Horizon violet bv proliferation dye

CD8⁺ T cells were isolated from single cell suspensions of lymph nodes and spleens of naive mice using anti-CD8 microbeads (Miltenyi Biotec) per the manufacturer’s protocol. The isolated CD8⁺ T cells were then labeled with BD Horizon Violet (BV) Proliferation Dye (BD Biosciences) before plating in complete RPMI in round bottom 96-well plates (5×10⁴ cells/well). Myeloid-derived suppressor cells were isolated from splenic tissue using the Myeloid-Derived Suppressor Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s protocol after red blood cell lysis (ACK lysis buffer, Lonza). Purified MDSCs were added, in the ratios denoted, to the isolated CD8⁺ T cells. Subsequently, Mouse T-Activator CD3/CD28 Dynabeads^® (Thermo Fisher) were added at a bead:T cell ratio of 1:2, and recombinant mouse IL-2 (Invitrogen) at 30U/mL was added and the cultures were incubated for 24 hours at 37C. Control wells were stimulated CD8⁺ T cells without isolated MDSCs (stim. control), or with MDSC fixed with 100% methanol for 20 minutes at −20°C, or stimulated CD8⁺ T cells plated with MDSCs isolated from tumor-bearing mice (suppression control). 4.5 hours prior to the end of the 24-hour incubation, Brefeldin A (Sigma) was added to the co-cultures. For testing the direct effect of RGX-104 on T cell activation, the same method was utilized except that no MDSCs were added to the culture.

For MDSC adoptive transfer experiments into tumor bearing mice, 2 × 10⁶ MDSCs were isolated as described above from C57BL/6 tumor-bearing mice and subsequently labeled with BD Horizon Violet (BV) Proliferation Dye (BD Biosciences) and then adoptively transferred via retro-orbital injection into wild-type mice. Mice were then treated with RGX-104 (100 mg/kg/day) or control diet for 36 hours. Spleens of recipient mice were then harvested and processed for flow cytometry analysis as described above. For MDSC adoptive transfer experiments in to WT and ApoE^−/− non-tumor bearing mice, 5 × 10⁶ MDSCs were isolated as described above from tumor-bearing mice (either wild-type C57BL/6 or Apoe^−/− mice, as indicated in the experiment) and subsequently labeled with BV cell tracker dye and then adoptively transferred via retro-orbital injection into either wild-type or Apoe^−/− mice as indicated. Mice were then treated with GW3965 (100 mg/kg/day) or control diet for 48 hours. Spleens of recipient mice were then harvested and processed for flow cytometry analysis as described above.