Fig 2: Increased CD11c+ CD80+ and CD8+IFN-?+ infiltrates in corneas of mice infected with KOS-ICP22?40 virus. Mice were ocularly infected as described in the legend of Fig. 5 with KOS-ICP22?40 or WT KOS, and corneas of infected mice were harvested on day 5 p.i. Corneas from three mice for each virus were combined, and single-cell suspensions were stained with combinations of anti-CD11c, anti-CD4, anti-CD8, anti-CD80, or anti-IFN-? and analyzed by flow cytometry. Experiments were repeated three times. Graphs represent average results from three independent experiments. Error bars represent SEM, and P values were calculated using Fisher’s exact test and considered significant for a P value of <0.05.

Fig 3: NAC1 deficiency increases tumor CTL infiltration following an ACT in immune-deficient mice. Human WT A2058 or NAC1 KO A2058 tumor cells (1×106) were s.c. injected into the flank of NSG mice, followed by treatment with or without the ACT of Tyrosinase-specific CD8+ T cells. (A) Graphical representation of the progression of tumor size. (B) The tumor weight was evaluated by scales and represented graphically. Values are mean±SEM of three independent experiments (n=5). (C) Indicated tumors were harvested and photographed at day 25. (D) CD8+ T cell infiltration (CD8+; green) in tumors (tyrosinase; red) was determined by immunofluorescence staining. Images are representative images taken from the tumors of three different mice. (E) Flow cytometric analysis of infiltrating CD8+ T cells (left panel) and quantitation of tumor infiltrating CD8+ T cells (right panel). Values are the mean±SEM of 3 independent experiments (n=5). (F) Flow cytometric analysis of infiltrating TIM-3+CD8+ T cells. (G) Flow cytometric analysis of infiltrating PD-1+CD8+ T cells. Values are the mean±SEM of 3 independent experiments (n=5). (H) Flow cytometric analysis of IFN-? production of infiltrating CD8+ T cells. (I) Flow cytometric analysis of the IL-2 production of infiltrating CD8+ T cells. (J) Flow cytometric analysis of Granzyme B production of infiltrating CD8+ T cells. Values are the mean±SEM of four independent experiments (n=4). (K) Schematic representation of the hypothesis.**p=0.01, ***p=0.001. ACT, adoptive cell transfer; CTL, cytotoxic T lymphocytes; LDHA, lactate dehydrogenase A; MFI, mean fluorescence intensity; NAC1, nucleus accumbens-associated protein-1.

Fig 4: Porins induce functional TIM-3+ PD-1+ CD8+ T cells in a therapeutic scheme. (A) Mice were inoculated with MO4 cells and immunized s.c. with OVA + Porin, OVA + CTB or PBS control, data is presented 21 days after the melanoma challenge. (B) CD8+ (green) T cells expressing IFN-? (red) from tumors (representative immunofluorescence micrographs from a zoomed area). (C) Flow cytometry of CD8+ PD-1+ TILs and (D) their percentages. (E) TIM-3 medium fluorescent intensity (MFI) from CD8+ and CD8+ PD-1+ TILs. (F) CD8+ (green) T cells expressing TIM-3 (red) and PD-1 (cyan) from tumors (representative immunofluorescence micrographs from a zoomed area) (Scale bar = 20µm). Statistical data were pooled from three independent experiments with three mice per experimental condition. **p < 0.01, ****p < 0.0001.

Fig 5: oHSV2-aPD1 Therapy Reduces Inhibitory Immune Cells in the SpleenC57BL/6J-Pdcd1 mice were subcutaneously inoculated with 2 × 105 B16R and treated as described in the Materials and Methods. Tumor-bearing mice were euthanized 5 days after the last treatment. (A–G) Spleens were collected and analyzed to determine levels of Tregs (defined as CD4+CD25+FoxP3+) (A), MDSCs (defined as CD45+CD11b+Gr-1+) (B), CD45+CD11b+F4/80+ (C), and CD8+ T cell exhaustion (D–G). Data are presented as individual replicates. *p < 0.05, **p < 0.01. ns, not significant.