结直肠癌中LC3与CD4~+、CD8~+、CD68~+免疫细胞浸润的相关性及其临床意义

目的观察自噬标志物LC3与肿瘤表面抗原NY-ESO-2、MAGE-D4在结直肠癌及癌旁正常黏膜组织中的表达以及CD4~+、CD8~+、CD68~+免疫细胞的浸润情况,分析LC3与NY-ESO-2、MAGE-D4以及CD4~+、CD8~+、CD68~+免疫细胞浸润的相关性,探讨结直肠癌细胞自噬对免疫细胞的影响及其临床意义。方法应用免疫组化En Vision法、Western blot法检测128例结直肠癌及癌旁正常黏膜组织中LC3、NY-ESO-2、MAGE-D4的表达以及CD4~+、CD8~+、CD68~+免疫细胞浸润情况,探讨各因子之间的相关性并结合临床病理特征进行分析。结果 (1)LC3在结直肠癌组织中的表达高于癌旁正常黏膜组织,NY-ESO-2在结直肠癌中低表达,MAGE-D4在结直肠癌中高表达,两者在癌旁正常黏膜组织中几乎不表达(P0.05);CD4~+、CD8~+、CD68~+免疫细胞在结直肠癌中的浸润量均高于癌旁正常黏膜组(P0.05);(2)结直肠癌中LC3与肿瘤表面抗原NY-ESO-2、MAGED4的表达以及CD4~+、CD8~+、CD68~+细胞的浸润量均呈正相关(P0.05);(3)NY-ESO-2的表达与CD4~+、CD8~+、CD68~+细胞的浸润量均呈正相关,MAGE-D4的表达与CD8~+、CD68~+细胞的浸润量均呈正相关(P0.05);(4)LC3、NY-ESO-2、MAGE-D4的表达及CD4~+、CD8~+免疫细胞的浸润量均与淋巴结转移呈负相关(P0.05);LC3、NY-ESO-2、MAGE-D4的表达及CD4~+、CD8~+、CD68~+细胞的浸润量与TNM分期呈负相关(P0.05);LC3的表达与结直肠癌的分化程度呈正相关;CD8~+细胞的浸润量与组织学分级呈正相关(P0.05)。结论结直肠癌中自噬标志物LC3的表达与肿瘤表面抗原NY-ESO-2、MAGE-D4的表达、免疫细胞CD4~+、CD8~+、CD68~+的浸润量具有相关性,自噬活性改变可能影响结直肠癌的细胞免疫。     

Trop-2在结直肠癌组织中的表达及其意义

目的 探讨人滋养层细胞表面抗原-2(Trop-2)在结直肠癌中的表达及其意义.方法 用免疫组化法检测84例结直肠癌组织标本及其相对应的癌旁正常组织中的Trop-2表达情况,分析其表达与临床病理特点的关系,进一步采用Westem blot检测34例手术标本的Trop-2表达情况.结果 Trop-2在结直肠癌组织中的表达高于癌旁正常组织(P ＜0.05);Trop-2表达量:直肠癌(RC)组＞左半结肠癌(LSCC)组＞右半结肠癌(RSCC)组(P＜0.05),Dukes'C+D组＞A+B组(P＜0.05),淋巴结转移组＞无淋巴结转移组(P＜0.05),远处转移组＞无远处转移组(P＜0.05);与性别、年龄、分化程度无关.结论 Trop-2在结直肠癌组织中高表达,与Dukes分期、淋巴结转移、远处转移和肿瘤所在部位有关.     

