TGF-beta1 production by CD4+ CD25+ regulatory T cells is not essential for suppression of intestinal inflammation

Naturally occurring CD4+ CD25+ regulatory T cells (Treg) are potent suppressors of CD4+ and CD8+ T cell responses in vitro and inhibit several organ-specific autoimmune diseases. While most in vitro studies suggest that CD4+ CD25+ Treg cells adopt a cytokine-independent but cell contact-dependent mode of T cell regulation, their precise mechanism of suppression in vivo remains largely unknown. Here we examine the functional contribution of Treg cell-derived TGF-beta1 and effector T cell responsiveness to TGF-beta in CD4+ CD25+ T cell-mediated suppression of inflammatory bowel disease (IBD). We show that CD4+ CD25+ Treg cells from either TGF-beta1+/+ or neonatal TGF-beta1-/- mice can suppress the incidence and severity of IBD as well as colonic IFN-gamma mRNA expression induced by WT CD4+ CD25- effector T cells. Furthermore, TGF-beta-resistant Smad3-/- CD4+ CD25+ Treg cells are equivalent to WT Treg cells in their capacity to suppress disease induced by either WT or Smad3-/- CD4+ CD25- effector T cells. Finally, anti-TGF-beta treatment exacerbates the colitogenic potential of CD4+ CD25- effector T cells in the absence of CD4+ CD25+ Treg cells. Together, these data demonstrate that in certain situations CD4+ CD25+ T cells are able to suppress intestinal inflammation by a mechanism not requiring Treg cell-derived TGF-beta1 or effector T cell/Treg cell responsiveness to TGF-beta via Smad3.     

Hodgkin's reed-sternberg cell line (KM-H2) promotes a bidirectional differentiation of CD4+CD25+Foxp3+ T cells and CD4+ cytotoxic T lymphocytes from CD4+ naive T cells

A recent report revealed that a large population of Hodgkin's lymphoma-infiltrating lymphocytes (HLILs) consisted of regulatory T cells. In this study, we cocultured CD4+ naive T cells with KM-H2, which was established as a Hodgkin's Reed-Sternberg cell line, to clarify their ability to induce CD25+ Forkhead box P3+ (Foxp3+) T cells. The characteristic analyses of T cells cocultured with KM-H2 revealed the presence of CD4+CD25+ T cells. They expressed CTLA-4, glucocorticoid-induced TNFR family-related gene, and Foxp3 and could produce large amounts of IL-10. Conversely, KM-H2 also generated CD4+ CTLs, which expressed Granzyme B and T cell intracellular antigen-1 in addition to Foxp3+ T cells. They exhibit a strong cytotoxic effect against the parental KM-H2. In conclusion, KM-H2 promotes a bidirectional differentiation of CD4+ naive T cells toward Foxp3+ T cells and CD4+ CTLs. In addition to KM-H2, several cell lines that exhibit the APC function were able to generate Foxp3+ T cells and CD4+ CTLs. Conversely, the APC nonfunctioning cell lines examined did not induce both types of cells. Our findings suggest that the APC function of tumor cells is essential for the differentiation of CD4+ naive T cells into CD25+Foxp3+ T cells and CD4+ CTLs and at least partly explains the predominance of CD25+Foxp3+ T cells in HLILs and their contribution to a better prognosis. Therefore, in APC-functioning tumors, including classical Hodgkin lymphomas, which generate Foxp3+ T cells and CD4+ CTLs, these T cell repertories play a beneficial role synergistically in disease stability.     

CD4+ T-cell subsets in intestinal inflammation

Intestinal CD4⁺ T cells are essential mediators of immune homeostasis and inflammation. Multiple subsets of CD4⁺ T cells have been described in the intestine, which represents an important site for the generation and regulation of cells involved in immune responses both within and outside of the gastrointestinal tract. Recent advances have furthered our understanding of the biology of such cells in the intestine. Appreciation of the functional roles for effector and regulatory populations in health and disease has revealed potential translational targets for the treatment of intestinal diseases, including inflammatory bowel disease. Furthermore, the role of dietary and microbiota-derived factors in shaping the intestinal CD4⁺ T-cell compartment is becoming increasingly understood. Here, we review recent advances in understanding the multifaceted roles of CD4⁺ T cells in intestinal immunity.     

