Decrease of tumoral CD39+CD8+T cells promoted by hypoxia confers resistance to immunotherapy in NSCLC

Decrease of tumoral CD39+CD8+T cells promoted by hypoxia confers resistance to immunotherapy in NSCLC | 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 Decrease of tumoral CD39 + CD8 + T cells promoted by hypoxia confers resistance to immunotherapy in NSCLC Jiajuan Wu, Jiawei Zhai, Leilei Lv, Yaoxin Zhang, Yu Shen, Qiuxia Qu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7102657/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 Understanding resistance to anti-PD-1 is crucial for the development of reversal strategies. Here, we analyzed a subset of tumor-infiltrating CD8 + T cells based on the expression of the ATP ectonucleotidase CD39, further explored its spatial distribution being priming as a contributor to anti-PD-1 therapeutic response or resistance. It is interesting that CD39 + CD8 + T cells were more enriched in peritumor compared to center, as well as being severed as a potential immuno-responsive biomarker for anti-PD-1 therapy. Correspondingly, it revealed more hypoxia actually existed in central TME, thereby reducing CD39 + CD8 + T cells infiltration and dampening the efficacy of anti-PD-1. As supported, multiple in vitro assays demonstrated that absence of CD39 limited functional restore of CD8 + T cells upon PD-1 blockade. Collectively, we illustrated the hypoxia was involved in establishing an immunosuppressive TME defined by spatial distribution of CD39 + CD8 + T cells and consequentially implied a novel approach of resistance to immunotherapy. CD39 PD-1 CD8+T cell HIF-α Tumor microenvironment lung cancer Immunotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Background Tumors employ diverse strategies to create a tolerogenic microenvironment, including producing immunosuppressive cytokines, competing for nutrients, and expressing inhibitory ligands, and others [1, 2] . Strategies to therapeutically target the tumor microenvironment (TME) have emerged as a promising approach for cancer treatment due to the critical roles of the TME in regulating tumor progression and modulating response to standard-of-care therapies [3, 4] . Correspondingly, the immunotherapy of cancer patients using anti-PD-1 and PD-L1 mAb has shown substantial clinical response albeit only in a subset of cancer patients, necessitating the understanding of mechanisms of TME mediated resistance. Tumor-infiltrating lymphocytes (TILs), particularly CD8 + T cells, present as exhausted in a wide variety of animal models and in humans with cancer or chronic viral infections [5, 6] . Both human and murine data support the existence of a highly heterogenous pool of CD8 + T cell that differ in their transcriptome, epigenome, proteome, and functionality [7, 8] . The spatial interactions among immune and cancer cells generate complex ecological dynamics that influence tumor progression and treatment response [9, 10] . The heterogenous distribution of immune infiltrates in the TME has been associated with clinical implications in several cancers, such as melanoma and biliary tract cancer [11] . Recently, the expression and function of CD39 and CD73 in human solid tumors has gained research interest [12] . CD39 is an ectonucleotidase presented by B cells, innate cells, regulatory T cells and activated CD4 and CD8 T cells, which, in coordination with CD73, can stimulate the local production of adenosine and create an immunosuppressive environment [13] . It has been reported that the expression of CD39 on CD8 + T cells is a candidate marker for exhausted TILs [14] . Moreover, CD39 is highly expressed on empirically defined neoantigen- and tumor-associated-antigen- reactive T cells in lung cancer and melanoma, proposed CD39 as a marker of tumor-reactive CD8 + T cells [15] . This suggests that CD39 could be a useful marker of tumor-specific CD8 + T cells, which could be exploited for the development of novel biomarkers or therapeutics. Here we report a new mechanism of resistance to anti-PD-1 therapy. It was shown that hypoxia mediated heterogenous distribution of the CD39 + CD8 + T cell subsets in lung cancer and revealed the CD39 + CD8 + T cell levels within the peripheral TME may act as a candidate biomarker for immunotherapy, as well as being used to select patients for anti-PD-1 therapy. On the other hand, absence of CD39 limited functional restore of CD8 + T cells upon PD-1 blockade, implying that resistance to immunotherapy and therapeutic failure could be partially attributed by reduce of CD39 + CD8 + T cells resulted from the PD-1 blockade. 2. Methods 2.1 Patients Fifty-three patients with lung cancer were admitted to the First Affiliated Hospital of Soochow University from 2019 to 2025. None had received anti-tumor therapy at the time of sample analysis. Clinic information included age, sex, histological subtype, pathological stage, and primary tumor size. This study was approved by the Institutional Review Boards of The First Affiliated Hospital of Soochow University (2019-070). The processing of clinical tissue samples is in strict compliance with the ethical standards of the Declaration of Helsinki. Informed consent was obtained from all participants. 2.2 Acquisition of tumor samples A bronchoscope was used to obtained tumor tissues from patients. Once the neoplasm was visualized under bronchoscopy, a superficial biopsy was performed directly. If lung cancer lesion (peripheral lesions) was invisible under bronchoscopy, intratumoral endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) was performed using a 22-gauge needle. 