MACS-enriched CD4+ T cells isolated from spleens and lymph nodes of CD45.1+CD45.2+ WT OT-II TCR transgenic mice and CD45.2+ DGKζ KO OT-II TCR transgenic mice were sorted for naïve T cells (CD4+CD45RB+CD25CD44lo). Sorted naïve CD45.1+CD45.2+ WT OT-II and CD45.2+ DGKζ KO OT-II cells were transferred intravenously into CD45.1+ WT hosts at a 1:1 ratio (400,000 WT OT-II:400,000 DGKζ OT-II). One day after transfer, mice were subjected to OVA sensitization. Five days after the second intraperitoneal OVA/Alum immunization, splenic CD4+ T cells were isolated by MACS enrichment and restimulated with PMA (100 ng/ml; Sigma-Aldrich) and ionomycin (1 μg/ml; Sigma-Aldrich) in the presence of brefeldin A (5 μg/ml; Cell Signaling Technology) for 5 hours. After restimulation, activated CD4+ T cells were washed twice with PBS and prepared for cell surface and intracellular cytokine staining.

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