FOXP3在乳腺癌中的表达及临床意义

目的 研究FOXP3+淋巴细胞和FOXP3蛋白在人乳腺癌细胞中的表达及其与患者临床病理特征的关系.方法 采用组织芯片平台,应用免疫组织化学方法 检测92例浸润性乳腺癌和26例癌旁乳腺组织中FOXP3+淋巴细胞和FOXP3蛋白的表达.结果 乳腺癌间质浸润的FOXP3+淋巴细胞阳性率高于癌旁组织(32.7% vs 7.7%,P<0.01);乳腺癌实质FOXP3蛋白阳性率也高于癌旁组织(52.2% vs 7.7%,P<0.01);乳腺癌间质浸润的FOXP3+淋巴细胞数和癌实质FOXP3蛋白表达无相关性(P>0.05);乳腺癌间质浸润的FOXP3+淋巴细胞与淋巴结转移、组织学分级、pTNM分期和c-erbB-2过表达呈正相关;乳腺癌实质FOXP3蛋白表达与淋巴结转移呈正相关(P<0.05);乳腺癌间质浸润的FOXP3+淋巴细胞和乳腺癌实质FOXP3蛋白表达与乳腺癌的预后无关(P>0.05).结论 FOXP3+淋巴细胞可能在乳腺癌的免疫逃逸中发挥一定作用;FOXP3表达异常可能与乳腺癌的转移关系密切;FOXP3可作为评估乳腺癌生物学行为的一种潜在标记物.     

CD4~+CD25~+FOXP3~+Treg细胞与急性移植物抗宿主病的关系

背景:CD4+CD25+FOXP3+Treg细胞具有免疫抑制作用,推测可能减少异基因造血干细胞后急性移植物抗宿主病的发生率。目的:观察粒细胞集落刺激因子动员前后,供者外周血CD4+CD25+FOXP3+Treg细胞比率变化,并探讨CD4+CD25+FOXP3+Treg细胞与异基因造血干细胞移植后发生急性移植物抗宿主病的关系。方法:以异基因造血干细胞移植受者90例及其供者为研究对象,供者皮下注射重组人粒细胞集落刺激因子(5μg/kg),1次/12 h,连续5 d后,采集干细胞;分别于动员前后采集供者外周血,流式细胞仪检测其中CD4+CD25+FOXP3+Treg细胞含量,及受者接受移植物中此类细胞含量;根据接受CD4+CD25+FOXP3+Treg细胞数分为高剂量组(细胞比例≥5%)及低剂量组(细胞比例5%),比较两组移植后急性移植物抗宿主病的发生率。结果与结论:供者在应用粒系集落刺激因子动员前后CD4+CD25+FOXP3+Treg细胞比例分别为11.3%和1.5%,差异有显著性意义(P0.05);急性移植物抗宿主病阳性组所接受移植物中该群细胞比例为3.4%,阴性组为15.7%,差异有显著性意义(P0.05);移植造血重建后高剂量组急性移植物抗宿主病的发生率为18.4%,低剂量组急性移植物抗宿主病的发生率为48.1%,二者差异有显著性意义(P0.05)。因而,粒系集落刺激因子应用能降低正常人外周血中CD4+CD25+FOXP3+Treg细胞比例;CD4+CD25+FOXP3+Treg细胞增加可以降低急性移植物抗宿主病的发生率。     

II型胶原蛋白对CIA大鼠外周血CD4~+CD25~+FOXP3~+调节性T细胞的影响

目的:观察Ⅱ型胶原蛋白对胶原诱导的关节炎(CIA)大鼠外周血CD4+CD25+FOXP3+调节性T细胞(Treg)的影响。方法:建立Ⅱ型胶原蛋白诱导的大鼠类风湿性关节炎(CIA)模型。采用流式细胞术分别检测正常对照组、模型组、Ⅱ型胶原蛋白治疗组和雷公藤多苷组大鼠外周血CD4+CD25+FOXP3+Treg的水平。结果:与正常对照组比较,模型组大鼠外周血CD4+T细胞亚群中CD25+FOXP3+Treg的水平明显下降(P<0.05或P<0.01);与模型组比较,Ⅱ型胶原蛋白治疗组大鼠外周血CD25+FOXP3+Treg显著升高(P<0.05或P<0.01);而CD25+FOXP3-T细胞和CD25-FOXP3+T细胞的水平则明显下降(P<0.05或P<0.01)。结论:II型胶原蛋白治疗CIA大鼠的可能途径是升高外周血CD4+CD25+FOXP3+Treg的水平。     

