Single-cell RNA-sequencing of blood and tonsillar CD4+ CD57+ and CD57- T cells

Gene-environment interactions are implicated in immunopathology, but few specific mechanisms have been identified. We defined two terminal-differentiation pathways for human CD4+ T cells each marked by CD57. Rare blood CD57+ CD4+ T cells marked by TCF1 downregulation (Thcyt) adopt a cytotoxic and terminal-effector transcriptome. By contrast, human CD57+ GC-Tfh cells exhibit a transcriptome of the precursor exhausted T cells marked by TCF7, TOX and ID3 and constitutive expression of CTLA4, which collectively confer resistance to becoming cytotoxic. By clarifying human CD4+ T cell terminal differentiation, we have revealed how CTLA4 mitigates the risk of maladaptive cytotoxicity during infection. Overall design: CD57+ CD4+ T cells (7AAD- CD19- CD3+ CD4+ CD8- CD57+) and CD57- CD4+ T cells (7AAD- CD19- CD3+ CD4+ CD8- CD57-) from tonsil and blood samples of a child were FACS-purified into 1.5-ml Eppendorf tubes with FACS buffer. Single cell gene expression and VDJ libraries were prepared by Biomolecular Resource Facility at ANU using Chromium Next GEM Single Cell 5' Kit v2 Dual Index and Single Cell Human TCR Amplification Kit (10x Genomics) according to the manufacturer's instructions. The library was sequenced in S1 flow cell with 2 x 50 bp paired-end reads by NovaSeq 6000 sequencer (Illumina). The 10X Cell Ranger package (10X Genomics, v6.0.1) was used to process data and align to human genome (GRCh38-2020-A).