CD25+ regulatory T cell inhibition enhances vaccine-induced immunity to neuroblastoma

Evidence that CD4CD25 regulatory T (Treg) cells play a role in the progression of cancer continues to mount. There is a great deal of interest as to whether transient elimination or functional inhibition of these cells can improve the efficacy of immunotherapy for cancer. Our goals in this study were to test whether treatment of mice with anti-CD25 monoclonal antibody (mAb) (PC61) could induce rejection of a murine neuroblastoma, whether anti-CD25 treatment could increase tumor immunity when administered just before cell-based vaccination, and to learn how anti-CD25 treatment influences the vaccine-induced antitumor response. Treatment of mice with anti-CD25 mAb induced rejection of the mouse neuroblastoma, Neuro-2a, as 90% of anti-CD25-treated mice survived challenge with a lethal dose of tumor cells. In vivo anti-CD25 mAb treatment before the first of 2 weekly vaccines significantly improved the survival of tumor-vaccinated/challenged mice (75% vs. 33% survival), whereas antibody treatment before each of the 2 vaccines did not, suggesting that excessive treatment with anti-CD25 mAb interferes with activated antitumor effector cells. A detailed phenotypic analysis of tissues from anti-CD25-treated mice indicated that the antibody partially depletes CD4Foxp3 Treg cells (25% to 40%) in A/J mice, and that the antibody may inhibit the remaining cells by inducing loss of CD25 expression and blocking CD25 molecules, partially confirming recent data from other investigators. Importantly, we found that in vivo anti-CD25 mAb treatment significantly decreased the contribution of asialo GM1 cells in the antitumor response. As we did not see a direct effect of anti-CD25 mAb on in vitro assays of immune cell function in spleen cells from treated animals, this indicates that inhibition of Treg cells amplifies the immune response in vivo in a manner that bypasses the requirement for innate immune activation, potentially mediated by natural killer cells, and allows for protective CD4 and CD8 cells to expand directly in response to cell-based vaccines.     

Regulatory CD4+CD25+ T cells restrict memory CD8+ T cell responses

CD4+ T cell help is important for the generation of CD8+ T cell responses. We used depleting anti-CD4 mAb to analyze the role of CD4+ T cells for memory CD8+ T cell responses after secondary infection of mice with the intracellular bacterium Listeria monocytogenes, or after boost immunization by specific peptide or DNA vaccination. Surprisingly, anti-CD4 mAb treatment during secondary CD8+ T cell responses markedly enlarged the population size of antigen-specific CD8+ T cells. After boost immunization with peptide or DNA, this effect was particularly profound, and antigen-specific CD8+ T cell populations were enlarged at least 10-fold. In terms of cytokine production and cytotoxicity, the enlarged CD8+ T cell population consisted of functional effector T cells. In depletion and transfer experiments, the suppressive function could be ascribed to CD4+CD25+ T cells. Our results demonstrate that CD4+ T cells control the CD8+ T cell response in two directions. Initially, they promote the generation of a CD8+ T cell responses and later they restrain the strength of the CD8+ T cell memory response. Down-modulation of CD8+ T cell responses during infection could prevent harmful consequences after eradication of the pathogen.     

同种异基因抗原特异性调节性T细胞的体外扩增

目的建立用人B淋巴细胞体外获得大量的同种异基因抗原特异性调节性T(Treg)细胞的方法,以此检测扩增细胞的表型及其免疫无能性和免疫抑制性。方法第1轮扩增将免疫磁珠分选的人CD4+CD25+T细胞和人B淋巴细胞按1∶4体外混合培养,并加入外源白介素-2(IL-2)和抗-CD28;将第1轮扩增得到的Treg细胞用抗-CD3/CD28包被的免疫磁珠和IL-2刺激,做第2轮扩增以获得更多数量的抗原特异性Treg细胞,分为添加和或未添加免疫抑制剂雷帕霉素(RAPA)2组(n=3)。结果经过2轮的扩增后,在第2轮扩增中未添加RAPA组扩增1×103倍,纯度80%;添加RAPA组扩增0.8×103倍,纯度90%。添加RAPA组得到的Treg细胞的Foxp3、CT-LA4、CD39表达水平高于未添加RAPA组,但是HLA-DR变化不大;未添加RAPA组扩增得到的Treg细胞分泌低水平的IL-2、IL-17、IL-4和IFN-γ,而添加RAPA组得到的Treg细胞几乎不分泌上述各种细胞因子,前者表现出部分免疫反应无能性,后者表现出完全的免疫反应无能性,两者都表现出免疫抑制性功能特征。人B淋巴细胞扩增得到的抗原特异性Treg细胞能够极大地抑制同源抗原引起的免疫反应,而对多克隆刺激的免疫反应抑制能力较弱。结论用人B细胞体外扩增抗原特异性Treg细胞,再通过抗-CD3/CD28包被的免疫磁珠进一步刺激可以体外获得大量的抗原特异性的Treg细胞,加入RAPA后可有效地提高Treg细胞的纯度和免疫抑制力且呈现抗原特异性。     

