Volume 18, Issue 5, Pages 912-920 (May 2010) Ad.Egr-TNF and Local Ionizing Radiation Suppress Metastases by Interferon-β- Dependent Activation of Antigen-specific CD8+ T Cells  Yuru Meng, Helena J Mauceri, Nikolai N Khodarev, Thomas E Darga, Sean P Pitroda, Michael A Beckett, Donald W Kufe, Ralph R Weichselbaum  Molecular Therapy  Volume 18, Issue 5, Pages 912-920 (May 2010) DOI: 10.1038/mt.2010.18 Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 IFN response and immune activation following Ad.Egr-TNF/IR treatment is TNFα-signaling dependent. B16F1 tumors were grown in WT C57/Bl6 mice or in TNFR1,2−/− mice. Tumors were irradiated or treated with Ad.Egr-TNF in combination with ionizing radiation (IR) as described in the Materials and Methods. Purification of RNA, hybridization with arrays (Mouse Genome 430 2.0 GeneChips; Affymetrix) and data analysis are described in the Materials and Methods. Expression values were normalized to untreated control tumors grown in WT mice. Stars indicate genes activated by type I and type II IFNs. (a) Two-dimensional unsupervised hierarchical clustering of genes upregulated in B16F1/WT, but not in B16F1/TNFR1,2−/− tumors. Genes were annotated according to Affymetrix Netaffix database (see Supplementary Table S1). The legend for expressional values is shown in heatmap. (b) Web of interaction of genes and their protein products presented in a. The network represents immune cell trafficking. Red color is proportional to the upregulation of any given gene in B16F1/WT relative to B16F1/TNFR1,2−/− tumors. Note that the central node is represented by IFNα/β. (c) The top five canonical pathways associated with genes presented in a. The five pathways are associated with immune function and include communication between innate and adaptive immune cells and IFN signaling. Network and pathway data were obtained with Ingenuity Pathway Analysis software. P values indicate significance of differences between expression of the given pathway in B16F1/WT relative to B16F1/TNFR1,2−/− tumors. IFN, interferon; IL, interleukin; TNF, tumor necrosis factor; WT, wild type. Molecular Therapy 2010 18, 912-920DOI: (10.1038/mt.2010.18) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Transcriptional and translational synthesis of IFNs in response to Ad.Egr-TNF/IR requires host TNFα/TNFR1,2. (a) Reverse transcriptase-PCR analysis revealed higher levels of mTNFα/IFNβ/γ gene expression in Ad.Egr-TNF/IR treated B16.SIY tumors relative to untreated control tumors in WT and TNFR1,2−/− mice. (b) Levels of IFNα, IFNβ, and IFNγ were measured in B16F1/WT and B16F1/TNFR1,2−/− tumors using enzyme-linked immunosorbent assay. All measurements were done 5 and 24 hours after IR when 32 and 48 hours after the 1st Ad.Egr-TNF treatments. Open bars indicate tumors grown in WT mice and solid bars indicate tumors grown in TNFR1,2−/− mice. *P < 0.05, Student's two-tailed t-test, indicate significance of differences between tumors grown in WT and TNFR1,2−/− mice. IFN, interferon; IL, interleukin; mTNF, murine tumor necrosis factor; WT, wild type. Molecular Therapy 2010 18, 912-920DOI: (10.1038/mt.2010.18) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Induction of chemokines in Ad.Egr-TNF/IR treated tumors resulted in greater numbers of antigen-specific CD8+ T cells infiltration. (a) Ad.Egr-TNF/IR enhances chemokine production in BM-DMϕ. BM-DMϕ were incubated with Ad.Egr-TNF, Ad.Null, or rhTNFα, rmTNF and cocultured with 10 Gy irradiated or unirradiated B16.SIY tumor cells in a transwell system for 6 hours. BM-DMϕ were collected, total RNA extracted, and reverse transcriptase (RT)-PCR performed to analyze the relative expression level of chemokines. Data shown are the mean ± SD of duplicate samples of one experiment which represents four individual experiments with similar results. BM-DMϕ monoculture served as control. (b) Groups of chemokine gene transcripts were detected at a relatively higher level in Ad.Egr-TNF/IR treated WT tumors. WT C57BL/6 mice (n = 6/group) were subcutaneously injected with 5 × 105 B16.SIY melanoma cells, Ad.Egr-TNF (2 × 109 particle units) and control Ad.Null (2 × 109 vector particles) virus were injected intratumorally on day 9 and day 10. A radiation dose of 20 Gy was locally delivered on day 10. At 7 days after IR, tumors were harvested, 1/3 of each tumor was lysed in Trizol, total RNA was purified, and multiple chemokine gene transcripts were detected by RT-PCR and normalized to GAPDH. (c) More SIY antigen–specific CD8+ T-cell infiltration in Ad.Egr-TNF/IR treated WT