Gene changes of CD4+ T cells infiltrating human breast cancer in the absence of tumor environment (Confirmation Set 24h)

CD4+ helper T (Th) cells are critical regulators of immune responses but their role in breast cancer is currently unknown. This work aims to characterize Th cells infiltrating invasive primary human breast tumors, analyze the influence by the tumor microenvironment and identify Th cell specific prognostic gene signatures. CD4+ T cells isolated from the tumor (TIL), axillary lymph node (LN) and blood (PB) of 10 patients were analyzed on Affymetrix U133 Plus 2.0 arrays. A confirmation set of 60 patients were studied by flow cytometry, qRT-PCR or immunohistochemistry and analyzed according to the extent of the tumor immune infiltrate. Gene expression profiles of freshly isolated TIL were also compared with TIL that had been rested overnight or with CD4+ T cells [non-stimulated (NS) or stimulated (S)] from healthy donor PB treated with tumor supernatant (SN). Analysis of CD4+ TIL with or without 24h ex-vivo rest, including donor blood memory CD4+ T cells treated in the same conditions as control CD4+ T cells isolated from primary tumors of 2 patients and memory CD4+ T cells from a healthy donor blood were immediately analyzed or incubated for 24h without stimulation before being analyzed on Affymetrix U133 Plus 2.0 arrays

CD4+辅助性T（Th）细胞是免疫应答的关键调控因子，但其在乳腺癌中的作用目前尚未明确。本研究旨在对浸润侵袭性原发性人乳腺肿瘤的Th细胞进行表征分析，解析肿瘤微环境对其的调控影响，并鉴定Th细胞特异性的预后基因特征。研究人员从10名患者的肿瘤、腋窝淋巴结（axillary lymph node, LN）及外周血（peripheral blood, PB）中分离CD4+ T细胞，其中肿瘤来源的CD4+ T细胞为肿瘤浸润淋巴细胞（Tumor Infiltrating Lymphocyte, TIL），并采用Affymetrix U133 Plus 2.0基因芯片对所有样本进行表达谱分析；另有60名患者组成的独立验证队列，通过流式细胞术、实时荧光定量聚合酶链反应（qRT-PCR）及免疫组织化学进行研究，并根据肿瘤免疫浸润程度开展分层分析。本研究同时将新鲜分离的TIL的基因表达谱，与经过过夜静置培养的TIL，以及经肿瘤上清液（tumor supernatant, SN）处理的健康供体外周血来源的CD4+ T细胞（未刺激组[NS]与刺激组[S]）的表达谱进行对比；此外，本研究还对经过或未经过24小时体外静置培养的CD4+ TIL进行了分析，同时纳入以与2名患者原发性肿瘤分离的对照CD4+ T细胞相同条件处理的供体外周血记忆性CD4+ T细胞，以及健康供体外周血来源的记忆性CD4+ T细胞，上述所有样本均被分为两个亚组：一组立即采用Affymetrix U133 Plus 2.0基因芯片进行表达谱检测，另一组在无刺激条件下孵育24小时后再进行相同的芯片分析。