Supplementary Materials 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@petermac.org (R.W.J.); jane.oliaro@petermac.org (J.O.)

Published 18 May 2018, Sci. Immunol. 3, eaar3451 (2018)

DOI: 10.1126/sciimmunol.aar3451

The 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

Other Supplementary Material for this manuscript includes the following:

(available at immunology.sciencemag.org/cgi/content/full/3/23/eaar3451/DC1)

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.

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

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.

Fig. S1. Additional validation and control for CRISPR screen. (A) MC38Ova chromium

release assay (4 h or 18 h), with Prf1+/+ or Prf1-/- OT-I T cells at the indicated E:T ratios. (B-

C) MC38Ova cells were cultured OT-I T cells for 18 hours followed by FACS staining for the

indicated proteins on both the MC38Ova cells (B) and the OT-I T cells (C). (D) B16Ova cells

were left untreated or treated with IFN (5 ng/ml) or OT-I T cells for 18 hours, followed by

FACS staining for MHCI. (E) After completion of the screen in Figure 1A, the surviving cells were used as targets in a chromium release assay (18 h) at the indicated E:T ratios.

Fig. S2. Confirmation of CRISPR gene deletion (A) Ado and Gosr1 were PCR amplified

from control or CRISPR knockout MC38Ova cells followed by Sanger sequencing. Indel

analysis was performed using TIDE. (B) Control and CRISPR knockout MC38Ova cells were

analyzed for expression of the indicated proteins by FACS or Western blot. (C) Comparison

of the screen hit results from figures 1C and 1J. (D) CAR-T cells were activated as indicated

followed by measurement of IFN by cytokine bead array (CBA). MDA-MB-231 cells were

treated with IFN (5 ng/ml, 18 h) followed by FACS analysis of PD-L1 expression. Log2

guide counts for ERBB2 after the screen in figure 1M are displayed on the right.

Fig. S3. Transcriptional analysis of cytokine-treated tumor cells. MC38 cells were treated

with IFNγ (10ng/ml), TNF (10 ng/ml) or supernatant from a MC38Ova – OT-I co-culture,

followed by 3’ RNA sequencing. Genes with synergistic induction are displayed in the heat

maps.

Fig. S4. Additional cytokine treatment data. (A) Prf1-/- OT-I T cells were used as effectors

against EO771Ova cells in a chromium release assay, in the presence of anti-TNF or anti-

IFNγ (25 μg/ml). EO771Ova cells were treated with the indicated concentrations of TNF or

IFNγ. After 18 hours, cell death was measured by chromium release. (B) OT-I T cells were

used as effectors against MC38Ova cells in a chromium release assay (18 h) , in the presence

of anti-FasL (25 μg/ml). (C) gRNA counts from the screen described in Figure 4F.

Fig. S5. Further adoptive T cell therapy screen data. (A) Three more mice from the

experiment described in Figure 5A. These mice received a single adoptive transfer of Prf1+/+

OT-I T cells. (B) In vitro proliferation of MC38Ova cells carrying the indicated gRNAs. In

vivo Growth rate of MC38ova tumours in NSG mice carrying the indicated gRNAs.

Fig. S6. Validation of Ado knockout TNF response in additional tumor lines. (A) Two

individual control or Ado knockout MC38Ova cell lines were treated with the indicated

concentrations of TNF. After 18 h cell death was measured by PI FACS analysis. (B) Two

individual control or Ado knockout MC38Ova cell lines were treated with the indicated

concentrations of perforin/granzyme B. After 6 h cell death was measured by chromium

release. (C) Two individual control or Ado knockout E0771 cell lines were treated with the

indicated concentrations of TNF. After 18 h cell death was measured by PI FACS analysis.

(D) Two individual control or Ado knockout AU565 cell lines were treated with the indicated

concentrations of TNF. After 18 h cell death was measured by PI FACS analysis. In all cases

relative killing from 3 independent experiments is pooled on the right.

Fig. S7. Confirmation of Gosr1 as an immune evasion gene. (A) Gosr1 was targeted with

two individual gRNAs in MC38Ova cells. These cells were then co-cultured with OT-I T cells

and cell death measured 18 hours later. Data is pooled from three independent experiments.

(B) Control or Gosr1 targeting gRNA was introduced into MC38Ova followed by

transplantation into NSG mice. Tumor bearing mice then received OT-I cellular therapy.

Tumor size on the indicated days is displayed (refer to Figure 8E). (C) TCGA data analyses

for Gosr1 expression in prognosis of different cancer types.

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

MC38Ova cells were labeled with CFSE and MC38Vec cells with CTV then mixed (50:50) and

seeded into chamber slides overnight. OT-I T cells were then added to adherent targets in

media containing 100 μM propidium iodide, and killing was monitored by time-lapse

microscopy.

Movie S2: Time-lapse imaging of T cells killing MC38Ova or MC38Vec cells in the

presence of anti-TNF antibody.

MC38Ova cells were labeled with CFSE and MC38Vec cells with CTV then mixed (50:50) and

seeded into chamber slides overnight. OT-I T cells were then added to adherent targets in

media containing 100 M propidium iodide and neutralizing α-TNF (50 μg/ml), and killing

was monitored by time-lapse microscopy.