自身免疫性肝炎患者外周血T细胞PD-1的表达及与肝功能指标的相关性

目的：研究自身免疫性肝炎（AIH）患者外周血CD4＋、CD8＋T细胞表面程序性死亡受体1（PD-1）的表达,并探讨其与肝功能指标的相关性。方法收集43例AIH患者（包括活动期31例和缓解期12例）,并选择18例健康体检者为对照组。采用流式细胞术检测外周血 CD4＋、CD8＋T细胞表达PD-1的细胞百分比。结果 AIH 组外周血 CD4＋、CD8＋T 细胞表达 PD-1的细胞百分比（分别为7.36±4.45％、6.71±3.84％）显著高于对照组（分别为3.78±2.32％、3.47±2.18％）（P均＜0.05）;AIH活动期组外周血 CD4＋、CD8＋T细胞表达PD-1的细胞百分比（分别为8.73±6.81％、7.44±2.75％）明显高于缓解期组（分别为5.42±2.14％、4.82±2.04％）和对照组（分别为3.78±2.32％、3.47±2.18％）（P均＜0.05）,且缓解期组高于对照组（P＜0.05）。 AIH患者外周血CD4＋、CD8＋T细胞 PD-1表达与 TBIL、ALT、AST及 IgG水平均成正相关关系（r分别为0.781、0.887、0.825、0.893,P均＜0.05）。结论 AIH患者外周血 CD4＋、CD8＋T 细胞表面 PD-1表达上调,且与肝功能指标成正相关,提示PD-1可能在AIH的发生及发展中发挥重要作用。     

Functional tumor specific CD8 + T cells in spleen express a high level of PD-1

The inhibitory effects of programmed cell death 1 (PD-1) receptor on tumor specific T cells were mainly investigated at tumor site. While PD-1 expression can be rapidly unregulated upon T cell activation at lymphoid tissues, little is known about where PD-1 signal exerts its inhibitory function in tumor-bearing host. To address this issue, we assessed the effects of PD-1 on vaccine induced activation of splenic CD8 + T cells in mice. The vaccine consisted of mice CD8 + T cell epitope peptide and poly IC. After vaccination, spleen or tumor tissues were dissociated, IFN-γ synthesis and PD-1 expression by CD8 + T cells were detected by flow cytometry. We found that CD8 + T cells could be successfully activated in spleen after immunization, characterized by the capability of producing IFN-γ when encountering relevant peptide. These activated splenic CD8 + T cells also expressed a high level of PD-1. Although PD-L1 expression in spleen parenchyma was also increased after vaccination, PD-1 blockade did not affect the activation of splenic CD8 + T cells, but enhanced the anti-tumor effects of peptide vaccine. This synergetic effect of peptide vaccine plus PD-1 blockade was coupled with increased aggregation of IFN-γ + CD8 + tumor infiltrated lymphocytes (TILs), rather than CD4 + TILs. The results indicated that for tumor-bearing host, PD-1 pathway exerted its inhibitory function at tumor site and PD-1 expression on the splenic CD8 + T cells correlated positively with IFN-γ synthesis.     

复方苦参注射液对胃癌术后化疗患者免疫功能的影响

目的：探讨复方苦参注射液对胃癌术后化疗患者的免疫增强作用。方法：将40例胃癌术后化疗患者随机分成两组，对照组给予氟尿嘧啶＋亚叶酸钙＋奥沙利铂化疗，试验组所用化疗方案与对照组相同，同时在化疗的第1～5天给予复方苦参注射液20mL／d。所有患者均用流式细胞仪测定其外周血T淋巴细胞亚群及自然杀伤细胞（NK细胞）数量，并进行对照分析。结呆：对照组化疗后外周血CD3^＋、CD4^＋、NK细胞数量及CD4^＋／CD8^＋细胞比值较化疗前显著下降（P〈0．05），CD8^＋细胞水平显著升高（P〈0．05）；试验组化疗后外周血CD3^＋、CD4^＋、CD8^＋、NK细胞数量及CD4^＋／CD8^＋细胞比值较化疗前无显著差异（P〉0．05）。化疗后试验组外周血CD3^＋、CD4^＋、NK细胞数量及CD4^＋／CD8^＋细胞比值均较对照组显著升高（P〈0．05），CD8^＋细胞水平与对照组无显著差异（P〉0．05）。结论：复方苦参注射液能有效增强胃癌术后化疗患者的免疫功能。     

