CD4+CD25+CD127low/-T淋巴细胞在类风湿关节炎患者外周血中的表达及意义

目的 检测调节性T淋巴细胞新旧标志在活动性类风湿关节炎(RA)患者外周血中的表达,探讨用CD127代替FOXP3对调节性T淋巴细胞进行研究的可能性.方法 选取活动性RA患者32例,其中未经缓解病情抗风湿药(DMARDs)治疗者20例,经DMARDs治疗效果不佳者12例,以25例原发性干燥综合征(pSS)患者和24名健康人作为对照,采用流式细胞术检测外周血中CD4+CD25high、CD4+CD25+CD127low/-及CD4*CD25+FOXP3+T淋巴细胞的比例,同时检测外周血C反应蛋白(CRP)、红细胞沉降率(ESR)、免疫球蛋白、补体等水平并进行相关性分析.结果 RA患者CD4+CD25highT淋巴细胞百分率与健康对照组相比差异无统计学意义(P>0.05).其CD4+CD25+FOXP3+T淋巴细胞与CD+CD25+CD127low/-T淋巴细胞的百分率均低于健康对照组(P<0.05),但CD4+D25+FOXP3+、CD4+CD25+CD127low/-T淋巴细胞在RA患者与pSS患者中的表达差异无统计学意义(P>0.05);这三群细胞的表达水平在RA未经治疗组和治疗效果不佳组之间差异均无统计学意义(P>0.05). RA患者CD4+CD25+FOXP3+T淋巴细胞与CD4+CD25+CD127low/-T淋巴细胞之间呈明显正相关(r=0.698,P=0.001),但这两群细胞与患者的CRP、ESR等水平及抗环瓜氨酸肽(CCP)抗体、类风湿因子(RF)均无相关性(P>0.05).结论 活动性RA患者外周血中,CD4+CD25+CD127low/-T淋巴细胞的百分率降低,并且与CD4+CD25+FOXP3+T淋巴细胞呈显著正相关性,提示CD127可能被作为Treg细胞中FOXP3的替代标记.     

强直性脊柱炎患者外周血CD4+CD25+调节性T细胞的研究

目的 观察强直性脊柱炎(AS)患者外周血CD4+CD25调节性T细胞(Treg)的数量、功能及肿瘤坏死因子(TNF)-a 抑制剂治疗的影响.方法 活动性AS患者25例,10例给予etanercept治疗12周,健康对照21名,分离外周血单个核细胞(PBMC),流式细胞术检测CD4+CD25high T细胞比例;实时定量聚合酶链反应检测FOXP3 mRNA表达;免疫磁珠法去除CD25+细胞,可溶性噻唑盐(WST-1)法检测T细胞增殖.结果 活动性AS组CD4+CD25high T细胞比例与对照组差异尤统计学意义,但FOXP3 mRNA表达明显低于对照组(P＜0.01),并与C反应蛋白(CRP)呈负相关(P＜0.01).两组的CD4+CD25+细胞体外均能抑制T细胞增殖(P均＜0.01). Etanercept治疗明显增加CD4+CD25highT细胞比例和FOXP3 mRNA表达(P均＜0.01),与CRP降低呈负相关(P＜0.05;P＜0.01).结论 AS患者外周血表达FOXP3的CD4+CD25+Treg细胞异常,可能参与AS发生和发展;Etanercept治疗上调Treg细胞,可能是抗TNF-α治疗的一个机制.     

