B-1 cells express transgelin 2: unexpected lymphocyte expression of a smooth muscle protein identified by proteomic analysis of peritoneal B-1 cells.

B-1 cells constitute a unique B cell subset that differs phenotypically, biochemically, and functionally from the predominant population of conventional B-2 cells. Functional differences include constitutive secretion of natural immunoglobulin and failure of BCR signaling to initiate proliferation. The origin of these differences remains uncertain. We hypothesized that unbiased analysis of differences in protein expression between highly pure populations of B-1 and B-2 cells might provide information not readily available through other means. To pursue this, we undertook 2D gel analysis of B-1 and B-2 cells combined with mass spectrometry. We identified the smooth muscle protein, transgelin 2, in peritoneal (but not splenic) B-1 cells and did not find it in splenic B-2 cells; these results were confirmed by Western blot analysis, which showed a more than 60-fold difference in transgelin 2 expression between peritoneal B-1 and splenic B-2 cells. In contrast, levels of transgelin 2 RNA differed to a much lesser extent (3-fold) in the two B cell populations, so transgelin 2 is an example of a molecule whose subset-specific expression is more readily detected by proteomic than transcriptomic analyses. Finally, transgelin 2 protein expression was induced in splenic B-2 cells; thus, transgelin 2 joins a number of other inducible molecules that are constitutively expressed by peritoneal B-1 but not splenic B-2 cells. Although the role of transgelin 2 in B-1 cell function remains uncertain, identification of this molecule demonstrates the value of examining protein expression in this B cell subset.

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 V(H)11 and V(H)12, 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.

Disruption of cyclin D3 blocks proliferation of normal B-1a cells, but loss of cyclin D3 is compensated by cyclin D2 in cyclin D3-deficient mice.

Peritoneal B-1a cells differ from splenic B-2 cells in the molecular mechanisms that control G(0)-S progression. In contrast to B-2 cells, cyclin D2 is up-regulated in a rapid and transient manner in phorbol ester (PMA)-stimulated B-1a cells, whereas cyclin D3 does not accumulate until late G(1) phase. This nonoverlapping expression of cyclins D2 and D3 suggests distinct functions for these proteins in B-1a cells. To investigate the contribution of cyclin D3 in the proliferation of B-1a cells, we transduced p16(INK4a) peptidyl mimetics (TAT-p16) into B-1a cells before cyclin D3 induction to specifically block cyclin D3-cyclin-dependent kinase 4/6 assembly. TAT-p16 inhibited DNA synthesis in B-1a cells stimulated by PMA, CD40L, or LPS as well as endogenous pRb phosphorylation by cyclin D-cyclin-dependent kinase 4/6. Unexpectedly, however, cyclin D3-deficient B-1a cells proliferated in a manner similar to wild-type B-1a cells following PMA or LPS stimulation. This was due, at least in part, to the compensatory sustained accumulation of cyclin D2 throughout G(0)-S progression. Taken together, experiments in which cyclin D3 was inhibited in real time demonstrate the key role this cyclin plays in normal B-1a cell mitogenesis, whereas experiments with cyclin D3-deficient B-1a cells show that cyclin D2 can compensate for cyclin D3 loss in mutant mice.