Tumor-associated neutrophils stimulate T cell responses in early-stage human lung cancer - PubMed (original) (raw)

Clinical Trial

. 2014 Dec;124(12):5466-80.

doi: 10.1172/JCI77053. Epub 2014 Nov 10.

Pratik S Bhojnagarwala, Jon G Quatromoni, Tom Li Stephen, Anjana Ranganathan, Charuhas Deshpande, Tatiana Akimova, Anil Vachani, Leslie Litzky, Wayne W Hancock, José R Conejo-Garcia, Michael Feldman, Steven M Albelda, Sunil Singhal

Clinical Trial

Tumor-associated neutrophils stimulate T cell responses in early-stage human lung cancer

Evgeniy B Eruslanov et al. J Clin Invest. 2014 Dec.

Abstract

Infiltrating inflammatory cells are highly prevalent within the tumor microenvironment and mediate many processes associated with tumor progression; however, the contribution of specific populations remains unclear. For example, the nature and function of tumor-associated neutrophils (TANs) in the cancer microenvironment is largely unknown. The goal of this study was to provide a phenotypic and functional characterization of TANs in surgically resected lung cancer patients. We found that TANs constituted 5%-25% of cells isolated from the digested human lung tumors. Compared with blood neutrophils, TANs displayed an activated phenotype (CD62L(lo)CD54(hi)) with a distinct repertoire of chemokine receptors that included CCR5, CCR7, CXCR3, and CXCR4. TANs produced substantial quantities of the proinflammatory factors MCP-1, IL-8, MIP-1α, and IL-6, as well as the antiinflammatory IL-1R antagonist. Functionally, both TANs and neutrophils isolated from distant nonmalignant lung tissue were able to stimulate T cell proliferation and IFN-γ release. Cross-talk between TANs and activated T cells led to substantial upregulation of CD54, CD86, OX40L, and 4-1BBL costimulatory molecules on the neutrophil surface, which bolstered T cell proliferation in a positive-feedback loop. Together our results demonstrate that in the earliest stages of lung cancer, TANs are not immunosuppressive, but rather stimulate T cell responses.

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Figures

Figure 7

Figure 7. The expression of costimulatory molecules on TANs and their role in stimulation of T cell proliferation.

(A) The expression of the costimulatory molecules on gated CD11b+CD15hi TANs and PBNs was analyzed by flow cytometry. The top panel summarizes the data for all the patients. Error bars represent mean ± SEM. Statistical analyses were performed with Student’s t test for paired data. (B–D) Neutrophil survival in the cell culture. Zombie Yellow Fixable Viability dye was used to discriminate viable CD15 neutrophils cultured alone (B) or in the coculture with resting T cells (C) and CD3/CD28–activated T cells (D). Representative dot plots from 1 of 5 experiments are shown. For all dot plots, numbers represent the percentage of cells in each quadrant. (E) The expression of costimulatory molecules was analyzed by flow cytometry on gated live CD11b+CD15hi PBNs (top) and TANs (bottom) after 2 days of coculture with activated (black histograms) or resting autologous T cells (gray histograms). Results from 1 of 5 representative experiments are shown. (F) The efficacy of blocking Abs in ablating the stimulatory effect of TANs on T cell proliferation. Autologous PBMCs were stimulated with plate-bound anti-CD3 Abs and mixed with TANs at a 1:1 ratio in the presence or absence of blocking Abs against the indicated receptors for 4 days. Numbers on histograms represent the percentage of proliferating cells. Mouse IgG1 Abs were used as isotype control Abs in the control group. Results from 1 of 3 representative experiments are shown.

Figure 6

Figure 6. Effect of TANs on T cell activation, cytokine production, and proliferation.

