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Bystander IFN-γ activity promotes widespread and sustained cytokine signaling altering the tumor microenvironment

Abstract

The cytokine interferon (IFN)-γ produced by tumor-reactive T cells is a key effector molecule with pleiotropic effects during anti-tumor immune responses. Although IFN-γ production is targeted at the immunologic synapse, its spatiotemporal activity within the tumor remains elusive. In the present study, we report that, although IFN-γ secretion requires local antigen recognition, IFN-γ diffuses extensively to alter the tumor microenvironment in distant areas. Using intravital imaging and a reporter for STAT1 translocation, we provide evidence that T cells mediate sustained IFN-γ signaling in remote tumor cells. Furthermore, tumor phenotypic alterations required several hours of exposure to IFN-γ, a feature that disfavored local IFN-γ activity over diffusion and bystander activity. Finally, single-cell RNA-sequencing data from melanoma patients also suggested bystander IFN-γ activity in human tumors. Thus, tumor-reactive T cells act collectively to create large cytokine fields that profoundly modify the tumor microenvironment.

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Fig. 1: T cell-derived IFN-γ induces phenotypic changes in the tumor microenvironment.
Fig. 2: Tumor antigen expression drives the selective accumulation and arrest of intratumoral T cells.
Fig. 3: Extensive bystander IFN-γ activity in the tumor microenvironment.
Fig. 4: T cells mediate widespread and sustained STAT1 activity in the tumor microenvironment.
Fig. 5: Sustained signaling is required to alter tumor cell phenotype.
Fig. 6: Assessing IFN-γ bystander activity in human melanoma samples.

Data availability

RNA-seq data reported in this paper are deposited in the National Center for Biotechnology Information GEO database (GSE140191). Previously published human single-cell RNA-seq data that were reanalyzed in the present study are available under accession codes GSE123139 and GSE103322. All other data supporting the findings of the present study are available from the corresponding author on reasonable request.

Code availability

The R and Matlab scripts used for the transcriptomic and image analysis are available on GitHub (https://github.com/PierreBSC/IFNG_Cancer_project).

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Acknowledgements

We thank members of P. Bousso’s laboratory for critical review of the manuscript. We thank the mouse facility and Technology Core of the Center for Translational Science (CRT) at Institut Pasteur for support in conducting the present study. The work was supported by Institut Pasteur, INSERM, a starting grant (Lymphocytecontact) and an advanced grant (ENLIGHTEN) from the European Research Council (to P. Bousso) and by the Bristol-Myers Squibb Foundation for Research in Immuno-Oncology.

Author information

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Authors

Contributions

R.T., I.M., M.C., F.L., Z.G., B.B. and C.C. conducted the experiments. R.T., I.M., M.C. and P. Bousso designed the experiments. I.A. and B.S. contributed to the sequencing analysis. R.T., I.M., M.C., P. Bost and P. Bousso analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to Philippe Bousso.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Tumor cell responses to IFN-γ in vitro.

a-b, Tumor cells respond to IFN-γ in an analogic manner in vitro. a E0771 mammary breast tumor cells were stimulated with indicated IFN-γ concentrations in vitro for 24h. H2-Kb (top), H2-Db (middle) and PD-L1 (bottom) surface expression was then analyzed by flow cytometry. Representative of two independent experiments. b, Representative histograms of H2-Db (left) and PD-L1 (right) surface expression in B16.F10 (top) or Eµ-myc (bottom) cells, after treatment with indicated IFN-γ concentrations for 24h. Representative of three independent experiments. c-f, Impact of IFN-γ and TNF-α on tumor cell death. B16.F10 (c,e) or Eµ-myc (d,f) cells were incubated with the indicated IFN-γ and/or TNF-α concentrations in vitro for 24h. Cells were then counted by flow cytometry. Representative of two independent experiments. Source data

Extended Data Fig. 2 Gating strategy for flow cytometry analysis of the tumor microenvironment.

The figure depicts the gating strategies for identifying tumor cells, OT-I T cells, NK cells, monocytes and neutrophils present in the bone marrow of tumor bearing mice.

