The frequency of interleukin-10- and interleukin-5-secreting CD4+ T cells correlates to tolerance of transplanted lung

Posttransplant bronchiolitis obliterans syndrome (BOS) results from a chronic immunological/inflammatory insult that leads to fibro-obliteration of the lumen of the allograft airways. The functional T-cell response that is associated with graft acceptance needs to be further clarified in humans. The aim of the present study was to assess the functional activity of peripheral CD4+ T lymphocytes in nine lung transplant recipients with BOS stage II or III (mean 5.4 years after transplant), in seven lung patients with stable clinical conditions (3.4 years posttransplant); and in six normal controls. Peripheral CD4+ T cells, obtained by magnetic bead vs negative purification, were studied using a computer-assisted enzyme-linked immunospot assay (ELISPOT) to assess the number of IFN-gamma-, interleukin (IL)5-, and IL10-gamma-producing cells (no./10(6) CD4+ cells) after allogeneic stimulation. The frequencies of IFN-gamma-producing CD4+ cells did not change significantly in stable patients compared to those with BOS. Interestingly in BOS, the number of IL5- and IL10-producing cells was significantly lower than in stable patients (P < or = .05), suggesting a possible role of these Th2 cytokines in the modulation of graft tolerance.     

Differentiation of human alloreactive CD4+ and CD8+ T cells in vitro

BACKGROUND: Recent studies have revealed that, during viral infection, ordered phenotypic and functional changes occur in human antigen-specific T cells. We analyzed whether a similar differentiation program is induced after alloantigen stimulation in vitro. METHODS: Peripheral blood mononuclear cells and purified CD4(+)CD45RA+, CD4(+)CD45RO+, and CD8+ T cells from healthy individuals were labeled with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE). Cells were co-cultured with allogeneic irradiated cells. Flow cytometric analysis was performed on days 3, 5, and 7 of culture using surface CD45RA, CD27, CD28, CCR7, and intracellular perforin and granzyme B markers in relation to CFSE dilution. RESULTS: Based on the decrease in CFSE fluorescence, both CD4+ and CD8+ T cells showed an early and vigorous response to allogeneic stimulation. Loss of CD45RA expression and up-regulation of CD27 and CD28 costimulatory molecules was an early event occurring in the first generations of dividing cells. Differentiation at later stages of proliferation was characterized by loss of CCR7 homing receptor expression, more pronounced in CD4+ than in CD8+ T cells, indicating the decreased ability of these cells to traffic to secondary lymphoid organs. Production of the cytotoxic effector molecules perforin and granzyme B increased with the number of cell divisions. CONCLUSIONS: Our data thus show that short-term phenotypic and functional changes of alloreactive T cells follow the differentiation pattern seen in the early stages of an antiviral immune response.     

CD4+CD25+调节性T细胞/辅助性T细胞17在脓毒症大鼠中的作用

目的 观察CD4+ CD25+调节T细胞(Treg)/辅助性T细胞17(Th17)细胞在脓毒症大鼠炎性免疫反应中的作用.方法 110只雄性SD大鼠随机分为正常对照组、假手术组、脓毒症(CLP)组,采用改良的盲肠结扎穿孔术(CLP)制作大鼠脓毒症模型.采用流式细胞术检测CD14+单核细胞表面人类白细胞抗原-DR基因(HLA-DR)表达率、Treg细胞及TH17细胞比例;酶联免疫吸附试验(ELISA)检测白细胞介素(IL)-6、IL-10、肿瘤坏死因子(TNF)-α、转化生长因子(TGF)-β、白细胞介素(IL)-17炎性因子蛋白表达.结果 与假手术组比较:(1)伴随着脓毒症病情的发展,大鼠出现明显的免疫抑制,CD14+单核细胞HLA-DR表达率＜30％,IL-10/TNF-α比值(27.41 ±7.04比6.63 ±2.60)明显增高(P＜0.01).(2)术后96 h脓毒症大鼠Treg细胞[(11.91±3.88)％比(6.57±2.60)％,P＜0.01]和Th17细胞[(5.14±0.29)％比(2.85±0.07)％,P＜0.01]表达明显增高.(3)术后96 h脓毒症组前炎性细胞因子IL-6[(42.31±15.89) ng/L比(6.32 ±3.18) ng/L,P＜0.01]、IL-10[(69.89 ±20.78) ng/L比(13.58±5.37) ng/L,P＜0.01]、TNF-α[(5.03±3.10) ng/L比(2.77±1.10) ng/L,P＜0.01]、TGF-β[(4.99±2.01) ng/L比(1.88±1.07) ng/L,P＜0.01]、IL-17[(92.77±11.64) ng/L比(7.58±2.30) ng/L,P＜0.01]表达明显增高.结论 伴随着脓毒症病情的发展,大鼠出现明显的免疫抑制;在大鼠脓毒症的发生发展中,Treg细胞介导的免疫抑制及Th17细胞介导免疫激活反应同时存在;脓毒症细胞因子微环境变化可能是导致Treg细胞/Th17细胞失衡的原因之一.     

