Splenic B-1 Cells Shows Diversified IgM Repertoire in Aged Atherosclerotic Mice.

Abstract B-1 cell derived IgM attenuates chronic inflammatory diseases like atherosclerosis. The spleen is a major lymphoid tissue where B-1 cells (both B-1a and B-1b) produce natural IgM. B-1 derived natural IgM has long been thought to contain restricted predominantly germline encoded V-D-J gene segments with low number of non-template encoded nucleotide (N)-additions, which are added at the junctions between V-D and D-J segments. However, differences in VDJ gene usage and N-addition frequency between peritoneal cavity (PerC) and spleen B-1 cells in aged atherosclerotic mice are unknown. B-1a and B-1b cells were single-cell sorted from PerC and spleen of 100-week-old, chow-fed ApoE−/− mice. Single cell sequencing of the immunoglobulin heavy chain variable region was performed. Splenic B-1 cells displayed increased diversity at the V-D and D-J junctions, as evidenced by increased average number of N-additions. Sequences containing >1 N-additions at both V-D and D-J junctions were 2% and 27% for PerC B-1a and B-1b sequences as compared to 44% and 44% of sequences in splenic B-1a and B-1b respectively. B-1 cells from PerC and spleen showed marked differences in VH, D and JH gene segment usage. Importantly, the most commonly expressed heavy chain complementarity determining region 3 (CDR-H3) amino acid sequence in spleen B-1a (AREVTTMYYFDY) and B-1b (AREDYYGSSYYFDY) cells was different from PerC B-1a (AGDYDGYWYFDV) and B-1b (AGDRDGYWYFDV) cells. Results provide clear evidence of a diversified IgM repertoire expressed by spleen B-1 cells compared to PerC B-1 cells during advanced atherosclerosis. The CDR-H3 sequences of splenic B-1 cells suggest underlying differences in antigen specificity which could further regulate disease progression.