Role of NK surface inhibitory receptor CD96 in chronic obstructive pulmonary disease and the effect of targeted CD96 intervention

Role of NK surface inhibitory receptor CD96 in chronic obstructive pulmonary disease and the effect of targeted CD96 intervention | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Role of NK surface inhibitory receptor CD96 in chronic obstructive pulmonary disease and the effect of targeted CD96 intervention Yuqing Fei, Renming Li, Pengcheng Liu, Wenjing Pei, Na Xin, Hu Xu, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8280945/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background CD96 is an inhibitory receptor found on natural killer (NK) cells, yet its mechanism of action in chronic obstructive pulmonary disease (COPD) remains unclear. This study aimed to examine the alterations and function of CD96 in COPD and to assess the impact of anti-CD96 intervention. Methods Peripheral blood samples were obtained from 44 patients with chronic obstructive pulmonary disease (COPD), along with a cigarette smoke (CS)-induced COPD mouse model. The expression of CD96 and IFN-γ in natural killer (NK) cells was measured using flow cytometry. Lung function, histopathological changes, airway remodeling, and levels of inflammatory mediators were evaluated in mice following 6 or 12 weeks of CS exposure, with or without the administration of anti-CD96 antibody. Results In patients with COPD, the expression of CD96 on NK cells was elevated, and the proportion of CD96⁺NK cells showed a positive correlation with the duration of hospitalization, CAT scores, and the incidence of acute exacerbations (≥ 2) within one year. In mice exposed to cigarette smoke, increased signaling through CD96 and CD155 was associated with a reduction in the FEV0.2/FVC ratio, disruption of emphysema-like structures, collagen deposition, airway wall thickening, and the upregulation of various inflammatory mediators, including IL-17A, IFN-γ, IL-6, and TNF-α. Treatment with anti-CD96 partially restored lung function, reduced alveolar injury, collagen deposition, and airway remodeling, and downregulated IL-17A along with several pro-inflammatory cytokines. Furthermore, this treatment preserved the IFN-γ production capacity of NK cells. The concurrent upregulation of CD96 and SHIP1 in NK cells, along with the enhancement of their function following CD96 blockade, suggests the involvement of a CD96-SHIP1-related inhibitory pathway in the disease process. Conclusion CD96-overexpressing NK cells and CD96/SHIP1-associated signaling are associated with disease severity and a chronic inflammatory state in COPD. The anti-CD96 intervention mitigated CS-induced lung injury in mice, indicating that CD96 may serve as a novel target for immunotherapy in COPD. Future research should further investigate its clinical translational potential in COPD patients and analyze its regulatory network by integrating multi-omics technology, thereby providing a more comprehensive molecular foundation for precision therapy. Chronic obstructive pulmonary disease NK cells CD96 SHIP1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Chronic obstructive pulmonary disease (COPD) is a preventable and treatable chronic airway disease marked by persistent airflow limitation and chronic respiratory symptoms, making it the third leading cause of death globally [ 1 ][ 2 ] . Key factors influencing the development of COPD include age, gender, smoking history, exposure to occupational and environmental particulate matter, early pulmonary dysplasia, and genetic predisposition [ 3 ][ 4 ][ 5 ][ 6 ] . Among these, immune dysregulation, particularly the synergistic imbalance between intrinsic and acquired immunity, plays a crucial role in the persistence of chronic inflammation and structural damage associated with COPD [ 7 ][ 8 ] . Label-free proteomics has identified that key molecules involved in Th17 cell differentiation and the IL-17-mediated signaling pathway are significantly upregulated in the plasma of COPD patients [ 9 ] . Additionally, the number of natural killer (NK) cells in the peripheral blood of COPD patients is significantly lower compared to that of healthy individuals. Although total IFN-γ levels are elevated [ 10 ] in patients with COPD, the ability of NK cells to secrete IFN-γ is significantly diminished. Th17 cells have been shown to recruit neutrophils and stimulate airway epithelial cells to secrete chemokines and matrix metalloproteinases via the release of IL-17. This process contributes to the persistence of inflammation and airway remodeling [ 11 ] . Elevated levels of IL-17 correlate closely with the frequency of acute exacerbations and the decline in lung function among COPD patients [ 12 ] . These findings suggest that the Th17/IL-17 axis may serve as a key driver of the sustained inflammatory response in COPD. Immune cell communication analysis was conducted on mice exposed to smoke, comparing the effects of various conditions on immune cell interaction pathways. For instance, AIR may activate the PECAM1-related pathway, while CS may activate the NECAB2-related pathway. This analysis utilized both relative proportions and absolute intensities. Results indicated that the information flow of CD96 was significantly greater in the CS condition compared to the AIR condition under the relative normalization dimension, suggesting that the CS condition markedly enhanced CD96 information transfer [ 9 ] . A flow assay of peripheral blood from patients with chronic obstructive pulmonary disease confirmed a significant upregulation of NK cell CD96 expression. CD96 is a type I transmembrane glycoprotein primarily expressed on the surface of T cells and NK cells, which competitively binds to the shared ligand CD155 with the costimulatory receptor CD226, exhibiting relatively high binding affinity [ 13 ] . The group utilized C57 mice to model chronic obstructive pulmonary disease. Following the modeling, alveolar lavage was conducted, and the collected lavage fluid underwent flow cytometry analysis. This analysis revealed a significant decrease in the proportion of NK cells, a notable increase in the number of CD96 + NK cells, and a substantial reduction in the number of IFN-γ + NK cells [ 9 ] . Existing literature indicates that a reduction in IFN-γ can enhance Th17 cell function, which subsequently increases IL-17 secretion and may represent a critical mechanism in the development of lentigo [ 14 ][ 15 ] . Peripheral blood was collected from healthy individuals and patients with chronic obstructive pulmonary disease (COPD) to assess the proportion of Th17 cells among CD4 + T cells using flow cytometry. The results indicated that both the proportion of Th17 cells and the levels of IL-17 were significantly elevated in the peripheral blood of COPD patients [ 9 ] . These findings suggest that the Th17/IL-17 axis may play a crucial role in driving the sustained inflammatory response associated with COPD. Furthermore, it is speculated that increased CD96 expression may serve as an upstream signal contributing to the Th17/IL-17-mediated development of chronic obstructive pulmonary disease. Further studies revealed that the expression of the NK cell phosphatase SHIP1 was significantly upregulated in patients with chronic obstructive pulmonary disease (COPD). Additionally, COPD-modeled mice with CD96 inhibition exhibited improved lung function compared to wild-type (WT) mice [ 9 ] . In summary, the research indicated that the CD96-SHIP1 signaling axis was upregulated in the NK cells of COPD patients. However, the potential of CD96 as a therapeutic target for COPD remains uncertain, and the role of targeted CD96 molecules in regulating immune cells and their downstream SHIP1 in the development of COPD is still not well understood. Consequently, this study integrated clinical samples with mouse models of COPD treated with CD96 monoclonal antibodies, focusing on the regulatory role of the CD96-SHIP1 signaling axis. The objective was to elucidate the mechanisms of disease progression mediated by CD96 and to evaluate the potential of targeted interventions, thereby providing a new theoretical foundation for the precise diagnosis and treatment of COPD. Methods 1 Animal Model and CS Exposure Six- to-eight-week-old SPF-grade C57BL/6 WT male mice were assigned to three groups: a healthy control group (WT), a smoke-exposed group (CS), and a smoke-exposed group treated with InVivoMAb anti-mouse CD96 (Bio X Cell, Cat# BE0337-50MG). The control group was exposed to filtered indoor air, while the anti-CD96 group received intraperitoneal injections of the CD96 inhibitor at a dose of 200 µg every 5 days. Both the CS and anti-CD96 groups were subjected to mainstream smoke from filtered cigarettes using a whole-body Exposure System. Each exposure consisted of 12 cigarettes, administered twice daily for 60 minutes per session, 5 days per week over a duration of 12 weeks. All experimental procedures received approval from the Institutional Animal Care and Use Committee of Anhui Medical University (Approval number: LLSC20210886). 2 Single-cell RNA sequencing and analysis Lung CD45⁺ immune cells were isolated and analyzed through single-cell RNA sequencing utilizing the 10x Genomics platform. Standard preprocessing, clustering, trajectory inference, regulator mapping, enrichment analysis, and intercellular communication modeling were conducted following established methodologies. 3 Lung function Anesthesia was induced in the mice through an intraperitoneal injection of 100 mg/kg sodium pentobarbital. Following the administration of anesthesia, pulmonary function was assessed by measuring several parameters, including respiratory rate (f), tidal volume (TV), total lung capacity (TLC), forced vital capacity (FVC), residual volume (RV), functional residual capacity (FRC), FEV0.20/FVC%, dynamic lung compliance (Cdyn), and airway resistance (RI). Each parameter was measured five times per animal. 4 Pathological image analysis Pathological sections of mice were randomly fixed at low magnification. Crosshairs were drawn at the center of each field of view, and the total length of the crosshairs (L) was measured. The number of alveolar septa intersecting the crosshairs (NS) was recorded, and emphysema analysis was conducted using the mean lining interval of lung tissue (MLI = L/NS). Following Masson staining, the relative collagen content of the airways was quantitatively analyzed using ImageJ, calculated as follows: [Relative collagen content = (collagen fiber positive area/total airway wall area) × 100%]. Image-Pro Plus was employed to determine the airway lumen area from the Masson-stained images. The selected airway cross-sections adhered to the following criteria: ① intact airway structures without evident folding or fracture; ② approximately circular or oval cross-sections (L/D ratio ≤ 1.5); and ③ no significant secretion or cellular obstruction within the lumen. 5 Flow Cytometry/financal capacity model To prepare 1 × 10^6 cells, incubate them in serum at 4℃ for 30 minutes. Subsequently, add a specific fluorescent antibody while maintaining the temperature at 4℃ to prevent light exposure during the labeling of NK cell extracellular molecules for an additional 30 minutes. Following this, employ a cell fixative and permeabilization solution to fix and permeabilize the cell membrane. Afterward, add a specific antibody to label the intracellular molecules of the NK cells. Finally, adjust the cell density to the appropriate level for flow cytometry analysis. 6 Real-time Quantitative PCR Detecting System(Q-PCR) Isolated NK cells were collected, and cellular RNA was extracted using Trizol homogenate for concentration and quality assessment. The RNA was then reverse transcribed to cDNA, followed by the construction of primers. PCR amplification was conducted with GAPDH as an internal reference to quantify the gene expression of airway inflammatory factors, including IL-33, IL-1β, IL-6, TNF, IL-10, and TGF-β, using fluorescence quantitative PCR. 7 Enzyme-Linked Immunosorbent Assay (ELISA) Following the instructions provided in the ELISA kit, plasma or alveolar lavage fluid samples were centrifuged, and the supernatant was extracted along with other necessary pre-treatments. The ELISA instrument was calibrated to a wavelength of 450 nm, and detection standards were employed to construct standard curves for measuring the concentrations of IFN-γ and IL-17. 8 Immunofluorescence technique For the immunofluorescence technique, frozen sections of mouse lung tissue were placed in a wet box and incubated with rat serum for 30 minutes. Subsequently, the sections were incubated overnight at 4℃ with an anti-CD155 primary antibody, followed by a 1-hour incubation at room temperature with a fluorescent secondary antibody, protected from light. After staining with DAPI, the expression and localization of CD155 in the lung tissues were observed under a confocal laser microscope after sealing the slices. 9 Clinical indicators 9.1 Research objects A total of 44 patients with chronic obstructive pulmonary disease (COPD) participated in this study, all of whom were recruited from the Second Affiliated Hospital of Anhui Medical University. Each enrolled patient met the diagnostic criteria for COPD established by the World Health Organization (WHO) and the Global Initiative for Chronic Obstructive Lung Disease (GOLD). Exclusion criteria encompassed other chronic respiratory diseases, autoimmune disorders, malignant tumors, severe cardiovascular conditions, and any diseases that could potentially impair immune function. Additionally, patients who had received corticosteroids or other immunosuppressive medications within the preceding month were excluded. All participants provided written informed consent. The study protocol was approved by the Ethics Committee of the Second Affiliated Hospital of Anhui Medical University (approval no. YX2025191). 