Antigen-specific T cell suppression by human CD4+CD25+ regulatory T cells

Anergic/suppressive CD4+CD25+ T cells have been proposed to play an important role in the maintenance of peripheral tolerance. Here we demonstrate that in humans these cells suppress proliferation to self antigens, but also to dietary and foreign antigens. The suppressive CD4+CD25+ T cells display a broad usage of the T cell receptor Vbeta repertoire,suggesting that they recognize a wide variety of antigens. They reside in the primed/memory CD4+CD45RO+CD45RB(low) subset and have short telomeres, indicating that these cells have the phenotype of highly differentiated CD4+ T cells that have experienced repeated episodes of antigen-specific stimulation in vivo. This suggests that anergic/suppressive CD4+CD25+ T cells may be generated in the periphery as a consequence of repeated antigenic encounter. This is supported by the observation that highly differentiated CD4+T cells can be induced to become anergic/suppressive when stimulated by antigen presented by non-professional antigen-presenting cells. We suggest that besides being generated in the thymus, CD4+CD25+ regulatory T cells may also be generated in the periphery. This would provide a mechanism for the generation of regulatory cells that induce tolerance to a wide array of antigens that may not be encountered in the thymus.     

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.     

Inhibition of phosphoantigen-mediated gammadelta T-cell proliferation by CD4+ CD25+ FoxP3+ regulatory T cells

Tumour growth promotes the expansion of CD4(+) CD25(+) FoxP3(+) regulatory T cells (Tregs) which suppress various arms of immune responses and might therefore contribute to tumour immunosurveillance. In this study, we found an inverse correlation between circulating Treg frequencies and phosphoantigen-induced gammadelta T-cell proliferation in cancer patients, which prompted us to address the role of Tregs in controlling the gammadelta T-cell arm of innate immune responses. In vitro, human Treg-peripheral blood mononuclear cell (PBMC) co-cultures strongly inhibited phosphoantigen-induced proliferation of gammadelta T cells and depletion of Tregs restored the impaired phosphoantigen-induced gammadelta T-cell proliferation of cancer patients. Tregs did not suppress other effector functions of gammadelta T cells such as cytokine production or cytotoxicity. Our experiments indicate that Tregs do not mediate their suppressive activity via a cell-cell contact-dependent mechanism, but rather secrete a soluble non-proteinaceous factor, which is independent of known soluble factors interacting with amino acid depletion (e.g. arginase-diminished arginine and indolamine 2,3-dioxygenase-diminished tryptophan) or nitric oxide (NO) production. However, the proliferative activity of alphabeta T cells was not affected by this cell-cell contact-independent suppressive activity induced by Tregs. In conclusion, these findings indicate a potential new mechanism by which Tregs can specifically suppress gammadelta T cells and highlight the strategy of combining Treg inhibition with subsequent gammadelta T-cell activation to enhance gammadelta T cell-mediated immunotherapy.     

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.     

Distinct roles of CTLA-4 and TGF-beta in CD4+CD25+ regulatory T cell function

Both CTLA-4 and TGF-beta have been implicated in suppression by CD4+CD25+ regulatory T cells (Treg). In this study, the relationship between CTLA-4 and TGF-beta in Treg function was examined. Blocking CTLA-4 on wild-type Treg abrogated their suppressive activity in vitro, whereas neutralizing TGF-beta had no effect, supporting a TGF-beta-independent role for CTLA-4 in Treg-mediated suppression in vitro. In CTLA-4-deficient mice, Treg development and homeostasis was normal. Moreover, Treg from CTLA-4-deficient mice exhibited uncompromised suppressive activity in vitro. These CTLA-4-deficient Treg expressed increased levels of the suppressive cytokines IL-10 and TGF-beta, and in vitro suppression mediated by CTLA-4(-/-) Treg was markedly reduced by neutralizing TGF-beta, suggesting that CTLA-4-deficient Treg develop a compensatory suppressive mechanism through CTLA-4-independent production of TGF-beta. Together, these data suggest that CTLA-4 regulates Treg function by two distinct mechanisms, one during functional development of Treg and the other during the effector phase, when the CTLA-4 signaling pathway is required for suppression. These results help explain contradictions in the literature and support the existence of functionally distinct Treg.     

