Amphiregulin promotes activated regulatory T cell-suppressive function via the AREG/EGFR pathway in laryngeal squamous cell carcinoma

Abstract

Background: Activated regulatory T cells (aTregs) play a vital role in promoting a tumor immunosuppressive microenvironment in laryngeal squamous cell carcinoma (LSCC). However, the regulatory factors that induce the generation of aTregs are not clear. Herein, we investigated the effect of amphiregulin (AREG) on the production of aTregs in the tumor microenvironment of LSCC.

Methods: Immunohistochemical (IHC) analysis was conducted to examine the expression of AREG and FOXP3, and their association with clinical parameters and patient outcomes was demonstrated. The expression level of EGFRs in three functional subsets of Tregs was assessed, and the induction of CD4⁺ T cells into aTregs in the presence or absence of AREG or Gefitinib was analyzed using flow cytometry.

Results: Our results showed a higher expression level of AREG was significantly related to advanced clinical stage and worse survival, particularly with increased infiltration of Tregs in LSCC tumor tissue. The in vitro study showed that AREG significantly promoted the differentiation of aTregs, and enhanced the inhibitory effect of Tregs on T cell proliferation, which could be reversed by epidermal growth factor receptor (EGFR) inhibitors. In addition, we found that EGFR was highly expressed in aTregs, but not in other subsets of Tregs. It is suggested that AREG might induce aTregs, and enhance the immunosuppressive function of Tregs via the AREG/EGFR signal pathway.

Conclusions: Collectively, this study revealed the role and mechanism of AREG in negative immune regulation, and targeting AREG might be a novel immunotherapy for LSCC.

Keywords: Amphiregulin; Epidermal growth factor receptor; Immunosuppressive microenvironment; Laryngeal squamous cell carcinoma; Regulatory T cells.

Fig. 1

The expression of AREG in LSCC specimens is correlated with clinical staging and patient overall survival. (A, B) Representative IHC images of AREG expression in paraffin-embedded LSCC tumor sections (n = 68). Scale bar: 50 μm in Fig. 1A, 20 μm in Fig. 1B. (C-E) IHC score was higher in advanced T stages (T3 and T4) and advanced clinical stages (III and IV) than in early T stages (T1 and T2) and early clinical stages (I and II). (F) Accumulation of AREG in the tumor microenvironment predicted poor survival of patients with LSCC (Kaplan-Meier method and log-rank test). Low AREG expression (n = 36) and high AREG expression (n = 32) groups are represented by red and blue lines, respectively. G) Higher Treg infiltration was founded in high AREG expression specimens than those in low AREG expression (n = 51). Data presented as mean ± SEM. *p < 0.05

Fig. 2

AREG upregulates the proportion of aTregs through EGFR. CD4⁺ T cells were isolated from PBMCs and cultured in the presence or absence of 100 ng/ml recombinant AREG or 200 ng/ml Gefitinib for 48 h and flow cytometry analysis were used to identify the subsets of Tregs. Generation proportion of Tregs subsets in different treatment groups control (A), AREG (B), AREG + Gefitinib (C) and the results were analyzed statistically (D). rTregs (I Tregs), aTregs (II Tregs), and nTregs (III Tregs); N = 5, **p < 0.01, ***p < 0.001

Fig. 3

EGFR was highly expressed in aTregs. The expression level of EGFR in three functional subsets of Tregs was detected by using the flow cytometry analysis. (A-D) Gating Strategy was shown. (E) The proportion of three functional subsets of Tregs in EGFR⁺CD4⁺T cells. (F-H) The expression level of EGFR was detected on rTregs (I Tregs), aTregs (II Tregs) an nTregs (III Tregs) respectively. All experiments were repeated three times

Fig. 4

AREG enhances the immunosuppressive effect of Tregs. CD4⁺CD25^hiCD127^lo T cells were sorted by magnetic cell separation from PBMCs and were cocultured with CD4⁺ T cells labeled with CFSE for 3 days in the presence or absence of 100 ng/ml recombinant AREG or 200 ng/ml Gefitinib. Proliferation of CD4⁺ T cells was determined in different treatment groups control by CFSE analysis (A) and the results were analyzed statistically (B). *p < 0.05, ***p < 0.001