Fig 2: DNMTi+ HDAC6i combination treatment improves response and changes the TME in the ID8 Trp53-/- mouse model of ovarian cancer. (A) C57Bl6 mice were injected with ID8 Trp53-/- cells and treated with 0.5 mg/mL Aza daily every other week and 25 mg/kg NextA daily every other week, alternating weeks as in Fig. 5A. The NextA + Aza group had significantly longer survival compared to the Mock group (p = 0.0128 by log-rank (Mantel-Cox) test)). Ascites were isolated at Week 9 and immunophenotyping was performed to measure: (B) Natural Killer (NK) cells as % NK of CD3- immune cells, (C) Natural Killer T cells as % NK of CD3+ immune cells, (D) Activated NK cells (as %IFNg+ of NK cells), (E) Activated NKT cells (as %IFNg+ of NK+ CD3+ cells), (F) Myeloid Derived Suppressor Cells (MDSCs), and (G) ratio of M1 (defined as CD11b+/Ly6C+ Ly6G-/F480+) to M2 (defined as CD11b+/Ly6C-Ly6G+/F480+) macrophages. T-tests were performed to test significance; *p < 0.05. Plots without * indicate non-significant differences.

Fig 3: Representative FACS contour plots showing Live CD45+CD3+TCRβ+ cells and the consequences of excessive concentration of Dispase used for gut digestion on the CD4 markerIdentical protocols were used to isolate intestinal immune cells from mouse #1 and #2 except for the concentration of Dispase as indicated. Lung and mesenteric lymph nodes were used as controls. Anti-CD45 BV786 (30-F11, BD Bioscience, CAT# 564225, 1/1000), anti-KLRG1 BUV395 (2F1, BD Bioscience, CAT# 740279, 1/200), anti-CD3 APC-eF780 (17A2, eBioscience, CAT# 47-0032-82, 1/200), anti-TCRβ PerCP-Cy5.5 (H57-597, BioLegend, CAT# 109228, 1/200), and anti-CD4 APC (GK1.5, eBioscience, CAT# 17-0041-83, 1/200) antibodies together with the fixable viability dye eF506 (eBioscience, CAT# 65-0866-14, 1/800) were used.

Fig 4: Smyd3 KO in the cancer cell compartment of MOC1 tumors induces an influx of CD8+ T-cells which are necessary for the anti-tumor efficacy of anti-PD-1 therapy(A–E) Multicolor flow cytometry of control (NC2) and Smyd3 KO (KO13) MOC1 tumors treated or not with anti-PD-1. C57BL/6 mice were injected with NC2 and KO13 cells in the right flank, and, once they reached an average tumor volume of 0.01cm3, treatment with anti-PD-1 was started. 34 days after tumor implantation, mice were sacrificed, and tumors were surgically resected and processed into single-cell suspensions. Cell suspensions were stained with antibodies and multicolor flow cytometry was conducted (day 34 post-tumor implantation, n = 4 per group). Data are represented as mean ± SEM. NC2: control, KO13: Smyd3 KO. (A) % of CD45−CD31-PDGFR-H-2Kb+ or PD-L1+ MOC1 cells, (B) % of CD3+ CD45+ cells, (C) % of CD3+CD8+ and CD3+CD8+/PD-1+ of CD45+ cells, (D) % CD3+CD4+ of CD45+ cells, (E) % of CD11b+/CD11c-/F4/80- of CD45+ cells. Unpaired t-tests between NC2 and KO13 or NC2 + anti-PD-1 and KO13 + anti-PD-1 conditions: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.(F) CD8+ T cell depletion abrogates the anti-tumor efficacy of anti-PD-1 in Smyd3 KO MOC1 tumors. C57BL/6 mice were injected with NC2 and KO10 cells in the right flank, and, once they reached an average tumor volume of 0.01cm3, mice were randomized into three groups, and treatment with anti-PD-1, anti-PD-1, and anti-CD8 or anti-PD-1 and anti-CD4 was started. Unpaired t-test, ∗p = 0.039.

Fig 5: T cells were among the biomarkers and therapeutic targets in patients with stroke and the establishment of a humanized T cell ischemic stroke model mimicking the clinical scenario(A) Dot plots showing human T cells/immune cells (CD3+/CD45+) collected from the peripheral blood in patients with stroke and healthy controls (control).(B) Statistical analyses of T cells were performed using unpaired t tests. Data are presented as the mean ± standard deviation (SD).(C) Receiver operating characteristic (ROC) curve of T cells.(D) Experimental design for single-cell RNA sequencing (scRNA-seq) in humanized mice; single cells were collected from two replicates (eight mice in each replicate) for each group.(E) Dot plots and statistical analyses showing hCD3/mCD3 cells collected from the peripheral blood before the injection of human peripheral blood mononuclear cells (PBMCs) and 5 weeks after injection.(F) Scatterplots of flow cytometric analysis of T cells (CD3+/CD45+) in the blood of control and transient middle cerebral artery occlusion (tMCAO) 3D mice with humanized T cells.(G) Uniform manifold approximation and projection (UMAP) plot of eight humanized T cell subpopulations in mice with humanized T cells. Each dot corresponds to a single cell, and cells are colored according to cell type.(H) Proportion of each subtype of humanized T cells in humanized mice shown as a bar plot.(I) Violin plots of marker genes in each subtype of humanized T cells.Statistical analyses (E and F) were performed using unpaired t tests. Data are presented as the mean ± SD.