Perturbation in B Cell-Subsets Can Explain the Observed Suppression of Uninvolved Immunoglobulins and Their Impact on T Tell-Subsets As Well As Patients Outcome in Myeloma

Multiple myeloma (MM), a plasma cell malignancy, is associated with immune dysfunction influenced by both direct and an indirect interaction between MM cells and its bone-marrow (BM) microenvironment. The most prominent immune deficiency lies in the humoral immune response, which manifests as low serum uninvolved immunoglobulin (UIg) levels, lack of responses to vaccination, and consequent infectious complications. The cellular and molecular processes driving suppressed normal B cell immunity remains unclear. Therefore, we investigated the hypothesis that observed low UIg levels and immune dysfunction are due to alterations in the developmental stages of the B cell-compartment with associated T and B cell subset alterations in MM BM (N=46) and peripheral blood mononuclear cells (PBMC, N=50) along with normal BM (N=17) and PBMC (N=33). We observed that B2 cells were significantly lower (42.7±4.3, p<0.05) and B1a cells were significantly higher (20.1±3.1, p<0.05) in myeloma BM compared with normal BM B2 cells (57±3.7) and B1a cells (10.5±2.5). Utilizing 12-color flow cytometry we integrated B cell-developmental stages in BM samples from normal donor) smoldering (SMM) (n=4), newly-diagnosed (NDMM) (n=10) and relapsed MM patients (RRMM) (n=6). We observed an altered B cell-development in BM with significantly lower proportion of pro-B cells (CD38⁺, CD34⁺, CD10⁺, CD19⁺ and IgM^-) with renewal capacity in SMM (12±3.1; p<0.05), NDMM (19.2±4.9;) and RRMM (3.1±1.6; p<0.05) compared to normal BM (25.1±3; N=3) suggesting that aberrations observed in B cell development may contribute to suppressed UIG in MM. Similarly, we observed that B2 cells were significantly lower (45.2±5.8, p<0.05) and B1a cells were significantly higher (30.3±5, p<0.05) in myeloma PBMC compared with normal PBMC B2 cells (69.6±2.9) and B1a cells (8.4±2). We next analyzed the impact of uniform treatment and response on B cell subsets in patients enrolled on IFM-DFCI 10-106 study. B cell subsets in PBMCs were evaluated at diagnosis and then following achieving complete response (CR) in 14 patients and those not achieving CR in 18 patients. We observed a significantly higher numbers of B2 cells at diagnosis (63.6±4.6l vs. 42.1±3.9; p<0.05), and significantly lower numbers of Bregs (19.8±2.6, vs. 30.4±2.6; p<0.05), IRA-B cells within B1a cells (8±2.2 vs. 32.8±6; p<0.05) and MZ cells within B2 cells (10.8±2.7 vs. 29.6±5; p<0.05) in patients achieving CR versus those not achieving CR respectively. The absolute B2 cell-numbers were also significantly higher (p<0.05) in patients achieving CR (123,872±29,226 cells/ml) versus those not achieving CR (33,174±17352 cells/ml). Importantly, the UIg levels were significantly higher (p<0.05) at diagnosis, in patients with higher B2 cells (682±108 mg/dL) compared with those with lower B2 cells (392±24 mg/d). Moreover, patients with higher B2 cells had longer EFS (115±28 weeks) compared to the patients with lower B2 cell numbers (82±15 weeks). In the T cell-compartment, amongst CD4 cells, Th2 cells were significantly higher (73±6.1) (p<0.05) in patients achieving CR compared with those not achieving CR (47.7±5.9); and amongst CD8 cells, PD-1 expressing CD8⁺ cells were significantly higher (63.2±8) (p<0.05) at diagnosis in patients achieving CR compared with those not achieving CR (39.5±6.1). We also observed that PD-1 expressing CD8⁺ cell numbers were significantly higher (p<0.05) in MRD-negative patients (68.4±11.3) compared with MRD-positive patients with (56.8 ± 8.5) or without achieving CR (35.5± 6.8). These results highlight possible B cell impact on immune T cell response in myeloma. Interestingly, our in vitro studies confirmed the above results when we activated normal PBMC via anti-CD3 antibody in the presence or absence of B cells, we observed that the absence of B cells significantly inhibited interferon-producing T cells compared to control (by 43%; p<0.05). In conclusion, we report a cellular explanation for observed suppression of B cell immunity in myeloma, specifically, suppression of uninvolved immunoglobulins. Here, for the first time we report significance of the B cell-subset in MM with possible impact also on T cell subsets as well as patients outcome. The observed relationship between B cell subset changes and ability to achieve complete response is being investigated in a large cohort of patients as potential bio marker of response and survival.