Transcriptional factor FOXO3a controls the persistance of memory CD4+ T cells in HIV infection

Persistence of memory CD4+ T cells in ECs was coupled with the inactivation of FOXO3a transcriptional activities, which we have previously identified as a critical regulator of TCM survival. Indeed, expression levels of transcriptional targets of FOXO3a, endowed with pro-apoptotic and anti-proliferative functions, were lower in TCM and TEM from ECs as compared to ST individuals. Silencing the transcriptionally active form of FOXO3a by siRNA rescued TCM and TEM of STs from Fas-mediated apoptosis. Moreover the expression of FOXO3a dominant negative form (FOXO3a Nt) rescued the long-term survival of TCM from STs as these cells persisted as long as those derived from ECs. Overall, these studies indicate that FOXO3a activation is an important mediator of the shortened survival and heighteined turnover of TCM and TEM in chronic HIV infection. Targeting this pathway may provide a strategy to preserve memory T cell numbers in HIV infection. Keywords: comparative gene profile, cell-type comparison Isolation of CD4+T cell sub-populations. Peripheral blood mononuclear cells (PBMCs) from healthy adult individuals were isolated by Ficoll-HyPaque (Pharmacia) density gradient. We first enriched for CD4+ T cells using negative immunomagnetic beads selection (Automacs, Myltenii), cells were then labeled with anti-CD4-APCcy7, anti-CD45RA-ECD, anti-CD27-FITC and anti-CCR7-PEcy7 and sorted into Naive cells described as CD4+, CD45RA+, CD27+ and CCR7+, Central Memory cells (TCM) described as CD4+, CD45RA-, CD27+ and CCR7+ cells and Effector Memory cells (TEM) described as CD4+, CD45RA-, CD27- and CCR7- cells. Sorting was performed using a BDAria (BD Pharmingen). Purity of the TCM and TEM sub-populations was ranging from 96 to 99%. All procedures were done at 4 degrees C to avoid any changes in cell phenotype or gene expression. Sample RNA was extracted using an RNA extraction kit (Quiagen), then amplified using the MessageAmp RNA kit (Ambion) as per the manufacturer’s instructions. The amplified RNA (aRNA) was then verified for quality and quantity using the Agilent Bioanalyser and measuring the OD. Universal human RNA (Stratagene) was also prepared in the same way. Sample probes were prepared by direct labelling with 3 µg of the aRNA Cy-5 (R values) fluorescent dye while the universal RNA probes were prepared by direct labelling of universal aRNA with Cy-3 (G values). All patient samples were hybridized against amplified universal RNA at 37 ºC for 18h on a custom human Immune array. Detailed information on the labeling and hybridization procedures can be obtained at the Microarray Centre web page (University Health Network).

HIV感染组（ECs）中记忆性CD4+T细胞的持久性与叉头框转录因子O3a（FOXO3a）转录活性的失活相关，而我们此前已证实该蛋白是中枢记忆性T细胞（central memory T cell, TCM）存活的关键调控因子。事实上，兼具促凋亡与抗增殖功能的FOXO3a转录靶基因，其表达水平在HIV感染组的TCM与效应记忆性T细胞（effector memory T cell, TEM）中，较血清阴性个体（ST）显著降低。通过小干扰RNA（small interfering RNA, siRNA）沉默转录活性形式的FOXO3a，可挽救血清阴性个体的TCM与TEM细胞免于Fas介导的细胞凋亡。此外，过表达FOXO3a显性负突变体（FOXO3a Nt）可延长血清阴性个体TCM细胞的长期存活能力，使其存活时长与HIV感染组来源的TCM细胞相当。综上，本系列研究表明，FOXO3a的激活是慢性HIV感染中TCM与TEM细胞存活期缩短、更新速率加快的重要介导因素。靶向该通路或可为维持HIV感染患者体内记忆性T细胞数量提供新的治疗策略。

关键词：比较基因谱、细胞类型比较

CD4+T细胞亚群的分离：采用聚蔗糖-泛影葡胺（Ficoll-HyPaque，Pharmacia品牌）密度梯度离心法分离健康成人外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）。我们首先采用阴性免疫磁珠分选法（Automacs系统，Myltenii品牌）富集CD4+T细胞，随后用抗CD4-APCcy7、抗CD45RA-ECD、抗CD27-FITC及抗CCR7-PEcy7抗体对细胞进行标记，通过流式分选将细胞分为初始T细胞（表型为CD4+、CD45RA+、CD27+、CCR7+）、中枢记忆性T细胞（TCM，表型为CD4+、CD45RA-、CD27+、CCR7+）及效应记忆性T细胞（TEM，表型为CD4+、CD45RA-、CD27-、CCR7-）。分选操作采用BD Aria流式细胞仪（BD Pharmingen品牌）完成。TCM与TEM细胞亚群的分选纯度介于96%至99%之间。所有实验操作均在4℃条件下进行，以避免细胞表型或基因表达发生改变。

样本总RNA采用RNA提取试剂盒（Qiagen品牌）提取，随后按照试剂盒说明书使用MessageAmp RNA扩增试剂盒（Ambion品牌）进行RNA扩增。采用安捷伦生物分析仪（Agilent Bioanalyser）并通过光密度（OD）值检测，对扩增后的RNA（aRNA）的质量与浓度进行验证。通用人类RNA（Stratagene品牌）亦采用相同方法进行制备。样本探针采用3μg扩增RNA直接标记Cy-5荧光染料（对应R值）进行制备，而通用RNA探针则通过将通用扩增RNA直接标记Cy-3荧光染料（对应G值）完成制备。所有患者样本均与扩增后的通用RNA在定制化人类免疫基因芯片上于37℃杂交18小时。关于标记与杂交流程的详细信息可登录大学健康网络（University Health Network）芯片中心官网获取。