Thy1.1 percp

Prior to perfusion, spleens were harvested from experimental mice. Single cell suspensions from spleens were treated with ACK erythrocyte lysis buffer. Mice were perfused with 25 mL of ice-cold PBS and brains and spinal cords were collected from perfused mice and dounce homogenized to obtain single cell suspensions. CNS cells were purified by centrifugation for 30 min in a 30% Percoll (GE Healthcare) solution as previously reported (25 (link)). Cells were incubated with the anti-Fc receptor antibody 2.4G2 prior to the addition of antibodies. The following antibodies were purchased from BD Biosciences: CD45-FITC, CD8α-APC-H7, CD19-APC-H7, CD19-BV510, B220-PE-TxRed, B220–PE-CF594, CD11b-AlexaFluor-700, MHC-v450. The following antibodies were purchased from eBioscience: MHCII-Pacific Blue, CD11c-PECy7. The following antibodies were purchased from BioLegend (San Diego, CA): CD138-PE, MHCII (I-A/I-E)-Pacific Blue, Thy1.1-PerCP, CD4-APC. Cells were acquired on a Gallios flow cytometer (Beckman Coulter) and analyzed with FloJo software (TreeStar) with doublets being excluded.

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.

To monitor antigen-specific donor-reactive CD8⁺ T cell responses, we used our previously described system in which OVA-specific transgenic T cells in naïve animals are adoptively transferred prior to skin transplantation with OVA-expressing skin. For the adoptive transfer, WT OT-I, Fcgr2b^−/− OT-I, and OT-II transgenic cells were harvested from the spleen and mesenteric lymph nodes. Cells were counted using a Nexcelom Cellometer Auto T4 (Nexecelom Bioscience, Lawrence, MA) and stained with CD8- BV785, CD4- PacBlue, Thy1.1- PerCP, Vα2- FITC, and Vβ5- PE (BioLegend). Frequency of OT-I and OT-II was determined via Vα2 and Vβ5 TCR co-expression. Cells were resuspended in 1X PBS and adoptively transferred into naïve hosts 24 hours prior to skin transplantation or B16-OVA melanoma inoculation. Where indicated, OT-I T cells were labeled with 5uM CellTrace Violet (CTV) dye (Life Technologies, Invitrogen) according to manufacturer’s instructions and then adoptively transferred into naïve hosts. Proliferation was measured on day 14 post transplantation via flow cytometry on a BS LSR II (BD Biosciences) and data were analyzed with FlowJo (Tree Star, San Carlos, CA) and Prism (GraphPad Software).