Most of metastatic cancer are accompanied by malignant effusion including peritoneal ascites and pleural fluid and portend poor oncological outcomes. Malignant effusion consist of cancer cells, mesothelial cells, and vasious immune cells including abundant T cells, natural killer (NK) cells, B cells, macrophages, and neutrophils. Each of these cells produces various chemokines and cytokines that lead to formation of tumor microenvironment in malignant effusion (Nakano et al. 2018). The malignant effusion including pleural and peritoneal cavities represent immunosuppressive environments which enables the tumor cells to escape from immune surveillance ( Donnenberg et al. 2019; Porcel et al. 2015).
Pro-inflammatory cytokine IL-6 play a central role in chronic inflammatory including cancers. The IL-6/JAK/STAT signaling pathway is aberrantly hyperactivated in many types of cancer. Blockade of the IL-6 signalling pathway has become a target for the therapy of diverse cancers. IL-6 was released by several cell populations in malignant effusion, including cancer cells, macrophages, mesothelial cells(Hayama et al. 2020; Kampan et al. 2018; Kim et al. 2013; Mace et al. 2016; Yamaguchi et al. 2000). In this study, the concentration of IL-6 in malignant effusion reached up to 3311pg/mL, whereas much lower (19.7 pg/mL) in blood, suggesting IL-6 was produced locally in the pleural or peritoneal cavity rather than exuding form blood. By contrast, pro-inflammatory Th1 cytokines including IL-2、TNF-α、IFN-γ had not noticeably altered, consistently with the study in malignant pleural effusion of non-small cell lung cancer (Hayama et al. 2020). Immune modulatory cytokine IL-10 has paradoxical effects on different types of immune response and is considered as a potential switcher of immunity. It can inhibit the production of pro-inflammatory cytokines and activation of T cells. It also has been reported to exert anti-tumor effects through promoting the proliferation and cytotoxicity of CD8+T cells (Rallis et al. 2020; Geginat et al. 2016; Emmerich et al. 2012). Our study showed that the concentration of IL-10 in malignant effusion (64.28 pg/mL) was slightly higher than that in the blood (5.02 pg/mL).
Trm cells are derived from precursors that entered tissues during the effector phase of immune responses and remained long-term within this compartment. In addition to protecting against local infections, Trm cells have been reported to suppress tumor growth and strongly correlate with favorable prognosis in cancer patients (Wang et al. 2020; Schenkel et al. 2014; Edwards et al. 2018; Bosmuller et al. 2016). The identification of Trm cells in peripheral tissues delineated two key markers that are expressed by the majority of these cells: CD69 and CD103 (αE integrin), both of which are gradually up-regulated during Trm cells development. CD69 can bind to S1PR1 and trap early activated T cells in secondary lymphoid organs until they are fully primed. Similarly, upregulation of CD69 both play functional roles in the development and retention of Trm cells. CD103 binds to E-cadherin expressed on epithelial cells to anchor Trm cells to epithelial tissues (Wang et al. 2020; Baeyens et al. 2015; Laura et al. 2015). In this study, we found that a substantial portion of T cells in malignant effusion were Trm cells highly expressing CD69 and/or CD103, especially CD8+T cells. Different from T cells in blood, 24.7% of CD4+T cells and 53.4% of CD8+T cells expressed CD69, and 25.8% of CD8+T cells expressed CD103. These results indicated that most tumor-infiltrating T cells in malignant effusion were resident in pleural and peritoneal cavities without recirculating.
In chronic infections and cancer, persistent antigen and/or inflammatory signals cause the deterioration of T cell function: a state called ‘exhaustion’. Exhausted T cells lose robust effector functions and express multiple inhibitory receptors. Different from T cell anergy, revitalization of exhausted T cells can reverse dysfunction and reinvigorate immunity. T cell exhaustion is a progressive process. Firstly, functions such as IL-2 production and cytokine polyfunctionality, as well as high proliferative capacity are lost. Secondly, the deficiency in the production of IFN-γ, TNF and chemokines, as well as in degranulation is followed. In addition, T cell exhaustion is also accompanied by a progressive increase in the amount and diversity of inhibitory receptors such as PD-1,Tim-3, et al. And finally, if the the antigen persists in the long-term, the antigen-specific T cells can be lost (Wherry et al. 2015; Blank et al. 2019). Our results indicated that CD4+T cells and CD8+T cells in malignant effusion were dysfunctional which expressed lower levels of IFN-γ, TNF-α, Granzyme B and Perforin than in blood. Higher and sustained expression of inhibitory receptors is a hallmark of exhausted T cells. The programmed death 1 (PD-1)/programmed death ligand 1(PD-L1) and/or PD-L2 axis is the more extensively studied inhibitory signalling pathway and correlated with early progression and shorter survival in cancer (Dermani, et al. 2018). CD4+T and CD8+T cells in malignant effusion expressed significantly higher levels of PD-1 than that in blood. Together, these data indicated that most CD4+T and CD8+T cells in malignant effusion were exhausted T cells and lost robust effector function.
In summary, our study showed that a substantial portion of T cells in malignant effusion were Trm cells without recirculating which highly expressed CD69 and/or CD103. Most CD4+T and CD8+T cells in malignant effusion were exhausted T cells which expressed lower levels of cytokines, cytotoxic molecules and markedly higher levels of inhibitory receptor PD-1 compared with in blood. To our best knowledge, our study is the first to identify the presence of Trm cells in malignant effusion. The function of these Trm cells in malignant effusion is needed to be further studied.