scRNAseq reveals distinct B cell states within the lung. (A) B cells were analyzed using scRNAseq, and unsupervised clustering was achieved using a hierarchical tree algorithm using Seurat’s Louvain algorithm and visualized by UMAP. (B) Comparison of Ighm and Ighd transcripts across all B cells. (C) Ridge plots of Ighd transcripts between clusters 0 and 7. (D) Flow cytometry of lungs demonstrates two main B cell subtypes, T1B and T2B, using fluorescently conjugated mAbs to detect cell surface expression of CD45, CD19, CD21, and CD24. Data represent n = 3 independent experiments using three mice total. (E) Reclustering of B cells to map T1B and T2B cell states. (F and G) Relative RNA expression of representative genes associated with B cell states, including Crlf3, Fcer2a (T2), and Egr1 and Vpreb3 (T1). (H) Violin plots of RNA transcripts grouped by B cell states. For scRNAseq data in A–C and E–H, 4,044 B cells were from seven pooled mice, and sequencing was performed once.