2.3 Isolation of lymphocytes from lung cancer tissue Fresh tissues were dissected and placed in RPMI medium, then disrupted mechanically using scissors. The tissues were then digested using a mixture of DNase I (0.3 mg/ml, Sigma-Aldrich) and Liberase™ TL (0.2 mg/ml, Roche) in serum-free RPMI 1640 medium (Gibco, USA) for 30 min at 37°C, and subsequently strained through a 70-µm cell strainer (Beyotime Biotechnology). With regard to fresh tissues obtained from intratumoral aspiration (EBUS-TBNA), the erythrocytes were lysed with 10 mL of ACK lysis buffer (Beyotime Biotechnology) at 37°C for 10 min. Then isolated cells were suspended in 1 ml of RPMI 1640 medium and stained for analysis. 2.4 Flow cytometry Single-cell suspensions containing 2×10⁶ cells/ml were prepared prior to immune staining. Following this, the cells were stained with several antibodies for 30 min 4°C in the dark: APC-Cyanine7-labeled anti-CD45 mAb (HI30), FITC-labeled anti-CD8α mAb (RPA-T8), PE-Cyanine7-labeled anti-CD39 mAb (A1), and APC-labeled anti-PD-1 mAb (EH12.2H7). All antibodies were purchased from Biolegend (USA). We used the Fluorescence Minus One (FMO) Control to help identify and gate cells, details are provided in the supplementary figure [16] . Lymphocytes were gated on SSC-A and FSC-A. CD39 + and PD-1 + TILs were gated on CD45 + CD8 + T cells. Gating strategies are shown in detail in Fig. 1 a and Fig. 4 a. Multi-colored flow cytometry was performed using Cytoflex (Beckman), and data were analyzed using FlowJo software (Tree Star). 2.5 Immunofluorescence analysis Frozen sections (5µm in thickness) were prepared from tumor tissue, embedded in optimum cutting temperature compound (Sakura Finetek). Then, sections were incubated with 3% BSA for 1 h at 37◦C. Following this, they were stained with rat anti-human CD39 mAb (1:150) overnight at 4◦C. After washing with PBS, primary antibodies were detected with rabbit anti-rat CD8 mAb (1:300). In some cases, rat anti-human HIF-α mAb (1:150) was stained. After washing, sections were subsequently embedded with DAPI. Sections were scanned using Nikon imaging system (Eclipse Ni-U).To count the number of target cells, six representative HPFS (×400 or ×200 amplification) were selected for each section. The number of target cells in each field of view was calculated, and then statistical analysis was conducted using Graphpad. 2.6 In vitro CD39 inhibition assay Single cells from lung cancer tissues were cultured with CD39 inhibition POM-1 (100 µmol/L, Sigma-Aldrich) or vehicle for 2h at 37°C and 5% CO 2 [13, 17] . After culture, the cells were stained with several antibodies for 30 min at 4°C in the dark: APC-Cyanine7-labeled anti-CD45 mAb (HI30), FITC-labeled anti-CD8α mAb (RPA-T8), and PE-Cyanine7-labeled anti-CD39 mAb (A1). Cells were then fixed (eBioscience™ Foxp3/ Transcription Factor Staining Buffer Set, ThermoFisher) for 30 min and stained with PE-labeled anti-granzyme B mAb (QA18A28) in a permeabilization buffer (ThermoFisher) for 30 min [18] . Multi-colored flow cytometry was performed using Cytoflex (Beckman) and data were analyzed using FlowJo software (Tree Star). 2.7 In vitro blockade culture Human lung cancer tissues were placed in RPMI 1640 medium containing 5% fetal bovine serum (FBS) and 1% antibiotics, and immediately transported to the laboratory at 4°C. After removing necrotic tissues, blood clots, and other debris, the tumor tissues were washed several times with PBS and cut into 1 mm2 fragments in the culture medium. An appropriate amount of 0.1% collagenase IV was added, and the mixture was incubated in a shaker water bath at 37°C. After 30 min, the digestion was terminated by adding culture medium. The cell suspension was filtered through a 100-µm cell strainer and centrifuged for 5 min, discarding the supernatant. The cells were resuspended in culture medium and randomly divided into two treatment groups: (i) control IgG; and (ii) human anti-PD-1 mAb (10 µg/mL, Biolegend). After incubation at 37°C with 5% CO₂ for 72 h, cells were stained with fluorochrome-conjugated antibodies for CD45, CD8, and CD39, followed by flow cytometric analysis. 2.8 Assessment of Immunotherapy Antitumor efficacy was assessed by reviewing computed tomography (CT) scans following four treatment courses. The same imaging modality was recommended for all patients. The change from baseline regarding the sum of target lesion diameters was measured according to RECIST 1.1. 2.9 Statistical Analysis Differences in mean ± standard error of mean (SEM) were evaluated with Student’s t-test, paired t-test, and Welch’s t-test. The correlation analysis was evaluated with Pearson correlation and linear regression. The association between response status and TIL frequency was analyzed using Fisher’s exact test. Receiver operating characteristic (ROC) curves for response status were constructed to assess the prognostic ability of TILs. All analyses were two-sided and performed at a significance level of 5% (p < 0.05) using GraphPad 8.0. 