Characterization of bronchoalveolar lavage samples from two Be-exposed subjects

Chronic beryllium disease (CBD) is a metal-induced hypersensitivity disorder characterized by CD4+ T cell alveolitis. Single-cell gene expression profiling of bronchoalveolar lavage (BAL) cells focused on T cells may provide insight into unique subsets of CD4+ T cells, functional pathways, and/or molecules that could potentially be developed into novel therapeutics for treating CBD and other granulomatous lung diseases. To understand the unbiased cellular composition of airway associated CD4+ T cells in subjects with beryllium exposure, we used BAL cells from one beryllium-exposed (BeS) and one CBD subject diagnosed using previously defined criteria, and these two samples comprised the disease cohort. Cells were thawed and rested in culture overnight in complete medium. Single cells were flow-sorted using propidium iodide to exclude dead cells. The sorted cells were resuspended in PBS buffer, recounted, and processed for 10X Genomics 3’v2 Kit as specified by the manufacturer’s protocol for each patient sample. Approximately 5000 cells were loaded in the wells of a 10X Genomics Chromium controller to create GEM emulsions, targeting a recovery of 3000 cells. Reverse transcription, cDNA amplification and RNA- sequencing library preparation were performed using the GEM emulsion. Libraries were sequenced on the Illumina Hi-Seq 4000 platform, using 75 bp paired end reads.慢性铍病（Chronic Beryllium Disease, CBD）是一种由金属诱导的超敏性疾病，以CD4+ T细胞性肺泡炎为典型特征。针对支气管肺泡灌洗（Bronchoalveolar Lavage, BAL）细胞中的T细胞开展单细胞基因表达谱分析，或可挖掘CD4+ T细胞的独特亚群、功能通路及相关分子，进而开发用于治疗CBD及其他肉芽肿性肺病的新型治疗手段。 为无偏解析铍暴露受试者气道相关CD4+ T细胞的细胞组成，本研究纳入1名铍暴露（beryllium-exposed, BeS）者与1名符合既往诊断标准的CBD患者的BAL细胞，构建疾病研究队列。将获取的细胞复苏后，置于完全培养基中静置培养过夜。采用碘化丙啶进行流式细胞分选以排除死细胞，随后将分选得到的细胞重悬于磷酸盐缓冲液（Phosphate Buffered Saline, PBS）中并重新计数，严格按照制造商说明书，使用10X Genomics 3'端v2试剂盒（10X Genomics 3’v2 Kit）对每份患者样本进行处理。 将约5000个细胞加载至10X Genomics Chromium控制器的反应孔中以生成GEM微滴，目标回收细胞数为3000个。通过GEM微滴完成逆转录、cDNA扩增及RNA测序文库制备。最终将文库置于Illumina HiSeq 4000测序平台，采用75 bp双端读长模式进行测序。