Fig 1: Membrane-bound IL-12 engineered TAG72-CAR T cells induce higher IFNγ, T-cell expansion, and anti-tumor activity in vitro.a–c Tumor cell killing of OV90 cells by TAG72-CAR T cells (E:T = 1:20) with the addition of varying concentrations of anti-IFNγR1 blocking antibody, isotype control, and recombinant human IL-12 cytokine measured by xCELLigence over 10 days (a, b). n = 2/group at each timepoint. Data are presented as mean values ±SD. At day 10, IFNγ levels in supernatants were quantified by ELISA (c). n = 2/group, representative of two independent experiments. Data are presented as mean values ±SD. d Illustration of TAG72-CAR/mbIL12 T cell. e Flow cytometric analysis of surface or intracellular expression of mbIL12 in TAG72-CAR T cells stimulated with varying concentrations of plate-bound TAG72. n = 2/group, representative of two independent experiments. Data are presented as mean values ±SD. f Intracellular flow cytometric analysis of phosphorylated STAT3 (pSTAT3, pY705) (left) and pSTAT4 (right) in TAG72-CAR and TAG72-CAR/mbIL12 T cells stimulated with varying concentrations of plate-bound TAG72 or recombinant huIL12 (10 ng/mL). g Intracellular flow cytometric analysis of pSTAT4 in TAG72-CAR T cells co-cultured with HT1080 (TAG72−) cells transduced with mbIL12. Cells were stimulated with Immunocult CD3/CD28 per manufacturer’s recommendation. Cells were gated on CAR T cells and evaluated for pSTAT4. h TAG72-CAR/mbIL12 T cells were co-cultured with OV90 cells (E:T = 1:3) and rechallenged with OV90 cells every 2 days. The remaining viable tumor cells and TAG72-CAR T-cell proliferation were quantified as described in Methods prior to each tumor cell rechallenge. n = 3/group, representative of two independent experiments. Data are presented as mean values ±SD. P values indicate differences between TAG72-CAR and TAG72-CAR/mbIL12 using a two-tailed Student’s t test.
Fig 2: IAV drives an increase in global CD3+ immune T cell infiltration and activation predominantly in the PVAT and periadventitial space of pregnant mice when compared to the arterial wall.Representative Immunofluorescence image of pregnant PBS and X-31 mice arterial wall and PVAT labelled with CD3 antibody (red) and counterstained with DAPI (blue). Data are representative of pregnant PBS, n = 5–6; pregnant X-31, n = 4–6; of at least two independent experiments.
Fig 3: Changes in serum transaminase levels, liver histology, cell apoptosis, and apoptosis-related proteins after IRI with or without IPC. (A) Serum alanine transaminase (ALT) and aspartate transaminase (AST) levels determined using enzyme-linked immunosorbent assay (*P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.05). (B-C) Liver histology examined using hematoxylin and eosin (HE) staining, and cell apoptosis examined using TdT-mediated biotin-16-dUTP nick-end labeling (TUNEL) assay (magnification, ×400; *P < 0.05, **P < 0.01 vs. control; ##P < 0.01 vs. IRI; n = 3 for each experiment). (D) Expression of apoptosis-related proteins measured using western blot analysis (*P < 0.05, ***P < 0.001 vs. control; ##P < 0.01 vs. IRI; n = 3 for each experiment). (E-F) CD68- and CD3-positive cells on immunohistochemical analysis (**P < 0.01, ***P < 0.001 vs. control; n = 3 for each experiment). (G) Detection of IL-6, CXCL-1, CXCL-2, TNF-a, IL-1β, and IFN-γ using quantitative polymerase chain reaction assays (*P < 0.05, **P < 0.01, ***P < 0.001 vs Control; #P < 0.05, ##P < 0.01, ###P < 0.001 vs IR). (H) Myeloperoxidase (MPO) concentration determined using enzyme-linked immunosorbent assay (*P < 0.05 vs Control; #P < 0.05 vs IR). (I) Western blot analysis of TIM4 expression (***P < 0.001 vs. control; n = 3 for each experiment). (J) TIM4 mRNA levels determined using by quantitative polymerase chain reaction assay (*P < 0.05, ***P < 0.001 vs Control; #P < 0.05 vs IR). (K) Flow cytometric analysis of TIM4-positive cells in serum samples.
Fig 4: Associations between TILs and underlying genomic changes.Violin plots showing the association between TP53 variants and a stromal CD3+ cells, b stromal CD4+ cells, c stromal FOXP3+ cells and d stromal PDL1 + TILs. The lower figures show the association between fraction of genome altered and e overall TILs density and f stromal CD3+ cells. The central red dots represent the mean, boxes represent the interquartile range, central line represent the median and whiskers shows the 95% confidence interval.
Fig 5: Role for T-cell immunity in virus-mediated tumor clearance. a Schematic representation of T-cell depletion study. b Confirmation of T-cell depletion by IP anti-CD4, anti-CD8, or isotype treatment 24 h prior to harvesting. Splenocytes were isolated 24 h after IP administration of anti-T-cell depletion antibodies in naïve FVB/N mice, and probed for CD3 + , CD4 + , and CD8 + T-cells as measured by flow cytometry. Live spenocytes were gated using forward and side scatter, and then gated on CD4-FITC and CD8-PE. Single-color positive quadrants (Q1 for CD8 + and Q3 for CD4 + ) were interrogated to determine depletion. c Survival of DB7 tumor-bearing FVB/N mice treated with saline control or HSV-P10 7 days post tumor cell implantation, and isotype or anti-CD4 antibodies 2, 4, and 7 days post virus injection. Significance in survival was assessed by Logrank (Mantel–Cox) test (n = 10, **p < 0.01). d Survival of DB7 tumor-bearing FVB/N mice treated with saline control or HSV-P10 7 days post tumor cell implantation, and isotype or anti-CD8 antibodies 2, 4, and 7 days post virus injection as indicated. Significance in survival was assessed by Logrank (Mantel–Cox) test (n = 10, **p < 0.01). e 51Cr release from uninfected DB7 tumor cells co-cultured with CD3 + splenocytes from untreated naive or immune mice from Fig. 5d. Data shown are averages ± s.d. Statistical significance was assessed by one-tailed Student’s T-test (n = 3, *p < 0.05)
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