3. Results 3.1 Patient characteristics The baseline characteristics of the 53 patients with lung cancer are summarized in Table 1 . The median age was 66 years (range, 37–87 years), and 83.01% of patients were male. Histological analysis revealed 30 squamous cell carcinomas, 14 adenocarcinomas, and 9 small cell carcinomas. Over half of the patients (50.95%) were in the late pathological stage (IIIB-IV), and 30 (56.6%) patients had a primary lesion larger than 3 cm. Table 1 Patient clinical characteristics. Characteristics Number(%) Sex Male 44(83.01) Female 9 (16.99) Age (years) < 65 20(37.74) ≥ 65 33(62.26) Types NSCLC 44(83.01) Adenocarcinomas 14(26.41) Squamous carcinomas 30(56.60) SCLC 9 (16.99) Stage II–IIIA 26(49.05) IIIB–IV 27(50.95) Size(cm) < 3 23(43.40) ≥ 3 30(56.60) 3.2 Infiltration of CD39 + CD8 + T cell subsets in human lung cancer To further explore the potential contribution of CD8 + T cells to the TME, we examined the expression of the ectonucleotidases CD39 on CD8 + T cell in lung cancer prior to any therapy (Fig. 1 A). We found that the percentage of infiltrated CD39 + CD8 + T cells was comparable between lung cancer patients from stage II-IIIA to IIIB-IV (36.12 ± 4.53% vs 28.46 ± 4.80%, p > 0.05). In addition, similar percentages of infiltrated CD39 + CD8 + T cells were observed between different ages, pathologic types, and different lesion sizes (Fig. 1 B). While it was noticed that the percentage of CD39 + CD8 + T cell subsets was higher in males than that in females (35.54 ± 3.71 vs 15.98 ± 4.39%, p < 0.05). 3.3 Decreased infiltration of CD39 + CD8 + T cells in center of lung cancer tissue To gain a more in-depth understanding of CD39 + CD8 + T cell infiltration, peripheral and central TME were assessed whether heterogenous reginal distribution was involved in its accumulation. Here, neoplasm-superficial biopsy and intratumoral EBUS-TBNAs were used to obtain peripheral and central lung cancer tissue samples, respectively (Fig. 2 A). As shown in Fig. 2 B, CD39 + CD8 + T cells were both found in the peripheral and central sites, whereas peripheral TME was more enriched with this T cell subset than that in the central TME (36.84 ± 3.58% vs 16.45 ± 6.34%, p < 0.01). 3.4 Heterogeneous infiltration of CD39 + CD8 + T cells is dependent on tumor size We next sought to determine whether this heterogenous distribution of CD39 + CD8 + T cells was correlated with tumor size. In Fig. 2 C, distinct infiltrated profile of CD39 + CD8 + T cells between peripheral and central region was only found in lesions with diameter larger than 3cm (39.98 ± 4.68% vs 3.59 ± 1.42%, p 0.05). As supported, immunofluorescence technique was further verified the distribution of tumoral CD39 + CD8 + T cells within TME, which was consistent with the findings obtained by flow cytometry (Fig. 3 ). 3.5 Reduced CD39 expression on intratumoral CD8 + T cell under hypoxia As CD39 + CD8 + T cells were heterogeneous resident in the context of compartment of TME characterized by location and size, we hypothesized that hypoxia has distinct effects on continuous experience of CD8 + T cell within TME. Then, immunofluorescence technique was again used to verify the distribution of HIF-α within central and peripheral TME. As shown in Fig. 4 A, central region exhibited significant enrichment of HIF-α compared to peripheral tumor. Therefore, we sought to a model mimic this stress in vitro, where fresh lung cancer derived cells were co-cultured under conditions of HIF-α (100ng/ml) for 3 d. Accordingly, it was found that HIF-α significantly decreased the expression of CD39 on CD8 + T cells, resulting in a significant decrease in the number of CD39 + CD8 + T cells (Fig. 4 B). Thus, these data show that a strong potential for the reginal hypoxia to serve as a mechanistic role in heterogenous CD39 + CD8 + T cell infiltration. 3.6 Abundance of peri-neoplastic CD39 + CD8 + T cells predicted efficacy of PD-1 blockade Of the total cases studied, 17 patients were administered anti-PD-1 therapy. We then investigated whether the quantification of CD39 + CD8 + T cells could serve as a predictive biomarker for response to anti-PD-1 therapy. Initial analysis revealed that random CD39 + CD8 + T cells displayed similar level between responders and non-responders (45.02 ± 19.13% vs 34.80 ± 17.83%, P > 0.05, Fig. 5 A). It only produced 80.00% sensitivity and 71.43% specificity, with a corresponding AUC of 0.6571 to predict therapeutic response to anti-PD-1 mAb (Fig. 5 A-B). Considering central tumor CD39 + CD8 + T cell vary according to lesion size and hypoxia, peri-neoplastic CD39 + CD8 + T cells was selected as candidate biomarker for anti-PD-1 therapy. In this cohort (n = 13), it was found that the fraction of CD39 + CD8 + T cells in the tumor samples from responders (n = 8) was significantly higher compared to non-responders (n = 5), (52.05 ± 11.81% vs 24.72 ± 4.433%, p ≤ 0.01). An AUC of 1.00 with a PPV of 1.00 and NPV of 1.00 suggests that the peri-neoplastic CD39 + CD8 + T cell fraction can serve as a potential predictive biomarker of therapeutic response to anti-PD-1 (Fig. 5 A-B). Next, it is generated by calculating the percentage change in the total diameter of the target lesions compared to the baseline. As shown in Fig. 5 C and D, unlike random CD39 + CD8 + T cells (r=-0.2355, p = 0.3628), the higher the degree of peri-neoplastic CD39 + CD8 + T cells infiltration correlated with the deeper the clinical remission after receiving anti-PD-1 treatment (r=-0.7793, p < 0.01). 