Adoptive cell transfer.

To monitor antigen-specific CD8⁺ T cell responses, we adoptively transferred OVA-specific transgenic T cells into naive prior to inoculation with B16 melanoma expressing OVA. For the adoptive transfer of tumor-specific CD8⁺ T cells, spleens and mesenteric lymph nodes from CD45.2⁺Thy1.2⁺ Fcgr2b^–/– OT-I mice or CD45.2⁺Thy1.1⁺ WT OT-I mice were processed into single-cell suspensions. Cells were counted using a Nexcelom Cellometer Auto T4 (Nexcelom Bioscience) and stained with CD8-BV785, CD4-PacBlue, Thy1.1-PerCP, CD45.2-PE/Dazzle, CD45.1-BV605, Vα2-FITC, and Vβ5-PE (all from BioLegend). The frequency of OT-I and OT-II cells was determined via Vα2 and Vβ5 TCR coexpression. Cells were then resuspended in 1× PBS and 1 × 10⁶ OT-I and 1x10⁶ OT-II cells were transferred intravenously into naive congenic CD45.1⁺ B6-Ly5.1/Cr hosts 24 hours prior to tumor inoculation.