Fig. 5: Cell-autonomous defect in B cell function of mature CBL UbLOF B cells.
a, Ig production by sorted primary B cell subsets from HDs and individuals with CBL-LOH from cryopreserved PBMCs after the indicated stimulations. Supernatants were collected after 5–7 days, and Ig levels were measured by enzyme-linked immunosorbent assay (ELISA). The line shows the mean of the displayed data points (one per individual); HDs (n = 13), participants with CBL-LOH (n = 5). The statistical significance of differences was assessed using multiple two-sided unpaired t-tests, with correction for multiple testing; *P < 0.05, **P < 0.005 and ***P < 0.0005. b,c, Ig production by plasma cells sorted from cryopreserved BM mononuclear cells of participants P1 and P2 with CBL-LOH and HDs (n = 2; b) and IgM production of WT, CBL-KO and rescue BJAB cell lines within 24 h of culture (c); data are shown as mean ± s.d. of n = 5 independent biological replicates. d, Western blot of WT and CBLY371C KI BJAB cells following BCR stimulation with monoclonal anti-IgM for the indicated time periods. Data are representative of three biological replicates. e, Primary monocytes from HDs (n = 15) and participants with CBL-LOH (n = 6) were sorted from fresh blood samples. After 24 h of nonstimulated culture, supernatants were collected, and B cells from HDs were stimulated with the supernatants for 24 h. Ig production was assessed by ELISA. Data are shown as mean ± s.d. Statistical significance was assessed using multiple two-sided Mann–Whitney tests adjusted for multiple testing; **P < 0.005 and ***P < 0.0005. f, Frequency of IgA+ and IgG+ B cells among memory B cells in patients (n = 3) and HDs (n = 13), as determined by flow cytometry. Statistical significance was assessed by unpaired, two-sided t-tests; **P < 0.005 and ***P < 0.0005. g, Frequency of spike+ B cells in patients (n = 4) and HDs (n = 10), as determined by flow cytometry with tetramer staining. Data are shown as mean ± s.d. Statistical significance was assessed by unpaired, two-sided t-tests; *P < 0.05 and **P < 0.005. h, Frequency of IgG+, IgA+ and IgG−IgA− B cells among spike+ B cells in patients (n = 4) and HDs (n = 6). Error bars indicate s.e.m. Statistical significance was assessed by unpaired, two-sided t-tests; *P < 0.05; EV, empty vector; Vinc, vinculin; wk, weeks; mo, months; yrs, years.
