Defining Fundamental B Cell-Subset Dysfunction in Myeloma

One of the most prominent features of multiple myeloma (MM) has been immune deficiency which predisposes patients to infectious complications and suppresses development of anti-MM immune responses. We and others have previously described the T cell dysfunction in Th1, Treg and Th17 cells, plasmacytoid dendritic cells and myeloid-derived suppressor cells (MDSC). However, the most fundamental and long identified deficiency is in the humoral immune response. Suppression of uninvolved immunoglobulins (UIgs) have been well described (i.e. suppression of serum IgA and IgM in IgG myeloma); and antibody responses to vaccination have been inadequate. However, very limited information is available regarding B cell function and how UIgs are suppressed in myeloma. We have now evaluated six different B cell subsets (B1a, B1b, B2, Breg, IRA-B, and MZ) in peripheral blood (PBMC) and bone marrow (BM) to understand alterations in B cell immune function in MM. We have observed significantly lower ratio of B2 (normal B cell-subset) and B1a (natural antibody-producing cells) subsets (10±4 vs 57±17; p < 0.05) and B2 and Breg (regulatory B cell-subset) subsets (14±4 vs 45±13; p< 0.05) in PBMC from MM patients (N=19) compared with healthy donor (N=33) respectively. Similar results were observed in BM samples from MM patients (N=18) compared with healthy donors (N=12); B2/B1a subset (2.4±0.6 vs 8±1.3; p < 0.05) and B2/Breg subset (8±1.4 vs 43.7±8.4; p< 0.05) respectively. To understand whether MM cells directly or indirectly alter B cell-subsets, we incubated myeloma cells (N=4) with healthy donor PBMCs, and analyzed B cell subsets after 3 days. We observed significant elevation in B1 subset (2.5 fold of control) and reduced B2 subset (89±3% of control). When we incubated PBMCs with IL-17A over-expressing MM cells (N=3), we observed further significant reduction in B2 subset (74% of control). When normal PBMCs are cultured in IL-17A (N=4) we observed significantly increased IL-10-producing Breg subset (28% of control). Similarly, co-culture of healthy B cells with MDSC led to significant increase (3.8 times) in Breg cell- population (N=3) compared with control group. To study the impact of B cell dysfunction on T cell function in MM, we activated normal PBMC via anti-CD3 antibody, in the presence or absence of B cells, and measured intra-cellular IFN-γ levels in CD69+ cells. We observed that the absence of B cells significantly inhibited interferon-producing T cells compared to control (by 43%; p<0.05). Importantly, following removal of CD25+ cells (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 influence memory T cells rather than naïve T cells for the production of IFN-γ. To evaluate impact of lenalidomide on this interaction, we stimulated purified normal donor CD45RO memory T cells with Th1 polarizing cocktail in the presence or absence of purified normal B cells or B cells from MM patient (MM-B) in presence of lenalidomide and observed thatlenalidomide significantly improved MM-B cell-mediated IFN-γ-producing Th1 responses (by 32%, p<0.05) compared to normal B cell-mediated Th1 responses. In an effort to evaluate whether any therapy may improve the B cell function, we cultured normal PBMCs in the presence of lenalidomide (N=9) and observed reduction in Breg subset by 40% of control. To evaluate the effect of therapy on B cell-subsets in MM, we analyzed B cell subsets in PBMC from newly-diagnosed and lenalidomide-treated MM patients and observed that lenalidomide-treated group showed significant (p<0.05) improvement in B cell subsets (increased B2 and lower B1 cells) even before clinical response. These results suggest that immunomodulatory agents may be able to re-program humoral immunity in these patients. In summary, we report that the myeloma cell driven skewed B cell subset distribution with consequent B cell dysfunction drives the observed abnormalities in humoral/cell mediated immunity. The current therapeutic interventions, besides providing deep clinical responses, may also improve B cell function with impact on long term outcome.