Single-cell RNA-seq analysis of longitudinal CD4+ T-cell samples reveals cell-type-specific changes during early stages of type 1 diabetes

Single-cell RNA sequencing (scRNA-seq) was performed on longitudinal CD4⁺ T-cell samples from 11 children who later developed type 1 diabetes and 11 matched controls. Samples were collected prior to or at the time of type 1 diabetes–specific autoantibody appearance (seroconversion), between ages 3 and 24 months (n = 73 samples). CD4⁺ T cells were isolated and profiled using the 10x Genomics Chromium Single Cell 3′ Reagent Kits v3.1. Individual samples were labeled with unique cell hashing antibodies and pooled into multiplexed runs, with up to 10 samples (from matched case–control pairs) combined per pool. In total, 9 multiplexed pools were processed on Chromium Next GEM Chips, targeting recovery of approximately 20,000 single cells per run (CH_run2-CH_run10). cDNA and hashtag oligo libraries were generated with dual indexing and sequenced on an Illumina NovaSeq 6000. Raw sequencing data were processed with the Cell Ranger pipeline, and computational demultiplexing was performed to assign each cell to its original sample. Downstream analysis identified 10 CD4⁺ T-cell subtypes across ~99,000 high-quality cells. No significant differences in cell-type proportions were observed between case and control pairs at individual time points. However, cell type–specific gene expression changes and enriched pathways were detected in specific cell types in cases. These early transcriptional and regulatory alterations in CD4⁺ T cells provide a valuable resource for understanding disease initiation and developing predictive biomarkers for type 1 diabetes.