CD14(+)S100A9(+) monocytic myeloid-derived suppressor cells and their clinical relevance in non-small cell lung cancer

Abstract

Rationale: Myeloid-derived suppressor cells (MDSCs) are a heterogeneous family of myeloid cells that suppress T-cell immunity in tumor-bearing hosts. Their clinical relevance remains unclear.

Objectives: To identify subtypes of myeloid-derived suppressor cells in patients with non-small cell lung cancer (NSCLC) and their clinical relevance.

Methods: CD11b(+)CD14(-) and CD11b(+)CD14(+) cells, determined and phenotyped by fluorescence-activated cell sorter analysis, in the peripheral blood mononuclear cells (PBMCs) of treatment-naive patients with advanced NSCLC were correlated with clinical data. T-cell activation in response to CD3/CD28 costimulation was determined by carboxy-fluorescein diacetate succinimidyl ester (CFSE) staining and ELISA analysis of IFN-γ. The percentage of CD11b(+)CD14(+)S100A9(+) cells in PBMCs was correlated with and tested as a predictor for treatment response in a cohort of patients prospectively receiving first-line cisplatin-based chemotherapy.

Measurements and main results: Patients with NSCLC had a significantly higher ratio of CD11b(+)CD14(+) cells than healthy subjects, which was correlated with poor performance status and poor response to chemotherapy. The depletion of these cells in the PBMC reversed the suppression of CD8(+) and CD4(+) T cells. Isolated CD11b(+)CD14(+) cells suppressed CD8(+) T-cell proliferation and IFN-γ production, and the former effect was attenuated by the inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine hydrochloride, arginase inhibitor N-hydroxy-nor-l-arginine (nor-NOHA), and blocking antibodies for IL-4Rα(+) and IL-10. CD11b(+)CD14(+) cells were monocyte-like, expressing CD33(+), CD15(-/low), IL-4Rα(+), and S100A9(+) and producing iNOS, arginase, and several cytokines. The ratio of S100A9(+) cells positively correlated with the suppressive ability of the CD11b(+)CD14(+) cells, was associated with poor response to chemotherapy, and predicted shorter progression-free survival.

Conclusions: CD14(+)S100A9(+) inflammatory monocytes in patients with NSCLC are a distinct subset of MDSCs, which suppress T cells by arginase, iNOS, and the IL-13/IL-4Rα axis. The amount of these inflammatory monocytes is associated with poor response to chemotherapy. Clinical trial registered with www.clinicaltrials.gov (NCT 01204307).

Trial registration: ClinicalTrials.gov NCT01204307.

Figures

Figure 1.

Clinical relevance of CD11b+CD14+ cells in patients with non–small cell lung cancer (NSCLC). (A) Representative dot plots of the peripheral blood mononuclear cells (PBMCs) of patients with NSCLC (CA) and normal healthy donors (NC). Nonlymphocyte mononuclear cells were gated, and CD11b+CD14+ and CD11b+CD14− cells were analyzed by flow cytometry. (B) Ratio of CD11b+CD14+ and CD11b+CD14− cells in the PBMCs of normal controls ( n = 17) and patients with NSCLC (n = 37). (C) The number of CD11b+CD14+ cells was increased in poor-performance patients. PS < 2, n = 9; PS ≥ 2, n = 6. (D) The number of CD11b+CD14+ cells was increased in patients with progressed disease after treatment. PR, n = 10; SD, n = 9; PD, n = 8. The data are the mean ± SEM; *P < 0.05; ***P < 0.001. FSC = forward scatter; PD = progressive disease; PR = partial response; PS = performance status; SD = stable disease; SSC = side scatter.

Figure 2.

