Fig. 2
From: Amyloid-β specific regulatory T cells attenuate Alzheimer’s disease pathobiology in APP/PS1 mice
Characterization of TCRAβ-Treg immunosuppressive function. A. Representative histograms of Treg suppressive function assay. CFSE-labelled-Tresp (CD4+ CD25-) cells co-incubated (3 days) with decreasing ratio of Treg:Tresp cells in the presence of anti-CD3/CD28 Dynabeads. Undivided CFSE+ Tresp (green peaks), divided CFSE+ Tresp (red peaks), Treg cells (blue peaks). B. Quantitation of the immunosuppressive function of TCRAβ-Tregs generated by electroporation of TCR−−-Tregs (TCR knockout Tregs) with 0.25 μL or 0.5 μL of TCRAβ plasmid (plasmid conc. = 1.5 μg/μL). Engineered TCRAβ-Tregs, Tregs or TCR knockout Treg (TCR−−-Tregs) were co-cultured with CFSE + Tresp cells (50 K cells/well) from non-Tg mice in the presence of anti-CD3/CD28 Dynabeads. Treg mediated immune suppression (%Inhibition) = [1- (% proliferation of Tresp:Treg dilution ÷ % proliferation of stimulated Tresp alone)] × 100. Linear regression analysis indicates r2 > 0.90, p < 0.03 for TCR−−-Tregs electroporated with 0.25 μL or 0.5 μL of TCRAβ plasmid. Regression of polyclonal Tregs were r2 > 0.50, p < 0.03. Table contains p-values for slopes and intercepts of Treg functions compared by linear regression analysis, n = 3. Data presented as mean ± SEM C. Supernatants of Treg (polyclonal), TCR−−-Tregs (TCR knockout Tregs), and TCRAβ-Tregs (TCR−−-Tregs + 0.5 μL TCRAβ plasmid) stimulated with PMA/ionomycin assessed by mouse cytokine array. Data represents mean intensities and statistical differences determined by two-way ANOVA tabulated in Supplementary Table 1. D. Quantification of Aβ-mediated Treg suppressive function. CFSE + Tresp cells from non-Tg mice were stimulated overnight with anti-CD3/CD28 Dynabeads. Pre-stimulated CFSE + Tresps were co-cultured with TCR Aβ-Tregs (TCR−−-Tregs + 0.5 μL TCR Aβ) or Tregs (polyclonal) or TCR knockout Treg (TCR−−-Tregs) at 1:1 ratio (50,000 cells/well) for 3 days with only MHC-Aβ-tetramer or control-tetramer. Treg–mediated % inhibition was calculated, and statistical differences were determined by one-way ANOVA followed by Turkey’s post hoc test. ***p < 0.001, n = 3. Data presented as mean ± SEM E. Representative histograms of Aβ-mediated Treg suppressive function of engineered Tregs co-incubated with pre-stimulated CFSE+ Tresp cells in the presence of Aβ-tetramer or control-tetramer. Undivided CFSE+ Tresp (green peaks), divided CFSE + Tresp (red peaks), Treg cells (blue peaks)