Abstract
T-cell-secreted interferon (IFN)-γ can exert pleiotropic effects on tumor cells that include induction of immune checkpoints and antigen presentation machinery components, and inhibition of cell growth. Despite its role as a key effector molecule, little is known about the spatiotemporal spreading of IFN-γ secreted by activated CD8+ T cells within the tumor environment. Using multiday intravital imaging, we demonstrate that T cell recognition of a minor fraction of tumor cells leads to sensing of IFN-γ by a large part of the tumor mass. Furthermore, imaging of tumors in which antigen-positive and antigen-negative tumor cells are separated in space reveals spreading of the IFN-γ response, reaching distances of >800 µm. Notably, long-range sensing of IFN-γ can modify tumor behavior, as shown by both induction of PD-L1 expression and inhibition of tumor growth. Collectively, these data reveal how, through IFN-γ, CD8+ T cells modulate the behavior of remote tumor cells, including antigen-loss variants.
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Data availability
Statistical source data for all figures and extended data figures including all independent repeats are provided online.
All other data supporting the findings of this study are available from the corresponding author upon reasonable request.
Change history
17 June 2020
A Correction to this paper has been published: https://doi.org/10.1038/s43018-020-0092-9
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Acknowledgements
We thank E. Beerling, M. Mertz, B. van den Broek, L. Fanchi, R. Mezzadra, D. Peters, A. Broeks and the staff at the NKI Animal facility, Flow Cytometry facility, Bioimaging facility and Molecular Pathology & Biobanking facility for technical support and input, and members of the Schumacher and van Rheenen laboratories for discussions. This work was supported by a Boehringer Ingelheim Fonds PhD Fellowship (to M.E.H.), a Swiss National Science Foundation APM fellowship (no. P300PB_177881; to D.S.T.), ERC CoG Cancer-recurrence (grant no. 648804), Cancer Genomics Netherlands and the Doctor Josef Steiner Foundation (to J.v.R.) and ERC AdG SENSIT (to T.N.M.S.).
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M.E.H., L.B., D.P. and I.N.P. performed experiments and analyzed data. L.B. performed multiphoton imaging, F.E.D. and M.E.H. designed and tested the IGS reporter. L.B. and M.E.H. designed imaging analyses. M.E.H. and M.T. performed LCK inhibitor experiments. D.S.T. provided and analyzed human tumor samples. M.E.H., L.B., F.E.D., J.v.R. and T.N.M.S. contributed to experimental design. M.E.H., L.B., J.v.R. and T.N.M.S. prepared the manuscript with input from all the coauthors.
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Extended data
Extended Data Fig. 1 In vitro IGS reporter cell characteristics.
a, Median Katushka fluorescence intensity of IGS reporter-modified CFP+ OVCAR5 cells upon incubation with recombinant IFNγ under the indicated conditions. Bar graph shows mean of n=3 technical replicates, representative data of two independent experiments are depicted. b, CFP+ IGS reporter modified OVCAR5 cells proficient or deficient for the IFNγR were incubated for 48h with the indicated concentrations of recombinant IFNγ or IFNα and Katushka expression was analyzed by flow cytometry. Bar graphs show mean of technical duplicates data obtained from one experiment. c, Percentage IR-Dye positive IGS reporter-modified CFP+ OVCAR5 cells upon incubation with recombinant IFNγ under the indicated conditions. Bar graph shows mean of n=3 technical replicates. Representative data of two independent experiments are depicted. d, Percentage of IR-Dye positive Ag−CFP+ or Ag−IFNγR−/− OVCAR5 cells after 72h incubation with 100 ng/mL IFNγ. Bar graph shows mean of n=3 technical replicates. Representative data of three independent experiments are depicted.
Extended Data Fig. 2 IFNγ-induced IGS reporter and PD-L1 expression in MDA-MB-231 cells.
a, Median Katushka fluorescence intensity of IGS reporter-modified CFP+ MDA-MB-231 cells upon incubation with recombinant IFNγ under the indicated conditions. Bar graph shows mean of n=3 technical replicates. Representative data from two independent experiments are depicted. b, Median fluorescence intensity of PD-L1 staining as a function of median Katushka fluorescence intensity of CFP+Ag− IGS MDA-MB-231 reporter cells incubated for 24h with recombinant IFNγ under the indicated conditions. Plot depicts representative data three technical replicates of two independent experiments. c, 20% GFP+ Ag+ cells and 80% CFP+ IGS reporter bystander MDA-MB-231 tumor cells (5 x 105 total) were subcutaneously injected in NSG-β2m-/- mice. Mice were treated with HBSS (control), 5 x 106 control CD8+ T cells or with 5 x 106 CDK4R>L-specific CD8+ T cells, and tumors were harvested at day 3 after treatment. Bar graphs depicting mean percentage plus SD of Katushka+ reporter cells in control and tumor-specific T cell treated mice, n=5 mice per group. Representative data of two independent experiments are depicted. Two tailed Mann-Whitney U test was performed, with: p= 0.3095 (ns); p= 0.0317 (*); p= 0.0079 (**). d, Percentage of PD-L1-expressing cells of IGS reporter-modified CFP+ MDA-MB-231 cells from tumors as described in c. Bar graphs depict mean percentage of PD-L1 positive Ag- IGS cells plus SD, n=5 mice per group. Representative data of n=2 independent experiments are depicted. Two tailed Mann-Whitney U test was performed, with: p> 0.9999 (ns); p= 0.0317 (*); p= 0.0079 (**).
Extended Data Fig. 3 Distance from Ag- IGS cells to the nearest Ag+ cell upon tumor cell co-injection.
