Figure 3.

Functional in vitro assays of CD27⁺ and CD27⁻ CTLs. [³H]thymidine uptake of CD27⁺ and CD27⁻ cells after sorting and in vitro expansion in response to (A) autologous LCLs pulsed with the noted concentrations of peptide, or to (B) titrated amounts of plate-bound anti-CD27 and 30 ng anti-CD3 mAb. (C) [³H]thymidine incorporation of CD27⁺ and CD27⁻ T cells of the clone 9G6-35 after three (R3) or six (R6) in vitro expansions in response to titrated amounts of plate-coated anti-CD3 mAb (OKT3). Proliferation index was determined as [[³H]thymidine uptake of stimulated samples]/[[³H]thymidine incorporation of unstimulated samples]. The [³H]thymidine uptake in unstimulated CD27⁺ and CD27⁻ subsets was comparable. (D) The percentage of annexin V⁺ cells is shown in CD27⁺ and CD27⁻ T cell subsets after stimulation for 16–18 h with 10 μg/ml of plate-bound anti-CD3 mAb and/or 10 μg/ml of soluble anti-FAS mAb in the presence or absence of 10 μg/ml of coated anti-CD27mAb. (E) CFSE-labeled CD27⁺ and CD27⁻ T cells of the clone number 9G6-35 were stimulated with 30 ng/ml of plate-bound anti-CD3 mAb (OKT3) in the presence or absence of 10 μg/ml of coated anti-CD27 mAb. CFSE expression is shown 7 d after stimulation. IL-2 production of CD27⁺ and CD27⁻ cells was assessed after 36 h of stimulation with (F) peptide-pulsed autologous LCLs or (G) titrated amounts of plate-bound anti-CD27 and 30 ng anti-CD3 mAb. (H) [³H]thymidine incorporation of CD27⁺ and CD27⁻ T cells of the clone 9G6-35 was measured after stimulation with 100 nM of autologous peptide-pulsed LCLs in the presence or absence of a blocking anti–IL-2Rβ mAb. The mean of triplicate samples is shown, with error bars indicating SEM. All in vitro stimulations presented in this figure were performed in the absence of IL-2 in the culture media.