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Fig. 5 | Journal for ImmunoTherapy of Cancer

Fig. 5

From: Intratumoral immunotherapy with TLR7/8 agonist MEDI9197 modulates the tumor microenvironment leading to enhanced activity when combined with other immunotherapies

Fig. 5

IT administration of MEDI9197 induces an increase in immune cell activation. SC implanted B16-OVA tumors were injected IT with 20 μg of MEDI9197 or Vehicle. The data for all graphs (except G) are shown in box-whiskers plots. Data is representative of at least 2 independent experiments. a and b Tumors were collected for qPCR analysis (performed in duplicate) (n = 5 per group). The data indicate fold-change in gene expression, relative to Vehicle, associated with type I IFN inducible (A) or T cell effector (B) genes. c-d, Percentage of T cells (CD3ε+ / CD4+ or CD8α+) in the CD45+ population and of activated T cells (CD69+) assessed by flow cytometry in individual tumors after MEDI9197 and Vehicle dosing (n = 4 mice/treatment group/day). e Ratio of effector T cells (Teff: CD45+/TCRβ+/CD44+/CD62L) and naïve/central memory T cells (Tnaive/CM: CD45+/TCRβ+/CD44+/−/CD62L+) for total T cells and CD8+ cells (CD45+, TCRβ+, CD8α+), measured by flow cytometry in tumors injected with MEDI9197 (n = 11) or Vehicle (n = 7) and collected 6–8 days post-dose. f The top graph shows the percentage of intracellular cytokines in CD8α+ T cells (IFNγ, TNFα, IL-2, or negative), the three graphs below show the percentage of single, double, and triple positive CD8α+ T cells from tumors collected 4–5 days post dosing with MEDI9197 (n = 7) and Vehicle (n = 6). Cytokines were analyzed by flow cytometry in PMA/ionomycin stimulated CD45+ enriched cells. g TILs were isolated from each tumor 11 days post-dose (n = 3–4/group) and used for ELISpot analysis for IFNγ and Granzyme B following stimulation with PMA/Ionomycin or class I-restricted peptides (H-2Kb or H-2Db). The results show the number of spots per 100,000 cells after background subtraction based on control peptide (H-2Db and H-2Kb) stimulated TILs. Statistical analyses were performed using multiple T tests of dCT values (a, b), 2-way ANOVA (c, d, and g) with Sidak’s multiple comparisons test (c, d), and the Mann Whitney test (e, f). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

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