Revolutionizing tumor immunotherapy:unleashing the power of progenitor exhausted T cells

In exploring persistent infections and malignancies, a distinctive subgroup of CD8^(+) T cells, progenitor exhausted CD8^(+) T(Tpex) cells, has been identified. These Tpex cells are notable for their remarkable self-renewal and rapid proliferation abilities. Recent strides in immunotherapy have demonstrated that Tpex cells expand and differentiate into responsive exhausted CD8^(+) T cells, thus underscoring their critical role in the immunotherapeutic retort. Clinical examinations have further clarified a robust positive correlation between the proportional abundance of Tpex cells and enhanced clinical prognosis. Tpex cells have found noteworthy applications in the formulation of inventive immunotherapeutic approaches against tumors. This review describes the functions of Tpex cells in the tumor milieu, particularly their potential utility in tumor immunotherapy. Precisely directing Tpex cells may be essential to achieving successful outcomes in immunotherapy against tumors.

外周血CD4^(+)PD-1^(+)Tcells及CD4^(+)T淋巴细胞ATP含量与复发性卵巢癌疗效的相关性分析

目的 探讨外周血CD4^(+)程序性细胞死亡受体-1(PD-1)^(+)T cells及CD4^(+)T淋巴细胞三磷酸腺苷(ATP)含量与复发性卵巢癌疗效的相关性。方法 选取30例复发性卵巢癌患者为复发组,30例未复发卵巢癌患者为非复发组;另选取30名同期体检健康者作为对照组。评估外周血CD4^(+)PD-1^(+)T cells及CD4^(+)T淋巴细胞ATP含量与复发性卵巢癌疗效的相关性。结果 复发组和非复发组的CD4^(+)PD-1^(+)T cells较对照组明显升高(P<0.05)。复发组和非复发组的CD4^(+)T淋巴细胞ATP含量较对照组明显降低(P<0.05)。复发组治疗后CD4^(+)PD-1^(+)Tcells显著低于治疗前(P<0.05),治疗后CD4^(+)T淋巴细胞ATP含量显著高于治疗前(P<0.05)。CD4^(+)PD-1^(+)T cells与复发性卵巢癌疗效成负相关(r=-0.393,P=0.039),CD4^(+)T淋巴细胞ATP含量与复发性卵巢癌疗效成正相关(r=0.449,P=0.031)。结论 复发性卵巢癌患者外周血CD4^(+)PD-1^(+)T cells及CD4^(+)T淋巴细胞ATP含量与疗效密切相关。

Analysis of CD4^+CD25^+ Regulatory T Cells and Foxp3 mRNA in the Peripheral Blood of Patients with Asthma

The changes of CD4^＋CD25^＋ regulatory T cells （CD4^＋CD25^＋ Treg） and Foxp3 mRNA in peripheral blood mononuclear cells （PBMCs） from patients with asthma were investigated in order to elucidate the possible roles of CD4^＋CD25^＋ Treg in the development of asthma. The peripheral blood samples were collected from 29 healthy controls （normal control group） and 78 patients with asthma which included 30 patients in exacerbation group, 25 patients in persistent group, and 23 patients in remission group. By using flow cytometry and RT-PCR, the CD4^＋CD25^＋ Treg ratio and Foxp3 mRNA in PBMCs were detected. The CD4^＋CD25^＋ Treg ratio and Foxp3 mRNA in PBMCs of exacerbation and persistent groups were lower than that of remission and normal control groups （P〈0.05）. Although the CD4^＋CD25^＋ Treg ratio and Foxp3 mRNA of remission group were also lower than that of normal control group, there was no significant difference between them （P〉0.05）. As compared with persistent group, exacerbation group had lower CD4^＋CD25^＋ Treg ratio and Foxp3 mRNA （P〈0.05）. It was indicated that the decrease of CD4^＋CD25^＋ Treg ratio and its function in PBMCs may be responsible for pathogenesis of asthma.