慢性乙型肝炎患者浆样树突状细胞体外诱导CD4+CD25+调节性T细胞的研究

目的 观察慢性乙型肝炎患者,乙型肝炎恢复者和健康人浆样树突状细胞(pDCs)体外诱导CD4+CD25+调节性T细胞(CD4+CD25+Treg)能力的差异,为阐明HBV感染慢性化的机制奠定基础.方法 采用免疫磁珠分离法体外分离46例慢性乙型肝炎患者,10例乙型肝炎恢复者和25名健康人外周血单个核细胞中pDCs,并将其分别与健康人CD4+CD45RA+初始T细胞共培养.采用HBcAg或破伤风毒素对去除CD25+细胞的外周血单个核细胞进行增殖刺激后,使用流式细胞仪及RT-PCR对pDCs-T共培养细胞中CD4+CD25+Treg的数量、表型及FOXP3基因表达情况进行测定;采用酶联免疫吸附法对共培养细胞上清液中的白细胞介素-10和转化生长因子β1进行了进一步检测.两组数据比较采用Mann Whitney U-test.结果 当细胞增殖刺激物为HBcAg时,细胞增殖幅度慢性乙型肝炎患者组为(7999.36±374.74)cpm,乙型肝炎恢复者组为(11 282.56±1174.46)cpm和健康人组为(12 304.58±1462.81)cpm,慢性乙型肝炎患者组细胞增殖幅度明显小于乙型肝炎恢复者组和健康人组,U=0～22.0,P值均<0.05·乙型肝炎恢复者组和健康人组间增殖幅度差异无统计学意义.当增殖刺激物为破伤风毒素时,细胞增殖幅度与阳性对照组之间,差异无统计学意义.CD4+CD25+Treg比例慢性乙型肝炎患者组为5.99%±1.85%,乙型肝炎恢复者组为3.04%±0.79%,健康人组为3.01%±1.53%,慢性乙型肝炎患者组中韵CD4+CD25+Treg比例明显高于乙型肝炎恢复者组和健康人组,U=6.0～71.5,P值均<0.05.3组人群pDCs-T共培养细胞的CD4+CD25+T细胞均检测到Fox p3 RNA,而在CD4+CD25 T细胞中,均未检测到Fox p3RNA.3组人群pDCs-T共培养细胞实验组上清液的白细胞介素-10和转化生长因子β1含量均明显高于阳性对照组.结论 pDCs以诱导CD4+CD25+Treg形式参与了乙型肝炎的慢性化.     

Most human thymic and peripheral-blood CD4+ CD25+ regulatory T cells express 2 T-cell receptors

Lack of allelic exclusion in the T-cell receptor (TCR) alpha locus gives rise to 2 different TCRs in 10% to 30% of all mature T cells, but the significance of such dual specificity remains controversial. Here we show that human CD4+ CD25+ regulatory T (Treg) cells express 2 distinct Valpha chains and thus 2 TCRs at least 3 times as often as other T cells. Extrapolating from flow cytometric analysis using Valpha2-, Valpha12-, and Valpha24-specific monoclonal antibodies (mAbs), we estimated that between 50% and 99% of the CD25+ Treg cells were dual specific, as compared with about 20% of their CD25- counterparts. Moreover, both TCRs were equally capable of transmitting signals upon ligation. Cells with 2 TCRs also expressed more FOXP3, the Treg-cell lineage specification factor, than cells with a single TCR. Our findings suggest that expression of 2 TCRs favors differentiation to the Treg-cell lineage in humans and raise the question of the potential functional consequences of dual specificity.     

IL-2 regulates FOXP3 expression in human CD4+CD25+ regulatory T cells through a STAT-dependent mechanism and induces the expansion of these cells in vivo

IL-2 plays a critical role in the maintenance of CD4+CD25+ FOXP3(+) regulatory T cells (Tregs) in vivo. We examined the effects of IL-2 signaling in human Tregs. In vitro, IL-2 selectively up-regulated the expression of FOXP3 in purified CD4+CD25+ T cells but not in CD4+CD25- cells. This regulation involved the binding of STAT3 and STAT5 proteins to a highly conserved STAT-binding site located in the first intron of the FOXP3 gene. We also examined the effects of low-dose IL-2 treatment in 12 patients with metastatic cancer and 9 patients with chronic myelogenous leukemia after allogeneic hematopoietic stem cell transplantation. Overall, IL-2 treatment resulted in a 1.9 median fold increase in the frequency of CD4+CD25+ cells in peripheral blood as well as a 9.7 median fold increase in FOXP3 expression in CD3+ T cells. CD56+CD3- natural killer (NK) cells also expanded during IL-2 therapy but did not express FOXP3. In vitro treatment of NK cells with 5-aza-2'-deoxycytidine restored the IL-2 signaling pathway leading to FOXP3 expression, suggesting that this gene was constitutively repressed by DNA methylation in these cells. Our findings support the clinical evaluation of low-dose IL-2 to selectively modulate CD4+CD25+ Tregs and increase expression of FOXP3 in vivo.     

