FIG. 2.

Detection of HLA-A2 transgenic murine CTLs by using ELISpot, tetramer, and nonradioactive cytotoxicity assays. (A) HLA-A2 transgenic mice were immunized three times with 50 μg of HIV-1 Gag_448-457 peptide alone or mixtures of 50 μg of peptides and 50 μg of gp96 in PBS. Seven days after the last inoculation, splenocytes were isolated from the immunized mice and were tested for 40 h by ELISpot assay. Bars represent the means, with standard deviations indicated. *, P < 0.05. (B) Splenocytes were stained with HLA-A2/k^b-β₂-microglobulin-10-mer peptide or HLA-A2/k^b-β₂-microglobulin-control peptide tetramer together with anti-CD8-FITC and anti-CD3-PE-Cy5 antibodies. The analyzed splenocytes were gated on CD3⁺, CD8⁺, and tetramer-positive cells. Numbers are percentages of tetramer-positive cells within CD8⁺ CD3⁺ T lymphocytes. (C) Splenocytes from the same mice were in vitro restimulated with 10 μM of the peptides. Five days later, a nonradioactive cytotoxicity assay was performed on T2 target cells pulsed with the same peptide.