Fig 1: Unsupervised clustering and dimension reduction analysis of B cells reveals similar frequencies of B cell subpopulations in AITD patients and controls(A) B cells were manually gated as CD45 + CD19 + CD3-CD14-and then analyzed using the unsupervised clustering and dimension reduction programs PhenoGraph and X-shift to generate distinct clusters based on B cell surface marker expression (PD-1, IgM, CD27, CD21, CD38, CXCR5, CD11c, CD86, and IgD). The UMAP plot was created using total B cells from concatenated adult AITD, adult healthy control, pediatric AITD, and pediatric healthy control samples with a minimum of 5,000 B cell events. Eight unique B cell subpopulations were identified. UMAP plots of each surface marker are displayed to demonstrate varying expression levels among the B cell subpopulations.(B) Heatmap of B cell surface marker expression for identification of the 8 B cell subpopulations identified using PhenoGraph/X-Shift. DN: double-negative (CD27-IgD-CD21-), DN2: double-negative CD11c+ (CD27-IgD-CD21-CD11c+).(C) Frequencies of B cell subpopulations in adult AITD (AA), adult healthy control (AH), pediatric AITD (PA), and pediatric healthy control (PH) subjects demonstrate AA patients have increased mature naive B cells and decreased class-switched B cells compared to AH. Data are represented as mean ± SEM. Samples were processed as depicted in Figure S1. See also Figure S7 for comparison of manually gated BND cells to algorithm determined BND cells. Statistical significance determined by Mann-Whitney non-parametric unpaired Student's t tests.