9.2 Specimen collection and processing Within 24 hours of admission, medical staff collected 2 ml of fasting peripheral venous blood from the patients and preserved it in EDTA anticoagulation tubes. All blood samples were sent to the laboratory for processing within 2 hours of collection. 9.3 Clinical data collection Baseline clinical data, including days of hospitalization and COPD Assessment Test (CAT) scores, were collected from all patients. The number of acute exacerbations occurring within one year of discharge was recorded through telephone follow-up after patients left the hospital. AECOPD definition: events characterized by worsening of dyspnea and/or cough and sputum within 14 days in patients with COPD, which may be accompanied by shortness of breath and/or tachycardia, are usually associated with an exacerbation of local or systemic inflammatory responses to respiratory tract infections, air pollution, or other causes [ 16 ] . Based on the frequency of acute exacerbations, patients were classified into two groups: those with ≤ 2 exacerbations and those with > 2 exacerbations. 10 Statistical Analysis All experimental data were derived from a minimum of three independent repeated tests. Normally distributed data are presented as Mean ± SEM, while non-normally distributed data are expressed as Median and Interquartile Range (IQR). Data organization and analysis were performed using software such as SPSS 22.0, GraphPad Prism 8.0, and FlowJo 10.0. For parametric tests of measurement data, an independent samples t-test was employed for comparisons between two groups, and one-way ANOVA was utilized for comparisons among multiple groups. Non-parametric tests were applied to hierarchical data, including normality tests and variance chi-square tests for measurement data. For data that did not meet the criteria for normal distribution, non-parametric tests such as the Mann-Whitney U test and Kruskal-Wallis test were used. A p-value of less than 0.05 was considered indicative of a statistically significant difference between the datasets. Results 1 Changes in immune cell signaling pathways under chronic obstructive pulmonary conditions 1.1 Immune cell interactions are more active in chronic lung conditions Comparative analysis of immune cell communication between CS and AIR conditions through the two dimensions of quantity difference and intensity difference(Fig. 1 A and 1 B), and found that the column values of CS group were higher in C and E graphs comparing with AIR group(Fig. 1 C and 1 E), indicating that the number of differential interactions between immune cells in CS group was significantly increased, which mainly involved multiple cell pairs such as NK-T cells, ILC-monocytes, macrophage-neutrophils, etc., and the number of signaling connections between cells was more frequent. The number of connections between cells was more frequent; the higher relative value of CS group in D and F graphs indicated that the relative strength of immune cell interactions in CS group was generally higher than that in AIR group, especially the signaling strength of NK cells and other immune cells was elevated significantly, suggesting that the signaling strength of inter-cellular communication was stronger(Fig. 1 D and 1 F). Meanwhile, the analysis of immune cell communication in mice under smoked condition revealed that CS condition significantly enhanced the messaging of CD96,speculating that the activity of immune cell interaction was related to the CS-related airway inflammation. 1.2 Analysis of CD96 pathway alterations in chronic lung mice using single-cell sequencing Visualization of single-cell sequencing and information flow analysis presented the abnormal activation of CD96 pathway in mice with chronic obstructive pulmonary disease (COPD). Figure 1 A shows that the clusters of cells with high expression of CD96 were more concentrated in the CS group, suggesting that the expression profiles of CD96 in immune cells were abnormally enriched; Fig. 1 B clearly shows that the information flow value of CD96 in the CS group was significantly higher than that in the AIR group, which is a direct evidence of the fact that the signaling of the CD96 pathway was significantly enhanced under the conditions of chronic obstructive pulmonary disease (COPD) directly proved that the signaling efficiency of CD96 pathway was significantly enhanced under the condition of chronic obstructive pulmonary disease. The above results indicated that the expression of CD96 was not randomly distributed, but highly enriched in specific immune cell subpopulations (e.g.NK cells, T cells) in the CS group, and the signaling activity of the CD96 pathway was significantly elevated in chronic obstructive pulmonary disease (COPD). 2 Cigarette smoke exposure consistently impairs lung function and worsens lung tissue destruction, which is partially mitigated by anti-CD96 antibodies. 2.1 Cigarette Smoke Persistently Impairs Lung Function, and Anti-CD96 Antibodies Partially Alleviate Smoke-Induced Lung Function Decline In comparison to the normal control group, the slope of the rising branch in the lower section of the flow-time curve was reduced and exhibited a staccato pattern in the CS group. Additionally, the expiratory phase was prolonged in this group. Conversely, the slope of the rising branch in the lower part of the flow-time curve increased, and the expiratory phase shortened in the anti-CD96 group relative to the CS group(Fig. 2 A- 2 C). The FEV0.20/FVC% in the CS group was significantly lower than that of the healthy control rats, while the anti-CD96 group demonstrated an increase in FEV0.20/FVC% compared to the CS group(Fig. 2 F). The administration of the anti-CD96 monoclonal antibody mitigated the decline in lung function induced by cigarette smoke in mice after 6 weeks of exposure, showing a significant improvement over the smoking group alone, although it did not fully restore lung function to the levels observed in healthy controls. After 12 weeks of smoking, lung function in mice significantly declined, indicating that prolonged exposure to cigarette smoke continued to adversely affect lung function. The beneficial effects of the anti-CD96 monoclonal antibody persisted at the 12-week mark, but the improvement was notably less pronounced than that observed at 6 weeks, potentially due to the effects of long-term smoke exposure or the timing of antibody administration. This study suggests that cigarette smoke consistently contributes to the decline in lung function, and the anti-CD96 monoclonal antibody may partially alleviate this decline induced by cigarette smoke. 2.2 Cigarette smoke persistently damages lung tissue structure in mice, and anti-CD96 antibody partially attenuates lung tissue destruction Pathological analysis revealed that the bronchial and alveolar structures in the wild-type (WT) group appeared intact, with no evident mucosal destruction or inflammatory cell infiltration(Fig. 3 B). In contrast, the cigarette smoke (CS) group exhibited disorganized lung architecture characterized by significant destruction, fractured alveolar walls, and deformed, necrotic bronchial mucosal epithelial cells. Additionally, cilia were uneven, shortened, inverted, or even detached, and the walls were thickened, accompanied by extensive inflammatory cell infiltration. The lumens of the airways were narrowed, twisted, or dilated. Prolonged fumigation time exacerbated the destruction of lung tissues(Fig. 3 B). Notably, the aforementioned lesions were significantly mitigated in the anti-CD96 group(Fig. 3 B). The mean linear intercept (MLI), which indicates the extent of alveolar destruction, revealed that the MLI in the CS group was significantly higher than that in the WT group at both 6 and 12 weeks of cigarette smoking. Furthermore, the MLI at 12 weeks was greater than that at 6 weeks, suggesting that prolonged exposure to cigarette smoke exacerbates alveolar structural damage. In contrast, the MLI in the anti-CD96 group was significantly lower than that in the CS group and comparable to that in the WT group at both time points(Fig. 3 C and 3 D). This finding indicates that the anti-CD96 antibody can consistently mitigate the alveolar structural damage induced by cigarette smoke. Collagen deposition in lung tissue was evaluated through relative collagen content. The CS group exhibited significantly higher levels than the WT group, indicating that CS induced collagen deposition and fibrosis in lung tissue. Additionally, collagen content in the anti-CD96 group was markedly lower than that in the CS group(Fig. 4 A- 4 I, 4 L), suggesting that the blockade of CD96 may mitigate collagen deposition and alleviate lung tissue fibrosis in mice exposed to smoke. Small airway remodeling serves as an indicator of the extent of airway hyperplasia and thickening [ 17 ] . The small airway wall thickness was significantly greater in the CS group compared to the WT group (P < 0.0001), accompanied by a reduction in lumen area. In contrast, the wall thickness in the anti-CD96 group was significantly lower than that in the CS group (P < 0.0001), with the lumen area significantly increased and approaching that of the WT group(Fig. 4 A-K). The difference between the WT and anti-CD96 groups was not statistically significant. These findings indicate that exposure to cigarette smoke leads to small airway wall thickening and lumen narrowing, while the anti-CD96 antibody appears to mitigate these pathological changes by modulating the CD96 pathway. Collectively, exposure to CS can result in alveolar destruction, collagen deposition, thickening of small airway walls, and narrowing of the lumen. In contrast, the anti-CD96 antibody mitigated these pathological changes to some degree, indicating that CD96 may serve as a potential therapeutic target for chronic airway and lung tissue injury. 3 CS stimulation activates Th1/Th17 and multiple inflammatory factors, which is partially reversed by anti-CD96 antibody IL-17A serves as a critical cytokine for Th17 cells, which play a role in inflammation and autoimmunity [ 18 ] , while IFN-γ is a pivotal cytokine for Th1 cells, mediating antiviral and antitumor immunity [ 19 ] . These cytokines represent the activation of the Th17 and Th1 pathways, respectively. At 6 weeks of smoking, the IL-17A level in the CS group was significantly elevated compared to the WT group, whereas the level in the anti-CD96 group was significantly reduced relative to the CS group. This difference persisted at 12 weeks of smoking, indicating that blocking CD96 could continuously inhibit the CS-induced increase in IL-17A(Fig. 2 K,Table 1 ). Additionally, the IFN-γ level in the CS group was higher than that in the WT group at both 6 and 12 weeks, and the level in the anti-CD96 group was significantly lower than that in the CS group at 12 weeks(Fig. 2 L). This finding suggests that anti-CD96 had a notable impact on IFN-γ levels, although its inhibitory effect on IFN-γ appeared to lag relative to that on IL-17A. Overall, these results indicate that CS stimulation can induce prolonged activation of the Th1 (IFN-γ) and Th17 (IL-17A) pathways over 6 and 12 weeks. Furthermore, the anti-CD96 antibody can rapidly inhibit the elevation of IL-17A while exhibiting a delayed inhibitory effect on IFN-γ elevation, suggesting that CD96 plays a regulatory role in the CS-induced immune response and that there is a temporal difference in the effects of CD96 on the Th1 and Th17 pathways. Gene expression of inflammation-related factors in lung tissues exhibited comparable alterations. For instance, treatment with CS resulted in the upregulation of IL-33 gene expression relative to the healthy control group. Inhibition of CD96 partially reversed the elevated IL-33 expression, with levels in the anti-CD96 group returning close to those of the wild-type (WT) group when compared to the CS group(Fig. 2 R). This finding suggests that CD96 molecules may play a role in regulating IL-33-associated immune pathways. The effects of CS status on IL-33 gene expression and the regulatory influence of CD96 antibody intervention were thus elucidated. Other factors, including pro-inflammatory cytokines such as IL-1β, IL-6, and TNF, as well as anti-inflammatory modulators like IL-10 and TGF-β, demonstrated similar trends. Notably, IL-6 expression in the CS group was significantly higher than that in the WT group; however, no significant difference was observed between the WT group and the anti-CD96 group, indicating that the CD96 inhibitor significantly regulated IL-6 levels. Furthermore, while TGF-β expression was markedly elevated in the CS group compared to the control group and reduced in the anti-CD96 group relative to the CS group, TGF-β levels remained extremely low in the WT group(Fig. 2 M- 2 Q). This suggests that TGF-β may exert a pro-fibrotic and immunosuppressive role in the disease. In summary, CS can persistently activate Th1/Th17 pathways and various inflammatory factor pathways. In contrast, the anti-CD96 antibody exhibits both early and late inhibitory effects on IL-17A and IFN-γ, while also partially reversing the upregulation of inflammatory factors. These findings suggest that CD96 plays a role in the regulation of CS-induced inflammation and may serve as a potential therapeutic target. Table 1 Sequences of the primers for real-time PCR 4 CS stimulation affects CD96 expression and thus NK cell immunoreactivity 4.1 Upregulation of CD96 expression is accompanied by diminished IFN-γ secretion by NK cells To establish the dosing regimen, the mean fluorescence intensity (MFI) of CD96 on the surface of NK cells was assessed via flow cytometry on days 3 and 10 following the administration of the anti-CD96 monoclonal antibody. Results indicated that the CD96 MFI in the anti-CD96 group was significantly lower than that in the wild-type (WT) group at day 3 post-treatment, suggesting a substantial reduction in CD96 expression on NK cells due to the inhibitor intervention. However, after 10 days of treatment, no significant difference in CD96 MFI was observed between the anti-CD96 and WT groups(Fig. 5 A), indicating that CD96 expression on NK cells had converged following 10 days of intervention with a 200 µg single dose of the CD96 inhibitor. Consequently, an administration interval of 5 days for the CD96 inhibitor was selected to maintain continuous inhibition of CD96 expression in the anti-CD96 group. After 12 weeks of smoke exposure, CD96 expression on the surface of NK cells was significantly elevated in the CS group compared to the WT group(Fig. 5 B), indicating that CS treatment upregulated CD96 expression. The functional capacity of NK cells to secrete IFN-γ was evaluated by measuring the percentage of IFN-γ-producing NK cells (%IFN-γ⁺NK cells). The WT group exhibited the lowest percentage, while the CS group showed a slight increase, and the anti-CD96 group had the highest percentage. Although the differences between the WT and CS groups, as well as between the CS and anti-CD96 groups, were not statistically significant, the difference between the WT and anti-CD96 groups was significant(Fig. 5 C). These findings suggest that blocking the CD96 pathway may enhance the IFN-γ secretion capacity of NK cells in the context of long-term smoking. 