Fig. 4: Active RANK signaling drives a plasma cell differentiation program via Blimp1 expression.

a Percentages of CD138⁺B220^low plasma cells of viable cells from SP (left) and BM (right) of six-week-old TC-RK (n = 5) and control mice (n = 4–5 per genotype). Pooled data from three different experiments. b Experimental set up of in vitro differentiation of naive B-cells using low concentrations of LPS (LPS^low, 100 ng/mL). B-cell stage and immunoglobulins in supernatants were analyzed by flow cytometry four days after stimulation. Graphic was created using BioRender.com. c Percentages of CD138⁺B220^low plasma cells after in vitro differentiation of naive B-cells derived from animals with indicated genotype (n = 4–5 per genotype) with LPS^low were determined by flow cytometry after four days. Biological replicates were pooled from two individual experiments. d Quantification of immunoglobulin isotypes in supernatants from in vitro differentiated naive B-cells, derived from animals with indicated genotype (n = 3–5 per genotype) and stimulated for four days with LPS^low, by flow cytometry-based multiplex immunoassay. Pooled data from two independent experiments. e Experimental set up of in vitro differentiation of naive B-cells derived from TC-RK mice using recombinant RANKL in presence or absence of LPS^low for four days. f Total TC-RK-derived B-cell count (n = 4) per well after in vitro stimulation with RANKL and LPS^low as indicated. Biological replicates were pooled from two individual experiments. g TC-RK-derived CD138⁺B220^low plasma cell count (n = 4) per well after in vitro differentiation using RANKL and LPS^low as indicated. Biological replicates were pooled from two individual experiments. h Geometric mean (geo. mean) of BLIMP1 from living cells after in vitro differentiation of TC-RK-derived B-cells (n = 4) using RANKL and LPS^low as indicated. Biological replicates were pooled from two individual experiments. i Macroscopic appearance of representative spleens (left) and dot plot graph (right) depicts SP weight in gram (g) of 20-week-old TC-RK (n = 10) and aged-matched control mice (n = 7–9 per genotype). j Percentages of CD138⁺B220^low plasma cells of viable cells from SP (left) and BM (right) from 20-week-old TC-RK (n = 6–7) and aged-matched control mice (n = 6–10 per genotype). Pooled data from more than three experiments. Statistical analysis was performed using Student’s t-test and one-way ANOVA with Tukey correction for multiple comparison. P values are indicated in respective graphs. All data are presented as mean ± standard deviation.