CD4~+CD25~+Foxp3~+调节性T细胞与LSCC发病的相关研究

为研究调节性T细胞在喉鳞状细胞癌(laryngeal squamous cell carcinoma,LSCC)、发展中的变化及其参与疾病进展的作用机制,收集2010～2011年上海市五官科医院收治的50例LSCC患者的肿瘤组织和外周血,应用流式细胞术检测CD4+CD25+Foxp3+Treg细胞及趋化因子受体CCR6的表达变化,Real-time PCR法检测转录因子Foxp3以及细胞因子mRNA的表达量。结果发现:LSCC患者外周血中CD4+CD25+Foxp3+Treg的百分比较正常人显著增加,并与临床分期相关;CD4+CD25+CCR6+Treg Foxp3的表达,以及肿瘤组织Foxp3mRNA的表达皆明显高于对照组,且与临床分期、淋巴结转移相关。同时发现,LSCC患者外周血中TGF-β和IL-10mRNA的检出水平分别高于对照组,但IFN-γ、IL-2、IL-12mRNA的水平低于对照组。提示此类Foxp3+Treg属于一类诱导性T抑制细胞(Foxp3+iTreg),可通过产生IL-10和TGF-β抑制LSCC患者的细胞免疫功能。Foxp3的检测可能对判断LSCC的预后有一定价值。     

胃癌及癌前病变组织中Nanog和Oct-4的表达及意义

目的 探讨肿瘤干细胞特异性标志物Nanog和Oct-4在各种胃组织中的表达及临床意义.方法 采用免疫组化SP法检测80例胃癌组织、80例癌旁组织及30例癌前病变组织中Nanog和Oct-4的表达及相关性.结果 胃癌组织中Oct-4的阳性率明显高于癌旁组织及癌前病变组织(P＜0.05),胃癌组织中Nanog的阳性率明显高于癌前病变组织(P＜0.05).Nanog 和Oct-4在低分化胃癌组中的表达明显高于高+中分化胃癌组(P＜0.05),浸润深度(T1 +T2)、淋巴结转移(N1)及无远处转移的胃癌组织中Nanog和Oct-4阳性率明显低于浸润深度(T3+T4)、淋巴结转移(N2+ N3)及伴远处转移的胃癌组织(P＜0.05或P＜0.01).胃癌组织中Nanog和Oct-4的表达呈正相关(rs=0.558 7,P＜0.01).结论 Nanog和Oct-4的表达与胃癌肿瘤干细胞的各种生物学行为相关,二者联合检测可作为胃癌敏感性、特异性的标志物和治疗靶点.     

可诱导 T 细胞共刺激分子在结肠癌组织中的表达情况及其与 远期生存的相关性研究

目的 探讨分析可诱导 T 细胞共刺激分子 (inducible T-cell co-stimulator, ICOS) 在结肠癌组织中 的表达情况及其与远期生存的相关性。 方法 选取 2018 年 5 月至 2019 年 5 月期间在大连大学附属新华医 院进行手术切除结肠组织的 206 例结肠癌患者作为研究对象, 取其结肠癌组织及癌旁组织标本, 使用荧光 定量 PCR 检测结肠癌组织及癌旁组织中可诱导 T 细胞共刺激分子表达水平, 分析其表达与临床病理特征的 关系及远期生存的相关性。 结果 结肠癌组织中 ICOS 表达水平 (8. 73 ± 2. 25) 显著低于癌旁组织的 (18. 54 ± 3. 26), 差异有统计学意义 (P< 0. 05); 结肠癌组织中 ICOS 表达水平与肿瘤直径、 远处转移、 临 床分期、 淋巴结转移有关 ( P < 0. 05), 与年龄、 性别、 肿瘤部位、 分化程度差异无统计学意义 ( P > 0. 05); ICOS 表达在 206 例结肠癌组织的中低表达 117 例, 高表达 89 例; ICOS 高表达和低表达与临床病理 因素进行 Logistic 二次回归分析结果显示, ICOS 表达与肿瘤大小、 临床分期、 淋巴结转移、 远处转移有关 (P< 0. 05), 与年龄、 性别、 肿瘤位置、 分化程度无关 (P > 0. 05); 随访截止至 2021 年 9 月 30 日, 206 例 结肠癌患者存活率 83. 01 % (171 / 206), 死亡率 16. 99 % (35 / 206), 中位生存时间为 (23. 5 ± 3. 3) 个 月。 ICOS 高表达的 89 例结肠癌患者的中位生存期为 (27. 4 ± 3. 2) 个月, 95 % CI 为 2. 234 ~ 6. 147; ICOS 低表达的 117 例结肠癌患者的中位生存期为 (16. 3 ± 4. 3) 个月, 95 % CI 为 1. 458 ~ 5. 237, ICOS 高表达结 肠癌患者中位生存期显著高于 ICOS 低表达者 (P< 0. 05)。 结论 ICOS 在结肠癌组织中呈较低表达, 其表 达水平与患者远期生存呈正相关关系, 其水平可作为结肠癌患者预后生存的重要评估指标。     