In all experiments, T cells were stimulated with plate-bound anti–CD3/CD28 Abs and incubated with TANs at a 1:1 ratio. (A) Expression of the CD62L, CD25, and CD107a markers on activated autologous T cells cultured with TANs for 20 hours. Representative dot plots from 1 of 3 experiments are shown. Numbers represent the percentage of CD4+ and CD8+ cells. (B) Flow cytometric analysis of autologous T cell proliferation in the presence of TANs using a transwell system. Activated CFSE-labeled T cells were mixed with TANs at a 1:1 ratio (TAN mix). To separate T cells and TANs, activated T cells were cultured in the bottom chamber and TANs were placed in the top chamber of the 24-well flat-bottom transwell culture plate (TAN transwell). Representative results from 1 of 3 experiments are shown. Numbers on histograms represent the percentage of proliferating T cells. (C and D) IFN-γ (C) and IL-10 (D) were measured by ELISA in 48- or 96-hour supernatants collected from cocultures of activated T cells with TANs or PBNs. Summary results from 8 lung cancer patients are shown in the graph (Wilcoxon matched-pairs rank test). (E) The percentage of IFN-γ– and IL-10–producing T cells cultured with TANs or PBNs was measured by intracellular cytokine staining at 48 hours of stimulation. The dot plots represent 1 of 3 independent experiments. Numbers represent the percentage of CD4+ and CD8+ cells.

Figure 5

Figure 5. T cell proliferation in the presence of TANs or PBNs.

(A) CFSE-labeled PBMCs isolated from a healthy donor were stimulated with plate-bound anti-CD3 Abs and mixed with TANs, neutrophils from distant lung tissue, or PBNs isolated from cancer patients at a 1:1 ratio for 4 days. Representative results from 1 of 12 experiments are shown. Numbers on histograms represent the percentage of proliferating T cells. (B) Autologous T cells were purified from PBMCs, stimulated with plate-bound anti-CD3 Abs and anti-CD28 Abs, and cultured alone or with PBNs, distant neutrophils, or TANs. 72 hours later, proliferation of T cells was assessed by incorporation of BrdU into DNA. Representative results from 1 of 5 experiments are shown. (C) Mixed lymphocyte reaction (MLR). CFSE-labeled T cells isolated from PBMCs of healthy donors were stimulated with allogeneic DCs in the absence or presence of PBNs or TANs for 5 days. Representative results from 1 of 5 experiments are shown. Numbers on dot plots represent the percentage of proliferating T cells. (D) Change in percentage of proliferating CFSElo CD4+ and CD8+ cells cultured alone versus cells cultured in the presence of TANs (Student’s t test, paired parametric test). Autologous PBMCs were stimulated with plate-bound anti-CD3 Abs and mixed with TANs at a 1:1 ratio for 4 days. Scatter plots show the correlation between tumor size and stimulatory activity of TANs defined as the ratio CFSElo (T cells +TANs)/CFSElo (T cells) (nonparametric Spearman correlation). Cumulative results from 16 independent experiments are presented.

Figure 4

Figure 4. Characterization of neutrophils isolated from tumor tissues and peripheral blood of patients with NSCLC.

(A and B) Neutrophil survival in vitro. TANs and PBNs were isolated from lung cancer patients with magnetic beads by positive selection. Cells were cultured in cell culture medium with or without TCM for 20 and 48 hours. TANs and PBNs were then assessed for apoptosis by FACS analysis of annexin V/PI staining at 1, 20, and 48 hours. Dot plots represent 1 of 6 similar experiments. Numbers represent the percentage of cells in each quadrant. Summary results from 6 lung cancer patients are also shown (*P ≤ 0.01, Wilcoxon matched-pairs rank test). (C) Production of H2O2 in TANs and PBNs isolated from lung cancer patients and healthy donors was measured using Amplex Red with horseradish peroxidase. Error bars represent mean ± SEM from 5 independent experiments (*P ≤ 0.001, Wilcoxon matched-pairs rank test). (D) Phagocytic capacity of TANs. TANs and PBNs were isolated and incubated with pHrodo Red E. coli BioParticles for 45 minutes to allow phagocytosis (internalized particles become fluorescent [red]). Histograms from 1 representative experiment are shown. Phagocytosis performed at 4°C and 37°C is shown by thin and thick lines, respectively; MFI values are as indicated in histograms. Summary results from 6 lung cancer patients are also shown (Wilcoxon matched-pairs rank test).

Figure 3

Figure 3. Characterization of TANs.