3.7 Absence of CD39 limited functional restore of CD8 + T cells upon PD-1 blockade To further confirm that immunotherapy is more effective in patients with higher CD39 + CD8 + T cells, we explored the correlation of CD39 expression and PD-1, whom is the target of anti-PD-1 mAb. As shown in Fig. 6 A, CD39 + CD8 + T cells expressed significantly higher levels of PD-1 compared to their CD39 − counterparts (54.21 ± 4.00% vs 16.18 ± 2.77%, p < 0.0001). Especially, CD39 + CD8 + T cells derived from the peri-neoplastic site were more PD-1 enriched than those from the central-neoplastic TME (59.83 ± 3.40% vs 44.83 ± 7.45%, p < 0.05). This pattern indicated that CD39 + subset might be the dominant CD8 + T cells population adapted by the PD-1 blockade. Correspondingly, we stimulated single cells isolated from human lung cancer tissues with anti-PD-1 mAb in vitro for 72 h to mimic clinical conditions. As shown in Fig. 6 B, anti-PD-1 treatment significantly reduced CD39 expression on CD8 + T cells, leading to a marked decrease in the proportion of CD39 + CD8 + T cells (p < 0.05). These data further support the notion that CD39 + subset could be a main targeted CD8 + T cell regulated by ICI. Next, we aimed to determine the function of CD39 expressed by CD8 + T cells by Polyoxometalate 1 (POM-1), which was used to block CD39 eATPase activity. As shown in Fig. 6 C, the CD39 + CD8 + T cells demonstrated increased cytotoxic potential, indicated by a higher frequency of granzyme B positive cells following POM-1 treatment (36.32 ± 2.69% vs 29.47 ± 3.41%, p < 0.01), while this effect was not found in CD39 − CD8 + T cells. Collectively, these data show that CD39 is not merely a marker to define which CD8 + T cell subset would be renewed capacity after receiving anti-PD-1 treatment, but serves a mechanistic role in rendering CD8 + T cells dysfunctionality. 4. Discussion This study provides the comprehensive characterization of spatial heterogeneity in CD39 + CD8 + T cells distribution within NSNLC. It was demonstrated that CD39 + CD8 + T cells are significantly enriched in peripheral tumor regions compared to central areas, and worked as a biomarker for anti-PD-1 therapy. Importantly, regional hypoxia was identified as a mechanism reducing CD39 + CD8 + T cells. Furthermore, our data reveal that the loss of CD39 limits functional restoration of CD8 + T cells following PD-1 blockade, together with downregulated CD39 expression driving by anti-PD-1 therapy itself, these findings not only advance our understanding of spatial immune architecture in tumors but also uncover a novel mechanism of immunotherapy resistance. Recent developments in studies of tumor heterogeneity have led to new insights into cancer management. For example, a higher level of intratumoral but not peritumoral CD8 + T cells in conjunction with a low level of myeloid cells was reported to be associated with favorable outcomes in melanoma patients treated with mitogen-activated protein kinase inhibitors [19] . In estrogen receptor (ER)-negative/human epidermal growth factor receptor 2 (Her2)-negative [20] and Her2-positive [21] breast cancer patients, a high degree of immune infiltration in tumor stroma was associated with increased survival and complete response rates. In lung cancer, recent evidence shows that PD-1 + Tim-3 + CD8 + tissue-resident memory T cells preferentially localized at invasive margins compared to tumor centers, with decreased spatial interactions independently associated with recurrence [22] . Tian et al. showed that CD8 + T cell infiltration markedly reduced in larger tumors [23] . These observations align with our findings, implying heterogeneity of CD39 + CD8 + T cell distribution within the TME of NSCLC extend concepts in spatial tumor immunology, establishing tumor size and location as a critical determinant of immune spatial organization. Then, we hypothesized that regional hypoxia correlates strongly infiltrated degree of CD39 + CD8 + T cells. As reported, hypoxic conditions promoted exhausted-like states through reactive oxygen species generation [24] . Scharping et al. elucidated the continuous TCR stimulation combined with hypoxia rapidly induces T cell exhaustion [25] . In NSCLC specifically, Giatromanolaki et al. confirmed that CD73 and CD39 expression correlates with HIF-α, creating an immunosuppressive feedback loop [26] . In our study, HIF-α treatment significantly reduces CD39 expression on CD8 + T cells, indicating hypoxia as a driver of decreased CD39 + CD8 + T cell frequency in tumor. And presumably, the more severe hypoxic conditions in larger tumor centers explain why spatial heterogeneity manifests only in tumors exceeding 3 cm. Despite available biomarker stratification, clinical responses vary [27] . Thus, the search for novel biomarkers with improved response predictions is ongoing. Previous study demonstrated that CD39 is highly expressed on neoantigen-reactive CD8 + T cells [28] , supporting CD39 as a marker of tumor-reactive T cells. Duhen et al. found that CD103 + CD39 + CD8 + T cells are enriched for tumor-specific TCR clones across multiple solid tumor types, while maintaining cytotoxic capacity through granzyme B and perforin [14] . Responsively, CD39 + CD8 + T cell proportion can serve as a potential biomarker that predicts response to PD-1 or PD-L1 blockade [29] . For example, Chow et al. used mass cytometry to analyze 440 lung cancer specimens, confirming that CD39 identifies CD8 + T cells as predictive of checkpoint blockade efficacy [16] . In our study, we suggested that peripheral CD39 + CD8 + T cells might be a better biomarker to predict response to anti-PD-1 therapy. Therefore, evaluating the proportion of regional CD39 + CD8 + T cells in TMEs may be a complementary approach to PD-L1 staining for patient stratification prior to immunotherapy. Given the higher prevalence of EGFR mutations in female NSCLC patients [30] , the observed gender difference likely reflects underlying molecular subtype distributions. Previous reports showing higher frequencies of CD39 + CD8 + T cells in EGFR wild-type tumors compared to EGFR-mutant tumors [31] . Our recent works further supports this, demonstrating that EGFR mutations suppress CD39 + CD8 + T cell infiltration in malignant pleura effusion [32] . However, the data on whether the accumulation of CD39 + CD8 + T cell was associated with the status of EGFR in the TME of lung cancer are not presented in this paper. However, as shown earlier, while CD39 expression reflects T cell reactivity to antigen exposure, we believe that modest response rates for anti-PD-1 therapy in EGFR-mutant NSCLC was associated to lack of these CD8 + T cells. It has been shown that anti-PD-1 can reinvigorate exhausted CD8 + T cells. However, this might be dependent on the initial cell activation status where tumor-reactive effector cells could be targeted by anti-PD-1 mAb. We found that CD39 + CD8 + T cells exhibit higher PD-1 compared to CD39 − counterpart. This CD39/PD-1 co-expression pattern carries important biological implications, indicated that CD39 + tumor-reactive T cell subset might be the dominant CD8 + T cells population adapted by the PD-1 blockade. Alternatively, CD39 + CD8 + T cells were depleted by anti-PD-1 mAb. This, in turn, leads to lack of pool in a substantial fraction of CD8 + T cells to response to PD-1 blockade, further support the notion that CD39 + subset could be a main targeted CD8 + T cell regulated by anti-PD-1 mAb. Actually, functional CD39 + CD8 + T cell is generated when CD39 is blocked by POM-1, inducing an effector phenotype. Through these experiments, it was found that CD39 + CD8 + T cells are not merely an indicator for anti-PD-1 therapeutic efficacy, but delineated a complex resistance network. This reveals a paradox: while CD39 + CD8 + T cells represent the primary targets of anti-PD-1 therapy, the treatment itself may deplete this population, absence of CD39 limited functional restore of CD8 + T cells upon PD-1 blockade. The clinical implications are profound, suggesting that PD-1 blockade alone may be insufficient to fully activate anti-tumor immunity, particularly in large or severely hypoxic tumors, suggesting that ameliorating hypoxia enhances immunotherapy responses. Several limitations warrant consideration when interpreting our results. First, the relatively small sample size, particularly the 13 patients receiving anti-PD-1 therapy, may limit statistical power and generalizability. Second, since most patients undergoing intratumoral EBUS-TBNA sampling did not receive subsequent anti-PD-1 therapy, we could not directly assess the relationship between central CD39 + CD8 + T cell levels and treatment response. Finally, our relatively brief in vitro experiments may not fully recapitulate long-term in vivo treatment effects. Therefore, our findings suggest several important avenues for future investigation. Large-scale prospective cohort studies should validate CD39 + CD8 + T cells as immunotherapy predictive biomarkers, particularly incorporating spatial distribution information into predictive models. Mechanistic studies dissecting the hypoxia-HIF-α-CD39 axis may reveal novel therapeutic targets and resistance mechanism. Declarations Ethics Approval and Consent to Participate This study was approved by the Institutional Review Boards of The First Affiliated Hospital of Soochow University (2019-070). The processing of clinical tissue samples is in strict compliance with the ethical standards of the Declaration of Helsinki. Informed consent was obtained from all participants. Consent for publication Not applicable Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by the National Natural Science Foundation of China (NSFC) grant 81874110 (to QQ), 81672280 (to CC), Natural Science Foundation of Jiangsu Province grant BK20181174 (to QQ), and Gusu Talent Project GSWS2019031(to QQ). Authors' contributions CC and QQX conceived the idea and designed and supervised the study. WJJ, ZJW and LLL performed the experiment and drafted the manuscript. ZYX, WJJ, ZJW, LLL and SY collected data. WJJ, ZJW and LLL analyzed data and performed statistical analysis. 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Nat Immunol, 2021, 22(2): 205-15. GIATROMANOLAKI A, KOUROUPI M, POULILIOU S, et al. Ectonucleotidase CD73 and CD39 expression in non-small cell lung cancer relates to hypoxia and immunosuppressive pathways [J]. Life Sci, 2020, 259: 118389. RöSNER E, KAEMMERER D, SäNGER J, LUPP A. Evaluation of PD-L1 expression in a large set of gastroenteropancreatic neuroendocrine tumours and correlation with clinicopathological data [J]. Transl Oncol, 2022, 25: 101526. HANADA K I, ZHAO C, GIL-HOYOS R, et al. A phenotypic signature that identifies neoantigen-reactive T cells in fresh human lung cancers [J]. Cancer Cell, 2022, 40(5): 479-93.e6. YEONG J, SUTEJA L, SIMONI Y, et al. Intratumoral CD39(+)CD8(+) T Cells Predict Response to Programmed Cell Death Protein-1 or Programmed Death Ligand-1 Blockade in Patients With NSCLC [J]. J Thorac Oncol, 2021, 16(8): 1349-58. SOO R A, REUNGWETWATTANA T, PERROUD H A, et al. Prevalence of EGFR Mutations in Patients With Resected Stages I to III NSCLC: Results From the EARLY-EGFR Study [J]. J Thorac Oncol, 2024, 19(10): 1449-59. SIMONI Y, BECHT E, FEHLINGS M, et al. Bystander CD8(+) T cells are abundant and phenotypically distinct in human tumour infiltrates [J]. Nature, 2018, 557(7706): 575-9. HUANG H, ZHU X, YU Y, et al. EGFR mutations induce the suppression of CD8(+) T cell and anti-PD-1 resistance via ERK1/2-p90RSK-TGF-β axis in non-small cell lung cancer [J]. J Transl Med, 2024, 22(1): 653. Additional Declarations No competing interests reported. 