Monitoring Antigen-specific CD8+ T Cells

To monitor antigen-specific donor-reactive CD8⁺ T cell responses, we used our previously described system in which OVA-specific transgenic T cells are adoptively transferred prior to skin transplantation with OVA-expressing skin or infection with OVA-expressing pathogens [13 (link),25 (link)]. For the adoptive transfer, WT OT-I (Thy1.1⁺, CD45.2⁺) or Fcgr2b^−/− OT-I (CD45.2⁺) transgenic cells were harvested from the spleen and processed into a single cell suspension. Cells were counted using a Nexcelom Cellometer Auto T4 (Nexecelom Bioscience, Lawrence, MA) and stained with CD8- BV785, CD4- PacBlue, CD45.2-PE-Dazzle, Thy1.1- PerCP, Vα2- FITC, and Vβ5- PE (Biolegend). Frequency of OT-I T cells was determined via Vα2 and Vβ5 TCR co-expression. Cells were resuspended in 1X PBS, and 10⁴ CD45.2⁺ OT-I T cells adoptively transferred into naïve CD45.1 hosts 24 hours prior to infection.