mRNA profile of CD8 T cells from chronic HBV patients

The low frequency of HBV-specific CD8+ T cells in the peripheral blood of CHB patients has limited studies of the mechanisms underlying HBV-induced T cell exhaustion. Similar to the expansion defect displayed in HBV-specific CD8+ T cells, TCR-induced proliferation of global CD8+ T cells is impaired in a fraction of chronic HBV (CHB) patients. Thus, examining the molecular regulation of global CD8+ T cell function in CHB patients may provide insight into the exhaustion of HBV-specific CD8+ T cells. We used microarrays to detail the global programme of gene expression of CD8 T cells from CHB patients who have not received anti-viral treatment. Fifteen milliliters of blood was drawn from eachof three CHB patients and three healthy donors. PBMCs were enriched using Ficoll, and CD8+ T cells were purified using positive selection beads to a purity of >95% (Miltenyi Biotec, Auburn, CA). Total RNA was extracted using a mirVana isolation kit (Life Technologies, Carlsbad, CA).

慢性乙型肝炎（chronic hepatitis B, CHB）患者外周血中乙型肝炎病毒（hepatitis B virus, HBV）特异性CD8+ T细胞的频率极低，这限制了对HBV诱导的T细胞耗竭机制的研究。与HBV特异性CD8+ T细胞所表现出的扩增缺陷类似，部分慢性HBV患者的总CD8+ T细胞的TCR诱导增殖能力存在受损情况。因此，探究慢性HBV患者总CD8+ T细胞功能的分子调控机制，或可为阐明HBV特异性CD8+ T细胞的耗竭过程提供新思路。本研究通过微阵列技术，详细解析了未接受抗病毒治疗的慢性HBV患者CD8+ T细胞的全局基因表达谱。我们从3名慢性HBV患者及3名健康供者体内各采集15mL外周血。采用Ficoll密度梯度离心法富集外周血单个核细胞（peripheral blood mononuclear cell, PBMCs），随后使用阳性分选磁珠纯化CD8+ T细胞，最终纯度可达95%以上（美天旎生物科技（Miltenyi Biotec），奥本，加利福尼亚州，美国）。总RNA的提取使用mirVana总RNA分离试剂盒（Life Technologies公司，卡尔斯巴德，加利福尼亚州，美国）完成。