CD8⁺ T cells from male H-Y mice possess a high activation threshold due to a defect in TCR signal transduction. (A) Purified CD8⁺ T cells (1 × 10⁴) from female H-2^b H-Y (CD8^hi), male H-2^b H-Y (CD8^lo), or male H-2^b/d H-Y (CD8^int) mice were cultured with irradiated B6-Tap-1^-/- splenocytes (5 × 10⁵) ± IL-2 and the indicated concentration of H-Y peptide. Proliferation was determined after 3 days, and the error bars represent the standard deviation of triplicate cultures. (B) Western blot analysis of CD8^hi, CD8^int, and CD8^lo cells immediately ex vivo or after stimulation for 10 minutes with anti-CD3 (10 μg/mL) or PMA (25 ng/mL) and ionomycin (500 ng/mL). Blots were probed with antiphospho-ZAP-70 and phospho-ERK and then stripped and reprobed with antibodies to unphosphorylated ZAP-70 and ERK2.