4.2 CD96 may be involved in mediating CS intervention-induced CD155 signaling activation Changes in CD155 signaling were evaluated through relative fluorescence intensity analysis. After 12 weeks of smoking, the relative fluorescence intensity of lung tissue in the CS group was significantly higher than that in the WT group (P < 0.05), indicating that CS stimulation activated the CD155 signaling pathway. Furthermore, the relative fluorescence intensity of CD155 in the anti-CD96 group was significantly lower than that in both the CS group and the WT group (P < 0.05 vs. WT, P < 0.01 vs. CS)(Fig. 5 D and 5 E). This finding suggests that blocking the CD96 pathway partially attenuated the CS-induced CD155 signaling, and the trends in CD96 signaling and CD155 signaling intensity showed some concordance. It is further speculated that CD96 may facilitate immune escape by sustaining the activity of the CD155 signaling pathway. These results imply that CD96 may play a role in the activation of CS-induced CD155 signaling, and the anti-CD96 antibody may enhance the local immune microenvironment by modulating this pathway. However, the direct molecular interactions between the two and the downstream mechanisms warrant further investigation. 5 Peripheral blood CD96 + NK cell ratio correlates with disease severity and acute exacerbation risk in patients with chronic obstructive pulmonary disease This study included a total of 44 patients diagnosed with chronic obstructive pulmonary disease (COPD). The ratio of CD96 + NK cells to total NK cells in peripheral blood was assessed using flow cytometry(Fig. 5 I). Correlation analysis revealed a significant positive correlation between this ratio and the duration of hospitalization (r = 0.440, p = 0.003), as well as with CAT scores (r = 0.380, p = 0.011)(Fig. 5 J and 5 K). These findings indicate that a higher ratio of CD96 + NK cells is associated with longer hospital stays and greater symptom burden. To further explore the relationship between the proportion of CD96 + NK cells and the frequency of acute exacerbations of COPD, patients were categorized into two groups: those with ≤ 2 exacerbations and those with > 2 exacerbations within one year post-discharge. Statistical analysis demonstrated that the proportion of NK cells represented by CD96 + NK cells in peripheral blood was significantly higher in the group with > 2 exacerbations compared to the ≤ 2 exacerbations group (p < 0.05). This suggests that a high ratio of CD96 + NK cells may serve as a predictor for an increased risk of future acute exacerbations. In this study, we observed that the inflammation-associated factor IL-17 was elevated in patients with chronic obstructive pulmonary disease (COPD). Additionally, Th17 differentiation and the IL-17 signaling pathway were upregulated(Fig. 5 F and 5 G), while CD96 and its downstream target SHIP1 were significantly increased in natural killer (NK) cells (P < 0.001)(Fig. 5 H). These findings suggest that CD96, an inhibitory receptor on NK cells, was markedly upregulated, and that Th17/IL-17 functionality was enhanced in COPD patients, thereby contributing to the progression of the disease. To further investigate the mechanisms underlying this immune disorder, we systematically validated these results in clinical COPD patients and a mouse model of COPD. Lung function assessments revealed that mice in the cigarette smoke (CS) exposure group exhibited significantly worse outcomes compared to the control group, with declines beginning at six weeks. Furthermore, lung function in the anti-CD96 treatment group showed improvement relative to the CS group, although this effect was less pronounced at twelve weeks than at six weeks. Histological analysis of the CS group at six weeks revealed alveolar wall destruction, inflammatory cell infiltration, and thickening of the small airway walls, with the mean linear intercept (MLI) being greater than that of the control group.MLI was significantly higher in the control group and worsened at 12 weeks. In contrast, alveolar disruption was reduced, and inflammatory infiltration decreased in the anti-CD96 group, where MLI was lower than in the CS group and approached levels observed in the WT group. Following smoking exposure, CD96 expression in NK cells was up-regulated, and IL-17A levels were significantly elevated in CS group mice at both 6 and 12 weeks. However, CD96 expression was suppressed in the anti-CD96 group, resulting in a significant reduction of IL-17A at 6 weeks, with continued efficacy observed at 12 weeks. The expression of pro-inflammatory factors, including IL-1β, IL-6, and TNF, was significantly up-regulated in the CS group, while the expression of these genes was partially reversed in the anti-CD96 group, approaching levels seen in the WT group. CD155 signaling was activated in the CS group, whereas signaling in the anti-CD96 group was significantly lower than that in the model group, indicating that CD96 may play a role in mediating the CD155 signaling pathway. Furthermore, a high ratio of CD96 + NK cells may predict an increased risk of acute exacerbation in patients over the following year. This article confirms the involvement of CD96 in the development of chronic obstructive pulmonary disease and suggests that inhibition of CD96 may serve as a potential therapeutic target for this condition. Discussion In this study, we established that the CD96-SHIP1 signaling axis serves as a critical regulatory node in the pathogenesis of COPD. We also identified CD96 as a potential therapeutic target and elucidated the mechanistic link between abnormal NK cell function and Th1/Th17 immune imbalance. Our core findings indicate that the expression of CD96 and SHIP1 on the surface of NK cells was upregulated. Furthermore, CD96 exerted a regulatory effect on IFN-γ and IL-17A, thereby confirming the therapeutic potential of targeting CD96. This work provides a new theoretical foundation and practical direction for the immunotherapy of COPD. This paper presents single-cell sequencing findings that reveal enhanced communication among immune cells under chronic obstructive lung conditions. The study employs dual visualization to assess both the dimensions of cellular interactions and the CD96 pathway. Results indicate that the CD96 pathway undergoes significant alterations in expression enrichment and signaling enhancement in immune cells derived from chronic obstructive lung mice. These findings provide direct single-cell-level evidence of the CD96 pathway's role as a critical regulatory mechanism in slow-onset lung disease. The conclusion that the upregulation of CD96 on the surface of NK cells facilitates the progression of COPD is substantiated by both clinical and experimental evidence. Clinical data indicated a significant association between the CD96 + NK cell ratio and prolonged hospitalization, elevated CAT scores, and an increased risk of acute exacerbation in patients. These findings suggest that CD96 + NK cells may function as a prognostic marker for COPD. Our experimental data indicated that the overall level of IFN-γ was elevated under CS conditions, while inhibition of CD96 resulted in a decrease in the overall level of IFN-γ, despite promoting IFN-γ secretion by NK cells. Thus, we concluded that the overexpression of CD96 impairs NK cell function by inhibiting IFN-γ secretion, although blocking CD96 partially restored the secretion of IFN-γ by NK cells. Previous studies have demonstrated that IFN-γ in COPD acts as a disease-promoting factor by activating macrophages, inducing protease and nitric oxide production, and contributing to the development of emphysema and chronic inflammation. Conversely, IFN-γ serves as a crucial effector molecule for NK cells and the Th1 response, playing a regulatory role in anti-infection processes and the inhibition of Th17 responses. This duality suggests that IFN-γ exhibits both beneficial and detrimental characteristics in the context of COPD. Our results indicated that exposure to CS elevated the overall level of IFN-γ, whereas the administration of anti-CD96 antibody diminished the global inflammatory burden. Concurrently, within NK cell subsets, CD96 blockade did not further impair, and may even enhance, their capacity for IFN-γ production. These findings corroborate existing studies and suggest that targeting CD96 may achieve a balance between reducing inflammation and preserving essential effector functions. The study identified an upregulation in the expression of the phosphatase SHIP1, which is known to antagonize NK cell activation. This finding suggests the potential existence of a CD96-SHIP1 cascade pathway that may inhibit NK cell-mediated immune surveillance functions. Importantly, this signaling axis interacts with the Th17/IL-17 pathway: blockade of CD96 rapidly diminished the elevation of IL-17A induced by cigarette smoke, while the inhibitory effect on IFN-γ exhibited a delay. This temporal discrepancy indicates a differential regulation of Th1 and Th17 responses by CD96. The underlying mechanism may relate to the 'double-edged sword' role of NK cell-derived IFN-γ in chronic obstructive pulmonary disease (COPD). Specifically, IFN-γ contributes to emphysema and chronic inflammation through macrophage activation (pro-disease role), while also exerting immunomodulatory effects by inhibiting the Th17 response (protective role). The primary insight of this study is that targeting CD96 effectively balances the reduction of systemic inflammation with the preservation of essential immune effector functions. While anti-CD96 antibodies decrease systemic IFN-γ levels, thereby mitigating the progression of chronic inflammation, they simultaneously enhance IFN-γ secretion specifically from NK cells. This dual effect directly addresses the 'double-edged sword' dilemma associated with IFN-γ in COPD. Such selective modulation carries significant implications; abnormal NK cell function contributes to impaired pathogen clearance and persistent inflammation in COPD patients, conditions that may be improved through targeted CD96 blockade. Furthermore, CD96 blockade reduced CS-induced activation of CD155 signaling, reinforcing the hypothesis that CD96 disrupts immune homeostasis by competing with CD226 for CD155 binding. Collectively, these findings unveil a novel immunopathological mechanism: activation of the CD96-SHIP1 axis may lead to alterations in NK cell function, enhance Th17-mediated inflammatory responses, and ultimately exacerbate lung tissue injury. The potential of CD96-targeted therapy was underscored by the findings of preclinical studies, which demonstrated that anti-CD96 antibodies enhanced lung function, reduced alveolar destruction and airway remodeling, and partially restored the expression of pro-inflammatory factors (IL-1β, IL-6, TNF) and collagen deposition. Notably, these therapeutic effects persisted even after 12 weeks of exposure to cigarette smoke, indicating the clinical translational potential for long-term management of COPD. Furthermore, the correlation between the percentage of CD96 + NK cells and disease severity reinforces the feasibility of utilizing CD96 as both a predictive biomarker and a therapeutic target for COPD, addressing an unmet need in the realm of precision medicine for this condition. This paper presents several limitations: first, the elucidation of the direct role of SHIP1 downstream of CD96 remains incomplete; second, there is insufficient data regarding CD96 expression in other immune cell subsets. While this study concentrated on NK cells, future research should investigate whether CD96 regulates SHIP1 through direct phosphorylation or via intermediate signaling molecules. Furthermore, the expression of CD96 on the surface of key effector T cells and neutrophils in COPD warrants further investigation to clarify the specific roles of various cell types in CD96-mediated pathology. Additionally, the observed difference in the efficacy of the anti-CD96 antibody at 6 weeks compared to 12 weeks of corticosteroid exposure may be attributed to the cumulative effects of