An Association between Immunosenescence and CD4^+CD25^+ Regulatory T Cells: A Systematic Review

Objective Age-related increment of the prevalence of CD4^＋CD25^＋ regulatory T （Treg） cells were described controversially, and whether such changes explain immune dysfunction in the elderly is still unclear. The aim of this systematic review is to evaluate the role of the Tregs in immunosenescence. Methods Medline and manual searches were performed to identify all published epidemiological and animal studies investigating the efficacy of the association between immunosenescence and Treg cells. Results It was founded that the frequency, phenotypic characteristics, and number/function of Tregs were altered significantly with aging. Medical conditions in individuals with advanced ageas well as apoptosis intensity of Treg cells had an impact on the accumulation of Tregs which in turn could deteriorate cytotoxic activity of CD8＋ T and NK cells and production of IL-2. The range of immune cells that could be suppressed by Treg cells was quite wide and covered CD4^＋CD25^＋ T cells, NK cells, dendritic cells and even monocytes. These changes were observed both in humans and experimental animals. Besides, it was believed that frequency of Tregs increased with age and was accompanied by intensified suppressive activity for Tregs in patients, for example, with Alzheimer disease （AD） and Parkinson disease （PD）. The impaired condition of CD4＋ T cells, so-called immunosenescence, rendered transplant recipients less responsive to an allogeneic kidney graft, an effect that was limited to transplant recipients who were aged over 60 years. Conclusions Treg cells are associated with immunosenescence. All these changes contribute to the aging-related decline of immune responses and lead to the higher risk of immune-mediated diseases, cancer or infections in aged individuals.

Depletion of CD4^+CD25^+ regulatory T cells can promote local immunity to suppress tumor growth in benzo[a]pyrene-induced forestomach carcinoma

AIM： To elucidate the distribution of CD4^＋CD25^＋ regulatory T cells （Tregs） in different lymphoid tissues and its local enhancement on tumor growth before and after depletion of CD4^＋CD25^＋ Tregs. METHODS： Female ICR mice were garaged with benzo[a]pyrene （BaP） to induce forestomach carcinoma. CD4^＋CD25^＋ Tregs were intraperitoneally depleted with monoclonal antibody PC61. These mice were divided into BaP-only, BaP ＋ IgG, BaP ＋ PC61, and control groups. The forestomach of mice was dissected for histological analysis, and tunnel test was performed for apoptosis of tumor cells. CD4^＋CD25^＋ Tregs were sorted from different lymphoid tissues and expression of Foxp3, IL-10, and chemokine receptors was analyzed by flow cytometry, semi-quantitative and veal-time polymerase chain reaction. RESULTS： The mice gavaged with only BaP showed increased forestomach papilloma and carcinoma at wk 16 and 32. The proportion of CD4^＋CD25^＋ Tregs was significantly higher in peri-stomach regional lymph nodes than in other lymphoid tissues. These CD4^＋CD25^＋ Tregs in regional lymph nodes expressed higher levels of Foxp3 and IL-10, enriched in the CD62L-subset, and CCR1 and CCR5 chemokine receptors. In mice gavaged with BaP ＋ PC61, the number of tumor nodules and tumor volume decreased significantly with massive infiltrating cells and apoptosis of tumor cells. In the draining regional lymph nodes, the number of CD4^＋CD25^＋ Tregs also decreased significantly. CONCLUSION： Inducible and activated CD4^＋CD25^＋ Tregs in the draining regional lymph nodes suppress host local immunity during tumor growth. Depletion of CD4^＋CD25^＋ Tregs can promote host local immunity to suppress tumor growth.

