Germinal center exclusion of autoreactive B cells is defective in human systemic lupus erythematosus
J. Clin. Invest. Amedeo Cappione, et al. 115:3205 doi:10.1172/JCI24179 [Go to this article.]

Figure 6
9G4 B cells display attenuated calcium responses. (A) Calcium responses in healthy PBL B cells after anti-IgM stimulation. B cells were purified by negative selection and stained with CD27-, CD3-, CD14-, and CD16-PE antibodies (to gate out non-B cells and memory B cells) and 9G4 at the time of calcium measurements (gating shown on the left). The responses of individual cells are depicted in the middle dot plots, and the median responses in the graphs at right. (B) Calcium responses in tonsillar naive B cells after anti-IgD stimulation. Cells were stained with CD38 mAb and CD27 mAb to gate out memory and GC B cells, as well as 9G4 at the time of calcium measurements. The attenuated response of 9G4⁺ cells is not due to IgM downmodulation, since similar results were seen with anti-IgD. (C) 9G4 B cells normally mobilize calcium in response to ionomycin and CD20 cross-linking. Yet anti-CD20 failed to restore full signaling upon subsequent BCR stimulation. Of note, CD40 costimulation by overnight incubation with CD40 ligand–expressing fibroblasts (dotted line) increased the response through both CD20 and the BCR, yet it failed to fully restore BCR signaling to the same level seen for 9G4^– cells. (D) 9G4 naive B cells have attenuated signaling compared with another positively selected naive B cell population. 9G4 tonsillar B cells were purified by magnetic selection using 9G4-FITC. Control VH4 naive B cells were purified using LC1. Calcium responses in the naive gated population were examined as described above. (E) The percentage of cells responding to BCR stimulation was similar in 9G4⁺ cells as compared with naive control B cells, indicating that the differences shown in D were due to lower average response per cell. (F) Calcium signaling through the BCR is similarly attenuated in PBL naive 9G4 B cells from SLE patients.