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-7102657","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":485944296,"identity":"c366c3ab-619a-456a-8cc9-1bd2020860ef","order_by":0,"name":"Jiajuan Wu","email":"","orcid":"","institution":"Clinical Immunology Institute, the First Affiliated Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Jiajuan","middleName":"","lastName":"Wu","suffix":""},{"id":485944297,"identity":"ffa6d756-f7c4-4d46-8363-9127abff363e","order_by":1,"name":"Jiawei Zhai","email":"","orcid":"","institution":"Respiratory Department, the First Affiliated Hospital of Soochow University,","correspondingAuthor":false,"prefix":"","firstName":"Jiawei","middleName":"","lastName":"Zhai","suffix":""},{"id":485944298,"identity":"d4a5b2a9-2f7d-4b52-b1bc-1e4c281f4bc7","order_by":2,"name":"Leilei Lv","email":"","orcid":"","institution":"Respiratory Department, the First Affiliated Hospital of Soochow University,","correspondingAuthor":false,"prefix":"","firstName":"Leilei","middleName":"","lastName":"Lv","suffix":""},{"id":485944299,"identity":"c6bfcdbb-532b-4c84-b52a-315effaaeb3b","order_by":3,"name":"Yaoxin Zhang","email":"","orcid":"","institution":"Respiratory Department, the First Affiliated Hospital of Soochow University,","correspondingAuthor":false,"prefix":"","firstName":"Yaoxin","middleName":"","lastName":"Zhang","suffix":""},{"id":485944300,"identity":"e4bddd0c-5137-4a79-8e78-d530fbe06114","order_by":4,"name":"Yu Shen","email":"","orcid":"","institution":"Clinical Immunology Institute, the First Affiliated Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Shen","suffix":""},{"id":485944301,"identity":"7e5d497e-8f0b-45ec-986c-d37590068fc4","order_by":5,"name":"Qiuxia Qu","email":"","orcid":"","institution":"Clinical Immunology Institute, the First Affiliated Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Qiuxia","middleName":"","lastName":"Qu","suffix":""},{"id":485944302,"identity":"44cff223-024f-4251-93e5-2de57a6c3294","order_by":6,"name":"Cheng Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYDACCSBmbAAS7ECCByRygGgtPAdI1iKRQKQW/tnNxx783GGTJx/5/JnEmxoGOb4bCYyfC/BZcudYumHvmbRiw9s5ZpJzjjEYS95IYJaegUeLgUSOmTRj2+HEjbNz2KR52BgSN9xIYGPmwasl/xtQy//EjTOPP5Pm+cdQT4QWoOGMbQcS50swmEnztjEkGBDSInEjzUyyty05cQNPjrHl3D4Jw5lnHjZL49PCPyP5mcTPNrvE+e3HH954881Gnu948sHP+LQgXHgAYisDNJqIAPJEqhsFo2AUjIIRCADTD0tvZ4VBJAAAAABJRU5ErkJggg==","orcid":"","institution":"Respiratory Department, the First Affiliated Hospital of Soochow University,","correspondingAuthor":true,"prefix":"","firstName":"Cheng","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2025-07-11 14:38:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7102657/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7102657/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86862182,"identity":"4ba85b51-7ce2-40b2-ae68-a242eb7b3db4","added_by":"auto","created_at":"2025-07-16 12:26:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":460051,"visible":true,"origin":"","legend":"\u003cp\u003eInfiltration of CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cell subsets in human lung cancer. (A) The gating strategy that identified CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells. (B) Comparisons of CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cell levels between different gender, ages, pathologic types, stages, and different lesion sizes. Results are expressed as the mean±SEM of independent experiments, *p\u0026lt;0.05 by unpaired t-test, ns: no significance.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7102657/v1/0f2eb270792c359349237af0.png"},{"id":86861215,"identity":"481044df-4d7f-4a7c-af06-575747debda4","added_by":"auto","created_at":"2025-07-16 12:18:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":346650,"visible":true,"origin":"","legend":"\u003cp\u003eHeterogeneity distribution of CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cell in lung cancer microenvironments. (A) Neoplasm-superficial biopsied and intratumoral EBUS-TBNA tissues representing the periphery and center of lung cancer tissue, respectively. (B) Representative dot plots of CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells from peripheral and central TME. (C) The ratio of CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells to CD8\u003csup\u003e+\u003c/sup\u003eT cells from peripheral TME (n=41) was greater than that from central TME (n=12). This spatial profile was only observed in subjects with primary lesions larger than 3 cm. Results are expressed as the mean±SEM of independent experiments, **p\u0026lt;0.01 by unpaired t-test, ****p\u0026lt;0.0001 by Welch’s t-test, ns: no significance.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7102657/v1/a45256d6bae57d2507113b00.png"},{"id":86861217,"identity":"44d921a1-459a-467f-888e-72e38d6d5102","added_by":"auto","created_at":"2025-07-16 12:18:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":303290,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution characteristics of CD39\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eCD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eT cells in different regions of the tumor. \u003c/strong\u003e(A-B) Representative images of the immunofluorescence staining with DAPI (blue), CD8 (red), CD39 (green), and Merge (double positive) on lung cancer tissues. Scans were imaged at 400 magnifications. Results are expressed as the mean±SEM of independent experiments, *p\u0026lt;0.05 by unpaired t-test, * ns: no significance.