Generation of allospecific human CD8⁺ T cells secreting either type I or type II cytokines. Allospecific CD8⁺ T cells of Tc1 or Tc2 phenotype were generated by in vitro allostimulation in the presence of either IL-12 (2.5 ng/mL) and TGF-β (5 ng/mL) or IL-4 (1,000 U/mL), respectively. On day 13 of culture, CD8⁺ cells were resuspended to a final concentration of 0.5 × 10⁶ cells/mL and restimulated in vitro with allogeneic (Allo) or autologous (Auto) bone marrow–derived stimulator cells (irradiated, 600 cGy; T-cell to stimulator ratio, 5:1). After 24 hours, the supernatants were harvested and tested in 2-site ELISAs for determination of cytokine production. (A) Lower limits of cytokine production (≪): IL-2, 1 Cetus U/mL; IFN-γ, 78 pg/mL; IL-5, 12.5 pg/mL; and IL-10, 10 pg/mL. (B) Representative ELISA result for IL-4 secretion from Tc1 and Tc2 populations (also generated on day 13 of culture). In addition to allogeneic and autologous stimulation, Tc1 and Tc2 cells were stimulated with PMA (5 ng/mL) and calcium ionophore (375 ng/mL) [P/CI]. Lower limit of detection for IL-4 ELISA, 10 pg/mL.