PD-1 expression by tumour-associated macrophages inhibits phagocytosis and tumour immunity (original) (raw)

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Acknowledgements

The authors thank S. Karten for assistance in editing the manuscript; and A. McCarty, T. Storm and T. Naik for technical support. Research reported in this publication was supported by the D. K. Ludwig Fund for Cancer Research (to I.L.W.); the A.P. Giannini Foundation and the Stanford Dean’s Fellowship (to M.N.M.); the Stanford Medical Scientist Training Program NIH-GM07365 (to B.M.G., B.W.D. and J.M.T.); a Cancer Research Institute Irvington Fellowship (to R.L.M.); and a Swiss National Science Foundation fellowship P300P3_155336 (to G.H.). The project described was supported, in apart, by ARRA Award Number 1S10RR026780-01 from the National Center for Research Resources (NCRR). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NCRR or the National Institutes of Health.

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Authors and Affiliations

  1. Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, 94305, California, USA
    Sydney R. Gordon, Roy L. Maute, Ben W. Dulken, Gregor Hutter, Benson M. George, Melissa N. McCracken, Jonathan M. Tsai, Rahul Sinha, Daniel Corey & Irving L. Weissman
  2. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, 4305, California, USA
    Sydney R. Gordon
  3. Ludwig Center for Cancer Stem Cell Research and Medicine, Stanford University School of Medicine, Stanford, 94305, California, USA
    Sydney R. Gordon, Roy L. Maute, Benson M. George, Melissa N. McCracken, Jonathan M. Tsai, Rahul Sinha, Daniel Corey & Irving L. Weissman
  4. Stanford Cancer Institute, Stanford University School of Medicine, Stanford, 94305, California, USA
    Sydney R. Gordon, Roy L. Maute, Benson M. George, Melissa N. McCracken, Jonathan M. Tsai, Rahul Sinha, Daniel Corey & Irving L. Weissman
  5. Department of Pathology, Stanford University Medical Center, Stanford, 94305, California, USA
    Sydney R. Gordon, Roy L. Maute, Benson M. George, Melissa N. McCracken, Jonathan M. Tsai, Rahul Sinha, Daniel Corey, Andrew J. Connolly & Irving L. Weissman
  6. Stanford Medical Scientist Training Program, Stanford University, Stanford, 94305, California, USA
    Ben W. Dulken, Benson M. George & Jonathan M. Tsai
  7. Department of Neurosurgery, Stanford University School of Medicine, Stanford, 94305, California, USA
    Gregor Hutter
  8. Department of Neurosurgery, University Hospital Basel, Basel, CH-4031, Switzerland
    Gregor Hutter
  9. Human Immune Monitoring Center Biobank, Stanford University School of Medicine, Palo Alto, 94304, California, USA
    Rohit Gupta
  10. Department of Immunobiology, Yale University School of Medicine, New Haven, 06519, Connecticut, USA
    Aaron M. Ring

Authors

  1. Sydney R. Gordon
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  2. Roy L. Maute
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  3. Ben W. Dulken
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  4. Gregor Hutter
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  5. Benson M. George
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  6. Melissa N. McCracken
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  7. Rohit Gupta
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  8. Jonathan M. Tsai
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  9. Rahul Sinha
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  10. Daniel Corey
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  11. Aaron M. Ring
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  12. Andrew J. Connolly
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  13. Irving L. Weissman
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Contributions

S.R.G. wrote the manuscript. S.R.G., R.L.M., M.N.M., A.M.R. and I.L.W. conceived and designed all experiments. S.R.G. performed TAM staining, made the HAC protein and conducted all in vivo studies, phagocytosis assays and analysis. B.W.D. and R.S. helped with FACS gating and TAM analysis. G.H. generated NSG _Ccr2_−/− mice. B.M.G. conducted bone marrow transplants. S.R.G., R.L.M. and M.N.M. generated cell lines. R.G. acquired human colon cancer samples. J.M.T. taught the immunofluorescence protocol. D.C. and A.J.C. characterized foamy TAMs. R.L.M. and I.L.W. supervised the research and edited the manuscript.

Corresponding author

Correspondence toIrving L. Weissman.

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Competing interests

S.R.G., R.L.M., M.N.M., A.M.R. and I.L.W. are inventors on a patent (15/502,439) that is related to the HAC protein. S.R.G. and M.N.M. provide paid consulting services to Ab Initio Biotherapeutics Inc., which licensed this patent. R.L.M. and A.M.R. are founders of Ab Initio Biotherapeutics Inc.

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Reviewer Information Nature thanks V. A. Boussiotis, M. De Palma and A. Mantovani for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 FACS gating strategy for TAMs.

Debris and doublets were removed, then TAMs were assessed as Hoechst−CD45+CD8a−CD19−Ter119−TCRβ−CD11b+F4/80+. TAM PD-1 gating is shown as well, based on the PD-1 isotype control. All other gates were determined on the basis of FMOs. T cells, gated as Hoechst−CD45+TCRβ+CD8a+, are shown as PD-1+ positive control.