Depletion of CD4+ CD25+ regulatory cells augments the generation of specific immune T cells in tumor-draining lymph nodes

Recent studies have identified a unique population of CD4+CD25+ regulatory T cells that is crucial for the prevention of spontaneous autoimmune diseases. Further studies demonstrated that depletion of CD4+CD25+ T cells enhances immune responses to nonself antigens. Because immune responses to malignant tumors are weak and ineffective, depletion of regulatory T cells has been reported to result in tumor regression. In the current study, using the weakly immunogenic MCA205 sarcoma and the poorly immunogenic B16/BL6/D5 (D5) melanoma, depletion of CD4+CD25+ T cells by the administration of anti-CD25 monoclonal antibodies (mAb), PC61 induced some tumor growth retardation, but all mice eventually succumbed to tumors. In our laboratory, immunotherapy by the transfer of tumor-immune T cells has demonstrated potent antitumor effects. A reliable source of tumor-reactive T cells has been lymph nodes (LN) draining progressive tumors. Therapeutic effector T cells can be generated by in vitro activation of draining LN cells with anti-CD3 mAb followed by culture in interleukin-2. In this system, PC61 mAb depletion of CD4+CD25+ T cells before or on day 8 of tumor growth resulted in increased sensitization in the draining LN. The therapeutic efficacy of activated tumor-draining LN cells from mAb depleted mice increased approximately three fold while maintaining specificity when tested in adoptive immunotherapy of established pulmonary metastases. Specific interferon-gamma secretion by LN T cells from mice treated with PC61 mAb 1 day before tumor inoculation increased significantly. However, this increase was not demonstrated with LN T cells from mice treated on day 8 despite their enhanced therapeutic reactivities. Our results indicate that although the antitumor immunity enhanced by the depletion of CD4+CD25+ T cells is insufficient to eradicate tumors, it augments the sensitization of immune T cells in the draining LN, thus, facilitating adoptive immunotherapy.     

CD4+CD25+调节性T细胞在特发性血小板减少性紫癜患者中的变化

目的探讨CD4^＋CD25^＋T细胞在特发性血小板减少性紫癜（ITP）患者发病机制中的作用。方法应用流式细胞术检测ITP患者外周血CD4^＋CD25^＋T细胞、CD4^＋CD25^highT细胞、CD4^＋FOXP3^＋T细胞、CD4^＋CD25^＋FOXP3^＋T细胞的数量；实时荧光定量PCR检测外周血FOXP3 mRNA的表达水平。将ITP患者和正常人CD4^＋CD25^highT细胞与自身CD4^＋CD25^-T细胞混合培养，检测CD4^＋CD25^high T细胞免疫抑制功能。结果ITP患者外周血中CD4^＋CD25^＋T细胞约占CD4^＋T细胞的（15．64±5．82）％，明显高于正常对照组（9．30±3．95）％（P〈0．01），CD4^＋CD25^high T细胞比例为（1．53±0．66）％，与对照组[（1．36±0．55）％]比较差异无统计学意义（P〉0．05）；CD4^＋FOXP3^＋T细胞和CD4^＋CD25^＋FOXP3^＋T细胞分别为（1．82±1．42）％和（1．25±0．94）％，均明显低于对照组[（3．90±1．37）％和（2．65±0．92）％]（P值均〈0．01）。ITP患者外周血FOXP3 mRNA表达水平较正常人明显下调（P〈0．01），CD4^＋CD25^high T细胞的抑制活性较正常人减弱（P〈0．01）。结论ITP患者中CD4^＋CD25^＋T细胞FOXP3表达水平降低，抑制活性减弱。     

