Fig. 5.

In vitro inhibition of mitochondrial respiration recapitulates the effects of BTKi on normal human B-cell activation and both inhibition of mitochondrial respiration and BTKi abrogate aberrant activation and costimulatory molecule expression of MS patient B cells. Purified healthy donor B cells were pre-treated with either vehicle, Rotenone (to inhibit mitochondrial respiration), 2-DG (to inhibit glycolysis), or the combination of Rotenone and 2-DG for 30 min, and then left unstimulated or stimulated with CD40L + αBCR + IL-4 for 2 days to assess expression of the activation marker CD69 and co-stimulatory molecules CD80, CD86; or for 5 days to assess proliferation by CFSE dilution, using flow cytometry. In the absence of stimulation (data not shown), average mean fluorescence intensities (MFIs) of B cell CD69, CD80 and CD86 expression in the ‘vehicle’ condition were approximately 1000, 75 and 125, respectively. Following stimulation which induced the expected proliferation and upregulation of activation and costimulatory molecules, inhibition of glycolysis was found to modestly limit B-cell proliferation (b) while having limited or no appreciable effect on B-cell activation (a) or activation-induced upregulation of costimulatory molecules (c and d, n = 10). In contrast, inhibiting mitochondrial respiration significantly decreased B-cell activation and proliferation, and limited the activation-induced upregulation of costimulatory molecules (a–d). To assess the effects of inhibiting mitochondrial respiration or BTK signaling on B-cell costimulatory molecule expression of MS patients, peripheral B cells were purified from either newly diagnosed (never-treated) MS patients, or healthy controls (HC) balanced for age and sex (Table S2 for participant demographics). The B cells were then treated with vehicle, BTKi or Rotenone and stimulated with CD40L + αBCR + IL-4 for 2 days. Expression of the costimulatory molecules CD80 (e) and CD86 (f) was measured by flow cytometry. Compared to HC, activated B cells of MS patients express abnormally higher levels of CD80 and CD86, and inhibition of BTK or inhibition of mitochondrial respiration with Rotenone, decrease the activation-induced B-cell expression of CD80 and CD86 by both HC and MS patient B cells, such that levels in MS patients are similar to those observed in HC (n = 13–16). a–d Repeated measure two-way ANOVA; a–d Mix-effects two-way ANOVA; Each line represents paired results from individual donors. ns not significant, *p < 0.05, **p < 0.01 ***p < 0.001 and ****p < 0.0001