For mechanistic studies, in-vitro human B-cell differentiation and generation of plasma cells are invaluable techniques. However, the heterogeneity of both T-cell-dependent (TD) and T-cell-independent (TI) stimuli and the disparity of culture conditions used in existing protocols make the interpretation of results challenging. The aim of the present study was to achieve the most optimal B-cell differentiation conditions using isolated CD19+ B cells and peripheral blood mononuclear cell (PBMC) cultures. We addressed multiple seeding densities, different durations of culturing, and various combinations of TD and TI stimuli including B-cell receptor (BCR) triggering. B-cell expansion, proliferation, and differentiation were analyzed after 6 and 9 days by measuring B-cell proliferation and expansion, plasmablast and plasma cell formation, and immunoglobulin (Ig) secretion. In addition, these conditions were extrapolated using cryopreserved cells and differentiation potential was compared.
This study demonstrates improved differentiation efficiency after 9 days of culturing for both B-cells and PBMC cultures using CD40L and IL-21 as TD stimuli and 6 days for CpG and IL-2 as TI stimuli. We arrived at optimized protocols requiring 2,500 and 25,000 B-cells per culture well for the TD and TI assays, respectively. The results of the PBMC cultures were highly comparable to the B-cell cultures, which allows dismissal of additional B-cell isolation steps prior to culturing. In these optimized TD conditions, the addition of anti-BCR showed a little effect on phenotypic B-cell differentiation; however, it interferes with Ig secretion measurements. The addition of IL-4 to the TD stimuli showed significantly lower Ig secretion. The addition of BAFF to optimized TI conditions showed enhanced B-cell differentiation and Ig secretion in B-cell but not in PBMC cultures. With this approach, efficient B-cell differentiation and Ig secretion were accomplished when starting from fresh or cryopreserved samples.
Our methodology demonstrates optimized TD and TI stimulation protocols for more in-depth analysis of B-cell differentiation in primary human B-cell and PBMC cultures while requiring low amounts of B cells, making them ideally suited for future clinical and research studies on B-cell differentiation of patient samples from different cohorts of B-cell-mediated diseases.
Copyright © 2022 Marsman, Verhoeven, Koers, Rispens, ten Brinke, van Ham and Kuijpers.

Angiogenic activity in solid tumors has been demonstrated to promote metastasis through the activation of certain proteins involved in the epithelial-mesenchymal transition-associated process. The molecular mechanism underlying multiple myeloma-induced angiogenesis involves angiogenic cytokines by plasma cells as well as their induction within the microenvironment. Integrin-linked kinase (ILK) is a highly evolutionarily conserved intracellular protein that was originally identified as an integrin-interacting protein, and extensive genetic and biochemical studies have identified ILK expression to be vital during tumor-driven angiogenesis. In the present study, it was identified that angiogenic factors were upregulated in mesenchymal stem cells (MSCs) that were co-cultured with multiple myeloma cell lines. It was also revealed that upregulated ILK expression significantly promoted the capillary-formation ability of MSCs. The concentrations of angiogenic factors were significantly decreased compared with non-targeting siRNA-transfected and control MSCs. MSCs may participate in inducing the angiogenic response in multiple myeloma depending on ILK expression.

Myeloma facilitates destruction of bone and marrow. Since physical activity encourages musculoskeletal preservation we evaluated whether low-intensity vibration (LIV), a means to deliver mechanical signals, could protect bone and marrow during myeloma progression. Immunocompromised-mice (n=25) were injected with human-myeloma cells, while 8 (AC) were saline-injected. Myeloma-injected mice (LIV; n=13) were subjected to daily-mechanical loading (15min/d; 0.3g @ 90Hz) while 12 (MM) were sham-handled. At 8w, femurs had 86% less trabecular bone volume fraction (BV/TV) in MM than in AC, yet only a 21% decrease in LIV was observed in comparison to AC, reflecting a 76% increase versus MM. Cortical BV was 21% and 15% lower in MM and LIV, respectively, than in AC; LIV showing 30% improvement over MM. Similar outcomes were observed in the axial skeleton, showing a 35% loss in MM with a 27% improved retention of bone in the L5 of LIV-treated mice as compared to MM. Transcortical-perforations in the femur from myeloma-induced osteolysis were 9× higher in MM versus AC, reduced by 57% in LIV. Serum-TRACP5b, 61% greater in MM versus AC, rose by 33% in LIV compared to AC, a 45% reduction in activity when compared to MM. Histomorphometric analyses of femoral trabecular bone demonstrated a 70% elevation in eroded surfaces of MM versus AC, while measures in LIV were 58% below those in MM. 72% of marrow in the femur of MM mice contained tumor, contrasted by a 31% lower burden in LIV. MM mice (42%) presented advanced-stage necrosis of tibial marrow while present in just 8% of LIV. Myeloma infiltration inversely correlated to measures of bone quality, while LIV slowed the systemic, myeloma-associated decline in bone quality and inhibited tumor progression through the hindlimbs.
Copyright © 2016 Elsevier Inc. All rights reserved.

The aim of the present study was to examine the frequencies of different subsets of B and follicular helper T (Tfh) lymphocytes in patients with immunoglobulin A nephropathy (IgAN), and investigate the potential underlying mechanism. A total of 27 patients with IgAN and 10 healthy controls (HC) were recruited for analysis of the frequencies of different subsets of B and Tfh cells. ELISA was used to analyze the concentration of serum interleukin (IL)‑21. The transcriptional levels of activation‑induced cytidine deaminase (AID) in the B cells were determined using reverse transcription‑quantitative polymerase chain reaction, while the translational levels of AID were analyzed using western blotting. The frequencies of circulating memory and activated B cells and Tfh cells were found to be significantly increased in the IgAN groups, compared with those of the HC group, although the number of plasma cells were not significantly different between the two IgAN groups. In addition, the serum levels of IL‑21 were found to be higher in the patients with IgAN, and correlated with 24‑h proteinuria. IL‑21 also enhanced the expression levels of AID in the B cells. The data of the present study revealed that the high levels of memory and activated B cells and Tfh cells were positively associated with the progression of IgAN, and that this may be mediated by the overexpression of AID, which is potentially regulated by IL‑21.