Surface markers of CD11b+CD14+ cells. (A) Representative histogram of various surface markers expressed on CD11b+/CD14+ and CD11b+/CD14− cells from normal subjects (NC) and patients with non–small cell lung cancer (NSCLC) (CA) analyzed by flow cytometry. The data for CD11b+/CD14− myeloid-derived suppressor cells (MDSCs) from patients with NSCLC are also shown. Gray lines, IgG control; black lines, surface marker staining as indicated. (B) Column bar graph analysis of the data as in (A) are presented as percentage of positively stained cells. Open bars, normal CD11b+CD14+ cells; dark gray bars, NSCLC CD11b+CD14+ cells; light gray with dots, normal CD11b+CD14− cells; dark gray with dots, NSCLC CD11b+CD14− cells; n = 6. (C) Microscopy analysis of the morphology of CD11b+CD14− and CD11b+CD14+ cells from patients with NSCLC. (D) ELISA analysis of cytokine profiles in the cultured supernatants of CD11b+CD14+ MDSCs isolated from patients with NSCLC. CD11b+/CD14+ cells from patients with NSCLC produced higher levels of tumor necrosis factor (TNF)-α (2.6 ± 0.4 ng/ml; n = 3; P < 0.05), IL-8 (137.9 ± 7.4 ng/ml; n = 3; P < 0.05), IL-10 (21.3 ± 5.7 ng/ml; n = 3; P < 0.05), IL-13 (89 ± 46.3 pg/ml; n = 3; P < 0.05) and hepatocyte growth factor (HGF) (110 ± 14 pg/ml; n = 3; P < 0.05) compared with normal subjects (0.8 ± 0.1 ng/ml, 36.4 ± 1.6 ng/ml, 5.3 ± 0.3 ng/ml, and 11.6 ± 0.5 pg/ml, 42.9 ± 1.4 pg/ml, respectively). n = 3. The data are the mean ± SEM; *P < 0.05.

Figure 3.

Immunosuppressive activity of CD11b+CD14+ and CD11b+CD14− cells. (A) Upper panel, representative histograms of flow cytometry analysis of CD8 T-cell proliferation in peripheral blood mononuclear cells (PBMCs) and CD11b− depleted or CD14− depleted PBMCs from healthy donors (NC) and patients with non–small cell lung cancer (NSCLC) (CA). Lower panel, effects of CD11b− and CD14− depletion on CD8 T-cell proliferation. Column bar graph analysis of the data as in the upper panel. CD8+ T-cell proliferation; empty bar, healthy donor (n = 6); solid bar, patients with NSCLC (n = 6). The data are the mean ± SEM and are presented as the percentage of maximal T-cell proliferation. (B) Upper panel, representative histograms of T-cell proliferation after adding CD11b+CD14+ or CD11b+CD14− cells into equal amount autologous effector cells (CD11b-depleted PBMCs). Lower panel, column bar graph analysis of the data as in the upper panel. Open bar, normal control (n = 3); solid bar, patients with NSCLC, (n = 6). The data are the mean ± SEM and are presented as the percentage of maximal T-cell proliferation. (C) Suppressive ability of CD11b+CD14+ and CD11b+CD14− cells from patients with NSCLC. CD11b+CD14+ or CD11b+CD14− myeloid-derived suppressor cells (MDSCs) from patients with NSCLC and CD11b+CD14+ cells from healthy donors were cocultured with carboxy-fluorescein diacetate succinimidyl ester (CFSE)-labeled autologous effector cells in variable proportions (1:4, 1:2, or 1:1), and T-cell proliferation was analyzed. The data were from three independent experiments. (D) Effects of NSCLC CD11b+CD14+ and CD11b+CD14− cells on CD3/CD28-stimulated IFN-γ production in the cultured supernatants of effector cells analyzed by ELISA. n = 4 independent experiments. The data are the mean ± SEM and are presented as the percentage of maximal IFN-γ production. (E) Proliferation assay analysis of CD8 T cells in CD11b-depleted PBMCs (effector cells) in the presence or absence of CD11b+CD14+ cells and CD14+HLA-DR−/low-enriched cells. n = 5. The data are the mean ± SEM and are presented as the percentage of maximal CD8+ T-cell proliferation; *P < 0.05.

Figure 4.

CD11b+CD14+ myeloid-derived suppressor cell (MDSC) suppression of T-cell proliferation is dependent on inducible nitric oxide synthase (iNOS), arginase 1, and the IL-13/IL-4Rα pathway. (A) Representative histograms of flow cytometry analysis of iNOS, arginase 1, and intracellular oxidative stress as determined by the DCFH method in CD11b+CD14+ cells from healthy donors (NC) and patients with non–small cell lung cancer (NSCLC) (CA) and CD11b+CD14− cells from patients with NSCLC. Gray lines, IgG control; black lines, iNOS, arginase 1 or 2′,7′-dichlorofluorescein (DCF) staining. (B) Open bar, CD11b+CD14+ cells from health donor; black bar, NSCLC CD11b+CD14+; gray bar, NSCLC CD11b+CD14−. Column bar graph analysis of the percentage of positive staining cells as in (A). The data are represented as the mean ± SEM, n = 5; *P < 0.05. (C) Effects of variable pharmacologic inhibitors, neutralizing antibodies, and IgG control on the CD14+ MDSC suppression of T-cell proliferation. The data are the mean ± SEM and are presented as the percentage of maximal T-cell proliferation. n = 6, *P < 0.05.

