As demonstrated by Nguyen et al., selection of the B cell repertoire is affected by the presence of IgM (46). lack (28). Together, these results demonstrate an important role for p18 in B-1a cell numbers, which in turn affects the production of autoantibodies and development of autoimmunity. However, the origin of B-1a cell expansion in B6.TC, B6.Slec1, and p18?/? mice could be due to an increase in proliferation of early-appearing fetal-derived B-1a cells or heightened production of later-appearing bone marrow-derived B-1a cells. As the repertoires of early- and later-appearing B-1a cells differ, these two possibilities can be distinguished. Herein, we investigated whether significant changes to the natural IgM repertoire occur in triple congenic B6.(B6.TC) lupus-prone mice. These mice carry the locus that drives B-1a cell expansion and present clinical autoimmune pathology that has been described for the NZM2410 pathology (29). B6.TC mice carry the NZM2410 susceptibility loci on a B6 genetic background ( 95%) that includes both heavy and light immunoglobulin chains, which allow to directly compare the lupus-prone B6.TC mice to the control B6 mice. Specifically, we found that the expansion of B-1a cells in B6.TC mice is associated with repertoire skewing toward VH11 and VH12 usage. Materials and Methods Mice B6.NZM-random insertion of Astragaloside A nucleotides at the VCD and DCJ junctions by the enzyme TdT. It is well-documented that peritoneal B-1a cells have limited N-addition due to the lack of TdT expression during fetal development (31). We analyzed N-addition at the DCJ and VCD junctions and determined CDR3 length. No significant differences were found when analyzing sequences with only unique CDR-H3 regions (Table ?(Table2).2). In contrast, analysis of all sequences, including the duplicates, demonstrated significant differences between B-1a cells from B6.TC and B6 mice. We found that the number of N-additions at the DCJ or VCD junctions of B6.TC Astragaloside A B-1a cells was significantly less than B6 B-1a cells ((B6.TC) lupus-prone mice demonstrated a large number of sequences that express identical CDR-H3 regions as compared to B-1a cells from healthy 8-week-old C57BL/6 (B6). This analysis demonstrates a significant increase in identical VH, DH, JH usage in B6.TC mice. Although it is not possible to determine whether the duplicate sequences observed herein result from a single clonal expansion or from analysis of Astragaloside A multiple cells with identical rearrangements, it has been well-documented over the years that B-1 cells have a limited repertoire (11, 14, 36C38), can undergo clonal expansion (39C42), and are self-replenishing (8). Therefore, these duplicate sequences are most likely due to expansion of single B-1a cells. Further analysis, including the duplicate sequences, reveals that the B6.TC B-1a cell repertoire displays early fetal/neonatal-like characteristics, which consists of an increase in use of JH1 [Figure ?[Figure4B;4B; Ref. (43)], few N-additions at both the VCD and DCJ junctions, and a shorter average CDR-H3 length (Table ?(Table2).2). In addition, the B6.TC repertoire overused VH11 and VH12 as compared to B6 (Figures ?(Figures11 and ?and2).2). Interestingly, VH11 and VH12 rearrangements are utilized almost exclusively by B-1a cells and target the cell membrane component PtC (19). Studies have shown VH11 in particular is a VH gene utilized during fetal development but not during adult development (44, 45). More recently, Yang et al. have shown overuse of VH11 in the normal healthy peritoneal B-1a cell pool (38). Our results demonstrate the most common CDR3 in peritoneal B-1a cells from our normal healthy 2-month old B6 mice is ARRDYGSSYWYFDV (VH1-55, DH1-1, JH1). Examining Yang et als most common CDR3 in peritoneal B-1a cells from their normal healthy 2-month old B6 mice, it is ARFYYYGSSYAMDY, (VH1-55, TF DH1-1, JH4), which does not share the exact same CDR3 as ours but does share the same VH and DH region. Our second most common CDR3 sequences (two are tied for second place) are identical to Yang et als first Astragaloside A and second most common CDR3 sequences ARFYYYGSSYAMDY and MRYGNYWYFDV (VH11-2, D2-8, JH1), respectively. The rank order of the sequences we identified is very similar to that of Yang et al. with only minor differences. Together, these results indicate that the B-1a cell repertoire in B6.TC mice.