EndoU is a novel regulator of AICD during peripheral B cell selection

Poe, Jonathan C.; Kountikov, Evgueni I.; Lykken, Jacquelyn M.; Natarajan, Abirami; Marchuk, Douglas A.; Tedder, Thomas F.

In these studies, a forward genetic screen was used to identify an evolutionarily conserved single-stranded RNA (ssRNA) binding protein, EndoU, as a novel regulator of AICD in CD22 /[B6] mice. EndoU was also overexpressed by anergic peripheral B cells from double-transgenic mice expressing BCRs specific for hen egg lysozyme (HEL) along with soluble HEL (sHEL) as the cognate auto-Ag (Ig Tg sHEL mice; Goodnow et al., 1989; Hippen et al., 2000; Shlomchik, 2008) . EndoU deficiency in Ig Tg sHEL mice also reversed AICD ex vivo and led to augmented anti-HEL auto-Ab responses in vivo. Thus, EndoU defines a new posttranscriptional regulatory pathway that controls B cell AICD, particularly in response to auto-Ag. [inbr] , CD22 / [inbr] ) mice were cultured in medium alone or containing F(ab') 2 anti-IgM Abs. After 48 h, the cells were analyzed for viability (7-amino-actinomycin d [7AAD] exclusion) and cell size (forward scatter [FSC] ) by flow cytometry. Percentages indicate viable B cell blasts (7AAD  FSC high ) within the gate shown. Results are representative of >10 mice of each genotype producing similar results. (B) The proliferation of purified spleen B cells from CD22 / [B6] or CD22 /[inbr] mice, or their WT littermates, was measured by [ 3 H]-thymidine incorporation. Values represent mean (±SEM) cpm for triplicate wells from one of three independent experiments producing similar results. **, P < 0.01, Student's t test. (C) Representative CD5 immunofluorescence staining of freshly isolated B220 + splenocytes from ≥5 mice/group of the indicated genotypes. Background staining using an isotype-matched control mAb is shown (Neg Ctl). (D) Spleen B cells from WT [B6] or CD22 /[B6] mice were cultured with F(ab') 2 anti-IgM Abs for 18 h. [ 32 P]-radiolabeled cDNA was generated from total RNA, followed by hybridization to GEArray signaling pathway gene arrays. Regions of the arrays containing the c-Myc gene (arrowheads) and other representative genes expressed in B cells, including the c-Myc binding partner Max, are shown. (E) Mean (±SEM) frequencies of B cell blasts in untreated or F(ab') 2 anti-IgM Ab-stimulated cultures (48 h) from four each Ig Tg and Ig Tg sHEL mice assessed in independent experiments. **, P < 0.01, Student's t test. (F) Representative CD5 expression by spleen B cells from Ig Tg sHEL mice and Ig Tg mice as assessed by flow cytometry. A nonreactive isotype-matched Ab was used as a negative control (Neg Ctl). (G) Purified spleen B cells from Ig Tg or Ig Tg sHEL mice were cultured alone or with F(ab') 2 anti-IgM Abs. After 18 h, the cells were analyzed for c-Myc expression by intracellular staining, with flow cytometry analysis. Percentages of c-Myc + B cells (those above the dashed line) are indicated in each plot. Bar graphs represent the mean (±SEM) percentage of c-Myc + B cells in BCR-stimulated cultures compared with unstimulated cultures from three mice of each genotype assessed in independent experiments. **, P < 0.01, Student's t test.