非小细胞肺癌不同胸腔积液严重程度及预后患者lncRNA MEG3表达及其与Th17/CD4^(+)T细胞的关系

目的:研究非小细胞肺癌(NSCLC)不同胸腔积液严重程度及预后患者lncRNA MEG3表达及其与Th17/CD4^(+)T细胞的关系。方法:选取2020年1月至2022年12月福建医科大学附属泉州第一医院收治的104例NSCLC恶性胸腔积液患者作为研究对象,根据胸腔积液量分为3组:少量胸腔积液组(35例)、中量胸腔积液组(42例)、大量胸腔积液组(27例)。根据患者疾病实际发展转归分为预后良好组(29例未出现复发和转移)和预后不良组(75例出现复发和转移)。另选取同期于福建医科大学附属泉州第一医院治疗的60例肺炎良性胸腔积液患者作为对照组。实时荧光定量PCR检测两组胸腔积液中MEG3表达。收集受试者外周静脉血,流式细胞术检测外周血Th17细胞、CD4^(+)T细胞比例,并计算Th17/CD4^(+)T。对比各组患者lncRNA MEG3及外周血Th17、CD4^(+)T细胞水平。Logistic回归分析NSCLC胸腔积液及预后的影响因素。结果:NSCLC组胸腔积液lncRNA MEG3表达及CD4^(+)T细胞百分比低于对照组,Th17细胞百分比、Th17/CD4^(+)T高于对照组(P<0.05)。大量胸腔积液组lncRNA MEG3表达及CD4^(+)T细胞百分比低于少量胸腔积液组、中量胸腔积液组,中量胸腔积液组lncRNA MEG3表达及CD4^(+)T细胞百分比低于少量胸腔积液组,大量胸腔积液组Th17细胞百分比、Th17/CD4^(+)T高于少量胸腔积液组、中量胸腔积液组,中量胸腔积液组Th17细胞百分比、Th17/CD4^(+)T高于少量胸腔积液组(P<0.05)。预后不良组lncRNA MEG3表达及CD4^(+)T百分比低于预后良好组,而Th17细胞百分比、Th17/CD4^(+)T高于预后良好组(P<0.05)。Logistic回归分析结果显示,lncRNA MEG3为NSCLC胸腔积液的保护因素,Th17/CD4^(+)T为危险因素(P<0.05);lncRNA MEG3为NSCLC预后的保护因素,Th17/CD4^(+)T为危险因素(P<0.05)。结论:NSCLC不同胸腔积液严重程度及预后患者lncRNA MEG3表达及Th17/CD4^(+)T不同,且lncRNA MEG3为NSCLC胸腔积液及预后的保护因素,Th17/CD4^(+)T为危险因素,可作为胸腔积液严重程度及预后诊断的有效生物标志物。

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。

MBD2 promotes Th2 differentiation in ovalbumin-induced CD4+T cells

Introduction:Allergen-specific CD4+T cells play a central role in autoimmune disorders,allergies and asthma,with Th2-type immunity being the typical functional response of CD4+T cells.This study aimed to investigate the role of MBD2 in regulating Th2 cell differentiation.Methods:Splenic mononuclear cells were extracted from C57BL/6 mice,and CD4+T cells were isolated using magnetic beads and confirmed through flow cytometry.Lentivirus was employed to construct MBD2-silenced CD4+T cells.In vitro experiments were performed to treat splenogenic mononuclear cells and CD4+T cells with Ovalbumin(OVA),and Th2 cell ratios and IL-4 levels were assessed using flow cytometry and ELISA.Results:The purity of the isolated CD4+T cells was 95.73%,confirming successful isolation of primary CD4+T cells.Compared to the control group,the Th2 cell ratio exhibited an increase in the Th2-induced group.Treatment with 5-Aza(concentrations,1-100μM)promoted Th2 cell differentiation and increased IL-4 levels.Notably,when combined with Th2 induction and 10μM 5-Aza treatment,silencing MBD2 further amplified Th2 cell ratios and elevated IL-4 levels in cell supernatants.Furthermore,OVA(concentration,200μg/mL)induced the differentiation of CD4+T cells into Th2 cells and increased IL-4 secretion.Interestingly,silencing MBD2 significantly increased the Th2 cell ratio and IL-4 levels in OVA-treated CD4+T cells.Conclusion:In summary,OVA promoted CD4+T cell differentiation into Th2 cells and enhanced IL-4 levels.MBD2 was identified as a mediator of Th2 cell differentiation in splenic-derived CD4+T cells,influenced by OVA or 5-Aza treatment.