tumors. Experiments performed as above, 1/3 of tumors were digested into single-cell suspension, stained with SIY tetramer, and anti-CD8 antibody to analyze frequency of CD8+ TIL. Gated on CD8+ cells, SIY/Kb PE tetramer–positive cells are shown. BM, bone marrow; IFN, interferon; IL, interleukin; rhTNF, recombinant human tumor necrosis factor; WT, wild type. Molecular Therapy 2010 18, 912-920DOI: (10.1038/mt.2010.18) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Ad.Egr-TNF/IR promote priming of Ag-specific CD8+ T-cell proliferation and greater IFNγ production than either Ad.Null or rhTNFα and rmTNF alone. (a) Ad.Egr-TNF enhanced proliferation of CD8+ T cells in the presence of 10 Gy irradiated or unirradiated or B16.SIY tumor cells. Carboxyfluorescein succinimidyl ester (CFSE)–labeled splenic CD8+ cells were cocultured with BM-DC treated with Ad.Egr-TNF control Ad.Null vectors, in the presence of irradiated (10 Gy) or unirradiated B16.SIY tumor cells, model tumor antigen SIY plus 20 U rIL-2 as a positive control. The same number of CD4+ T cells were added as described in the Materials and Methods. (b) Ad.Egr-TNF enhanced the IFNγ production of CD8+ T cells in the presence of 10 Gy irradiated or unirradiated B16.SIY tumor cells. Coculture supernatant was collected from the above experiments and IFNγ production was analyzed by enzyme-linked immunosorbent assay. Data are shown as the mean ± SD of duplicate samples and are representative of three separate experiments. (c) Ad.Egr-TNF promoted BM-DC maturation in the presence of 10 Gy irradiated or unirradiated B16.SIY tumor cells. Ad.Egr-TNF or control vectors were added to BM-DC at 7 day of induction in the presence of 10 Gy irradiated or unirradiated B16.SIY tumor cells. After 24 hours, BM-DC was analyzed for CD86 expression by fluorescence-activated cell sorting. BM, bone marrow; DC, dendritic cells; IFN, interferon; rIL, recombinant interleukin; TNF, tumor necrosis factor. Molecular Therapy 2010 18, 912-920DOI: (10.1038/mt.2010.18) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Ad.Egr-TNF/IR inhibit local tumor growth and DLN metastases and stimulate CD8+ T-cell proliferation. (a) Enhanced inhibition of local B16.SIY tumor growth following treatment with Ad.Egr-TNF/IR compared to IR alone, or Ad.Null/IR. WT C57BL/6 (n = 12/group) were subcutaneously injected with 5 × 105 B16.SIY melanoma cells, Ad.Egr-TNF [2 × 109 particle units (pu)] and control Ad.Null (2 × 109 pu) virus were intratumorally injected on day 9 and day 10. A radiation dose of 20 Gy was delivered on day 10. (b) The same experimental conditions noted above except the primary tumors were surgically removed 5 days after IR. Mice were killed on day 35 for DLN tumor clonogenic assay (n = 4–5/group). (c) 5 × 105 B16.SIY melanoma cells were injected subcutaneously. Virus and IR were administrated as above. On the same day of IR 5 × 106 carboxyfluorescein succinimidyl ester (CFSE)–labeled naive 2C T cells were transferred by intravenous injection. At 4 days after IR, DLNs were harvested and single-cell suspension were acquired, gating on CD8+ and 1B2+ cells, CFSE dilution of 2C T cells were analyzed. DLN, draining lymph node; IFN, interferon; TNF, tumor necrosis factor; WT, wild type. Molecular Therapy 2010 18, 912-920DOI: (10.1038/mt.2010.18) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Depletion of CD8+ T cells reversed the suppressive effects of Ad.Egr-TNF/IR on B16.SIY tumor growth and DLN metastasis. (a) B16.SIY tumor suppression after Ad.Egr-TNF/IR treatment is partially dependent on CD8+ T cells. WT C57BL/6 (n = 12/group) were subcutaneously injected with 5 × 105 B16.SIY melanoma cells, Ad.Egr-TNF [2 × 109 particle units (pu)] and control Ad.Null (2 × 109 pu) virus were injected intratumorally on day 9 and day 10. A radiation dose of 20 Gy was delivered locally on day 10. Mice were treated with CD8+-depleting antibodies before and after IR in addition to Ad.Egr-TNF/IR. (b,c) Depletion of CD8+ cells reverses the suppressive effect of Ad.Egr-TNF/IR on B16.SIY tumor regrowth and DLN metastases, Lymph node metastases was measured by direct observation of the size and color of DLNs, and by clonogenic assay of separated cells. (d) Schematic showing effects of Ad.Egr-TNF/IR on tumor microenvironment, the subsequent activation/proliferation and migration of CD8+ T cells. DLN, draining lymph node; IFN, interferon; TNF, tumor necrosis factor; WT, wild type. Molecular Therapy 2010 18, 912-920DOI: (10.1038/mt.2010.18) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions