Fig. 2. Validation of SUGAR-seq.

(A) TILs from day 14 B16-Ova tumors were FACS gated as CD8⁺ TCR⁺ CD3⁺, followed by selecting CD44⁺ CD62L^+/− and then analyzed for N-glycan abundance through detection of FITC-conjugated L-Pha. (B) L-Pha^high and L-Pha^low CD8⁺ TILs from (A) (right-hand panel) were analyzed for expression of PD-1 and TIM3 by FACS. Bar charts represent the frequency of PD-1 TIM3–positive or PD-1 TIM3–negative CD8⁺ TILs across L-Pha^low or L-Pha^high subsets. Data points represent individual mice. *P < 0.01. (C) TILs (CD45⁺ CD8⁺) from day 14 B16-Ova tumors were FACS analyzed for the indicated surface proteins, L-Pha staining, and intracellular proteins as indicated. TSNE clustering is depicted. Bar charts represent the mean fluorescence intensity (MFI) of the indicated TFs in CD45⁺ CD8⁺ TILs across L-Pha^low or L-Pha^high subsets. Data points represent individual mice. *P < 0.01.