中国HIV早期感染者CD+4CD+25Foxp3+调节性T淋巴细胞变化研究

目的 研究1年以内感染HIV的感染者(早期感染者,EHI)体内CD+4 CD+25 Foxp3+调节性T淋巴细胞水平及其与疾病进展相关性.方法 随机选取51例HIV感染者,依据感染时间及CD+4 T淋巴细胞水平分为3组:EHI组30例、HIV组15例、AIDS组6例,20名健康人作为对照组,各组对象的年龄、性别具有可比性.用EDTA抗凝管采集全血,应用FACSAria流式细胞仪及Foxp3染色试剂盒,检测外周血单个核细胞CD+4CD+25Foxp3+调节性T淋巴细胞表达水平,分析EHI者及全部HIV感染者CD+4 CD+25Foxp3+调节性T淋巴细胞表达水平与CD+4T淋巴细胞数量、病毒调定点、病毒载量及淋巴细胞活化水平间的相关性.结果 健康对照组、EHI组、HIV组及AIDS组CD+4C+25Foxp3+T淋巴细胞百分率逐级上升,其中EHI组CD+4CD+25Foxp3+T淋巴细胞百分率[3.79(2.11～5.43)%]低于AIDS组[8.09(4.90～8.90)%],差异有统计学意义(Z=-2.29,P=0.022);EHI组CD+4 CD+25Foxp3+T淋巴细胞百分率与病毒调定点正相关(r=0.479,P=0.038),与CD4T淋巴细胞计数呈负相关(r=-0.455,P=0.011),与CD+3 HLA+T淋巴细胞呈正相关(r=0.533,P=0.002).结论 中国EHI者CD+4 CD+25Foxp3+调节性T淋巴细胞百分率高与高病毒调定点及低CD+4 T淋巴细胞数量相关,提示CD+4 CD+25Fox3+调节性T淋巴细胞是加速HIV感染早期疾病进展的因素之一.     

Human CD4+ CD25hi Foxp3+ regulatory T cells are derived by rapid turnover of memory populations in vivo

While memory T cells are maintained by continuous turnover, it is not clear how human regulatory CD4+ CD45RO+ CD25hi Foxp3+ T lymphocyte populations persist throughout life. We therefore used deuterium labeling of cycling cells in vivo to determine whether these cells could be replenished by proliferation. We found that CD4+ CD45RO+ Foxp3+ CD25hi T lymphocytes were highly proliferative, with a doubling time of 8 days, compared with memory CD4+ CD45RO+ Foxp3- CD25- (24 days) or naive CD4+ CD45RA+ Foxp3- CD25- populations (199 days). However, the regulatory population was susceptible to apoptosis and had critically short telomeres and low telomerase activity. It was therefore unlikely to be self regenerating. These data are consistent with continuous production from another population source. We found extremely close TCR clonal homology between regulatory and memory CD4+ T cells. Furthermore, antigen-related expansions within certain TCR Vbeta families were associated with parallel numerical increases of CD4+ CD45RO+ CD25hi Foxp3+ Tregs with the same Vbeta usage. It is therefore unlikely that all human CD4+ CD25+ Foxp3+ Tregs are generated as a separate functional lineage in the thymus. Instead, our data suggest that a proportion of this regulatory population is generated from rapidly dividing, highly differentiated memory CD4+ T cells; this has considerable implications for the therapeutic manipulation of these cells in vivo.     

Conversion of CD4+ CD25- cells into CD4+ CD25+ regulatory T cells in vivo requires B7 costimulation, but not the thymus

The CD4+ CD25+ regulatory T cells play a critical role in controlling autoimmunity, but little is known about their development and maintenance. In this study, we investigated whether CD4+ CD25- cells can convert to CD4+ CD25+ regulatory T cells in vivo under natural conditions. CD4+ CD25- cells from CD45.1+ mice were sorted and transferred into congenic CD45.2+ mice. Converted CD4+ CD25+ cells could be detected in lymphoid organs as early as 1 wk after transfer and by 6 wk after transfer, 5-12% of transferred CD4+ cells expressed CD25. Converted CD4+ CD25+ cells themselves failed to proliferate after stimulation, but could suppress proliferation of responder cells in vitro, and also expressed high levels of Foxp3 mRNA. In addition, CD4+ CD25- cells transferred into thymectomized congenic mice converted to CD4+ CD25+ cells that also suppressed responder cell proliferation in vitro, and expressed high levels of Foxp3 mRNA. Finally, CD4+ CD25- cells transferred into B7-/- mice failed to convert into CD4+ CD25+ cells that exhibit the regulatory phenotype. These data indicate that CD4+ CD25- cells convert into CD4+ CD25+ regulatory T cells spontaneously in vivo and suggest that this conversion process could contribute significantly to the maintenance of the peripheral CD4+ CD25+ regulatory T cell population.     