Organophosphorous pesticide metabolite (DEDTP) induces changes in the activation status of human lymphocytes by modulating the interleukin 2 receptor signal transduction pathway

Diethyldithiophosphate (DEDTP) is a metabolite formed by biotransformation of organophosphorous (OP) compounds that has a longer half-life than its parental compound. Here we evaluate the effects of DEDTP on human CD4+ T lymphocytes. In vitro exposure to DEDTP (1-50 μM) decreased [³H]thymidine incorporation in resting cells and increased CD25 surface expression without altering cell viability. DEDTP treatment inhibited anti-CD3/anti-CD28 stimulation-induced CD4+ and CD8+ T cell proliferation determined by CFSE dilution. Decreased CD25 expression and intracellular IL-2 levels were correlated with this defect in cell proliferation. IL-2, IFN-γ and IL-10 secretion were also reduced while IL-4 secretion was not altered. Increased phosphorylation of SOCS3 and dephosphorylation of STAT5 were induced by DEDTP after as little as 5 min of exposure. In addition, DEDTP induced phosphorylation of ERK, JNK and p38 and NFAT nuclear translocation. These results suggest that DEDTP can modulate phosphorylation of intracellular proteins such as SOCS3, which functions as a negative regulator of cytokine signalling, and that DEDTP exposure may thus cause T cells to fail to respond to further antigen challenges.     

慢性丙型肝炎患者免疫细胞PD-L1表达特点及临床意义

目的 观察慢性丙型肝炎(CHC)患者外周血CD4~+T细胞和CD8~+T细胞表面程序性死亡配体1(PD-L1)的表达水平以及与HCV RNA载量的关系.方法 流式细胞仪检测外周血CD4~+T细胞和CD8~+T细胞表面PD-L1的表达,荧光定量PCR检测HCV RNA载量,基因芯片检测HCV基因型.结果 CHC患者外周血CD4~+T细胞和CD8~+T细胞表面PD-L1的表达水平较健康人明显上调,与HCV RNA和ALT无相关性.结论 PD-L1在CD4~+T细胞和CD8~+T细胞表面的高表达,抑制免疫细胞清除HCV的能力,导致感染的慢性化.     

PD-1 regulates CXCR5+ CD4 T cell-mediated proinflammatory functions in non-small cell lung cancer patients

PD-1 inhibitors have been used to revive exhausted T cell responses in non-small cell lung cancer (NSCLC) and other malignancies. CXCR5⁺ T follicular helper (Tfh) cells are characterized by constitutive high PD-1 expression and have been associated with the formation of tertiary lymphoid structures and implicated in antitumor immunity. In this study, we investigated the effect of PD-1 and PD-1 inhibition on CXCR5⁺ CD4 T cells. Data showed that CXCR5⁺ CD4 T cells in both healthy subjects and NSCLC patients presented markedly higher PD-1 expression than CXCR5⁻ CD4 T cells. Both CXCR5⁻ and CXCR5⁺ CD4 T cells from NSCLC patients presented higher PD-1 expression than their counterparts in healthy subjects. PD-1⁺ CXCR5⁺ CD4 T cells were functional, could express IL-21, IL-10, and CXCL13 upon stimulation, demonstrated auxiliary effects toward CD8 T cell-mediated IFN-γ production and proliferation, and promoted IgM and IgG production. However, the potency of PD-1⁺ CXCR5⁺ CD4 T cells was lower than the potency of PD-1⁻ CXCR5⁺ CD4 T cells. PD-1 blocking could significantly enhance the effector functions of PD-1⁺ CXCR5⁺ CD4 T cells. Overall, this study demonstrated that PD-1⁺ CXCR5⁺ CD4 T cells could promote CD8 T cell and B cell inflammation and could be modulated by PD-1 inhibition.     

体外阻断CD137-CD137L途径抑制移植物抗宿主病的免疫效应

目的探讨体外阻断活化的供鼠T淋巴细胞CD137-CD137L共刺激途径抑制供鼠淋巴细胞的免疫反应。方法应用雄性供鼠BALB/c脾淋巴细胞为反应细胞,雌性受鼠C57BL/6脾淋巴细胞为刺激细胞,进行混合淋巴细胞培养(MLC)。设单抗实验组(加抗CD137L单抗)和对照组(不加抗CD137L单抗),初次和再次MLC第1、3、5、7天采用流式细胞术检测CD3+CD4+T细胞、CD3+CD8+T细胞;RT-PCR法检测培养的细胞IL-2、IFN-γ、IL-4、IL-10的水平。结果实验组CD3+CD8+T细胞明显低于对照组(P<0.01);实验组IL-2、IFN-γ水平明显低于对照组(P<0.01);实验组IL-10表达明显高于对照组(P<0.01)。结论体外MLC中,应用抗CD137L单抗孵育供鼠脾T淋巴细胞主要抑制供鼠CD3+CD8+T细胞的增殖,抑制Ⅰ类细胞因子IFN-γ及IL-2的表达,促进Ⅱ类细胞因子IL-10的表达。     