Only the CD62L+ subpopulation of CD4+CD25+ regulatory T cells protects from lethal acute GVHD

CD4+CD25+ regulatory T (Treg) cells are potent modulators of alloimmune responses. In murine models of allogeneic bone marrow transplantation, adoptive transfer of donor CD4+CD25+ Treg cells protects recipient mice from lethal acute graft-versus-host disease (aGVHD) induced by donor CD4+CD25- T cells. Here we examined the differential effect of CD62L+ and CD62L- subsets of CD4+CD25+ Treg cells on aGVHD-related mortality. Both subpopulations showed the characteristic features of CD4+CD25+ Treg cells in vitro and did not induce aGVHD in vivo. However, in cotransfer with donor CD4+CD25- T cells, only the CD62L+ subset of CD4+CD25+ Treg cells prevented severe tissue damage to the colon and protected recipients from lethal aGVHD. Early after transplantation, a higher number of donor-type Treg cells accumulated in host mesenteric lymph node (LN) and spleen when CD4+CD25+CD62L+ Treg cells were transferred compared with the CD62L- subset. Subsequently, CD4+CD25+CD62L+ Treg cells showed a significantly higher capacity than their CD62L- counterpart to inhibit the expansion of donor CD4+CD25- T cells. The ability of Treg cells to efficiently enter the priming sites of pathogenic allo-reactive T cells appears to be a prerequisite for their protective function in aGVHD.     

Interaction with activated monocytes enhances cytokine expression and suppressive activity of human CD4+CD45ro+CD25+CD127low regulatory T cells

Objective

Despite the high frequency of CD4+ T cells with a regulatory phenotype (CD25+CD127^low FoxP3+) in the joints of patients with rheumatoid arthritis (RA), inflammation persists. One possible explanation is that human Treg cells are converted into proinflammatory interleukin‐17 (IL‐17)–producing cells by inflammatory mediators and thereby lose their suppressive function. The aim of this study was to investigate whether activated monocytes, which are potent producers of inflammatory cytokines and are abundantly present in the rheumatic joint, induce proinflammatory cytokine expression in human Treg cells and impair their regulatory function.

Methods

The presence and phenotype of CD4+CD45RO+CD25+CD127^low T cells (memory Treg cells) and CD14+ monocytes in the peripheral blood (PB) and synovial fluid (SF) of patients with RA were investigated by flow cytometry. Memory Treg cells obtained from healthy control subjects underwent fluorescence‐activated cell sorting and then were cocultured with autologous activated monocytes and stimulated with anti‐CD3 monoclonal antibodies. Intracellular cytokine expression, phenotype, and function of cells were determined by flow cytometry, enzyme‐linked immunosorbent assay, and proliferation assays.

Results

In patients with RA, the frequencies of CD4+CD45RO+CD25+CD127^low Treg cells and activated CD14+ monocytes were higher in SF compared with PB. In vitro–activated monocytes induced an increase in the percentage of IL‐17–positive, interferon‐γ (IFNγ)–positive, and tumor necrosis factor α (TNFα)–positive Treg cells as well as IL‐10–positive Treg cells. The observed increase in IL‐17–positive and IFNγ‐positive Treg cells was driven by monocyte‐derived IL‐1β, IL‐6, and TNFα and was mediated by both CD14+CD16− and CD14+CD16+ monocyte subsets. Despite enhanced cytokine expression, cells maintained their CD25+FoxP3+CD39+ Treg cell phenotype and showed an enhanced capacity to suppress T cell proliferation and IL‐17 production.

Conclusion

Treg cells exposed to a proinflammatory environment show increased cytokine expression as well as enhanced suppressive activity.

     

