Defective B-cell death induction and granzyme B release by WKO nTreg cells. (A) Percentage of apoptotic B220⁺ cells after coculture of activated nTreg and CD8⁺ cells from WKO or WT mice with LPS-induced B-cell blasts at a ratio of 5:1 in the presence or absence of anti-CD3 antibody and LPS (0.5 μg/mL). Bar graph represents the results of 11 independent experiments expressed as mean ± SD. *P < .0001. **P < .05. NS indicates not significant. (B) Granzyme B (Gzmb) and perforin (Prf1) expression in WKO and WT preactivated nTreg and CD8⁺ cells. Open histograms represent isotype controls; and solid histograms, Gzmb- and Prf1-specific staining. Representative data from at least 3 independent experiments are shown. (C) ELISA determination of granzyme B (Gzmb) release in culture media by preactivated WT and WKO nTreg and CD8⁺ cells after restimulation with 2 μg/mL anti-CD3 antibody. Results are mean ± SD of 5 independent experiments. *P = .0260. Statistical significances of differences among samples were calculated using the 2-tailed Mann-Whitney test. (D) Percentage of CD107a⁺ after culture of WKO and WT-activated nTreg and CD8⁺ cells (5 × 10⁴) with 2 μg/mL soluble anti-CD3 and irradiated T cell–depleted spleen cells (5 × 10⁴) for 8 hours in the presence of FITC-anti–mouse CD107a or FITC-conjugated isotype control and GolgiStop (BD Biosciences PharMingen). After restimulation, the cells were stained with anti-CD107a, anti-CD4, and anti-CD8 and analyzed by flow cytometry. Results are mean ± SD of 3 independent experiments. *P = .0317. Statistical significances of differences among samples were calculated using the 2-tailed Mann-Whitney test. (E-F) Defective suppression of B-cell proliferation and LPS-induced B-cell blast killing by WKO-OT2 nTreg cells. (E) B cells (5 × 10⁴) from WT mice were stimulated with LPS (3 μg/mL) and cocultured with equal number of preactivated nTreg cells from both WT or WKO mice in the presence of OVA_323-339 peptide (10μM), and irradiated T-depleted APCs. Proliferation was then assessed as in Figure 1. (F) B-cell blasts were pulsed with OVA_323-339 peptide (10μM) for 2 hours and cultured alone or with WKO or WT OT-2 nTreg cells at an effector-to-target ratio of 1:1 for 8 hours in the presence of LPS (0.5 μg/mL). Statistical significance of differences between samples was calculated using the Mann-Whitney test with 95% confidence intervals. Results are mean of triplicate cultures and are representative of at least 2 experiments.