ICOS expression is up-regulated upon alloactivation and during GVHD. (A) In vivo–alloactivated T cells up-regulate ICOS expression. B6 splenocytes were labeled with CFSE and transferred into a sublethally irradiated (750 cGy) syngeneic B6 or allogeneic C3FeB6F1 recipients. Transferred cells were analyzed after 72 hours for ICOS expression. A horizontal line represents the fluorescence intensity of negative controls for both isotype-stained and unstained cells. T cells were stained for donor T-cell origin and were analyzed by gating on Ly9.1^-, CD3⁺, and CD4⁺, or CD8⁺. Data from 1 mouse is representative of 2 independent experiments (n = 3 per experiment). (B) ICOS is highly expressed on alloreactive T cells during GVHD. C3FeB6F1 mice were lethally irradiated (1300 cGy split) and received transplants with TCD allo-HSCs and 1 × 10⁶ T cells from B6 mice. Donor T cells were harvested from recipients' spleens 7 and 14 days after transplantation (GVHD day 7, day 14) and analyzed for ICOS cell surface expression in CD44^hi cells. As controls, splenocytes from B6 mice not receiving transplants were used (control), and insets show CD44 subpopulations. Gray histograms represent the isotype control, and the black histograms represent staining with anti-ICOS monoclonal antibody. Shown are representative data of 1 mouse each from 1 independent experiment (normal mouse and day 7, n = 5) and 2 independent experiments (day 14, n = 5 to 8).