Inability to mediate prolonged reduction of regulatory T Cells after transfer of autologous CD25-depleted PBMC and interleukin-2 after lymphodepleting chemotherapy

CD25CD4 regulatory T cells (Treg) regulate peripheral self-tolerance and possess the ability to suppress antitumor responses, which may explain the poor clinical response of cancer patients undergoing active immunization protocols, and provides the rationale for neutralizing Treg cells in vivo to strengthen local antitumor immune responses. Because interleukin-2 (IL-2) mediates tumor regression in about 15% of treated patients but simultaneously increases Treg cells, we hypothesized that transient elimination of Treg cells will enhance the clinical effectiveness of IL-2 therapy. In the current study, 5 patients with metastatic melanoma who were refractory to prior IL-2 received a lymphodepleting preparative regimen followed by the adoptive transfer of autologous lymphocytes depleted of CD25 Treg cells and high-dose IL-2 administration. CD25 cells were eliminated from patient leukapheresis samples using a clinical-grade, large-scale immunomagnetic system, leaving CD8 and CD25CD4 T cells intact. In the early aftermath of CD25 Treg cell-depleted cell infusion, CD25FOXP3+ CD4 Treg cells rapidly repopulated the peripheral blood of treated patients with 18% to 63% of CD4 T cells expressing FOXP3. Recovering CD25CD4 T cells exhibited suppressive activity against CD25CD4 effector T-cell proliferation in vitro. No patient experienced objective tumor regression or autoimmunity. Our results indicate that in vivo transfer of autologous CD25-depleted mononuclear populations to lymphopenic patients in combination with high-dose IL-2 is not sufficient to mediate prolonged reduction of Treg cells after IL-2 administration.     

Partial reduction of human FOXP3+ CD4 T cells in vivo after CD25-directed recombinant immunotoxin administration

The regulation of tolerance to self-proteins and the suppression of T-cell responses have in part been attributed to the activity of CD25+CD4+ T regulatory (Treg) cells. Further, Treg cells can inhibit the antitumor effectiveness of adoptive immunotherapy and active immunization approaches in preclinical models. In an effort to selectively eliminate Treg cells from human peripheral blood mononuclear cell to potentially bolster antitumor responses, we have evaluated the Treg-cell depleting capacity of the CD25-directed immunotoxin, RFT5-SMPT-dgA. In preclinical studies, incubation of human peripheral blood mononuclear cell with RFT5-SMPT-dgA mediated a partial reduction in the levels of CD25+, Foxp3-expressing CD4+ T cells in vitro. Administration of RFT5-SMPT-dgA to 6 patients with metastatic melanoma induced a transient but robust reduction in the number of CD25high CD4 T cells in vivo (a 97.5% mean reduction at nadir; from 69.4 +/- 12.4 cells/miroL to 1.7 +/- 0.3 cells/microL). The reduction in FOXP3+ CD4 T-cell number was less comprehensive (a 71.3% mean reduction at nadir; from 66.6 +/- 16.5 cells/microL to 14.2 +/- 3.9 cells/tL). This resulted in the selective persistence of a stable number of CD25(low/neg) FOXP3+ CD4+ T cells in vivo. No objective antitumor responses were seen in any patient. Our results indicate that the CD25-directed, RFT5-SMPT-dgA immunotoxin can mediate a transient, partial reduction in Treg-cell frequency and number in vitro and in vivo and suggest that comprehensive eradication of human Treg cells in vivo may require the ability to target and eliminate FOXP3+ CD4+ T cells expressing both high and low levels of CD25.     

人CD4+FOXP3+T细胞的异质性研究进展

CD4+ CD25+调节性T细胞(Treg)是一群具有免疫调节功能的细胞,对维持自身免疫耐受和免疫自稳必不可少,FOXP3特异性表达于Treg细胞,是Treg细胞发育、活化、发挥功能的关键.目前,CD4+ FOXP3+T淋巴细胞常被用来定义Treg细胞进行科学研究,而最近研究表明,人CD4+ FOXP3+T淋巴细胞存在表型和功能上的异质性,这其中包括具有免疫抑制功能的CD4+ FOXP3+ Treg,还包含没有抑制功能的其它类型T细胞,而这些不同功能的细胞亚群可以通过表型的差异加以区分.本文就近年来关于CD4+ FOXP3 +T细胞亚群表型特征和功能的异质性研究作一综述.     