Increase of CD4^+CD25^+ T cells in Smad3^(-/-) mice

AIM： To investigate the changes of lymphocyte subpopulations, especially CD4^＋CD25^ T regulatory cells in Smad3^-/- mice. METHODS： Hematological changes and changes of lymphocyte subpopulations were detected in Smad3＂/- mice using cell counter and flow cytometry, respectively, and compared to their littermate controls. RESULTS： The numbers of neutrophils and lymphocytes in peripheral blood were significantly increased in Smad3^-/- mice compared to littermate controls. CD19^＋ expressing cells in blood and spleen, and CD8^＋ T cells in thymus were all markedly decreased in Smad3^-/- mice. More important, Smad3^-/- mice had an increased population of CD4^＋CD25^＋ T cells in peripheral lymphoid tissues, including thymus, spleen, and lymph nodes. CONCLUSION： These observations suggest that the changes of lymphocyte subpopulations might play a role in susceptibility to inflammation of Smad3^-/- mice.

Influence of Danshen Injection on airway inflammation and CD4^+ CD25^+ regulatory T cells of asthmatic rats

Objective： To investigate the influence of Danshen Injection on airway inflammation and CD4^＋CD25^＋ regulatory T cells（CD4^＋CD25^＋ Tr） of asthmatic rats, and elucidate the possible mechanism of Danshen Injection in treatment of asthma. Methods： 30 Wister rats were randomly divided into control group, asthma group and Danshen Injection treated group. Bronchoalveolar lavage fluids （BALF） were collected, and cytology studies were conducted. Lung tissues were obtained and pathologic analyses were done with hematoxylin and eosin stain （HE）. Flow cytometry was used to detect the CD4^＋CD25^＋ Tr ratio in peripheral blood mononuclear cells （PBMCs）. Results： Total cell, the percentage of lymphocytes, neutrophils and eosinophils （Eos） in BALF of Danshen Injection-treated group were lower than that in asthma group （P〈0.05, P〈0.01）. Compared with asthma group, less infiltration of inflammatory cells in lung tissues was observed in Danshen Injection-treated group. CD4^＋CD25^＋ Tr of asthma group was lower than that of control and Danshen Injection treated group （P〈0.05）. Conclusion： Danshen Injection can suppress airway inflammation of asthmatic rats, probably by increasing the number of CD4^＋CD25^＋ Tr.

Glutamine deprivation impairs function of infiltrating CD8^(+)T cells in hepatocellular carcinoma by inducing mitochondrial damage and apoptosis

BACKGROUND The functions of infiltrating CD8^(+)T cells are often impaired due to tumor cells causing nutrient deprivation in the tumor microenvironment.Thus,the mechanisms of CD8^(+)T cell dysfunction have become a hot research topic,and there is increased interest on how changes in metabolomics correlate with CD8^(+)T cell dysfunction.AIM To investigate whether and how glutamine metabolism affects the function of infiltrating CD8^(+)T cells in hepatocellular carcinoma.METHODS Immunohistochemical staining and immunofluorescence were performed on surgically resected liver tissues from patients.Differentially expressed genes in infiltrating CD8^(+)T cells in hepatocellular carcinoma were detected using RNA sequencing.Activated CD8^(+)T cells were co-cultured with Huh-7 cells for 3 d.The function and mitochondrial status of CD8^(+)T cells were analyzed by flow cytometry,quantitative real-time polymerase chain reaction,and transmission electron microscopy.Next,CD8^(+)T cells were treated with the mitochondrial protective and damaging agents.Functional alterations in CD8^(+)T cells were detected by flow cytometry.Then,complete medium without glutamine was used to culture cells and their functional changes and mitochondrial status were detected.RESULTS There were a large number of infiltrating PD-1+CD8^(+)T cells in liver cancer tissues.Next,we cocultured CD8^(+)T cells and Huh-7 cells to explore the regulatory effect of hepatoma cells on CD8^(+)T cells.Flow cytometry results revealed increased PD-1 expression and decreased secretion of perforin(PRF1)and granzyme B(GZMB)by CD8^(+)T cells in the co-culture group.Meanwhile,JC-1 staining was decreased and the levels of reactive oxygen species and apoptosis were increased in CD8^(+)T cells of the co-culture group;additionally,the mitochondria of these cells were swollen.When CD8^(+)T cells were treated with the mitochondrial protective and damaging agents,their function was restored and inhibited,respectively,through the mitochondrial damage and apoptotic pathways.Subsequently,complete medium without glutamine was used to culture cells.As expected,CD8^(+)T cells showed functional downregulation,mitochondrial damage,and apoptosis.CONCLUSION Glutamine deprivation impairs the function of infiltrating CD8^(+)T cells in hepatocellular carcinoma through the mitochondrial damage and apoptotic pathways.