Figure 5.

S100A9 as a marker of CD11b+CD14+ myeloid-derived suppressor cells (MDSCs). (A) Representative histograms of flow cytometry analysis of S100A8 and S100A9 expression in CD11b+CD14+ cells from a patient with non–small cell lung cancer (NSCLC) (CA) and normal subject (NC). Gray lines, IgG control; black lines, S100A8 or S100A9 staining. (B) Column bar graph analysis of S100A8 and S100A9 expression determined by fluorescence-activated cell sorter scan. The data are the mean ± SEM and are presented as the percentage of positively stained cells (left panel) and mean fluorescence intensity (MFI, right panel). Open bars, normal CD11b+CD14+ cells; solid bars, NSCLC CD11b+CD14+ cells; n ≥ 4; *P < 0.05. (C) Correlation of S100A9+ cells in CD11b+CD14+ cells with the T-cell suppression ability of CD11b+CD14+ cells from patients with NSCLC. n = 8; Spearman r = −0.85; P = 0.01. (D) Correlation of the MFI of S100A9 to T-cell suppression ability from patients with NSCLC. Spearman r = −0.89; n = 6; *P = 0.033.

Figure 6.

Clinical relevance of CD11b+CD14+S100A9+ in non–small cell lung cancer (NSCLC). (A) Relationship of the percentage of CD11b+CD14+S100A9+ and chemotherapy response. Partial response (PR), stable disease (SD), progressive disease (PD), n = 9, 10, and 5, respectively. PR versus PD and SD versus PD, both P < 0.05. (B) Correlation of the percentage of CD11b+CD14+S100A9+ cells in the peripheral blood mononuclear cells (PBMCs) with progression-free survival after platinum-based doublet chemotherapy. Spearman r = −0.83; n = 24; *P < 0.0001. (C) Correlation of the percentage of CD11b+CD14+ cells in the PBMCs with progression-free survival (PFS) after platinum-based doublet chemotherapy. Spearman r = −0.58; n = 24; *P = 0.001. (D) Kaplan-Meier curve of PFS according to the median percentage of CD11b+CD14+S100A9+. Dark line, CD11b+CD14+S100A9+ ≤ 20% in PBMCs; dashed gray line, CD11b+CD14+S100A9+ greater than 20%. Median survival 9.2 versus 3 months; hazard ratio (HR), 0.06; 95% confidence interval (CI), 0.02–0.23; log-rank test P < 0.0001. (E) Kaplan-Meier curve of PFS according to the median percentage of CD11b+CD14+. Dark line, CD11b+CD14+ < 19.1% in PBMC; dashed gray line, CD11b+CD14+ > 19.1% in PBMC. Median survival 9 versus 2.9 months; hazard ratio, 0.30; 95% confidence interval, 0.10–0.88; log-rank test P = 0.03. (F) Representative images of migrated carboxy-fluorescein diacetate succinimidyl ester (CFSE)-labeled CD14+ cells that were photographed with fluorescence microscopy after overnight culture in the presence of A549 cells in the bottom chamber in migration assays. S100A9 or anti-RAGE blocking antibody or IgG control was added in the CD14+ cell-seeded upper chamber. (G) Column bar graph analysis of the quantification of migrated cells as in (F) from four independent experiments. The data are presented as the mean ± SEM of cell numbers/field; *P < 0.05. (H) MTT assay analysis of A549 cells viability in response to cisplatin (0, 50, 100 mM) in the presence or absence of coculture with NSCLC CD14+ cells. The data are presented as the mean ± SEM of the percentage of cell viability relative to the corresponding control cells without cisplatin treatment. n = 3; ***P < 0.001 compared with the corresponding CD14+ cells controls at each cisplatin concentration. CA = patients with NSCLC; NC = healthy donors.