Vasoactive intestinal peptide generates CD4+CD25+ regulatory T cells in vivo

CD4+CD25+ regulatory T (Treg) cells control the immune response to a variety of antigens, including self-antigens, and several models support the idea of the peripheral expansion of CD4+CD25+ Treg cells. Although hormones such as estrogen and alpha-melanocyte-stimulating hormone have been recently reported to expand the CD4+CD25+ Foxp3-expressing Treg cell compartment, little is known about the endogenous factors and mechanisms controlling the peripheral expansion of CD4+CD25+ Treg cells. In this study, we report on the capacity of the vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide, to induce functional Treg cells in vivo. The administration of VIP together with specific antigen to T cell receptor (TCR)-transgenic (Tg) mice results in the expansion of the CD4+CD25+, Foxp-3/neuropilin 1-expressing T cells, which inhibit responder T cell proliferation through direct cellular contact. In addition to the increase in the number of CD4+CD25+ Treg cells, VIP induces more efficient suppressors on a per-cell basis. The VIP-generated CD4+CD25+ Treg cells transfer suppression, inhibit delayed-type hypersensitivity in TCR-Tg hosts, and prevent graft-versus-host disease in irradiated hosts reconstituted with allogeneic bone marrow.     

Cure of innate intestinal immune pathology by CD4+CD25+ regulatory T cells

CD4+CD25+ regulatory T (T(R)) cells are a naturally occurring population of T cells that suppress the development of a variety of pathological immune responses. However, as human inflammatory diseases are usually not diagnosed until after the onset of clinical symptoms, it is of great interest to determine whether CD4+CD25+ T(R) cells can reverse established pathology. To examine this question we have utilized a murine model of human inflammatory bowel disease (IBD), where pathology is triggered by infection of immune deficient RAG-/- mice with the pathogenic bacterium Helicobacter hepaticus. Here we demonstrate that adoptively transferred CD4+CD25+ T(R) cells can cure established intestinal inflammation that is mediated by innate immune activation in H. hepaticus-infected RAG-/- mice. CD4+CD25+ T(R) cell-mediated amelioration of innate intestinal pathology was accompanied by a reversal in systemic innate immune activation, but did not involve any detectable anti-bacterial effects, as bacterial colonization levels were unchanged. Cure of established pathology was not achieved using subpopulations of CD4+CD25- T cells, further emphasizing the enhanced regulatory activity of CD4+CD25+ T(R) cells.     

IL-2抑制小鼠CD4~+ CD62L~+ T细胞分化为Th17细胞

目的研究IL-2对小鼠脾脏CD4+CD62L+T细胞在体外向Th17细胞分化的作用。方法免疫磁珠法分选C57BL/6小鼠脾脏CD4+CD62L+T细胞,于抗体包被的培养板中培养3 d,实验分为对照组和IL-2处理组。对照组为经典Th17诱导分化培养基,IL-2处理组在对照组基础上于培养体系中添加IL-2。CFSE染色检测细胞增殖,Annexin V-PI法检测细胞凋亡,ELISA检测培养上清中IL-17A的浓度,荧光定量PCR检测Rorγt mRNA的表达,流式细胞术检测CD4~+IL-17~+Th17的生成比例以及Rorγt的表达。结果磁珠分选的小鼠脾脏CD4+CD62L+nave T细胞纯度高于95%。与对照组相比,IL-2处理组细胞数目明显增多,增殖能力明显增强(P0.05),细胞凋亡比例降低(P0.05);IL-2处理组培养上清中IL-17A的浓度明显降低(P0.05),且CD4+IL-17+细胞比例下降,其特异性转录因子Rorγt的表达水平也显著降低(P0.05)。结论 IL-2在CD4+CD62L+T细胞分化为Th17的过程中,能够促进T细胞的增殖并且抑制Th17的分化。     

Activated CD4+ CD25+ T cells suppress antigen-specific CD4+ and CD8+ T cells but induce a suppressive phenotype only in CD4+ T cells