Role of LAP^+CD4^+ T cells in the tumor microenvironment of colorectal cancer

AIM To investigate the abundance and potential functions of LAP^+CD4^+ T cells in colorectal cancer(CRC). METHODS Proportions of LAP^+CD4^+ T cells were examined in peripheral blood and tumor/paratumor tissues of CRC patients and healthy controls using flow cytometry. Expression of phenotypic markers such as forkhead box(Fox)p3, cytotoxic T-lymphocyte-associated protein(CTLA)-4, chemokine CC receptor (CCR)4 and CCR5 was measured using flow cytometry. LAP^-CD4^+ and LAP^+CD4^+ T cells were isolated using a magnetic cellsorting system and cell purity was analyzed by flow cytometry. Real-time quantitative polymerase chain reaction was used to measure expression of cytokines interleukin (IL)-10 and transforming growth factor(TGF)-β.RESULTS The proportion of LAP^+CD4^+ T cells was significantly higher in peripheral blood from patients (9.44% ± 3.18%) than healthy controls (1.49% ± 1.00%, P < 0.001). Among patients, the proportion of LAP^+CD4^+ T cells was significantly higher in tumor tissues(11.76% ± 3.74%) compared with paratumor tissues (3.87% ± 1.64%, P < 0.001). We also observed positive correlations between the proportion of LAP^+CD4^+ T cells and TNM stage(P < 0.001), distant metastasis(P < 0.001) and serum level of carcinoembryonic antigen(P < 0.05). Magnetic-activated cell sorting gave an overall enrichment of LAP^+CD4^+ T cells (95.02% ± 2.87%), which was similar for LAP^-CD4^+ T cells(94.75% ± 2.76%). In contrast to LAP^-CD4^+ T cells, LAP^+CD4^+ T cells showed lower Foxp3 expression but significantly higher levels of CTLA-4, CCR4 and CCR5(P < 0.01). LAP^+CD4^+ T cells expressed significantly larger amounts of IL-10 and TGF-β but lower levels of IL-2, IL-4, IL-17 and interferon-γ, compared with LAPCD4+ T cells.CONCLUSION LAP^+CD4^+ T cells accumulated in the tumor microenvironment of CRC patients and were involved in immune evasion mediated by IL-10 and TGF-β.

Isolation and identification of CD4^+CD25^+ regulatory T cells in rat

Objective： To establish a stable and high efficient method for collection of CD4^＋CD25^＋ regulatory T cells from rats in vitro. Methods： CD4^＋CD25^＋ regulatory T cells were isolated from the rat splenic cells through two steps by magic cell sorting （MACS） system. The first step was negative selection of CD4^＋T cells by cocktail antibodies and anti-IgG magic microbeads, and the second step was positive selection of CD25^＋T cells by anti-CD25 PE and anti-PE magic microbeads. The purity and viability of separated cells were measured by flow cytometry （FACS） and Trypan blue staining. The suppressive ability of seperated cells on the proliferation of CD4^＋CD25^- T cells was assessed by cell proliferation assay. Results： The purity of negatively enriched CD4^＋ T cells was 79%-87% （83.6%±2.5% ） , and the purity of positively enriched CD4^＋CD25^＋ T cells was 86%- 93% （ 90.2±1.8% ） with the viability of 92%～95% （92.8% ± 3.4% ）. The enriched cells significantly suppressed the proliferation of CD4^＋CD25^- T cells in mixed lymphocyte culture （P 〈 0.05）. Conclusion： An effective method can be established for enrichment of CD4^＋CD25^＋ regulatory T cells in two steps by MACS, with satisfied cell purity, viability and function.

