CD4^＋CD25^＋Treg细胞对B细胞的作用机制分析

Immuological mechanism for regulatory T cells acting on B lymphocytes in vitro

目的:研究CD4+CD25+调节性T细胞对B细胞作用机制。方法:用尼龙毛柱法分离B细胞,用MACS磁珠分选法从Wistar大鼠脾脏淋巴细胞中分离CD4+CD25+Treg细胞,然后将分离纯化的Wistar大鼠Treg细胞负载SD大鼠抗原。以Trans Well Millcell-PCF分隔培养槽分隔或共培养Treg细胞与不同淋巴细胞组成的反应体系,然后向共培养体系中加或不加入Anti-TGF-β1、Anti-CTLA4的阻断方法,培养5天后用ELISA法检测不同组上清中的IgA和IgG分泌水平。结果:分隔培养组上清中的IgG和IgA水平分别为(4·7±1·1)、(10·5±1·7)μg/ml,与阳性对照组中IgG的含量(5·1±1·0)μg/ml以及IgA的(11·2±1·6)μg/ml相比没有显著差异(P>0·05)。而与阳性对照组相比较,共培养组上清中的IgG和IgA水平明显降低(P<0·01),分别达到了(2·2±0·8)μg/ml和(5·4±0·9)μg/ml。在共培养体系中加入Anti-TGF-β1后,上清中的IgG和IgA的分泌水平分别达到(3·1±0·5)μg/ml和(6·9±0·8)μg/ml;加入Anti-CTLA4后,上清中的IgG和IgA的分泌水平分别达到(3·2±0·6)μg/ml和(7·2±0·9)μg/ml;两种阻断剂同时加入后,上清中的IgG和IgA的分泌水平分别达到(3·5±0·5)μg/ml和(7·4±0·6)μg/ml,三组分泌水平都较共培养组明显升高(P<0·05),但仍明显低于阳性对照组的水平(P<0·05)。结论:CD4+CD25+Treg通过细胞-细胞间接触机制直接抑制B淋巴细胞反应,在这一过程中TGF-β1和CTLA4都发挥了一定作用。

Objective： To study the immuological mechanism for regulatory T cells acting on effect B lymphocytes in vitro.Methods： B cells of Wistar rats were enriched by Nyloon wool column. CD4^＋CD25^＋ regulatory T cells of Wistar rats were purified by the MACS and Treg cells isolated were loaded with antigen of SD rats.Treg cells were added to mixed lymphocyte reaction system（MLR） in co-cultured system （CCS）and Trans Well Millicell-PCF separate-cultured system（SCS）.The inhibitory effects of Treg cells on MLR were detected after adding Anti-TGF-β1 or Anti-CTLA4 to the reacting systems.After Treg cells were co-cultured with B cells for 5 days,concentrations of IgG and IgA in supernatants were determined by ELISA.Results： The secretion level of IgG and IgA in SCS was not obviously reduced, compared with the secretion level in positive control（ P 〉 0.05）. But the secretion level of IgG and IgA in CCS was obviously lower than in the positive control（ P 〈 0.01） .When Anti-TGF-β1 or/and Anti-CTLA4 were added into CCS,the secretion level of IgG and IgA was higher than in CCS（P 〈 0.05）and lower in the positive control. Conclusion： Cell-to-cell contact mechanism plays primal role in the regulation of B cells by CD4^＋CD25^＋ regulatory T cells. TGF-β1 and CTLA4 have partial effect in Tregs suppression of B cells.