TGF-beta1 modulates Foxp3 expression and regulatory activity in distinct CD4+ T cell subsets

Although forkhead box p3 (Foxp3) expression is restricted to naturally occurring CD4(+) regulatory T cells (T(REG)), little is known about the various signals that regulate it in T cells. As TGF-beta has been reported to modulate Foxp3 expression in T cells, we investigated its effects on the induction or maintenance of regulatory functions in different CD4(+) T cell subsets. TGF-beta1 priming was able to promote differentiation of T(REG) cells from nonregulatory CD4(+)CD25(-) T cells in a concentration-dependent manner through Foxp3 induction. As CD4(+)CD25(-) T cells remain a highly heterogeneous population with variable degrees of antigen experience, we then examined the effect of TGF-beta1 on naive CD4(+)CD25(-)CD45RB(HIGH) T cells. Freshly isolated or TGF-beta1-treated CD4(+)CD25(-)CD45RB(HIGH) T cells never displayed any regulatory functions or significant Foxp3 expression following TCR activation. In stark contrast, freshly isolated CD4(+)CD25(-)CD45RB(LOW) cells, albeit expressing low levels of Foxp3 mRNA and protein, were unable to suppress CD4(+) effector T cell proliferation but acquired regulatory activity and de novo Foxp3 expression following TGF-beta1 exposure. Furthermore, suppression was IL-10-dependent, as anti-IL-10 receptor antibody treatment abrogated this suppression completely, consistent with the ability of TGF-beta1-treated CD4(+)CD25(-)CD45RB(LOW) to synthesize IL-10 upon restimulation in vitro. Last, we show that TGF-beta1 treatment or blockade did not lead to enhanced expansion or function of naturally occurring CD4(+)CD25(+) T(REG) cells, although it maintained Foxp3 mRNA and protein expression. Altogether, TGF-beta1 promotes the induction of IL-10-secreting CD4(+) T(REG) cells from CD4(+)CD25(-)CD45RB(LOW) precursors through de novo Foxp3 production and maintains natural T(REG) cell peripheral homeostasis by sustaining Foxp3 expression.     

CD25(+)CD4(+) regulatory T cells exert in vitro suppressive activity independent of CTLA-4

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is constitutively expressed on CD25(+)CD4(+) regulatory T cells (Treg) and is suggested to play a role in Treg-mediated suppression. However, the results of analysis with anti-CTLA-4 have been controversial. We addressed this issue by analyzing mice over-expressing or deficient in CTLA-4. For over-expression, CTLA-4 transgenic mice expressing a full-length (FL) or a truncated (TL) mutant of CTLA-4 were analyzed. FL T cells expressed similar levels of CTLA-4 to Treg, whereas TL T cells expressed much higher levels on the cell surface. The number of Treg in both mice was decreased, although Foxp3 expression was not altered. Treg from both mice exerted suppressive activity, whereas CD25(-) T cells from FL mice showed no suppression. Furthermore, CD25(+)CD4 thymocytes from young CTLA-4-deficient mice were analyzed and found to exhibit suppressive activity. These results indicate that Treg exert in vitro suppressive activity independent of CTLA-4 expression.     

Natural and TGF-beta-induced Foxp3(+)CD4(+) CD25(+) regulatory T cells are not mirror images of each other

Foxp3(+) CD4(+) CD25(+) regulatory cell (Treg) subsets that maintain immunologic homeostasis have been considered to be a homogeneous population of naturally occurring, thymus-derived CD4(+)CD25(+) cells (nTregs). However, similar Foxp3+ Tregs can be induced from CD25(-) precursors in vivo, and ex vivo with interleukin 2 (IL-2) and transforming growth factor beta (TGF-beta) (iTregs). These two subsets differ in their principal antigen specificities and in the T-cell receptor signal strength and co-stimulatory requirements needed for their generation. However, whether iTregs have any unique functions in vivo has been unclear. Although IL-6 can convert nTregs to Th17 cells, iTregs induced by IL-2 and TGF-beta are resistant to this cytokine and thereby might retain suppressive function at inflammatory sites. Thus, nTregs and iTregs may have different roles in the adaptive immune response.     

