Research ArticleCANCER IMMUNOLOGY

Tumor immune evasion arises through loss of TNF sensitivity

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Science Immunology  18 May 2018:
Vol. 3, Issue 23, eaar3451
DOI: 10.1126/sciimmunol.aar3451
  • Fig. 1 Immune evasion occurs through loss of TNF, IFN-γ, or antigen presentation pathways.

    (A) Experimental design of CRISPR/Cas9 screening. (B) OT-I T cell–MC38Ova killing and TNF secretion assays in the presence or absence of a neutralizing α–PD-1 antibody (50 μg/ml). (C) MC38Ova cells were subjected to three rounds of exposure to OT-I T cells, as described in (A), followed by sequencing for the top enriched genes. (D) The screen from (C) was carried out in parallel, in the presence of α–PD-1 (50 μg/ml), followed by sequencing for the top enriched genes. (E) Comparison of top scoring genes in screens described in (C) and (D). (F) Normalized sgRNA counts from the screen described in (C). unt, untreated. (G) Validation of screen hits using the indicated individual sgRNAs in MC38Ova upon an 18-hour exposure to OT-I T cells. (H) Protein network analysis of the screen hits from (D). (I) GO term analysis from the screen hits from (D). (J) The screen described in (A) was performed using B16Ova cells. After three rounds of exposure to OT-I T cells, the cells were sequenced for the top enriched genes. WT, wild type. (K) Protein network analysis from the top hits identified in (J). (L) GO term analysis from the top hits identified in (J). (M) The screen described in (A) was performed using MDA-MB-231 cells. After three rounds of exposure to HER2-directed human CAR T cells, the cells were sequenced for the top enriched genes.

  • Fig. 2 NK cell immune evasion occurs through loss of TNF pathway members.

    (A) Experimental design of CRISPR/Cas9 screening for resistance to NK cell killing. (B) NK cell–MC38 killing assay (18 hours) at the indicated E:T ratios. (C) NK cells were cocultured with MC38 cells at the indicated E:T ratios. After 6 hours, cytokines were measured by cytometric bead array. (D) MC38 cells were subjected to three rounds of exposure to primary mouse NK cells, as described in (A), followed by sequencing for the top enriched genes. (E) Protein network analysis from the top hits identified in (D). (F) GO term analysis from the top hits identified in (D). TRAIL, TNF-related apoptosis-inducing ligand. (G) Comparison of the top scoring genes in screens described in (D) and the OT-I T cell screen described in Fig. 1C. (H) Schematic representation of immune evasion from NK attack. DISC, death-inducing signaling complex.

  • Fig. 3 Immune evasion occurs independently of perforin-mediated killing.

    (A) MC38Ova/Vector chromium release assay (18 hours) at the indicated E:T ratios with Prf1−/− OT-I T cells. B16Ova/Vector coculture (48 hours) with Prf1−/− OT-I T cells (2:1 E:T ratio). DAPI, 4′,6-diamidino-2-phenylindole. (B) MC38Ova screen as in Fig. 1A, but using Prf1−/− OT-I T cells. (C) B16Ova screen, as in Fig. 1J, but using Prf1−/− OT-I T cells. (D) Comparison of top enriched genes in screens described in (B) and (C). (E) Selected sgRNA counts from (B) and (C). (F) Protein network analysis of the top scoring genes from (B). (G) GO term analysis from the top scoring genes from (B). (H) Comparison of top enriched genes in screens described in Figs. 1C and 2C. KO, knockout. (I) Protein network analysis of the top scoring genes from (B). (J) GO term analysis of the top scoring genes from (C). (K) Comparison of top enriched genes in screens described in Fig. 1J and (C).

  • Fig. 4 T cell–derived cytokines drive immune evasion.