Gemcitabine and checkpoint blockade exhibit synergistic anti-tumor effects in a model of murine lung carcinoma

Lung cancer is leading cause of cancer death in the world. Chemotherapy is currently one of the standard treatments for lung cancer. Gemcitabine is a pyrimidine nucleoside drug which has been approved by FDA to treat lung cancer. However, acquired resistance inevitable develops after Gemcitabine treatment, limiting clinical efficacy. Lewis lung carcinoma (LLC) cells were treated with Gemcitabine and cell apoptosis and programmed cell death-ligand 1 (PD-L1) expression were analyzed by flow cytometry. LLC mouse model was established to analysis the proportion and programmed cell death-1 (PD-1) expression of CD8 + T cells. Anti-tumor effect by treating with Gemcitabine and anti-PD-1 antibody was measured through in vivo LLC mouse model. Gemcitabine treatment induces tumor cell apoptosis and PD-L1 expression. Further study showed that Gemcitabine treatment also increases CD8⁺ and CD4⁺ T cells proportion, PD-1 and PD-L1 expression in LLC mouse model. Combination therapy with Gemcitabine and αPD-1 not only has strong anti-tumor effect, but also could inhibit postsurgical recurrence of LLC. Our findings demonstrated that the combination therapy of Gemcitabine and αPD-1 is an effective therapeutic strategy for lung cancer.     

PD-1 blockade augments humoral immunity through ICOS-mediated CD4+ T cell instruction

Successful applications of PD-1/PD-L1 blockade in multiple cancers highlight the efficacy of immunotherapy mediated by enhancing CD8⁺ T cell immunity both in mouse and human. How PD-1 blockade affects humoral immunity remains unclear. Herein we demonstrated that treatment of anti-PD-1 antibody led to the increase in both total IgG and OVA-specific IgG in OVA-immunized mice. However, no effect was observed on Ab affinity maturation. Accumulation of germinal center (GC) and memory B cells was observed in the spleens together with elevated percentages of plasma cells in the spleens and bone marrow. More interestingly, dramatic infiltration of CD4⁺ T cells was apparent in GCs after PD-1 blockade with a significant increase in the expression of ICOS. When CD4⁺ T cells and B cells from OVA-immunized mice were co-cultured with neutralizing anti-PD-1 Ab in vitro, PD-1 blockade recapitulated the up-regulation of ICOS expression on CD4⁺ T cells with the activation of ERK signaling. Suppression of ERK activation not only reduced ICOS expression on CD4⁺ T cells but also attenuated IgG production upon PD-1 blockade. Taken together, PD-1 blockade enhances humoral immunity. This process partially relies on more accumulation of CD4⁺ T cells in GCs with the up-regulation of ICOS expression and the promotion of B cell terminal differentiation. The regulatory pattern of PD-1 blockade illustrated here provides a new mechanism of how immune checkpoint molecules regulating humoral immune responses.     

乙型肝炎患者外周血淋巴细胞表达的变化

目的 分析乙型肝炎患者外周血淋巴细胞亚群的表达.方法 对112例乙型肝炎患者采用荧光定暈PCR技术和流式细胞术分别检测血中HBV DNA及外周血淋巴细胞亚群CD3~+T细胞、CD4~+T细胞、CD8~+T细胞、CD3~-/CD16~+CD56~+(NK细胞)、CD3~- CD19~+(B细胞)表达百分比,进行各淋巴细胞亚群百分比统计学分析.结果 与急性乙型肝炎(AHB)组比较,慢性乙型肝炎(CHB)组、肝炎肝硬化(LC)组CD3~+T细胞、CD4~+T细胞、CD8~+T细胞、CD4~+/CD8~+比值均有显著性下降,LC组下降最显著(P<0.01).而LC组B细胞百分比较CHB、AHB组高(P<0.01).NK细胞百分比较CHB、AHB组均存在降低趋势(P<0.01).结论 乙型肝炎患者体内存在T细胞亚群失衡和细胞免疫功能紊乱;外周血T细胞亚群随病情进展而减少.     