Control of NK cell functions by CD4+CD25+ regulatory T cells

Regulatory T cells (Treg) are key players in the maintenance of peripheral tolerance. As a result of suppressive effects on CD4+ and CD8+ effector T cells, Treg control the adaptive immune system and prevent autoimmunity. In addition, they inhibit B lymphocytes, dendritic cells, and monocytes/macrophages. It is interesting that several recent papers show that CD4+CD25+ Treg are also able to inhibit NK cells. Thus, Treg exert their control on immune responses from the onset (triggering of innate immune cells) to the effector phase of adaptive immunity (B and T cell-mediated responses). That Treg inhibit NK cells suggests that their uncontrolled activation might break self-tolerance and induce "innate" autoimmune pathology. Conversely, Treg-mediated suppression of NK cell functions might have negative effects, as these cells are important in defense against infections and cancer. It is conceivable that Treg might dampen efficient activation of NK cells in these diseases.     

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.     

CD4-mediated functional activation of human CD4+CD25+ regulatory T cells

Naturally occurring CD4(+)CD25(+)FoxP3(+) regulatory T cells (CD25(+) Tregs) constitute a specialized population of T cells that is essential for the maintenance of peripheral self-tolerance. The immune regulatory function of CD25(+) Tregs depends upon their activation. We found that anti-CD4 antibodies activate the suppressive function of human CD25(+) Tregs in a dose-dependent manner. We demonstrate that CD4-activated CD25(+) Tregs suppress the proliferation of CD4(+) and CD8(+) T cells, their IL-2 and IFN-gamma production as well as the capacity of CD8(+) T cells to re-express CD25. By contrast, anti-CD4 stimulation did not induce suppressive activity in conventional CD4(+) T cells. These results identify CD4 as a trigger for the suppressive function of CD25(+) Tregs and suggest a possible CD4-mediated exploitation of these cells.     

卵巢癌细胞培养上清诱导CD4+CD25-T细胞分化为CD4+CD25+调节性T细胞

目的研究卵巢癌细胞培养上清液是否能诱导外周血CD4^＋CD25^- T细胞转变为CD4^＋CD25^＋调节性T细胞。方法将外周血CD4^＋CD25^- T细胞分离后，对照组用CD3和CD28单抗活化，实验组在对照基础上加用卵巢癌细胞株SKOV3培养上清，72h后分离各组的CD25^＋和CD25^-T细胞，溴化脱氧尿嘧啶掺入标记法测定增殖能力及对静息的自体同源CD4^＋CD25^- T细胞的增殖抑制能力，流式细胞仪测定细胞糖皮质激素诱发型TNF受体（glucocorticoid-induced TNFR,GITR）与CTLA-4分子的表达，RT-PCR检测细胞卿mRNA的表达。结果与对照组相反，实验组的CD4^＋CD25^＋T细胞具有免疫抑制功能，自身增殖能力下降，GITR和CTLA-4分子的表达和CD4^＋CD25^＋调节性T细胞相似，并被诱导表达转录因子Foxp3 mRNA。结论卵巢癌细胞分泌的可溶性物质能诱导外周血CD4^＋CD25^-T细胞转化为CD4^＋CD25^＋调节性T细胞。     

MicroRNAs are implicated in the suppression of CD4CD25 conventional T cell proliferation by CD4CD25 regulatory T cells

CD4⁺CD25⁺ regulatory T cells (Tregs) are critical for sustaining immunological homeostasis. CD4⁺CD25⁻ conventional T cells (Tcons) are the progenitors of populations including Th1, Th2, Th17, Tfh, and Treg cells. Suppression of Tcons proliferation by Tregs requires cell–cell contact and/or is mediated by immunosuppressive soluble factors. However, upon receiving suppressive signals from Tregs, the exact molecular responses in Tcons remain elusive. Here, by using microRNA (miRNA) microarray preliminary screening and quantitative RT-PCR (qRT-PCR) validation, we showed that paralleled with the suppression of the Tcons proliferation, miR-146a was induced but miR-106b and miR-21 were reduced in Tcons upon receiving suppressive signals from Tregs. Moreover, our results showed that either increase of miR-146a or decrease of miR-106b and miR-21 by using miRNA mimics or inhibitors in Tcons significantly enhanced the suppression triggered by Tregs. However, decrease of miR-146a or increase of miR-106b and miR-21 in Tcons impaired the suppression triggered by Tregs. Collectively, our findings demonstrate the roles of miR-146a, miR-106b and miR-21 in Tcons in regulating Treg-triggered immune-suppression.     

