ICOS在健康人外周血调节性T细胞上的生物学特征初探

Role of ICOS on in-vitro cultured human PBMC Treg

目的:研究ICOS在健康人外周血Treg离体培养中的作用,了解m TOR对离体培养Treg ICOS表达的调控。方法:MACS分选Treg后经anti-CD3+anti-CD28磁珠刺激后第3、7天流式检测Treg表面ICOS表达情况;anti-CD3+anti-CD28抗体或anti-CD3+ICOSL-Fc刺激3 d,流式检测Treg ICOS表达情况;雷帕霉素处理离体培养Treg,流式分析其对Treg ICOS表达作用。CFSE标记人PBMC细胞,并与离体培养Treg混合培养,流式检测Treg的接触抑制活性。结果:anti-CD3+anti-CD28磁珠刺激Treg 3 d,ICOS+Treg中活、死细胞的比例分别为(92.00±2.69)%和(2.20±0.56)%,在ICOS-Treg中比例为(90.30±3.53)%和(1.77±0.78)%,两者无显著差异;培养第7天,ICOS+Treg细胞比例从第3天(40.20±1.83)%降至(11.60±1.10)%;anti-CD3+ICOSL-FC刺激Treg 3 d后,ICOS MFI为(403.30±74.42),anti-CD3+anti-CD28刺激组为(2 410.0±746.4),anti-CD3+ICOSL-FC刺激后Treg ICOS表达相比anti-CD3+anti-CD28组显著下降;雷帕霉素处理离体培养Treg细胞3 d后,ICOS表达下调。此外,雷帕霉素处理的离体培养Treg均能有效抑制PBMC中Tcon的分裂增殖。结论:ICOS表达高低对人外周血Treg存活率影响无显著差异,m TOR信号并非调控人Treg离体培养ICOS表达的唯一因素,CD28协同信号对调控离体培养人Treg。表达比ICOSL信号更为重要,雷帕霉素处理的离体培养人Treg仍具有细胞接触抑制活性。

Objective： To investigate the role of mTOR in regulation of ICOS expression in human blood regulatory T cells. Methods： Isolation of Treg cells from human PBMC using MACS beads. We detected the ICOS expression on purified Treg cells and Treg cells viability using flow cytometry in anti-CD3 plus anti-CD28（ antibody or beads） or anti-CD3 plus ICOSL-Fc for 3 days and7 days. CFSE labeling human PBMC cells and in vitro cultured Treg mixed,Treg contact inhibition activity was detected by flow analysis. Results： After in vitro stimulation of Treg cells in the presence of anti-CD3＋anti-CD28 for 3 days,there was no significant statistic difference in viability between ICOS＋（ 92. 00±2. 69） % and ICOS-（ 90. 30±3. 53） % Treg-cells. After cultured for 7 days,the decreased ICOS＋Treg cells percentage within total Treg cells from（ 40. 20±1. 83） % to（ 11. 60± 1. 10） % compared with that of 3 days.Further more,the ICOS expression level between stimulated with anti-CD28 or ICOSL-Fc condition group,compared with the ICOS MFI in the condition of anti-CD3 plus anti-CD28 treatment for 3 days was（ 2410. 0±746. 4） obviously higher than（ 403. 30±74. 42）,that of the group treated with anti-CD3 plus ICOSL-FC. Rapamycin could partially suppress Treg cells ICOS expression,but unaffected the Treg suppression ability. Conclusion： ICOS expression level may not important for in vitro cultured human PBMC Treg cells survival although m TOR signling is important for regulation ICOS expression on in-vitro cultured Treg cells,but the ICOS expression on Treg regulated by multiply signaling pathways. CD28 signaling is the key stimulation factor for ICOS upregulation on in-vitro cultured Treg cells compared to ICOSL signaling.