Fig 2: Tumor specific T cell and antibody analysis 10 and 30 days post AOaV-1 administration, respectively. Retro-orbital blood samples were taken 10 days after mice received an i.v. injection of 1 × 108 PFU AOaV-1 or PBS. Red blood cells were lysed, and the remaining cells were stained and analyzed via flow cytometry. Graphed are the number of activated tumor specific TNF-α+IFN-γ+ CD8+ (A) and CD4+ (B) T cells in the blood (n = 6 mice per group). (C) Tumor specific antibodies in the serum from treated mice were quantified by flow cytometry and the mean fluorescent intensity (FITC channel) graphed (n = 6 mice per group). AOaV-1, avian orthoavulavirus-1; AAV, Adeno-associated virus; PBS, phosphate-buffered saline mock control group; 3TSR, thrombospondin-1 type I repeats; * p < 0.05; ** p < 0.01; ns = not significant. Error bars denote standard deviation.

Fig 3: CD8+ T cells have intrathymic deletion of histone deacetylase 3 (HDAC3) in dLck-HDAC3 cKO, but CD4+ T cells initiate deletion at the recent thymic emigrant (RTE) stage.(a) Profile of primary splenic T-cell populations from wild-type (WT) and dLck-Cre HDAC3 cKO mice including total T cells (TCRß+), total CD4+ (TCRß+ CD4+), and total CD8+ (TCRß+ CD8+), as well as memory (CD44hi CD62Llo) and naive (CD44lo CD62Lhi) from each of the CD4+ and CD8+ populations. Bar graph depicts mean ± standard deviation (SD). Total cell number from three independent experiments (n = 5 mice/group). Statistical significance was determined for the indicated comparisons with Mann–Whitney tests between each WT and cKO population. (b) Expression of HDAC3 in thymocyte populations from WT, dLck-Cre HDAC3 cKO and CD4-Cre HDAC3 cKO mice. Thymic populations are gated as in Figure 1—figure supplement 1, and quantification of normalized HDAC3 MFI (median fluorescent intensity) ± SD from three independent experiments is shown on the right (n = 2–4 mice/group). Statistical significance was determined for the indicated comparisons with a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. (c) Profile of HDAC3 deletion in dLck-Cre HDAC3 cKO mice. Splenocytes were gated on key populations including total CD4+ (CD4+), naive CD4+ (CD4+ CD44lo CD62Lhi), and memory CD4+ (CD4+CD44hiCD62Llo). Naive cells were further gated in mature naive T cells (MNTs; CD45RBhiCD55hi) or recent thymic emigrants (RTEs; CD45RBloCD55lo). HDAC3-positive and -negative population frequencies are shown below. Bar chart on right quantifies the mean frequency ± SD of HDAC3+ or HDAC3- events within each population (n = 8 mice/group from three independent experiments).

Fig 4: Induction of Surface-Expressed MHC on ID8 Cancer Cells Is Required to Detect T Cell Responses following Antigen-Agnostic Immunotherapy(A) C57BL/6 mouse-derived ID8 ovarian cancer cells did not express MHC class I or class II (top panel) unless stimulated with 50 U rIFNγ for 48 h (bottom panel). (B) Pre-stimulation of ID8 cells with rIFNγ was required for the detection of tumor-specific CD8+ T cells in the cancer cell-leukocyte co-culture assay (one-way ANOVA with Dunnett’s multiple comparison test; n = 14/group). (C) Pre-stimulation of ID8 cells with rIFNγ was also needed to detect tumor-specific CD4+ T cells (one-way ANOVA with Dunnett’s multiple comparison test; n = 11/group). Co-culture with unstimulated ID8 tumor cells failed to result in IFNγ expression from either CD8+ or CD4+ T cells. ns, not significant.

Fig 5: NK cells correlate with cDC1s and T cells in the human ovarian TME and are linked to better overall survival. (A) Gene signatures used to identify cDC1, NK, CD8+ and CD4+ T cells from the ICGC human ovarian cancer dataset. (B) Bivariate correlation comparing cDC1s and NK cells in the ICGC human ovarian cancer dataset. (C) Bivariate correlation comparing CD8+ T cells and NK cells in the ICGC human ovarian cancer dataset. (D) Bivariate correlation comparing CD8+ T cells and cDC1s in the ICGC human ovarian cancer dataset. (E) Bivariate correlation comparing CD4+ T cells and cDC1s in the ICGC human ovarian cancer dataset. (F) Log-rank test of ICGC patient samples binned according to the highest and lowest thirds of NK-cell signature expression. (G) Log-rank test of ICGC patient samples binned according to the highest and lowest thirds of cDC1-cell signature expression. (H) Log-rank test of ICGC patient samples binned according to the highest and lowest thirds of CD4+ T-cell signature expression. (I) Log-rank test of ICGC patient samples binned according to the highest and lowest thirds of CD8+ T-cell signature expression. (J) Bivariate correlation comparing the KLRG NK-cell signature and the cDC1 signature in the ICGC human ovarian cancer dataset. (K) Log-rank test of ICGC patient samples binned according to the highest and lowest thirds of KLRG1 NK-cell signature expression. cDC1, classical type 1 dendritic cell; ICGC, International Cancer Genome Consortium; KLRC, killer cell lectin-like receptor; NK, natural killer; TME, tumor microenvironment.