Study Of Clec9A-mediated Antigen Cross-presentation In Activating CD8⁺ Cytotoxic T Lymphocyte Immune Response In COPD

Objective:CD8⁺cytotoxic T lymphocyte（CD8⁺CTL）mediated lung tissue injury plays an important role in the pathogenesis of the chronic obstructive pulmonary disease（COPD）.C-type lectin domain family 9member A（Clce9A）is a DAMPs receptor that is highly restricted on conventional type 1 dendritic cells（c DC1s）.Functionally,Clec9A binds to filaments actin（F-actin）exposed on the surface of necrotic cells and promotes cross-presentation of necrotic cell antigen to CD8⁺T cells.This study aims to explore the role of Clec9A-mediated cross-presentation of necrotic cell antigen in the development of COPD airway inflammation.Methods:（1）A total of 207 patients with COPD,and 29 patients with non-obstructive chronic bronchitis（NOCB）admitted to Wuhan No.1Hospital from September 2020 to November 2021 were enrolled.Twenty-three smokers without COPD were recruited as smoker controls（SC）and 90 healthy subjects were selected as the healthy controls（HC）.The expression levels of Clec9A,F-actin,IFN-γ,and Granzyme-B in serum and bronchoalveolar perfusion fluid（BALF）of COPD patients and matched controls were determined by ELISA.The m RNA and protein levels of Clec9A in peripheral blood mononuclear cells（PBMC）from healthy individuals and patients with COPD were examined by real time-PCR and western blot.TUNEL assay was employed to identify the dead cells in BALF between patients with COPD and NOCB.The percentages of Clec9A⁺DC and CD8⁺CTL（CD8⁺Granzyme-B⁺T cells and CD8⁺IFN-γ⁺T cells）in circulating and BALF from patients with COPD or matched controls were determined by flow cytometry.Correlation analysis was performed between the percentages of Clec9A⁺DC and CD8⁺CTL in BALF from patients with COPD.（2）We constructed a mice model to develop COPD-like pathology with cigarette smoke exposure.Male C57BL/6 mice were exposed for nine months and collected lung tissues.The presence of Clec9A⁺DC and CD8⁺T cells were determined by immunofluorescence.Results:（1）Compared with the matched controls,COPD patients showed increased percentages of CD8⁺CTL both in circulating and BALF.Meanwhile,the BALF values of soluble cytotoxic mediators/total protein were significantly higher than that in paired serum.（2）Compared with the healthy subjects,the serum levels of soluble Clec9A were significantly increased in patients at different stages of COPD.The m RNA and protein levels of Clec9A in PBMCs were both markedly increased in patients with COPD at GOLD stagesⅢ-Ⅳthan that in healthy subjects.Furthermore,the percentage of Clec9A⁺DC in BALF was significantly increased in patients with COPD at GOLD stagesⅢ-Ⅳcompared with NOCB patients.（3）Accumulated dead cells were observed in the BALF of COPD patients than that in NOCB patients.The BALF values of F-actin also increased in COPD patients compared with NOCB patients.（4）There were positive correlations between the percentages of BALF-derived Clec9A⁺DC and CD8⁺Granzyme-B⁺T cells or CD8⁺IFN-γ⁺T cells in patients with COPD,respectively.In addition,the ratios of Clec9A/TP were strongly correlated with the ratios of Granzyme-B/TP or IFN-γ/TP in BALF,respectively.（5）After nine months of cigarette smoke exposure,significant COPD-like changes were observed in the lung tissues of mice,including increased inflammatory response and alveolar fusion,as well as increased infiltration of Clec9A⁺DC and CD8⁺T cells.Conclusions:Clec9A-dependent cross-presentation induces activation of CD8⁺CTL,which contributes to the progression of COPD.Targeting Clec9A has a promising application in the treatment of persistent airway inflammation in COPD.