Tumor-Infiltrating PD-L1+ Neutrophils Induced by GM-CSF Suppress T Cell Function in Laryngeal Squamous Cell Carcinoma and Predict Unfavorable Prognosis.

PURPOSE: Chronic inflammation contributes to tumor initiation, progression, and immune escape. Neutrophils are the major component of inflammatory response and participate in the tumorigenesis process. However, compared to other immune cells in the tumor microenvironment of laryngeal squamous cell carcinoma (LSCC), neutrophils, especially the tumor-associated neutrophils (TANs), have not yet been comprehensively explored. The mechanism for regulating the crosstalk between TANs and tumor cells still remains unclear. MATERIALS AND METHODS: The distribution profiles and phenotypic features of neutrophils and other inflammatory immune cell populations from a large LSCC patient cohort were systemically analyzed. Co-culturing of peripheral blood associated neutrophils (PANs) and TANs with PBMCs was performed, and the immunosuppression effect on T-cells was examined. RESULTS: LSCC microenvironment is highly inflammatory with remarkable TANs infiltration, which is often associated with unfavorable prognosis and advanced clinical stage. We find that TANs in LSCC display morphologically immature and lower apoptosis, exhibit distinctively immunosuppressive phenotype of high PD-L1, and suppress CD8+ T lymphocytes proliferation and activation. We subsequently discover that PD-L1+TANs induced by LSCC-derived GM-CSF potently impair CD8+ T-cells proliferation and cytokines production function, which are partially blocked by a PD-L1-neutralizing antibody. Clinical data further support GM-CSF as an unfavorable prognostic biomarker and reveal a potential association with inflammatory immune cell infiltration, in particular neutrophils. CONCLUSION: Tumor-infiltrating PD-L1+ neutrophils induced by LSCC-derived GM-CSF suppress T cell proliferation and activation in the inflammatory microenvironment of LSCC and predict unfavorable prognosis. These TANs cripple antitumor T cell immunity and promote tumor progression. Our findings provide a basis for targeting PD-L1+TANs or GM-CSF as a new immunotherapeutic strategy for LSCC.

Stromal interleukin-33 promotes regulatory T cell-mediated immunosuppression in head and neck squamous cell carcinoma and correlates with poor prognosis.

Regulatory T cells (Tregs) mediate immunosuppressive signals that can contribute to the progression of head and neck squamous cell carcinoma (HNSCC). Interleukin-33 (IL-33) is defined as an 'alarmin', an endogenous factor that is expressed during tissue and cell damage, which has been shown to promote Treg proliferation in non-lymphoid organs. However, the interaction between IL-33 and Tregs in the HNSCC tumor microenvironment remains uncertain. In this study, we examined IL-33+ and Foxp3+ cells by immunohistochemistry in 68 laryngeal squamous cell cancer patients, followed by functional analysis of IL-33 in Tregs. In addition, the suppressive function of Tregs was assessed by cell proliferation assays. The level of stromal IL-33 was significantly upregulated in advanced versus early stage HNSCC patients and positively correlated with Foxp3+ Treg infiltration as well as a poor prognosis. ST2 is regarded as the only receptor of IL-33. Infiltrated ST2-expressing Tregs were responsive to IL-33, and the percentage of Tregs was increased upon IL-33 stimulation. Functional investigation demonstrated that IL-33 increased the proportion of Foxp3+GATA3+ Tregs and improved the suppressive functions of Tregs by inducing IL-10 and TGF-ß1 as well as decreasing the proliferation of responder T cells. Blockade of ST2 abrogated the immunosuppression caused by IL-33. Our data demonstrate that stromal IL-33 both expands the Treg population and enhances their functions in the tumor microenvironment. Furthermore, stromal IL-33 has prognostic value for tumor progression. Thus, stromal IL-33 is a potential target for future HNSCC immunotherapy.

A positive-feedback loop between tumour infiltrating activated Treg cells and type 2-skewed macrophages is essential for progression of laryngeal squamous cell carcinoma.

BACKGROUND: Foxp3+ regulatory T (Treg) cells and M2 macrophages are associated with increased tumour progression. However, the interaction between Treg cells and M2 macrophages remains unclear. METHODS: The expression of FoxP3 and CD163 was detected by immunohistochemistry in 65 cases of laryngeal squamous cell carcinoma (LSCC). In vitro, the generation of activated Treg (aTreg) cells and M2 macrophages by interactions with their precursor cells were analysed by flow cytometry and ELISA. In vivo, the antitumour effects were assessed by combined targeting aTreg cells and M2 macrophages, and intratumoural immunocytes were analysed by flow cytometry. RESULTS: In LSCC tissue, accumulation of aTreg cells and M2 macrophages predicted a poor prognosis and were positively associated with each other. In vitro, aTreg cells were induced from CD4+CD25- T cells by cancer cell-activated M2-like macrophages. Consequently, these aTreg cells skewed the differentiation of monocytes towards an M2-like phenotype, thereby forming a positive-feedback loop. Combined targeting aTreg cells and M2 macrophages led to potent antitumour immunity in vivo. CONCLUSIONS: The positive-feedback loop between aTreg cells and M2 macrophages is essential to maintain or promote immunosuppression in the tumour microenvironment and may be a potential therapeutic target to inhibit tumour progression.

Expression profiles of histone lysine demethylases during cardiomyocyte differentiation of mouse embryonic stem cells.

AIM: Histone lysine demethylases (KDMs) control the lineage commitments of stem cells. However, the KDMs involved in the determination of the cardiomyogenic lineage are not fully defined. The aim of this study was to investigate the expression profiles of KDMs during the cardiac differentiation of mouse embryonic stem cells (mESCs). METHODS: An in vitro cardiac differentiation system of mESCs with Brachyury (a mesodermal specific marker) and Flk-1(+)/Cxcr4(+) (dual cell surface biomarkers) selection was used. The expression profiles of KDMs during differentiation were analyzed using Q-PCR. To understand the contributions of KDMs to cardiomyogenesis, the mESCs on differentiation d 3.5 were sorted by FACS into Brachyury(+) cells and Brachyury(-) cells, and mESCs on d 5.5 were sorted into Flk-1(+)/Cxcr4(+) and Flk-1(-)/Cxcr4(-) cells. RESULTS: mESCs were differentiated into spontaneously beating cardiomyocytes that were visible in embryoid bodies (EBs) on d 7. On d 12-14, all EBs developed spontaneously beating cardiomyocytes. Among the 16 KDMs examined, the expression levels of Phf8, Jarid1a, Jhdm1d, Utx, and Jmjd3 were increased by nearly 2-6-fold on d 14 compared with those on d 0. Brachyury(+) cells showed higher expression levels of Jmjd3, Jmjd2a and Jhdm1d than Brachyury(-) cells. A higher level of Jmjd3 was detected in Flk-1(+)/Cxcr4(+) cells, whereas the level of Jmjd2c was lower in both Brachyury(+) cells and Flk-1(+)/Cxcr4(+) cells. CONCLUSION: KDMs may play important roles during cardiomyogenesis of mESCs. Our results provide a clue for further exploring the roles of KDMs in the cardiac lineage commitment of mESCs and the potential interference of cardiomyogenesis.