Research ArticleCancer

Human tumor-associated monocytes/macrophages and their regulation of T cell responses in early-stage lung cancer

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Science Translational Medicine  13 Feb 2019:
Vol. 11, Issue 479, eaat1500
DOI: 10.1126/scitranslmed.aat1500
  • Fig. 1 Frequencies of tumor-infiltrating macrophage/monocytes.

    (A) Frequencies of tumor myeloid CD11b+ cell populations and morphology of tumor CD14+ cells. Scale bar, 10 μm. SSC-A, side scatter area. (B) CD14+ MMLC frequencies in tumor, distant lung tissue, and blood of patients with LC, as well as in noncancerous lung transplant (LT) and HD blood. (C) The expression of macrophage-associated markers on CD11b+CD14+ cells in digested tumors and blood of patients with LC. (D) The proportion of CD14+ cells expressing macrophage markers. MFI, mean fluorescence intensity. (E) CD68+ cells in tumor islet and tumor stroma with CD68 (brown) and cytokeratin (red). Scale bar, 100 μm. (F) HLA-DR expression in HD blood and on gated CD14+ cells. (G and H) The proportion of CD14 cells expressing low and high HLA-DR. (I) The morphology of tumor HLA-DRhi and HLA-DRint CD14+cells. Scale bars, 10 μm. Kruskal-Wallis (B and D: CD206 and CD163; G and H, left), Wilcoxon paired test (E), and Friedman tests (G and H, right), one-way analysis of variance [ANOVA; D: CD115 and interferon (IFN) regulatory factor 8 (IRF8)]. The red line represents mean ± SEM; ***P < 0.001, **P < 0.01, and *P < 0.05. The number of patients included in each analysis is indicated on the graphs.

  • Fig. 2 Mixed phenotype of NSCLC-associated macrophages.

    (A) Flow cytometric analysis of HLA-DR expression and macrophage markers on CD11b+CD14+ cells in tumor and peripheral blood mononuclear cells (PBMCs) of patients with LC. (B) The correlation between the proportions of CD14+ cells expressing HLA-DR and macrophage markers among all live CD11b+CD14+ cells in tumors. Spearman test (HLA-DRhiCD206, HLA-DRhiCD40, HLA-DRhiCD86, and HLA-DRhiCD163) and Pearson test (HLA-DRhiCD80). (C) Cumulative flow cytometry results showing the frequency of cells coexpressing HLA-DR and macrophage markers among all live CD11b+CD14+ cells in tumors, distant lung tissue of patients with LC, and noncancerous lung transplant (LT). (D) Cumulative flow cytometry results showing TAM frequencies among all nucleated cells in tumors, distant lung tissue of patients with LC, and noncancerous lung transplant. TAMs were defined as CD11b+CD14+CD66bHLA-DRhiCD206hiCD40hi cells. (E) Flow cytometric analysis of the expression of macrophage markers on TAMs and M1/M2 macrophages differentiated in vitro. Representative histograms from one of five experiments are shown. (F) Heat map of the proportions of tumor CD14+ cells expressing macrophage markers (rows) across studied patients (columns). Kruskal-Wallis with Dunn’s multiple comparisons test [C (HLA-DRhiCD206) and D], one-way ANOVA with Turkey’s multiple comparisons tests (C: HLA-DRhiCD40, HLA-DRhiCD80, HLA-DRhiCD86). The red line represents mean ± SEM; ***P < 0.001, **P < 0.01, and *P < 0.05. The number of patients included in each analysis is indicated on the graphs.

  • Fig. 3 Expression of T cell cosignaling receptors on MMLCs.