lung injury or alterations in antibody pharmacokinetics; this issue could be addressed in future studies by optimizing the dosing regimen. Conclusions This study collectively confirms that the CD96-SHIP1 axis serves as a crucial driver of abnormal NK cell function and Th1/Th17 imbalance in COPD. The findings not only validate the potential of CD96 as an intervenable target for reducing inflammation and preserving lung function, but also identify CD96 + NK cells as a promising prognostic marker. By correlating cellular and molecular mechanisms with clinical outcomes, this research enhances our understanding of the immunopathogenesis of COPD and establishes a foundation for the development of CD96-targeted therapies aimed at improving patient prognosis. Abbreviations COPD chronic obstructive pulmonary disease NK natural killer CS a smoke-exposed group WT a healthy control group IFN-γ Interferon-gamma PECAM1 platelet endothelial cell adhesion molecule-1 NECAB2 N-terminal EF-hand calcium binding protein 2 IL-17 Interleukin-17 SHIP1 SH2-containing inositol phosphatase-1 f respiratory rate TV tidal volume TLC total lung capacity FVC forced vital capacity RV residual volume FRC functional residual capacity Cdyn dynamic lung compliance RI airway resistance NS crosshairs MLI the mean lining interval of lung tissue GOLD Global Initiative for Chronic Obstructive Lung Disease CAT COPD Assessment Test Declarations Ethics approval and consent to participate All animal experiments were approved by the Institutional Animal Care and Use Committee of Anhui Medical University (Approval No.LLSC20210886). Human sample collection was approved by the Ethics Committee of the Second Affiliated Hospital of Anhui Medical University (Approval No.YX2025191), and written informed consent was obtained from all participants.The studies adhered to the principles outlined in the Declaration of Helsinki. Consent for publication All authors unanimously consent to submit the manuscript to Respiratory Research for publication. We agree that if the manuscript is accepted after peer review,Respiratory Research shall have the right to publish the manuscript in its print edition, electronic edition, and other derivative versions (including but not limited to online databases, digital archives, etc.). We also consent to the journal’s editorial department making necessary editorial revisions (including language polishing, format adjustment, etc.) to the manuscript to meet the publication standards of the journal, provided that the core content and academic views of the manuscript are not altered without the authors’consent. Availability of data and materials The datasets used and analysed during the current study are available from the corresponding author on reasonable request.The datasets generated during and analysed during the current study are available in the PubMed repository,https://pubmed.ncbi.nlm.nih.gov/ [1-19] . Competing interests The authors declare no competing interests. Funding This study was supported by the Major Difficult Disease Collaborative Research Project of Traditional Chinese and Western Medicine (Grant No. 2021zdynjb07), the Natural Science Foundation of Anhui Province (Grant No. 2208085MH194), the Anhui Provincial Key Research and Development Program (Grant No. 202304295107020022), the Science Research Project of Universities in Anhui Province (Grant No. 2023AH040374), the National Natural Science Foundation of China (Grant No. 82371756), and the Science Fund of Universities of Anhui Province for Distinguished Young Scholars (Grant No. 2023AH020032). Authors' contributions YQF conceived and designed the study. JY, CXM, and DHZ supervised the project and provided critical revisions. RML, PCL, WJP, NX, HX, LDL, CX and SGZ performed the experiments. DHZ secured funding and provided resources. RML and PCL conducted data analysis and bioinformatics processing. YQF drafted the initial manuscript.All authors read and approved the final manuscript. Acknowledgements We extend our gratitude to all members of the Department of Respiratory and Critical Care Medicine at Anhui Medical University for their technical support and insightful discussions. Additionally, we thank Novozymes Ltd (Beijing, China) for providing single-cell RNA sequencing and preliminary bioinformatics services. References Chair CV, agusti,A.Anzueto A, Criner,PFrith PBJBG, HalpinM.Han D. F,Martinez,M.MontesdeOca,A.Papi,L.Pavord,N.Roche,D.Sin,D.Singh, R.Stockley,M.Victorina lopezVarela, J.Wedziha. Global Initiative for Chronic Obstructive Lung DiseaseGlobal Strategy for the diagnosis, management and prevention of Chronic Obstructive LungDisease.2021 Reports. 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Ding T, Zhao S, Gu Y, He G, Lang Y, Rao X, Chen J, Ou-Yang Y. IL-17A regulates airway remodelling in COPD through the PI3K/AKT/mTOR pathway. Sci Rep. 2025;15(1):16546. Pasquarelli-do-Nascimento G, Machado SA, de Carvalho JMA, Magalhães KG. Obesity and adipose tissue impact on T-cell response and cancer immune checkpoint blockade therapy. Immunother Adv. 2022;2(1):ltac015. Table 1 Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8280945","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":570908121,"identity":"b8439c42-5fea-4c75-856d-a3ef2b53e2d8","order_by":0,"name":"Yuqing Fei","email":"","orcid":"","institution":"The Second Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuqing","middleName":"","lastName":"Fei","suffix":""},{"id":570908122,"identity":"a462c099-6315-4d17-8768-81cf9e0f5648","order_by":1,"name":"Renming Li","email":"","orcid":"","institution":"The Second Affiliated Hospital of Anhui 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07:41:09","extension":"html","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":120601,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8280945/v1/ccb0a18545b82e910fbebc2d.html"},{"id":100360747,"identity":"3e66f24a-74ac-4658-873b-793d7c25fbf0","added_by":"auto","created_at":"2026-01-16 07:41:34","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":425845,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eContribution of CD96in the pathway to communication and signal gene expression patterns in the AIR and CS groups.\u003c/strong\u003eThe visualization analysis derived from single-cell sequencing elucidates the communication characteristics of immune cells and the fundamental alterations in the CD96 pathway under chronic obstructive pulmonary disease (CS exposure) compared to healthy controls (AIR). This analysis provides compelling evidence for the ‘more active interaction of immune cells in the state of chronic obstructive pulmonary disease’ and the ‘abnormal activation of the CD96 pathway.’ \u003cstrong\u003e(A)\u003c/strong\u003e UMAP Distribution Map illustrates the expression level of CD96, represented by a color gradient ranging from 0.00 to 0.15, and depicts the distribution pattern of CD96 in lung CD45⁺ immune cells along the horizontal axis (UMAP_1) and vertical axis (UMAP_2). \u003cstrong\u003e(B)\u003c/strong\u003e Information Flow Comparison Chart presents the information flow (information transmission efficiency) on the horizontal axis and the groups (AIR vs. CS) on the vertical axis, demonstrating that the information flow value of CD96 in the CS group is significantly higher than that in the AIR group. \u003cstrong\u003e(C,D,E,F)\u003c/strong\u003eFigures C and E depict the ‘quantity of differential interaction’ analysis, with the vertical axis representing the number of differential interactions, while Figures D and F illustrate the ‘intensity of differential interaction’ analysis, where the vertical axis denotes the relative intensity value. These analyses focus on key inflammation-related pathways, including ANNEXIN signaling and ANXA1-FPR2/FPR1, and encompass essential immune cell subsets such as NK cells, T cells, ILC cells, monocytes, and macrophages.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8280945/v1/c4d38fe279ca741ea6d19872.jpg"},{"id":100007197,"identity":"24d93519-40ab-4791-88c9-90d644b03ca4","added_by":"auto","created_at":"2026-01-12 05:49:59","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":516430,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLung function test and CS stimulation upregulates multiple inflammatory factors.\u003c/strong\u003eCS is an abbreviation for \"Cigarette Smoke,\" which denotes a model of cigarette smoke exposure. Anti-CD96 refers to the investigation of the CD96 molecule's function through its blockade.\u003cstrong\u003e(A,B,C)\u003c/strong\u003eMouse lung function flow-time curves.\u003cstrong\u003e(D,E,F,G,H) \u003c/strong\u003eChanges in mouse body weight,peak expiratory flow rate (PEF), FEV0.20/FVC%, dynamic lung compliance (Cdyn) and airway resistance (RI) were assessed after 12 weeks of smoke exposure.\u003cstrong\u003e(I,J)\u003c/strong\u003eThe comparison of pulmonary function indicators, including FEV0.20/FVC% and PEF, among mice in the WT group, CS group, and anti-CD96 group was conducted at 6 weeks and 12 weeks of modeling, respectively.\u003cstrong\u003e(K,L)\u003c/strong\u003eChanges in the concentrations of IL-17A and IFN-γ in the peripheral blood of mice across each group of smokers were assessed during Week 6 and Week 12. \u003cstrong\u003e(M,N,O,P,Q,R)\u003c/strong\u003eQPCR analysis was conducted to compare the effects of each group on the gene expression of various inflammatory cytokines.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8280945/v1/0ecea88aadf6c79a384d9174.jpg"},{"id":100361449,"identity":"93fd4f31-ee48-441f-9cf0-dda5f02aedf6","added_by":"auto","created_at":"2026-01-16 07:45:11","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1017213,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePathological changes.(A)\u003c/strong\u003eProgression of emphysema in mice.\u003cstrong\u003e(B)\u003c/strong\u003eThe manifestations of bronchial, alveolar and mucosal destruction and inflammatory cell infiltration in each group of mice.\u003cstrong\u003e(C,D)\u003c/strong\u003eThe mean linear intercept (MLI) of lung tissue serves as a traditional indicator of lung morphology. It is determined by the intersection of a random straight line with the alveolar wall, thereby reflecting both the size and structural integrity of the alveoli.The MLI of mice in each group was assessed at 6 weeks and 12 weeks.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8280945/v1/c04533c4fe82ca659bf81d2a.jpg"},{"id":100360780,"identity":"f9e5d121-84ab-4f1e-8b5b-fbc8adc78c5d","added_by":"auto","created_at":"2026-01-16 07:43:43","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1077776,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMasson.(A,B,C,D,E,F,L)\u003c/strong\u003eRelative collagen content of mice in each group.\u003cstrong\u003e(G,H,I,J,K)\u003c/strong\u003eThe thickness of alveolar walls and the area of lumens in each group of mice.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8280945/v1/c1d17fa4d1a8766a662c2abc.jpg"},{"id":100361391,"identity":"7dd9909b-0723-4819-80bf-d9dcb71041b2","added_by":"auto","created_at":"2026-01-16 07:45:04","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":611121,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eThe MFI of CD96 on the surface of NK cells was measured by flow cytometry on the 3rd and 10th days following the injection of the anti-CD96 monoclonal antibody.\u003cstrong\u003e(B,C)\u003c/strong\u003eAfter 12 weeks of smoke exposure, the MFI of CD96 and %IFN-γ⁺NK cells on the surface of NK cells in each group was compared.\u003cstrong\u003e(D,E)\u003c/strong\u003eThe changes of CD155 signals in each group after 12 weeks of smoke exposure were evaluated by relative fluorescence intensity.\u003cstrong\u003e(F,G)\u003c/strong\u003eFlow cytometry was employed to assess the proportion of Th17 cells within CD4\u003csup\u003e+\u003c/sup\u003e T cells and to measure the concentration of IL-17 in the peripheral blood of both healthy individuals and patients with chronic obstructive pulmonary disease.\u003cstrong\u003e(H)\u003c/strong\u003eThe expression of phosphatase SHIP1 on NK cells in patients with chronic obstructive pulmonary disease (COPD) is upregulated.\u003cstrong\u003e(I,J,K)\u003c/strong\u003eThe association between the proportion of CD96\u003csup\u003e+\u003c/sup\u003e NK cells in the peripheral blood of patients with chronic obstructive pulmonary disease, the duration of hospital stay, and the COPD Assessment Test (CAT) score.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8280945/v1/fa65146f03025ca891c58462.jpg"},{"id":101943265,"identity":"b6939fad-667d-4fa8-b44c-2018cc118286","added_by":"auto","created_at":"2026-02-05 09:41:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5088569,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8280945/v1/c6eb603a-88bf-4f80-a044-c8bd7360faf0.pdf"},{"id":100007195,"identity":"80ee3bfd-b266-4063-af87-894dbe80baaf","added_by":"auto","created_at":"2026-01-12 05:49:59","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":138417,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8280945/v1/eac42aa9f965ba92797eab92.