CD4+CD25+ regulatory T cells mediate acquired transplant tolerance

The Holy Grail of clinical organ transplantation is the safe induction of allograft tolerance. Transplant tolerance has been successfully induced in animal models. Since T cells play a pivotal role in graft rejection, modulating T cell function has been the primary focus of studies aimed at inducing transplant tolerance. Rodent models of transplant tolerance induction include central deletion and peripheral mechanisms involving activation-induced cell death (AICD), anergy, immune deviation, and production of regulatory T cells. These mechanisms are not mutually exclusive. Although clonal deletion and anergy limit self-reactive T cells in the thymus, these mechanisms alone are not sufficient for controlling self-reactive T cells in the periphery. There is now evidence that the adult animal harbors two functionally distinct populations of CD4(+) T cells; one mediates autoimmune disease and the other dominantly inhibits it. The latter cells express CD4, CD25 and CTLA-4. These thymus-derived T cells have recently been shown to mediate the induction and maintenance of transplant tolerance. These CD4(+)CD25(+) T cells are similar in origin, phenotype, and function to those that maintain natural self-tolerance and T cell homeostasis in the periphery. Against this background, is it possible that alloantigen specific regulatory T cells might be generated and expanded ex vivo before organ transplantation and then infused to induce long-term tolerance, perhaps without the need for chronic immunosuppression?     

大鼠CD4^＋CD25^＋调节性T细胞的分离及功能鉴定

目的：研究利用免疫磁珠分选法稳定分离正常大鼠脾脏CD4^＋CD25^＋调节性T细胞的方法。方法：采用免疫磁珠两步法分离大鼠脾组织CD4^＋CD25^＋T细胞。首先采用藻红蛋白（PE）标记的抗CD25抗体和抗PE多功能磁珠试剂盒阳性分选CD25^＋T细胞,再用抗异硫氰酸荧光素（FITC）标记抗体和抗IgG磁珠阳性分选获得CD4^＋CD25^＋T细胞。分离后的细胞经流式细胞仪检测分离纯度,台盼蓝染色检测细胞存活率,体外增殖实验检测其对CD4^＋CD25^-T细胞的免疫抑制作用。结果：两次阳性分选后获得的CD4^＋CD25^＋T细胞纯度为（90.4±1.6）%,细胞存活率为（92.6±2.4）%。体外增殖实验表明,CD4^＋CD25^＋T细胞能明显抑制CD4^＋CD25^-T细胞的增殖（P〈0.01）。结论：采用免疫磁珠法两次阳性分选,可稳定地获得纯度理想并有免疫抑制功能的大鼠CD4^＋CD25^＋T细胞。     

Expansion of CD25+CD4+ regulatory T cells by natural mature dendritic cells

Because of the anergy of CD25+CD4+ regulatory T cells, it is unclear how the number of these regulatory T cells is sustained and expanded in normal physiologic circumstances. In the present study, we examined the effect of natural allogeneic mature dendritic cells (DCs) on the proliferation and function of CD25+CD4+ T cells. Our data showed that natural allogeneic mature DCs stimulated CD25+CD4+ T-cell growth vigorously, whereas immature DCs had little effect on the proliferation of CD25+CD4+ T cells. After expansion by mature DCs, CD25+CD4+ T cells maintained their expression of Foxp3 and suppressed the proliferation of CD25- CD4+ T cells similar to freshly isolated CD25+CD4+ T cells. Our results introduce a potentially critical role played by natural allogeneic mature DCs, which exist in normal physiologic circumstances, in controlling CD25+CD4+ regulatory T-cell expansion and function.     

