Cyclin D3 Is Required for Peritoneal B-1a Lymphocyte Proliferation.

Cyclins D2 and D3 are coordinately induced in response to mitogenic stimulation of splenic B-2 lymphocytes. It is recognized that the cyclin D2:cdk4:pRb pathway plays an essential role in B-2 cell proliferation. By contrast, little is known about the function of cyclin D3 in B cells. Peritoneal CD5+ B-1 cells represent a distinct subset of B lymphocytes and exhibit several unique phenotypic and functional characteristics, which distinguish them from B-2 cells. B-1 cells are associated with several human disease states characterized by B-cell expansion. Notably, B-1 cells represent the normal counterpart for a subset of human chronic lymphocytic leukemia (CLL), the cells of which express CD5. In mice, monoclonal expansions of CD5+ B-1 cells, that resemble CLL, develop as a function of age. These associations with malignant diseases impel the necessity to understand cell cycle control in B-1 cells. In contrast to splenic B-2 cells, stimulation of ex vivo B-1 cells results in an early and transient induction of cyclin D2; however, cyclin D2 complexes account for a relatively minor amount of the total pRb phosphorylation. Evidence for a second cyclin functioning in this capacity is supported by the finding that stimulated cdk4/6-mediated phosphorylation of pRb increases dramatically in mid-to-late G1-phase coincident with the induction of cyclin D3. Thus, in contrast to B-2 cells, the non-overlapping expression of cyclins D2 and D3 points to distinct roles for these D-type cyclins in B-1 cells. We hypothesize that the timing and duration of cyclin D3 induction suggest a unique role in mediating the G1/S transition in B-1 cells. To investigate this possibility, we transduced TAT-p16 peptidyl mimetics into ex vivo B-1 cells immediately prior to cyclin D3 induction to specifically target and inactivate cyclin D3-cdk complexes. We report here that transduction of TAT-p16 mimetics selectively disrupted cyclin D3-cdk4 complexes, whereas cdk2-containing complexes remained intact. Transduction of wild-type TAT-p16 mimetics, but not mutant TAT-p16 mimetics blocked S-phase entry in response to several B-cell mitogens. In addition, we found that transduction of TAT-p16 peptidyl mimetics inhibited pRb phosphorylation by D-type cyclin-cdk4/6 holoenzymes in B-1 cells stimulated with PMA, LPS or CD40L. These results provide the first evidence of a subset-specific functional role for cyclin D3 in B cells and suggest that cyclin D3, in its capacity as a regulator of cdk4/6-mediated pRb phosphorylation, is required for B-1 cell proliferation.