CD5+/Mac-1− peritoneal B cells: A novel B cell subset that exhibits characteristics of B-1 cells

The peritoneal cavity of mice is enriched for B-1 B cells, a lymphocyte subset that differs from conventional B-2 cells phenotypically, functionally, and developmentally. According to current paradigms, all peritoneal B-1 cells express Mac-1 whereas B-2 cells do not and thus these populations are often purified by FACS sorting or magnetic bead isolation based on B cell expression of Mac-1 or lack thereof. However, in the course of studying B220+/Mac-1− peritoneal B-2 cells, we discovered that this population is actually heterogeneous, with approximately 30–40% of these B220+/Mac-1− cells expressing the B-1 cell marker CD5. It was unclear whether this B220+/CD5+/Mac-1− peritoneal B cell population represented aberrantly CD5 expressing B-2 cells or Mac-1− B-1 cells. To address this issue we tested CD5+/Mac-1− peritoneal B cells for several traits that distinguish B-1 and B-2 cells. We found that CD5+/Mac-1− peritoneal B cells resembled CD5+ B-1 cells and not B-2 cells in terms of expression of several additional surface markers (IgM, IgD, CD23, CD43, and CD80). Further, CD5+/Mac-1− peritoneal B cells expressed high levels of VH11 and VH12, two Ig variable genes that are expressed mainly by B-1 but not B-2 cells. In addition, CD5+/Mac-1− peritoneal B cells responded to PMA, a mitogen that stimulates B-1 cells but not B-2 cells, and not to anti-Ig, that stimulates B-2 cells but not B-1 cells. ELISPOT analyses of freshly isolated CD5+/Mac-1− peritoneal B cells revealed that they secreted IgM constitutively, like B-1 cells and unlike B-2 cells. These results indicate that CD5+/Mac-1− peritoneal B cells are a new subset of B-1 cells, here termed B-1c, and stress the importance of using multiple surface markers to identify and purify specific B cell populations.