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7102657/v1/5f9d337d5ae59f822e5fd14d.png"},{"id":86861221,"identity":"bc05e786-f5e5-4890-b37b-ad1194394fab","added_by":"auto","created_at":"2025-07-16 12:18:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":342217,"visible":true,"origin":"","legend":"\u003cp\u003eHIF-α can reduce the expression of CD39 on CD8\u003csup\u003e+\u003c/sup\u003eT cells derived from lung cancer. (A) Comparison of the expression levels of HIF-α in the peri- and central regions of the tumor. (B) Representative flow cytometry of CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells: (i) Untreated group; (ii) HIF-α treatment group. Immunofluorescence co-localization representative map (200X). Unpaired t-test, **P\u0026lt;0.01; Paired t-test, *P\u0026lt;0.05.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7102657/v1/cb147766f7d8e9aa1c0e65bf.png"},{"id":86861220,"identity":"0fbc486f-a8d7-4984-b5a2-5219e74949e1","added_by":"auto","created_at":"2025-07-16 12:18:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":278676,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells in human lung cancer tissues predicting the efficacy of PD-1 antibody. (A) Comparison of CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells between non-responders and responders. (B) ROC curve analysis of the proportion of CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells for predicting the efficacy of immunotherapy. (C) Waterfall plot of the total diameter of the target lesion based on RECIST v1.1 versus baseline changes (Fisher's exact test). (D) Pearson correlation and linear regression analysis were used to evaluate the relationship between the infiltration level of CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells and the response to anti-PD-1 treatment.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7102657/v1/44ff4b7948ad0f524d3d2419.png"},{"id":86861226,"identity":"3b017f95-38da-4fef-b666-6cf050b31e84","added_by":"auto","created_at":"2025-07-16 12:18:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":580237,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAbsence of CD39 limited functional restore of CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eT cells upon PD-1 blockade. \u003c/strong\u003e(A) CD39\u003csup\u003e+\u003c/sup\u003e subset might be the dominant CD8\u003csup\u003e+\u003c/sup\u003eT cells population adapted by the PD-1 blockade. CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells exhibited higher PD-1\u003csup\u003e+\u003c/sup\u003ecells compared to their CD39\u003csup\u003e-\u003c/sup\u003ecounterparts, PD-1 expression was more abundant in peritumoral CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells. (B) Anti-PD-1 treatment reduced CD39 expression on CD8\u003csup\u003e+\u003c/sup\u003eT cells, leading to a marked decrease in the proportion of CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells (p\u0026lt;0.05), further support the notion that CD39\u003csup\u003e+\u003c/sup\u003e subset could be a main targeted CD8\u003csup\u003e+\u003c/sup\u003eT cell regulated by ICI. (C) POM-1 treatment selectively elevated GZMB expression in CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7102657/v1/e6e9e47fb0d4a4f1746a89ec.png"},{"id":96915513,"identity":"de3e069a-2777-431a-a58b-2a3a3c003fc7","added_by":"auto","created_at":"2025-11-27 14:07:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3303938,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7102657/v1/5b94ea8f-7f01-4179-8674-4ecda4dd89d8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eDecrease of tumoral CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells promoted by hypoxia confers resistance to immunotherapy in NSCLC\u003c/p\u003e","fulltext":[{"header":"1. Background","content":"\u003cp\u003eTumors employ diverse strategies to create a tolerogenic microenvironment, including producing immunosuppressive cytokines, competing for nutrients, and expressing inhibitory ligands, and others\u003csup\u003e[1, 2]\u003c/sup\u003e. Strategies to therapeutically target the tumor microenvironment (TME) have emerged as a promising approach for cancer treatment due to the critical roles of the TME in regulating tumor progression and modulating response to standard-of-care therapies\u003csup\u003e[3, 4]\u003c/sup\u003e. Correspondingly, the immunotherapy of cancer patients using anti-PD-1 and PD-L1 mAb has shown substantial clinical response albeit only in a subset of cancer patients, necessitating the understanding of mechanisms of TME mediated resistance.