Research ArticleCancer

In vivo imaging reveals a tumor-associated macrophage–mediated resistance pathway in anti–PD-1 therapy

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Science Translational Medicine  10 May 2017:
Vol. 9, Issue 389, eaal3604
DOI: 10.1126/scitranslmed.aal3604
  • Fig. 1. aPD-1 mAb labeling facilitates tracking of tissue biodistribution.

    (A) The rat anti-mouse PD-1 29F.1A12 clone, conjugated to AF647 via NHS ester linkage, efficiently binds PD-1+ T cells (here, EL4 cells) as detected by flow cytometry (gray histogram). Isotype control staining is shown in white. (B) MC38 tumors were equally responsive to single-dose AF647–aPD-1 and unlabeled aPD-1, whereas tumor sizes increased 72 hours after control immunoglobulin G2a (IgG2a) treatment. (C) Fluorescence reflectance imaging of three tumors compared to tumor-draining (tdLN) and nondraining (ndLN) lymph nodes 24 hours after AF647–aPD-1 treatment (AF647: λex = 620 to 650 nm, λem = 680 to 710 nm). Scale bar, 5 mm. (D) Quantified AF647–aPD-1 in each tissue demonstrating tumor accumulation. Values represent SEM and n = 3 unless otherwise noted. ***P < 0.001, unpaired, two-tailed t test.

  • Fig. 2. In vivo temporal aPD-1 mAb pharmacokinetics reveals drug accumulation in TAMs.

    (A) Diagram depicting intravital imaging setup with labeled aPD-1, MC38 tumor cells, T cells, and TAMs. FMX-PacBlue, ferumoxytol–Pacific Blue. (B) Treatment with single-dose aPD-1 mAb can achieve remission in the MC38–H2B-mApple tumor model. Tumors are outlined in gray. Scale bars, 2 mm. (C) Z-projections of an MC38–H2B-mApple tumor in a DPE-GFP mouse injected intravenously with AF647–aPD-1 after 15 min (top) or 24 hours (bottom). (D) Micrographs of T cells (magenta outline) identified as GFP+ cells and TAMs (yellow outline) identified by Pacific Blue signal. Outlines are overlaid on micrographs of the corresponding AF647–aPD-1 channel. Scale bars, 30 μm. (E) Intravital microscopy biodistribution studies indicate early aPD-1 binding to T cells and long-term accumulation in TAMs. Data are representative of five independently treated DPE-GFP mice and normalized to autofluorescent signal.

  • Fig. 3. In vivo imaging reveals aPD-1 mAb transfer from CD8+ T cells to TAMs.

    (A) Quantified AF647 signal on T cells and TAMs from a representative DPE-GFP mouse demonstrates collection of AF647–aPD-1 in T cells at 15 min and in TAMs at 24 hours after injection of therapy. (B) Early time-course intravital microscopy images acquired in an IFN-γ–YFP reporter mouse with an MC38–H2B-mApple tumor. Yellow arrows indicate the site of aPD-1 mAb binding to an IFN-γ+ CD8 T cell at 6 to 15 min and macrophage internalization at times >21 min. Scale bar, 30 μm. (C) Quantification of aPD-1 mAb on IFN-γ–expressing CD8+ lymphocytes and TAMs reveals a narrow window of target binding. (D) Flow cytometry histograms pregated for 7-AAD (7-aminoactinomycin D–negative)/CD45+ show AF647–aPD-1 signal (x axis, logarithmic scale) on immune cell populations at 0.5 and 24 hours after administration (blue). Cell populations from untreated control animals (red) were used as reference. DC, dendritic cell; Granul./Mono., granulocyte/monocyte. (E) aPD-1 mAb binds to CD8+ lymphocytes early but accumulates in TAMs at later time points. **P < 0.01; ****P < 0.0001, unpaired, two-tailed t test.

  • Fig. 4. aPD-1 mAb transfer to macrophages is mediated by FcγRs.