抗CD40抗体增强宫颈癌肽疫苗的治疗作用

目的：探讨抗CD40抗体对宫颈癌肽疫苗治疗作用的影响。方法将乳头状病毒结构性蛋白 E7表位肽（ E749－57,H－2Db限制性,CD8 T细胞表位）与Toll样受体7配体（Gardiquimod）组成疫苗,C57BL／6小鼠给予疫苗及抗CD40抗体免疫后取得外周血及脾组织CD8 T细胞,利用流式细胞仪分析特异性CD8 T细胞数量,Elisa检测CD8 T细胞与TC－1细胞（表达HPV E7蛋白的小鼠宫颈癌细胞）共孵育后干扰素（ INF－γ）表达水平。另取15只皮下荷瘤小鼠,监测免疫后肿瘤生长速度。结果与单用疫苗组相比,抗 CD40抗体明显增加小鼠外周血肿瘤特异性 CD8 T细胞数量（16．50±0．8185％ vs 9．747±1．834％,P＝0．0282）,且内源性CD8 T细胞体外与TC－1细胞共孵育可以分泌INF－γ,体内显著抑制皮下肿瘤生长（ P＜0．001）。结论抗CD40抗体增强肽疫苗诱发的免疫应答,有潜力成为一种良好的佐剂。     

急性乙型肝炎外周血淋巴细胞亚群动态分析

目的 探讨急性乙型肝炎(AHB)患者外周血免疫细胞包括T淋巴细胞亚群、B淋巴细胞和自然杀伤(NK)细胞在疾病发展中的动态变化及其意义.方法 流式细胞技术检测17例AHB患者(AHB组)外周血淋巴细胞亚群动态变化和36例慢性乙型肝炎(CHB组)患者及32例正常对照组淋巴细胞亚群分布特点;动态检测AHB患者CD4~+/CD8~+变化,并探讨其与ALT改变及与HBVDNA清除的相关性.结果 AHB组人院4周内外周血CD3~+、CD4~+和CD8~+T细胞频率显著高于CHB组和对照组,NK细胞4周内均低于其余2组(P<0.05).AHB疾病早期ALT高水平异常时CD4~+/CD8~+明显低下;随着ALT恢复正常,CD4~+/CD8~+比值呈逐渐升高趋势.结论 AHB患者外周血免疫细胞的分布与对照组和CHB患者不同,淋巴细胞亚群的动态变化与疾病发展可能有一定的相关性.     

Forced expression of programmed death-1 gene on T cell decreased the incidence of type 1 diabetes

Programmed death-1 (PD-1) is a co-inhibitory receptor of the CD28/CTLA-4 family which is expressed on activated T cells and inhibits T cell activation after binding to PD-1 ligands. In animal models, PD-1 regulates autoimmune disease and induces tolerance in pancreas. In this study the effects of PD-1 on type 1 diabetes were examined using PD-1 transgenic mice (Tg). The incidence of autoimmune diabetes induced by multiple low dose of streptozotocin (STZ) was reduced in PD-1 Tg mice. Although the expression of CTLA-4, PD-1 and FoxP3, which are inhibitory molecules of activated T cells, is reduced only on STZ injected wild type (WT) mice, CD4, CD8 and regulatory T cell populations were not changed in all experimental groups. When splenocytes were re-stimulated in ex vivo, the production of IL-2 and IFN-γ and the T cell proliferation were increased in all STZ injected mice, but the increment rate was less in PD-1 Tg groups. Interestingly, macrophages were observed in splenocytes of STZ injected PD-1 Tg at somewhat lower level than macrophage in diabetic wild type mice. In this research, we found out that total numbers of T cell in the experimental groups are not changed, but T cell function is changed, and FoxP3 expression is decreased in pancreas and spleen of diabetes-induced groups. Macrophage frequency might also affects on type 1 diabetes. Although more experimental evidence needs to be provided, these results suggest that ligation of PD-1 and PD-L1/2 may have an effect on macrophages as well as does T cells.     

