Generation of HA-1–specific CTLs restricted by nonself HLA-A2 molecules using T2 cells pulsed with HA-1 peptide.

(A) Subclones that showed more than 30% specific reactivity against HA-1 peptide–pulsed T2 cells as compared with unpulsed T2 cells are designated as “HA-1–specific” (n = 17 of 211). Subclones that did not discriminate between peptide-pulsed and unpulsed T2 cells are designated as “alloreactive” (n = 182 of 211). The results are presented as the mean cytotoxic activity. The error bars represent the SEM. (B) HA-1^A2 tetramer staining of the “HA-1–specific” T-cell cultures, showing PE-conjugated HA-1^A2 tetramers (x axis) and PE-Cy-5–conjugated anti-CD8 antibody (y axis). Appropriate gates were set on vital lymphocytes according to their typical forward- and side-scattering characteristics. (C) The specificity of HA-1^A2 tetramer staining. Three CD8⁺ HA-1–specific CTL clones and 15 CD8⁺ allo–HLA-A2–specific CTL clones were stained with HA-1^A2 tetramers. Appropriate gates were set on vital lymphocytes according to their typical forward- and side-scattering characteristics. The histograms represent the tetramer staining of CD8⁺ T cells. Representative examples are shown for each group of CTL clones.