B cell-malignancies exhibit considerable immune dysfunction particularly in multiple myeloma (MM). We have previously demonstrated that in T cell-compartment, regulatory T helper cells are dysfunctional in multiple myeloma (MM) while Th17 cells are significantly elevated and IL-17 produced by them is associated with MM cell growth and survival as well as suppressed immune responses and bone disease. We have here investigated the B cell-subsets and their ability to re-program anti-tumor immunity in MM. We have first characterised four different B cell-subsets (B1a, B1b, B2 and regulatory B cells) using 10-color flow cytometric analysis in both peripheral blood and bone-marrow (BM) samples from MM patients compared with normal healthy donors. We observe that CD5+ B1a-B cells are significantly elevated in peripheral blood of MM patients (N=7) compared to healthy donor (N=15) (42±8% vs 13±3%, respectively, p<0.05); while normal B cells (B2 cells) are significantly reduced in peripheral blood (29.8±6.5, p<0.05) and in the BM samples (11±4.8, N=4, p<0.05) of MM patients compared to healthy donors (59±3, and 60.2±2, N=10, respectively). We also observed that both B1b (47.9±18 vs. 22.8±4) and regulatory B cells (7.1±4.5 vs. 1.54±0.3) are elevated in BM samples of MM compared to healthy donors, however there were no differences in B1b and regulatory B cells in the peripheral blood of MM compared to healthy donor samples. Interestingly, in myeloma we observe higher levels of activated B cell subsets but lower levels of memory B cell subsets compared to healthy donors. These results, particularly very low levels of normal B cells in MM patients, may explain the decreased levels of uninvolved immunoglobulin in MM.
As removal of B cell population has been shown to re-program T helper cell populations, we next investigated impact of B cell population on T cell activation. We activated normal PBMC via the anti-CD3 antibody, in the presence or absence of B or CD25+ cells and measured intra-cellular IFN-γ levels in CD69+ cells. We found that the absence of B cells significantly inhibited interferon-producing T cells compared to PBMC (by 43%; p<0.05). Importantly, following removal of CD25+ cells, which consists of both Tregs and activated memory T cells, with or without B cells, we did not observe any difference in the inhibition of IFN-γ, indicating that B cells are influencing memory T cells rather than naïve T cells for the production of IFN-γ. This prompted us to identify the phenotypic signature of regulatory T cell populations when purified memory T cells are polarized with the regulatory T cell cocktail in presence or absence of B cells. We observed that B cells reduce FOXP3 expression by 18 %(N=5) and establish cognitive interactions with T cells. This occurred by increasing the expression of GITR (154%) and CTLA4 (54%); while reducing PD1 (−24%) and OX40 (−21%) expression on T cells without affecting HLA expression. We have also observed these improvements by B cell modulation on T cells in MM. Our results indicate that targeting these re-programmable capabilities of B cells to modulate T helper cell populations may enable us to improve T cell function in MM; and may improve immune function in MM and also allow us to enhance responses to vaccinations.
Ghobrial:Millennium: Advisory Board Other; Novartis: Advisory Board, Advisory Board Other. Richardson:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees. Treon:Onyx: Research Funding; Celgene: Research Funding; Pharmacyclics: Research Funding; Cephalon: Consultancy; Avila: Consultancy. Anderson:Celgene, Millennium, BMS, Onyx: Membership on an entity's Board of Directors or advisory committees; Acetylon, Oncopep: Scientific Founder, Scientific Founder Other.
Asterisk with author names denotes non-ASH members.