CD4(+) CD25(+) regulatory T cells are increasingly recognized as central players in the regulation of immune responses. In vitro studies have mostly employed allogeneic or polyclonal responses to monitor suppression. Little is known about the ability of CD4(+) CD25(+) regulatory T cells to suppress antigen-specific immune responses in humans. It has been previously shown that CD4(+) CD25(+) regulatory T cells anergize CD4(+) T cells and turn them into suppressor T cells. In the present study we demonstrate for the first time in humans that CD4(+) CD25(+) T cells are able to inhibit the proliferation and cytokine production of antigen specific CD4(+) and CD8(+) T cells. This suppression only occurs when CD4(+) CD25(+) T cells are preactivated. Furthermore, we could demonstrate that CD4(+) T-cell clones stop secreting interferon-gamma (IFN-gamma), start to produce interleukin-10 and transforming growth factor-beta after coculture with preactivated CD4(+) CD25(+) T cells and become suppressive themselves. Surprisingly preactivated CD4(+) CD25(+) T cells affect CD8(+) T cells differently, leading to reduced proliferation and reduced production of IFN-gamma. This effect is sustained and cannot be reverted by exogenous interleukin-2. Yet CD8(+) T cells, unlike CD4(+) T cells do not start to produce immunoregulatory cytokines and do not become suppressive after coculture with CD4(+) CD25(+) T cells.     

Urothelial antigen-specific CD4+ T cells function as direct effector cells and induce bladder autoimmune inflammation independent of CD8+ T cells

The role of CD4(+) T cells in bladder autoimmune inflammation has not been identified because of the lack of a proper animal model. We investigated CD4(+) T-cell responses to bladder urothelial ovalbumin (OVA), a model self-antigen (Ag), in transgenic URO-OVA mice. The expression of bladder urothelial OVA rendered mice unresponsive to OVA and resulted in quick clearance of Ag-specific CD4(+) T cells. Adoptive transfer of naive OVA-specific CD4(+) T cells led to exogenous T-cell proliferation, activation, and bladder infiltration but no inflammatory induction. In contrast, adoptive transfer of preactivated OVA-specific CD4(+) T cells induced bladder inflammation. Studies further demonstrated that CD4(+) T cells induced bladder inflammation in URO-OVA mice depleted of CD8(+) T cells or deficient in the recombinase activating gene-1 (Rag-1(-/-)). These results indicate that urothelial Ag-specific CD4(+) T cells can function as direct effector cells to induce bladder autoimmune inflammation independent of CD8(+) T cells.     

Competition controls the rate of transition between the peripheral pools of CD4+CD25- and CD4+CD25+ T cells

Recent reports have hinted that it is possible to regenerate CD4+CD25+ regulatory T cells (Treg) from CD4+CD25- cells, a phenomenon termed conversion. We evaluated the relative contribution of this process to the Treg pool by transferring purified populations of CD4+ T cells into T cell-deficient mice. We report that conversion of CD25- cells into the CD4+CD25+Treg pool is minor if other bona fide CD25+ Tregs are present. Moreover, in the same hosts, the loss of CD25 expression by a population of Tregs also decreases in the presence of co-injected CD4+CD25- cells. Thus, the rate of exchange between CD25- and CD25+ T-cell populations is determined by the presence or absence of T-cell competitors. Our results attest for the role of competition in the contribution of different T-cell subsets for the regeneration of the peripheral CD4+ T-cell pool during lymphopenia.     

Effect of intestinal microbiota on the induction of regulatory CD25+ CD4+ T cells

When oral tolerance was induced in either specific pathogen-free (SPF) or germ-free (GF) mice, ovalbumin (OVA) feeding before immunization induced oral tolerance successfully in SPF mice. On the other hand, OVA-specific immunoglobulin G1 (IgG1) and IgE titres in OVA-fed GF mice were comparable to those in phosphate-buffered saline-fed GF mice, thus demonstrating that oral tolerance could not be induced in GF mice. The frequencies of CD25(+) CD4(+)/CD4(+) cells in the mesenteric lymph node (MLN) and the absolute number of CD25(+) CD4(+) cells in the Peyer's patches and MLN of naive GF mice were significantly lower than those in naive SPF mice. In an in vitro assay, the CD25(+) CD4(+) cells from the naive SPF mice suppressed more effectively the proliferation of responder cells in a dose-dependent manner than those from the GF mice. In addition, the CD25(+) CD4(+) regulatory T (T(reg)) cells from the naive SPF mice produced higher amounts of interleukin (IL)-10 and transforming growth factor (TGF)-beta than those from the GF mice. When anti-TGF-beta neutralizing antibody, but not anti-IL-10 neutralizing antibody, was added to the in vitro proliferation assay, the suppressive effect of the CD25(+) CD4(+) T(reg) cells from the SPF mice was attenuated to the same level as that of the CD25(+) CD4(+) cells from the GF mice. In conclusion, the TGF-beta-producing CD25(+) CD4(+) T(reg) cells from the MLN of SPF mice played a major role in oral tolerance induction. In addition, as the regulatory function of the CD25(+) CD4(+) cells from the naive GF mice was much lower than that of the CD25(+) CD4(+) T(reg) cells from the SPF mice, indigenous microbiota are thus considered to contribute to the induction and maintenance of CD25(+) CD4(+) T(reg) cells.     

