Unc93b1^3d/3d mutation paradoxically increases accumulation of CD19^lowMYD88^L265P B cells in vivo. (A) Anti-IgM plus anti-CD40 activated Unc93b1^wt/wt or Unc93b1^3d/3d B cells were transduced with the indicated vectors that also encoded EGFP, washed, and cultured in triplicate without mitogen (day 0) for 3 days. Mean and standard deviation number of EGFP⁺ (left) and EGFP⁻ (right) cells were compared with the starting number on day 0 of the culture. Data are representative of 3 independent experiments. (B) Cell division measured by cell trace violet (CTV) dilution on days 1 and 3 of culture without antigen or CD40 stimulation, gated on EGFP⁺ cells expressing the indicated vectors. (C) Flow cytometric analysis of the spleens of Rag1^−/− recipient mice 11 days after transplantation of transduced B cells. Plots show concatenated data from 3 recipients per treatment: B220 vs IgM plots show the mean percentage of live spleen lymphocytes falling within the indicated IgM⁺ B-cell gate; CD19 vs EGFP plots are gated on the IgM⁺ B cells and show the percentage of EGFP-expressing cells with either high or low levels of CD19 expression. (D) Total number of live EGFP⁺ cells in the spleen of each recipient mouse. (E) Percentage of CD19^low cells among live EGFP⁺ cells in the spleen of each recipient mouse. Total number of live CD19^high EGFP⁺ (F) and CD19^low EGFP⁺ (G) cells in the spleen of each recipient mouse. Data are representative of 3 independent experiments. Statistical analysis by unpaired Student t-test. ***P < .001.