CD4^+CD25^(high) Regulatory Cells in Peripheral Blood of NSCLC Patients

The proportion and changes of CD4^＋CD25^high regulatory T cells （Trs） in peripheral blood of non-small cell lung cancer （NSCLC） patients were analyzed and their clinical significance explored. The peripheral blood was collected from 61 patients with NSCLC and 15 healthy controls. By using monoclonal antibodies, the blood samples were evaluated with the flow cytometry for lymphocyte subsets （CD3^＋, CD4^＋ and CD8^＋） and CD4^＋CD25^high Tr cells. The results showed that the proportion of CD4^＋CD25^high Tr cells in NSCLC group was significantly higher than in control group [（4.36 ±2.07） % vs （2.04±1.03） %, P〈0.01]. The proportion of CD4^＋CD25^ high Tr cells in late stage was higher than that in early stage [stages Ⅰ ＋Ⅱ （2.264±0.6） %; stage Ⅲ（3.284± 1.38） %; stage IV （6.06 4±4.08） %] （P〈0.05）. Kaplan-Meier survival analysis revealed that the prognosis of the patients who had higher proportion of CD4^＋CD25^high Tr cells in peripheral blood was worse （P=0.0026）. In conclusion, the relative increase in CD4^＋CD25^high Tr cells in peripheral blood may be related to im- munosuppression and tumor progression in patients with NSCLC. This finding suggests that CD4^＋CD25^high Tr cells in peripheral blood of NSCLC may be positive for prognosis analysis. The use of depletion of the CD4^＋CD25^high Tr cell therapy to treat NSCLC patients may be an effective strategy.

Peripheral CD4^(+)CD8^(+) double positive T cells:A potential marker to evaluate renal impairment susceptibility during systemic lupus erythematosus

Lupus nephritis(LN) has a high incidence in systemic lupus erythematosus(SLE) patients, but there is a lack of sensitive predictive markers. The purpose of the study was to investigate the association between the CD4^(+)CD8^(+)double positive T(DPT) lymphocytes and LN. The study included patients with SLE without renal impairment(SLE-NRI), LN, nephritic syndrome(NS), or nephritis. Peripheral blood lymphocyte subsets were analyzed by flow cytometry. Biochemical measurements were performed with peripheral blood in accordance with the recommendations proposed by the National Center for Clinical Laboratories. The proportions of DPT cells in the LN group were significantly higher than that in the SLE-NRI group(t=4.012, P<0.001), NS group(t=3.240,P=0.001), and nephritis group(t=2.57, P=0.011). In the LN group, the risk of renal impairment increased significantly in a DPT cells proportion-dependent manner. The risk of LN was 5.136 times(95% confidence interval, 2.115–12.473) higher in cases with a high proportion of DPT cells than those whose proportion of DPT cells within the normal range. These findings indicated that the proportion of DPT cells could be a potential marker to evaluate LN susceptibility, and the interference of NS and nephritis could be effectively excluded when assessing the risk of renal impairment during SLE with DPT cell proportion.