CD70+ antigen-presenting cells control the proliferation and differentiation of T cells in the intestinal mucosa

One unresolved issue in gut immunity is how mucosal T lymphocytes are activated and which antigen-presenting cell (APC) is critical for the regulation of this process. We have identified a unique population of APCs that is exclusively localized in the lamina propria. These APCs constitutively expressed the costimulatory molecule CD70 and had antigen-presenting functions. After oral infection of mice with Listeria monocytogenes, proliferation and differentiation of antigen-specific T cells occurred in the gut mucosa in situ and blockade of CD70 costimulation abrogated the mucosal T cell proliferation and effector functions. Thus, a potent CD70-dependent stimulation via specialized tissue-specific APCs is required for the proliferation and differentiation of gut mucosal T cells after oral infection.     

CD4+CD25+ T cells as immunoregulatory T cells in vitro

We have further characterized the in vitro phenotype and function of anergic and suppressive CD4(+)25(+) T cells. Following TCR ligation, DO.11.10 CD4(+)25(+) T cells suppress the activation of OT-1 CD8(+)25(-) T cells in an antigen nonspecific manner. Although suppression was seen when using a mixture of APC from both parental strains, it was very much more marked when using F1 APC. APC pretreated with, and then separated from CD4(+)25(+) T cells did not have diminished T cell costimulatory function, suggesting that APC are not the direct targets of CD4(+)25(+) T cell regulation. CTLA-4 blockade failed to abrogate suppression by CD4(+)25(+) T cells in mixing experiments. Although CD4(+)25(+) T cells failed to respond following cross-linking of TCR, they could be induced to proliferate following the addition of exogenous IL-2, allowing the generation of a T cell line from CD4(+)25(+) T cells. After the first in vitro restimulation, CD4(+)25(+) T cells were still anergic and suppressive following TCR engagement. However, after three rounds of restimulation, their anergic and suppressive status was abrogated.     

Conventional CD4+ T cells regulate IL-22-producing intestinal innate lymphoid cells

The innate and adaptive immune systems in the intestine cooperate to maintain the integrity of the intestinal barrier and to regulate the composition of the resident microbiota. However, little is known about the crosstalk between the innate and adaptive immune systems that contribute to this homeostasis. We find that CD4+ T cells regulate the number and function of barrier-protective innate lymphoid cells (ILCs), as well as production of antimicrobial peptides (AMPs), Reg3γ and Reg3β. RAG1−/− mice lacking T and B cells had elevated ILC numbers, interleukin-22 (IL-22) production, and AMP expression, which were corrected by replacement of CD4+ T cells. Major histocompatibility class II−/− (MHCII−/−) mice lacking CD4+ T cells also had increased ILCs, IL-22, and AMPs, suggesting that negative regulation by CD4+ T cells occurs at steady state. We utilized transfers and genetically modified mice to show that reduction of IL-22 is mediated by conventional CD4+ T cells and is T-cell receptor dependent. The IL-22-AMP axis responds to commensal bacteria; however, neither the bacterial repertoire nor the gross localization of commensal bacteria differed between MHCII+/− and MHCII−/− littermates. These data define a novel ability of CD4+ T cells to regulate intestinal IL-22-producing ILCs and AMPs.     