Combined TIM-3 and PD-1 blockade restrains hepatocellular carcinoma development by facilitating CD4+ and CD8+T cellmediated antitumor immune responses

BACKGROUND Immune checkpoint inhibitors(ICIs)targeting programmed cell death protein 1(PD-1)and T cell immunoglobulin and mucin domain-containing protein 3(TIM-3)are beneficial to the resumption of anti-tumor immunity response and hold extreme potential as efficient therapies for certain malignancies.However,ICIs with a single target exhibit poor overall response rate in hepatocellular carcinoma(HCC)patients due to the complex pathological mechanisms of HCC.AIM To investigate the effects of combined TIM-3 and PD-1 blockade on tumor development in an HCC mouse model,aiming to identify more effective immunotherapies and provide more treatment options for HCC patients.METHODS The levels of PD-1 and TIM-3 on CD4+and CD8+T cells from tumor tissues,ascites,and matched adjacent tissues from HCC patients were determined with flow cytometry.An HCC xenograft mouse model was established and treated with anti-TIM-3 monoclonal antibody(mAb)and/or anti-PD-1 mAb.Tumor growth in each group was measured.Hematoxylin and eosin staining and immunohistochemical staining were used to evaluate T cell infiltration in tumors.The percentage of CD4+and CD8+T cells in tissue samples from mice was tested with flow cytometry.The percentages of PD-1+CD8+,TIM-3+CD8+,and PD-1+TIM-3+CD8+T cells was accessed by flow cytometry.The levels of the cytokines including tumor necrosis factor alpha(TNF-α),interferon-γ(IFN-γ),interleukin(IL)-6,and IL-10 in tumor tissues were gauged with enzyme-linked immunosorbent assay kits.RESULTS We confirmed that PD-1 and TIM-3 expression was substantially upregulated in CD4+and CD8+T cells isolated from tumor tissues and ascites of HCC patients.TIM-3 mAb and PD-1 mAb treatment both reduced tumor volume and weight,while combined blockade had more substantial anti-tumor effects than individual treatment.Then we showed that combined therapy increased T cell infiltration into tumor tissues,and downregulated PD-1 and TIM-3 expression on CD8+T cells in tumor tissues.Moreover,combined treatment facilitated the production of T cell effector cytokines TNF-α and IFN-γ,and reduced the production of immunosuppressive cytokines IL-10 and IL-6 in tumor tissues.Thus,we implicated that combined blockade could ameliorate T cell exhaustion in HCC mouse model.CONCLUSION Combined TIM-3 and PD-1 blockade restrains HCC development by facilitating CD4+ and CD8+T cell-mediated antitumor immune responses.

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.

中药调控CD^(+)_(4)T细胞干预湿疹炎症应答的研究进展

湿疹是一种变态反应性炎症性皮肤病,近期研究表明以CD^(+)_(4)T细胞亚群如Th1/Th2、Th17/调节性T细胞(regulatory T cells,Treg)细胞分化失衡导致湿疹发病中出现过度炎症应答,而中药以改善CD^(+)_(4)T细胞亚群分化平衡干预湿疹免疫微环境具有较好的调节作用。以湿疹炎症应答前沿研究动态,探讨中药在改善调控CD^(+)_(4)T细胞亚群分化干预湿疹致病的作用优势,为中药在改善湿疹临床症状及机制研究提供一定的理论指导。

Effects of estrogen on CD4^+ CD25^+ regulatory T cell in peripheral blood during pregnancy