Analysis of the distance between Ag- IGS tumor cells and the nearest Ag+ tumor cell for the imaging experiments depicted in Fig. 3. a, Representative image of a tumor with intermingled Ag- and Ag+IGS cells. Scale bar is 100 μm b, Plots show the min., max., and mean of 25th and 75th percentile plus the median for n=4 mice. c, Percentage of Ag-IGS reporter cells in the indicated distance bins to the nearest Ag+ cell, depicted mean plus SD for n=4 mice. Data obtained from three independent experiments.
Extended Data Fig. 4 CD8+ T cell dependent Katushka signaling in IGS reporter cells in vivo.
a, Flow cytometric analysis of Katushka expression in CFP+ IGS reporter (left panel) and GFP+ Ag+ (right panel) cells derived from mixed tumors described in Fig. 2b. Data from mice treated with HBSS are depicted in blue, data from mice treated with CDK4R>L-specific CD8+ T cells are depicted in red, n=4 mice per condition, data obtained from one experiment. b, Representative images of tumors before and 120h after injection of CDK4R>L-specific CD8+ T cells (left panel, three independent experiments with n=1 mouse each) or HBSS (right panel, two independent experiments with n=1 mouse each), for the imaging experiments described in Fig. 3. SHG: Second-harmonic generation. Scale bar is 200 μm.
Extended Data Fig. 5 Analysis of T cell mediated loss of Ag-presenting tumor cells over time.
a, Relative GFP+ volume in tumors from imaging experiments described in Fig. 2 quantified over time. Mean and SEM are depicted for n=5 mice (n=5 mice for time 0, 16, 24, and 32 h; n=4 for 40, 48, and 72 h; n=3 for 120 h, from data obtained in all independent experiments. b, The distance between CFP+ bystander tumor cells and the closest GFP+ Ag+ tumor cell was determined at indicated time points from tumors described in Fig. 2 for n=2 mice. Data are obtained from two independent experiments, boxplot presenting the minimum, 25th percentile, median, 75th percentile and the maximum For total sample size per timepoint see Source Data ED_Fig5_source table.
Extended Data Fig. 6 CD8+ T cell quantification in Ag+ and Ag- IGS reporter tumor areas.
a, Quantification of mOrange2+ CD8+ T cells in tumors with spatially separated GFP+Ag+ (green) and CFP+Ag- IGS reporter cell (cyan) islands obtained by sequential injection, as described in Fig. 4d, e. Number of mOrange2+ T cells was determined in multiple three-dimensional stacks of 2.5*107 μm3 in either Ag+ or Ag- areas. Symbols represent individual mice, and mean and SD for n=4 mice are depicted, obtained from two independent experiments. Normal distribution was confirmed by D’Agostino and Pearson omnibus normality test. Two tailed unpaired t-tests were performed, p=0.0003 (***). b, Estimate of the ratio of tumor cells to T cells in Ag+ and Ag- areas under the assumption that the diameter of an average tumor cell is 24 μm. c, Purified CD8+ T cells were activated with plate-bound anti-CD3/anti-CD28 antibodies for 2h. Subsequently, cells were either left untreated or were treated with 5nM LCKi inhibitor for the indicated times. Cells were washed to remove previously secreted IFNγ, and fresh control medium or medium containing 5nM LCK inhibitor was added to the cells. After 3h incubation, supernatants were collected and IFNγ concentrations were analysed. Bar graph shows mean IFNγ concentrations of n=3 technical replicates. Representative data of four independent experiments are depicted. d, As in c, depicting the IFNγ concentration in supernatants obtained from 2h LCK inhibitor treated cell cultures as a percentage of IFNγ concentration in control, non treated cell cultures. Dots represent four independent experiments, using different T cell donors in each experiment.
Extended Data Fig. 7 Distinct β2m positive and negative areas in human cancers.
a, Immunohistochemical staining of β2m and b, β2m and CD8 proteins on FFPE tissue of indicated human tumors. Heterogeneous β2m signal was observed in 16/51 tumors analyzed, one representative slide per tumor (obtained from resection material) was assessed and representative images are depicted in a. a. Scale bars are 100 μm. Note that CD8+ T cells in tumors predominantly localize to β2m high regions, representative images are depicted in b. Scale bars are 250 μm.
Extended Data Fig. 8 CD8+ T cell mediated killing of bystander OVCAR5 tumor cells.
A mixture of GFP+ Ag+, CFP+ Ag- IFNγR proficient and CFP+ Ag- IFNγR deficient OVCAR5 cells (2:1:1 ratio) was treated with CDK4R>L-specific CD8+ T cells at a 2:1 T cell: tumor cell ratio, or left untreated, and cell survival was analyzed by staining with IR-Dye and subsequent flow cytometry. a, Representative plots depicting the percentage of IR-Dye+ cells for the indicated groups. b, Quantification of a, bar graph shows mean of n=3 technical replicates. Representative data of two independent experiments are depicted.
Supplementary information
Source data
Source data Fig. 1
Statistical source data of all independent experiments.
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Statistical source data of all independent experiments.
Source data Extended data Fig. 1
Statistical source data of all independent experiments.
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Source data Extended data Fig. 3
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Statistical source data of all independent experiments.
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Source data Extended data Fig. 8
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Hoekstra, M.E., Bornes, L., Dijkgraaf, F.E. et al. Long-distance modulation of bystander tumor cells by CD8+ T-cell-secreted IFN-γ. Nat Cancer 1, 291–301 (2020). https://doi.org/10.1038/s43018-020-0036-4
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DOI: https://doi.org/10.1038/s43018-020-0036-4
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