Supplementary Figure 2: Stimulation of memory B cells is affected by an α4β7 -reactive gp120.
a) % β7 expression on CD27- and CD27+ B cells p=0.0017 (Wilcoxon matched-pairs signed rank test) (n=13). Histograms of two representative donors to show the variability in β7 expression on CD27+ B cells. b) FACS analysis of CD80 surface expression induced by either a TI or a TD stimulation for 96h, in the presence or absence of an α4β7-reactive gp120 (R66M). Values reported are average % reactivity for five donors normalized to CD80 expression at 96h upon B cell stimulation (100%), p<0.0001 (two way ANOVA) (error bars, s.e.m ) (n=5). c) FACS analysis of CD86 surface expression. Values reported are average % reactivity for five donors normalized to CD86 expression at 96h upon B cell stimulation (100%) in the conditions described in panel b. d) FACS analysis of FcRL4 surface expression. Values reported are average % reactivity for five donors normalized to FcRL4 expression at 96h upon B cell stimulation (100%) in the conditions described in panel b. e) CFSE assay of TI stimulation of B cell proliferation (1st panel), in the presence of: an α4β7-reactive gp120 (2nd panel), an α4β7-reactive gp120 and an anti-TGF-β1 mAb (3rd panel), soluble TGF-β1 (4th panel), and soluble TGF-β1 plus an anti-TGF-β1 mAb (5th panel) of a representative donor. Cells were cultured for 96h. f) Division Index (FlowJo) for experiments described in panel e, indicating the average number of cell divisions, p<0.001 (two-way ANOVA) (n=5). Treatments are listed below the x-axis. g) CFSE assay of one representative donor as described in panel e but employing a TD inductive signal to induce proliferation of memory B cells. h) Division Index (FlowJo) for experiment in panel g of five donors indicating the average number of cell divisions, (NS) (two-way ANOVA) (n=5).
