Fig 2: The addition of a TIGIT inhibitor to RT enhances DC numbers and function in MC38 tumor-bearing mice. Mice were inoculated with MC38 cells on day 0. On day 10, mice were given either isotype IgG, anti-TIGIT therapy, RT, or RT plus anti-TIGIT therapy. Tumors and TdLNs were excised on day 10. a. CD155 expression in CD11c + DCs purified from MC38 TdLNs (red lines), non-draining LNs (green lines), and healthy mouse LNs (purple lines). Representative flow cytometry histogram (left) and quantification of CD155 mean fluorescent intensity in DCs (right). b. Representative flow cytometry contour plots of CD155 expression on DCs after RT in tumors and TdLNs (n = 5); Quantitation of CD155 MFI on DCs is shown on the right. c. Bar graphs show the DC density in tumor tissues as measured by flow cytometry. (d-e). Intracellular IL-10 and IL-12 (interleukin 10 and 12) levels on gated CD11c + DCs from tumor tissues. f. Bar graphs show the DC density in TdLNs as measured by flow cytometry. (g-h). Intracellular IL-10 and IL-12 levels on gated CD11c + DCs from TdLNs. For intracellular cytokine staining, cells were stimulated with Cell Activation Cocktail (with Brefeldin A) (1:500) for 4–5 h before being harvested for cell surface staining, after which cells were fixed and permeabilized and stained with IL-10, and IL-12. Results are shown as the means ± SEM (standard errors of the mean) for one experiment (n = 5). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. DCs, dendritic cells; IgG, immunoglobulin G; LNs, lymph nodes; NS, not statistically significant; RT, radiotherapy; TdLNs, tumor draining lymph nodes; TIGIT, T cell immunoreceptor with immunoglobulin and ITIM (immunoreceptor tyrosine-based inhibitory motif) domains

Fig 3: CD103 + DCs are critical for effective anti-tumor responses to RT combined with anti-TIGIT therapy. a. Frequency of CD103 + DCs among total myeloid cells infiltrating MC38, B16-F10, and LLC tumor models. Myeloid cells were gated on CD11b + and/or CD11c + cells within CD45 + cells. b. Quantification of CD155 on CD103 + DCs purified from MC38 TdLNs, non-draining LNs, and healthy mouse LNs on day 10 following tumor challenge. The gating strategy is shown in Fig. S9. c. MC38 tumor-bearing WT or BATF3−/− mice were treated with RT plus anti-TIGIT therapy or RT alone on day 10 after the tumor challenge. Data are shown as mean ± SEMs (standard errors of the mean) for two independent experiments (n = 4–5). (d-e). The production of IFN-γ (interferon gamma) and TNF-α (tumor necrosis factor alpha) by CD8 + T-cells in tumor tissues d and TdLNs e was analyzed herein. f. WT and BATF3.−/− mice were inoculated with MC38 cells and treated with RT and anti-TIGIT therapy as described in Fig. 3a. Moreover, 200 μg of anti-CD8 monoclonal antibodies (mAb) was administered as described in Fig. 5a. Tumor growth was monitored after RT. Data are shown as means ± SEM (standard errors of the mean) of two independent experiments (n = 5). *p < 0.05; ***p < 0.001; ****p < 0.0001. BATF3, basic leucine zipper transcription factor ATF-like 3; DCs, dendritic cells; LNs, lymph nodes; mAb, monoclonal antibodies; NS, not statistically significant; RT, radiotherapy; TdLNs, tumor draining lymph nodes; TIGIT, T cell immunoreceptor with immunoglobulin and ITIM (immunoreceptor tyrosine-based inhibitory motif) domains; WT, wild type

Fig 4: Flt3L injections improved the response of WT mice to treatment with RT and TIGIT mAb. a. WT MC38 tumor-bearing mice (n = 5 mice/group) were treated with local tumor RT in a single 15 Gy dose given on day 10. The mice received anti-TIGIT mAb on days 10, 13, 16, and 19. Some mice received Flt3L (10 ng/mouse/injection) on days 1–9 post tumor inoculation, which was injected intraperitoneally for nine consecutive days after tumor inoculation. b. Tumor growth is shown for each group. The data are presented as mean tumor growth ± SEM (standard errors of the mean) of two independent experiments (n = 5). Fractions indicate the number of mice showing complete tumor regression over the total in each group. c. Survival times were followed until day 90 for each group. Data shown are from one of two independent experiments performed with similar results. (d-e). Representative multiple immunofluorescence images of DCs infiltrating MC38 tumors treated with nine daily injections of Flt3L (10 ng/mouse/injection), which was injected intraperitoneally into the mice for nine consecutive days after tumor inoculation or isotype IgG starting on day 1 after tumor inoculation. Tumor biopsies were performed on day 18 after the tumor inoculation, DCs were identified as F4/80-CD11c + cells. f. The absolute number of CD103 + /CD11c + DCs standardized to tumor weight. g. On day 18, the mice were sacrificed and the percentages of CD8 + cells among the CD45 + cells were analyzed using flow cytometry. h. The production of IFN-? (interferon gamma) and TNF-a (tumor necrosis factor alpha) by CD8 + T-cells was also analyzed. See Fig. S2 for the gating strategy. Data are shown as means ± SEM (standard errors of the mean) for two independent experiments (n = 4–5). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. DCs, dendritic cells; IgG, immunoglobulin G; FLT3L, factor FMS-like tyrosine kinase 3 ligand; mAb, monoclonal antibody; RT, radiotherapy; TIGIT, T cell immunoreceptor with immunoglobulin and ITIM (immunoreceptor tyrosine-based inhibitory motif) domains; WT, wild type

Fig 5: The effects of liposomal honokiol (Lip-HNK) on the progression of glioblastomas in a G422 xenograft model. G422 glioma cells were administered subcutaneously into the flanks of ICR mice, which were then treated with Lip-HNK (12.5 or 25 mg/kg per day) or temozolomide (TMZ) (50 mg/kg). The weights of the tumors (A) and tumor volume changes (B) were measured. Moreover, visualization of the infiltrating M1 macrophages in tumor sections was achieved using immunofluorescence staining of CD11c and F4/80 (C). Visualization of the tumor sections infiltrated by M2 macrophages was achieved using immunofluorescence staining of F4/80 and CD206 (D). The F4/80+CD11c+ area (E) and the F4/80+CD206+ area (F) (% of F4/80+ tumor area) were quantified as the area of intersecting fields. Five randomly selected fields from mouse xenograft tumor sections were examined. *, P<0.05; **, P<0.01; ***, P<0.001.