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Role of NK surface inhibitory receptor CD96 in chronic obstructive pulmonary disease and the effect of targeted CD96 intervention","fulltext":[{"header":"Background","content":"\u003cp\u003eChronic obstructive pulmonary disease (COPD) is a preventable and treatable chronic airway disease marked by persistent airflow limitation and chronic respiratory symptoms, making it the third leading cause of death globally \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e][\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Key factors influencing the development of COPD include age, gender, smoking history, exposure to occupational and environmental particulate matter, early pulmonary dysplasia, and genetic predisposition \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e][\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e][\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e][\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Among these, immune dysregulation, particularly the synergistic imbalance between intrinsic and acquired immunity, plays a crucial role in the persistence of chronic inflammation and structural damage associated with COPD \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e][\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Label-free proteomics has identified that key molecules involved in Th17 cell differentiation and the IL-17-mediated signaling pathway are significantly upregulated in the plasma of COPD patients\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Additionally, the number of natural killer (NK) cells in the peripheral blood of COPD patients is significantly lower compared to that of healthy individuals. Although total IFN-γ levels are elevated \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e in patients with COPD, the ability of NK cells to secrete IFN-γ is significantly diminished.\u003c/p\u003e \u003cp\u003eTh17 cells have been shown to recruit neutrophils and stimulate airway epithelial cells to secrete chemokines and matrix metalloproteinases via the release of IL-17. This process contributes to the persistence of inflammation and airway remodeling\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Elevated levels of IL-17 correlate closely with the frequency of acute exacerbations and the decline in lung function among COPD patients\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. These findings suggest that the Th17/IL-17 axis may serve as a key driver of the sustained inflammatory response in COPD.\u003c/p\u003e \u003cp\u003eImmune cell communication analysis was conducted on mice exposed to smoke, comparing the effects of various conditions on immune cell interaction pathways. For instance, AIR may activate the PECAM1-related pathway, while CS may activate the NECAB2-related pathway. This analysis utilized both relative proportions and absolute intensities. Results indicated that the information flow of CD96 was significantly greater in the CS condition compared to the AIR condition under the relative normalization dimension, suggesting that the CS condition markedly enhanced CD96 information transfer\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. A flow assay of peripheral blood from patients with chronic obstructive pulmonary disease confirmed a significant upregulation of NK cell CD96 expression. CD96 is a type I transmembrane glycoprotein primarily expressed on the surface of T cells and NK cells, which competitively binds to the shared ligand CD155 with the costimulatory receptor CD226, exhibiting relatively high binding affinity\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe group utilized C57 mice to model chronic obstructive pulmonary disease. Following the modeling, alveolar lavage was conducted, and the collected lavage fluid underwent flow cytometry analysis. This analysis revealed a significant decrease in the proportion of NK cells, a notable increase in the number of CD96\u003csup\u003e+\u003c/sup\u003eNK cells, and a substantial reduction in the number of IFN-γ\u003csup\u003e+\u003c/sup\u003eNK cells\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Existing literature indicates that a reduction in IFN-γ can enhance Th17 cell function, which subsequently increases IL-17 secretion and may represent a critical mechanism in the development of lentigo \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e][\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePeripheral blood was collected from healthy individuals and patients with chronic obstructive pulmonary disease (COPD) to assess the proportion of Th17 cells among CD4\u003csup\u003e+\u003c/sup\u003e T cells using flow cytometry. The results indicated that both the proportion of Th17 cells and the levels of IL-17 were significantly elevated in the peripheral blood of COPD patients\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. These findings suggest that the Th17/IL-17 axis may play a crucial role in driving the sustained inflammatory response associated with COPD. Furthermore, it is speculated that increased CD96 expression may serve as an upstream signal contributing to the Th17/IL-17-mediated development of chronic obstructive pulmonary disease.\u003c/p\u003e \u003cp\u003eFurther studies revealed that the expression of the NK cell phosphatase SHIP1 was significantly upregulated in patients with chronic obstructive pulmonary disease (COPD). Additionally, COPD-modeled mice with CD96 inhibition exhibited improved lung function compared to wild-type (WT) mice\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn summary, the research indicated that the CD96-SHIP1 signaling axis was upregulated in the NK cells of COPD patients. However, the potential of CD96 as a therapeutic target for COPD remains uncertain, and the role of targeted CD96 molecules in regulating immune cells and their downstream SHIP1 in the development of COPD is still not well understood. Consequently, this study integrated clinical samples with mouse models of COPD treated with CD96 monoclonal antibodies, focusing on the regulatory role of the CD96-SHIP1 signaling axis. The objective was to elucidate the mechanisms of disease progression mediated by CD96 and to evaluate the potential of targeted interventions, thereby providing a new theoretical foundation for the precise diagnosis and treatment of COPD.\u003c/p\u003e"},{"header":"Methods","content":"\n\u003ch3\u003e1 Animal Model and CS Exposure\u003c/h3\u003e\n\u003cp\u003eSix- to-eight-week-old SPF-grade C57BL/6 WT male mice were assigned to three groups: a healthy control group (WT), a smoke-exposed group (CS), and a smoke-exposed group treated with InVivoMAb anti-mouse CD96 (Bio X Cell, Cat# BE0337-50MG). The control group was exposed to filtered indoor air, while the anti-CD96 group received intraperitoneal injections of the CD96 inhibitor at a dose of 200 \u0026micro;g every 5 days. Both the CS and anti-CD96 groups were subjected to mainstream smoke from filtered cigarettes using a whole-body Exposure System. Each exposure consisted of 12 cigarettes, administered twice daily for 60 minutes per session, 5 days per week over a duration of 12 weeks. All experimental procedures received approval from the Institutional Animal Care and Use Committee of Anhui Medical University (Approval number: LLSC20210886).\u003c/p\u003e\n\u003ch3\u003e2 Single-cell RNA sequencing and analysis\u003c/h3\u003e\n\u003cp\u003eLung CD45⁺ immune cells were isolated and analyzed through single-cell RNA sequencing utilizing the 10x Genomics platform. Standard preprocessing, clustering, trajectory inference, regulator mapping, enrichment analysis, and intercellular communication modeling were conducted following established methodologies.\u003c/p\u003e\n\u003ch3\u003e3 Lung function\u003c/h3\u003e\n\u003cp\u003eAnesthesia was induced in the mice through an intraperitoneal injection of 100 mg/kg sodium pentobarbital. Following the administration of anesthesia, pulmonary function was assessed by measuring several parameters, including respiratory rate (f), tidal volume (TV), total lung capacity (TLC), forced vital capacity (FVC), residual volume (RV), functional residual capacity (FRC), FEV0.20/FVC%, dynamic lung compliance (Cdyn), and airway resistance (RI). Each parameter was measured five times per animal.\u003c/p\u003e\n\u003ch3\u003e4 Pathological image analysis\u003c/h3\u003e\n\u003cp\u003ePathological sections of mice were randomly fixed at low magnification. Crosshairs were drawn at the center of each field of view, and the total length of the crosshairs (L) was measured. The number of alveolar septa intersecting the crosshairs (NS) was recorded, and emphysema analysis was conducted using the mean lining interval of lung tissue (MLI\u0026thinsp;=\u0026thinsp;L/NS). Following Masson staining, the relative collagen content of the airways was quantitatively analyzed using ImageJ, calculated as follows: [Relative collagen content = (collagen fiber positive area/total airway wall area) \u0026times; 100%]. Image-Pro Plus was employed to determine the airway lumen area from the Masson-stained images. The selected airway cross-sections adhered to the following criteria: ① intact airway structures without evident folding or fracture; ② approximately circular or oval cross-sections (L/D ratio\u0026thinsp;\u0026le;\u0026thinsp;1.5); and ③ no significant secretion or cellular obstruction within the lumen.\u003c/p\u003e\n\u003ch3\u003e5 Flow Cytometry/financal capacity model\u003c/h3\u003e\n\u003cp\u003eTo prepare 1 \u0026times; 10^6 cells, incubate them in serum at 4℃ for 30 minutes. Subsequently, add a specific fluorescent antibody while maintaining the temperature at 4℃ to prevent light exposure during the labeling of NK cell extracellular molecules for an additional 30 minutes. Following this, employ a cell fixative and permeabilization solution to fix and permeabilize the cell membrane. Afterward, add a specific antibody to label the intracellular molecules of the NK cells. Finally, adjust the cell density to the appropriate level for flow cytometry analysis.\u003c/p\u003e\n\u003ch3\u003e6 Real-time Quantitative PCR Detecting System(Q-PCR)\u003c/h3\u003e\n\u003cp\u003eIsolated NK cells were collected, and cellular RNA was extracted using Trizol homogenate for concentration and quality assessment. The RNA was then reverse transcribed to cDNA, followed by the construction of primers. PCR amplification was conducted with GAPDH as an internal reference to quantify the gene expression of airway inflammatory factors, including IL-33, IL-1β, IL-6, TNF, IL-10, and TGF-β, using fluorescence quantitative PCR.\u003c/p\u003e\n\u003ch3\u003e7 Enzyme-Linked Immunosorbent Assay (ELISA)\u003c/h3\u003e\n\u003cp\u003eFollowing the instructions provided in the ELISA kit, plasma or alveolar lavage fluid samples were centrifuged, and the supernatant was extracted along with other necessary pre-treatments. The ELISA instrument was calibrated to a wavelength of 450 nm, and detection standards were employed to construct standard curves for measuring the concentrations of IFN-γ and IL-17.\u003c/p\u003e\n\u003ch3\u003e8 Immunofluorescence technique\u003c/h3\u003e\n\u003cp\u003eFor the immunofluorescence technique, frozen sections of mouse lung tissue were placed in a wet box and incubated with rat serum for 30 minutes. Subsequently, the sections were incubated overnight at 4℃ with an anti-CD155 primary antibody, followed by a 1-hour incubation at room temperature with a fluorescent secondary antibody, protected from light. After staining with DAPI, the expression and localization of CD155 in the lung tissues were observed under a confocal laser microscope after sealing the slices.\u003c/p\u003e\n\u003ch3\u003e9 Clinical indicators\u003c/h3\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e9.1 Research objects\u003c/h2\u003e \u003cp\u003e A total of 44 patients with chronic obstructive pulmonary disease (COPD) participated in this study, all of whom were recruited from the Second Affiliated Hospital of Anhui Medical University. Each enrolled patient met the diagnostic criteria for COPD established by the World Health Organization (WHO) and the Global Initiative for Chronic Obstructive Lung Disease (GOLD). Exclusion criteria encompassed other chronic respiratory diseases, autoimmune disorders, malignant tumors, severe cardiovascular conditions, and any diseases that could potentially impair immune function. Additionally, patients who had received corticosteroids or other immunosuppressive medications within the preceding month were excluded. All participants provided written informed consent. The study protocol was approved by the Ethics Committee of the Second Affiliated Hospital of Anhui Medical University (approval no. YX2025191).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e9.2 Specimen collection and processing\u003c/h2\u003e \u003cp\u003eWithin 24 hours of admission, medical staff collected 2 ml of fasting peripheral venous blood from the patients and preserved it in EDTA anticoagulation tubes. All blood samples were sent to the laboratory for processing within 2 hours of collection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e9.3 Clinical data collection\u003c/h2\u003e \u003cp\u003eBaseline clinical data, including days of hospitalization and COPD Assessment Test (CAT) scores, were collected from all patients. The number of acute exacerbations occurring within one year of discharge was recorded through telephone follow-up after patients left the hospital. AECOPD definition: events characterized by worsening of dyspnea and/or cough and sputum within 14 days in patients with COPD, which may be accompanied by shortness of breath and/or tachycardia, are usually associated with an exacerbation of local or systemic inflammatory responses to respiratory tract infections, air pollution, or other causes\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Based on the frequency of acute exacerbations, patients were classified into two groups: those with \u0026le;\u0026thinsp;2 exacerbations and those with \u0026gt;\u0026thinsp;2 exacerbations.