Clinical-scale generation of dendritic cells in a closed system

Immunotherapy of malignant diseases based on dendritic cells (DCs) pulsed with tumor antigens is a promising approach. Therefore, there is a demand for large-scale, clinical-grade ex vivo generation of DCs. Here, a procedure is presented that combines monocyte selection and tissue culture in closed systems under current good manufacturing practice conditions. Leukocytes from three patients with urologic cancers were collected by leukapheresis and subjected to immunomagnetic enrichment. From leukapheresis products containing 1.6 +/- 0.2 x 1010 (mean +/- SEM) leukocytes with a frequency of CD14+ monocytes of 18.7 +/- 2.3%, monocytes were enriched to 94.3 +/- 2.2%. CD14+ cell recovery was 67.0 +/- 4.7%. After 6 days of culture in Teflon bags in X-Vivo 15 medium supplemented with autologous plasma, GM-CSF, and IL-4, cells showed an immature DC phenotype and efficient antigen uptake. Following an additional 3 days of culture in the presence of GM-CSF, IL-4, IL-1beta, IL-6, TNFalpha, and PGE(2), cells (82.0 +/- 5.8% CD83+) displayed a mature DC morphology and phenotype, including expression of CD11b, CD11c, CD18, CD25, CD40, CD54, CD58, CD80, CD86, HLA class I, and HLA-DR as well as expression of CCR7 but not CCR5. The mature DC phenotype remained stable for at least 5 days in the absence of cytokines. Yield of DC was 14.0 +/- 4.7% and viability was 91.9 +/- 3.5%. Mature DCs effectively clustered with naive T cells and potently induced allogeneic T-cell proliferation and IL-2 and IFNgamma but not IL-4 production. Thus, this procedure allows large-scale generation of stably mature, Th1 responses inducing DCs under cGMP conditions in a closed system from cancer patients and is therefore well suited for immunotherapy.     

人CD4~+FOXP3~+T细胞的异质性研究进展

CD4+CD25+调节性T细胞(Treg)是一群具有免疫调节功能的细胞,对维持自身免疫耐受和免疫自稳必不可少,FOXP3特异性表达于Treg细胞,是Treg细胞发育、活化、发挥功能的关键。目前,CD4+FOXP3+T淋巴细胞常被用来定义Treg细胞进行科学研究,而最近研究表明,人CD4+FOXP3+T淋巴细胞存在表型和功能上的异质性,这其中包括具有免疫抑制功能的CD4+FOXP3+Treg,还包含没有抑制功能的其它类型T细胞,而这些不同功能的细胞亚群可以通过表型的差异加以区分。本文就近年来关于CD4+FOXP3+T细胞亚群表型特征和功能的异质性研究作一综述。     

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.     

Defective regulatory and effector T cell functions in patients with FOXP3 mutations

The autoimmune disease immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) is caused by mutations in the forkhead box protein P3 (FOXP3) gene. In the mouse model of FOXP3 deficiency, the lack of CD4+ CD25+ Tregs is responsible for lethal autoimmunity, indicating that FOXP3 is required for the differentiation of this Treg subset. We show that the number and phenotype of CD4+ CD25+ T cells from IPEX patients are comparable to those of normal donors. CD4+ CD25high T cells from IPEX patients who express FOXP3 protein suppressed the in vitro proliferation of effector T cells from normal donors, when activated by "weak" TCR stimuli. In contrast, the suppressive function of CD4+ CD25high T cells from IPEX patients who do not express FOXP3 protein was profoundly impaired. Importantly, CD4+ CD25high T cells from either FOXP3+ or FOXP3- IPEX patients showed altered suppression toward autologous effector T cells. Interestingly, IL-2 and IFN-gamma production by PBMCs from IPEX patients was significantly decreased. These findings indicate that FOXP3 mutations in IPEX patients result in heterogeneous biological abnormalities, leading not necessarily to a lack of differentiation of CD4+ CD25high Tregs but rather to a dysfunction in these cells and in effector T cells.     

