Fig 1: Combining vaccinia virus expressing IL-2 with immune checkpoint therapy. B6 mice were inoculated with 5 × 105 MC38-luc cells and treated with PBS, vvDD, vvDD-IL-2-FG, or vvDD-IL-2-RG at 2 × 108 PFU per mouse 9 days post-tumour inoculation. Tumour-bearing mice were sacrificed 5 days post-treatment and primary tumours were collected and analysed using RT-qPCR to determine the expressions of PD-1 (a), PD-L1 (b), and CTLA-4 (c) in the tumour microenvironment. Five mice were used for each treatment group in one experiment and data are combined from three independent experiments. In a separate experiment, B6 mice were i.p. inoculated with 5 × 105 MC38-luc cells and treated with vvDD/vvDD-IL-2-RG or PBS 9 days post-tumour inoculation. Anti-PD-1 Ab (200 µg per injection), anti-PD-L1 Ab (200 µg per injection), or anti-CTLA-4 Ab (100 µg per injection) were i.p. injected into mice as scheduled (nine mice per group) (d), and a log-rank (Mantel-Cox) test was used to compare survival rates (e, f). In some experiments, to measure the abscopal effect, B6 mice were also s.c. inoculated with 5 × 105 MC38-luc 4 days after i.p. tumour inoculation, and treated with i.p. vvDD-IL-2-RG alone or combined with α-PD-1/PD-L1 Ab as scheduled (ten mice per group) (g) to monitor s.c. tumour growth (h, i). *P<0.05; **P<0.01; ***P<0.001; and ****P<0.0001. ns: not significant
Fig 2: The TME Is Dynamically Modified Post-dual Therapy(A–F) MC38-luc tumor-bearing mice were sacrificed 1 day after the first or second injection of CpG in different groups (n = 6 in each group), and tumor nodules on both sides were collected and digested for qRT-PCR to explore the cancer-immune set point. The expression levels of all markers are relative to the housekeeping gene, HPRT1. (A–C) Results show the levels of perforin (A), granzyme B (B), and IFN-γ (C), 1 day after the first injection of CpG in the treated side. (D–F) 1 day after the second injection of CpG, the levels of CD8 (D), TGF-β (E), and CD105 (F) from the untreated side are also presented. (G–N) MC38-luc tumor-bearing mice were euthanized 1 day after finishing the whole treatment, and tumor nodules from both sides were collected and lysed for flow cytometry to analyze the TME. Data are quantified (n = 8 in each group). (G–L) Quantities of activated TNF-α+CD8+ T cells (G) and IFN-γ+CD4+ T cells (H); the ratio of more severely exhausted PD-1+CTLA-4+CD8+ T cells (I); and quantities of NKs (defined as CD45+CD3−NK1.1+) (J), macrophages (defined as CD45+CD11b+ F4/80+) (K), and Tregs (defined as CD45+CD4+FOXP3+) (L) from both sides are shown. (M and N) On the untreated side, the number of myeloid-derived suppressor cells (MDSCs) (defined as CD45+CD11b+Gr-1+) (M) and DCs (defined as CD45+CD11c+) (N) is also presented. Values are presented as mean ± SD. Two-way ANOVA was used to analyze the data from (G) to (L). One-way ANOVA was used to analyze the statistical significance in other panels (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; and ns, not significant).
Fig 3: Tumor microenvironment (TME) is dynamically modified post combination therapy. Tumors are from the experiment depicted in Figure 5C, which were used to isolate RNA for RT-qPCR analyses. Expression levels of all markers are expressed relative to the housekeeping gene HPRT1. The expression levels of (A) CD4, (B) CD8, (C) IFN-γ, (D) TNF-α, (E) PD-1, and (F) CTLA-4 are presented. The symbols for the statistics are, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.