Dendritic cell-expanded, islet-specific CD4+ CD25+ CD62L+ regulatory T cells restore normoglycemia in diabetic NOD mice

Most treatments that prevent autoimmune diabetes in nonobese diabetic (NOD) mice require intervention at early pathogenic stages, when insulitis is first developing. We tested whether dendritic cell (DC)-expanded, islet antigen-specific CD4+ CD25+ suppressor T cells could treat diabetes at later stages of disease, when most of the insulin-producing islet beta cells had been destroyed by infiltrating lymphocytes. CD4+ CD25+ CD62L+ regulatory T cells (T reg cells) from BDC2.5 T cell receptor transgenic mice were expanded with antigen-pulsed DCs and IL-2, and were then injected into NOD mice. A single dose of as few as 5x10(4) of these islet-specific T reg cells blocked diabetes development in prediabetic 13-wk-old NOD mice. The T reg cells also induced long-lasting reversal of hyperglycemia in 50% of mice in which overt diabetes had developed. Successfully treated diabetic mice had similar responses to glucose challenge compared with nondiabetic NOD mice. The successfully treated mice retained diabetogenic T cells, but also had substantially increased Foxp3+ cells in draining pancreatic lymph nodes. However, these Foxp3+ cells were derived from the recipient mice and not the injected T reg cells, suggesting a role for endogenous T reg cells in maintaining tolerance after treatment. Therefore, inoculation of DC-expanded, antigen-specific suppressor T cells has considerable efficacy in ameliorating ongoing diabetes in NOD mice.     

Role of IFN-gamma in induction of Foxp3 and conversion of CD4+ CD25- T cells to CD4+ Tregs

IFN-gamma is an important Th1 proinflammatory cytokine and has a paradoxical effect on EAE in which disease susceptibility is unexpectedly heightened in IFN-gamma-deficient mice. In this study, we provide what we believe is new evidence indicating that IFN-gamma is critically required for the conversion of CD4+ CD25- T cells to CD4+ Tregs during EAE. In our study, the added severity of EAE in IFN-gamma knockout mice was directly associated with altered encephalitogenic T cell responses, which correlated with reduced frequency and function of CD4+ CD25+ Foxp3+ Tregs when compared with those of WT mice. It was demonstrated in both human and mouse systems that in vitro IFN-gamma treatment of CD4+ CD25- T cells led to conversion of CD4+ Tregs as characterized by increased expression of Foxp3 and enhanced regulatory function. Mouse CD4+ CD25- T cells, when treated in vitro with IFN-gamma, acquired marked regulatory properties as evidenced by suppression of EAE by adoptive transfer. These findings have important implications for the understanding of the complex role of IFN-gamma in both induction and self regulation of inflammatory processes.     

CD8 is required during positive selection of CD4-/CD8+ T cells

Interactions between self-MHC molecules and T cells are necessary for the proper development of mature T cells, in part due to an absolute requirement for self-MHC-TCR interactions. Recently, we showed that CD4-mediated interactions also participate in shaping the T cell repertoire during thymic maturation. We now examine the possible role of the CD8 molecule during in vivo T cell development. Our results demonstrate that perinatal thymi treated with intact anti-CD8 mAb fail to generate CD8 single-positive T cells, while the generation of the other main phenotypes remains unchanged. Most importantly, the use of F(ab')2 anti-CD8 mAb fragments gave identical results, i.e., lack of generation of CD4-/CD8+ cells, with no effect on the generation of CD4+/CD8+. Furthermore, selective blocking of one CD8 allele with F(ab')2 mAbs in F1 mice expressing both CD8 alleles did not interfere with the development of CD4-/CD8+ cells, demonstrating that the absence of CD8+ T cells in homozygous mice is not due to depletion, but rather is caused by a lack of positive selection. This is most likely attributable to a deficient CD8-MHC class I interaction. Our findings strongly advocate that CD8 molecules are vital to the selection process that leads to the development of mature single-positive CD8 T cells.     