    (A) RNA-seq (triplicate samples) of MC38Ova cells that were cocultured with OT-I T cells for 6 hours. (B) Volcano plot of top regulated genes from (A). (C) GSEA from (A). (D) MC38Ova cells were treated with supernatant (6 hours) derived from an overnight MC38Ova coculture with OT-I T cells (1:1 E:T ratio) followed by 3′ RNA-seq. GSEA is displayed. (E) MC38Ova killing assay with Prf1−/− OT-I T cells ± α-TNF or α–IFN-γ (25). MC38Ova killing assay using supernatant from an MC38Ova/vec coculture with OT-I T cells ± α-TNF or α–IFN-γ (25 μg/ml). MC38 Ova killing assay upon TNF or IFN-γ treatment at the indicated concentrations for 18 hours. (F) Custom sgRNA pool screen in MC38Ova cells. Cells were treated with TNF (1 ng/ml) three consecutive times. (G) Comparison of the top scoring genes from (F) to the OT-I T cell screen described in Fig. 1C. (H and I) MC38 and B16 cells carrying the custom gRNA library were treated with IFN-γ overnight and then cell-sorted for MHC-I negatives (repeated three times). Cells were then sequenced for gRNA enrichment. (J) Selected gRNAs from the MC38 screen in (H).

  • Fig. 5 T cell–derived TNF kills tumor cells in a bystander effect.

    (A) MC38Ova cells were labeled with CFSE and MC38Vec with Cell Trace Violet and then mixed (50:50) in the presence or absence of neutralizing α-TNF (50 μg/ml). OT-I T cells were then overlaid, and killing was monitored by time-lapse microscopy. Percentage of MC38Vec PI-positive cells that occurred in the presence or absence of T cells ± α-TNF. Pooled experimental data, n = 3; *P < 0.05, Student’s t test. (B) MC38Vec cells were labeled with chromium and then mixed (50:50) with unlabeled MC38Ova ± α-TNF (50 μg/ml). Prf1+/+ or Prf1−/− OT-I T cells were then added for 18 hours. *P < 0.05, Student’s t test. (C) MC38Ova cells (1 × 106) were injected subcutaneously into Prf1 knockout mice. Mice were treated with anti–PD-1 (200 μg) twice per week for the time span indicated. Tumor growth and overall survival are displayed.

  • Fig. 6 Immune evasion occurs through loss of TNF signaling, IFN-γ signaling, or antigen presentation in vivo.

    (A) MC38Ova custom sgRNA library–containing cells (1 × 106) (Fig. 3F) were subcutaneously injected into NSG mice. Prf1+/+ or Prf1−/− OT-I T cells (5 × 106) were adoptively transferred either once (Prf1+/+) or again 5 days later (Prf1+/+ and Prf1−/−). Tumors were then isolated followed by polymerase chain reaction of sgRNAs and sequencing for sgRNA enrichment. (B) Overlapping hits from (A). (C) Control, Tap1-, Tnfrsf1a-, or Jak1-depleted MC38Ova cells (1 × 106) were injected subcutaneously into NSG mice. On day 12, 5 × 106 Prf1+/+ OT-I T cells were adoptively transferred. This was repeated on days 16 and 25. Tumor size on day 28 is displayed. (D) Schematic representation of immune evasion pathways. (E) The Cancer Genome Atlas (TCGA) survival plots for colorectal adenocarcinoma for two genes from each of the immune evasion pathways. HR, hazards ratio; CI, confidence interval.

  • Fig. 7 Ado loss alters cellular metabolism.

    Control or Ado knockout MC38 cells were subjected to amine derivatization/liquid chromatography–MS (cysteamine, cystamine, taurine, and glutathione) and GC-MS polar metabolomics (cystamine, taurine, cysteine, and hypotaurine). ATP, adenosine 5′-triphosphate; ADP, adenosine 5′-diphosphate; AMP, adenosine 5′-monophosphate. *P < 0.01, **P < 0.001, ***P < 0.0001, ****P < 0.00001. NS, not significant.

  • Fig. 8 Ado loss drives TNF-dependent immune evasion.

    (A and B) Control or Ado knockout MC38 cells were left untreated or treated with TNF (10 ng/ml) for 1 or 6 hours, followed by 3′ RNA-seq. The top regulated genes are displayed with comparisons of control gRNA and Ado knockout (two individual gRNAs for each, performed in duplicate). Representative box plots for the top three TNF-induced genes are displayed. (C) Ado was targeted in MC38Ova with two individual sgRNAs. Prf1+/+ OT-I T cells were then added, and cell death was measured at 18 hours. Data are pooled from three independent experiments. (D) Cells from (C) were treated with TNF (1 ng/ml). At 18 hours, cell death was measured by PI uptake. Data are representative of three independent experiments. (E) Control or Ado-depleted MC38Ova cells were injected subcutaneously into NSG mice. On day 12, and again on day 16, 5 × 106 Prf1+/+ OT-I T cells were adoptively transferred. (F) Schematic representation of bystander immune evasion. *P < 0.05, Student’s t test. (G) TCGA data analyses of Ado expression and disease-free survival for different cancer types.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/23/eaar3451/DC1

    Fig. S1. Additional validation and control for CRISPR screen.

