Inherited CD4 deficiency: short-read single-cell RNA sequencing (scRNASeq) of stimulated TCRab+ memory T cells
Description
To identify transcriptional modules perturbed by inherited CD4 deficiency, single-cell RNA sequencing was performed on isolated memory (defined as CD45RA-CCR7+/-) CD3+CD8-TCRαβ+ and/or CD3+CD4-CD8-TCRαβ+ double negative (DN) T cells from healthy donors (n=4), FAS deficient (n=1), STAT3GOF (n=1) and CD4 deficient (P1-P5) patients. Isolated cells were cultured with TAE beads + IL-2 (50 IU/mL, #IL002, Millipore) for 20h. Cells were washed 3 times in cold PBS + 2% HI FBS and incubated with TotalSeq anti human hashtag antibodies (A0251-A0256, #394601, #394603, #394605, #394607, #394609, #394611, BioLegend) for 30 min on ice. Hashtagged cells were washed 3 times in cold PBS + 2% HI FBS and passed through a 35µm nylon mesh cell strainer (#352235, Falcon) to remove aggregates. Cells were resuspended at a concentration of 1000 cells/µl in cold PBS + 2% HI FBS, pooled, and loaded onto a 10X Genomics Chromium B or G chips. Single-cell capture, reverse transcription, and library preparation were performed with the Chromium Single-Cell 3’ Reagent Kits (v3 or v3.1), in accordance with the manufacturer’s instructions. The quality of the cDNA and feature barcode library was assessed with a TapeStation (Agilent). Transcriptome sequencing was performed on the S4 flow cells of an Illumina NovaSeq 6000 sequencer, while hashtag library sequencing was performed on NextSeq 500/550 system with High Output Kit v2.5 (Illumina). Cells from P1/P2/P3 and P4/P5 were processed in two batches of experiments, along with cells from two healthy donors per batch. For comparison, CD3+CD4-CD8 TCRαβ+ double-negative T cells from two healthy donors, one FAS-deficient patient, and one patient with a STAT3 GOF mutation were isolated and processed similarly. Hashtag demultiplexing was performed using Seurat with default parameter settings. Hash tag doublets were excluded from subsequent analyses. Cells were further filtered based on standard QC metrics. Batch correction, unsupervised clustering, pseudobulk PCA, differential expression (DE) analysis, and gene-set enrichment analysis (GSEA) were performed as described previously (Ogishi et al., 2022). All analyses were conducted in R (version 4.2.2, https://www.R-project.org/).