Suppression of T cell activation mediated by BAD-1 binding of CD47.

(A) BAD-1 inhibition of CD69 expression is dependent on CD47. Jurkat T cells were activated by anti-CD3 antibody alone (5 µg/ml) or in the presence of BAD-1 or TSP1 (10 µg/ml) for 2 hours in vitro. RNA was isolated and relative CD69 mRNA expression determined by real-time PCR. (B) JinB8 T cells lacking CD47 were activated by anti-CD3 antibody alone or in the presence of BAD-1 or TSP1 for 2 hours in vitro. RNA was isolated and relative CD69 mRNA expression determined as above. (C) Un-transfected JinB8 cells and cells transfected with CD47 and CD47-S64A were activated by anti-CD3 antibody alone or in the presence of BAD-1 or TSP-1 for 2 hours in vitro. RNA was isolated and relative CD69 mRNA expression determined as above. Values are percent activation relative to stimulated cells ± SD of 4 experiments for data in panels A–C; *, p<0.05. (D) CD4⁺ T cells from 1807 TCR Tg mice were exposed to increasing amounts of BAD-1 for 90 minutes, washed and added to co-culture of BAD-1 null B. dermatitidis yeast and DC for 96 hours. After incubation, the T cells were analyzed by flow cytometry for expression of CD69. (E) BAD-1 (50 µg/ml) was incubated with 1807 cells at 37°C for 90 min and analyzed by FACS with anti-BAD-1 mAb conjugated to FITC. In top panel, the numbers are the mean fluorescence intensity of mAb binding to control-treated (grey) vs. BAD-1-treated (black) cells. After incubating 1807 Tg cells as above, the T cells were added to wells containing DC and Blastomyces cell wall/membrane antigen (10 µg/ml). After 24 hours the cells were stained for the activation markers shown and analyzed by FACS. (F) Supernates were collected from co-cultures in panel D and assayed by ELISA for IL-17A and IFN-γ. *, p<0.05. vs. control. Results are representative of 2–4 independent experiments for panels D and E.