CTLA-4 x Ig converts naive CD4+CD25- T cells into CD4+CD25+ regulatory T cells

CTLA-4 x Ig was originally designed as an immunosuppressive agent capable of interfering with the co-stimulation of T cells. In the present study, we demonstrate that CTLA-4 x Ig, in combination with TCR ligation, has the additional capacity to convert naive CD4+CD25- T cells into Foxp3+ regulatory T (T(reg)) cells, as well as to expand their numbers. The CD4+CD25+Foxp3+ T(reg) generated by CTLA-4 x Ig treatment in vitro potently suppress effector T cells. Extending this in vivo, we show that systemic administration of CTLA-4 x Ig increases the percentage of CD4+CD25(hi)Foxp3+ cells within mixed lymphocyte reaction-induced murine lymph nodes. Significantly, the in vitro conversion of naive CD4+CD25- T cells into T(reg) cells is antigen-presenting cell (APC) dependent. This finding, together with the further observation that this conversion can also be driven in vitro by an antibody that engages B7-2 ligand, suggests that CTLA-4 x Ig-driven T(reg) induction may be predicated upon active CTLA-4 x Ig to B7-2 signaling within APC, which elicits from them T(reg)-inducing potential. These findings extend CTLA-4 x Ig's functional repertoire, and at the same time, reinforce the concept that T cell anergy and active suppression are not entirely distinct processes and may be linked by some common molecular triggers.     

Human CD4+CD25+ regulatory T cells

In this report, we review studies of human CD4+CD25+ regulatory T cells (T-reg). Although lagging a few years behind the discovery of these cells in the mouse, the equivalent population of CD4+CD25+ regulatory T cells has also been isolated from human peripheral blood, thymus, lymph nodes and cord blood. In general, the characteristics of this T cell subset are strikingly similar between mouse and man. In the recent explosion of research reports on human CD4+CD25+ cells, although the majority of the characteristics ascribed to these cells appear to be consistent, contrasting results have been found primarily in regards to potential involvement of TGFbeta and production of IL-10. One explanation for this variability may reside in the fact that markedly different techniques are used to isolate human CD4+CD25+ T-reg cells and thus may result in the comparison of T-reg populations that differ in cellular composition and/or activation state. Another potential explanation for differences in human T-reg function may rest on the extreme variability of the culture conditions and TCR stimuli that have been used to test the functional properties of these cells in vitro. The strength of the TCR signal provided to the culture greatly affects the functional outcome of the co-culture and can result in the difference between suppression and full activation. Surprisingly, it appears that stronger stimulation has a greater and more rapid effect on the T-resp cell than on the T-reg cell as it causes T-resp cells to quickly become resistant to suppression. Thus, the details of in vitro culture conditions may at least partially account for disparate findings in regard to the functional characterization of human CD4+CD25+ cells. Here we review the evidence regarding the identification of human CD4+CD25+ regulatory T cells and their possible mechanism(s) of function.     

Neuropilin-1+T细胞与CD4+CD25+调节性T细胞的比较

目的:分析Neuropilin-1 T细胞(Nrp-1 T细胞)与经典CD4 CD25 调节性T细胞(Treg)的关系并比较二者的免疫调节作用。方法:流式细胞术分析BALB/c小鼠脾脏T细胞上Nrp-1与CD4、CD25的表达关系并分选Nrp-1 T细胞及CD4 CD25 Treg,通过B16-F10-luc-G5黑色素肿瘤细胞体外培养实验并利用萤光成像系统,观察比较两种细胞对NK细胞杀伤B16-F10-luc-G5黑色素瘤细胞的影响。结果:CD4 CD25 Treg中表达Nrp-1的比例为(27.28±1.17)%,明显高于CD4 CD25-T细胞的(1.63±0.08)%(P<0.01);在体外实验中,Nrp-1 T细胞与CD4 CD25 Treg均能抑制NK细胞杀伤B16-F10-luc-G5黑色素瘤细胞,Nrp-1 T细胞组的肿瘤细胞数目在6、24、48、72h分别为984±15、1015±14、1261±21、1323±38,高于CD4 CD25 Treg组的931±4、983±8、1201±18、1256±18,两组肿瘤细胞数目在各时间点均有统计学意义(P<0.01)。结论:经典CD4 CD25 Treg中表达Nrp-1的细胞比例较高,Nrp-1 T细胞有负性免疫调节作用,抑制功能比CD4 CD25 Treg更强,可以作为一类新的Treg亚群。