In vitro expanded human CD4+CD25+ regulatory T cells are potent suppressors of T-cell-mediated xenogeneic responses

BACKGROUND: Cellular rejection of xenografts is predominantly mediated by CD4 T cells. Little is known of the effectiveness of CD4CD25 T regulatory (Treg) cells at suppressing this strong T-cell mediated immune response. In this study, we evaluated the activity of fresh Treg cells and expanded Treg cells to suppress the xeno immune response in vitro. METHODS: Human Treg cells were preferentially expanded by CD3/CD28 expand beads, interleukin (IL)-2, and rapamycin. Human CD4CD25 T cells were stimulated with irradiated porcine peripheral blood mononuclear cells in the presence or absence of fresh or expanded human Treg cells for 5 days before proliferation assay. In a separate experiment, the porcine xenoantigen-stimulated CD4CD25 T cells were separated from Treg cells by transwells and assessed for cytotoxicity of porcine peripheral blood mononuclear cells target cells. Cytokine-producing cells and cytokine release in the cocultures were examined by enzyme-linked immunosorbent spot and enzyme-linked immonosorbent assay, respectively. RESULTS: Human Treg were expanded up to 3,500-fold after 14 days in culture. The addition of fresh Treg suppressed the T-cell mediated xenoimmune response. Compared with fresh Treg cells, expanded Treg cells were more potent at suppressing CD4CD25 T-cell-mediated antiporcine xenogeneic responses. This suppression required cell contact. However, the enhanced suppression by expanded Treg cells was associated with increased secretion of IL-4 and IL-10 when compared with their nonexpanded Treg counterparts. CONCLUSION: This study shows that expanded human Treg cells were capable of suppressing antiporcine xenogeneic responses in vitro and involve both contact dependent and cytokine mediated mechanisms.     

Roles of CD4+ and CD8+ T cells in discordant skin xenograft rejection

An essential role of murine CD4+ T cells in immune reactivity and skin graft rejection in discordant xenogeneic combinations have been reported. Our study was conducted to further clarify the roles of CD4+ and CD8+ T cells in discordant skin xenograft rejection, by using CD4 and CD8 knockout [C57BL/6 Cr Slc (B6; H-2b) background] mice. When human skins were grafted on CD8 knockout mice or B6 mice, both hosts rejected human skin grafts within 12 days after grafting. By contrast, survival of human skin grafts was significantly prolonged in CD4 knockout mice (mean survival times=19.3+/-(SD) 1.6 days; median 19 days). Fully allogeneic C3H/He Slc (H-2k) skin grafts were rejected within 14 days in CD4 knockout mice, suggesting that non-CD4+ T cells in CD4 knockout mice were immunocompetent for allograft rejection. In spleens of these recipient mice, CD8+ T cells seemed to be activated 10 days after human skin grafting. Immunohistological analysis revealed the infiltration of CD8+ T cells at the site of transplanted human skin on CD4 knockout mice. To further examine the role of CD8+ T cells in CD4 knockout mice, human skin grafting was performed on day 0 followed by administration of anti-CD8 monoclonal antibody on days 0, 5, and 14. The administration of anti-CD8 monoclonal antibodies caused the significant prolongation of human skin graft survival. These results indicate the following two conclusions: (1) CD4+ T cells have an essential role in rejecting discordant human skin xenografts rapidly and (2) however, CD8+ T cells also are capable of rejecting discordant human skin xenografts.     

树突状细胞和CD4+CD25+调节性T细胞在免疫耐受中的作用及关系

诱导免疫耐受具有高效、低毒、经济等优点,在器官移植中有着极其重要的意义.在建立免疫耐受中,树突状细胞(DC)起到克隆清除、克隆无能、表达T细胞抑制因子、选择性激活辅助T细胞和诱导调节性T细胞尤其是CD4+CD25+调节性T细胞的产生等作用.而CD4+CD25+调节性T细胞主要以DC为作用靶点,影响其分化成熟,抑制DC向...     