小鼠脾细胞CD4~+ CD25~+调节性T细胞的表型特征

观察CD4+CD25+T和CD4+CD25-T细胞的表型和细胞因子的表达。自小鼠脾脏制备单个细胞悬液,分离CD4+T细胞、CD4+CD25+和CD4+CD25-T细胞,进行细胞表面标记,激活后进行细胞内细胞因子染色,利用流式细胞仪在单个细胞水平上分析细胞表面分子、转录因子和细胞因子表达之间的关系。结果:在CD4+T细胞中,约有7.8%的细胞同时表达CD25分子。与CD4+CD25-T细胞相比,CD4+CD25+T细胞CD44的表达略有增加,CD45RB的表达明显下降,CTLA-4和Foxp3明显增加。以同时表达CTLA-4和Foxp3的细胞为主,其次为单独表达Foxp3的细胞。细胞因子的研究结果表明,与CD4+CD25-T细胞相比,CD4+CD25+T细胞IL-2、IFN-γ明显减少,而只产生IL-10的细胞略有增加。CD4+CD25+调节性T细胞无论在表型、转录因子的表达以及细胞因子表达方面均于非调节性T细胞不同。     

Suppressive properties of human CD4+CD25+ regulatory T cells are dependent on CTLA-4 expression

It has been demonstrated that T cells with regulatory properties are present within the peripheral blood CD4(+)CD25(+) T cell compartment. Here, we describe an original method to purify human CD4(+)CD25(+)CD152(+) T lymphocytes as living cells by forcing the exportation of CTLA-4 molecules stored in intracellular vesicules at the cell surface. By doing so, we demonstrate that CD4(+)CD25(+) T cells contain a smaller and more homogeneous population enriched in cells with in vitro regulatory activity. Moreover, we show that this enrichment in regulatory T cells is associated with an increased expression of Foxp3 and that CD4(+)CD25(+)CD152(+) T lymphocytes display a much stronger suppressive activity in controlling in vitro proliferation of alloantigen-specific T cells than CD4(+)CD25(+)CD152(-) T lymphocytes purified in parallel. Lastly, by purifying such cells expressing CTLA-4, we demonstrate that indeed CTLA-4 is involved in CD4(+)CD25(+)CD152(+) T cell regulatory activity, while suppressive cytokines are not.     

Human CD19(+)CD25(high) B regulatory cells suppress proliferation of CD4(+) T cells and enhance Foxp3 and CTLA-4 expression in T-regulatory cells

Studies in both animal models and humans have shown a subset of B cells behaving as immuno-regulatory cells, being a source of inhibitory cytokines such as IL-10 and TGF-β. Our aims were to establish the presence of human B regulatory (Breg) cells and to assess their ability to suppress proliferation of CD4(+) T cells and to mediate T regulatory (Treg) cells' properties. For this purpose, human Breg, CD4(+) T and Treg cells were purified using magnetic microbeads. CFSE-labeled CD4(+) T cells were stimulated and cultured alone or with Breg cells. Their proliferative response was determined 72 hours later based on the CFSE staining. In parallel, Treg cells were cultured alone or with Breg cells in different conditions for 24 hours, and then stained and analyzed for Foxp3 and CTLA-4 expression. We found that, the co-culture of Breg cells (defined as CD25(high) CD27(high) CD86(high) CD1d(high) IL-10(high) TGF-β(high)) with autologous stimulated CD4(+) T cells decreased significantly (in a dose-dependent way) the proliferative capacity of CD4(+) T cells. Furthermore, Foxp3 and CTLA-4 expression in Treg cells were enhanced by non-stimulated and further by ODN-CD40L stimulated Breg cells. The regulatory function of Breg cells on Treg cells was mainly dependent on a direct contact between Breg and Treg cells, but was also TGF-β but not IL-10 dependent. In conclusion, human Breg cells decrease the proliferation of CD4(+) T cells and also enhance the expression of Foxp3 and CTLA-4 in Treg cells by cell-to-cell contact.     

Generation of highly suppressive adaptive CD8(+)CD25(+)FOXP3(+) regulatory T cells by continuous antigen stimulation

