Development of self-reactive Pik3cd GOF B cells in the BM. (A) Four different types of BM chimeric mice were constructed by transplanting mixtures of CD45.1⁺ RAG.SW_HEL and CD45.2⁺ nontransgenic (non-tg) BM into recipient mice that did or did not express membrane HEL^3X. The CD45.1⁺ RAG.SW_HEL BM was either WT or Pik3cd^GOF, while the CD45.2⁺ BM was WT for both Rag1 and Pik3cd; thus, WT T and B cells with endogenous repertoires were derived from the CD45.2⁺ BM. In chimeras where SW_HEL cells are denoted as self-reactive, the recipients and all CD45.2⁺ blood cells expressed membrane HEL^3X, while control chimeras were identical except that they lacked the membrane HEL^3X gene (“non self-reactive”). BM was harvested from chimeric mice 8–15 wk after reconstitution. SW_HEL cells were identified as CD45.1⁺B220⁺ cells. (B) Plots show early BCR-negative cells stained with CD43 and CD24 to identify pre-pro-, pro-, and pre-B cells. Numbers are each population as a percentage of CD45.1⁺B220⁺ cells (mean ± SEM of all mice). (C) Plots show HEL-binding cells gated on CD24 and IgD to determine percentages of immature/transitional and mature B cell populations. Representative pseudocolor plots are shown for each condition. Numbers give each population as a percentage of CD45.1⁺B220⁺ cells (mean ± SEM of all mice). (D) Graphs show indicated populations as a percentage of total BM cells (the center line shows the median, box limits show the upper and lower quartiles, and whiskers show the minimum and maximum). All panels represent n = 17–21 mice per condition combined from nine experiments. Significant differences were determined using two-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.