脓毒症患者自然调节性T细胞及可溶性CD25水平研究

目的 通过前瞻性研究探讨脓毒症患者外周血自然调节性T细胞(Treg)百分比和血浆可溶性CD25分子(IL-2sRa)水平的变化及其临床价值.方法 将2009年2月至2010年2月上海交通大学附属瑞金医院重症医学科所收治的符合ACCP/SCCM于1997年提出的SIRS与脓毒症诊断标准的37例脓毒症患者与15例非感染SIRS患者,以及上海交通大学附属瑞金医院体检中心24例排除感染及免疫系统基础疾病患者,根据诊断标准分为脓毒症组、非感染SIRS组与正常对照组.其中脓毒症组男性26例,女性11例,年龄(61.67±11.87)岁;非感染SIRS组男性8例,女性7例,年龄(67.06±12.57)岁;正常对照组男14例,女10例,年龄(56.54±6.37)岁.脓毒症组肺部感染28例,腹腔感染6例,其他4例;非感染SIRS组为择期清洁手术后24 h内无感染征象者.所有入选对象均排除免疫系统基础疾病和(或)近期(30 d内)曾使用或正在使用强效免疫抑制剂患者.流式细胞分析术检测三组外周血Treg细胞百分比,ELISA检测血浆IL-2sRa,IL-4,IFN-γ水平.所得数据采用方差分析及非参数检验的Kruskal-Wallis H方法.结果 ①Treg细胞在脓毒症组、SIRS组和正常对照组占外周血CD4+CD25+T细胞的比例分别为:(66.82±21.79)%,(51.79±21.79)%、(56.45±10.68)%,脓毒症组Treg细胞显著高于SIRS组和正常对照组(P=0.001).②与SIRS组(381.664±189.83)和对照组(164.132±56.37)相比,脓毒症组可溶性CD25分子(IL-2sRa)水平(425.619±270.12)显著增高(P=0.000).③IL-2sRa浓度与Treg细胞占CD4+CD25+T细胞的比例相关分析示:Spearman相关系数=0.390,P=0.003(P＜0.05),两者存在正相关关系.结论 Treg细胞在脓毒症患者外周血的表达水平特征性增高.而外周血IL-2sRa水平可以反映Treg细胞表达水平,也有助于简化判断脓毒症的免疫状态的方法.

Abstract：

Objective To explore the CD4 + CD25 + Foxp3 + regulatory T cell percentage and plasma levels of soluble CD25 molecules in peripheral blood of septic patients and their clinical value through prospective study. Method A total of 37 septic patients and 15 non-infectious SIRS patients, who conformed to the criteria of SIRS and sepsis which proposed by American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference ( ACCP/SCCM ) in 1997, were collected in ICU of Ruijin Hospital ( Shanghai Jiaotong University) from February 2009 to February 2010. Twenty-four health people were from Medical Center of Ruijin Hospital, who were excluded infection and (or) autoimmune diseases. There were 26 male and 11 female in sepsis group, average age ( 61.67 ± 11.87 ) years old; 8 male and 7 female in SIRS group, average age (67.06 ± 12.57)years old; 14 male and 10 female in health control, average age (56.54 ± 6.37 )years old. All selected patrents were excluded the autoimmune diseases and (or) patients within recent (30 days) had used or now used immunosuppressive agents. We therefore measured the Treg cell percentage in peripheral blood by Flow Cytometry and the plasma levels of IL-2sRa, IL-4, IFN-γ by ELISA. The data were analyzed by analysis of variance or nonparametric Kruskal-Wallis H test. Results ① The percentage of CD4 + CD25 + Foxp3 + regulatory T cells among septic patients, SIRS patients, and control group was: ( 66.82 ± 21.79 ) %, ( 51.79 ± 21.79 ) %, ( 56.45 ± 10. 68 ) %, respectively. septic patients showed the highest percentages of CD4 + CD25 + Foxp3 + regulatory T cell among CD4 + CD25 + T cells(P ＜ 0.05 ). ② The plasma levels of soluble CD25 in septic patients (425. 619 ± 270.12 ) were significantly higher than SIRS patients (381. 664 ± 189.83) and the control group ( 164. 1 32 ± 56.37 ) ( P ＜ 0.05 ). ③ The correlation analysis between the concentration of soluble CD25 molecules in plasma and the ratio of CD4 + CD25 + Foxp3 + regulatory T cells to CD4 + CD25 + T cells showed Spearman correlation coefficient =0.390, P = 0.003 ( P ＜ 0.05 ). Conclusion: the expression of natural regulatory T cells characteristically increased in septic patients. And the levels of soluble CD25 in peripheral blood were related to the percentages of natural regulatory T cells, which simplified the assessment of the immune status in Septic patients.     