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e10 Statistical Analysis\u003c/h3\u003e\n\u003cp\u003eAll experimental data were derived from a minimum of three independent repeated tests. Normally distributed data are presented as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, while non-normally distributed data are expressed as Median and Interquartile Range (IQR). Data organization and analysis were performed using software such as SPSS 22.0, GraphPad Prism 8.0, and FlowJo 10.0. For parametric tests of measurement data, an independent samples t-test was employed for comparisons between two groups, and one-way ANOVA was utilized for comparisons among multiple groups. Non-parametric tests were applied to hierarchical data, including normality tests and variance chi-square tests for measurement data. For data that did not meet the criteria for normal distribution, non-parametric tests such as the Mann-Whitney U test and Kruskal-Wallis test were used. A p-value of less than 0.05 was considered indicative of a statistically significant difference between the datasets.\u003c/p\u003e"},{"header":"Results","content":"\n\u003ch3\u003e1 Changes in immune cell signaling pathways under chronic obstructive pulmonary conditions\u003c/h3\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e1.1 Immune cell interactions are more active in chronic lung conditions\u003c/h2\u003e \u003cp\u003eComparative analysis of immune cell communication between CS and AIR conditions through the two dimensions of quantity difference and intensity difference(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), and found that the column values of CS group were higher in C and E graphs comparing with AIR group(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), indicating that the number of differential interactions between immune cells in CS group was significantly increased, which mainly involved multiple cell pairs such as NK-T cells, ILC-monocytes, macrophage-neutrophils, etc., and the number of signaling connections between cells was more frequent. The number of connections between cells was more frequent; the higher relative value of CS group in D and F graphs indicated that the relative strength of immune cell interactions in CS group was generally higher than that in AIR group, especially the signaling strength of NK cells and other immune cells was elevated significantly, suggesting that the signaling strength of inter-cellular communication was stronger(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Meanwhile, the analysis of immune cell communication in mice under smoked condition revealed that CS condition significantly enhanced the messaging of CD96,speculating that the activity of immune cell interaction was related to the CS-related airway inflammation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e1.2 Analysis of CD96 pathway alterations in chronic lung mice using single-cell sequencing\u003c/h2\u003e \u003cp\u003eVisualization of single-cell sequencing and information flow analysis presented the abnormal activation of CD96 pathway in mice with chronic obstructive pulmonary disease (COPD). Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA shows that the clusters of cells with high expression of CD96 were more concentrated in the CS group, suggesting that the expression profiles of CD96 in immune cells were abnormally enriched; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB clearly shows that the information flow value of CD96 in the CS group was significantly higher than that in the AIR group, which is a direct evidence of the fact that the signaling of the CD96 pathway was significantly enhanced under the conditions of chronic obstructive pulmonary disease (COPD) directly proved that the signaling efficiency of CD96 pathway was significantly enhanced under the condition of chronic obstructive pulmonary disease. The above results indicated that the expression of CD96 was not randomly distributed, but highly enriched in specific immune cell subpopulations (e.g.NK cells, T cells) in the CS group, and the signaling activity of the CD96 pathway was significantly elevated in chronic obstructive pulmonary disease (COPD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e2 Cigarette smoke exposure consistently impairs lung function and worsens lung tissue destruction, which is partially mitigated by anti-CD96 antibodies.\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cigarette Smoke Persistently Impairs Lung Function, and Anti-CD96 Antibodies Partially Alleviate Smoke-Induced Lung Function Decline\u003c/h2\u003e \u003cp\u003eIn comparison to the normal control group, the slope of the rising branch in the lower section of the flow-time curve was reduced and exhibited a staccato pattern in the CS group. Additionally, the expiratory phase was prolonged in this group. Conversely, the slope of the rising branch in the lower part of the flow-time curve increased, and the expiratory phase shortened in the anti-CD96 group relative to the CS group(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The FEV0.20/FVC% in the CS group was significantly lower than that of the healthy control rats, while the anti-CD96 group demonstrated an increase in FEV0.20/FVC% compared to the CS group(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). The administration of the anti-CD96 monoclonal antibody mitigated the decline in lung function induced by cigarette smoke in mice after 6 weeks of exposure, showing a significant improvement over the smoking group alone, although it did not fully restore lung function to the levels observed in healthy controls. After 12 weeks of smoking, lung function in mice significantly declined, indicating that prolonged exposure to cigarette smoke continued to adversely affect lung function. The beneficial effects of the anti-CD96 monoclonal antibody persisted at the 12-week mark, but the improvement was notably less pronounced than that observed at 6 weeks, potentially due to the effects of long-term smoke exposure or the timing of antibody administration. This study suggests that cigarette smoke consistently contributes to the decline in lung function, and the anti-CD96 monoclonal antibody may partially alleviate this decline induced by cigarette smoke.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e2.2 Cigarette smoke persistently damages lung tissue structure in mice, and anti-CD96 antibody partially attenuates lung tissue destruction\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePathological analysis revealed that the bronchial and alveolar structures in the wild-type (WT) group appeared intact, with no evident mucosal destruction or inflammatory cell infiltration(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In contrast, the cigarette smoke (CS) group exhibited disorganized lung architecture characterized by significant destruction, fractured alveolar walls, and deformed, necrotic bronchial mucosal epithelial cells. Additionally, cilia were uneven, shortened, inverted, or even detached, and the walls were thickened, accompanied by extensive inflammatory cell infiltration. The lumens of the airways were narrowed, twisted, or dilated. Prolonged fumigation time exacerbated the destruction of lung tissues(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Notably, the aforementioned lesions were significantly mitigated in the anti-CD96 group(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eThe mean linear intercept (MLI), which indicates the extent of alveolar destruction, revealed that the MLI in the CS group was significantly higher than that in the WT group at both 6 and 12 weeks of cigarette smoking. Furthermore, the MLI at 12 weeks was greater than that at 6 weeks, suggesting that prolonged exposure to cigarette smoke exacerbates alveolar structural damage. In contrast, the MLI in the anti-CD96 group was significantly lower than that in the CS group and comparable to that in the WT group at both time points(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). This finding indicates that the anti-CD96 antibody can consistently mitigate the alveolar structural damage induced by cigarette smoke.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCollagen deposition in lung tissue was evaluated through relative collagen content. The CS group exhibited significantly higher levels than the WT group, indicating that CS induced collagen deposition and fibrosis in lung tissue. Additionally, collagen content in the anti-CD96 group was markedly lower than that in the CS group(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI,\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL), suggesting that the blockade of CD96 may mitigate collagen deposition and alleviate lung tissue fibrosis in mice exposed to smoke.\u003c/p\u003e \u003cp\u003eSmall airway remodeling serves as an indicator of the extent of airway hyperplasia and thickening\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. The small airway wall thickness was significantly greater in the CS group compared to the WT group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), accompanied by a reduction in lumen area. In contrast, the wall thickness in the anti-CD96 group was significantly lower than that in the CS group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), with the lumen area significantly increased and approaching that of the WT group(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-K). The difference between the WT and anti-CD96 groups was not statistically significant. These findings indicate that exposure to cigarette smoke leads to small airway wall thickening and lumen narrowing, while the anti-CD96 antibody appears to mitigate these pathological changes by modulating the CD96 pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCollectively, exposure to CS can result in alveolar destruction, collagen deposition, thickening of small airway walls, and narrowing of the lumen. In contrast, the anti-CD96 antibody mitigated these pathological changes to some degree, indicating that CD96 may serve as a potential therapeutic target for chronic airway and lung tissue injury.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e3 CS stimulation activates Th1/Th17 and multiple inflammatory factors, which is partially reversed by anti-CD96 antibody\u003c/h3\u003e\n\u003cp\u003eIL-17A serves as a critical cytokine for Th17 cells, which play a role in inflammation and autoimmunity\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e, while IFN-γ is a pivotal cytokine for Th1 cells, mediating antiviral and antitumor immunity\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. These cytokines represent the activation of the Th17 and Th1 pathways, respectively. At 6 weeks of smoking, the IL-17A level in the CS group was significantly elevated compared to the WT group, whereas the level in the anti-CD96 group was significantly reduced relative to the CS group. This difference persisted at 12 weeks of smoking, indicating that blocking CD96 could continuously inhibit the CS-induced increase in IL-17A(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK,Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Additionally, the IFN-γ level in the CS group was higher than that in the WT group at both 6 and 12 weeks, and the level in the anti-CD96 group was significantly lower than that in the CS group at 12 weeks(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL). This finding suggests that anti-CD96 had a notable impact on IFN-γ levels, although its inhibitory effect on IFN-γ appeared to lag relative to that on IL-17A. Overall, these results indicate that CS stimulation can induce prolonged activation of the Th1 (IFN-γ) and Th17 (IL-17A) pathways over 6 and 12 weeks. Furthermore, the anti-CD96 antibody can rapidly inhibit the elevation of IL-17A while exhibiting a delayed inhibitory effect on IFN-γ elevation, suggesting that CD96 plays a regulatory role in the CS-induced immune response and that there is a temporal difference in the effects of CD96 on the Th1 and Th17 pathways.\u003c/p\u003e \u003cp\u003eGene expression of inflammation-related factors in lung tissues exhibited comparable alterations. For instance, treatment with CS resulted in the upregulation of IL-33 gene expression relative to the healthy control group. Inhibition of CD96 partially reversed the elevated IL-33 expression, with levels in the anti-CD96 group returning close to those of the wild-type (WT) group when compared to the CS group(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eR). This finding suggests that CD96 molecules may play a role in regulating IL-33-associated immune pathways. The effects of CS status on IL-33 gene expression and the regulatory influence of CD96 antibody intervention were thus elucidated. Other factors, including pro-inflammatory cytokines such as IL-1β, IL-6, and TNF, as well as anti-inflammatory modulators like IL-10 and TGF-β, demonstrated similar trends. Notably, IL-6 expression in the CS group was significantly higher than that in the WT group; however, no significant difference was observed between the WT group and the anti-CD96 group, indicating that the CD96 inhibitor significantly regulated IL-6 levels. Furthermore, while TGF-β expression was markedly elevated in the CS group compared to the control group and reduced in the anti-CD96 group relative to the CS group, TGF-β levels remained extremely low in the WT group(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eM-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eQ). This suggests that TGF-β may exert a pro-fibrotic and immunosuppressive role in the disease.\u003c/p\u003e \u003cp\u003eIn summary, CS can persistently activate Th1/Th17 pathways and various inflammatory factor pathways. In contrast, the anti-CD96 antibody exhibits both early and late inhibitory effects on IL-17A and IFN-γ, while also partially reversing the upregulation of inflammatory factors. These findings suggest that CD96 plays a role in the regulation of CS-induced inflammation and may serve as a potential therapeutic target.