Extended Data Fig. 3 NK cells are dispensable for tumor phenotypic changes upon T cell transfer.

a, Experimental set-up. Rag2-/-γc-/- recipient mice were injected i.v with OVA-expressing Eµ-myc B lymphoma cells. On day 12-13, in vitro activated OT-I CD8+ T cells were injected i.v. Two days later, the recipient bone marrow was harvested and analyzed by flow cytometry. b, Representative examples of histograms showing H2-Kb (left) and PD-L1 (right) surface expression on tumor cells isolated from the bone marrow of mice that were left untreated (filled grey) or injected with OT-I T cells (line, blue). c, H2-Kb (left), H2-Db (middle) and PD-L1 (right) surface expression on tumor cells isolated from mice treated or not with OT-I T cells, as assessed by flow cytometry. Each dot represents one mouse with n=4 mice per group. Red lines indicate mean values. (* P<0.05, two-tailed Mann-Whitney U-test).

Extended Data Fig. 4 OT-I T cells arrest on antigen-positive tumor cells independently of IFN-γ.

a, Rag2-/- mice were injected with a 1:1 mixture of OVA-expressing and non-expressing Eµ-myc B lymphoma cells, labeled with CFP and YFP, respectively. On day 12-13, mice were injected with activated GFP+ OT-I T cells. Two days later, intravital imaging of the bone marrow was performed. The graph shows the relationship between mean T cell velocity and straightness in the OVA+ (left) and OVA- (right) tumor areas. Each dot represents one T cell track (OVA- areas n= 103 tracks, OVA+ areas n=75 tracks). b-d, Rag2-/- mice were injected with a 1:1 mixture of OVA-expressing and non-expressing Eµ-myc B lymphoma cells, labeled with CFP and GFP, respectively. On day 12-13, mice were injected with activated WT or IFN-γ-deficient OT-I T cells transduced to express the mCherry fluorescent protein. Two days later, intravital imaging of the bone marrow was performed. b, Representative image of OVA+ (blue) tumor patches infiltrated with IFN-γ-deficient OT-I T cells (red). Scale bar: 50 µm. Right. Time lapse images (corresponding to the dashed squares) showing IFN-γ-deficient OT-I T cells (pointed by arrows) forming stable contacts with OVA+ Eµ-myc cells. Scale bar: 15 µm. c-d, Both WT and IFN-γ-deficient OT-I T cells decelerate in OVA+ tumor areas. Graphs show mean velocities (c) and arrest coefficient (d), for individual WT or IFN-γ-deficient T cells in OVA+ (blue) and OVA- (orange) tumor areas. Each dot represents one track. Red lines indicate mean values. Data shown in b-d, are representative of two independent experiments with n=3 mice per group. (* P<0.05; ** P<0.01; *** P<0.001; two-tailed Mann-Whitney U-test). e, OT-I T cells infiltrate antigen-negative tumors in the bone marrow. Rag2-/- mice were injected with either Eµ-myc alone or a 1:1 mixture of Eµ-myc and OVA-expressing Eµ-myc B lymphoma cells (labeled with different fluorescent proteins). On day 12-13, recipients were injected with OT-I T cells or left untreated. Two days later, the bone marrow of the mice was recovered and analyzed by flow cytometry. The graph shows that OT-I T cells can efficiently infiltrate tumors that contain only antigen-negative cells. Each dot represents one mouse with n=4 mice per group. Red lines indicate mean values.

