Anti mouse cd8

For CD8 depletion study, mice were given anti-mouse CD8 (clone: 53–6.7, BioXCell) for 3 consecutive days following with booster shots every 3 days after. Flow cytometry analysis of splenocytes and lymphocytes were performed with anti-mouse CD3 (clone: 17A2, Biolegend), anti-mouse CD4 (clone: RM4–5, Biolegend), anti-mouse CD8 (clone: KT15, Serotec) to confirm success of depletion.

Tumors removed from the intestine were covered with Tissue-Tek O.C.T Compound in a cryomold and stored at −80°C. Frozen blocks were sectioned and fixed with 75% acetone/25% methanol for 5 minutes. The slides were washed thrice with PBS and 10% goat serum was added for 1 hour at room temperature followed by an anti-mouse PD-L1 (abcam; clone: MIH6; 1/100 dilution) or anti-mouse CD8 (AbD Serotec; clone KT15; 1/200 dilution) overnight at 4°C. After washing, the slides were incubated with AlexaFluor 488 anti-rat IgG (abcam; 1:1000 dilution) for 1 hour at room temperature. Positive cells were counted in three 20X microscopic fields and expressed as the mean percent of total cells.