Effect of Mg^(2+)level on the functions of CD8^(+)T lymphocytes and NK cells in patients with COVID-19

Objective:To investigate the changes of Mg^(2+) levels in serum and peripheral blood mononuclear cells(PBMCs)of patients with COVID-19 and its effects on the functions of CD8^(+)T lymphocytes and NK cells.Methods:A total of 165 COVID-19 patients hospitalized in Ezhou Central Hospital from January 20 to February 20,2020 were divided into mild/common group(98 cases)and severe/critical group(67 cases).At the same time,34 healthy persons were selected as the control group.Peripheral blood was collected and PBMCs were isolated,the level of Mg^(2+) in serum and PBMCs was detected.The subsets of CD8^(+)T lymphocytes and NK cell and the expression levels of their surface inhibitory molecular PD-1 and activator molecular NKG2D were detected by flow cytometry.The correlation between Mg^(2+) concentration and the expression levels of PD-1 and NKG2D was also analyzed.Results:Compared with the control group,the concentration of Mg^(2+) in serum and PBMCs,the counts of CD8^(+)T lymphocytes and NK cell in patients with mild/common and severe/critical groups were significantly reduced(P<0.05),while the expression level of surface inhibitory molecular PD-1 were significantly increased(P<0.05),while the expression level of the activation molecule NKG2D were significantly decreased(P<0.05).However,the changes of the above indicators in patients with severe/critical group were greater than those in the mild/common group(P<0.05).In addition,the Mg^(2+) concentration in COVID-19 patients was negatively correlated with the expression level of PD-1 on CD8^(+)T lymphocytes and NK cells(P<0.05),and positively correlated with the expression levels of NKG2D(P<0.05).Conclusion:The concentration of Mg^(2+) in the serum and PBMCs of COVID-19 patients is significantly reduced,which may cause the function of CD8^(+)T lymphocytes and NK cells to be inhibited.

乙肝疫苗无应答者CD4^+T细胞TCR Vβ基因克隆化特征的研究

目的:研究乙肝疫苗接种者CD4+T细胞TCR Vβ基因克隆化特征,分析乙肝疫苗有应答者和无应答者TCR Vβ基因单克隆改变的差异性。方法:采用多引物PCR技术扩增80例乙肝疫苗接种者CD4+T细胞TCR Vβ基因22个家族的CDR3区基因片段,对PCR产物分别应用琼脂糖凝胶电泳和基因扫描两种方法检测。结果:80例乙肝疫苗接种者中有58例产生抗体,有22例未产生抗体。TCR Vβ基因单克隆改变主要集中在Vβ2、Vβ8、Vβ9、Vβ11和Vβ17五个家族上,乙肝疫苗无应答组这五个Vβ基因单克隆改变频率明显低于乙肝疫苗有应答组(P<0.01或P<0.05)。结论:CD4+T细胞TCR Vβ基因克隆化改变是影响机体对乙肝疫苗免疫应答效果的重要因素之一。

CML患者CD4^+和CD8^+ T细胞的TCR Vβ基因谱系和克隆性分析

目的:了解慢性粒细胞白血病慢性期(CML-CP)患者外周血CD4+和CD8+T细胞中TCRVβ亚家族T细胞的基因表达和克隆性。方法:利用RT-PCR分别扩增19例CML-CP患者外周血单个核细胞(PBMCs)、CD4+和CD8+细胞TCRVβ亚家族基因的CDR3,阳性产物进一步经基因扫描分析其克隆性。结果:CML患者外周血CD4+和CD8+细胞分别表达部分(1～21个)Vβ亚家族,均以Vβ13的表达率最高,其次为Vβ9,多数患者的一些Vβ亚家族T细胞呈克隆性增殖,主要出现于CD8+细胞中,分别以Vβ21(CD4+)和Vβ11(CD8+)亚家族的克隆性增殖多见。结论:CML患者外周血CD4+和CD8+细胞的TCRVβ谱系均存在限制性分布,患者存在的克隆性增殖CD4+和CD8+T细胞可能与宿主抗CML抗原反应有关,它们可能在抗CML效应中起主要作用。