CD4+CD25+ T cells regulate virus-specific primary and memory CD8+ T cell responses

Naturally occurring CD4+CD25+ regulatory T cells appear important to prevent activation of autoreactive T cells. This article demonstrates that the magnitude of a CD8+ T cell-mediated immune response to an acute viral infection is also subject to control by CD4+CD25+ T regulatory cells (Treg). Accordingly, if natural Treg were depleted with specific anti-CD25 antibody before infection with HSV, the resultant CD8+ T cell response to the immunodominant peptide SSIEFARL was significantly enhanced. This was shown by several in vitro measures of CD8+ T cell reactivity and by assays that directly determine CD8+ T cell function, such as proliferation and cytotoxicity in vivo. The enhanced responsiveness in CD25-depleted animals was between three- and fourfold with the effect evident both in the acute and memory phases of the immune response. Surprisingly, HSV infection resulted in enhanced Treg function with such cells able to suppress CD8+ T cell responses to both viral and unrelated antigens. Our results are discussed both in term of how viral infection might temporarily diminish immunity to other infectious agents and their application to vaccines. Thus, controlling suppressor effects at the time of vaccination could result in more effective immunity.     

探讨CD4+CD25+Foxp3+调节性T淋巴细胞在HCV早期感染的作用

目的研究HCV抗原阳性抗体阴性的早期感染者体内的CD4+CD25+Foxp3+调节性T淋巴细胞水平及其与疾病进展相关性,观察CD4+CD25+Foxp3+调节性T淋巴细胞在HCV早期感染的作用。方法20例HCV抗原阳性抗体阴性的早期感染的病例,22例抗原阴性与抗体阳性HCV感染病例及25例抗原阳性与抗体阳性慢性HCV感染病例和21例健康人作对照组。应用流式细胞技术检测比较各组外周血中的有核细胞中的CD4+CD25+Foxp3+调节性T淋巴细胞;并同时检测丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST),分析与其的相关性。结果HCV感染病例的外周血中CD4+CD25+Foxp3+调节性T淋巴细胞表达率(百分率)较健康对照组高(t值分别为2.231,3.764;P均<0.05);抗原阳性抗体阴性组与抗原阳性抗体阳性组比较差异有统计学意义(t=2.180,P=0.035);抗原阴性抗体阳性组与健康对照组比较差异无统计学意义(t=0.437,P=0.665);抗原阳性抗体阴性组CD4+CD25+Foxp3+调节性T淋巴细胞表达率与肝脏炎症指标的ALT和AST有相关性(rALT=0.571,rAST=0.521,P<0.05)。结论CD4+CD25+Foxp3+调节性T淋巴细胞水平及其与疾病进展有重要相关性,可能是早期感染HCV并加速其慢性化的其中重要影响因素之一。     

不同树突状细胞对CD4~+CD25~+Foxp3~+调节性T细胞体外扩增的影响

目的利用不同种类树突状细胞(dendritic cells,DC)体外扩增获得表型和功能稳定的CD4+CD25+Foxp3+调节性T细胞(Treg)。方法免疫磁珠法(MACS)分离Balb/c小鼠CD4+CD25+调节性T细胞,利用与调节性T细胞同基因或异基因成熟DC、未成熟DC和调节性DC刺激其扩增,流式细胞术测定其纯度和表型。以CD4+CD25-T细胞作为反应细胞,验证扩增前后Treg细胞的免疫抑制功能。结果MACS分离的CD4+CD25+调节性T细胞纯度达到(95.38±1.82)%,同基因和异基因DC都能有效刺激Treg细胞体外扩增,其中同基因成熟DC扩增效果最为明显。而且同基因成熟DC扩增后CD4+CD25+调节性T细胞纯度达到(94.16±1.88)%,而且高表达Foxp3分子。当CD4+CD25+调节性T细胞与效应T细胞比例为1∶1时,能够有效的抑制效应T细胞的增殖,而且,同基因成熟DC扩增的CD4+CD25+调节性T细胞的抑制效果比新分离的Treg效果更好。结论同基因成熟DC能够体外扩增表型和功能稳定的Treg细胞。     