    (A) Cumulative results showing the proportion of CD14+ cells expressing T cell cosignaling molecules in tumors, distant lung tissue, peripheral blood of patients with LC, and noncancerous lung transplant (LT). (B) Expression of PD-L1 (brown) in lung tumor tissue. Scale bar, 100 μm. (C) Expression of PD-L1 and CD14 in digested tumor, distant tissue, and PMBCs. (D) Cumulative results showing the intensity of PD-L1 expression on CD14+ and EpCam+ cells in tumors. (E) Expression of HLA-DR and PD-L1 on live-gated CD11b+CD14+CD66b cells in tumor and PBMCs. (F) Correlation between the proportions of CD14+ cells expressing HLA-DR and PD-L1 among all live CD11b+CD14+cells in tumors. (G) Summary results showing the proportion of HLA-DRhiPD-L1hi cells among all CD11b+CD14+CD66b cells in tumor, distant lung tissue of patients with LC, and noncancerous lung transplant. (H) Representative dot plots demonstrating the expression of PD-L1 and indicated T cell costimulatory receptors on live-gated CD11b+CD14+ cells in tumor and PBMC. (I) Heat map of the proportions of CD14+ cells expressing T cell cosignaling receptors across studied patients. One-way ANOVA (A: CD40hiCD14, CD54hiCD14, 4-1BBLhiCD14, PD-L1hiCD14, B7-H3hiCD14, and PD-L2hiCD14), Kruskal-Wallis test [A (CD86hiCD14) and G], paired t test (D), and Spearman test (F) were performed. The red line represents mean ± SEM; ***P < 0.001, **P < 0.01, and *P < 0.05. The number of patients included in each analysis is indicated on the graphs.

  • Fig. 4 Effects of tumor CD14 cells on antigen-nonspecific T cell responses.

    (A) Neutral (blue box), stimulatory (green box), and suppressive (red box) effects of tumor CD14+ cells from different patients on the IFN-γ production by autologous CD8+ T cells stimulated with plate-bound anti-CD3 Abs. (B) Summary results of IFN-γ production by stimulated autologous CD8+ T cells in the presence of CD14+ cells isolated from tumor, distant tissue, and blood. Stimulatory index (SI) is a ratio (CD8 cells + CD14 cells)/(CD8 cells). (C) Proliferation of stimulated autologous CD8+ T cells in the presence of CD14+ cells from tumor, distant tissue, and blood. CFSE, carboxyfluorescein succinimidyl ester; BrdU, bromodeoxyuridine. (D) Cumulative results of autologous CD8+ T cell proliferation stimulated with anti-CD3 Abs in the presence of CD14+ cells from tumor, distant tissue, and blood. SI is a ratio (CD8 + CD14)/(CD8). (E and F) Representative dot plots and cumulative results showing the effect of CD14+ cells on IFN-γ production by CD8+ T cells in digested tumor, distant tissue, and PBMCs that were depleted for CD14+ cells before stimulation with plate-bound anti-CD3 Abs. SI is a ratio (IFN-γ+CD8+ cells in total digest)/(IFN-γ+CD8+ cells in CD14 cell depleted digest). One-way ANOVA (B) and Friedman (D and F) tests were performed for two groups, where SI is >1 and <1, *P < 0.05. The number of patients included in each analysis is indicated on the graphs.

  • Fig. 5 Effects of tumor CD14 cells on the effector phase of NY-ESO-1–specific T cell responses.

    (A) Activation and IFN-γ production by Ly95 cells stimulated with A549 or A549/A2–NY-ESO-1 tumor cells. (B) Suppressive (red box), neutral (blue box), and stimulatory (green box) effects of tumor CD14 cells from different patients on Ly95 T cell response. (C) Cumulative results showing the different effects of tumor CD14+ cells on Ly95 cell response. SI is a ratio (Ly95 + A549-NY-ESO-1 + CD14)/(Ly95 + A549-NY-ESO-1). One-way ANOVA test for two groups, where SI is >1 and <1, *P < 0.05. (D) IFN-γ production by Ly95 cells that were either simultaneously mixed with tumor CD14 cells and A549/A2–NY-ESO-1 cells (Tumor CD14 day 0) or preincubated with CD14 cells for 24 hours before stimulation with A549/A2–NY-ESO-1 cells (Tumor CD14 day 1). Representative results from one of five experiments are shown. (E and F) Effect of CD14 cells on the tumoricidal activity of Ly95 cells toward A549/A2–NY-ESO-1 tumor cells. Tumoricidal index is a ratio (Ly95 + A2-NY-ESO-1 + CD14)/(Ly95 + A2-NY-ESO-1). Friedman test for two groups, where SI is >1 and <1, *P < 0.05. (G and H) IFN-γ production by Ly95 T cells stimulated with A549/A2–NY-ESO-1 cells or tumor HLA-A2+CD14 cells pulsed with NY-ESO-1 peptide (Tumor CD14). One-way ANOVA, *P < 0.05 and ***P < 0.001. The number of patients included in each analysis is indicated on the graphs.

