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. 2021 Aug 18;297(3):101097. doi: 10.1016/j.jbc.2021.101097

Figure 1.

Figure 1

Regulation of T cell GITR expression by TRAF3.A, splenic T cells isolated from LMC or T-Traf3−/− mice as indicated above the histograms were unstimulated (-TCR) or stimulated with plate bound Ab to CD3 (0.5 μg/ml) and soluble Ab to CD28 (5 μg/ml) (+TCR) for 16 h. Washed cells were then stained with anti-GITR or an isotype control antibody (Iso) and analyzed by flow cytometry. Mean fluorescence intensity (MFI) ± SEM of three experiments are shown to the right of a representative experiment. B and C, unstimulated WT or Traf3−/− subclones of the mouse T cell line 2B4 were analyzed as in (A). A representative result (B) and MFI ± SEM (n = 6) (C) are shown. D–G, unstimulated primary splenic T cells (D, conventional T cells; E, regulatory T cells; F, pan T cells) were analyzed for GITR surface expression as in A–C. One representative of three replicate experiments is shown. Quantification of MFI ± SEM (n = 4) is shown in G. LMC: filled; T-Traf3−/−: open. For A, C, and G, ∗∗p < 0.01, ∗p < 0.05, and n.s indicates no significant difference by unpaired t test. H–I, GITR relative mRNA levels, 2−ΔΔCt normalized to GAPDH (y axis), were determined for RNA isolated from 2B4 (H) or primary splenic T cells (I) by quantitative RT-PCR, as described in Experimental procedures. Cells in H were unstimulated (−TCR) or stimulated for 4 h with Abs to CD3 and CD28, as in (A) (+TCR). Error bars in F and G represent mean values ± SEM; ∗∗p < 0.01 by unpaired t test. N = 12.