CP-25 alleviates rat collagen induced arthritis by regulating T cell subsets

Rheumatoid arthritis(RA) is a chronic,systemic and autoimmune disease. Abnormally activated T cells play a key role in the pathological mechanism of RA, and the differentiated T cell subsets also have imbalance in RA. Paeoniflorin-6-oxygen-benzenesulfonate(code CP-25) is a new active monomer derived from paeoniflorin. OBJECTIVE To investigate the therapeutic effect of CP-25 on collagen induced-arthritis(CIA) rats and regulation on T cell subsets. METHODS Rats with CIA were establisghed and administered with CP-25(50 mg·kg~(-1)) in tragastric administration, and Iguratimod(25 mg·kg~(-1)) was given as positive control drug. The therapeutic effects of CP-25 on the CIA of rats and its regulatory effects on the T cell subsets were evaluated by measuring the foot paw volume, the number of swollen joints,the histopathology of the joints and spleen and the T cell subgroup. The proliferation of T and B cells, cytokines and T lymphocyte subsets were analyzed by CCK-8, ELISA and flow cytometry. RESULTS CP-25 has therapeutic effect on CIA in rats. CP-25(50 mg·kg~(-1)) can significantly reduce the paw volume and joint swelling of CIA rats,reduce the white myelosis of spleen and synovial tissue inflammatory cell infiltration, inhibit the proliferation of T cells. The ratio of CD3~+T cells, CD3~+CD4~+T cells, CD3~+CD4~+CD25~+T cell subsets, Th1, Th2 and Th17 cells increased significantly in CIA rats. CP-25 reduce CD3~+T cels, CD3~+CD4~+T cels, CD3~+CD4~+CD25~+T cel subgroups,downregulate the ratio of Th1, Th2 and Th17 cells, and the above indicators were not statistically significant with the positive control group. CONCLUSION CP-25 has antiinflammatory effects on rats with CIA, which may be related to regulating activated T cell subsets and the differentiation of T cell subsets.     

靶向CD19嵌合抗原受体T细胞体外构建、扩增及抗肿瘤活性研究

目的  探索特异性靶向CD19的嵌合抗原受体（chimeric antigen receptor, CAR）T细胞的构建与制备方法,并研究其体外杀伤靶细胞的效果。方法  通过基因合成和分子克隆手段构建anti-CD19-CAR片段并将其插入plenti6.3慢病毒载体,利用293FT悬浮细胞系包装慢病毒并转染人外周血单个核细胞来源的CD3⁺ T细胞,通过流式细胞术鉴定转染效率,并通过实时无标记细胞分析法和细胞计数试剂盒8检测anti-CD19-CAR-T细胞体外杀伤效果。结果  获得了表达抗CD19的单链抗体基因的慢病毒,病毒滴度可达3.2×10⁸ 噬斑形成单位/ml。经慢病毒转染的CD3⁺ T细胞在体外培养14 d后,细胞扩增效率达(60.2±11.5)倍,anti-CD19-CAR-T细胞阳性率达90.57%。经检测,anti-CD19-CAR-T细胞均可有效杀伤CD19⁺靶细胞。结论  建立了基于无血清悬浮细胞系的anti-CD19-CAR慢病毒包装系统,成功构建靶向CD19抗原的CAR-T细胞,能特异性杀伤CD19+肿瘤细胞。     

Effect of coronary percutaneous revascularization on interferon-gamma and interleukin-10 producing CD4+ T cells during acute myocardial infarction

T lymphocytes play an important role in the induction and progression of acute coronary syndromes (ACS). To gain insight into how different T cell subsets can influence ACS, we analyzed the frequencies of circulating CD4+ T cells producing either pro-inflammatory interferon(IFN)-gamma or anti-inflammatory interleukin (IL)-10 in subjects presenting with ST-elevation myocardial infarction (STEMI). The effect of coronary bare metal (BS) and paclitaxel-eluting stent (PES) on the balance between CD4+IFN-gamma+ and CD4+IL-10+ lymphocytes was also investigated. Peripheral blood mononuclear cells (PBMC) were isolated from 38 consecutive patients with STEMI before and 48 hrs or 6 days after implantation of either BS or PES. Twenty patients with no history of coronary artery disease were included as basal controls. PBMC were stimulated in vitro with anti-CD3/anti-CD28 monoclonal antibodies, and CD4+IFN- gamma+ or CD4+IL-10+ T cells were detected by flow cytometry intracellular staining. The frequency of peripheral CD4+IL-10+ T cells was significantly higher in STEMI patients as compared with controls. Conversely, the frequency of CD4+IFN-gamma+ T lymphocytes did not differ between STEMI and subjects without history of coronary artery disease. Six days after the revascularization procedure, the percentage of CD4+IL-10+ T cells was significantly decreased in BS but not in the PES group, whereas the relative percentage of CD4+IFN-gamma+ T lymphocytes were diminished in both groups as compared with baseline levels. Our data indicate that STEMI is associated with a peripheral expansion of CD4+IL-10+ T lymphocytes, and that primary coronary revascularization with implantation of either BS or PES is followed by a reduction in circulating CD4+IFN-gamma+ T lymphocytes. PES implantation, however, appears to inhibit the relative decrease of the IL-10 producing lymphocyte as observed in BS implanted patients, shifting the balance between pro-inflammatory and anti-inflammatory T cell populations in favor of the latter.     

