Perforin, granzyme B, and IFNγ expression and ex vivo cytotoxic activity of CD8 T-cell subsets. (A) The percentage of EMRA CD8 T cells and CX3CR1⁺ CD8 T cells expressing perforin was compared with the percentage of CXCR1⁺ CD8 T cells expressing perforin. Significantly more CXCR1⁺ CD8 T cells expressed perforin than either EMRA CD8 T cells (77% ± 9% versus 48% ± 12%) or CX3CR1⁺ CD8 T cells (77% ± 9% versus 44% ± 8%). Data represent the mean (± SD) from at least 3 independent experiments. (B) Comparison of perforin and granzyme B mRNA expression levels of bulk CD8 T cells, EMRA CD8 T cells, and CXCR1⁺ CD8 T cells. The mRNA levels of perforin and granzyme B were 2-fold and 0.25-fold higher, respectively, in the CXCR1⁺ CD8 T-cell subset than in the EMRA CD8 T-cell subset. Data represent the mean (± SD) from 3 independent experiments. (C) Direct ex vivo cytotoxic activity of bulk CD8 T cells, CX3CR1⁺ CD8 T cells, EMRA CD8 T cells, and CXCR1⁺ CD8 T cells was compared in 6 individuals, by means of a sensitive caspase-activity assay. Representative data from 4 independent experiments are shown, and data are expressed as the percentage of caspase-positive target cells induced by each indicated group. The relative difference between CXCR1⁺ CD8 T cells and EMRA CD8 T cells and between CXCR1⁺ CD8 T cells and CX3CR1⁺ CD8 T cells is indicated in the figure (Δ). Statistical analysis of the pooled data (n = 6 individuals; 2 of them tested twice) confirmed that cytotoxicity was significantly higher in the CXCR1⁺ CD8 T-cell subset (42% ± 15% caspase-positive target cells) than in bulk CD8 T cells (21% ± 14% caspase-positive target cells), EMRA CD8 T cells (30% ± 12% caspase-positive target cells), or CX3CR1⁺ CD8 T cells (28% ± 6% caspase-positive target cells) (P < .005). In the absence of anti-CD3 antibody stimulation, fewer than 5% of target cells were caspase-positive. (D) IFNγ production of anti-CD3–activated EM CD8 T cells, EMRA CD8 T cells, and CXCR1⁺ CD8 T cells was analyzed by intracellular cytokine staining. Four independent experiments are shown, and data are expressed as the percentage of IFNγ⁺ cells within the indicated CD8 T-cell subset. The relative difference between cytotoxic activity of CXCR1⁺ CD8 T cells and EMRA CD8 T cells and between CXCR1⁺ CD8 T cells and EM CD8 T cells is indicated in the figure (Δ). Statistical analysis of the pooled data (n = 4) confirmed that IFNγ production was significantly higher in the CXCR1⁺ CD8 T-cell subset (37% ± 7% IFNγ⁺ CD8 T cells) than in EMRA CD8 T cells (25% ± 5% IFNγ⁺ CD8 T cells) and EM CD8 T cells (19% ± 4% IFNγ⁺ CD8 T cells) (P < .005). No IFNγ production was observed in absence of anti-CD3 antibody stimulation (data not shown).