    Fig. S2. Confirmation of CRISPR gene deletion.

    Fig. S3. Transcriptional analysis of cytokine-treated tumor cells.

    Fig. S4. Additional cytokine treatment data.

    Fig. S5. Further adoptive T cell therapy screen data.

    Fig. S6. Validation of Ado knockout TNF response in additional tumor lines.

    Fig. S7. Confirmation of Gosr1 as an immune evasion gene.

    Movie S1. Time-lapse imaging of T cells killing MC38Ova or MC38Vec cells.

    Movie S2. Time-lapse imaging of T cells killing MC38Ova or MC38Vec cells in the presence of anti-TNF antibody.

    Table S1. Raw data.

    Table S2. CRISPR screen data, B16 perforin knockout OT-I screen.

    Table S3. CRISPR screen data, B16 OT-I screen.

    Table S4. CRISPR screen data, MC38 OT-I screen.

    Table S5. CRISPR screen data, MDA CAR T cell screen.

    Table S6. CRISPR screen data, OT-I IgG versus T0.

    Table S7. CRISPR screen data, OT-I PD-1 versus T0.

    Table S8. CRISPR screen data, MC38 NK cell.

  • Supplementary Materials

    Supplementary Material for:

    Tumor immune evasion arises through loss of TNF sensitivity

    Conor J. Kearney, Stephin J. Vervoort, Simon J. Hogg, Kelly M. Ramsbottom, Andrew J. Freeman, Najoua Lalaoui, Lizzy Pijpers, Jessica Michie, Kristin K. Brown, Deborah A. Knight, Vivien Sutton, Paul A. Beavis, Ilia Voskoboinik, Phil K. Darcy, John Silke, Joseph A. Trapani, Ricky W. Johnstone,* Jane Oliaro*

    *Corresponding author. Email: ricky.johnstone{at}petermac.org (R.W.J.); jane.oliaro{at}petermac.org (J.O.)

    Published 18 May 2018, Sci. Immunol. 3, eaar3451 (2017)
    DOI: 10.1126/sciimmunol.aar3451

    This PDF file includes:

    • Fig. S1. Additional validation and control for CRISPR screen.
    • Fig. S2. Confirmation of CRISPR gene deletion.
    • Fig. S3. Transcriptional analysis of cytokine-treated tumor cells.
    • Fig. S4. Additional cytokine treatment data.
    • Fig. S5. Further adoptive T cell therapy screen data.
    • Fig. S6. Validation of Ado knockout TNF response in additional tumor lines.
    • Fig. S7. Confirmation of Gosr1 as an immune evasion gene.
    • Legends for movies S1 and S2

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). Time-lapse imaging of T cells killing MC38Ova or MC38Vec cells.
    • Movie S2 (.mov format). Time-lapse imaging of T cells killing MC38Ova or MC38Vec cells in the presence of anti-TNF antibody.
    • Table S1 (Microsoft Excel format). Raw data.
    • Table S2 (Microsoft Excel format). CRISPR screen data, B16 perforin knockout OT-I screen.
    • Table S3 (Microsoft Excel format). CRISPR screen data, B16 OT-I screen.
    • Table S4 (Microsoft Excel format). CRISPR screen data, MC38 OT-I screen.
    • Table S5 (Microsoft Excel format). CRISPR screen data, MDA CAR T cell screen.
    • Table S6 (Microsoft Excel format). CRISPR screen data, OT-I IgG versus T0.
    • Table S7 (Microsoft Excel format). CRISPR screen data, OT-I PD-1 versus T0.
    • Table S8 (Microsoft Excel format). CRISPR screen data, MC38 NK cell.

    Files in this Data Supplement:

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