EV augmentation of T lymphocyte proliferation. PBMCs were stained with CFSE and cultured for 7 days, stained with CD3, CD4, and CD8 monoclonal antibodies, and proliferation was quantified as the percentage of CFSE^low cells on CD3⁺CD4⁺ or CD3⁺CD8⁺ T cells. (A) T-cell proliferation was not detectable when PBMCs were stimulated with EVs alone. (B) Proliferation of CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells stimulated with PHA or PHA plus day 0 EV. (C) T-cell proliferation changes induced by EVs derived from RBCs stored up to 42 days. Proliferation of CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells is shown as percent augmentation relative to PHA alone stimulation. Six independent experiments were performed at each time point. Six PBMC preparations and 6 independent EV isolates were used for this experiment (*P < .05, day 7 vs day 42 EVs). (D) Proliferation of CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells in response to day 0 autologous or allogeneic EVs did not show a significant difference. A total of 4 independent autologous and allogeneic experiments were performed.