The contained self-reactive peripheral T cell repertoire: size, diversity and cellular composition

To further characterize the self-reactive T cell population and determine if there are sub-compartments within the naïve T cell pool that are more likely to respond in the absence of Treg cell-mediated control, we focused on CD5. CD5 is a membrane protein that is associated with the TCR and its expression is correlated with higher signaling intensity, presumably due to higher TCR self-MHC/peptide affinity, during positive selection. FACS was used to purify three distinct populations of naïve CD4+ T cells from the bottom, middle and top 10 percent based on CD5 expression; CD5low, CD5int and CD5high. Naïve (CD44low/int CD62Lhigh) CD4+ CD5low, CD5int and CD5high T cell populations were FACS purified from spleens from three female (Fe) and three male (Ma) mice, to account for possible gender differences. RNA was extracted using the RNEasy Plus Micro Kit (Qiagen).

为进一步表征自身反应性T细胞群，并探究初始T细胞库内是否存在亚群，在缺乏调节性T细胞（Regulatory T cell, Treg）介导的调控时更易发生免疫应答，本研究聚焦于CD5分子。CD5是一种与T细胞受体（T cell receptor, TCR）相结合的膜蛋白，其表达水平与更强的信号转导强度呈正相关，这一关联推测源于阳性选择过程中，TCR与自身主要组织相容性复合体（Major Histocompatibility Complex, MHC）-肽复合物的亲和力更高。研究人员采用荧光激活细胞分选术（Fluorescence-Activated Cell Sorting, FACS），根据CD5的表达水平，从初始CD4阳性T细胞中分离出三个亚群：CD5low、CD5int与CD5high，分别对应CD5表达量最低、中等与最高的10%细胞群。为排除性别差异可能带来的实验影响，研究人员从3只雌性（Fe）与3只雄性（Ma）小鼠的脾脏中，分选得到初始型（CD44low/int CD62Lhigh）CD4阳性CD5low、CD5int及CD5high T细胞群。采用RNEasy Plus Micro试剂盒（Qiagen公司）提取RNA。