不同的免疫抑制方案对肾移植受者免疫调节性T细胞及表面因子的影响

目的比较钙调磷酸酶抑制剂[环孢素A（CsA）和他克莫司（FK506）]与西罗莫司（SRL）对CD4^＋CD25^＋免疫调节性T细胞（CD4^＋CD25^＋Tregs）及其细胞表面标志性因子细胞毒T淋巴细胞相关抗原4（CTLA-4）和叉状头／翅膀状螺旋转录因子（Foxp3）的影响，为临床合理选择免疫抑制方案提供依据。方法选择2004年1月至4月行。肾移植的患者30例，男女不限，年龄20～50岁，随机分为3组，每组10例。（1）CsA组：免疫抑制方案为CsA＋霉酚酸酯（MMF）＋泼尼松（Pred）；（2）FK506组：免疫抑制方案为FK506＋MMF＋Pred；（3）SRL组：免疫抑制方案为SRL＋MMF＋Pred；3组的免疫诱导治疗方案均相同。各组分别于术前、术后半年、1年、2年取血样检测CD4^＋CD25^＋high／CD4^＋T细胞比值及CD4^＋CD25^highT细胞表面CTLA-4和Foxp3的表达率；比较3组治疗方案对各项指标的影响。结果应用3种不同的免疫抑制方案后，各组受者CD4^＋CD25^high／CD4^＋T细胞比值及CD4^＋CD25^highT细胞表面CTLA-4和Foxp3的表达率均明显低于术前，虽然半年后有所上升，但CsA和FK506组各项指标上升明显较SRL组慢，SRL组各项指标恢复水平明显优于钙调磷酸酶抑制剂组。结论SRL与钙调磷酸酶抑制剂比较，对CD4^＋CD25^＋Treg细胞影响明显较轻，可能对器官移植术后移植免疫耐受的诱导和维持有促进作用。     

Foxp3 is critical for human natural CD4+CD25+ regulatory T cells to suppress alloimmune response

Naturally occurring CD4+CD25+ regulatory T cells (nTregs) that express high level of Foxp3 actively suppress pathological and physiological immune responses, contributing to the maintenance of immunological self-tolerance and immune homeostasis. Although Foxp3 is required for nTreg development and appears to be necessary for mature murine Treg function, the precise role of Foxp3 in regulating natural human Treg function in alloimmune response is unclear. In this study, we used siRNA-mediated gene silencing to knockdown Foxp3 expression in natural human Tregs and investigated the importance of Foxp3 in maintaining human nTreg suppressive function. We showed that Foxp3 knockdown resulted in impaired phenotype and nonresponsiveness, downregulated expression of function molecules, and reduced production of suppressive cytokines in nTregs. These changes correlated with diminished nTreg activity in suppressing proliferation of effector CD4+CD25- T cells, their cytotoxicity against allogeneic target cells and production of effector cytokines in response to allogeneic stimulation. Thus, this study shows that ongoing Foxp3 expression is required for natural human Tregs to maintain their phenotype and suppressive function in the alloimmune response.     