Continuous antigen stimulation of CD4(+)CD25(-) T cells leads to generation of adaptive CD4(+)CD25(+)FOXP3(+) regulatory T (T(R)) cells. Here, we show that highly suppressive adaptive CD8(+)CD25(+)FOXP3(+) T cells can be generated in the same manner by continuous antigen stimulation in the presence of CD14(+) monocytes. During the course of stimulation, acquisition of immunosuppressive properties develops in parallel with up-regulation and expression of cytotoxic molecules. The CD8(+) T(R) cells inhibit CD4(+) and CD8(+) T cell proliferation and cytokine production, but do not alter the expression of granzyme A and granzyme B or perforin in CD8(+) effector T cells. Although, the CD8(+) T(R) cells express prostaglandin E(2), IL-10 and TGF-beta, the mechanism of suppression was independent of these soluble factors. In contrast to adaptive CD4(+) T(R) cells, the CD8(+) T(R) cells suppress mainly by a contact-dependent mechanism as evident from transwell experiments. However, neither blocking antibodies to CTLA-4, CD80 nor CD86 could reverse CD8(+) T(R)-mediated suppression, indicating that other mechanism(s) must be employed by these cells.     

Characteristics of rat CD4(+)CD25(+) T cells and their ability to prevent not only diabetes but also insulitis in an adoptive transfer model in BB rats

Human and mouse CD4(+)CD25(+) T cells have been intensively studied through the last decade. However, little is known about this subset in other species. This study describes the phenotype of rat CD4(+)CD25(+) Foxp3(+) T cells and the site in which they exert regulation in a transfer-induced autoimmune diabetes model. Several proteins and mRNAs are up-regulated in unstimulated rat CD4(+)CD25(+) T cells compared with CD4(+)CD25(-) T cells, including Foxp3, Lag-3, CD80, interleukin 10 (IL-10) and CTLA-4. To investigate CD4(+)CD25(+) T cells in vivo, we transferred three million diabetogenic T cells either alone or in combination with two million CD4(+)CD25(+) T cells to 30-day-old BB rats. The pancreas and the pancreatic lymph nodes were examined as two potential regulatory sites. Time-course analysis of pancreatic histology following diabetogenic T-cell transfers revealed insulitis from about 14 days after transfer. By contrast, rats receiving both diabetogenic T cells and CD4(+)CD25(+) T cells had no insulitis at any time. Moreover, the frequency of diabetogenic T cells in the pancreatic lymph nodes 2 days after transfer was significantly reduced in rats receiving both subsets. These data indicate that the primary site of T-cell regulation is in the draining lymph nodes and not the pancreas in our model.     

CD5 plays an inhibitory role in the suppressive function of murine CD4(+) CD25(+) T(reg) cells

A subset of CD4(+) T cells, the CD4(+) CD25(+) regulatory T (T(reg)) cells in the lymphoid organs and peripheral blood are known to possess suppressive function. Previous in vitro and in vivo studies have indicated that T cell receptor (TCR) signal is required for development of such 'natural regulatory (T(reg)) cells' and for activation of the effector function of CD4(+) CD25(+) regulatory T cells. CD5 is a cell surface molecule present on all T cells and a subtype of B lymphocytes, the B-1 cells, primarily localized to coelomic cavities, Peyer's patches, tonsils and spleen. CD5 acts as a negative regulator of T cell and B cell signaling via recruitment of SHP-1. Here, we demonstrate that T(reg) cells obtained from CD5(-/-) mice are more potent than those from wild type mice in suppressing the in vitro cell proliferation of anti-CD3 stimulated CD4(+) CD25(-) responder T cells. This phenomenon was cell contact and GITR dependent. Lack of CD5 expression on T(reg) cells (from spleen, lymph node and thymus) did not affect the intracellular levels of Foxp3. However, CD5(-/-) T(reg) thymocytes were able to elicit a higher Ca(2+) response to TCR + co-stimulatory signals than the wild type cells. CD5(-/-) mice expressed more Foxp3 mRNA in the colon than wild type mice, and additionally, the severity of the dextran sulfate sodium (DSS)-induced colitis in CD5(-/-) mice was less than the wild type strain. We suggest that manipulation of CD5 expression or the downstream signaling components of CD4(+) CD25(+) T(reg) cells as a potential strategy for therapeutic intervention in cases of auto-immune disorders.     