Objective To investigate the effects of estrogen(E_2)level on regulatory T cells(Treg)in peripheral blood during pregnancy.Methods:A total of 30 healthy non-pregnant women were selected as control group,90 pregnant women of early,middle and late pregnancy and 30 postpartum women at 1 month after parturition were selected as experimental groups including early pregnancy group,middle pregnancy group and late pregnancy group;the proportions of CD4^+CD25^+Treg and CD4^+CD25^+CD127^-Treg among CD4 T cells were detected by flow cytometry;the serum estrogen content in peripheral blood was detected by electrochemical immune luminescence method.Results:E_2 level was coincident with the change of Tregs number during pregnancy.The estrogen content in peripheral blood increased gradually from early pregnancy to late pregnancy,then decreased significantly after parturition,and the level at 1 month after parturition down to the level in non-pregnancy group(P>0.05);the level of E_2 in pregnancy groups were significantly higher than those in non-pregnancy group(P<0.01);and there were significant differences among early pregnancy group,middle pregnancy group and late pregnancy group(P<0.05).The proportions of CD4^+CD25^+Treg and CD4^+CD25^+CD127^-Treg in pregnancy groups were significantly higher than those in non-pregnancy group(P<0.05),but decreased significantly after parturition,and there was no significant difference between non-pregnancy group and postpartum women group(P>0.05):the proportions in middle and late pregnancy groups were significantly higher than those in early pregnancy group(P<0.05).but decreased slightly in late pregnancy group,there was no significant difference between late pregnancy group and middle pregnancy group(P>0.05).There was correlation between Tregs number with estrogen level during pregnancy.The proportion of CD4^+CD25^+Treg and CD4^+CD25^+CD 127^-Treg were positively correlated with estrogen level.Conclusions:High proportion of CD4^+CD25^+Trcg and CD4^+CD25^+CD127^-Treg is closely related to the high level of E,during pregnancy.It suggested that high level of estrogen may induce an increase of CD4^+CD25^+Treg in peripheral blood.and then influence the immune function of pregnant women.The results of this experiment might play an important role of estrogen in immune-modulation during pregnancy.

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.

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.

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.

Changes of CD4^+CD25^+ Regulatory T Cells in Patients with Acute Coronary Syndrome and the Effects of Atorvastatin

The function of CD4＋CD25＋ regulatory T lymphocytes （Treg） in patients with acute coronary syndrome （ACS） and the effects of atorvastatin were investigated. Forty-eight patients with ACS were randomly divided into two groups： group C receiving conventional therapy （n=24）, and group C＋A receiving conventional therapy＋atorvastatin （10 mg/day, n=24）. T lymphocytes from ACS patients （before and 2 weeks after the treatment） or 18 healthy subjects were separated and the flow cytometry was used to measure the percentage of Treg. The inhibitory ability of Treg on effector T cells was determined by mixed lymphocyte reaction （MLR）. ELISA was used to measure the serum levels of cytokines （IL-10, TGF-β1 and IFN-γ） before and after treatment. The results showed that as compared with normal control group, Treg percentage was decreased significantly （P〈0.01）, the inhibitory ability of Treg on the T lymphocytes proliferation was reduced （P〈0.01）, IFN-γ levels were increased and IL-10 and TGF-β1 levels were lowered in ACS patients. After treatment with atorvastatin, Treg percentage and the inhibitory ability of Treg on T lymphocytes proliferation were significantly increased in ACS patients. Serum IFN-γ was decreased significantly, while IL-10 and TGF-β1 were elevated significantly as compared with the non-atorvastatin group. The number of Treg was positively correlated with serum TGF-β1, but negatively with serum IFN-γ and CRP. It was concluded that ACS was associated with decreased number and defected function of Treg, which may play an important role in initiating immune-inflammatory response in ACS. The inhibitory effects of atorvastatin on inflammation in ACS may be due to its beneficial effects on Treg and restoration of immune homeostasis.

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.

