Figure 2. The residual DKO B-1a cells exhibit an altered BCR repertoire.

(a,b) Peritoneal B-1a cells were sorted from four wild-type and four DKO mice, and the sorted cells of each mouse were individually analyzed by RNA-seq. (a) Volcano plot showing expression changes (log₂-transformed values; horizontal axis) between wild-type (WT) and DKO cells and adjusted P values (vertical axis) for V gene segments of the immunoglobulin heavy-chain (Igh) and κ light-chain (Igk) loci. (b) Pie charts showing the distribution of RNA-seq reads in V segments of the Igh and Igk genes for B-1a cells from two pairs of wild-type and DKO mice (left) and for B-1b cells from one wild-type and DKO mouse (right). The V genes are named according to the IMGT nomenclature. (c) Peritoneal cells from wild-type, Bhlhe41^–/– and DKO mice were stained with antibodies against CD19, B220, CD5, IgM, and V_H12 or with FITC-loaded PtC-containing liposomes. Expression of CD5 and the V_H12 BCR, as well as binding of liposomes is shown for CD19⁺B220^lo B-1 cells. (d) Frequencies and absolute numbers of V_H12⁺ B-1 cells (left) and liposome-binding B-1 cells (right). Horizontal bars indicate mean value, error bars represent SD. NS P > 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, as determined by the Student’s t-test. Five age-matched mice were used per genotype. One representative result of four (V_H12 staining) and two (liposome staining) independent experiments is shown.