Preferential replication of FIV in activated CD4(+)CD25(+)T cells independent of cellular proliferation

Studies attempting to identify reservoirs of HIV-1 latency have documented that the virus persists as both a latent and productive infection in subsets of CD4(+) cells. Reports regarding establishment of a stable HIV-1 infection in quiescent T cells in vitro, however, are controversial. In the present study, we investigated the susceptibility of naive and activated CD4(+) cell subsets (distinguished by differential expression of CD25) to feline immunodeficiency virus (FIV) infection, their ability to replicate the virus, and potentially act as a reservoir for virus persistence in infected animals. While both CD4(+)CD25(+) and CD4(+)CD25(-) cells are susceptible to FIV infection in vitro and in vivo, only CD4(+)CD25(+) cells produce infectious virions when cultured with interleukin-2 (IL-2). Latently infected CD4(+)CD25(-) cells produce infectious virions following ConcanvalinA (ConA) stimulation, which correlates with upregulated surface expression of CD25. In contrast to CD4(+)CD25(-) cells, CD4(+)CD25(+) cells remain unresponsive to mitogen stimulation and are relatively resistant to apoptosis whether or not infected with FIV. The ability of CD4(+)CD25(+) cells to replicate FIV efficiently in the presence of IL-2 but remain anergic and unresponsive to apoptotic signaling suggests that these cells may provide a reservoir of productive FIV infection. On the contrary, CD4(+)CD25(-) cells seem to establish as latent viral reservoirs capable of being reactivated after stimulation.     

Origin and T cell receptor diversity of Foxp3+CD4+CD25+ T cells

Foxp3(+)CD4(+)CD25(+) regulatory T cells can differentiate from Foxp3(-)CD4(+) medullary thymocytes and Foxp3(-)CD4(+) naive T cells. However, the impact of these two processes on size and composition of the peripheral repertoire of regulatory T cells is unclear. Here we followed the fate of individual Foxp3(+)CD4(+)CD25(+) thymocytes and T cells in vivo in T cell receptor (TCR) transgenic mice that express a restricted but polyclonal repertoire of TCRs. By utilizing high-throughput single-cell analysis, we showed that Foxp3(+)CD4(+) peripheral T cells were derived from thymic precursors that expressed a different TCRs than Foxp3(-)CD4(+) medullary thymocytes and Foxp3(-)CD4(+) T cells. Furthermore, the diversity of TCRs on Foxp3(+)CD4(+) regulatory T cells exceeded the diversity of TCRs on Foxp3(-)CD4(+) naive T cells, even in mice that lack expression of tissue-specific antigens. Our results imply that higher TCR diversity on Foxp3(+) regulatory T cells helps these cells to match the specificities of autoreactive and naive T cells.     

Decrease of CD4(+)CD25(high)Foxp3(+) regulatory T cells and elevation of CD19(+)BAFF-R(+) B cells and soluble ICAM-1 in myasthenia gravis

Myasthenia gravis (MG) is caused by T-cell-dependent autoantibodies against muscle acetylcholine receptors (AChR) at the neuromuscular junction. Here, we adopted ELISA and flow cytometry techniques to measure the levels of Th1, Th2, Th3 cytokines, inflammatory cytokine and chemokine sICAM-1 and to analyze the phenotypes of CD4(+) and CD8(+) regulatory cells as well as the expression of BAFF-R on CD19(+) B cells in peripheral blood from 75 MG patients and 50 healthy controls. There were no differences in the levels of IL-2, IL-4, IL-10, IL-13, IFN-gamma, TNF-alpha, TGF-beta and sCTLA-4 in both sera and culture supernatants between MG patients and healthy controls. The level of IL-12 was decreased in culture supernatants from MG patients, and the level of sICAM-1 was increased in both sera and culture supernatants from MG patients. Although the populations of CD8(+)CD28(-) and CD8(+)CD122(+) regulatory T cells were not different between MG patients and healthy controls, MG patients exhibited the decrease of CD4(+)CD25(high)Foxp3(+) regulatory T cells and the increase of CD19(+)BAFF-R(+) B cells, revealing that MG patients should display the dysfunction of T cell balance and the activation of B cell maturation.     

Decreased frequencies of CD4+CD25+Foxp3+ cells and the potent CD103+ subset in peripheral lymph nodes correlate with autoimmune disease predisposition in some strains of mice