清络通痹颗粒对胶原性关节炎大鼠T细胞亚群及滑膜细胞的影响

目的：观察清络通痹颗粒(QLT)对胶原性关节炎大鼠T细胞亚群及滑膜细胞TNF-α,IL-1的影响。方法：建立胶原性关节炎(CIA)大鼠模型,同时设正常对照组．给药自d 7始,持续21 d。采用流式细胞术、酶联免疫吸附(EuSA)法、RT-PCR技术,观察QLT对CIA大鼠外周血T细胞亚群及滑膜细胞分泌TNF-α,IL-1和TNF-αmRNA,IL-1 mRNA表达的影响。结果：QLT 14．4 g·kg~(-1)可明显升高CIA大鼠外周血CD4~+T细胞,降低CD8~+T细胞,升高CD4~+/CD8~+比值(P＜0．05,P＜0．01)；QLT 7．2和14．4 g·kg~(-1)降低滑膜细胞分泌TNF-α,IL-1(P＜0．05,P＜0．01)及TNF-αmRNA,IL-1 mRNA的表达。结论：QLT可调节CIA大鼠T细胞亚群的比例,减少炎性细胞因子的产生,提示这可能是其治疗类风湿关节炎主要作用机制的一个重要方面。     

慢性乙型肝炎患者不同免疫状态下外周血T细胞表面程序性死亡受体1的表达

目的 检测慢性乙型肝炎(CHB)患者不同免疫状态下外周血CD4+、CD8+T细胞表面程序性死亡受体1(PD-1)表达,探讨其与血清HBV DNA载量、ALT之间的关系.方法 收集CHB患者免疫耐受期(A组,24例)、免疫活化期(B组,64例)、HBeAg阴性(C组,23例)和11例健康对照者(D组)外周血,采用流式细胞术检测CD4+、CD8+T细胞表面PD-1表达,以实时荧光定量PCR检测血清HBV DNA,同时检测ALT.结果 B、C组CD4+、CD8+细胞表面PD-1表达显著高于A、D组(P＜0.05).C组CD8+T细胞表面PD-1表达显著高于B组(P＜0.05).相关性分析发现,不同免疫状态下CHB患者CD4+、CD8+T细胞表面PD-1表达水平与HBV DNA载量及ALT均无相关性.结论 CHB患者外周血T细胞表面PD-1表达与机体免疫状态有关.肝脏炎症损伤是影响PD-1表达的重要因素,HBV DNA载量不是主要影响原因.     

WT1特异性 CD8+ T 细胞治疗乳腺癌的实验研究

摘要： 目的 通过检测 Wilms’肿瘤基因 1 （WT1） 特异性 CD8+ T 细胞对乳腺癌细胞的杀伤活性, 探讨正常外周血中提取的 WT1 特异性 CD8+ T 细胞用于治疗乳腺癌的可行性。方法 运用流式细胞仪检测 20 例人白细胞抗原（HLA） -A2 血清阳性健康志愿者外周血中 WT1 特异性 CD8+ T 细胞的浓度, 通过 WT1/主要组织相容性复合体（MHC） 连锁状多聚体磁珠分选 WT1 特异性 CD8+ T 细胞并进行培养, 细胞毒性实验检测 WT1 特异性 CD8+ T 细胞的功能。结果 20 例 HLA-A2 血清阳性健康志愿者外周血中均可检测到 WT1 特异性 CD8+ T 细胞, 12 例健康外周血中 WT1 特异性 CD8+ T 细胞的浓度＞0.5%, 其中 4 例＞1%。WT1/MHC 连锁状多聚体磁珠分选后, 其浓度明显增加至 80%左右。WT1 特异性 CD8+ T 细胞可特异性杀伤 WT1 多肽负载的乳腺癌细胞株。结论 健康志愿者外周血中可检测到 WT1 特异性 CD8+ T 细胞, 该细胞可能对乳腺癌细胞具有特异性杀伤作用, 提示 WT1 特异性 CD8+ T 细胞用于乳腺癌过继性免疫治疗的可能性。