Tnfaip3 curtails the effects of MYD88^L265P on B cells. (A) EGFP⁺ B cells expressing the indicated vectors were sorted from three independent cultures on day 1, and mRNA from each cell was converted to cDNA and analyzed on microarrays. Shown are the mean and SD arbitrary units of Tnfaip3 mRNA and a constitutively expressed B cell transcript, Cd79a, from each sample. *, P < 0.05. (B) WT or Tnfaip3^lsv homozygous mutant polyclonal B cells were transduced with the indicated vectors and cultured in triplicates in the absence of mitogenic stimuli for 5 d. The number of EGFP⁺ transduced B cells and EGFP⁻ nontransduced control B cells in each culture was compared with the starting number on day 0 (mean and SD). Data are representative of three independent experiments. (C) Western blot analysis of EGFP⁺ WT or Tnfaip3^lsv polyclonal B cells expressing the indicated vectors, sorted on day 1 of culture without mitogenic stimuli, measuring phosphorylated p65 NF-κB. Tubulin was used as a loading control. (D) Relative expression of CD23, CD25, CD95, and CD86 on EGFP⁺ WT or Tnfaip3^lsv polyclonal B cells expressing MYD88^L265P or empty vector compared with nontransduced EGFP⁻ B cells in the same culture. Mean and SD for triplicate cultures per group. Statistical analysis by Student’s t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data are representative of two independent experiments. (E) WT or Tnfaip3^lsv homozygous HEL-specific B cells were activated by HEL in vivo and anti-CD40 in vitro, transduced with indicated vectors, and cultured in triplicates in the absence of mitogenic stimuli for 7 d, and the number of EGFP⁺ transduced B cells and EGFP⁻ nontransduced B cells in each culture compared with starting number on day 0 (mean and SD). Data are representative of two independent experiments. (F) Division of HEL-specific B cells measured by CTV dilution on days 1, 3, and 5 of triplicate cultures in the absence of antigen or anti-CD40 stimuli. Cells were gated on WT or Tnfaip3^lsv transduced with MYD88^L265P or nondividing B cells transduced with empty:EGFP vector (gray histogram) showing the median and SD CTV intensity. Data are representative of two independent experiments.