Think B lymphocytes just produce antibodies? Think again! Although, of course, B cells are vital for the humoral immune response, many studies in recent years have begun to uncover antibody-independent actions of B cells: regulating T cells and thus also playing a part in cellular immunity. For example, B cell depletion therapy, a new treatment for autoimmune disorders, has been found to influence T cells in addition to antibody titers. B cells can affect T cells in opposing ways – both enhancing and suppressing T cell immune responses – and accomplish this via costimulation, production of cytokines, and antigen presentation. In two recent publications, Griffin and Rothstein describe their discovery that CD11b (cluster of differentiation 11b) distinguishes ‘orchestrator B1 cells’, with characteristic T-cell interacting properties, from other human B1 cells; this may be of relevance in the study and treatment of autoimmune diseases.
WB analysis of CD11b in RAW 264.7 cell lysate
In the first paper, cells were immunofluorescently stained with labeled antibodies for various CD molecules, including fluorescently conjugated CD11b antibody, and analyzed by flow cytometry, revealing that human B1 cells (CD20+CD27+CD43+) can be divided into two separate groups based on the presence/absence of CD11b. Around 1 in 8-10 of the B1 cells were the CD11b+ cells expressing this epitope. In addition to CD11b, CD11b+ cells were also differentiated from other B cells by their staining for CD14 and CD11c. Probing B1 cells with labeled CD11b antibody enabled purification of the two types of B1 cells by cell sorting. Further to the cell surface markers, microarray analysis showed that the expression of numerous other genes demarcated CD11b+ cells from CD11b– cells and other B cell types.
Sort-purified CD11b+ and CD11b– B1 cells were then tested for stimulation of T cells and spontaneous secretion of IgM, known characteristics of B1 cells. Although both cell types secreted IgM and stimulated proliferation of T cells (measured by DNA replication of allogeneically mismatched CD4+ T cells) to a much greater extent than non-B1 cells, CD11b– B1 cells secreted significantly more IgM than CD11b+ cells, while CD11b+ cells were much more effective at driving T cell proliferation than the CD11b– B1 cells, demonstrating that these two cell types are each largely responsible for only one of these activities. CD11b+ cells were found to have high levels of the costimulatory molecule CD86, and neutralization of CD86 (with a specific antibody) impaired the T cell stimulation, indicating that up-regulated CD86 plays a key role in this T-cell interaction. Finally, the circulating B cells of lupus (a complex autoimmune disease) sufferers and normal controls were studied. In the lupus patients, several changes in the CD11b+ B1 cells were discovered: not only was the number of CD11b+ cells significantly larger (around four times greater), but those CD11b+ cells had elevated expression of CD86, and stimulated T cells much more strongly, compared to the CD11b+ B1 cells from normal controls, suggesting that CD11b+ may participate in lupus pathogenesis.
In the second of these publications, Griffin and Rothstein assessed human B cells for their ability to spontaneously produce and secrete the cytokine interleukin-10 (IL-10). Firstly, they immunofluorescently stained peripheral blood B cells with directly conjugated antibodies for multiple CD molecules and intracellular IL-10, and examined them by flow cytometry. Gating into different B cell types revealed that the proportion of cell staining positive IL-10 cells was high for CD11b+ B1 cells but very low for CD11b– B1, naive or memory B cells. The B cells were then sorted, cultured separately, and tested for IL-10 secreted spontaneously (i.e. without treatment with any reagents to induce this). Again, the CD11b+ B1 cells had much higher IL-10 levels than the other B cell types. The different B cell groups were cocultured with stimulated (by immobilized CD3 antibody) naive CD4+ T cells, and T-cell activation was evaluated (in terms of intracellular TNF-α staining). Only the CD11b+ group greatly inhibited the TNF-α expression by these T cells, and this was caused by the IL-10 (as demonstrated by the effect of an IL-10-neutralizing antibody).
Novus Biologicals offers CD11b reagents in the form of antibodies, peptides and proteins and RNAi kits for your research needs. We also stock antibody labeling kits for conjugating antibodies to a wide variety of labels.
Written by Carly Hammond
