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. 2018 Jan 10;8:1938. doi: 10.3389/fimmu.2017.01938

Figure 2.

Figure 2

Interferon regulatory factor 5 (IRF5) is required for toll-like receptor 9/B cell receptor -induced antibody secreting cell differentiation. Isolated human naive B cells were nucleofected with 500 nM of mock, scrambled or IRF5 siRNA and mock-stimulated or stimulated with the indicated cocktails. (A) IRF5 transcript expression was quantified through qPCR on RNA isolated 48 h postnucleofection and 12 h poststimulation with anti-IgM+ CpG-B (two-way ANOVA with Tukey’s post hoc test; n = 4 independent donors). (B) Protein lysates were prepared from nucleofected B cells 72 h postnucleofection and 24 h poststimulation with anti-IgM+ CpG-B. Western blot is one representative experiment out of three performed on n = 3 independent donors. (C) Representative histograms of IRF5 expression 48 h postnucleofection. Viable cells were analyzed through live/dead staining discrimination. (D) Plotted MFI of IRF5 from (C) (one-way ANOVA with Tukey’s post hoc test; n = 4 independent donors). (E) Plotted percentage of B cell viability assessed 72 h post-nucleofection, as determined through trypan blue exclusion. Data are from n = 3 independent donors. (F) Representative dot plots from B cell apoptosis quantified following staining with Annexin V and 7 amino-actinomycin D (7-AAD). Early apoptotic events are characterized as Annexin V+ 7AAD−, whereas late apoptotic events are Annexin V+ 7AAD+. Quantitation is shown in (G). (G) Average apoptotic B cells from (F) 96 h post-nucleofection and 48 h post-stimulation (Two-way ANOVA with Tukey’s post hoc test; n = 3 independent donors). (H) Isolated naive B cells were nucleofected with 500 nM of mock, scrambled or IRF5 siRNA and stimulated with either CD40L or the combination of CD40L, IL21, anti-IgM, and CpG-B for 7 days. Plasmablast differentiation was quantified through gating of CD19+CD20+IgD−CD27+CD38+ B cells; final CD27+CD38+ gating is shown. Flow cytometry contour plots are representative of one experiment from a single donor. (I) Average number of plasmablasts from (H) following culture for 7 days (two-way ANOVA with Tukey’s post hoc test; n = 9 independent donors). (J) Average number of IgD+CD38lo B cells from (H) following stimulation (two-way ANOVA with Tukey’s post hoc test; n = 9 independent donors). Error bars represent SD. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.