初发系统性红斑狼疮患者外周血CD4+调节性T细胞CD73表达

目的 研究CD73在初发活动性系统性红斑狼疮(SLE)患者外周血CD4+调节性T细胞的表达情况,探讨其在SLE发病中的作用.方法 采用流式细胞术检测29例初发未经治疗的活动期SLE患者(SLE组)和22例健康人(健康对照组)外周血CD4+CD25+CD73+T细胞百分率及CD4+CD73+、CD4+CDhi25、CD4+CD25+T细胞中叉头状转录因子3(FOXP3)蛋白表达,同时对CD73表达水平与SLE活动指标进行相关性分析.结果 SLE组患者外周血CD4+ CD25+ CD73+T细胞百分率低于健康对照组[(1.25±1. 32)%vs(2.35±1.09)%,P＜0.01].SLE组和健康正常对照组,CD73在C4+ CDhi25T细胞的表达水平[(29.05±12.53)%、(43.35±10.09)%]高于CD4+ CD25+T细胞[(17.48±6.92)%、(29.98±10.39)%,P＜0.001];FOXP3蛋白在CD4+ CD73+ T细胞[(65.36±14.40)%、(63.80±14.05)%]、CD4+ CDhi25 T细胞的表达水平[(67.30±13.04)%、(56.30±9.21)%]明显高于CD4+ CD25+ T细胞[(45.70±12.74)%、(43.98±5.17)%,P＜0.001],在CD4+ CD73-T细胞几乎不表达,而在CD4+ CD73+ T细胞、CD4+ CDhi25 T细胞中的表达差异无统计学意义(P值均大于0.05).CD73在CD4+ CD25+ T细胞的表达水平与SLE疾病活动指数、ESR、C反应蛋白、抗补体C1q、抗核小体抗体均无相关性(P＞0.05).结论 CD73可作为调节性T细胞新的表面标记,其在调节性T细胞中的异常表达可能参与SLE的发病机制.     

Local and systemic induction of CD4+CD25+ regulatory T-cell population by non-Hodgkin lymphoma

Regulatory T (Treg) cells contribute to immune evasion by malignancies. To investigate their importance in non-Hodgkin lymphoma (NHL), we enumerated Treg cells in peripheral blood mononuclear cells (PBMCs) and involved tissues from 30 patients. CD25(+)FoxP3(+)CD127(low)CD4(+) Treg cells were increased markedly in PBMCs (median = 20.4% CD4 T cells, n = 20) versus healthy controls (median = 3.2%, n = 13, P < .001) regardless of lymphoma subtype, and correlated with disease stage and serum lactate dehydrogenase (R(s) = 0.79, P < .001). T-cell hyporesponsiveness was reversed by depleting CD25(+) cells, or by adding anti-CTLA-4, supporting the view that Treg cells explain the systemic immunosuppression seen in NHL. A high proportion of Treg cells was also present in involved tissues (median = 38.8% CD4 T cells, n = 15) versus reactive nodes (median = 11.6%, n = 2, P = .02). When autologous CD25(-) PBMC fractions were incubated with tumor cells from patients (n = 6) in vitro, there was consistent strong induction and then expansion of cells with the CD4(+)CD25(+)FoxP3(+) phenotype of classic "natural" Treg cells. This population was confirmed to be suppressive in function. Direct cell-cell interaction of tumor cells with CD25(-) PBMCs was important in Treg induction, although there was heterogeneity in the mechanisms responsible. We conclude that NHL cells are powerful inducers of Treg cells, which may represent a new therapeutic target.     

结肠癌细胞上清液对CD4~＋FOXP3~＋调节性T淋巴细胞形成影响

目的观察结肠癌细胞上清液是否能促进正常CD4＋T淋巴细胞转换成调节性T淋巴细胞,并且探讨TGFβ1在促细胞转换中的重要作用。方法应用流式细胞仪染色CD4＋FOXP3＋,ELISA检测结肠癌上清液中TGFβ1浓度。结果分选的CD4＋CD25-细胞纯度高达96.58%±0.59%,这群细胞中表达CD4＋FOXP3＋较低,为0.34%±0.03%。健康正常人CD4＋FOXP3-细胞分别与人结肠癌细胞colo320HSR及DLD-1上清液共培养后,CD4＋FOXP3＋细胞表达增高（colo320HSR组为4.78%±0.41%,DLD-1组为4.48%±0.29%）;这两组与RPMI 1640培养液阴性对照组CD4＋FOXP3＋细胞2.54%±0.41%相比差异有统计学意义（P〈0.05）。ELISA法检测colo320HSR细胞上清液TGFβ1浓度为（1 228.80±10.65）ng/mL,DLD-1细胞上清液TGFβ1浓度（624.50±12.26）ng/mL,而RPMI 1640无血清培养液中未检测到TGFβ1。在与结肠癌细胞上清液培养同时给与TGFβ抗体后,CD4＋FOXP3＋表达较未加入抗体组明显降低（colo320HSR组2.20%±0.09%,P=0.011;DLD-1组1.67%±0.34%,P=0.033）;而RPMI1640阴性对照组CD4＋FOXP3＋细胞表达下降不显著（1.80%±0.58%,P=0.159）。结论人结肠癌细胞上清液可以促进正常CD4＋T淋巴细胞（CD4＋FOXP3-细胞）转换为调节性T淋巴细胞（CD4＋FOXP3＋）,TGFβ1在肿瘤微环境中调节T淋巴细胞形成可能有一定的作用。识别人调节性T淋巴细胞（CD4＋FOXP3＋）的起源将为肿瘤治疗提供重要信息。     

