CAJ | 학술논문

目的:观察BTLA分子在T细胞上的表达并探讨其在各个阶段不同时相对T细胞活化的抑制.方法:分离人外周血单个核细胞,经阴性选择磁珠分离纯化获得T淋巴细胞.检测T细胞上BTLA、CTLA-4和PD-1的表达;用CD3抗体刺激T细胞活化,比较BTLA、CTLA-4和PD-1在T细胞活化过程中的动态表达.CD3抗体联合CD28抗体活化T细胞,在不同的活化时间,MTT法检测BTLA单抗8H9对T细胞增殖的影响.GM-CSF和IL-4体外诱导单核细胞分化成未成熟DC,CD40抗体刺激DC成熟,流式榆测HVEM在DC上的表达.用DC诱导T细胞活化,加入游离8H9或抗HVEM抗体,阻断HVEM和BTLA结合,MTT法检测T细胞增殖.结果:静止T细胞组成性高表达BTLA,不表达CTLA-4和PD-1分子.T细胞活化后,BTLA分子表达有所降低,然后迅速回升至高水平.CTLA-4、PD-1分子在活化后两大几乎不表达,第三天开始表达并逐渐上升.8H9可以抑制CD3和C028抗体活化的T细胞增殖.CD3和CD28抗体预先活化T细胞24小时或48小时后,再加入8H9仍然具有抑制效应,但不如在T细胞活化之初加入8H9的抑制效应.单核细胞诱导的不成熟DC上高表达HVEM,当DC成熟后,HVEM表达降低.用游离8H9或HVEM抗体阻断DC表面HVEM与T细胞表面BTLA结合,48小时之内均明显增强了DC诱导的T细胞增殖.结论:BTLA信号可以提高T细胞的活化阈值,在T细胞活化的起始和早期阶段发挥重要的负性调控作用.
목적:관찰BTLA분자재T세포상적표체병탐토기재각개계단불동시상대T세포활화적억제.방법:분리인외주혈단개핵세포,경음성선택자주분리순화획득T림파세포.검측T세포상BTLA、CTLA-4화PD-1적표체;용CD3항체자격T세포활화,비교BTLA、CTLA-4화PD-1재T세포활화과정중적동태표체.CD3항체연합CD28항체활화T세포,재불동적활화시간,MTT법검측BTLA단항8H9대T세포증식적영향.GM-CSF화IL-4체외유도단핵세포분화성미성숙DC,CD40항체자격DC성숙,류식유측HVEM재DC상적표체.용DC유도T세포활화,가입유리8H9혹항HVEM항체,조단HVEM화BTLA결합,MTT법검측T세포증식.결과:정지T세포조성성고표체BTLA,불표체CTLA-4화PD-1분자.T세포활화후,BTLA분자표체유소강저,연후신속회승지고수평.CTLA-4、PD-1분자재활화후량대궤호불표체,제삼천개시표체병축점상승.8H9가이억제CD3화C028항체활화적T세포증식.CD3화CD28항체예선활화T세포24소시혹48소시후,재가입8H9잉연구유억제효응,단불여재T세포활화지초가입8H9적억제효응.단핵세포유도적불성숙DC상고표체HVEM,당DC성숙후,HVEM표체강저.용유리8H9혹HVEM항체조단DC표면HVEM여T세포표면BTLA결합,48소시지내균명현증강료DC유도적T세포증식.결론:BTLA신호가이제고T세포적활화역치,재T세포활화적기시화조기계단발휘중요적부성조공작용.
Objective:To observe the expression of BTLA on T cells during activation and further analyze its inhibitory effects on T cell activation in different phases.Methods: T cells from PBMC were enriched by negative selection using magnetic beads.Expression of BTLA,CTLA-4 and PD-1 on freshly isolated human T cells and kinetics expression of BTLA,CILA-4 and PD-1 on CD3 mAb stimulated T cells were examined by flow cytometry.T cells were stimulated by anti-CD3 mAb combined with anti-CD28 mAb in the presence of anti-BTLA mAb 8H9,then T cell proliferation was tested by MTT assay in the different culture time.Immature DCs were generated from monocytes cultured in the medium containing GM-CSF and IL-4, and further driven to maturation by anti-CD40 mAb.Expression of HVEM on DCs was measured by flow cytometry.T cells were co-cultured with DCs in the presence of soluble 8H9 or anti-HVEM antibody to block HVEM-BTLA interaction,T cell proliferation was measured by MTT assay.Results:Freshly isolated T cells exhibited high levels of BTLA expression, but not CTLA-4 and PD-1.After T cell activation, BTLA expression decreased on first 2 days, with rapidly increasing to high levels.Unlike BTLA, expression of CTLA-4 and PD-1 was gradually increased during T cell activation.8H9 significantly inhibited the proliferation of T cell stimulated by CD3 mAb and CD28 mAb.8H9 could still exhibit inhibitory effect on T cell proliferation after 24 h or 48 h of preactivation by CD3 mAb plus CD28 mAb stimulation.HVEM was highly expressed on immature DCs, and down-regulated on mature DCs.Blockade of BTLA by soluble 8H9 or anti-HVEM antibody enhanced DC-mediated T cell proliferation within 48 h.Conclusion: BTLA signal enhances the threshold of T cell activation and plays importantly negative regulatory role in the initiation and early phase of T cell activation.