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。     

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。     

骨髓增生异常综合征患者CD4+CD25+FoxP3+Treg细胞特点及环孢素对其的影响

目的探讨骨髓增生异常综合征(myelodysplastie syndrome,MDS)-难治性贫血(refractory anemia,RA)及难治性血细胞减少伴有多系发育异常(refractory cytopenia with multiple dysplasia,RCMD)患者CD4+CD25+FOXP3+Treg细胞与白细胞介素(interleukin,IL)-10表达水平,评估环孢素对MDS患者CD4+CD25+FOXP3+Treg细胞的影响。方法选择2016年1月至2018年1月于新疆维吾尔自治区人民医院25例MDS-RA及RCMD患者(MDS组)和13名健康对照(健康对照组),采用流式细胞术及酶联免疫吸附试验检测外周血标本CD4+CD25+FOXP3+Treg与IL-10表达水平,检测MDS-RA及RCMD患者在应用以环孢素为基础的免疫抑制方案治疗前、治疗6个月后及MDS组治疗有效及无效的CD4+CD25+FOXP3+Treg与IL-10的表达情况。结果全部25例患者中,13例(52%)有效,12例(48%)无效。MDS组CD4+CD25+FOXP3+Treg占CD4+T细胞比例显著高于健康对照组[(0.37±0.10)%与(0.12±0.06)%,t=2.02,P<0.001]。MDS组IL-10水平显著高于健康对照组[(7.16±1.27)μg/L与(2.75±1.06)μg/L,t=2.03,P<0.001]。MDS治疗有效组CD4+CD25+FOXP3+Treg细胞比例低于无效组[(0.15±0.06)%与(0.26±0.08)%,t=1.71,P<0.001],有效组IL-10水平低于无效组[(3.22±1.01)μg/L与(4.25±1.22)μg/L,t=2.06,P=0.030]。25例MDS患者外周血CD4+CD25+FOXP3+Treg比例与IL-10表达水平呈正相关关系(r=0.35,P=0.02)。结论MDS患者存在CD4+CD25+FOXP3+Treg细胞与IL-10的表达升高,环孢素治疗后降低。     

流式细胞术检测RA患者外周血CD4^＋CD25^＋FOXP3^＋ Treg细胞及其GITR表达

目的研究FOXP3和糖皮质激素诱导的肿瘤坏死因子受体(GITR)在类风湿关节炎(RA)患者外周血Treg细胞的表达并探讨其临床意义。方法用五色流式细胞术(FCM)检测40例健康体检者、61例RA患者外周血CD4 CD25 FOXP3 调节性T细胞(Treg)及其中GITR的表达情况。结果RA患者组FOXP3的平均荧光强度(MFI)低于健康对照组(P<0.05),CD4 CD25 FOXP3 Treg占CD4 T细胞的比例、CD4 CD25 FOXP3 Treg中GITR的表达率、GITR MFI和健康对照组相比无统计学差异(P>0.05);活动期RA患者的FOXP3 MFI和GITR MFI均显著低于非活动期组(P<0.01和P=0.032),CD4 CD25 FOXP3 Treg占CD4 T细胞中的比例、CD4 CD25 FOXP3 Treg中GITR的表达率与非活动期组相比无统计学差异(P>0.05)。结论RA患者的FOXP3表达低下,活动期组的FOXP3表达低于非活动期组,为其作为RA病情变化的判断指标提供进一步的证据。     

再生障碍性贫血患者CD4+ CD25+ CD127low调节性T细胞及Notch1表达水平的变化

目的 测定再生障碍性贫血(AA)患者治疗前后外周血CD4+ CD25+ CD127low调节性T细胞(Treg)的数量及叉头翼状螺旋转录因子(FOXP3)mRNA、Notch1 mRNA的表达水平,探讨Treg在AA发病中的作用及其机制.方法 流式细胞术检测29例初发AA患者、14例环孢素(CsA)治疗后恢复期及11例治疗后未恢复期患者外周血中CD4+ CD25+ CD127low T细胞、CD4+ CD25+ T细胞的数量,并与正常对照比较;采用RT-PCR检测FOXP3 mRNA和Notch1 mRNA的表达水平,分析两者相关性.结果AA初发组及治疗后未恢复组患者外周血中活化CD4+ CD25+ T细胞占CD4+ T细胞比例分别为(4.3±0.7)%、(4.2±0.6)%,明显高于正常对照组[(2.4±0.8)%](P＜0.05).CsA治疗后恢复组患者比例下降为(2.6±0.7)%(P＜0.05),与对照组比较差异无统计学意义.AA初发组及未恢复组CD4+ CD25+ CD127low T细胞在CD4+ T细胞中的比例分别为(2.4±1.2)%、(2.5±1.1)%,较正常对照组[(7.1±2.7)%]及恢复组[(5.3±1.0)%]明显降低(P值均＜0.01);但后两组比较差异无统计学意义.AA初发组患者FOXP3 mRNA及Notch1 mRNA分别为(0.260±0.011)和(0.018±0.005),较正常对照[(1.307±0.011)和(0.308±0.028)]表达明显下调(P值均＜0.01),治疗后分别为(1.287±0.012)和(0.281±0.013),表达较初发组显著提高(P值均＜0.01),与对照组比较差异无统计学意义(P值均＞0.05).AA患者CD4+ CD25+ CD127low T细胞、FOXP3均与Notch1表达呈正相关性(P值均＜0.01).结论AA患者外周血CD4+ CD25+ CD127low Treg减少,其抑制作用减弱,导致自身反应性T细胞过度活化,抑制造血.其作用机制之一可能与靶细胞表面Notch1分子表达降低相关.     

