Socs1 KD tumors exhibit a more T cell–inflamed phenotype and increased CD8+ T cell activation. Socs1 KD tumors exhibit a more T cell–inflamed phenotype.

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Socs1 KD tumors exhibit a more T cell–inflamed phenotype and increased CD8+ T cell activation. Socs1 KD tumors exhibit a more T cell–inflamed phenotype and increased CD8+ T cell activation. LLC-NT or LLC-sh21 cells were orthotopically injected into the left lung lobes of mice and established primary tumors. After 2 wk of tumor growth with no treatment, the mice were euthanized and their tumor-bearing lung lobes were isolated for either flow cytometry or FFPE and T cell staining by immunofluorescence. (A, B) Representative images of T cell staining from (A) an LLC-NT tumor or (B) an LLC-sh21 tumor (scale bars: 100 μm), showing CD8+ T cell staining. DAPI is shown in blue, CD3 in green, CD8 in red, and a merge of all channels in yellow. (C, D) Quantification of CD3+ T cells and (D) CD8+ T cells per high-power field (HPF) in LLC-NT versus LLC-sh21 tumors. There were six tumors from LLC-NT mice, and six tumors from LLC-sh21 mice. T cell numbers per HPF were averaged over four experiments (six random fields per tumor × four staining experiments = 24 fields averaged/tumor in total). Quantification of T cells was performed by two blinded observers (BB & AN). For flow cytometry, single-cell suspensions were made from tumor-bearing lung lobes. There were three experimental replicates × three tumor-bearing pooled lung lobes, for a total of nine lung lobes per the experimental conditions of “LLC-NT” or “LLC-sh21”. (E, F) For the “T Cell Phenotypic Panel,” single-cell suspensions were assessed for the following: (E) the percentage of PD-1–expressing CD8+ T cells or (F) double-positive PD-1/CD69–expressing CD8+ T cells gated as a percentage of all CD8+ T cells. For the “T Cell Stimulation Panel,” single-cell suspensions were stimulated with brefeldin A, monensin, and a cellular stimulation cocktail (PMA/Ionomycin) for 5 h to determine intracellular cytokine production at the time of harvest. Cell suspensions were either unstimulated (treated with brefeldin and monensin alone) or stimulated (treated with brefeldin, monensin, and PMA/Ionomycin). (G, H) Single-cell suspensions were assessed for the following: (G) the percentage of single-positive IFNγ-expressing CD8+ T cells or (H) double-positive IFNγ/TNFα–expressing CD8+ T cells gated as a percentage of all CD8+ T cells. Error bars represent the mean of the data ± SEM after a t test (C–F) or a two-way ANOVA (G–H) (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). Bonnie L Bullock et al. LSA 2019;2:e201900328 © 2019 Bullock et al.