CD5+B-1 cell responses to infection are facilitated by toll-like receptor stimulation

Abstract B-1 lymphocytes are neonatally-derived, self-reactive, innate-like B cells that produce natural IgM at steady state. B-1 cells also actively respond to infections with induced local IgM production. These two distinct functions of B-1 cells have been linked to the expression CD5, an inhibitor of BCR signaling, with CD5+ cells thought to contribute to steady state IgM, and CD5− cells to local IgM production during infection. The mechanisms underlying such “division of labor” are poorly understood. We aim to test our hypothesis that CD5+ B-1 cells are stimulated by innate signals via TLR, during infection, resulting in their activation and differentiation into CD5− IgM antibody-secreting cells. For that we first cultured CD5+ B-1 cells with TLR9 agonist, ODN-CpG. The cells proliferated, secreted IgM and showed increased Nur77 expression, Akt phosphorylation and interaction of Syk with CD79a, signs of BCR signaling. They lost expression of the BCR-inhibitor CD5 and upregulated the plasma cell marker CD138+. Following influenza infection of mice, CD5+ B-1 cells migrated to the mediastinal lymph nodes (medLN) where they lost CD5 and became IgM secreting cells. Both, global TLR−/− mice and chimeric mice lacking TLR only on B-1 cells had reduced numbers of B-1 derived plasma cells and antibody secreting cells in the medLN compared to controls. Our findings show that TLR-mediated activation of CD5+ B-1 cells results in the rapid reorganization of the BCR-complex, followed by loss of CD5 and that TLR stimulation of CD5+ B-1 is required for differentiation of B-1 cells to infection in vivo. Overall our data suggest that CD5+ and CD5− B-1 cells are not distinct subsets, but rather that CD5 expression indicates the activation state of B-1 cells.