Human gammadelta T cells from G-CSF-mobilized donors retain strong tumoricidal activity and produce immunomodulatory cytokines after clinical-scale isolation

Human gammadelta T cells are a small fraction of T cells that have been shown to exert major histocompatibility (MHC)-unrestricted natural cytotoxicity against a variety of solid tumors and some subsets of leukemias and lymphomas. They are also involved in the immune response to certain bacterial, viral, and parasitic infections and expand significantly in CMV- or HSV-infected organ allografts. They are able to mediate antibody-dependent cytotoxicity and are not alloreactive, which makes them attractive candidates for cell-based immunotherapy. However, their frequency in peripheral blood is low and ex vivo expansion of gammadelta T cells is labor-extensive, does not always yield cells with full innate cytotoxic power, and has the potential for microbial contamination. Therefore, the authors developed a clinical-scale, automated cell purification method for the efficient enrichment of gammadelta T cells from leukapheresis products. Six leukapheresis products were purified for gammadelta T cells using a single-step immunomagnetic method. Purity and phenotype were assessed by flow cytometry. A standard Europium release assay was performed to determine the cytotoxic capacity of the cells. Cytokine production was measured using a multiplex sandwich immunoassay. The mean percentage of gammadelta T cells in the final product was 91%, with an average recovery of 63%. The cells showed a high co-expression of CD8, CD56, CD28, and CD11b/CD18. In some products an unusually high proportion of Vgamma9Vdelta1 T cells was found. The isolated cells were cytotoxic against the neuroblastoma cell line NB1691 and the erythroleukemic line K562 in vitro. They were able to produce a variety of immunomodulatory cytokines such as IFNgamma, TNFalpha, and MIP-1beta, but also GM-CSF and G-CSF when co-incubated in culture with and without various stimuli. In summary, the authors describe a rapid, automated, and efficient method for the large-scale enrichment of human gammadelta T cells. The cytotoxic properties of the cells were preserved. This method yields sufficient purified gammadelta T cells for use in adoptive immunotherapy as well as laboratory investigations and animal studies.     

为过继性免疫治疗培养淋巴细胞时的细胞因子作用和基因表达特征

在过继免疫性细胞培养时需要加入不同的细胞因子。本研究旨在比较不同细胞因子及EBV抗原肽联合细胞因子刺激淋巴细胞后淋巴细胞分化方向和相关的基因表达变化特征。分别用不同细胞因子及EBV抗原肽联合细胞因子刺激淋巴细胞,在培养的当天和第1、3、7、10天,用流式细胞术检测各组细胞中CD3+(总T细胞)、CD3+CD4+(辅助T细胞)、CD3+CD8+(细胞毒性T细胞)、CD3+CD8+CD45RO+(记忆型T细胞)、CD3+CD8+CD45RA+(初始型T细胞)、CD3+CD30+(Th2辅助细胞)、CD19+(B细胞)、CD56+(NK细胞)、CD4+CD25+(初始调节T细胞)、CD4+CD25+FOXP3+(精确调节T细胞)在培养前后总细胞中的百分比变化;用RT-PCR技术检测管家基因mad1、pten和辅助T细胞转录调控基因t-bet(Th1)、gata3(Th2)、细胞因子ifn-γ(Th1)、il-4(Th2)基因表达量。结果表明:EBV多肽组CTL细胞成为优势细胞,临床治疗有确切疗效;比较加入EBV多肽组和不同细胞因子培养组结果显示,EBV抗原肽可以更有效刺激CTL生成,ifn-γ基因表达量明显增加;辅...     

