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Cancer Immunology Research
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IRF1 Inhibits Antitumor Immunity through the Upregulation of PD-L1 in the Tumor Cell

Lulu Shao, Weizhou Hou, Nicole E. Scharping, Frank P. Vendetti, Rashmi Srivastava, Chandra Nath Roy, Ashley V. Menk, Yiyang Wang, Joe-Marc Chauvin, Pooja Karukonda, Stephen H. Thorne, Veit Hornung, Hassane M. Zarour, Christopher J. Bakkenist, Greg M. Delgoffe and Saumendra N. Sarkar
Lulu Shao
1Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania.
2Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
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Weizhou Hou
1Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania.
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Nicole E. Scharping
3Tumor Microenvironment Center, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania.
4Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
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Frank P. Vendetti
5Department of Radiation Oncology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
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Rashmi Srivastava
1Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania.
2Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
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Chandra Nath Roy
1Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania.
2Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
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Ashley V. Menk
3Tumor Microenvironment Center, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania.
4Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
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Yiyang Wang
3Tumor Microenvironment Center, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania.
4Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
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Joe-Marc Chauvin
4Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
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Pooja Karukonda
5Department of Radiation Oncology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
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Stephen H. Thorne
1Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania.
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Veit Hornung
6Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany.
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Hassane M. Zarour
4Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
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Christopher J. Bakkenist
5Department of Radiation Oncology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
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Greg M. Delgoffe
3Tumor Microenvironment Center, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania.
4Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
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Saumendra N. Sarkar
1Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania.
2Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
4Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
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  • For correspondence: saumen@pitt.edu
DOI: 10.1158/2326-6066.CIR-18-0711 Published August 2019
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    Figure 1.

    Knockout of IRF1 in tumor cells causes a loss of in vivo tumorigenicity. A–C, Cell growth rates of MC38, B16-F10, and CT26 WT and IRF1-KO in vitro. Cells were seeded and cultured in 10 wells of cell culture plates, and cell numbers were counted daily (n = 3). For each data point mean and SEM were plotted. D–F, Tumor growth rates of MC38 (n = 5), B16-F10 (n = 5), and CT26 (n = 5) WT and IRF1-KO cells in C57BL/6 and/or BALBc mice. Mice were subcutaneously or intradermally injected with 106 (MC38 and CT26) or 5 × 105 (B16-F10) cells, followed by tumor growth measurements. Representative results from at least twice repeated experiments are shown. PR, partial response; CR, complete response.

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    Figure 2.

    Host CD8+ T cells are required for the loss of tumorigenicity in IRF1-deficient tumor cells. A, Tumor growth of MC38 IRF1-KO cells in CD8+ T-cell depletion C57BL/6 mice. 106 of MC38 WT or IRF1-KO cells were subcutaneously injected into C57BL/6 mice (n = 8). IRF1-KO cell injection groups of mice were intraperitoneally injected with 250 μg of anti-CD8 or rat IgG2b isotype control 2 days before tumor cell injection, then followed by antibody or isotype control injections twice a week. A group of WT MC38 cell–injected mice were intraperitoneally injected with anti-CD8 as control. Representative results from at least twice-repeated experiments are shown. B, MC38 IRF1-KO cells induced WT tumor regression. Following complete regression of MC38 IRF-1 KO tumors (n = 8), mice were rechallenged with MC38 WT tumor cells on day 47 of postinjection. Seven of 8 mice showed complete tumor regression. Representative results from at least twice-repeated experiments are shown.

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    Figure 3.

    Loss of IRF1 in tumor does not influence the frequencies of tumor-infiltrating lymphocytes populations. A, A total of 5 × 105 of B16-F10 WT or IRF1-KO cells were intradermally injected into C57BL/6 mice (n = 13, 12). Tumors were collected around size 100–200 mm3 on days 12–14 postinjection. B, Representative flow cytogram (left) and tabulated (right) percentage of exhausted T cells and PD-1 intermediate expression CD8+ T cells (activated T cells) in B16-F10 WT and IRF1-KO tumor groups. C, Representative flow cytogram (left) and tabulated (right) percentage of Tregs in B16-F10 WT and IRF1-KO tumor groups. The ratio of CD8+ T cells: Tregs (D) and the frequencies of PD1+, Tim3+, LAG3+ in CD8+ T cells (E) in the two groups. For each column mean and SEM were plotted. Statistical significance was calculated by unpaired Student t test.

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    Figure 4.

