Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor that is upregulated on activated T cells for the induction of immune tolerance1,2. Tumour cells frequently overexpress the ligand for PD-1, programmed cell death ligand 1 (PD-L1), facilitating their escape from the immune system3,4. Monoclonal antibodies that block the interaction between PD-1 and PD-L1, by binding to either the ligand or receptor, have shown notable clinical efficacy in patients with a variety of cancers, including melanoma, colorectal cancer, non-small-cell lung cancer and Hodgkin’s lymphoma5,6,7,8,9. Although it is well established that PD-1–PD-L1 blockade activates T cells, little is known about the role that this pathway may have in tumour-associated macrophages (TAMs). Here we show that both mouse and human TAMs express PD-1. TAM PD-1 expression increases over time in mouse models of cancer and with increasing disease stage in primary human cancers. TAM PD-1 expression correlates negatively with phagocytic potency against tumour cells, and blockade of PD-1–PD-L1 in vivo increases macrophage phagocytosis, reduces tumour growth and lengthens the survival of mice in mouse models of cancer in a macrophage-dependent fashion. This suggests that PD-1–PD-L1 therapies may also function through a direct effect on macrophages, with substantial implications for the treatment of cancer with these agents.
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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.
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.
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
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.
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. Source data
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. Source data
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. Source data
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. Source data
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. Source data
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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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