  • Fig. 6 Effects of TAMs and tumor-expressed PD-L1 on the effector phase of tumor-specific T cell responses.

    (A) Representative dot plots demonstrating the NY-ESO-1–specific Ly95 cell response (IFN-γ production and killing activity) to control A549 and PD-L1+/− A549/A2–NY-ESO-1 tumor cells. (B) Cumulative results showing the NY-ESO-1–specific Ly95 cell IFN-γ production to A549/A2–NY-ESO-1 and A549/A2–NY-ESO-1 PD-L1hi tumor cells in the presence or absence of PD-L1–blocking Abs. Repeated measures (RM) one-way ANOVA with Turkey’s multiple comparisons tests were performed for two groups, where SI is >1 and <1, ***P < 0.001. (C) The effects of tumor PDL-1hi and PD-L1lo CD14 cells isolated from different patients on the IFN-γ and granzyme B production by Ly95 cells cocultured with A549/A2–NY-ESO-1 tumor cells in the presence or absence of PD-L1–blocking Abs. (D) The proportions of HLA-DRhiPD-L1hi cells among tumor CD14+ cells that had an SI of more than 1 (SI > 1) and less 1 (SI < 1) (mean ± SEM). Unpaired t test, **P < 0.01. (E) Heat map demonstrating the relationship of the SIs of tumor CD14+ cells and their relative proportions expressing T cell costimulatory and coinhibitory receptors (rows) across the studied patients (columns). The number of patients included in each analysis is indicated on the graphs.

  • Fig. 7 Role of TAM-derived PD-L1 in the regulation of tumor-specific T cell responses.

    (A) The gating strategy for sorting CD14+HLA-DRhiPD-L1hi TAMs and CD14+HLA-DRintPD-L1 T-Mos from tumors. (B and C) Representative dot plots and cumulative results showing the IFN-γ and granzyme B production by Ly95 cells cocultured with A549/A2–NY-ESO-1 tumor cells in the presence or absence of CD14+HLA-DRhiPD-L1hi and CD14+HLA-DRintPD-L1 cells sorted from tumors. SI is a ratio (Ly95 + A549-NY-ESO-1 + CD14)/(Ly95 + A549-NY-ESO-1), paired t test, **P < 0.01. (D) TAMs and T-Mos were preincubated with long NY-ESO145–174 peptide at a concentration of 10 μg/ml for 2 hours before mixing with Ly95 cells. NY-ESO-1–specific IFN-γ production was measured by flow cytometry in live-gated CD8+TCRVβ13.1+ and CD8+TCRVβ13.1 cells. Mann-Whitney test, *P < 0.05. (E) Representative dot plots and cumulative results demonstrating the cytotoxic activity of Ly95 T cells toward NY-ESO-1 peptide–pulsed HLA-A2+CD14 macrophages differentiated from blood monocytes in the presence of M-CSF (Mo-Mph) or M-CSF and tumor-conditioned media (TCM; TCM Mo-Mph) in vitro. (F) Representative dot plots and cumulative results demonstrating the cytotoxic activity of Ly95 T cells toward NY-ESO-1 peptide–pulsed HLA-A2+CD14 cells isolated from tumors. Wilcoxon paired test, *P < 0.05. The number of patients included in each analysis is indicated on the graphs.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/11/479/eaat1500/DC1

    Materials and Methods

    Fig. S1. Phenotypic characterization of tumor-infiltrating MMLCs.

    Fig. S2. Production of indicated monocyte chemotactic proteins by tumor, distant lung tissue dissociates, and PBMCs.

    Fig. S3. HLA-DRhiCD14+ TAM coexpress M1/M2 markers, as well as T cell coinhibitory and costimulatory receptors.

    Fig. S4. The accumulation of nonclassical CD14intCD16hi monocytes in tumor, distant lung tissue, and blood.