CD4+CD25+ T regulatory cells control anti-islet CD8+ T cells through TGF-beta-TGF-beta receptor interactions in type 1 diabetes

Pancreatic lymph node-derived CD4+CD25+ T regulatory (Treg) cells inhibit in situ differentiation of islet-reactive CD8+ T cells into cytotoxic T lymphocytes, thereby preventing diabetes progression. The mechanism by which these Treg cells suppress anti-islet CD8+ T cells is unknown. Here, we show by using a CD8+ T cell-mediated model of type 1 diabetes that transforming growth factor (TGF)-beta-TGF-beta receptor signals are critical for CD4+CD25+ Treg cell regulation of autoreactive islet-specific cytotoxic T lymphocytes. Transgenic expression of tumor necrosis factor alpha from birth to 25 days of age in the islets of B6 mice that constitutively express CD80 on their beta cells results in accumulation of CD4+CD25+TGF-beta+ cells exclusively in the islets and pancreatic lymph nodes, which delays diabetes progression. In contrast, expression of tumor necrosis factor alpha until 28 days of age prevents islet accumulation of CD4+CD25+TGF-beta+ Treg cells, resulting in acceleration to diabetes. Furthermore, adoptive transfer experiments demonstrated that CD4+CD25+ Treg cells could not control na?ve or activated islet-reactive CD8+ T cells bearing a dominant negative TGF-beta receptor type II. Our data demonstrate that, in vivo, TGF-beta signaling in CD8+ T cells is critical for CD4+CD25+ Treg cell suppression of islet-reactive CD8+ T cells in type 1 diabetes.     

IL-2-independent generation of FOXP3(+)CD4(+)CD8(+)CD25(+) cytotoxic regulatory T cell lines from human umbilical cord blood

OBJECTIVE: Since the existence of mouse naturally occurring CD4(+)CD25(+) T regulatory (Treg) cells was demonstrated, a variety of human Treg subsets have been identified as distinct T cell populations. Here we show the establishment of novel Treg cell lines possessing unique characteristics. METHODS: Novel Treg cell lines, designated HOZOT, were generated by coculturing human umbilical cord blood cells with mouse stromal cell lines in the absence of exogenous IL-2 or other cytokines. HOZOT were characterized and compared with CD4(+)CD25(+) Treg cells in terms of the CD phenotype, FOXP3 expression, suppressor activity against allogeneic MLR, anergy property, and IL-10 production. RESULTS: HOZOT were generated and expanded as normal lymphoblastoid cells with cytotoxic activity against the cocultured stromal cells. HOZOT consisted of three subpopulations as defined by phenotype: CD4(+)CD8(+), CD4(+)CD8(dim), and CD4(-)CD8(+). All three subpopulations showed both suppressor and cytotoxic activities. While HOZOT's expression of FOXP3, CD25, GITR, and cytoplasmic CTLA-4 implied a similarity to naturally occurring CD4(+)CD25(+) Treg cells, these two Treg cells differed in IL-2 responsiveness and IL-10 production. CONCLUSIONS: Our studies introduce a new method of generating Treg cells in an IL-2-independent manner and highlight a unique Treg cell type with cytotoxic activity and a phenotype of FOXP3(+)CD4(+)CD8(+)CD25(+).     

Functional CD4+CD25high regulatory T cells are enriched in the colonic mucosa of patients with active ulcerative colitis and increase with disease activity