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSequences of the primers for real-time PCR\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003e4 CS stimulation affects CD96 expression and thus NK cell immunoreactivity\u003c/h3\u003e\n\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Upregulation of CD96 expression is accompanied by diminished IFN-γ secretion by NK cells\u003c/h2\u003e \u003cp\u003eTo establish the dosing regimen, the mean fluorescence intensity (MFI) of CD96 on the surface of NK cells was assessed via flow cytometry on days 3 and 10 following the administration of the anti-CD96 monoclonal antibody. Results indicated that the CD96 MFI in the anti-CD96 group was significantly lower than that in the wild-type (WT) group at day 3 post-treatment, suggesting a substantial reduction in CD96 expression on NK cells due to the inhibitor intervention. However, after 10 days of treatment, no significant difference in CD96 MFI was observed between the anti-CD96 and WT groups(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), indicating that CD96 expression on NK cells had converged following 10 days of intervention with a 200 \u0026micro;g single dose of the CD96 inhibitor. Consequently, an administration interval of 5 days for the CD96 inhibitor was selected to maintain continuous inhibition of CD96 expression in the anti-CD96 group.\u003c/p\u003e \u003cp\u003eAfter 12 weeks of smoke exposure, CD96 expression on the surface of NK cells was significantly elevated in the CS group compared to the WT group(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), indicating that CS treatment upregulated CD96 expression. The functional capacity of NK cells to secrete IFN-γ was evaluated by measuring the percentage of IFN-γ-producing NK cells (%IFN-γ⁺NK cells). The WT group exhibited the lowest percentage, while the CS group showed a slight increase, and the anti-CD96 group had the highest percentage. Although the differences between the WT and CS groups, as well as between the CS and anti-CD96 groups, were not statistically significant, the difference between the WT and anti-CD96 groups was significant(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These findings suggest that blocking the CD96 pathway may enhance the IFN-γ secretion capacity of NK cells in the context of long-term smoking.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e4.2 CD96 may be involved in mediating CS intervention-induced CD155 signaling activation\u003c/h2\u003e \u003cp\u003eChanges in CD155 signaling were evaluated through relative fluorescence intensity analysis. After 12 weeks of smoking, the relative fluorescence intensity of lung tissue in the CS group was significantly higher than that in the WT group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating that CS stimulation activated the CD155 signaling pathway. Furthermore, the relative fluorescence intensity of CD155 in the anti-CD96 group was significantly lower than that in both the CS group and the WT group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. WT, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. CS)(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). This finding suggests that blocking the CD96 pathway partially attenuated the CS-induced CD155 signaling, and the trends in CD96 signaling and CD155 signaling intensity showed some concordance. It is further speculated that CD96 may facilitate immune escape by sustaining the activity of the CD155 signaling pathway. These results imply that CD96 may play a role in the activation of CS-induced CD155 signaling, and the anti-CD96 antibody may enhance the local immune microenvironment by modulating this pathway. However, the direct molecular interactions between the two and the downstream mechanisms warrant further investigation.\u003c/p\u003e \u003cp\u003e \u003cb\u003e5 Peripheral blood CD96\u003c/b\u003e \u003csup\u003e \u003cb\u003e+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eNK cell ratio correlates with disease severity and acute exacerbation risk in patients with chronic obstructive pulmonary disease\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThis study included a total of 44 patients diagnosed with chronic obstructive pulmonary disease (COPD). The ratio of CD96\u003csup\u003e+\u003c/sup\u003eNK cells to total NK cells in peripheral blood was assessed using flow cytometry(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). Correlation analysis revealed a significant positive correlation between this ratio and the duration of hospitalization (r\u0026thinsp;=\u0026thinsp;0.440, p\u0026thinsp;=\u0026thinsp;0.003), as well as with CAT scores (r\u0026thinsp;=\u0026thinsp;0.380, p\u0026thinsp;=\u0026thinsp;0.011)(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK). These findings indicate that a higher ratio of CD96\u003csup\u003e+\u003c/sup\u003eNK cells is associated with longer hospital stays and greater symptom burden. To further explore the relationship between the proportion of CD96\u003csup\u003e+\u003c/sup\u003eNK cells and the frequency of acute exacerbations of COPD, patients were categorized into two groups: those with \u0026le;\u0026thinsp;2 exacerbations and those with \u0026gt;\u0026thinsp;2 exacerbations within one year post-discharge. Statistical analysis demonstrated that the proportion of NK cells represented by CD96\u003csup\u003e+\u003c/sup\u003eNK cells in peripheral blood was significantly higher in the group with \u0026gt;\u0026thinsp;2 exacerbations compared to the \u0026le;\u0026thinsp;2 exacerbations group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This suggests that a high ratio of CD96\u003csup\u003e+\u003c/sup\u003eNK cells may serve as a predictor for an increased risk of future acute exacerbations.\u003c/p\u003e \u003cp\u003eIn this study, we observed that the inflammation-associated factor IL-17 was elevated in patients with chronic obstructive pulmonary disease (COPD). Additionally, Th17 differentiation and the IL-17 signaling pathway were upregulated(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG), while CD96 and its downstream target SHIP1 were significantly increased in natural killer (NK) cells (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001)(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). These findings suggest that CD96, an inhibitory receptor on NK cells, was markedly upregulated, and that Th17/IL-17 functionality was enhanced in COPD patients, thereby contributing to the progression of the disease. To further investigate the mechanisms underlying this immune disorder, we systematically validated these results in clinical COPD patients and a mouse model of COPD. Lung function assessments revealed that mice in the cigarette smoke (CS) exposure group exhibited significantly worse outcomes compared to the control group, with declines beginning at six weeks. Furthermore, lung function in the anti-CD96 treatment group showed improvement relative to the CS group, although this effect was less pronounced at twelve weeks than at six weeks. Histological analysis of the CS group at six weeks revealed alveolar wall destruction, inflammatory cell infiltration, and thickening of the small airway walls, with the mean linear intercept (MLI) being greater than that of the control group.MLI was significantly higher in the control group and worsened at 12 weeks. In contrast, alveolar disruption was reduced, and inflammatory infiltration decreased in the anti-CD96 group, where MLI was lower than in the CS group and approached levels observed in the WT group. Following smoking exposure, CD96 expression in NK cells was up-regulated, and IL-17A levels were significantly elevated in CS group mice at both 6 and 12 weeks. However, CD96 expression was suppressed in the anti-CD96 group, resulting in a significant reduction of IL-17A at 6 weeks, with continued efficacy observed at 12 weeks. The expression of pro-inflammatory factors, including IL-1β, IL-6, and TNF, was significantly up-regulated in the CS group, while the expression of these genes was partially reversed in the anti-CD96 group, approaching levels seen in the WT group. CD155 signaling was activated in the CS group, whereas signaling in the anti-CD96 group was significantly lower than that in the model group, indicating that CD96 may play a role in mediating the CD155 signaling pathway. Furthermore, a high ratio of CD96\u0026thinsp;+\u0026thinsp;NK cells may predict an increased risk of acute exacerbation in patients over the following year. This article confirms the involvement of CD96 in the development of chronic obstructive pulmonary disease and suggests that inhibition of CD96 may serve as a potential therapeutic target for this condition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we established that the CD96-SHIP1 signaling axis serves as a critical regulatory node in the pathogenesis of COPD. We also identified CD96 as a potential therapeutic target and elucidated the mechanistic link between abnormal NK cell function and Th1/Th17 immune imbalance. Our core findings indicate that the expression of CD96 and SHIP1 on the surface of NK cells was upregulated. Furthermore, CD96 exerted a regulatory effect on IFN-γ and IL-17A, thereby confirming the therapeutic potential of targeting CD96. This work provides a new theoretical foundation and practical direction for the immunotherapy of COPD.\u003c/p\u003e \u003cp\u003eThis paper presents single-cell sequencing findings that reveal enhanced communication among immune cells under chronic obstructive lung conditions. The study employs dual visualization to assess both the dimensions of cellular interactions and the CD96 pathway. Results indicate that the CD96 pathway undergoes significant alterations in expression enrichment and signaling enhancement in immune cells derived from chronic obstructive lung mice. These findings provide direct single-cell-level evidence of the CD96 pathway's role as a critical regulatory mechanism in slow-onset lung disease.\u003c/p\u003e \u003cp\u003eThe conclusion that the upregulation of CD96 on the surface of NK cells facilitates the progression of COPD is substantiated by both clinical and experimental evidence. Clinical data indicated a significant association between the CD96\u003csup\u003e+\u003c/sup\u003eNK cell ratio and prolonged hospitalization, elevated CAT scores, and an increased risk of acute exacerbation in patients. These findings suggest that CD96\u003csup\u003e+\u003c/sup\u003eNK cells may function as a prognostic marker for COPD.\u003c/p\u003e \u003cp\u003eOur experimental data indicated that the overall level of IFN-γ was elevated under CS conditions, while inhibition of CD96 resulted in a decrease in the overall level of IFN-γ, despite promoting IFN-γ secretion by NK cells. Thus, we concluded that the overexpression of CD96 impairs NK cell function by inhibiting IFN-γ secretion, although blocking CD96 partially restored the secretion of IFN-γ by NK cells. Previous studies have demonstrated that IFN-γ in COPD acts as a disease-promoting factor by activating macrophages, inducing protease and nitric oxide production, and contributing to the development of emphysema and chronic inflammation. Conversely, IFN-γ serves as a crucial effector molecule for NK cells and the Th1 response, playing a regulatory role in anti-infection processes and the inhibition of Th17 responses. This duality suggests that IFN-γ exhibits both beneficial and detrimental characteristics in the context of COPD. Our results indicated that exposure to CS elevated the overall level of IFN-γ, whereas the administration of anti-CD96 antibody diminished the global inflammatory burden. Concurrently, within NK cell subsets, CD96 blockade did not further impair, and may even enhance, their capacity for IFN-γ production. These findings corroborate existing studies and suggest that targeting CD96 may achieve a balance between reducing inflammation and preserving essential effector functions.\u003c/p\u003e \u003cp\u003eThe study identified an upregulation in the expression of the phosphatase SHIP1, which is known to antagonize NK cell activation. This finding suggests the potential existence of a CD96-SHIP1 cascade pathway that may inhibit NK cell-mediated immune surveillance functions. Importantly, this signaling axis interacts with the Th17/IL-17 pathway: blockade of CD96 rapidly diminished the elevation of IL-17A induced by cigarette smoke, while the inhibitory effect on IFN-γ exhibited a delay. This temporal discrepancy indicates a differential regulation of Th1 and Th17 responses by CD96. The underlying mechanism may relate to the 'double-edged sword' role of NK cell-derived IFN-γ in chronic obstructive pulmonary disease (COPD). Specifically, IFN-γ contributes to emphysema and chronic inflammation through macrophage activation (pro-disease role), while also exerting immunomodulatory effects by inhibiting the Th17 response (protective role).\u003c/p\u003e \u003cp\u003eThe primary insight of this study is that targeting CD96 effectively balances the reduction of systemic inflammation with the preservation of essential immune effector functions. While anti-CD96 antibodies decrease systemic IFN-γ levels, thereby mitigating the progression of chronic inflammation, they simultaneously enhance IFN-γ secretion specifically from NK cells. This dual effect directly addresses the 'double-edged sword' dilemma associated with IFN-γ in COPD. Such selective modulation carries significant implications; abnormal NK cell function contributes to impaired pathogen clearance and persistent inflammation in COPD patients, conditions that may be improved through targeted CD96 blockade. Furthermore, CD96 blockade reduced CS-induced activation of CD155 signaling, reinforcing the hypothesis that CD96 disrupts immune homeostasis by competing with CD226 for CD155 binding. Collectively, these findings unveil a novel immunopathological mechanism: activation of the CD96-SHIP1 axis may lead to alterations in NK cell function, enhance Th17-mediated inflammatory responses, and ultimately exacerbate lung tissue injury.