Downregulation of CD4+CD25+ regulatory T cells may underlie enhanced Th1 immunity caused by immunization with activated autologous T cells

Regulatory T cells （Treg） play important roles in immune system homeostasis, and may also be involved in tumor immunotolerance by suppressing Th1 immune response which is involved in anti-tumor immunity. We have previously reported that immunization with attenuated activated autologous T cells leads to enhanced anti-tumor immunity and upregulated Thl responses in vivo. However, the underlying molecular mechanisms are not well understood. Here we show that Treg function was significantly downregulated in mice that received immunization of attenuated activated autologous T cells. We found that Foxp3 expression decreased in CD4＋CD25＋ T cells from the immunized mice. Moreover, CD4＋CD25＋Foxp3＋ Treg obtained from immunized mice exhibited diminished immunosuppression ability compared to those from naive mice. Further analysis showed that the serum of immunized mice contains a high level ofanti-CD25 antibody （about 30 ng/ml, p〈0.01 vs controls）. Consistent with a role ofanti-CD25 response in the downregulation of Treg, adoptive transfer of serum from immunized mice to naive mice led to a significant decrease in Treg population and function in recipient mice. The triggering of anti-CD25 response in immunized mice can be explained by the fact that CD25 was induced to a high level in the ConA activated autologous T cells used for immunization. Our results demonstrate for the first time that immunization with attenuated activated autologous T cells evokes anti-CD25 antibody production, which leads to impeded CD4＋CD25＋Foxp3＋ Treg expansion and function in vivo. We suggest that dampened Treg function likely contributes to enhanced Thl response in immunized mice and is at least part of the mechanism underlying the boosted anti-tumor immunity.

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.

CD28/CTLA-4/B7 and CD40/CD40L costimulation and activation of regulatory T cells

Costimulatory signals are crucial for T cell activation. Attempts to block costimulatory pathways have been effective in preventing unwanted immune reactions. In particular, blocking the CD28/cytotoxic T lymphocyte antigen(CTLA)-4/B7 interaction(using CTLA-4Ig) and the CD40/CD40 L interaction(using anti-CD40 L antibodies) prevents T cell mediated autoimmune diseases, transplant rejection and graft vs host disease in experimental models. Moreover, CTLA-4Ig is in clinical use to treat rheumatoid arthritis(abatacept) and to prevent rejection of renal transplants(belatacept). Under certain experimental conditions, this treatment can even result in tolerance. Surprisingly, the underlying mechanisms of immune modulation are still not completely understood. We here discuss the evidence that costimulation blockade differentially affects effector T cells(Teff) and regulatory T cells(Treg). The latter are required to control inappropriate and unwanted immune responses, and their activity often contributes to tolerance induction and maintenance. Unfortunately, our knowledge on the costimulatory requirements of Treg cells is very limited. We therefore summarize the current understanding ofthe costimulatory requirements of Treg cells, and elaborate on the effect of anti-CD40 L antibody and CTLA-4Ig treatment on Treg cell activity. In this context, we point out that the outcome of a treatment aiming at blocking the CD28/CTLA-4/B7 costimulatory interaction can vary with dosing, timing and underlying immunopathology.

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.

LAP^+CD4^+T细胞在结直肠癌肿瘤微环境中的分布情况及其临床意义

目的:检测LAP+CD4+T细胞在结直肠癌患者肿瘤微环境中的分布情况,分析其与CD4+CD25+Treg及患者临床病理特征的相关性,初步探讨LAP+CD4+T细胞在结直肠癌发生、发展中的作用。方法:收集2014年1月至2014年3月期间广西医科大学第一附属医院结直肠肛门外科收治并行手术治疗的50例结直肠癌患者临床病理资料,采集所有患者术前外周血、术中肿瘤组织和距离癌组织边缘10 cm以上的相应癌旁组织标本,同时采集本科室25例健康志愿者外周血作为对照组;流式细胞术检测各标本中LAP+CD4+T细胞和CD4+CD25+Treg的分布比例,比较结直肠癌患者与健康志愿者外周血中LAP+CD4+T细胞分布比例差异、结直肠癌肿瘤组织与相应癌旁组织中LAP+CD4+T细胞分布比例差异;分析LAP+CD4+T细胞和CD4+CD25+Treg与临床病理特征的相关性。结果:50例结直肠癌患者和25位健康志愿者外周血及组织标本LAP+CD4+T细胞占CD4+T细胞比例,结直肠癌患者外周血[(9.44±3.18)%]高于正常人外周血[(1.49±1.00)%,P=0.000]、结直肠癌患者肿瘤组织[(11.76±3.74)%]高于相应癌旁组织[(3.87±1.64)%,P=0.000];其中肿瘤组织中LAP+CD4+T细胞的分布比例最高。肿瘤组织中LAP+CD4+T细胞与CD4+CD25+Treg细胞成正相关(r=0.327,P=0.02);LAP+CD4+T细胞占CD4+T细胞的比例与肿瘤的TNM分期、远处转移及CEA水平相关(P<0.05)。结论:LAP+CD4+T细胞易聚集于结直肠癌肿瘤组织中,可能参与了结直肠癌的发生、发展,起到促进肿瘤生长、转移的作用。