    Fig. S5. Production of factors involved in monocyte recruitment and macrophage differentiation/polarization in tumor, distant lung dissociates, and blood.

    Fig. S6. The expression of T cell coinhibitory and costimulatory receptors on MMLCs.

    Fig. S7. Effects of tumor CD14+ cells on NY-ESO-1–specific T cell responses.

    Fig. S8. The expression of key T cell suppressive genes in CD14+ MMLCs and their correlation with the ability of tumor MMLCs to regulate tumor-specific T cell responses.

    Fig. S9. Production of key T cell suppressive factors by CD14+ MMLCs and their correlation with the ability of tumor MMLCs to regulate tumor-specific T cell responses.

    Fig. S10. Common T cell suppressive mechanisms in the regulation of tumor-specific Ly95 response by early-stage tumor MMLCs.

    Fig. S11. Role of TAM-derived PD-L1 in the regulation of tumor-specific T cell responses.

    Fig. S12. Cross-presentation of NY-ESO-1 by TAMs and role of TAM-expressed PD-L1 in the regulation of cytotoxic activity of Ly95 cells.

    Fig. S13. Correlation analysis of the presence of MMLCs in lung tumor with overall survival.

    Fig. S14. Correlation analysis of the accumulation of MMLC populations with the frequency and function of tumor-associated neutrophils, Tregs, and CD8 cells in tumor.

    Fig. S15. Correlation analysis of the ability of tumor CD14+ cells to regulate T cell responses with accumulation of CD8+ T cells, Tregs, and IFN-γ production by CD8+ T cells in tumor.

    Table S1. Patient characteristics.

    Table S2. Correlation analysis of the phenotypic and functional characteristics of tumor CD14+ cells with clinical parameters of patients with LC.

    Data file S1. Primary data.

    References (42, 43)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Phenotypic characterization of tumor-infiltrating MMLCs.
    • Fig. S2. Production of indicated monocyte chemotactic proteins by tumor, distant lung tissue dissociates, and PBMCs.
    • Fig. S3. HLA-DRhiCD14+ TAM coexpress M1/M2 markers, as well as T cell coinhibitory and costimulatory receptors.
    • Fig. S4. The accumulation of nonclassical CD14intCD16hi monocytes in tumor, distant lung tissue, and blood.
    • Fig. S5. Production of factors involved in monocyte recruitment and macrophage differentiation/polarization in tumor, distant lung dissociates, and blood.
    • Fig. S6. The expression of T cell coinhibitory and costimulatory receptors on MMLCs.
    • Fig. S7. Effects of tumor CD14+ cells on NY-ESO-1–specific T cell responses.
    • Fig. S8. The expression of key T cell suppressive genes in CD14+ MMLCs and their correlation with the ability of tumor MMLCs to regulate tumor-specific T cell responses.
    • Fig. S9. Production of key T cell suppressive factors by CD14+ MMLCs and their correlation with the ability of tumor MMLCs to regulate tumor-specific T cell responses.
    • Fig. S10. Common T cell suppressive mechanisms in the regulation of tumor-specific Ly95 response by early-stage tumor MMLCs.
    • Fig. S11. Role of TAM-derived PD-L1 in the regulation of tumor-specific T cell responses.
    • Fig. S12. Cross-presentation of NY-ESO-1 by TAMs and role of TAM-expressed PD-L1 in the regulation of cytotoxic activity of Ly95 cells.
    • Fig. S13. Correlation analysis of the presence of MMLCs in lung tumor with overall survival.
    • Fig. S14. Correlation analysis of the accumulation of MMLC populations with the frequency and function of tumor-associated neutrophils, Tregs, and CD8 cells in tumor.
    • Fig. S15. Correlation analysis of the ability of tumor CD14+ cells to regulate T cell responses with accumulation of CD8+ T cells, Tregs, and IFN-γ production by CD8+ T cells in tumor.
    • Table S1. Patient characteristics.
    • Table S2. Correlation analysis of the phenotypic and functional characteristics of tumor CD14+ cells with clinical parameters of patients with LC.
    • References (42, 43)

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    Other Supplementary Material for this manuscript includes the following:

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