Effect of allogeneic platelet MHC class I antigens on in vitro cytotoxicity in antibody- and T-cell–mediated ITP. (A) Representative flow cytometric dot plot analysis of cytotoxic killing against CD61⁺/H-2^d PU5-1.8 target cells by nondepleted splenocytes from either a naïve BALB/c mouse (left panel), or from CD61 KO mice immunized either against BALB/c (CD61⁺/H-2^d) platelets (middle panel), or against C57BL/6 (CD61⁺/H-2^b) platelets (right panel). Splenocytes were incubated with CFSE-labeled CD61⁺/H-2^d PU5-1.8 cells for 4 hours in vitro, then stained with the vital dye 7-AAD and acquired on a flow cytometer. Results are shown at an Effector:Target ratio of 10:1. (B) Cumulative cytotoxicity of splenocytes from naïve BALB/c mice (●; N = 19), CD61 KO mice transfused weekly with 10⁸ platelets from either BALB/c (CD61⁺/H-2^d [○; N = 10]), or C57BL/6 (CD61⁺/H-2^b [△; N = 10]) mice. (C) Cumulative cytotoxicity of splenocytes from naïve BALB/c mice (●; N = 19) or SCID mice transferred with the corresponding CD61 KO immune splenocyte populations as in (B) at 4 weeks post-transfer. Results in (B) and (C) are expressed as Percent Lysis at the indicated Effector:Target ratios (**P < .01 for ○ vs △). (D) Splenocytes from BALB/c CD61 KO mice immunized against platelets from BALB/c (CD61⁺/H-2^d) mice were incubated with either syngeneic BALB/c WT platelets (CD61⁺/H-2^b) or allogeneic C57 BL/6 WT platelets (CD61⁺/H-2^b) for 4 hours in vitro, and then examined for their cytotoxicity against PU5-1.8 target cells. Results in (D) are expressed as Percent Lysis at an Effector:Target ratio of 5:1 (N = 3) (*P < .05).