慢性特发性血小板减少性紫癜患者外周血树突细胞的变化及意义

目的 探讨慢性特发性血小板减少性紫癜(ITP)患者体内树突细胞(DC)数量和功能的变化.方法 慢性ITP患者予以大剂量地塞米松(HD-DXM)冲击治疗,剂量40 mg/d,口服,连续4 d,观察短期疗效.用流式细胞术检测患者外周血中髓系DC(mDC)和浆细胞样DC(pDC)在治疗前后绝对数量的变化及与CD4+CD25+调节性T(Treg)细胞的相关性;流式细胞术检测外周血总Dc表面协同刺激分子的表达.将慢性ITP患者外周血单核细胞来源的DC、CD4+T淋巴细胞与自身血小板或健康人同源异基因血小板混合培养,观察CD4+T淋巴细胞增殖情况,检测DC血小板相关抗原的呈递功能.结果 与正常对照组比较,慢性ITP患者外周血pDC和mDC绝对数量均无明显变化(P值均》0.05);而外周血CD4+FOXP3+T细胞数明显降低(P《0.01),pDc和mDC与CD4+FOXP3+T细胞数之间均存在负相关性(r=-0.396,P=0.045和r=-0.410,P=0.037).HD-DXM治疗初始反应率达92.3%,pDC绝对数量较治疗前减少达75.5%(P《0.01);而mDC较治疗前虽增加了24.3%(P《0.05),但mDC上CD11c表达的平均荧光强度(MFI)从治疗前340±30降至199±21(P《0.01);CD4+F0xP3+T细胞数较治疗前显著增加(P《0.01),治疗后pDC与CD4+F0XP3+T细胞存在负相关(r=-0.524,P=0.006),而mDC与CD4+F0XP3+T细胞间无相关性(r=-0.360,P=0.071).慢性ITP患者外周血DC表面协同刺激分子CD86表达的MFI明显高于正常对照组(P《0.05);CD86、CD40、CD80表达阳性率及CD40、CD80表达的MFI与正常对照组比较差异均无统计学意义(P值均》0.05).慢性ITP患者CD4+T淋巴细胞增殖反应强度高于对照组(P《0.05),DC对血小板相关抗原呈递功能亢进.结论 DC可能参与了慢性ITP的免疫发病机制,并与CD4+CD25+Treg细胞有一定的关联.     

骨髓源性而非脾源性树突状细胞能够诱导产生调节性T细胞

越来越多的证据表明,树突状细胞(DC)能够通过刺激CD4+T细胞产生调节性T细胞(Treg)而参与免疫耐受。本研究旨在探索骨髓源性DC(bone marrow-derived DC,BM-DC)或脾源性DC(spleen derived DC,spDC)诱导CD4+CD25+FOXP3+Treg产生的可能性。以GM-CSF和IL-4从C57BL/6小鼠骨髓前体细胞诱导产生骨髓未成熟DC(immature DC,imDC);6 d后,imDC经脂多糖(LPS)进一步刺激16 h得到成熟树突状细胞(mature DC,mDC);同时用免疫磁珠从小鼠脾脏分选得到spDC,以这3种DC分别与新鲜分离的BALB/c小鼠脾CD4+T细胞混合培养,诱导CD4+CD25+FOXP3+Treg的产生;用流式细胞仪检测CD4+T细胞中FOXP3的表达,对比分析3种DC诱导产生CD4+CD25+FOXP3+Treg的能力。结果表明:经C57BL/6小鼠骨髓imDC、mDC的刺激,BALB/c小鼠CD4+T细胞中的FOXP3表达率从(8.57±1.14)%分别提高到(15.80±1.35)%、(17.93±1.45)%(P<0.01);经spDC的刺激,BALB/c小鼠的CD4+T细胞中的FOXP3表达率则从(8.57±1.14)%下降到(3.95±0.79)%(P<0.05)。结论:体外诱导的BM-DC而非spDC能够诱导Treg的产生,具有介导免疫耐受的作用。     