BACKGROUND: Factors determining the extension and degree of inflammation in the colonic mucosa of patients with ulcerative colitis (UC) are largely unknown, but CD4+CD25high regulatory T cells (Tregs) have been implicated to play an important role in suppressing inflammation. Therefore, the aims of this study were to determine whether colonic Tregs have suppressive effects on colonic effector T cells in UC and to analyze the association between segmental colonic Treg distribution and disease activity. MATERIALS AND METHODS: The suppressive activity of colonic CD4+CD25high Tregs from patients with active UC was determined in coculture assays measuring proliferation and cytokine production. The frequency of Tregs and the expression of the Treg marker FOXP3 were analyzed with flow cytometry and RT-PCR in isolated cells and the whole mucosa from patients with active and inactive disease, as well as healthy mucosa. RESULTS: Colonic CD4+CD25high T cells from patients with UC suppressed the proliferation and cytokine secretion of colonic effector CD4+ T cells. Healthy controls but not patients with UC had lower Treg frequencies in the sigmoid than in the ascending colon. Patients with UC with active disease had increased frequency of colonic Tregs. The frequency of Tregs was positively correlated with colonic disease activity and serum C-reactive protein. CONCLUSIONS: Colonic CD4+CD25high Tregs are able to suppress colonic effector T cell activity in vitro, and the Treg frequency in the inflamed intestine increases with disease activity in patients with active UC. This suggests that Tregs may be outnumbered by other inflammatory cells or that their suppressive activity may be influenced by the in vivo environment.     

TNF downmodulates the function of human CD4+CD25hi T-regulatory cells

CD4+CD25+ T-regulatory cells (Tregs) play an essential role in maintaining immunologic homeostasis and preventing autoimmunity. However, little is known about the exogenous factors that regulate their differentiation and function. Here, we report that TNF inhibits the suppressive function of both naturally occurring CD4+CD25+ Tregs and TGFbeta1-induced CD4+CD25+ T-regulatory cells. The mechanism of this inhibition involves signaling through TNFRII that is constitutively expressed selectively on unstimulated Tregs and that is up-regulated by TNF. TNF-mediated inhibition of suppressive function is related to a decrease in FoxP3 mRNA and protein expression by the Tregs. Notably, CD4+CD25hi Tregs isolated from patients with active rheumatoid arthritis (RA) expressed reduced levels of FoxP3 mRNA and protein and poorly suppressed the proliferation and cytokine secretion of CD4+ effector T cells in vitro. Treatment with anti-TNF antibody (infliximab) increased FOXP3 mRNA and protein expression by CD4+CD25hi Tregs and restored their suppressive function. Thus, TNF has a novel action in modulating autoimmunity, by inhibiting CD4+CD25+ Treg activity.     

CD4+CD25+ T cells in skin lesions of patients with cutaneous leishmaniasis exhibit phenotypic and functional characteristics of natural regulatory T cells

Endogenous regulatory T (Treg) cells are involved in the control of infections, including Leishmania infection in mice. Leishmania viannia braziliensis is the main etiologic agent of cutaneous leishmaniasis (CL) in Brazil, and it is also responsible for the more severe mucocutaneous form. Here, we investigated the possible involvement of Treg cells in the control of the immune response in human skin lesions caused by L. viannia braziliensis infection. We show that functional Treg cells can be found in skin lesions of patients with CL. These cells express phenotypic markers of Treg cells--such as CD25, cytotoxic T lymphocyte-associated antigen 4, Foxp3, and glucocorticoid-induced tumor necrosis factor receptor--and are able to produce large amounts of interleukin-10 and transforming growth factor- beta . Furthermore, CD4+CD25+ T cells derived from the skin lesions of 4 of 6 patients with CL significantly suppressed in vitro the phytohemagglutinin-induced proliferative T cell responses of allogeneic peripheral-blood mononuclear cells (PBMCs) from healthy control subjects at a ratio of 1 Treg cell to 10 allogeneic PBMCs. These findings suggest that functional Treg cells accumulate at sites of Leishmania infection in humans and possibly contribute to the local control of effector T cell functions.     

Expression of the putatively regulatory T-cell marker FOXP3 by CD4(+)CD25+ T cells after pediatric hematopoietic stem cell transplantation

FOXP3 has been proposed to be critical for the regulatory function of CD4(+)CD25+ T cells and it has been reported that its expression correlates with protection from graft-versus-host-disease (GvHD) after allogeneic hematopoietic stem cell transplantation (HSCT). Here, by monitoring 28 pediatric HSCT recipients, we found that the levels of FOXP3-mRNA expression in highly enriched CD4(+)CD25+ cells were identical to those in healthy controls irrespective of GvHD status. Moreover, FOXP3-mRNA was abundant in recently in vitro stimulated CD4(+)CD25+ cells that lacked regulatory function. Together these findings suggest that FOXP3-mRNA expression primarily reflects CD4(+)CD25+ cell frequency rather than defining the regulatory potential of CD4(+)CD25+ T cells and GvHD risk after HSCT.