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.