B cells expressing autoreactive BCRs are increased in PIK3CD GOF patients. (A and B) B cells from healthy controls or PIK3CD GOF patients were stained with the anti-idiotypic antibody 9G4, which binds the VH4-34 heavy chain. The general increase in 9G4 staining on B cells likely represents binding of serum VH4-34 antibodies to B cells, while the very brightly staining population corresponds to B cells endogenously expressing surface VH4-34 antibodies. (A) Representative staining on total B cells is shown. (B) Percentage of VH4-34^hi cells in each B cell population, as determined by flow cytometry. Each point represents an individual healthy control (HC; n = 31) or patient (n = 18–19). (C) mRNA was prepared from sorted transitional or naive B cell populations, and IGHM cDNA was amplified, deep-sequenced, and analyzed to calculate the percentage of unique clones that used the IGHV4-34 element. Each point represents an individual healthy control (n = 8–9) or patient (n = 6); line shows mean. Significant differences were determined by Mann-Whitney (B) or ANOVA Tukey (C) test. ****, P < 0.0001; **, P < 0.01. See also Fig. S2.