The CD4(+)CD25(+)Foxp3(+) cells are essential for regulation of the immune response, and the integrin, CD103 (α(E)β(7)), identifies a potent subset of these cells. Defects in CD4(+)CD25(+)Foxp3(+) cells are thought to contribute to susceptibility to autoimmune disease in predisposed individuals. Studies evaluating the quality and quantity of CD4(+)CD25(+)Foxp3(+) regulatory cell populations in the context of autoimmune disease susceptibility have been inconclusive, and few if any, have analyzed the CD103 subset. In this study, we analyzed regulatory T cells (Tregs) from different strains of mice with varying degrees of susceptibility to autoimmune disease. We found no differences in the ability of CD4(+)CD25(+) or the CD103(+) subset of Tregs from young female (NZB?×?NZW)F1 (BWF1), SJL, C57BL/6, or BALB/c mice to suppress CD4(+)CD25(-?) responders in vitro. Analysis of CD4(+)Foxp3(+) and CD4(+)CD25(+)CD103(+) cell frequencies in lymphoid organs revealed that BWF1 mice had dramatically lower percentages of both populations in the lymph node (LN) than the other strains, and lower percentages in the spleen in all but the C57BL/6 strain. We next determined whether these findings extended to another autoimmune-prone strain. Similar to BWF1 mice, percentages of CD4(+)Foxp3(+) and CD4(+)CD25(+)CD103(+) cells were significantly lower in predisease NOD mice. The low frequencies of CD4(+)Foxp3(+) and CD4(+)CD25(+)CD103(+) cells in BWF1 and NOD mice were not due to deficiencies in either thymic production or homeostatic proliferation. These data indicate that decreased percentages of CD4(+)Foxp3(+) cells and particularly, CD4(+)CD25(+)CD103(+) cells in LN correlate with the predisposition to spontaneous development of autoimmune disease.     

Induction of antigen-specific immunologic tolerance by in vivo and in vitro antigen-specific expansion of naturally arising Foxp3+CD25+CD4+ regulatory T cells

Naturally arising CD25(+)CD4(+) regulatory T (T(R)) cells can be exploited to establish immunologic tolerance to non-self antigens. In vivo exposure of CD25(+)CD4(+) T cells from normal naive mice to alloantigen in a T cell-deficient environment elicited spontaneous expansion of alloantigen-specific CD25(+)CD4(+) T(R) cells, which suppressed allograft rejection mediated by subsequently transferred naive T cells, leading to long-term graft tolerance. The expanded T(R) cells, which became CD25(low) in the absence of other T cells, stably sustained suppressive activity, maintained expression levels of other T(R) cell-associated molecules, including Foxp3, CTLA-4 and GITR, and could adoptively transfer tolerance to normal mice. Furthermore, specific removal of the T(R) cells derived from originally transferred CD25(+)CD4(+) T(R) cells evoked graft rejection in the long-term tolerant mice, indicating that any T(R) cells deriving from CD25(-)CD4(+) naive T cells minimally contribute to graft tolerance and that natural T(R) cells are unable to infectiously confer significant suppressive activity to other T cells. Similar antigen-specific expansion of T(R) cells can also be achieved in vitro by stimulating naturally present CD25(+)CD4(+) T cells with alloantigen in the presence of IL-2. The expanded CD25(+)CD4(+) T cells potently suppressed even secondary MLR in vitro and, by in vivo transfer, established antigen-specific long-term graft tolerance. Thus, in vivo or in vitro, direct or indirect ways of antigen-specific expansion of naturally arising Foxp3(+)CD25(+)CD4(+) T(R) cells can establish antigen-specific dominant tolerance to non-self antigens, and would also be instrumental in re-establishing self-tolerance in autoimmune disease and antigen-specific negative control of pathological immune responses.     

大鼠CD4+ CD25+ T调节细胞的分离培养及其功能分析

目的:研究大鼠CD4 CD25 T调节细胞(Tr)的分离培养,并对其功能进行初步分析。方法:无菌条件下切取大鼠脾脏分离脾淋巴细胞。用免疫磁珠细胞分离系统(MACS)分选CD4 CD25 T细胞,并以流式细胞术检测其纯度后,对其进行扩增。采用混合淋巴细胞反应研究CD4 CD25 Tr细胞对CD4 CD25-T细胞的免疫抑制作用。用ELISA法检测培养上清中IL-2、IFN-γ及IL-10水平的差异。结果:MACS分离的CD4 CD25 T细胞的纯度达86%~93%。该细胞与CD4 CD25-T细胞相比能特异性地表达Foxp3基因。体外培养中能明显抑制效应T细胞增殖及其分泌IFN-γ、IL-2,但其自身能分泌Th2型细胞因子IL-10。结论:采用MACS系统阴性加阳性分选,可高效快速的获得理想纯度和免疫抑制功能的大鼠CD4 CD25 T调节细胞,该细胞对CD4 CD25-T细胞具有明显的免疫抑制作用,并能特异性的表达Foxp3基因。