\u003c/p\u003e \u003cp\u003eThe potential of CD96-targeted therapy was underscored by the findings of preclinical studies, which demonstrated that anti-CD96 antibodies enhanced lung function, reduced alveolar destruction and airway remodeling, and partially restored the expression of pro-inflammatory factors (IL-1β, IL-6, TNF) and collagen deposition. Notably, these therapeutic effects persisted even after 12 weeks of exposure to cigarette smoke, indicating the clinical translational potential for long-term management of COPD. Furthermore, the correlation between the percentage of CD96\u003csup\u003e+\u003c/sup\u003e NK cells and disease severity reinforces the feasibility of utilizing CD96 as both a predictive biomarker and a therapeutic target for COPD, addressing an unmet need in the realm of precision medicine for this condition.\u003c/p\u003e \u003cp\u003eThis paper presents several limitations: first, the elucidation of the direct role of SHIP1 downstream of CD96 remains incomplete; second, there is insufficient data regarding CD96 expression in other immune cell subsets. While this study concentrated on NK cells, future research should investigate whether CD96 regulates SHIP1 through direct phosphorylation or via intermediate signaling molecules. Furthermore, the expression of CD96 on the surface of key effector T cells and neutrophils in COPD warrants further investigation to clarify the specific roles of various cell types in CD96-mediated pathology. Additionally, the observed difference in the efficacy of the anti-CD96 antibody at 6 weeks compared to 12 weeks of corticosteroid exposure may be attributed to the cumulative effects of lung injury or alterations in antibody pharmacokinetics; this issue could be addressed in future studies by optimizing the dosing regimen.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study collectively confirms that the CD96-SHIP1 axis serves as a crucial driver of abnormal NK cell function and Th1/Th17 imbalance in COPD. The findings not only validate the potential of CD96 as an intervenable target for reducing inflammation and preserving lung function, but also identify CD96\u003csup\u003e+\u003c/sup\u003eNK cells as a promising prognostic marker. By correlating cellular and molecular mechanisms with clinical outcomes, this research enhances our understanding of the immunopathogenesis of COPD and establishes a foundation for the development of CD96-targeted therapies aimed at improving patient prognosis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCOPD\u0026nbsp;chronic obstructive pulmonary disease\u003c/p\u003e\n\u003cp\u003eNK\u0026nbsp;natural killer\u003c/p\u003e\n\u003cp\u003eCS\u0026nbsp;a smoke-exposed group\u003c/p\u003e\n\u003cp\u003eWT\u0026nbsp;a healthy control group\u003c/p\u003e\n\u003cp\u003eIFN-\u0026gamma; Interferon-gamma\u003c/p\u003e\n\u003cp\u003ePECAM1 platelet endothelial cell adhesion molecule-1\u003c/p\u003e\n\u003cp\u003eNECAB2 N-terminal EF-hand calcium binding protein 2\u003c/p\u003e\n\u003cp\u003eIL-17 Interleukin-17\u003c/p\u003e\n\u003cp\u003eSHIP1 SH2-containing inositol phosphatase-1\u003c/p\u003e\n\u003cp\u003ef\u0026nbsp;respiratory rate\u003c/p\u003e\n\u003cp\u003eTV\u0026nbsp;tidal volume\u003c/p\u003e\n\u003cp\u003eTLC\u0026nbsp;total lung capacity\u003c/p\u003e\n\u003cp\u003eFVC\u0026nbsp;forced vital capacity\u003c/p\u003e\n\u003cp\u003eRV\u0026nbsp;residual volume\u003c/p\u003e\n\u003cp\u003eFRC\u0026nbsp;functional residual capacity\u003c/p\u003e\n\u003cp\u003eCdyn\u0026nbsp;dynamic lung compliance\u003c/p\u003e\n\u003cp\u003eRI\u0026nbsp;airway resistance\u003c/p\u003e\n\u003cp\u003eNS\u0026nbsp;crosshairs\u003c/p\u003e\n\u003cp\u003eMLI\u0026nbsp;the mean lining interval of lung tissue\u003c/p\u003e\n\u003cp\u003eGOLD\u0026nbsp;Global Initiative for Chronic Obstructive Lung Disease\u003c/p\u003e\n\u003cp\u003eCAT COPD Assessment Test\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Institutional Animal Care and Use Committee of Anhui Medical University (Approval No.LLSC20210886). Human sample collection was approved by the Ethics Committee of the Second Affiliated Hospital of Anhui Medical University (Approval No.YX2025191), and written informed consent was obtained from all participants.The studies adhered to the principles outlined in the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors unanimously consent to submit the manuscript to Respiratory Research for publication. We agree that if the manuscript is accepted after peer review,Respiratory Research shall have the right to publish the manuscript in its print edition, electronic edition, and other derivative versions (including but not limited to online databases, digital archives, etc.). We also consent to the journal\u0026rsquo;s editorial department making necessary editorial revisions (including language polishing, format adjustment, etc.) to the manuscript to meet the publication standards of the journal, provided that the core content and academic views of the manuscript are not altered without the authors\u0026rsquo;consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analysed during the current study are available from the corresponding author on reasonable request.The datasets generated during and analysed during the current study are available in the PubMed repository,https://pubmed.ncbi.nlm.nih.gov/\u003csup\u003e[1-19]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Major Difficult Disease Collaborative Research Project of Traditional Chinese and Western Medicine (Grant No. 2021zdynjb07), the Natural Science Foundation of Anhui Province (Grant No. 2208085MH194), the Anhui Provincial Key Research and Development Program (Grant No. 202304295107020022), the Science Research Project of Universities in Anhui Province (Grant No. 2023AH040374), the National Natural Science Foundation of China (Grant No. 82371756), and the Science Fund of Universities of Anhui Province for Distinguished Young Scholars (Grant No. 2023AH020032).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYQF conceived and designed the study. JY, CXM, and DHZ supervised the project and provided critical revisions. RML, PCL, WJP, NX, HX, LDL, CX and SGZ performed the experiments. DHZ secured funding and provided resources. RML and PCL conducted data analysis and bioinformatics processing. YQF drafted the initial manuscript.All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extend our gratitude to all members of the Department of Respiratory and Critical Care Medicine at Anhui Medical University for their technical support and insightful discussions. Additionally, we thank Novozymes Ltd (Beijing, China) for providing single-cell RNA sequencing and preliminary bioinformatics services.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChair CV, agusti,A.Anzueto A, Criner,PFrith PBJBG, HalpinM.Han D. 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Effects of pm2.5 exposure on the notch signaling pathway and immune imbalance in chronic obstructive pulmonary disease, Environmental pollution (Barking, Essex: 1987) 226 (2017), 163\u0026ndash;173.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao X, Feng X, Liu P, Ye J, Tao R, Li R, Shen B, Zhang X, Wang X, Zhao D. Abnormal expression of CD96 on natural killer cell in peripheral blood of patients with chronic obstructive pulmonary disease. Clin Respir J. 2022;16(8):546\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAgarwal AR, Kadam S, Brahme A, Agrawal M, Apte K, Narke G, Kekan K, Madas S, Salvi S. Systemic immuno-metabolic alterations in chronic obstructive pulmonary disease (copd). Respir Res. 2019;20(1):171.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQiu S, Jiang Q, Li Y. The association between pan-immune-inflammation value and chronic obstructive pulmonary disease: data from NHANES 1999\u0026ndash;2018. Front Physiol. 2024;15:1440264.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZou Y, Chen X, Liu J, Zhou DB, Kuang X, Xiao J, Yu Q, Lu X, Li W, Xie B, Chen Q. Serum IL-1β and IL-17 levels in patients with COPD: associations with clinical parameters. Int J Chron Obstruct Pulmon Dis. 2017;12:1247\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin A, Ye P, Li Z, Jiang A, Liu Z, Cheng Q, Zhang J, Luo P. Natural Killer Cell Immune Checkpoints and Their Therapeutic Targeting in Cancer Treatment. Research (Wash D C). 2025;8:0723.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu W, Li R, Sun Y. Increased ifn-γ-producing th17/th1 cells and their association with lung function and current smoking status in patients with chronic obstructive pulmonary disease. BMC Pulm Med. 2019;19(1):137.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGlencross DA, Ho TR, Cami\u0026ntilde;a N, Hawrylowicz CM, Pfeffer PE. Air pollution and its effects on the immune system. Free Radic Biol Med. 2020;151:56\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen Y, Yang LL, Ning GL, Teng XB, Shi JF, Cui SS, Cao ZX, Zhang YB, Han MF. Analysis of exhaled nitric oxide and its influencing factors in patients with chronic obstructive pulmonary disease. Front Med (Lausanne). 2025;12:1611947.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReza MI, Ambhore NS. Inflammation in Asthma: Mechanistic Insights and the Role of Biologics in Therapeutic Frontiers. Biomedicines. 2025;13(6):1342.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing T, Zhao S, Gu Y, He G, Lang Y, Rao X, Chen J, Ou-Yang Y. IL-17A regulates airway remodelling in COPD through the PI3K/AKT/mTOR pathway. Sci Rep. 2025;15(1):16546.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePasquarelli-do-Nascimento G, Machado SA, de Carvalho JMA, Magalh\u0026atilde;es KG. Obesity and adipose tissue impact on T-cell response and cancer immune checkpoint blockade therapy. Immunother Adv. 2022;2(1):ltac015.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Chronic obstructive pulmonary disease, NK cells, CD96, SHIP1","lastPublishedDoi":"10.21203/rs.3.rs-8280945/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8280945/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCD96 is an inhibitory receptor found on natural killer (NK) cells, yet its mechanism of action in chronic obstructive pulmonary disease (COPD) remains unclear. This study aimed to examine the alterations and function of CD96 in COPD and to assess the impact of anti-CD96 intervention.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003ePeripheral blood samples were obtained from 44 patients with chronic obstructive pulmonary disease (COPD), along with a cigarette smoke (CS)-induced COPD mouse model. The expression of CD96 and IFN-γ in natural killer (NK) cells was measured using flow cytometry. Lung function, histopathological changes, airway remodeling, and levels of inflammatory mediators were evaluated in mice following 6 or 12 weeks of CS exposure, with or without the administration of anti-CD96 antibody.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn patients with COPD, the expression of CD96 on NK cells was elevated, and the proportion of CD96⁺NK cells showed a positive correlation with the duration of hospitalization, CAT scores, and the incidence of acute exacerbations (\u0026ge;\u0026thinsp;2) within one year. In mice exposed to cigarette smoke, increased signaling through CD96 and CD155 was associated with a reduction in the FEV0.2/FVC ratio, disruption of emphysema-like structures, collagen deposition, airway wall thickening, and the upregulation of various inflammatory mediators, including IL-17A, IFN-γ, IL-6, and TNF-α. Treatment with anti-CD96 partially restored lung function, reduced alveolar injury, collagen deposition, and airway remodeling, and downregulated IL-17A along with several pro-inflammatory cytokines. Furthermore, this treatment preserved the IFN-γ production capacity of NK cells. The concurrent upregulation of CD96 and SHIP1 in NK cells, along with the enhancement of their function following CD96 blockade, suggests the involvement of a CD96-SHIP1-related inhibitory pathway in the disease process.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eCD96-overexpressing NK cells and CD96/SHIP1-associated signaling are associated with disease severity and a chronic inflammatory state in COPD. The anti-CD96 intervention mitigated CS-induced lung injury in mice, indicating that CD96 may serve as a novel target for immunotherapy in COPD. Future research should further investigate its clinical translational potential in COPD patients and analyze its regulatory network by integrating multi-omics technology, thereby providing a more comprehensive molecular foundation for precision therapy.\u003c/p\u003e","manuscriptTitle":"Role of NK surface inhibitory receptor CD96 in chronic obstructive pulmonary disease and the effect of targeted CD96 intervention","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-12 05:49:54","doi":"10.21203/rs.3.rs-8280945/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b84fef29-8170-476e-8052-96b0ff72e8a3","owner":[],"postedDate":"January 12th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-05T00:39:09+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-12 05:49:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8280945","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8280945","identity":"rs-8280945","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}