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. Author manuscript; available in PMC: 2021 Jun 28.
Published in final edited form as: Cancer Immunol Res. 2019 Jan 25;7(3):401–413. doi: 10.1158/2326-6066.CIR-18-0546

Figure 3. Immune correlative and functional effects of NIR-PIT and PD-1 mAb in mice bearing a unilateral MC38-luc tumors.

Figure 3.

(A) MC38-luc tumors (day 10, n = 5/group) treated with NIR-PIT with and without PD-1 mAb and controls were harvested, digested into single-cell suspensions, and analyzed for tumor-infiltrating lymphocytes (TILs) via flow cytometry. Presented as absolute number of infiltrating cells per 1.5 × 104 live cells analyzed. PD-1 expression shown as inset (MFI, mean fluorescence intensity). *p < 0.05, **p < 0.01, ***p < 0.001, t test with ANOVA. Representative data from one of two independent expeirments shown. (B) Multiplex immunofluorescence was used to validate flow cytometric data. Representative 400× images shown. Quantification of infiltrating TILs from 5 high power fields (HPF) per tumor, n = 3/group. **p < 0.01, ***p < 0.001, t test with ANOVA. (C) TILs were isolated from tumors treated as above (n = 5/group) via an IL2 gradient, enriched via negative magnetic selection, and stimulated with irradiated splenocytes pulsed with peptides representing known MHC class I-restricted epitopes from selected tumor-associated antigens. IFNγ production was determined by ELISA from supernatants collected 24 hours after stimulation. Supernatants from splenocytes (APCs) alone, TILs (T) alone, and a MHC-class I-restricted epitope from ovalbumin (OVA, SIINFEKL) were used as controls. *p < 0.05, **p < 0.01, ***p < 0.001, t test with ANOVA. (D) Flow cytometric analysis of tumor-infiltrating DCs and macrophages, with quantification of macrophage polarization based on MHC class II expression. **p < 0.01, ***p < 0.001, t test with ANOVA. (E) Flow cytometric analysis of tumor infiltrating neutrophilic myeloid cells (PMN-myeloid) and regulatory T-cells (Tregs). *p < 0.05, **p < 0.01, t test with ANOVA. (F) Flow cytometric analysis of PD-L1 expression on CD45.2–CD31–PDGFR– tumor cells and CD45.2+CD31– immune cells. **p < 0.01 compared to control, t test with ANOVA. N = 5/group.