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. 2017 Nov;66(11):1900-1911.
doi: 10.1136/gutjnl-2016-313075. Epub 2017 Mar 8.

Tumour-activated neutrophils in gastric cancer foster immune suppression and disease progression through GM-CSF-PD-L1 pathway

Ting-Ting Wang  1 , Yong-Liang Zhao  2 , Liu-Sheng Peng  1 , Na Chen  1 , Weisan Chen  3 , Yi-Pin Lv  1 , Fang-Yuan Mao  1 , Jin-Yu Zhang  1 , Ping Cheng  1 , Yong-Sheng Teng  1 , Xiao-Long Fu  2 , Pei-Wu Yu  2 , Gang Guo  1 , Ping Luo  1 , Yuan Zhuang  1 , Quan-Ming Zou  1
Affiliations

Tumour-activated neutrophils in gastric cancer foster immune suppression and disease progression through GM-CSF-PD-L1 pathway

Ting-Ting Wang et al. Gut. 2017 Nov.

Abstract

Objective: Neutrophils are prominent components of solid tumours and exhibit distinct phenotypes in different tumour microenvironments. However, the nature, regulation, function and clinical relevance of neutrophils in human gastric cancer (GC) are presently unknown.

Design: Flow cytometry analyses were performed to examine levels and phenotype of neutrophils in samples from 105 patients with GC. Kaplan-Meier plots for overall survival were performed using the log-rank test. Neutrophils and T cells were isolated, stimulated and/or cultured for in vitro and in vivo regulation and function assays.

Results: Patients with GC showed a significantly higher neutrophil infiltration in tumours. These tumour-infiltrating neutrophils showed an activated CD54+ phenotype and expressed high level immunosuppressive molecule programmed death-ligand 1 (PD-L1). Neutrophils activated by tumours prolonged their lifespan and strongly expressed PD-L1 proteins with similar phenotype to their status in GC, and significant correlations were found between the levels of PD-L1 and CD54 on tumour-infiltrating neutrophils. Moreover, these PD-L1+ neutrophils in tumours were associated with disease progression and reduced GC patient survival. Tumour-derived GM-CSF activated neutrophils and induced neutrophil PD-L1 expression via Janus kinase (JAK)-signal transducer and activator of transcription 3 (STAT3) signalling pathway. The activated PD-L1+ neutrophils effectively suppressed normal T-cell immunity in vitro and contributed to the growth and progression of human GC in vivo; the effect could be reversed by blocking PD-L1 on these neutrophils.

Conclusions: Our results illuminate a novel mechanism of PD-L1 expression on tumour-activated neutrophils in GC, and also provide functional evidence for these novel GM-CSF-PD-L1 pathways to prevent, and to treat this immune tolerance feature of GC.

Keywords: GASTRIC CANCER.

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Conflict of interest statement

Competing interests: None declared.