Fig 4: (A) Experimental timeline of the dose-efficacy study, n=4. CD45.2+ mice that had received an adoptive transfer of both OTI (CD45.1+CD3+CD8+) and OTII (CD45.1+CD3+CD4+) T cells via i.v. injection, were treated with saline or a low, mid or high dose of OVA-p(GluNAc) s.c. in all four hocks or i.v. in the tail vein (as benchmark) either once on day 1 or twice on days 1 and 8. 9 days following the last dose, on day 10 (for the groups that received one dose) or on day 17 (for the groups that received two doses), all mice were administered an OVA+LPS challenge s.c., and 5 days later, the dLNs and spleen were examined for an OVA-specific response. Stars above horizontal bars represent p values with respect to the i.v. groups (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001) and † indicate p values with respect to the saline group (†p ≤ 0.05, ††p ≤ 0.01). (B) OTI CD8+ T cells recovered from dLNs at time of sacrifice. Plot legends are as follows: 1-1 (1 μg s.c., once), 15 (5 μg s.c., once), 1-20 (20 μg s.c., once), 1-5 i.v. (5 μg i.v., once), 2-1 (1 μg s.c., twice), 2-5 (5 μg s.c., twice), 2-20 (20 μg s.c., twice) and 2-5 i.v. (5 μg i.v., twice). (C–R) Data are representative of three pooled experiments performed at the optimal high 20 μg dose of OVA as unconjugated OVA or OVA-p(GluNAc) injected twice on days 1 and 8, followed by an OVA+LPS challenge on day 17 and sacrifice on day 22, n=8-20. (C) OTI CD8+ T cells recovered from dLNs. (D) PD-1+ OTI CD8+ T cells in dLNs. (E) Lag-3+ OTI CD8+ T cells in spleen. (F) Tim-3+ OTI CD8+ T cells in spleen. (G) MFI of the TCR on OTI CD8+ T cells in dLNs. (H) IFNγ MFI of IFNγ secreting OTI cells after a 6-h ex vivo restimulation with OVA257-264 peptide. (I) IL-10 producing OTI CD8+ T cells after a 6-h ex vivo restimulation with OVA257-264 peptide. (K) OTII CD4+ T cells recovered from dLNs. (L) Foxp3+CD25+ OTII CD4+ Tregs induced in dLNs. (M) Foxp3+ST2+ OTII CD4+ Tregs induced in dLNs. (N) Lag3+ OTII CD4+ T cells in dLNs. (O) CTLA-4+ OTII CD4+ T cells in dLNs. (P) MFI of the TCR on OTII CD4+ T cells in dLNs. (P) IL-13 levels in the supernatant of LN cells restimulated with 100 μg/mL OVA protein for 4 days, measured by LegendPlex assay. (Q) OTII CD4+ T cells from the dLNs (left) or spleen (right) that secreted IFNγ, IL-2, TNFα, or a combination of two or all three cytokines after a 6-h ex vivo restimulation with OVA323-339 peptide. (R) IL-10 producing OTII CD4+ T cells after a 6-h ex vivo restimulation with OVA323-339 peptide. Data represent mean ± SD. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 by one-way ANOVA using Tukey’s post hoc test.
Fig 5: Pulsatile Treatment of Selumetinib Induces CTLA-4 and PD-1 Expression In VivoHKP1 transplantable lung-tumor-bearing mice were treated with selumetinib (25 mg/kg, BID) as presented in (A). After 2 weeks of treatment, lungs were collected and analyzed by flow cytometry.(A) Schema of selumetinib treatment in HKP1 lung-tumor-bearing mice in vivo.(B) Frequency of CD3+ T cell subsets in lung tumors by flow cytometry.(C) Ki-67 of diverse cell populations in lung tumors by flow cytometry.(D) Scatterplots of PD-1 and CTLA-4 marker (left) and co-inhibitory marker expression from CD3+ T cell subsets of lung tumors by flow cytometry (right). Gating controls are samples without either PD-1 or CTLA-4 antibodies.*p < 0.05; **p < 0.01; ***p < 0.001, Mann-Whitney test. Samples were biological replicates. The experiment was performed 3 times, and representative results are shown here.
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