Onset of Regulatory B Cells Occurs at Initial Stage of B Cell Dysfunction in Multiple Myeloma
Authors: Zou Zhongqing , Guo Tingting , Cui Jian , Zhang Li , Pan Ling 
Published: November 2019

Introduction :Multiple myeloma(MM) is caused by aggregation of clonal plasma cells, clinically presenting as evolvement in order from monoclonal gamma globulin disease (MGUS), smoldering MM (SMM), symptomatic MM to plasma cell leukemia. Regulatory B cells (Bregs), only a small immunosuppressive subgroup of B cells, have been recently identified in the setting of autoimmune diseases, immune thrombocytopenia (ITP) and gastric cancer . The role of Bregs in MM remains poorly defined. Here, the study was carried out on how Bregs correlate with evolution of MM, as well as how Bregs would be influenced by bortezomib, which is currently the first-line anti-MM agent.
Methods :All patients met the International Myeloma Working Group (IMWG) Criteria for the Diagnosis of MM. Mononuclear cells (MNCs) were isolated from bone marrow(BM)but not peripheral blood(PB)at specified time points. Cell numbers were quantified by hemocytometer. The ratios of Bregs and B cells were delected by flow cytometry (FCM). Propidium iodide was widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic. The results were expressed as the mean ± SD. Comparisons between 2 groups were performed with Student's t-test. Multiple groups (≥3) were analyzed by one-way ANOVA, and paired groups were analyzed by two-way ANOVA or Student t test. Data were graphed and analyzed using GraphPad Prism 6.0. P &lt; 0.05 was considered statistically significant.
Results: The study firstly found that Bregs' ratio increased at very beginning stage of MM and headed for extinction during progression of MM. It showed that Bregs' ratios were 11.7 ± 6.3%, 11 ± 8.6%, 15.8 ± 6.8%, and 4.9 ± 2.1% at stage of MGUS (n=5), SMM (n=4), newly diagnosed MM (NDMM) (n=9), and relapsed or refractory MM (RRMM) (n=6), respectively (p&lt;0.05). We then obtained mononuclear cells from NDMM samples. It revealed a positive correlation on both ratios and absolute numbers between Breg subgroup and B cell groups when B cells' ratio was higher than 5% of BMMNCs, whereas Bregs' ratio was rarely detected when B cells' ratio was lower than 5%. Subsequently, a retest was made to observe how Breg subgroup would be influenced by using bortezomib to target B-cell reservoir in MM. We added bortezomib of different concentrations to MNCs from NDMM samples within culture medium RPMI1640 (Ruikos Biotechnologies) for 24 hours (n=6). Bregs and B cells were totally killed when treated with high-dose bortezomib (n=3), while partially killed with low-dose bortezomib (n=3). To further explore how bortezomib could influence Bregs, another 6 samples were enrolled to demonstrate that the apoptotic absolute number of Bregs significantly increased after treatment of high-dose bortezomib (135615 ± 92085 v.s. 81132 ± 52908, P ≤ 0.05).
Conclusions : Bregs were strongly entwined with the whole group of preserved B cell in MM. Bregs began to increase at very beginning stage of MM when B cells were preserved, accompanying with transition from MGUS to diagnostic MM. Bregs would substantially decreased while B-cell reservoir diminished during MM progression or by B cell targeted bortezomib.

Disclosures
Zou: the National Natural Science Foundation of China: Research Funding. Guo:the National Natural Science Foundation of China: Research Funding. Cui:the National Natural Science Foundation of China: Research Funding. Zhang:the National Natural Science Foundation of China: Research Funding. Pan:the National Natural Science Foundation of China: Research Funding.