Fig 2: Reduced number of T cells in non-tumoural regions in b16 melanoma bearing Gsk-3b cKO(A–D) Immunohistochemistry of lung tissue sections from B16 tumor bearing mice. Non-tumor areas are shown following staining for (A) CD4+, (B) CD8+, (C) PD1+ and (D) FOXP3+ expression. End panels depict number of positive cells quantified from 5 different areas using ImageScope Software.Data are representative of two independent experiments. Groups compared using unpaired t test. ∗∗∗p < 0.0001; ∗∗p < 0.001; ∗p < 0.01; ns, no significant difference relative to controls.

Fig 3: Regulatory T-cell recruitment to HNSCC tumor sites is inhibited by dietary black raspberry extract administration. (A) Multiplex confocal microscopy images of representative tongues taken from each mouse group. For each image, individual stains and composite images are shown. Each tongue was stained for DAPI (blue), CD4 (red), and FoxP3 (green). FoxP3+ CD4+ cells were quantified using ImageJ. (B) Western blot demonstrating FoxP3, and GAPDH protein content of tongue lysates isolated from sentinel, 4NQO, and 4NQO+BRB-E groups. Average protein intensity of Western blot bands was measured for several samples using ImageJ. (C) RT-qPCR data of tongue homogenate taken from sentinel or cancer-bearing mice receiving the control diet or the black raspberry extract supplemented feed for Foxp3, Il2r, and Ctla4. Data are represented as fold change relative to the Bactin control. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; #p-value < 0.1 for comparisons of BRB-E treatment groups to the 4NQO-exposed control diet group using Student’s t-test.

Fig 4: Therapeutic effects of different treatments on tumor development and progression in a murine subcutaneous model of HCC. A Experimental protocols for murine subcutaneous model of liver (HCC) and orthotopic model of breast cancer (TNBC) generated by injecting ASPH-expressing BNL cells subcutaneously and 4T1 cells orthotopically (mammary fat pad), respectively. For each model, Balb/C mice were randomly divided to 4 groups (n = 6/group): control, PD1 blockade (anti-PD-1 mAb), ASPH-based λ phage vaccine, and combination (PD-1 blockade + vaccine). B Growth curves of HCC tumors. Note the substantial anti-tumor effect of combination therapy (also see Fig. S1B). C Macroscopic appearance of resected HCC tumors. D Tumor weights of HCC after 49 days in different groups. E In vitro cytotoxicity of splenocytes derived from mice against target BNL cells. F Antigen (ASPH) specific CD4+ and CD8+ T cell activation as demonstrated by upregulation of IFN-γ. G Histological features of ASPH expression in HCC tumors. H-I Number of CD3+ tumor infiltrating lymphocytes (TILs) in HCC tumors. J Anti-ASPH antibody titers in serum derived from different groups. K Percentage of immunosuppressive CD4+/CD25+/FOXP3+ Tregs among TILs in response to therapy. L-M Number of IFN-γ producing CD4+ and CD8+ T cells, respectively, in response to therapy. N Overall survival of mice harboring HCC in response to therapy. *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.001

Fig 5: Tumor rejection in Gsk-3b cKO is associated with higher numbers of tumor-infiltrating T cells(A–D) Immunohistochemistry of lung tissue sections from B16 tumor bearing mice. Intra-tumoural areas are shown following staining for (A) CD4+, (B) CD8+, (C) PD1+ and (D) FOXP3+ expression. End panels depict number of positive cells quantified from 5 different areas using ImageScope Software.Data representative of two independent experiments. Groups compared using unpaired t test. ∗∗∗p < 0.0001; ∗∗p < 0.001; ∗p < 0.01; ns, no significant difference relative to controls.