Extended Data Fig. 5 T cell-derived IFN-γ can control bystander tumor cells.

a-b, Estimating the intratumoral IFN-γ concentration upon T cell transfer. a, The ratio of OT-I T cells to OVA+Eµ-myc tumors in the bone marrow (measured at day 2) is graphed as a function of the total number of transferred T cells. Results are shown as mean+SD with n=5 mice per group. b, The fold change in H2-Db levels measured in Eµ-myc tumors in vitro is graphed as a function of IFN-γ concentration. The graph was used to infer the putative IFN-γ concentration in vivo (dashed red line) upon transfer of 20x106 T cells. c-f, Control of tumor burden by T cell-derived IFN-γ. c-d, Recipient female Rag2-/- mice were injected with H-Y+ Eµ-myc B lymphoma cells. After 3 weeks, mice were either injected i.v. with recombinant IFN-γ (1µg) twice (one day apart) or left untreated. On day 2, tumor burden c, and phenotype d, in the bone marrow were assessed by flow cytometry. Each dot represents one mouse (n=3 and n=4 mice for the untreated and the treated group, respectively). Red lines indicate mean values. (* P<0.05, two-tailed unpaired t-test). e-f, Functional activity of intratumoral IFN-γ. e, Experimental set-up. Rag2-/- mice were injected with a 1:1 mixture of Eµ-myc and OVA-expressing Eµ-myc B lymphoma cells (labeled with different fluorescent proteins). On day 12-13, recipients were injected with either WT or IFN-γ-deficient OT-I T cells or left untreated. Five days later, the bone marrow was recovered and analyzed by flow cytometry. f, Graphs show the residual numbers of antigen-positive (top) and antigen-negative (bottom) tumors following transfer of the indicated T cell populations. Each dot represents one mouse (n=6 mice per group). Red lines indicate mean values. (* P<0.05, ** P<0.01, two-tailed Mann-Whitney U-test). Representative of two independent experiments. Of note, small deviation from the initial ratio seen in the absence of T cells is most likely due to minor differences in tumor cell division time.

Extended Data Fig. 6 Automated procedure for quantifying STAT1 translocation in tumor cells imaged in vitro or in vivo.

a, The diagram recapitulates the various steps used for image processing and quantification (see methods for details). b, Representative example of cell segmentation from the STAT1-GFP signal in tumor cells. Scale bar: 50 µm.

Extended Data Fig. 7 Tumor-reactive CD8+ T cells mediate widespread and sustained STAT1 activity in the tumor microenvironment.

a, Experimental set-up. Recipient Rag2-/-γc-/- mice were injected with OVA+ Eµ-myc B lymphoma cells expressing the STAT1-GFP reporter and a nuclear mCherry protein. After 3 weeks, activated CD8+ T cells bearing the OT-I TCR were injected i.v. One day later, recipients were subjected to intravital imaging of the bone marrow. b, STAT1-GFP is largely excluded from the nucleus in tumor cells developing in the absence of T cells. Representative two-photon images (scale bar: 20µm), highlighting two specific regions (insets, scale bar: 10µm). c, Detection of nuclear STAT1-GFP in T cell-infiltrated tumors. Representative two-photon images (scale bar: 20µm), highlighting two specific regions (insets, scale bar: 10µm). d, Translocation score was computed from two-photon images obtained in mice left untreated (no T cells) or transferred with OT-I T cells (n=10 cells per group, box plot showing the median, first and third quartile and min and max values, *** P<0.001, two-tailed Mann-Whitney U-test). Data in b-d are representative of two independent experiments. e-h STAT1 signaling is detected at distance from antigen-positive tumor cells. e, Experimental set-up. Rag2-/- mice were injected with Eµ-myc cells expressing the STAT1 reporter either alone or mixed at a 1:1 ratio with OVA+ Eµ-myc (expressing mCFP). Two weeks later, all mice were adoptively transferred with activated OT-I T cells. After 2 days, mice were subjected to intravital imaging of the bone marrow. f, STAT1-GFP is largely excluded from the nucleus in tumor cells developing in the absence of antigen-positive tumors. Representative two-photon images (scale bar: 20µm), highlighting two specific regions. Scale bar, 20 μm. g, Detection of STAT1 translocation in antigen-negative tumor cells developing in the presence of antigen-positive tumor cells. Representative two-photon images (scale bar: 20µm), highlighting two specific regions. Scale bar, 20 μm. h, Translocation score was computed from two-photon images obtained in mice bearing STAT1-GFP-expressing tumor cells alone, or bearing mosaic tumors containing both STAT1-GFP and OVA+ tumor cells. (n=10 cells per group, box plot showing the median, first and third quartile and min and max values, ** P<0.01, two-tailed Mann-Whitney U-test). Data in f-h are representative of n=3 mice per group.

