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.

B cells after BCR ligation (Fig. 3, B and C) , the effect of reduced EndoU expression on CD22 /[B6] B cell AICD was assessed. EndoU-deficient mice were generated by appropriately targeting the EndoU gene on B6 Chr 15 (Fig. 4, A-C) . PCR-based SNP genotyping of genomic DNA from targeted mice confirmed that the targeted EndoU locus was B6 in origin. The chimeric mice were then bred onto a B6 genetic background to generate EndoU /[B6] mice that were homozygous B6:B6 throughout the 93.3-98.3 Mbp Chr 15 region (unpublished data). The absence of EndoU protein in homozygous gene-targeted mice was confirmed by Western blot analysis, which identified an appropriately sized protein in WT mice (Fig. 4 D) . EndoU from CD22 /[B6] B cells and thymocytes migrated more slowly on SDS-PAGE gels (50 kD band) than EndoU from CD22 /[inbr] thymocytes (48 kD band, Figs. 3 C and 4 E), due to a nonsynonymous SNP present within EndoU exon 2 which switches a single Glu (B6 mice) to Lys (129 mice). This was verified using A/J mice that share the EndoU SNP sequence with 129 mice, with EndoU from both CD22 /[inbr] and A/J mice migrating identically (Fig. 4 F) (Fig. 5 A) . AICD was also reversed in B cells from EndoU / CD22 /[B6] mice with normal BCR-induced blast development (Fig. 5 B) and proliferation (Fig. 5 C) . c-Myc expression was also normalized in BCRstimulated EndoU / CD22 /[B6] B cells relative to CD22 /[B6] B cells (Fig. 5, D and E) . EndoU may preferentially regulate and high CD5 and HSA expression, with low spleen B cell numbers (Fig. 6, E and F) . The frequency of IgM a-high B cells in augmented-responder mice (11% ± 2.1%) was significantly greater than the frequency of IgM a-high B cells present in Ig Tg sHEL mice (2.2 ± 0.5%, P < 0.01). Thus, the normalized phenotype of high-responder EndoU / Ig Tg sHEL mice and the IgM a-low CD5 high HSA high phenotype of augmentedresponder EndoU / Ig Tg sHEL B cells suggests differential sensitivities to auto-Ag exposure.