急性髓系白血病患者外周血CD4+CD25highFOXP3+调节性T细胞变化及临床意义

目的:观察急性髓系白血病(Acute myelogenous leukemia,AML)患者外周血中调节性T细胞(Regulatory T cells,Treg细胞)的变化,探讨其在AML发病中的作用及临床意义.方法:应用流式细胞术检测31例初诊AML患者(初诊组)、23例经化疗取得完全缓解患者(CR组)及20例健康人群(对照组)外周血CD4+CD25highFOXP3+ Treg细胞、CD4+FOXP3+ T细胞占CD4+细胞的比例,同时还分析了外周血CD4+/CD8+比值、NK细胞及血清乳酸脱清酶(LDH)水平.结果:与对照组相比较,AML患者初诊组及CR组外周血CD4+CD25highFOXP3+ Treg细胞和CD4+FOXP3+ T细胞均升高(P＜0.01).与初诊组相比较,CR组CD4+CD25highFOXP3+ Treg细胞无显著降低(P＞0.05),CD4+FOXP3+T细胞明显下降(P＜0.01).CD4+CD25highFOXP3+ Treg细胞的升降与CD4+FOXP3+T细胞呈正相关(r=0.86;P＜0.01).CD4+CD25highFOXP3+ Treg细胞及CD4+FOXP3+ T细胞比例与CD4+/CD8+比值呈负相关(r分别为-0.54、-0.52;P＜0.01)、与NK细胞比例呈负相关(r分别为-0.41、-0.43;P＜0.05),而与LDH水平呈正相关(r分别为0.51、0.57;P＜0.05).结论:CD4+CD25highFOXP3+ Treg细胞增多可能是AML患者免疫功能受抑的重要原因之一,其变化对于AML的预后判断有一定的意义.CD4+FOXP3+ T细胞的作用类似于CD4+CD25highFOXP3+ Treg细胞,其在AML疗效评价方面可能更有价值.     

抗ICOS抗体体外对哮喘大鼠血液和淋巴液CD4~+CD25~+Foxp3~+调节性T细胞数量及其功能影响

目的:研究抗ICOS抗体对哮喘大鼠外周血和淋巴液来源CD4+CD25+Foxp3+调节性T细胞(Treg)数量及其功能的影响。方法:抗ICOS抗体处理血液和淋巴液中单个核细胞(MNC),流式细胞仪检测MNC中CD4+CD25+Foxp3+T细胞百分率,酶联免疫吸附试验(ELISA)检测MNC培养液上清IL-10和TGF-β1含量。结果:末次激发后各个时间点收集MNC,体外培养96 h,各组淋巴液来源MNC体外培养体系中CD4+CD25+Foxp3+Treg细胞百分率均显著高于血液(P<0.05),哮喘组淋巴液和血液来源MNC体外培养体系中CD4+CD25+Foxp3+Treg细胞百分率均显著低于正常对照组(P<0.05),抗ICOS抗体组淋巴液和血液来源MNC体外培养体系中CD4+CD25+Foxp3+Treg细胞百分率显著低于哮喘组(P<0.05)。末次激发后0 h收集淋巴液来源和血液来源MNC培养上清中抗ICOS抗体组IL-10显著低于哮喘组和正常对照组(P<0.05);末次激发后不同时间点收集MNC培养上清中各组TGF-β1无明显差别。结论:用抗ICOS抗体阻断ICOS/ICOSL信号通路加重哮喘大鼠Treg细胞缺陷,并在哮喘激发早期0 h抑制血液和淋巴液来源MNC体外培养体系中CD4+CD25+Foxp3+Treg细胞分泌IL-10,但对于TGF-β1分泌无显著影响。     

