Fig 2: IL-15 costimulation and p38-MAPK blockade specifically empowers DC vaccines to generate IL-17+ T cells and high avidity antibodies in vivo in addition to IFN-γ+ and IL-4+ T cells. (A–C) Representative ELISpot wells and min/max box and whisker plots (n=9–18) depicting the number of antigen-specific IFN-γ+ (A), IL-4+ (B), and IL-17+ (C) T cells per million splenocytes following vaccination with PBS or with cDCs or Th17-DCs that were either pulsed or not pulsed with tumor lysate antigen (Ag). (D–E) The min/max box and whisker plots showing total tumor (D) and high avidity (E) antigen-specific- IgG antibody levels (n=12–18) in the blood. The results shown are derived from three independent experiments. Each symbol is a unique replicate. P values shown in (A–C) compare Ag+DC and Ag+Th17 DC. P values were calculated with one-way ANOVAs followed by the Tukey’s multiple comparisons test (A–C) or Fisher’S LSD test (D–E). ANOVA, analysis of variance; cDC, conventional dendritic cell.

Fig 3: Vaccination induces infiltration of T cells into tumor tissue and extends the lifespan of mice bearing OC. (A) Kaplan-Meier survival analysis of mice (n=13–16/group) immunized with PBS control, non-antigen pulsed cDCs (cDCs), non-antigen pulsed Th17-inducing DCs (Th17-DCs), antigen-pulsed cDCs (Ag+cDCs) or antigen-pulsed Th17-DCs (Ag+Th17 DCs). (B) Kaplan-Meier survival analysis of mice (n=14–25/group) immunized with PBS control, or antigen-pulsed Th17-DCs (Ag+Th17 DCs) with or without CD4 (αCD4) or CD8 (αCD8) T cell depletion. (C) The mean (±SE, n=6) levels of IL-17 in the ascites fluid of representative tumor-bearing mice at sacrifice in the same groups depicted in (A). (D) CD3, CD4 and CD8 IHC analysis in tumor tissue harvested at sacrifice in mice treated with PBS, antigen-pulsed cDCs or antigen-pulsed Th17-DCs. (E–G) The min/max box and whisker plots depicting the levels of CD3 (E), CD4 (F), and CD8 (G) T cells per field analyzed. (H) Min-max box and whiskers plots of levels of antigen-specific antibodies (n=12–18) in blood and ascites. (I) Kaplan-Meier survival analysis of either wild-type (WT) or IL-17 knockout (KO) mice (n=5–10/group) immunized with PBS or antigen-pulsed Th17-DCs (Th17-DCs). Inset p values (**p<0.05, ****p<0.0001) for (A, B) were calculated using Mantel-Cox log rank test. P values for (C–H) were calculated with one-way ANOVA followed by Fisher’s LSD test. Light blue inset lines on the x-axis in A, D, I show approximate treatment period. ANOVA, analysis of variance; cDC, conventional dendritic cell; IHC, immunohistochemistry; PBS, phosphate buffered saline; MDSC, myeloid derived suppressor cells.

Fig 4: Th17-DC vaccination synergizes with immune checkpoint blockade in a CD4 T cell dependent manner. (A) PD-L1 staining of the tumor cells derived from the peritoneal cavity on Day 42. (B) The min/max box and whisker plots (n=10/group) depicting PD-1 expression on surface of CD4 and CD8 T cells derived from mice vaccinated with either PBS or antigen-pulsed Th17 DC vaccines. P values were calculated with an unpaired Student’s t-test. C) A Kaplan-Meier curve comparing survival in tumor-bearing mice (n=8/group) immunized with PBS, antigen-pulsed Th17-DC vaccines, anti-PD-1 (αPD-1), the combination of anti-PD-1 and Th17-DC vaccine (*p<0.05, ****p<0.0001). P values were calculated using Mantel-Cox log rank test. Light blue inset line on the x-axis shows approximate treatment period. (D) The min/max box and whisker plots (n=9–18) depicting the levels of splenic antigen-specific IFN-γ+, IL-4+ and IL-17+T cells at day 42 in tumor-bearing mice immunized and treated with αPD-1 as in (B). (E) Levels of serum antibodies targeting tumor antigens at day 42. P values in (D) and (E) were calculated with one-way ANOVA followed by Fisher’s LSD test. (F) The min/max box plots (n=2–7/group) number of cells recovered from the peritoneal cavity at day 42 in mice immunized with PBS, Th17-DC vaccine, αPD-1 or combination Th17-DC vaccine and αPD-1. NS=Not significant, P value calculated by Mann-Whitney U test. (G) The min/max box plots depicting the distribution of peritoneal immune cells in the lymphocyte (lymphs), monocyte (DCs/Macs), and granulocyte (Gran) gates from mice treated as in (F) (n=4–6/group). (H) The min/max box plots depicting the relative levels of total and activated (CD69+) CD4+ and CD8+ T cells and B cells and NK cells in the lymphocyte gate at day 42 following treatment of mice as described in (E) (n=4–6/group). (I) The relative levels of CD11b+CD11c+DCs, CD11b+F4/80+macrophages (Macs) and CD11b+GR-1+ MDSCs in the monocyte gate at day 42 following treatment of mice as described in A (n=4–6/group). (J) The relative levels of CD11b+Ly6G+ (neutrophils, Neut), CD11b+CD193+SiglecF+ (eosinophils, Eosin) and CD11b+CD200 R3+FcεRIα+ (Basophils, Baso) in the granulocyte gate at day 42 following treatment of mice as described in A (n=4–6/group). P values for (F–J) were calculated using a one-way ANOVA followed by Fisher’s LSD post hoc test. (K) The min/max box plots of IL-10 levels (pg/mL) in ascites fluid of moribund mice (n=3–10) following treatment as described in (C). P values were calculated with unpaired Student’s t-test. ANOVA, analysis of variance; cDC, conventional dendritic cell; PBS, phospate buffered saline; MDSCs, myeloid derived suppressor cells.