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. 2022 Aug 9;13:932742. doi: 10.3389/fimmu.2022.932742

Figure 4.

Figure 4

Black raspberry extract enhances antitumoral CD8 effector T-cell activity in the HNSCC tumor microenvironment. (A) Representative immunofluorescent images of tongue sections showing tumor-infiltrating CD8+ cells (red) in non-carcinogen-induced mice (sentinel) and carcinogen-induced mice fed the control AIN76 diet (4NQO) or the BRB-E-supplemented diet (4NQO+BRB-E). Tissues were counterstained with DAPI (blue) and quantified using ImageJ. (B) Representative immunofluorescent images of Granzyme B+ cells (green) in tongue sections of non-carcinogen-induced mice (sentinel) and carcinogen-induced mice fed the control AIN76 diet (4NQO) or the BRB-E-supplemented diet (4NQO+BRB-E). Tissues were counterstained with DAPI (blue) and analyzed using ImageJ. (C and D) Representative flow cytometric plots and population distributions of CD8+ Granzyme B+ cells in the spleens (C) and lymph nodes (D) of control feed or berry-treated tumor-bearing mice. Data are presented as percentage of total CD8+ T cells. (E and F) Representative flow cytometric plots and population distributions of CD8+ IFN-γ+ cells in the spleens (E) and lymph nodes (F) of control feed or berry-treated tumor-bearing mice. Data are presented as percentage of total CD8+ T cells. (G) Gating strategy for identification of NK cells from the draining cervical lymph nodes or spleens of HNSCC tumor-bearing or sentinel mice by flow cytometry. Proportions of NK cells in spleens and lymph nodes of non-carcinogen-induced mice (sentinel) and carcinogen-induced mice fed the control AIN76 diet (4NQO) or the BRB-E-supplemented diet (4NQO+BRB-E) are shown as bar graphs. (H–K) Representative flow plots demonstrating Granzyme B-positive staining of CD49b/DX5+ natural killer cells, isolated from spleens (H) and lymph nodes (I) of non-carcinogen-induced mice (sentinel) and carcinogen-induced mice fed the control AIN76 diet (4NQO) or the BRB-E-supplemented diet (4NQO+BRB-E). Representative flow plots demonstrating CD49b/DX5+ IFN-γ + natural killer cells isolated from spleens (J) and lymph nodes (K) of non-carcinogen-induced mice (sentinel) and carcinogen-induced mice fed the control AIN76 diet (4NQO) or the BRB-E-supplemented diet (4NQO+BRB-E). *p-value < 0.05; **p-value < 0.01 for comparisons of BRB-E treatment groups to the 4NQO-exposed control diet group using Student’s t-test.