Single-cell RNA-sequencing of CD7 knockout CD8 T cells

The goal of this study is to identify transcriptional changes in antigen-specific CD8 T cells caused by CD7 deletion. Overall design: CD7-/- mice were crossed to CD45.1+ P14 mice, which have a transgenic TCR specific for gp33, an epitope of lymphocytic choriomeningitis virus (LCMV). CD7 knockout (KO) and littermate wildtype (WT) P14 cells were co-transferred to C57BL/6J mice, which were subsequently infected with 2e6 PFU of LCMV clone 13 following transient CD4 T cell depletion. >45 days after infection, WT and KO P14 cells were isolated from liver and spleens of infected animals, and naive P14 WT and KO cells were isolated from the blood of the donor mice. Individual cell types were labeled with hashing antibodies, pooled, and subjected to the 10x Genomics Chromium scRNA-seq workflow. Mouse hashing antibody sequences: Naive WT: Totalseq-C hash 1 (ACCCACCAGTAAGAC); Naive KO: Totalseq-C hash 2 (GGTCGAGAGCATTCA); Spleen WT P14s from infected mice: Totalseq-C hash 3 (CTTGCCGCATGTCAT); Spleen KO P14s from infected mice: Totalseq-C hash 4 (AAAGCATTCTTCACG); Liver WT P14s from infected mice: Totalseq-C hash 5 (CTTTGTCTTTGTGAG); Liver KO P14s from infected mice: Totalseq-C hash 6 (TATGCTGCCACGGTA)

本研究旨在明确CD7缺失所诱导的抗原特异性CD8 T细胞转录组变化。总体实验设计如下：将CD7敲除（CD7-/-）小鼠与CD45.1+ P14小鼠杂交，后者携带针对淋巴细胞脉络丛脑膜炎病毒（LCMV）表位gp33的转基因T细胞受体（TCR）。将CD7敲除（KO）型与同窝野生型（WT）P14细胞共转移至C57BL/6J小鼠体内，随后在短暂耗竭CD4 T细胞后，以2×10^6 PFU的LCMV克隆13毒株感染受体小鼠。感染45天以上，从感染小鼠的肝脏与脾脏中分离WT及KO型P14细胞，并从供体小鼠血液中分离未致敏（naive）WT与KO型P14细胞。使用细胞哈希抗体（hashing antibodies）对各细胞群进行标记后混合，随后采用10x Genomics Chromium单细胞RNA测序（scRNA-seq）流程开展实验。小鼠细胞哈希抗体序列如下：
未致敏WT：Totalseq-C 标记1（ACCCACCAGTAAGAC）；
未致敏KO：Totalseq-C 标记2（GGTCGAGAGCATTCA）；
感染小鼠脾脏WT P14细胞：Totalseq-C 标记3（CTTGCCGCATGTCAT）；
感染小鼠脾脏KO P14细胞：Totalseq-C 标记4（AAAGCATTCTTCACG）；
感染小鼠肝脏WT P14细胞：Totalseq-C 标记5（CTTTGTCTTTGTGAG）；
感染小鼠肝脏KO P14细胞：Totalseq-C 标记6（TATGCTGCCACGGTA）