Murine DN T-reg cells impart prolonged cardiac allograft survival

Recent studies have demonstrated that both mouse and human alpha beta TCR(+)CD3(+)NK1.1(-)CD4(-)CD8- double-negative regulatory T (DN Treg) cells can suppress Ag-specific immune responses mediated by CD8+ and CD4+ T cells. To identify molecules involved in DN Treg cell function, we generated a panel of murine DN Treg clones, which specifically kill activated syngeneic CD8+ T cells. Through serial cultivation of DN Treg clones, mutant clones arose that lost regulatory capacity in vitro and in vivo. Although all allogeneic cardiac grafts in animals preinfused with tolerant CD4/CD8 negative 12 DN Treg clones survived over 100 days, allograft survival is unchanged following infusion of mutant clones (19.5 +/- 11.1 days) compared with untreated controls (22.8 +/- 10.5 days; p < 0.001). Global gene expression differences between functional DN Treg cells and nonfunctional mutants were compared. We found 1099 differentially expressed genes (q < 0.025%), suggesting increased cell proliferation and survival, immune regulation, and chemotaxis, together with decreased expression of genes for Ag presentation, apoptosis, and protein phosphatases involved in signal transduction. Expression of 33 overexpressed and 24 underexpressed genes were confirmed using quantitative real-time PCR. Protein expression of several genes, including Fc epsilon RI gamma subunit and CXCR5, which are >50-fold higher, was also confirmed using FACS. These findings shed light on the mechanisms by which DN Treg cells down-regulate immune responses and prolong cardiac allograft survival. Type 2 experiment where functional DN T-reg cells (CN4 or TN12) were cy-3 labeled and co-hybridized with non-functional mutant cell lines (CN4.8 or CN12.8). Used 100 ug total-RNA each channel and no amplifaction. A transcript identification design type characterizes the length and position of transcripts and allows identification of all forms of transcripts in the genome. Keywords: transcript_identification_design Using regression correlation

近期研究证实，小鼠及人类的αβ T细胞受体（TCR）阳性、CD3阳性、NK1.1阴性、CD4阴性、CD8阴性的双阴性调节性T（DN Treg）细胞，能够抑制CD8+与CD4+ T细胞介导的抗原特异性免疫应答。

为鉴定参与DN Treg细胞功能发挥的分子，我们构建了一组小鼠源性DN Treg细胞克隆，该克隆可特异性杀伤活化的同基因CD8+ T细胞。通过对DN Treg克隆进行连续传代培养，获得了在体外与体内均丧失调节能力的突变克隆。

尽管预先输注耐受型CD4/CD8双阴性12号DN Treg克隆的动物，其所有同种异体心脏移植物的存活期均超过100天，但输注突变克隆后，移植物存活期为（19.5±11.1天），与未处理对照组（22.8±10.5天；p<0.001）相比无显著差异。

我们比较了功能正常的DN Treg细胞与功能丧失的突变克隆之间的全基因表达差异。结果发现1099个差异表达基因（校正P值<0.025%），这些基因涉及细胞增殖与存活、免疫调节及趋化作用的上调，同时抗原呈递、细胞凋亡及信号转导相关蛋白磷酸酶编码基因的表达出现下调。

我们通过实时定量聚合酶链式反应（quantitative real-time PCR）验证了33个上调基因与24个下调基因的表达水平。此外，通过荧光激活细胞分选术（Fluorescence Activated Cell Sorting, FACS）验证了FcεRIγ亚基与CXCR5等50倍以上差异表达基因的蛋白质表达水平。

上述发现为DN Treg细胞下调免疫应答、延长同种异体心脏移植物存活期的作用机制提供了新的见解。本研究采用的2型实验方案为：将功能正常的DN Treg细胞（CN4或TN12）进行Cy3荧光标记，并与功能丧失的突变细胞系（CN4.8或CN12.8）进行共杂交；每个通道使用100μg总RNA，未进行扩增。

转录本鉴定设计类型可表征转录本的长度与位置，并能够鉴定基因组中所有形式的转录本。

关键词：transcript_identification_design、回归相关性（regression correlation）