase of FOXP3 on CD4⁺ T cells without affecting the number of CD4⁺CD25⁺FOXP3⁺ Treg cells with parallel increase in the percentage of non-conventional CD4⁺CD25⁻FOXP3⁺ Treg cells; ii) increase intracellular levels of TGF-β1 in CD4⁺CD25⁻FOXP3⁺ Treg cells and of IL-10 in both CD4⁺CD25⁺FOXP3⁺ and CD4⁺CD25⁻FOXP3⁺ Treg cells; iii) enhance the suppressive activity of CD4⁺CD25⁺FOXP3⁺ and CD4⁺CD25⁻FOXP3⁺ Treg cells in terms of inhibition of effector T cell activity and increased secretion of IL-10; iv) decrease Th1 response as demonstrated by inhibition of T-bet activation and down-regulation of IFN-γ and IL-12p70 production; v) decrease Th17 differentiation by down-regulating pSTAT3 activation and IL-17A, IL-23, IL-21, IL-22 and IL-6 production.

Conclusion

These data show that GXMGal improves Treg functions and increases the number and function of CD4⁺CD25⁻FOXP3⁺ Treg cells of RA patients. It is suggested that GXMGal may be potentially useful for restoring impaired Treg functions in autoimmune disorders and for developing Treg cell-based strategies for the treatment of these diseases.