VEGFR2 is selectively expressed by FOXP3high CD4+ Treg

CD25⁺ FOXP3⁺CD4⁺ T cells (Treg) have been considered to play an important role in immune tolerance against several tumor antigens. It has also been indicated that high‐level expression of FOXP3 (FOXP3^high) is sufficient to confer suppressive activity to normal non‐Treg. Here, we showed for the first time that vascular endothelial growth factor receptor 2 (VEGFR2) is selectively expressed by FOXP3^high but not FOXP3^low Treg. Such VEGFR2⁺ Treg exist in several tissues including PBMC and malignant effusion‐derived lymphocytes. In conclusion, VEGFR2 may be a novel target for controlling Treg with highly suppressive function.     

CD4~＋ CD25~＋ Foxp3~＋调节性T细胞与自身免疫性疾病

自身免疫性疾病系由于机体免疫系统失衡,产生针对自身组织的免疫应答并导致自身组织、器官损害的一类疾病。调节性T淋巴细胞（regulatory T cell,Treg）具有免疫应答低下和免疫抑制特性,在维持机体免疫耐受和免疫应答稳态方面具有非常重要的作用,Treg的异常与多种自身免疫性疾病有关[1]。Foxp3特异性表达于CD4＋CD25＋Treg细胞,与其发育、成熟以及抑制功能关系密切。但是目前关于该转录因子的表达调控机制却不清楚。本文拟就CD4＋CD25＋Foxp3 Treg细胞的研究进展及与多种自身免疫性疾病的关系作一综述。     

FOXP3的研究进展

在免疫调节机制中,CD4^＋CD25^＋Treg细胞和FOXP3转录因子成为近十年来的一个研究热点。CD4^＋CD25^＋Treg细胞在免疫调节中的重要作用已经得到了肯定,FOXP3被证实是CD4^＋CD25^＋Treg细胞的发育和功能起关键作用的调节子。目前认为,至少在鼠类,FOXP3是CD4^＋CD25^＋Treg细胞区别于CD4^＋CD25^-T细胞的一个较特异性的标志。影响FOXP3诱导表达的因素很多,FOXP3介导的免疫调节机制也涉及多种分子,对FOXP3的更深入研究有助于发挥其在免疫调节中的重要作用。     

CXCL4 promoted the production of CD4+CD25+FOXP3+treg cells in mouse sepsis model through regulating STAT5/FOXP3 pathway

Abstract

Background: CXCL4 plays an essential role in the regulation of multiple immune diseases. However, the underlying role of CXCL4 is still not clear in sepsis. Aim: In the present study, we aimed to investigate the function of CXCL4 in sepsis.

Methods: Sepsis model was constructed on mouse. Flow cytometry was used to determine the ratio of CD4⁺CD25⁺FOXP3⁺Treg cells. ELISA assays were used to determine the levels of CXCL4, IL-6, IL-10, and TNF-α respectively. Western blot was used to examine protein contents.