血必净注射液对脂多糖刺激大鼠调节性T细胞凋亡及其介导效应T细胞免疫功能的影响

目的 探讨中药血必净注射液对体外脂多糖(LPS)刺激CD4+CD25+调节性T细胞(Treg)凋亡及其介导效应T细胞(Teff)增殖反应、分泌白细胞介素-2(IL-2)功能的影响.方法 40只雄性清洁级Wistar大鼠,实验前禁食12 h.免疫磁珠法分选大鼠脾脏CD4+CD25+Treg,分为对照组(n=8)、抗CD3/CD28组(n=8)、抗CXB/CD28+LPS组(/1,=8)、抗CD3/CD28+血必净组(n=8)和抗CD3/CD28+LPS+血必净组(n=8).培养3 d后流式细胞术检测CD4+CD25+Treg凋亡率、叉头翼状螺旋转录因子(Foxp3)和T淋巴细胞毒性相关抗原4(CTLA-4)表达,酶联免疫吸附试验检测IL-10分泌.同时,CD4+CD25+Treg与CD4+CD25-T细胞1:1培养,刀豆素A刺激68 h后分析Treg对Teff增殖的抑制效应,并检测上清中IL-2/可溶性IL-2受体水平.结果 抗CD3/CD28诱导Treg凋亡率为(33.70±3.06%),显著高于对照组(12.84±0.84)%;抗CD3/CD28+LPS+血必净组(45.13±2.70)%凋亡率明显高于抗CD3/CD28+LPS组[(29.41±1.63)%,P＜0.01].同时,CD4+CLY25+Treg roxp3和CTLA-4表达及IL-10分泌随凋亡增加而降低.抗CD3/CD28+血必净组(31.26%)Teff增殖反应平均抑制率显著低于对照组(54.48%,P＜0.05),抗CD3/CD28+LPS+血必净组抑制率与抗CD3/CD28+LPS组比较亦明显下降(P＜0.01).此外,抗CD3/CD28+血必净组和抗CD3/CD28+LPS+血必净组IL-2分泌水平均显著高于抗CD3/CD28+LPS组(P＜0.01).结论 LPS刺激可明显上调大鼠Treg对Teff的抑制功能,血必净注射液能通过促进Treg凋亡而有效改善其对Teff的抑制效应.     

抗CD3单克隆抗体对人CD4^＋CD25^＋T淋巴细胞凋亡和自噬的影响

目的观察抗CD3单克隆抗体对分离培养的人外周血CD4^＋CD25^＋T淋巴细胞自噬、凋亡及其分泌的代表性因子转化生长因子β（TGF-β）的影响。方法采用密度梯度离心法及尼龙棉柱法分离32例健康者外周血T淋巴细胞,磁性细胞分离器（MACS）分离得到CD4^＋CD25^＋T淋巴细胞,分别利用电镜及流式细胞仪观察、检测各组（抗CD3单克隆抗体1mg/L组、IgG1同型抗体对照组）干预72h后细胞的凋亡率、自噬率,用ELISA法检测细胞培养上清液中细胞因子TGF-β的水平。结果抗CD3单克隆抗体组CD4^＋CD25^＋T淋巴细胞自噬率、凋亡率及TGF-β水平均增加（均P〈0.01）,凋亡率和自噬率之间无相关性（P〉0.05）。结论抗CD3单克隆抗体可促进CD4^＋CD25^＋T淋巴细胞凋亡和自噬及TGF-β分泌,且自噬与凋亡间相互独立。     

CD4~+CD25~+FOXP3~+调节性T细胞在结核病发生中的作用研究

目的:探讨CD4+CD25+FOXP3+调节性T细胞在结核病发生中的作用。方法:采用回顾性分析的方法,分析我院收治的60例结核病患者(观察组)和60例健康体检者(对照组)的临床资料。结果:观察组治疗前CD4+CD25+FOXP3+调节性T细胞比例明显高于对照组,治疗2、6个月末观察组患者CD4+CD25+FOXP3+调节性T细胞比例明显降低,观察组CD4+CD25+FOXP3+调节性T细胞比例与CD4+T细胞、CD8+T细胞、IFN-γ和IL-4呈现明显的负相关(P<0.05)。结论:针对CD4+CD25+FOXP3+调节性T细胞在结核病发生机制中的作用,开展从免疫治疗和结核病疫苗设计提供新的作用靶点和诊疗依据,值得临床推广应用。