Human leukocyte antigen class I-restricted immunosuppression by human CD8+ regulatory T cells requires CTLA-4-mediated interaction with dendritic cells

We previously reported autoreactive CD8(+) regulatory T cells (Tregs) that were expanded and cloned from human peripheral blood by coculture with autologous dendritic cells (DC). Here we demonstrate that these CD8(+) Tregs require human leukocyte antigen (HLA)-class I restricted activation and then mediate cell-contact-dependent suppression of CD4(+) T cells. CD8(+) Tregs interacted with DC to suppress T-cell responses but DC were not irreversibly altered by this interaction because they could subsequently stimulate CD4(+) T cells normally. The ability of DC to form conjugates with CD4(+) T cells was reduced in the presence of CD8(+) Tregs. Suppression was blocked by Abs to CD80 and CTLA-4, implicating CTLA-4:CD80 interactions in the function of CD8(+) Tregs. CD8(+) Tregs rapidly express very high levels of surface CTLA-4 following activation compared with conventional T cells. Related to this, the expression of TRAT1 mRNA (T-cell receptor interacting molecule, or TRIM) was highly upregulated in microarray analysis of CD8(+) Tregs compared with conventional cytotoxic or nonregulatory CD8(+) T cells. TRIM acts to chaperone CTLA-4 transport to the cell surface; this function would be required to account for the phenotypic and functional properties of CD8(+) Tregs.     

CD4+CD25+免疫调节性T细胞对CD4+T细胞介导的移植物抗宿主病预防作用的研究

目的研究CD4+CD25+免疫调节性T(Treg)细胞在小鼠骨髓移植后,对移植物抗宿主病的预防作用及其作用机制.方法用C3H(H-2k)小鼠骨髓作为供体,提取C3H(H-2k)小鼠CD4+T及CD4+CD25+T细胞,C3H×B6(H-2k/b)F1小鼠为骨髓移植的受者.在受者接受致死量全身放射后,输注供者去除T细胞的骨髓(ATBM),使其造血功能重建(ATBM组).于不同的实验组给予CD4+(CD4组)T细胞,CD4+CD25+T(CD25组)或二者同时输注(CD4/CD25组).观察各组小鼠移植物抗宿主病(GVHD)的发生率.结果所有10只ATBM组小鼠至骨髓移植后60天仍全部存活,无GVHD发生;所有10只CD4组小鼠在骨髓移植10天内全部死于GVHD(P＜0.01);所有5只CD25组小鼠于骨髓移植后60天仍全部存活,无明显GVHD发生(P＞0.05);同样,所有6只CD4/CD25组小鼠至骨髓移植后仍全部存活,无明显GVHD发生(P＞0.05).结论在同种异基因小鼠的骨髓移植模型中,CD4+CD25+T不诱导GVHD的发生,并有预防CD4+T细胞介导的GVHD发生的作用.     

CD4+CD25high regulatory T cells in human autoimmune diabetes

In mouse models, CD4+CD25+ T cells are involved in maintenance of peripheral tolerance. In humans, a subset of CD4+CD25+ T cells expressing high levels of CD25 (CD4+CD25high) with characteristics identical to murine CD4+CD25+ was recently described. We evaluated the characteristics of CD4+CD25high T cells in peripheral blood of type 1 diabetic subjects (T1D) and normal controls (NC). In contrast to a previous report, we found no difference in the number of CD4+CD25high and CD4+CD25+ T cells between T1D and NC. We confirmed previous studies that demonstrated that human CD4+CD25high cells can suppress the proliferation of co-cultured CD4+CD25- cells stimulated in conditions of sub-maximal cross-linking by anti-CD3 either with or without anti-CD28. However, we did not observe statistical differences between the normal controls and the chronic diabetic subjects we tested. Culturing of these cell populations did not appear to affect their ability to suppress proliferation in both groups. In conclusion, we found no significant differences in number or in vitro regulatory function of CD4+CD25high in chronic human T1D subjects.     