Results: Our results suggested that the serum level of CXCL4 was upregulated in patients with sepsis and positively associated with the ratio of human CD4⁺CD25⁺FOXP3⁺Treg cells. To further examine the role of CXCL4 in sepsis, we constructed the mouse sepsis model. Our results indicated that the mouse antibody of CXCL4 treatment reduced the expression of urine creatinine and urea nitrogen in sepsis model. Moreover, the frequency of CD25⁺FOXP3⁺ mouse regulatory T cells (Tregs) cells was decreased in mouse CD4⁺ T cells in the presence of mouse CXCL4 antibody. Further, the mouse recombinant protein CXCL4 was used to culture normal mouse CD4⁺ T cells in vitro. Our finding indicated that the recombinant protein CXCL4 promoted the percentage of mouse CD25⁺FOXP3⁺Treg cells and enhanced the phosphorylation of STAT5 in mouse CD4⁺ T cells in a dose-dependent manner. However, these effects were significantly reversed by the STAT5 inhibitor (p?<?.001). Conclusion: our findings not only indicated the function and signalling pathway of CXCL4 in CD4⁺ T cells but also provided novel insight and target in sepsis treatment.     

Combination of rapamycin and IL-2 increases de novo induction of human CD4(+)CD25(+)FOXP3(+) T cells

The immune system protects itself from autoreactivity by maintaining a balance between effector and Treg responses. Peripheral induction of Treg is one mechanism by which this balance may be maintained. Thus, it is important to understand factors that influence de novo generation of CD4⁺CD25⁺FOXP3⁺ Treg. Here, we focus on the effects of cytokines and the cell cycle inhibitor rapamycin. The cytokines IL-2 and IL-7, but not IL-4, increased initial activation induced FOXP3 expression, increased proliferation and sustained expression of FOXP3⁺ cells throughout the culture. Addition of rapamycin to cultures containing IL-2 further increased the frequency and absolute number of functional CD4⁺CD25⁺FOXP3⁺ Treg. This increase was not due to selective proliferation of FOXP3 cells, but was instead, the result of an increase in the frequency of FOXP3⁺ cells induced in G0 through delayed activation while the addition of IL-2 promoted survival and proliferation of the FOXP3⁺ population. Thus, combination of rapamycin and IL-2 may provide improved treatment options in transplantation and autoimmunity by promoting induction, survival, and expansion of functional iTreg from CD4⁺CD25⁻ cells.     

Developmental changes of FOXP3-expressing CD4+CD25+ regulatory T cells and their impairment in patients with FOXP3 gene mutations

FOXP3 is required for the generation and function of CD4(+)CD25(+) regulatory T (Treg) cells. To elucidate the biological role of Treg cells, we used a monoclonal anti-FOXP3 antibody to examine the frequencies of Treg cells during child development. The percentages of CD4(+)CD25(+)FOXP3(+) T cells were constant shortly from after birth through adulthood. CD4(+)CD25(+)FOXP3(+) T cells in cord blood showed the naive CD45RA(+)CD45RO(-) phenotype, whereas adult CD4(+)CD25(+)FOXP3(+) T cells expressed mostly the memory CD45RA(-)CD45RO(+) phenotype. The age-dependent dominance of memory CD4(+)CD25(+)FOXP3(+) T cells implies functional differences between naive and memory Treg cells. Notably, four patients with FOXP3 gene mutations revealed a paucity of CD4(+)CD25(+)FOXP3(+) T cells. Importantly, one patient with a frame shift mutation, who showed typical symptoms of IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked), exhibited marked T cell activation, whereas others with missense mutations, who were clinically milder, did not. This observation suggests a possible genotype-phenotype correlation in IPEX.     

Lewis肺癌细胞通过TLR9对CD4~+CD25~+Treg细胞影响的研究

目的:本研究以Lewis肺癌细胞为研究对象,探讨肿瘤细胞通过TLRs对CD4+CD25+Treg细胞的影响。方法:我们采用流式细胞术检测了Lewis肺癌细胞与脾淋巴细胞共培养系统中CD4+CD25+Treg细胞数量变化;通过RT-PCR方法检测了共培养对Foxp3和TLR1-9mRNA表达的影响;采用TLR9受体阻断剂氯喹阻断Lewis肺癌TLR9的表达。结果:与对照组相比,共培养组CD4+CD25+Treg细胞数量及Foxp3 mRNA表达均明显增高(P0.05);Lewis肺癌细胞与淋巴细胞共培养后可影响多种TLRs表达,其中TLR9 mRNA表达与对照组相比明显增高(P0.05),阻断Lewis肺癌细胞TLR9可明显降低CD4+CD25+Treg细胞数量及Foxp3 mRNA表达(P0.05)。结论:Lewis肺癌细胞可通过TLR9促进CD4+CD25+Treg细胞产生及功能增强,参与诱导肿瘤的免疫耐受,从而促进肿瘤的发生和发展。     