Figures

Figure 1
Figure 1
Neutrophils accumulate in gastric cancer (GC) with disease progression and poor patient survival. (A) Neutrophil percentage in CD45+ cells or (C) the total number of neutrophils per million total cells in each tissue of patients with GC by gating on CD45+CD66b+ cells or counting. Cumulative results from 105 patients with GC and 18 healthy donors are shown. (B) Dot plots of surface molecule staining for neutrophils gating on CD45+ cells. (D) Representative analysis of CD15+ (brown) neutrophil distributions in tissues of patients with GC by immunohistochemical staining. Scale bars: 100 micrometers. (E and F) Neutrophil percentage (E) or neutrophil number (F) among tumour, node, metastases (TNM) stages was compared. Kaplan-Meier plots for overall survival by median neutrophil percentage (5.24%) (E) or median neutrophil number (3008 per million) (F). The horizontal bars in panels A, C, E and F represent mean values. Each ring in panels A, C, E and F represents one patient. *p<0.05, **p<0.01, n.s. p>0.05 for groups connected by horizontal lines. Neu (%), neutrophil percentage; Neu (NO.), neutrophil number.
Figure 2
Figure 2
Expression of programmed death-ligand 1 (PD-L1) on neutrophils is correlated with the activation pattern of neutrophils in gastric cancer (GC). (A) Statistics analysis of CD54+ neutrophil percentage or (B) PD-L1+ neutrophil percentage in total neutrophils in each samples of patients with GC (n=23). (B) Expression of molecules CD54 and PD-L1 on neutrophils. Colour histograms represent staining of neutrophil activation marker CD54 and immunosuppressive functional molecule PD-L1; black, isotype control. (C) The correlations between CD54+ neutrophils and PD-L1+ neutrophils in human tumours were analysed. Results are expressed as percentage of CD54+ neutrophils and PD-L1+ neutrophils in neutrophils or the number of CD54+ neutrophils and PD-L1+ neutrophils per million total cells in tumour tissues of patients with GC. (D) Co-expression of molecules CD54 and PD-L1 on tumour-infiltrating neutrophils. The horizontal bars in panels A represent mean values. Each ring or dot in panels A or C represents one patient. *p<0.05, **p<0.01 for groups connected by horizontal lines.
Figure 3
Figure 3
Tumour environment sustains neutrophil survival, activation and programmed death-ligand 1 (PD-L1) expression. (A and B) Dot plots and statistics analysis of annexin V− viable non-apoptotic neutrophils exposed to 50% tumour tissue culture supernatants (TTCS) and 50% non-tumour tissue culture supernatants (NTCS) from autologous gastric cancer (GC) patients (A); or neutrophils to exposed to 20%, 40% or 80% TTCS from GC patients for 16 hours (B) (n=6). (C) Expressions of CD54 and PD-L1 on neutrophils exposed to 50% TTCS and 50% NTCS from autologous GC patients, or to medium control for 12 hours; or exposed to 50% TTCS from patients with GC for 3, 6 or 12 hours. black, isotype control. MFI: mean fluorescence intensity. *p<0.05, **p<0.01 for groups connected by horizontal lines.
Figure 4
Figure 4
Tumour-infiltrating and tumour-conditioned neutrophils suppress T cell immunity through programmed death-ligand 1 (PD-L1). (A) Carboxyfluorescein succinimidyl ester (CFSE)-labelled peripheral CD3+ T cells of patients with gastric cancer (GC) were co-cultured for 5 days with autologous neutrophils from non-tumour or tumour tissues with or without anti-PD-L1 antibody. Representative data and statistical analysis of T cell proliferation and interferon (IFN)-γ production were shown (n=5). (B) PD-L1+ neutrophil percentage or PD-L1+ neutrophil number among tumour, node, metastases (TNM) stages was compared. (C) Kaplan-Meier plots for overall survival by median PD-L1+ neutrophil percentage (24%) or median PD-L1+ neutrophil number (1147 per million). (D and E) CFSE-labelled peripheral CD3+ T cells of donors were co-cultured for 5 days with tumour tissue culture supernatants (TTCS)-, or non-tumour tissue culture supernatants (NTCS)-conditioned autologous (D) or allogeneic (E) blood neutrophils with or without anti-PD-L1 antibody. Representative data and statistical analysis of T cell proliferation and IFN-γ production were shown (n=5). The horizontal bars in panel B represent mean values. Each ring in panels B represents one patient. *p<0.05, **p<0.01 for groups connected by horizontal lines.
Figure 5
Figure 5