脐血淋巴细胞的免疫学特性研究

目的探讨脐血中T淋巴细胞的免疫功能,为脐血移植后移植物抗宿主病(GVHD)、移植物抗白血病反应(GVL)及免疫功能重建提供理论依据.方法用免疫荧光抗体直接标记淋巴细胞分化抗原,应用流式细胞仪进行检测;用微量全血法进行单细胞内的细胞因子检测从而判断脐血T淋巴细胞分泌细胞因子(IFN-γ、IL-4)的能力;用植物血凝素(PHA)作为刺激剂,观察T淋巴细胞CD25表达的动态变化.结果脐血T淋巴细胞CD4+/CD8+为1.93±0.91,较成人高(P＜0.01),CD8+细胞百分比低于成人外周血(P＜0.01),而且90%以上T淋巴细胞表达初始型CD45RA,显著高于成人外周血淋巴细胞(P＜0.01);CD25、CD28、HLA-DR二者间差异无显著性.脐血CD4+、CD8+细胞分泌IFN-γ、IL-4的水平显著低于成人T淋巴细胞(P＜0.01).脐血T淋巴细胞经PHA刺激可以活化,但活化的时相与成人略有不同.结论脐血淋巴细胞各细胞亚群的构成比例及发育成熟度与成人均不相同,脐血CD4+CD8+淋巴细胞中,以代表初始型、未接触抗原刺激的CD45RA+细胞为主,而且分泌细胞因子的能力低下,可能是脐血移植后急、慢GVHD发生率低、强度弱的原因之一.脐血淋巴细胞经适当刺激可以活化,有望用于过继性免疫治疗.     

地塞米松联合IL-2对供鼠体内调节性T细胞选择性扩增作用及对急性移植物抗宿主病反应的抑制作用

目的 通过地塞米松(Dex)联合IL-2处理小鼠,建立体内扩增调节性T细胞(Treg细胞)的方法 .观察用该方法 处理的小鼠脾细胞移植后受鼠急性移植物抗宿主病(aGVHD)的发生与转归.方法 使用Dex和IL-2处理雄性C57BL/6N供鼠3 d后提取脾单个核细胞(MNC),用流式细胞术(FCM)分析CD4~+ CD25~+、CD25~+ FOXP3~+细胞的变化.,以以上方法 处理的雄性C57BL/6N小鼠为供者,对雌性BALB/c受鼠进行非清髓异基因淋巴细胞移植.移植后观测受鼠的生存率、生存时间、组织病理学变化,以及用PCR和FCM分析测定受鼠嵌合体等来评价aGVHD的发生.结果 经过Dex和IL-2联合处理,供鼠脾CD4~+ CD25~+ FOXP3~+ Treg细胞数量[(24.2±7.6)%]明显高于对照组[(4.0±0.8)%](P=0.01).Dex联合IL-2组供鼠Treg细胞与效应T细胞(Teff)的比值(0.43±0.15)显著高于对照组(0.14±0.01)(P=0.01).经过Dex联合IL-2处理供鼠后进行异基因淋巴细胞移植,受鼠aGVHD明显减轻,中位生存时间＞60 d,与对照组的中位生存时间12 d相比明显延长(P=0.0045).对照组出现典型的aGVHD表现,移植后2周Dex联合IL-2组和对照组的总体死亡率分别为29.4%和71.4%(P＜0.05).结论 经IL-2和糖皮质激素处理供鼠后进行主要组织相容性抗原复合物完全不相合脾淋巴细胞移植能明显减轻aGVHD,显著延长生存时间,可能与供者体内诱导扩增的Treg细胞增加有关.     

基于免疫磁珠分选的调节性T细胞亚群嵌合性定量分析

为了探讨免疫分选偶联短串重复序列（STR）方法用于调节性T细胞（Treg）嵌合性定量监测的可行性,以淋巴细胞不同比例混合制备14组人工嵌合体血样,以单个核细胞为起点,以免疫磁珠阴性和阳性选择法获取CD4＋CD25＋Treg细胞,分选后的Treg细胞再进行16位点的STR长度多态性分析。结果显示,从分选后的Treg细胞提取的DNA量能满足STR检测要求,当嵌合体中受体比例≥10%时,16个STR位点均能检出受体和供体特异性等位基因,按照STR峰面积计算的受体比例与理论值相符;当受体比例≤1%时,仅有部分位点能检出受体特异性等位基因,实测嵌合率与理论值存在差异。结论：建立了基于免疫分选的Treg细胞嵌合性定量分析方法,对造血嵌合体的检出灵敏度和准确性在1%-10%,可用于造血干细胞移植术后嵌合体监测。