Phenotypic and functional characteristics of CD4+ CD39+ FOXP3+ and CD4+ CD39+ FOXP3neg T-cell subsets in cancer patients

Human CD4(+) CD39(+) regulatory T (Treg) cells hydrolyze exogenous adenosine triphosphate (ATP) and participate in immunosuppressive adenosine production. They contain two T-cell subsets whose role in mediating suppression is not understood. Frequencies of both CD4(+) CD39(+) subsets were evaluated in peripheral blood lymphocytes of 57 cancer patients and in tumor infiltrating lymphocytes (TILs) of 6 patients. CD4(+) CD39(+) and CD4(+) CD39(neg) T cells isolated using immunobeads and cell sorting were cultured under various conditions. Their conversion into CD39(+) FOXP3(+) CD25(+) or CD39(+) FOX(neg) CD25(neg) cells was monitored by multiparameter flow cytometry. Hydrolysis of exogenous ATP was measured in luminescence assays. Two CD4(+) CD39(+) cell subsets differing in expression of CD25, FOXP3, CTLA-4, CD121a, PD-1, latency associated peptide (LAP), glycoprotein A repetitions predominant (GARP), and the cytokine profile accumulated with equal frequencies in the blood and tumor tissues of cancer patients. The frequency of both subsets was significantly increased in cancer. CD39 expression levels correlated with the subsets' ability to hydrolyze ATP. Conventional CD4(+) CD39(neg) T cells incubated with IL-2 + TGF-β expanded to generate CD4(+) CD39(+) FOXP3(+) Treg cells, while CD4(+) CD39(+) FOXP3(neg) CD25(neg) subset cells stimulated via the TCR and IL-2 converted to FOXP3(+) CTLA4(+) CD25(+) TGF-β-expressing Treg cells. Among CD4(+) CD39(+) Treg cells, the CD4(+) CD39(+) FOXP3(neg) CD25(neg) subset serves as a reservoir of cells able to convert to Treg cells upon activation by environmental signals.     

The maintenance of human CD4+ CD25+ regulatory T cell function: IL-2, IL-4, IL-7 and IL-15 preserve optimal suppressive potency in vitro

CD4+ CD25+ regulatory T cells (Tregs) have far-reaching immunotherapeutic applications, the realization of which will require a greater understanding of the factors influencing their function and phenotype during ex vivo manipulation. In murine models, IL-2 plays an important role in both the maintenance of a functional Treg population in vivo and the activation of suppression in vitro. We have found that IL-2 maintains optimal function of human CD4+ CD25+ Tregs in vitro and increases expression of both forkhead box protein 3, human nomenclature (FOXP3) and the distinctive markers CD25, cytotoxic T lymphocyte antigen-4 (CTLA-4) and glucocorticoid-induced tumor necrosis factor receptor superfamily member number 18 (GITR). Although IL-2 reduced spontaneous apoptosis of Tregs, this property alone could not account for the optimal maintenance of the regulatory phenotype. The inhibition of phosphatidylinositol 3-kinase (PI3K) signaling by LY294002, a chemical inhibitor of PI3K, abolished the maintenance of maximal suppressive potency by IL-2, yet had no effect on the up-regulation of FOXP3, CD25, CTLA-4 and GITR. Other common gamma chain (gammac) cytokines-IL-4, IL-7 and IL-15-had similar properties, although IL-4 showed a unique lack of effect on the expression of FOXP3 or Treg markers despite maintaining maximal regulatory function. Taken together, our data suggest a model in which the gammac cytokines IL-2, IL-4, IL-7 and IL-15 maintain the optimal regulatory function of human CD4+ CD25+ T cells in a PI3K-dependent manner, offering new insight into the effective manipulation of Tregs ex vivo.