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.

Effect of CD4^+CD25^+ regulatory T cells in the development of anterior chamber-associated immune deviation

AIM: To investigate whether CD4(+)CD25(+) regulatory T (Treg) cells play a role in the development of anterior chamber-associated immune deviation (ACAID). METHODS: The dynamic changes in the frequency of CD4(+)D25(+) T cells, CD4(+)D25(+) FoxP3(+) T cells and CD4(+)CD25(+) PD-1(+) T cells from spleens of mice with ACAID were analyzed by flow cytometry. Foxp3 mRNA expression in purified CD4(+)CD25(+) T cells was analyzed using real-time PCR. The suppressive effect of purified CD4(+)CD25(+) T cells on the proliferation of CD4(+)CD25(-) T cells was evaluated by [H-3] thymidine incorporation. A blocking experiment was performed to further address the role of CD4(+)CD25(+) T cells in ACAID. The expression of IL-10 in purified CD4(+)CD25(+) T cells was evaluated by ELISA. RESULTS: Increased frequencies of CD4(+)CD25(+) T cells, CD4(+)CD25(+) Foxp3(+) T cells and CD4(+)CD25(+) PD-1(+) T cells were observed in ACAID. The CD4(+)CD25(+) T cells from mice with ACAID showed enhanced suppressive effect on the proliferation of CD4(+)CD25(-) T cells. Treatment of BALB/c mice with anti-CD25 antibody after injection of OVA into the anterior chamber significantly inhibited the induction of ACAID. Furthermore, purified CD4(+)CD25(+) T cells from ACAID mice secreted IL-10. CONCLUSION: Our results demonstrate that Treg cells are induced in the mice undergoing ACAID. These Treg cells may play a role in the development of ACAID.

Immunotherapy of rat glioma without accumulation of CD4^+CD25^+FOXP3^+ regulatory T cells

Immunotherapy may be used for the treatment of glioblastoma multiforme; however, the induced immune response is inadequate when either T cells or dendritic cells are used alone. In this study we established a novel vaccine procedure in rats, using dendritic cells pulsed with C6 tumor cell lysates in combination with adoptive transfer of T lymphocytes from syngenic donors. On day 21 after tumor inoculation, all the rats were sacrificed, the brains were harvested for calculation of glioma volume, cytolytic T lymphocyte responses were measured by cytotoxic assay, and the frequency of regulatory T lymphocytes （CD4＋CD25~FOXP3~） in the peripheral blood was investigated by flow cytometric analysis. The survival rate of rats bearing C6 glioma was observed. Results showed that the co-immunization strategy had significant anti-tumor potential against the pre-established C6 glioma, and induced a strong cytolytic T lymphocyte response in rats. The frequency of peripheral blood CD4＊CD25＊FOXP3＊ regulatory T lymphocytes was significantly decreased following the combination therapy, and the rats survived for a longer period. Experimental findings indicate that the combined immunotherapy of glioma cell lysate-pulsed dendritic cell vaccination following adoptive transfer of T cells can effectively inhibit the growth of gliomas in rats, boost anti-tumor immunity and produce a sustained immune response while avoiding the accumulation of CD4＋CD25＋FOXP3＋ regulatory r lymphocytes.