妊娠期母鼠给予葡萄球菌肠毒素B对成年子代CD3^+TCR Vβ8^+T细胞的影响

目的观察妊娠期母鼠给予葡萄球菌肠毒B(SEB)对成年子代CD3+TCR Vβ8+T细胞的影响。方法在妊娠16 d时给予SD大鼠尾静脉注射15μg SEB,同时设立PBS对照组。孕鼠自然分娩后子代鼠生长至成年,流式细胞仪检测成年子代鼠胸腺及外周血中CD3+TCR Vβ8+T细胞;并观察成年子代鼠再次给予SEB时胸腺及外周血中CD3+TCR Vβ8+T细胞的应答变化。结果妊娠期母鼠给予SEB可导致雌、雄性成年子代鼠胸腺CD3+TCR Vβ8+T细胞的比例(雌性:1.760,雄性:1.098),较对照组的(雌性:2.714,雄性:2.088)明显减少(P<0.05),其外周血中CD3+TCR Vβ8+T细胞的变化与胸腺中类似。PBS组的雌雄性成年子代鼠给予SEB后其胸腺及外周血中CD3+TCR Vβ8+T细胞较同窝对照组明显减少(P<0.05);而SEB组的雌雄性成年子代鼠给予SEB后与其同窝PBS对照组比较,胸腺中CD3+TCR Vβ8+T细胞无变化(P>0.05),但外周血中CD3+TCR Vβ8+T细胞则明显增加(P<0.05)。结论妊娠期母鼠给予SEB改变其成年子代大鼠CD3+TCR Vβ8+T细胞对再次SEB刺激的应答方式。

EGTA对SLET细胞TCR/CD3复合物介导的[Ca^(2+)]i反应的影响

目的 :证实SLE患者T细胞功能异常是否与其生物化学信号传导异常有关以及EGTA对其影响 ,探讨SLE的发病机理。方法 :用CD3单抗与羊抗鼠二抗IgG相交联刺激T细胞并用EGTA干预后 ,分别粘附细胞仪连续观察 10minT细胞 [Ca2 + ]i的变化 ,并评价 [Ca2 + ]i反应与CD3分子的相关性。结果 :正常人和SLE患者T细胞 [Ca2 + ]i反应的基准值相似 (P =0 .10 5 ) ;SLE患者高峰值、平台值T细胞的 [Ca2 + ]i反应明显高于正常对照 (P <0 .0 0 1,P <0 .0 0 1) ;加入EGTA后二者 [Ca2 + ]i反应有显著差异 :二者的T细胞CD3阳性率无差异 (P =0 .6 6 5 )。结论 :SLE患者T细胞TCR/CD3介导的信号转导途径存在异常 ,且不受EGTA的影响 ,可能是贮存库 [Ca2 + ]i释放的增加所致。

