Figure 6: Themis2 sets the calcium, PLC-γ2 and Erk response to low-avidity, but not high-avidity, antigen.
From: Themis2 lowers the threshold for B cell activation during positive selection
(a) Median calcium flux in mixed naive CD45.2 Themis2−/− and CD45.1 wild-type IgHEL splenic B cells in response to stimulation with sHEL or anti-IgM F(ab')2. Data are representative of four experiments. (b) Peak median height and time to peak height of calcium flux in response to varying concentrations of sHEL. Symbols represent individual samples. (c) Mean phospho-specific antibody binding to intracellular signaling molecules downstream of the BCR, 5 min after stimulation of mixed MACS sorted IgHEL CD45.2 Themis2−/− (open circles) and CD45.1 wild-type (closed circles) B cells with sHEL. (d) Time course of activation of p-Erk and pCD19 in CD45.2 Themis2−/− and CD45.1 wild-type IgHEL B cells following stimulation with 100 ng/ml sHEL. Results derived from triplicate samples and bars indicate 95% confidence limits with unpaired t tests. *P < 0.05, **P < 0.01 and ***P < 0.001. Graphs are representative of at least three separate experiments in each case. (e) Mean SOS fluorescence intensity in confocal images from mixed wild-type and Themis2−/− IgHEL B cells stimulated for 10 min with 5 ng/ml or 1 μg/ml HEL, and identified as wild-type or Themis2−/− by counterstaining with B220 alone (KO, Themis2−/−) or B220 and CD45.1 (WT, wild type). (f) Mean anti-SHP1 and SHP2 phospho-specific antibody binding following stimulation of 50:50 mixtures of CD45.1 wild-type and CD45.2 Themis2−/− IgHEL B cells with sHEL. Data show means from triplicate samples and graphs are representative of three or more experiments.
