Search

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 Disclosures No relevant conflicts of interest to declare.

We have previously demonstrated that Th17 cells, which produce IL-17A, are significantly elevated in peripheral blood and bone marrow (BM) of patients with Multiple Myeloma (MM) and IL-17A promotes MM cell growth and survival, both in vitro and in vivo via IL-17A receptor. We have recently evaluated and observed that anti-IL-17A monoclonal antibody (mAb) significantly inhibited MM cell growth in vitro, while IL-17A induced proliferation of MM cells compared to control. We have also observed significant down-regulation of IL-6 production by anti-IL-17A mAb in MM-BMSC co-culture. Importantly, the administration of anti-IL-17A mAb weekly for 4 weeks in the SCIDhu model of human myeloma, where MM cells grow within the human microenvironment in mice led to a significant inhibition of tumor growth compared to the control mice. This remarkable activity of anti-IL17 mAb raised the question of whether the myeloma cells themselves are a possible source of IL-17. In this study, we used transcriptome sequencing (RNA-Seq) data to evaluate the expression of IL-17A in primary CD138+ myeloma cells (N=17) compared to normal plasma cells (NPC) (N=5). Whereas none of the NPCs expressed IL-17A, it was significantly over-expressed in majority of MM cells. In addition, these data also showed that the expression of other IL-17 family members (IL-7B, C, D, E & F) and Th17-associated pro-inflammatory cytokines (IL-21, IL-22 & IL-23) were not significantly elevated in primary myeloma cells compared to normal donor plasma cells. We further validated these observations by IL-17 immunoblot showing IL17 expression in all MM cell lines and 10 out of 14 primary patient MM cells; confirmed IL-17 expression in MM cells by quantitative RT-PCR, and flow cytometry and by immuno-histochemistry and confocal microscopy. We observed that IL-17 knock down by IL-17-specific siRNA inhibited MM cell growth as well as their ability to induce IL-6 production in co-cultures with BMSC. Finally, expression profile data from 172 uniformly treated patients showed that patients with lower IL-17A expression had superior overall survival compared to those with higher expression. These data confirms that MM cells express IL-17 and targeting it with a mAb will abrogate the autocrine loop making it an attractive therapeutic target. Disclosures: No relevant conflicts of interest to declare.

Abstract 3986 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 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 Disclosures: 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.