Figure 3.

(A) Tregs do not suppress CD3 Ab-induced CD3ζ phosphorylation. CFSE-labeled CD8⁺ T cells were co-cultured with Tregs or non Tregs (ratio 4:1) for 6 hours and then were activated with CD3 Ab for 5 min, fixed, permeabilized and stained with anti-CD3ζpY142 Ab and were analyzed by flow cytometry. Histogram shows expression of CD3ζ phosphorylated at tyrosine 142 in CD8⁺ T cells (CFSE-gated cells). (B) Tregs do not suppress CD3 Ab-induced ZAP70 phosphorylation in CD8⁺ T cells. Cells were cultured as for Figure 3A and then, Tregs and non- Tregs were removed from the mixtures using Dynabeads^® CD4, and the remaining CD8⁺ T cells were either lysed immediately (non-activated, NA) or activated as above (Act) and then, lysed to test for phosphorylated form of Zap70 (pZap70). (C and D) Tregs utilize βGBP to suppress CD3 Ab-induced ERK phosphorylation of CD8⁺ T cells. Flow cytometry specifically gated on CD8⁺ T cells (C) or western blotting on lysates from purified CD8⁺ T cells (D) that were cultured with either Tregs or non-Tregs (ratio 4:1) in the presence of βGBP-neutralizing Ab (βGBP Ab) or control isotype-matched IgG (Cnt Ab) for 6 hours prior to activation with soluble CD3 Ab (5 min). For Figure 3D Tregs and non-Tregs were removed from cell mixtures prior to activation as described in Materials and Methods. *P<0.05 is for comparison with βGBP Ab in CD8⁺Treg group (D). Lower panel in Figure 3D are data after quantitative densitometry. (E) Western blotting of lysates from CD8⁺ T cells incubated with titrated amounts of βGBP (μg/ml) for 3 hours prior to stimulation with CD3 Ab. The results have been independently reproduced at least three times using different donor blood from normal humans, and mean ± SD of the individual experiments in duplicates.