JAML costimulation activates effector CD8 and γδ T cells. (A–E) CD8 and γδ T cells from WT mice were expanded ex vivo and then analyzed by flow cytometry before and after overnight costimulation via CD28 or JAML. (A) Representative flow cytometry plots of JAML expression by expanded CD8 T cells before costimulation. (B) Representative histogram and quantification of CD69 MFI (fold change compared with unstimulated condition) by CD8 T cells after costimulation. (C) Representative flow cytometry plots and frequency of TNFα and IFNγ production by expanded CD8 T cells after costimulation. (D) Representative histogram of JAML expression by expanded γδ T cells. (E) Representative histogram and quantification of CD69 MFI (fold change compared with unstimulated condition) by γδ T cells after costimulation. (F) Representative flow cytometry plots and frequency of TNFα and IFNγ production by expanded γδ T cells after costimulation. Data are expressed as mean ± SEM and are representative of three independent experiments. (B and C)n = 3 replicates per condition. (E and F)n = 3–4 replicates per condition. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ANOVA with post hoc Tukey’s test (B, C, E, and F).