    PD-L1 expression in vitro and in vivo is specifically affected by IRF1 loss. B16-F10 WT and IRF-1 KO cells were treated with mouse IFNγ for 0, 2, 4, 6, and 8 hours, and collected for the detection of expression of PD-L1. A and B, The mRNA expression of PD-L1 and ICAM1 were detected using TaqMan RT-PCR. C, Total protein expression of PD-L1 was examined by immunoblotting. D, The cell surface expression of PD-L1 was assessed by flow cytometry. E, A total of 5 × 105 of B16-F10 WT or IRF1-KO cells were intradermally injected into C57BL/6 mice (n = 5). Tumors were collected on day 12 of postinjection. The percentage of and geometric mean (MFI) of PD-L1high in CD45− cells (tumor cells) were tested by flow cytometry. In A and B, each data point represents mean and SEM from 3 independent replicates. Representative results from twice-repeated experiment are shown in C–E.

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    Figure 5.

    IRF1-deficient tumor cells affect infiltrating T-cell functions ex vivo and are more vulnerable to CD8+ T-cell cytotoxicity in vitro. A, Intracellular cytokine production of infiltrating CD8+ T cells in B16-F10 WT and IRF1-KO tumors (n = 9, 8). Single-cell suspension was prepared after tumor collection. The mixture of tumor cells and infiltrated lymphocytes was stimulated overnight with PMA and ionomycin, then cytokine secretion was blocked with GolgiPlug for 4 hours. Representative flow-cytogram for intracellular cytokine expression is shown. For each type of cytokine expression, cumulative mean and SEM were plotted from twice-repeated experiments, statistical significance was calculated by unpaired Student t test, and presented as *, P < 0.05. B, The percentage of dead tumor cells at different Pmel T: tumor cell ratios. A total of 1 × 104 tumor cells in each 96-well plate were cocultured with different amount of Pmel CD8+ T cells overnight. Cells were harvested and examined for viability using Zombie Aqua Fixable Viability Kit. The experiment was repeated 3 times. For each data point, mean and SEM from twice-repeated experiments were plotted, statistical significance calculated by two-way ANOVA with Sidak multiple comparison test, and presented as *, P < 0.05; **, P < 0.01.

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    Figure 6.

    Doxycycline (DOX)-induced PD-L1 expression rescues tumorigenicity of IRF1-KO cells in mice. B16-F10 WT cells were treated with mouse IFNγ for 4 hours for positive control. B16-F10 WT, IRF1-KO, and IRF1-KO pInducer20-PD-L1 cells were treated with doxycycline for 48 hours and collected for the detection of PD-L1expression. A, Total protein expression of PD-L1 was examined by immunoblotting. B, The cell surface expression of PD-L1 was assessed by flow cytometry. Representative histograms from twice-repeated experiments are shown. C, A total of 5 × 105 of B16-F10 WT, IRF1-KO, or IRF1-KO pInducer20-PD-L1 cells were intradermally injected into C57BL/6 mice (n = 5). One group of either B16-F10 IRF1-KO or IRF1-KO pInducer20-PD-L1 cell–injected mice were fed with doxycycline-containing food (200 mg/kg) from 3 days before tumor cell injection, and then continued being fed with doxycycline-containing food. Tumor growth measurements were taken with all groups of mice.

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Cancer Immunology Research: 7 (8)
August 2019
Volume 7, Issue 8
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IRF1 Inhibits Antitumor Immunity through the Upregulation of PD-L1 in the Tumor Cell
Lulu Shao, Weizhou Hou, Nicole E. Scharping, Frank P. Vendetti, Rashmi Srivastava, Chandra Nath Roy, Ashley V. Menk, Yiyang Wang, Joe-Marc Chauvin, Pooja Karukonda, Stephen H. Thorne, Veit Hornung, Hassane M. Zarour, Christopher J. Bakkenist, Greg M. Delgoffe and Saumendra N. Sarkar
Cancer Immunol Res August 1 2019 (7) (8) 1258-1266; DOI: 10.1158/2326-6066.CIR-18-0711

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IRF1 Inhibits Antitumor Immunity through the Upregulation of PD-L1 in the Tumor Cell
Lulu Shao, Weizhou Hou, Nicole E. Scharping, Frank P. Vendetti, Rashmi Srivastava, Chandra Nath Roy, Ashley V. Menk, Yiyang Wang, Joe-Marc Chauvin, Pooja Karukonda, Stephen H. Thorne, Veit Hornung, Hassane M. Zarour, Christopher J. Bakkenist, Greg M. Delgoffe and Saumendra N. Sarkar
Cancer Immunol Res August 1 2019 (7) (8) 1258-1266; DOI: 10.1158/2326-6066.CIR-18-0711
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