Extended Data Figure 2 TAM characterization.

a, No primary control for immunofluorescence images is shown. Cytospinned TAMs were stained with fluorescently conjugated secondary antibodies only. n = 2, two experimental repeats. 20× magnification; scale bar, 20 μm. Red, 594; green, 488; blue, Hoechst. b, Mouse PD-1− TAMs trend towards an M1 (CD206−MHC IIhigh) expression profile, rather than M2 (CD206+MHC IIlow or negative). TAMs that did not adhere to either of these expression profiles were not classified as M1 or M2. n = 5, experiment conducted once. Paired one-tailed _t_-test. c, Human PD-1− TAMs are predominantly M1 (CD206−CD64+) rather than M2 (CD206+CD64−). n = 10, two experimental repeats. Paired one-tailed _t_-test. d, In mice, there is a highly significant correlation between tumour volume and the percentage of PD-1+ TAMs. n = 20, two experimental repeats. An exponential growth equation is shown. e, Donor chimaerism six weeks after bone marrow transplantation. Granulocytes (Gr1high), 99%; myeloid cells (CD11b+), 92%; B cells (CD19+), 97%; T cells (TCRβ+), 74%. n = 8, experiment conducted once. Data are mean ± s.e.m.; **P < 0.01; n.s., not significant.

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Extended Data Figure 3 Ex vivo phagocytosis assay with FACS-sorted TAMs.

Sorted PD-1− and PD-1+ TAMs from CT26 tumours were assayed with pHrodo green Staphylococcus aureus bioparticles. These particles are GFPlow at neutral pH, and GFPhigh in the acidic phagosome. a, Representative histogram showing difference in GFP fluorescence of PD-1− versus PD-1+ TAMs in the phagocytosis assay, and in comparison to S. aureus bioparticles alone. Bioparticles alone are clearly GFPlow, but have an obvious upshift in fluorescence when they are phagocytosed. b, Representative histograms showing the flow cytometry gating strategy for phagocytosis by PD-1− and PD-1+ TAMs. GFPhigh TAMs were considered to be phagocytosing. c, Analysis of phagocytosis shows that PD-1+ TAMs phagocytosed significantly less than PD-1− TAMS. n = 4, two experimental repeats. Paired one-tailed _t_-test. Data are mean ± s.e.m.; ****P < 0.0001.

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Extended Data Figure 4 Immunocompromised mice also exhibit tumour-specific expression of PD-1 on macrophages.

a, Analysis of PD-L1-overexpressing CT26/YFP+ tumours in BALB/c _Rag2_−/−_γc_−/− mice shows that TAMs specifically express PD-1. n = 4, two experimental repeats. Paired one-way ANOVA with multiple comparisons correction. b, There is a highly significant correlation between BALB/c _Rag2_−/−_γc_−/− tumour volume and the percentage of PD-1+ TAMs. n = 9, two experimental repeats. Best fit line is shown. c, Analysis of DLD-tg(hPD-L1)-GFP-luc+ tumours shows that TAMs specifically express PD-1. n = 5, two experimental repeats. Paired one-way ANOVA with multiple comparisons correction. d, There is a highly significant correlation between NSG tumour volume and the percentage of PD-1+ TAMs. n = 1, two experimental repeats. Best fit line is shown. Data are mean ± s.e.m.; *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.

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Extended Data Figure 5 In vivo phagocytosis analysis.

a, Representative FACS plots showing gating strategy for in vivo phagocytosis. Here, total phagocytosis was analysed by first gating on TAMs, and then gating on YFP+ cells. Total TAM PD-1 expression from the same tumour sample is shown side by side to demonstrate that high PD-1 expression inversely correlates with phagocytosis. b, Analysis of PD-1− TAM phagocytosis shows that the presence or absence of PD-L1 does not affect PD-1− TAM phagocytosis. PD-L1 overexpression, n = 7; PD-L1 knockout, n = 9, two experimental repeats. Paired one-tailed _t_-test. c, TAM PD-1 expression is not affected by the presence or absence of PD-L1. PD-L1 overexpression, n = 7; PD-L1 knockout, n = 9, two experimental repeats. Paired one-tailed _t_-test. Data are mean ± s.e.m.; n.s., not significant.

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Extended Data Figure 6 In vivo TAM depletion.

TAMs were depleted with anti-CSF1R treatment in NSG-_Ccr2_−/− mice. a, TAM depletion protocol does not affect the number of granulocytes (Gr1high) in DLD-tg(PD-L1)-GFP-luc+ tumours. n = 10, experiment conducted once. Unpaired one-tailed _t_-test. b, TAM depletion protocol eliminates almost all TAMs in tumours. n = 10, experiment conducted once. Unpaired one-tailed _t_-test. Data are mean ± s.e.m.; ****P < 0.0001; n.s., not significant.

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Gordon, S., Maute, R., Dulken, B. et al. PD-1 expression by tumour-associated macrophages inhibits phagocytosis and tumour immunity.Nature 545, 495–499 (2017). https://doi.org/10.1038/nature22396

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