Single-cell transcriptome landscape of circulating CD4+ T cell populations in autoimmune diseases.

CD4+ T cells are key mediators of various autoimmune diseases; however, their role in disease progression remains unclear due to cellular heterogeneity. Here, we evaluated CD4+ T cell subpopulations using decomposition-based transcriptome characterization and canonical clustering strategies. This approach identified 12 independent gene programs governing whole CD4+ T cell heterogeneity, which can explain the ambiguity of canonical clustering. In addition, we performed a meta-analysis using public single-cell datasets of over 1.8 million peripheral CD4+ T cells from 953 individuals by projecting cells onto the reference and cataloging cell frequency and qualitative alterations of the populations in 20 diseases. The analyses revealed that the 12 transcriptional programs were useful in characterizing each autoimmune disease and predicting its clinical status. Moreover, genetic variants associated with autoimmune diseases showed disease-specific enrichment within the 12 gene programs. The results collectively provide a landscape of single-cell transcriptomes of CD4+ T cell subpopulations involved in autoimmune disease.

Protocol to evaluate cell lineage stability of mouse natural and induced regulatory T cells using bisulfite sequencing.

The establishment of regulatory T cells (Treg)-specific demethylation regions (TSDRs) is essential for the Treg-lineage stability. Here, we present a protocol using bisulfite sequencing to assess Treg-lineage stability. The protocol describes the isolation of lymphocytes and DNA extraction, followed by bisulfite conversion in unmethylated CpG DNA, bisulfite PCR and cloning, and sequencing to define the TSDR methylation. This protocol uses lymph nodes and spleen tissues and can be adapted to assess the methylation status of Tregs in other tissue types.