Modulation of monocyte/macrophage function by human CD4+CD25+ regulatory T cells

The suppressive effects of CD4+CD25+ regulatory T cells (Tregs) on T cells have been well documented. Here we investigated whether human CD4+CD25+ Tregs can inhibit the proinflammatory properties of monocytes/macrophages. Monocytes and T cells were isolated from peripheral blood of healthy volunteers by magnetic cell separation and cocultured for 40 h. Monocytes were analyzed directly for cytokine production and phenotypic changes or repurified and used in T-cell stimulation and lipopolysaccharide challenge assays. Coculture with CD4+CD25+ Tregs induced minimal cytokine production in monocytes, whereas coculture with CD4+CD25- T cells resulted in large amounts of proinflammatory (tumor necrosis factor-alpha, interferon-gamma, interleukin-6) and regulatory (interleukin-10) cytokines. Importantly, when these CD4+CD25+ Treg-treated monocytes were repurified after coculture and challenged with lipopolysaccharide, they were severely inhibited in their capacity to produce tumor necrosis factor-alpha and interleukin-6 compared with control-treated monocytes. In addition, monocytes that were precultured with CD4+CD25+ Tregs displayed limited upregulation of human leukocyte antigen class II, CD40 and CD80, and downregulation of CD86 compared with control-treated monocytes. This altered phenotype had functional consequences, as shown by the reduction in T cell-stimulatory capacity of Treg-treated monocytes. Together, these data demonstrate that CD4+CD25+ Tregs can exert direct suppressive effects on monocytes/macrophages, thereby affecting subsequent innate and adaptive immune responses.     

天然CD4+CD25+Treg细胞的研究进展

天然CD4+ CD25+ Treg细胞在针对自身抗原和外来抗原的免疫应答中起关键控制作用,其缺乏或功能性的缺陷将导致多重病理性的失调.本文就近年在其产生、作用机制以及与免疫耐受的诱导关系等方面的研究进展进行了综述.     

免疫磁珠法分离人外周血CD4+CD25+调节性T细胞

目的 建立人外周血单个核细胞中CD4 CD25 调节性T细胞(regulatery T cells,Treg)免疫磁性细胞分离力(megnetic activated cell sorting,MACS),并鉴定其分离效率.方法 采用免疫磁珠两步法(即阴性分选和阳性分选2步)分离人周血单个核细胞中的CD4 CD25 调节性T细胞,首先采用生物素标记的鸡尾酒抗体和抗生物素标记的磁珠阴性分选CIM细胞,再用抗CD25 的磁珠阳性分选CD4 CD25 T细胞.分离后的细胞经抗体染色后再通过流式细胞仪检测其分离纯度;内因子染色检测其转录因子FOXV3的表达频率;台盼蓝染色检测细胞的存活率;3H-TdR掺入法检测其对CD4 CD25-T细脆殖抑制效应.结果 阴性分选CD4 T细胞的纯度为(92.2±1.7)%,阳性分选后CD4 CD25 Treg细胞的纯度(95.1±1.2)%.胞内因子染色FOXF3在CD4 CD25 Treg细胞中的表达率为(80.4±1.2)%,台盼蓝染色细胞存活率为(95.6±3.3)%.3H-TdR掺入法检测其对CIM CD25-T细胞具有明显的抑制作用.结论 采用免疫磁性细胞分离技术能够高效、快地得到一群纯度高并且细胞活力好的CD4 CIY25 Treg,为进一步研究其功能提供了方便.     

Altered proximal T cell receptor (TCR) signaling in human CD4+CD25+ regulatory T cells

CD4+CD25+ regulatory T cells play an important role in peripheral tolerance. Upon T cell receptor (TCR)-mediated activation, the cells fail to proliferate but are induced to have a suppressor function. The intracellular signaling events that lead to their responses have not been elucidated. In this study, we have examined the proximal TCR signaling events in freshly isolated human CD4+CD25+ regulatory T cells after TCR ligation. In contrast to CD4+CD25- T cells, TCR ligation of CD4+CD25+ regulatory T cells by anti-CD3 cross-linking resulted in a lower calcium influx and extracellular signal-regulated kinase 1/2 phosphorylation. Examination of the CD3zeta chain phosphorylation status indicated that CD4+CD25+ regulatory T cells have poor phosphorylation of the protein and consequently, reduced recruitment of zeta-associated protein-70 to the TCR immunoreceptor tyrosine motif. The adaptor protein, Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa, which relays signals to downstream signaling components, also showed reduced phosphorylation, which correlated with reduced VAV guanine nucleotide exchange factors association. Consistent with other findings, the defect is accompanied with impaired actin cap formation, implicating a failure of actin remodeling of the cells. Together, our results demonstrate that CD4+CD25+ regulatory T cells have altered TCR proximal signaling pathways, which could be critical for inducing the distinct behavior of these cells.     

Development and function of CD25+CD4+ regulatory T cells

The essential role played by CD25(+)CD4(+) regulatory T cells (T(R) cells) in the control of physiological as well as pathological immunity is now well established, but many aspects of their biology still remain unclear. One of the unresolved issues regards their development: where does this occur, what signals are required, and how do T(R) cells fit into the larger taxonomy of the T-cell family? Recent data has begun to shed light on the development and function of these important cells.