Inherited CD4 deficiency: short-read single-cell RNA sequencing (scRNASeq) of stimulated TCRab+ memory T cells

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/).

为识别由遗传性CD4缺乏所扰动的转录模块，对从健康供体（n=4）、FAS缺乏（n=1）、STAT3GOF（n=1）和CD4缺乏（P1-P5）患者中分离的内存T细胞（定义为CD45RA-CCR7+/- CD3+CD8-TCRαβ+和/或CD3+CD4-CD8-TCRαβ+双阴性（DN）T细胞）进行了单细胞RNA测序。分离的细胞与TAE磁珠+ IL-2（50 IU/mL，#IL002，Millipore）共同培养20小时。细胞经三次冷PBS+2% HI FBS洗涤后，在冰上与TotalSeq抗人标签抗体（A0251-A0256，#394601，#394603，#394605，#394607，#394609，#394611，BioLegend）孵育30分钟。标记后的细胞经三次冷PBS+2% HI FBS洗涤，并通过35µm尼龙网细胞过滤器（#352235，Falcon）以去除聚集物。细胞以1000细胞/µl的浓度在冷PBS+2% HI FBS中重悬，混合，并加载至10X Genomics Chromium B或G芯片上。单细胞捕获、逆转录和文库制备使用Chromium Single-Cell 3’试剂套件（v3或v3.1）进行，遵循制造商的说明。使用TapeStation（Agilent）评估cDNA和特征条形码文库的质量。转录组测序在Illumina NovaSeq 6000测序仪的S4流细胞上进行，而标签文库测序在NextSeq 500/550系统上进行，使用High Output Kit v2.5（Illumina）。P1/P2/P3和P4/P5的细胞分两批实验处理，每批实验还包括两个健康供体的细胞。为了比较，从两个健康供体、一名FAS缺乏患者和一名STAT3 GOF突变患者中分离的CD3+CD4-CD8 TCRαβ+双阴性T细胞以相似的方式进行分离和处理。使用Seurat和默认参数设置进行标签去复用。排除后续分析中的标签双体。根据标准质量控制指标进一步筛选细胞。进行批量校正、无监督聚类、伪bulk PCA、差异表达（DE）分析和基因集富集分析（GSEA），如前所述（Ogishi等，2022）。所有分析均在R（版本4.2.2，https://www.R-project.org/）中完成。