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.

Ig Tg sHEL mice. (A) Serum anti-HEL auto-Ab levels in the indicated mice at 9 wk of age, with WT littermates assessed as negative controls (WT Ctl). Symbols represent ELISA ODs for individual mice shown on a log 10 scale. Horizontal bars represent means for each genotype. For EndoU / Ig Tg sHEL mice, high-responder littermates (HR) were independently compared statistically with Ig Tg and EndoU / Ig Tg littermates lacking the sHEL transgene. Likewise, augmented-responder (AR) littermates were independently compared with Ig Tg sHEL mice. Mean values significantly different between groups indicated by lines are represented by asterisks (*, P < 0.05; **, P ≤ 0.01, Student's t test). (B) PCR genotyping of EndoU / Ig Tg sHEL mice was performed using forward and reverse primers as described in the Materials and methods section, followed by 2.5% agarose gel analysis. The arrow in the top gel indicates the location of the band showing the presence of the Ig Tg transgene. (C) Serum anti-HEL auto-Ab levels from littermate mice were assessed at 5 wk and again at 12 wk of age. Triangles connected by lines represent individual high-responder EndoU /  Ig Tg sHEL mice. A littermate EndoU / Ig Tg mouse assessed at these ages (connected circles) and a WT control mouse assessed at 12 wk (diamond) are shown for comparison. (D) Serum anti-HEL auto-Ab responses were assessed as in A for representative augmented-responder EndoU / Ig Tg sHEL mice at 9 wk of age and again at 12 mo (#1), 8 mo (#2), 6 mo (#3), and 5 mo (#4). 9-wk-old high-responder EndoU / Ig Tg sHEL littermate mice (HR) are shown for comparison. (E) Total spleen B cell number, expression of the Ig Tg BCR (IgM a ) and CD5 on freshly isolated spleen B cells, and c-Myc expression and B cell blast development after BCR stimulation for Ig Tg , EndoU / Ig Tg , Ig Tg sHEL, high-responder EndoU / Ig Tg sHEL, and augmented-responder EndoU / Ig Tg sHEL mice. Means (±SEM) represent ≥3 mice assessed for each genotype in all assays. Assessment of B cell c-Myc expression and blast development was as described in Fig. 5 . For statistical analysis, Ig Tg sHEL mice were compared against littermate Ig Tg mice and high-responder EndoU / Ig Tg sHEL and augmented-responder EndoU / Ig Tg sHEL mice were compared against EndoU / Ig Tg littermate mice (*, P < 0.05; **, P ≤ 0.01, Student's t test). (F) Spleen cells from the indicated genotypes were stained with fluorescently labeled IgM a , CD5, HSA, and B220 Abs and analyzed by flow cytometry. Histogram overlays represent gating on B220 + cells. Results are representative of three mice of each genotype assessed in independent experiments producing similar results.