CD4+CD25-T淋巴细胞转化为CD4-CD8-调节T细胞的体外实验研究

目的 探讨体外诱导和纯化CD4+ CD25-T淋巴细胞(effector T cell,Teff)转化为CD4-CD8-双阴性调节T细胞(double negative regulatory T cell,DN Treg)的最适条件.方法 采用免疫磁珠分选方法提取C57BL/6小鼠的CD4+ CD25-T淋巴细胞、DBA/2小鼠的成熟树突状细胞共培养,加入不同剂量的IL-2,通过流式细胞检测CD4-CD8-T细胞的转化比例并确定最适条件,免疫磁珠阴性选择分选提纯转化的CD3+ CI4-CD8-T细胞,流式细胞仪检测转化的CD4-CD8-调节T细胞对CD4+ CD25-效应T细胞增殖抑制情况.结果 CD4+ CD25-T淋巴细胞与DBA/2小鼠的树突状细胞共培养6d后检测CD4-CD8-调节T细胞的转化比率为6.21％ ±2.03％,实验组加入不同浓度IL-2的转化率:A组(25 ng/ml)为14.77％±2.15％,B组(50 ng/ml)为21.29％±2.68％,C组(75 ng/ml)为43.45％ ±4.45％,D组(100 ng/ml)为28.59％±3.05％,IL-2浓度在75 ng/ml时,转化获得率最高(C组与对照组、实验A、B、D组比较分别t=10.700,8.288,6.158,3.932,均P＜0.05);分离提纯CD4-CD8-双阴性调节细胞纯度达到98.10％,CD4-CD8-双阴性调节细胞与CFSE染色的CD4+ CD25-T淋巴细胞、小鼠树突状细胞共培养6d,实验组增殖指数为1.15明显低于对照组2.07.结论 小鼠CD4+ CW25-T淋巴细胞在体外,成熟树突状细胞刺激下可转化为CD4-CD8-双阴性调节T细胞,IL-2可显著提高其转化率.     

Both CD45RA+ and CD45RO+ human CD4+ T cells drive direct xenogeneic T‐cell responses against porcine aortic endothelial cells

Kim CH, Oh K, Kim D‐E, Lee SB, Yang JH, Lee G, Cho J, Lee D‐S. Both CD45RA⁺ and CD45RO⁺ human CD4⁺ T cells drive direct xenogeneic T‐cell responses against porcine aortic endothelial cells. Xenotransplantation 2010; 17: 224–232. © 2010 John Wiley & Sons A/S. Abstract: Background: Xenogeneic cellular immune responses are mediated by either direct or indirect pathways depending on the participation of donor or host antigen presenting cells, respectively. The contribution of direct response of human T cells, especially memory T cells, to porcine antigen presenting cells is currently unknown. Here, we sought to determine whether human peripheral blood memory/activated phenotype T cells are directly responsive to porcine endothelial cells. Methods: Porcine aortic endothelial cells (PAECs) were prepared from Yorkshire or miniature pigs. Highly purified human T cells, including naïve and memory/activated phenotype cells, were incubated with PAECs with or without the addition of exogenous cytokines. T‐cell proliferation and T‐cell receptor (TCR) Vβ usage in response to PAECs were analyzed. Results: Both CD8⁺ and CD4⁺ T cells responded directly to PAECs and exhibited exclusive responsiveness to SLA class I and class II molecules, respectively. Naïve and memory/activated phenotype CD4⁺ T cells responded against PAECs, whereas only naïve phenotype CD8⁺ T cells contributed to such a response. In addition, both populations of xenogeneic human CD4⁺ T cells exhibited similar and diverse Vβ usage. Conclusion: Due to the considerable contribution of human CD45RO⁺CD4⁺ T cells to the xenoreactivity against PAECs, effective control of xenogeneic memory/activated T‐cell responses would significantly affect long‐term survival of transplanted grafts.     

调节性CD4~+CD25~+T细胞的分离纯化及免疫功能研究

目的 制备调节性CD~+ CD25~+T细胞(Treg)分析其免疫功能,诱导局部免疫耐受防治同种异体复合组织移植(CTA)排斥反应.方法 采用免疫磁珠法(MACS)从雄性大鼠脾脏细胞分离CD4~+CD25~+Treg(1×10~6),2%锥虫蓝染色检测活性、流式细胞术分析其纯度,在5 mg/L抗CD3的刺激下观察其反应性、增殖及其与200 U/ml细胞介素(IL)-2的关系.结果 从8只雄性大鼠脾脏分选出的CD4~+CD25~+Treg活性平均为(97.90±0.36)%及纯度为(96.05±0.41)%,CD3刺激呈低反应,按比例培养抑制率为89%,IL-2可使CD4~+CD25~-抑制逆转.结论 MACS能快速分选出较高纯度的CD4~+CD25~+Treg,并且活性良好在体外具有免疫无能及免疫抑制作用,能满足动物CTA排斥反应研究的需要.