    (A) Ex vivo flow cytometry histograms of MC38 tumors stained with phycoerythrin (PE)–aPD-1 show that CD8+ T cells but not TAMs express cell surface PD-1. (B) Coculture of bone marrow–derived macrophages (Mø) and AF647–aPD-1–coated EL4 lymphocytes was used to quantify the AF647–aPD-1 puncta in macrophages preblocked with Fcγ receptor (FcγR) inhibitors or the phagocytosis inhibitor dynasore. *P < 0.05; ****P < 0.0001, one-way analysis of variance (ANOVA). n.s., not significant. (C) Representative images of macrophages labeled with PKH red and observed by time-lapse imaging for aPD-1 mAb (yellow) transfer from neighboring lymphocytes (cerulean) after 30 min. FcγRII/III inhibition blocked antibody transfer (insets). Scale bars, 10 μm. (D and E) Flow cytometry was used to estimate AF647–aPD-1 transfer to F4/80+ bone marrow–derived macrophages in coculture assays when the mAb was added directly (green), bound to EL4 cells (red), or bound to EL4 cells in the presence of FcγRII/III neutralizing antibody (blue). Results in (D) are a representative histogram of AF647 signal in macrophages, and (E) is the geometric mean fluorescence intensity (G-MFI) value of conditions presented in (D). Data are from three independent experiments. *P < 0.05; ** P < 0.01, two-way ANOVA with Tukey’s multiple comparisons test. Values represent SEM for three separate experiments.

  • Fig. 5. Nivolumab shares similar glycan patterns with mouse aPD-1 and is transferred to macrophages via FcγRs.

    (A) HPLC analysis of the glycan patterns found on the mouse aPD-1 mAb and nivolumab shows the G0F (“2”) isoform to be predominant, but glycosylation is not uniform. (B) AF647-labeled nivolumab (yellow) was used to stain the surface of aCD3-stimulated PKH green–labeled human CD8+ T cells (cerulean) co-incubated with PKH red–labeled peripheral blood mononuclear cell–derived macrophages in the presence or absence of Fc Block. Scale bars, 20 μm. (C) Quantification of AF647+ puncta per macrophage confirms that nivolumab is transferred via FcγRs. Values represent SEM for four separate experiments.

  • Fig. 6. Disrupting Fc binding affects macrophage uptake of aPD-1 and improves treatment efficacy.

    (A) PKH green–labeled EL4 cells (cerulean) stained with native AF647–aPD-1 or deglycosylated AF647–aPD-1 cocultured with PKH red–labeled bone marrow–derived macrophages (Mø). Images are representative of three separate experiments. Scale bars, 10 μm. (B) Fluorescence-activated cell sorting plots of mouse Mø (gated on F4/80+) cocultured with PD-1+ EL4 cells labeled with either AF647–aPD-1 mAb (blue) or deglycosylated AF647–aPD-1 mAb (red). (C) aPD-1 mAb deglycosylation substantially reduces the transfer from EL4 cells to Mø (n = 3). **P < 0.01, unpaired two-tailed t test. (D) Intravital images 10 and 120 min after AF647–aPD-1 drug extravasation in a representative IFN-γ–YFP reporter mouse MC38–H2B-mApple tumor pretreated with the FcγRII/III blocking antibody (2.4G2). (E) Quantified AF647–aPD-1 from three 2.4G2-treated mice shows prolonged aPD-1 binding to tumor T cells, relative to mice treated with AF647–aPD-1 alone (data repeated from Fig. 3C for comparison). (F) MC38 tumor growth curves of mice (n ≥ 5) treated with isotype control (black), aPD-1 (blue), and aPD-1 plus 2.4G2 (red). *P < 0.05, one-way ANOVA with Tukey’s multiple comparison test.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/389/eaal3604/DC1

    Materials and Methods

    Fig. S1. AF647–aPD-1 mAb quantification in various tissues at 24 hours after injection.

    Fig. S2. Specificity of T cell reporter mice and dextran nanoparticles.

    Fig. S3. Representative intravital microscopy time course of AF647–aPD-1 from an IFN-γ–YFP mouse.

    Fig. S4. T cell and macrophage motility before and after aPD-1 treatment.

    Fig. S5. Assessment of aPD-1 binding to tumor cells.

    Fig. S6. aPD-1 transfer to tumor macrophages in vivo.

    Fig. S7. Distribution of AF647–aPD-1 across tumor models.

    Fig. S8. PD-1 expression by CD8+ T cells in the MC38 tumor microenvironment.

    Fig. S9. aPD-1 mAb transfer from T cells to macrophages in vitro.