(A) Heat map comparing the phenotypes of TANs and PBNs. A single-cell suspension was obtained from freshly harvested tumor tissues, and expression of the indicated markers was assessed using flow cytometry. TANs were gated on CD11b+CD15hi cells and further analyzed for the expression of indicated markers. PBNs were treated similarly to TANs. Expression of each marker was analyzed in 10–18 patients. The intensity key for the heat map is shown below. (B) The cytokine/chemokine production by TANs, PBNs, and total tumor dissociates of AC and SCC. TANs and PBNs were isolated from tumor tissues and peripheral blood of lung cancer patients (n = 5) using magnetic beads. Purified neutrophils and unseparated cells from digested tumor were cultured for 24 hours in the cell culture medium, and cell-free supernatants were collected and frozen. The indicated factors were detected using the Cytokine Human 30-Plex assay. The presence of each secreted factor was heat-mapped on the basis of the concentration in tested supernatants, as indicated below.

Figure 2

Figure 2. TANs acquire an activated phenotype and novel repertoire of chemokine receptors.

(A) Expression of the activation markers CD62L and CD54 on CD15hiCD66b+ PBNs. PBNs were isolated from lung cancer patients using anti-CD15 beads. Results represent 1 of 5 experiments. (B) Digestion protocol did not elicit premature activation of resting PBNs. Results represent 1 of 5 experiments. (C) PBNs acquire an activated CD62LloCD54+ phenotype after treatment with TCM in plates with ultralow attachment surface. Each experiment was repeated at least 5 times. (D) A single-cell suspension was obtained from freshly harvested tumor tissues. TANs were gated on CD11b+CD15hiCD66b+ cells and further analyzed for the expression of activation markers. TANs displayed an activated CD62LloCD54+ phenotype. Results represent 1 of 12 experiments. (E) Expression of the activation markers on gated CD11b+CD15hi TANs, distant lung neutrophils (Distant N), and PBNs. (F) Expression of CCR5, CCR7, CXCR3, and CXCR4 was analyzed on gated CD11b+CD15hiCD66b+ TANs, distant lung neutrophils, and PBNs of cancer patients. Bottom: Representative dot plots. Numbers represent the percentage of cells in each quadrant. Top: Summary of all patient data. Error bars represent mean ± SEM. Statistical analyses were performed with repeated-measures 1-way ANOVA with Tukey’s multiple comparison test for CD62L, CD54, CXCR2, CXCR1, and CCR5, and Kruskal-Wallis and Dunn’s multiple comparison tests for CCR7, CXCR3, and CXCR4 (*P ≤ 0.001, **P ≤ 0.01, ***P ≤ 0.05).

Figure 1

Figure 1. Neutrophils infiltrate NSCLC tissue.

(A–C) Lung cancer tissue sections were stained using 2-color immunohistochemistry for MPO, HLA-DR, and CD3 to visualize neutrophils, APCs, and T lymphocytes, respectively. Representative images are shown. Original magnification, ×10 (A and C, top), ×20 (B, top), ×40 (bottom). (D) Representative dot plots of total tumor cells that define the phenotype of TANs in NSCLC. TANs were defined as CD15hiCD66b+CD11b+CD16intArg1+MPO+IL-5Ra–. Results represent 1 of 20 experiments. Numbers represent the percentage of TANs. (E) Comparative immunohistochemical analysis of TAN density (cells/mm2) in AC (n = 45) and SCC (n = 25) performed by counting of MPO+ cells in the tumor stroma and the tumor islets. Statistical analyses were performed with Student’s t test for unpaired data. (F) The frequency of TANs in AC (n = 31) and SCC (n = 11) determined by flow cytometry as the percentage of CD11b+CD15hiCD66b+ cells among all cells in tumor. Cumulative results from 42 independent experiments are shown in the scatter plot. Student’s t test for unpaired data. (G) The ratio of TANs to other CD15–CD11b+ cells in AC (n = 22) and SCC (n = 9). Mann-Whitney test for unpaired data. For all scatter plots, error bars represent mean ± SEM. (H) PBNs were analyzed for migration in the Neuro Probe ChemoTx system. Each experiment was run in triplicate and repeated at least 3 times. Results of 1 representative experiment are shown. Error bars represent mean ± SEM. Statistical analysis was performed with Kruskal-Wallis and Dunn’s multiple comparison tests (*P ≤ 0.05, **P ≤ 0.01). fMLP, _N_-formyl-methionyl-leucyl-phenylalanine.

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