Journal: bioRxiv

Article Title: Cytokine-mediated activation of exhausted T cells

doi: 10.64898/2026.09.28.755149

Figure Lengend Snippet: ( A ) UMAP of WT tumor infiltrating CD8 + T cells (TILs) from the published scRNA-seq dataset of B16 tumors from retaining original UMAP embedding and cell-state annotations. ( B ) Feature plot depicting TIA score within this dataset. ( C ) Experimental setup for results depicted in ( D ) and ( E ) TILs were isolated from LLC-OVA tumor bearing mice at d20 post cell transfer and stimulated in vitro. ( D - E ) TILs were stimulated with IL-12+IL-18, OVA peptide (OVA), anti-CD3 (αCD3), or left unstimulated (unstim) and IFNγ production within total CD8 + T cells ( D ) and PD-1 + (left) or PD-1 hi (right) CD8 + T cells ( E ) isolated from subcutaneous tumors was analyzed by flow cytometry. Results are pools of 2 experiments using 5-6 mice per group. Each dot represents one mouse; paired samples from the same mouse are connected by lines. Differences between groups were determined using two-way ANOVA ( D and E ). p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).

Article Snippet: Red blood cells were lysed with ACK buffer (155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM Na2EDTA, pH: 7.4) for 3 min. For stimulations, single cell suspensions were incubated in U-bottom plates with the indicated cytokines (10 ng/mL, BioLegend) 16-20h overnight with 5 μg/mL Brefeldin A (BioLegend) for the last 4h; re-stimulated with gp 33-41 (gp33, 1ug/ml, KAVYNFATC, EMC microcolletions GmbH) and gp 61-80 (GLKGPDIYKGVYQFKSVEFD, 1ug/ml, EMC microcolletions GmbH) for 4 hours, or with anti-CD3 (2 μg/mL, clone 145-2C11; BioXCell) for 4 hours, or left unstimulated.

Techniques: Isolation, In Vitro, Flow Cytometry