Extended Data Fig. 8 Prolonged STAT1 translocation in tumor cells with sustained exposure to IFN-γ.

a, Experimental set-up. Eµ-myc cells expressing STAT1-GFP and nuclear mCherry were cultured for one hour in presence of recombinant IFN-γ. Cells were washed and either incubated with anti-IFN-γ mAb (to limit cytokine exposure), or re-incubated with IFN-γ (to prolong cytokine exposure). After an additional hour, cells were imaged. b, STAT1 translocation score was computed automatically for all individual cells (unstimulated n=593 cells. IFN-γ n=583, anti-IFN-γ n=570 cells). Each dot represents one cell. Red lines indicate mean values. (**** P<0.0001, *** P<0.001, two-tailed Mann-Whitney U-test).

Extended Data Fig. 9 Determination of an IFNG signature in monocyte/macrophage cluster.

15 over-dispersed gene sets were determined for the monocyte/macrophage cluster as explained in materials and methods. Graph shows the contribution of IFNG or type I IFN-related genes for each gene sets. Arrow highlights a gene signature that is specific of IFN-γ signaling (hereafter called IFNG signature).

Extended Data Fig. 10 Distribution of IFN-γ signature in tumor cells from head and neck squamous cell carcinoma patients.

a, Gene contribution to the IFNG signature in the tumor cells. b, Distribution of IFNG signature in tumor cells from 13 different patients with head and neck squamous cell carcinoma patients. Although the mean of the distribution varies from patient to patient, a relatively uniform distribution is observed in most patients.

Supplementary information

Reporting Summary

Supplementary Video 1

Tumor antigen expression drives the selective accumulation and arrest of intratumoral T cells. Rag2−/− mice were injected with a 1:1 mixture of OVA-expressing and OVA-non-expressing Eµ-myc B lymphoma cells, labeled with CFP and YFP, respectively. On days 12–13, mice were injected with activated GFP+ OT-I T cells. After 2 d, intravital imaging of the bone marrow was performed. The video illustrates that CD8+ T cells (green) specifically accumulate and arrest in antigen-expressing cellular patches (blue) of mosaic tumors but not in antigen-negative patches (orange). It represents two independent experiments with n = 3 mice per group.

Supplementary Video 2

Tumor-reactive (H-Y-specific) CD8+ T cells mediate widespread and sustained STAT1 activity in the tumor microenvironment. Recipient Rag2−/− mice were injected with male Eµ-myc B lymphoma cells expressing the STAT1–GFP reporter and a nuclear mCherry protein. After 3 weeks, activated CD8+ T cells bearing the anti-H-Y MataHari TCR were injected i.v. After 3 d, recipients were subjected to intravital imaging of the bone marrow. The video shows widespread STAT1 activity (yellow) in the tumor in the presence, but not the absence, of T cells. T cells are shown in blue. It represents two independent experiments with n = 3 mice per group.

Supplementary Video 3

Tumor-reactive (OVA-specific) CD8+ T cells mediate widespread and sustained STAT1 activity in the tumor microenvironment. Recipient Rag2−/−γc−/− mice were injected with OVA+ Eµ-myc B lymphoma cells expressing the STAT1–GFP reporter and a nuclear mCherry protein. After 3 weeks, activated CD8+ T cells bearing the OVA-specific OT-I TCR were injected i.v. After 3 d, recipients were subjected to intravital imaging of the bone marrow. The video shows widespread STAT1 activity (yellow) in the tumor in the presence, but not the absence, of T cells. T cells appear bright yellow. It represents two independent experiments.

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Thibaut, R., Bost, P., Milo, I. et al. Bystander IFN-γ activity promotes widespread and sustained cytokine signaling altering the tumor microenvironment. Nat Cancer 1, 302–314 (2020). https://doi.org/10.1038/s43018-020-0038-2

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