    Fig. S10. Assessment of aPD-1 internalization after binding to PD-1.

    Fig. S11. aPD-1 degradation by macrophages.

    Fig. S12. PD-1 localization following aPD-1 internalization after binding to PD-1.

    Fig. S13. Comparative analysis of mAb glycosylation patterns between mouse and human PD-1 antibodies.

    Fig. S14. Assessment of cell membrane component exchange during aPD-1 transfer.

    Fig. S15. Confirmation of deglycosylation and antigen binding affinity for rat anti-mouse PD-1.

    Fig. S16. Impact of Fc blockade on aPD-1 treatment efficacy.

    Fig. S17. Proposed resistance mechanism and potential improvement of aPD-1 mAb therapy.

    Movie S1. Intravital microscopy imaging of AF647–aPD-1 injection in a DPE-GFP mouse bearing an MC38–H2B-mApple tumor in a dorsal skinfold chamber.

    Movie S2. Intravital microscopy imaging of AF647–aPD-1 injection in an IFN-γ–enhanced YFP GREAT mouse bearing an MC38–H2B-mApple tumor in a dorsal skinfold chamber.

    Movie S3. Live imaging of mouse T cell aPD-1 transfer to macrophages.

    Movie S4. Live imaging of human T cell aPD-1 transfer to macrophages.

    References (4652)

  • Supplementary Material for:

    In vivo imaging reveals a tumor-associated macrophage–mediated resistance pathway in anti–PD-1 therapy

    Sean P. Arlauckas, Christopher S. Garris, Rainer H. Kohler, Maya Kitaoka, Michael F. Cuccarese, Katherine S. Yang, Miles A. Miller, Jonathan C. Carlson, Gordon J. Freeman, Robert M. Anthony, Ralph Weissleder, Mikael J. Pittet*

    *Corresponding author. Email: mpittet{at}mgh.harvard.edu

    Published 10 May 2017, Sci. Transl. Med. 9, eaal3604 (2017)
    DOI: 10.1126/scitranslmed.aal3604

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. AF647–aPD-1 mAb quantification in various tissues at 24 hours after injection.
    • Fig. S2. Specificity of T cell reporter mice and dextran nanoparticles.
    • Fig. S3. Representative intravital microscopy time course of AF647–aPD-1 from an IFN-γ–YFP mouse.
    • Fig. S4. T cell and macrophage motility before and after aPD-1 treatment.
    • Fig. S5. Assessment of aPD-1 binding to tumor cells.
    • Fig. S6. aPD-1 transfer to tumor macrophages in vivo.
    • Fig. S7. Distribution of AF647–aPD-1 across tumor models.
    • Fig. S8. PD-1 expression by CD8+ T cells in the MC38 tumor microenvironment.
    • Fig. S9. aPD-1 mAb transfer from T cells to macrophages in vitro.
    • Fig. S10. Assessment of aPD-1 internalization after binding to PD-1.
    • Fig. S11. aPD-1 degradation by macrophages.
    • Fig. S12. PD-1 localization following aPD-1 internalization after binding to PD-1.
    • Fig. S13. Comparative analysis of mAb glycosylation patterns between mouse and human PD-1 antibodies.
    • Fig. S14. Assessment of cell membrane component exchange during aPD-1 transfer.
    • Fig. S15. Confirmation of deglycosylation and antigen binding affinity for rat anti-mouse PD-1.
    • Fig. S16. Impact of Fc blockade on aPD-1 treatment efficacy.
    • Fig. S17. Proposed resistance mechanism and potential improvement of aPD-1 mAb therapy.
    • Legends for movies S1 to S4
    • References (4652)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). Intravital microscopy imaging of AF647–aPD-1 injection in a DPE-GFP mouse bearing an MC38–H2B-mApple tumor in a dorsal skinfold chamber.
    • Movie S2 (.mp4 format). Intravital microscopy imaging of AF647–aPD-1 injection in an IFN-γ–enhanced YFP GREAT mouse bearing an MC38–H2B-mApple tumor in a dorsal skinfold chamber.
    • Movie S3 (.mov format). Live imaging of mouse T cell aPD-1 transfer to macrophages.
    • Movie S4 (.mov format). Live imaging of human T cell aPD-1 transfer to macrophages.

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