Tumour-derived GM-CSF induces neutrophil activation and neutrophil programmed death-ligand 1 (PD-L1) expression via Janus kinase (JAK)-signal transducer and activator of transcription 3 (STAT3) pathway. (A) Expression of CD54 and PD-L1 on neutrophils exposed to GM-CSF for 3, 6, 12 hours. black, isotype control. (B) GM-CSF concentration between autologous tumour and non-tumour tissues (n=16) or between autologous tumour tissue culture supernatants (TTCS) and non-tumour tissue culture supernatants (NTCS) (n=7) was analysed. (C) The correlations between GM-CSF and PD-L1+ neutrophils or CD54+ neutrophils in human tumours were analysed. Results are expressed as the number of PD-L1+ neutrophils or CD54+ neutrophils per million total cells and GM-CSF concentration in tumour tissues. (D) Expression of CD54 and PD-L1 on neutrophils exposed to TTCS with anti-GM-CSF antibody or NTCS with GM-CSF for 12 hours. black, isotype control. (E) Expression of CD54 and PD-L1 on neutrophils exposed to TTCS or GM-CSF with or without JAK signal transduction inhibitor AG490 or STAT3 phosphorylation inhibitor FLLL32 alone or both for 12 hours. (F) STAT3 and p-STAT3 in neutrophils exposed to autologous TTCS, NTCS, or TTCS with anti-GM-CSF antibody were analysed by western blot. Each dot in panel C represents one patient. *p<0.05, **p<0.01 for groups connected by horizontal lines.
Figure 6
Figure 6
Blockade of neutrophil-associated programmed death-ligand 1 (PD-L1) on T cell immunity inhibits tumour growth and gastric cancer (GC) progression in vivo. (A) Mice were injected with human GC cells (SGC)-7901 cells, as described in Materials and methods. The control animals (●) received no further injections. The experimental treatments entailed injections with T cells in combination with untreated neutrophils (N) (formula image) or TTCS (tumour tissue culture supernatants)-conditioned neutrophils (TCN) (formula image), or TCN pre-treated with an anti-PD-L1 antibody (formula image) or a control IgG (formula image). The illustrated data represent tumour volumes (5 mice in each group). The day of tumour cell injection was counted as day 0. *p<0.05, for groups injections with TCN pre-treated with an anti-PD-L1 antibody (formula image), compared with groups injections with TCN pre-treated with a control IgG (formula image). The tumours were excised and photographed 24 day after injecting the tumour cells. (B and C) Interferon (IFN)-γ-producing T cell response (B) in spleens and proliferating cell nuclear antigen (PCNA) (brown) expression or CD3+ T cell infiltration (red) (C) in tumours of mice injected with T cells in combination with TCN, or TCN pre-treated with an anti-PD-L1 antibody or a control IgG on day 24 after tumour cell injection were compared. (D) The correlations between T cells and neutrophils in human tumours were analysed. Results are expressed as percentage of T cells and neutrophils in CD45+ cells or the number of T cells and neutrophils per million total cells in tumour tissues of patients with GC. Scale bars: 100 μ. Arrows indicate staining-positive cells. *p<0.05 for groups connected by horizontal lines.
Figure 7
Figure 7
Tumour-activated neutrophils suppress T cell immunity through programmed death-ligand 1 (PD-L1)-PD-1 interaction, and a proposed model of cross-talk among neutrophils, T cells and tumour cells leading to neutrophil-mediated immunosuppression in gastric cancer (GC). (A and B) carboxyfluorescein succinimidyl ester (CFSE)-labelled fluorescence activating cell sorter (FACS)-sorted peripheral PD-1+ T cells of donors were co-cultured for 5 days with tumour tissue culture supernatants (TTCS)-conditioned or non-tumour tissue culture supernatants (NTCS)-conditioned autologous (A) or allogeneic (B) blood neutrophils with or without anti-PD-L1 antibody. Representative data and statistical analysis of T cell proliferation and interferon (IFN)-γ production were shown (n=3). (C) The correlations between PD-1+ T cells and PD-L1+ neutrophils in human tumours were analysed. Results are expressed as percentage of PD-1+ T cells in T cells and PD-L1+ neutrophils in neutrophils or the number of PD-1+ T cells and PD-L1+ neutrophils per million total cells in tumour tissues of patients with GC. (D) Release of GM-CSF induces the activation of intratumorous neutrophils, a process that is accompanied by the induction of PD-L1 expression on these cells as a result of Janus kinase (JAK)-signal transducer and activator of transcription 3 (STAT3) signalling pathway activation. These activated neutrophils exert their pro-tumour effect, contributing to immunosuppression and GC progression by inhibition T cell immunity in a PD-L1-PD-1-dependent manner.
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