CD8^+CIK细胞表面NKG2D及TCR抗原识别受体的功能分析

目的探讨CD8^+细胞因子诱导的杀伤细胞(CIK细胞)亚群表面T细胞受体(TCR)和NKG2D(即CD314)抗原识别受体在发挥抗肿瘤特别是卵巢癌免疫效应中的功能。方法 CIK细胞由卵巢癌志愿者外周血单个核细胞(PBMCs)制备而成,采用磁性细胞分选(MACS)技术从培养1周的CIK细胞中富集CD8^+亚群并继续培养扩增,获得高度均质性的CD8^+CIK细胞后进行表型检测。将CD8^+CIK细胞分别在CD3抗体和NKG2D抗体包被的培养板中进行培养,以CD137为CD8^+CIK细胞激活标志物,应用流式细胞术(FCM)检测CD137表达,评估CD8^+CIK细胞的激活状况。并检测经CD3抗体、NKG2D抗体刺激或与红白血病细胞株人类白细胞抗原(HLA)-主要组织相容性复合体-Ⅰ类相关分子(MIC)A/B+K562细胞共培养对CD8^+CIK细胞分泌γ-干扰素(IFN-γ)的影响。选用K562细胞作为CD8^+CIK细胞作用的靶细胞,应用NKG2D抗体阻断NKG2D与MICA/B相互作用,测试激活的CD8^+CIK细胞在NKG2D阻断状态下对靶细胞K562的杀伤能力。结果 TCR能够提供CD8^+CIK细胞的激活信号,而NKG2D不具备该功能。外周血单个核细胞(PBMC)中CD8^+细胞占24.2%,该细胞群中NKG2D阳性率为16.3%。CIK培养后CD8^+细胞比例增至88.1%,这些CD8^+CIK细胞均表达NKG2D,但其中仍含10%左右的CD8-细胞;CD3抗体组中CD137阳性率为88.8%;CD3抗体刺激后24h,约50%的CD8^+CIK细胞呈IFN-γ染色阳性,而NKG2D抗体刺激后,IFN-γ阳性率(4.8%和5.6%)高于对照组(2.9%),但明显低于CD3抗体;CD8^+CIK细胞经CD3抗体刺激24h后IFN-γ检测阳性率为41.9%,但与K562细胞按1:1比例共培养后阳性率仅为0.5%。CD8^+CIK细胞对K562细胞具有杀伤功能,即能够以HLA非限制的方式杀伤靶细胞。而在CD3-TCR复合物激活的CD8^+CIK细胞上阻断NKG2D与MICA/B相互作用后,CD8^+CIK细胞杀伤活性受到抑制。结论 CD8^+CIK细胞的激活主要通过CD3-TCR受体复合物,而非通过NKG2D受体。CD8^+CIK细胞的NKG2D受体与靶细胞表面相应配体结合后可以介导HLA非限制的抗癌功能,但NKG2D受体的此种作用比较有限。

孕鼠接触葡萄球菌肠毒素B对子代新生鼠TCR Vβ8^+T细胞的影响

目的:观察孕鼠接触葡萄球菌肠毒素B(SEB)对其子代新生鼠胸腺及外周血TCR Vβ8^+T细胞的影响。方法:在妊娠16 d时给予SD大鼠尾静脉注射15μg SEB(SEB组),同时设立注射磷酸盐缓冲溶液对照组。孕鼠自然分娩后获取出生后0~5 d的子代新生鼠胸腺及外周血,流式细胞仪检测其TCR Vβ8^+T细胞比例。结果:与对照组比较,SEB组出生后0~3 d的新生鼠胸腺中CD4^+Vβ8^+T细胞比例均减少(P<0.05~P<0.01),出生后4~5 d 2组差异均无统计学意义(P>0.05);出生后0~5 d,SEB组胸腺中CD8^+Vβ8^+T细胞比例均低于对照组(P<0.05~P<0.01);出生后0~5 d,SEB组CD4^+Vβ8^+及CD8^+Vβ8^+T细胞的绝对数均较对照组明显减少(P<0.01)。新生鼠出生后0~5 d,SEB组外周血CD4^+Vβ8^+T细胞比例均较对照组明显减少(P<0.01);CD8^+Vβ8^+T细胞比例亦均较对照组减少(P<0.05~P<0.01);出生后0~5 d,SEB组外周血CD4^+Vβ8^+及CD8^+Vβ8^+T细胞绝对数均较对照组明显减少(P<0.01)。结论:妊娠期大鼠接触SEB可减少子代新生鼠胸腺及外周血TCR Vβ8^+T细胞,并保留至成年期。

SLE患者T细胞TCR/CD3复合物介导的[Ca^(++)]i反应与患者临床特征相关性的研究

为研究SLE患者原发的T细胞功能异常与TCR/CD3复合物介导生物化学信号转导异常相关,及与其临床特征的相关性。用尼龙柱分离肝素化的静脉血得到T细胞悬液,用CD3单抗与羊抗鼠二抗IgG相交联刺激T细胞,粘附细胞仪连续观察10min,观察T细胞犤Ca++犦i的变化,并分析其与患者的临床特征相关性。发现正常人和SLET细胞的[Ca++]i反应的基准值相似(P=0.105),SLE患者T细胞高峰值和平台值均明显高于正常对照(P<0.001,P<0.001),且与患者临床特征无关。