PRT-treated platelets induce only weak activation of splenic T cells and inhibit T-cell responses to subsequent untreated platelets. The responses of T cells were examined from the spleen at days 2, 4, 7, and 14. (A) Cells were gated on CD3⁺, followed by CD4⁺ or CD8⁺ events. FSC, forward scatter. (B) CD4⁺ and CD8⁺ T cells were then evaluated for frequency of CD25⁺, CD69⁺, CD44⁺, and ICOS⁺/CD28⁺. (C) Heat map shows fold changes in frequencies of each group compared with the nontransfused group for CD4⁺ T cells (top) or CD8⁺ T cells (bottom) (out of total CD3⁺ events) and CD69⁺ and CD25⁺ events (out of total CD4⁺ or CD8⁺ events, respectively). CD8⁺ T cells did not meet threshold of 100 positive events for CD25 and were therefore excluded (X). (D-E) Frequencies of CD44 expression (D) and ICOS⁺/CD28⁺ (E) out of total CD4⁺ or CD8⁺ T-cell events are plotted. Notation indicates significance between groups. Combined data from 5 independent experiments (n = 3-5 per group) are shown, and bars indicate mean and standard deviation. *P < .05, **P < .01, ***P < .001, ****P < .0001.