\u003c/p\u003e\u003cp\u003eTumor-infiltrating lymphocytes (TILs), particularly CD8\u003csup\u003e+\u003c/sup\u003eT cells, present as exhausted in a wide variety of animal models and in humans with cancer or chronic viral infections\u003csup\u003e[5, 6]\u003c/sup\u003e. Both human and murine data support the existence of a highly heterogenous pool of CD8\u003csup\u003e+\u003c/sup\u003eT cell that differ in their transcriptome, epigenome, proteome, and functionality\u003csup\u003e[7, 8]\u003c/sup\u003e. The spatial interactions among immune and cancer cells generate complex ecological dynamics that influence tumor progression and treatment response\u003csup\u003e[9, 10]\u003c/sup\u003e. The heterogenous distribution of immune infiltrates in the TME has been associated with clinical implications in several cancers, such as melanoma and biliary tract cancer\u003csup\u003e[11]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eRecently, the expression and function of CD39 and CD73 in human solid tumors has gained research interest\u003csup\u003e[12]\u003c/sup\u003e. CD39 is an ectonucleotidase presented by B cells, innate cells, regulatory T cells and activated CD4 and CD8 T cells, which, in coordination with CD73, can stimulate the local production of adenosine and create an immunosuppressive environment \u003csup\u003e[13]\u003c/sup\u003e. It has been reported that the expression of CD39 on CD8\u003csup\u003e+\u003c/sup\u003eT cells is a candidate marker for exhausted TILs\u003csup\u003e[14]\u003c/sup\u003e. Moreover, CD39 is highly expressed on empirically defined neoantigen- and tumor-associated-antigen- reactive T cells in lung cancer and melanoma, proposed CD39 as a marker of tumor-reactive CD8\u003csup\u003e+\u003c/sup\u003eT cells\u003csup\u003e[15]\u003c/sup\u003e. This suggests that CD39 could be a useful marker of tumor-specific CD8\u003csup\u003e+\u003c/sup\u003eT cells, which could be exploited for the development of novel biomarkers or therapeutics.\u003c/p\u003e\u003cp\u003eHere we report a new mechanism of resistance to anti-PD-1 therapy. It was shown that hypoxia mediated heterogenous distribution of the CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cell subsets in lung cancer and revealed the CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cell levels within the peripheral TME may act as a candidate biomarker for immunotherapy, as well as being used to select patients for anti-PD-1 therapy. On the other hand, absence of CD39 limited functional restore of CD8\u003csup\u003e+\u003c/sup\u003eT cells upon PD-1 blockade, implying that resistance to immunotherapy and therapeutic failure could be partially attributed by reduce of CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells resulted from the PD-1 blockade.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Patients\u003c/h2\u003e\u003cp\u003eFifty-three patients with lung cancer were admitted to the First Affiliated Hospital of Soochow University from 2019 to 2025. None had received anti-tumor therapy at the time of sample analysis. Clinic information included age, sex, histological subtype, pathological stage, and primary tumor size. This study was approved by the Institutional Review Boards of The First Affiliated Hospital of Soochow University (2019-070). The processing of clinical tissue samples is in strict compliance with the ethical standards of the Declaration of Helsinki. Informed consent was obtained from all participants.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Acquisition of tumor samples\u003c/h2\u003e\u003cp\u003eA bronchoscope was used to obtained tumor tissues from patients. Once the neoplasm was visualized under bronchoscopy, a superficial biopsy was performed directly. If lung cancer lesion (peripheral lesions) was invisible under bronchoscopy, intratumoral endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) was performed using a 22-gauge needle.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Isolation of lymphocytes from lung cancer tissue\u003c/h2\u003e\u003cp\u003eFresh tissues were dissected and placed in RPMI medium, then disrupted mechanically using scissors. The tissues were then digested using a mixture of DNase I (0.3 mg/ml, Sigma-Aldrich) and Liberase\u0026trade; TL (0.2 mg/ml, Roche) in serum-free RPMI 1640 medium (Gibco, USA) for 30 min at 37\u0026deg;C, and subsequently strained through a 70-\u0026micro;m cell strainer (Beyotime Biotechnology). With regard to fresh tissues obtained from intratumoral aspiration (EBUS-TBNA), the erythrocytes were lysed with 10 mL of ACK lysis buffer (Beyotime Biotechnology) at 37\u0026deg;C for 10 min. Then isolated cells were suspended in 1 ml of RPMI 1640 medium and stained for analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Flow cytometry\u003c/h2\u003e\u003cp\u003eSingle-cell suspensions containing 2\u0026times;10⁶ cells/ml were prepared prior to immune staining. Following this, the cells were stained with several antibodies for 30 min 4\u0026deg;C in the dark: APC-Cyanine7-labeled anti-CD45 mAb (HI30), FITC-labeled anti-CD8α mAb (RPA-T8), PE-Cyanine7-labeled anti-CD39 mAb (A1), and APC-labeled anti-PD-1 mAb (EH12.2H7). All antibodies were purchased from Biolegend (USA). We used the Fluorescence Minus One (FMO) Control to help identify and gate cells, details are provided in the supplementary figure\u003csup\u003e[16]\u003c/sup\u003e. Lymphocytes were gated on SSC-A and FSC-A. CD39\u003csup\u003e+\u003c/sup\u003e and PD-1\u003csup\u003e+\u003c/sup\u003e TILs were gated on CD45\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells. Gating strategies are shown in detail in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003ea. Multi-colored flow cytometry was performed using Cytoflex (Beckman), and data were analyzed using FlowJo software (Tree Star).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Immunofluorescence analysis\u003c/h2\u003e\u003cp\u003eFrozen sections (5\u0026micro;m in thickness) were prepared from tumor tissue, embedded in optimum cutting temperature compound (Sakura Finetek). Then, sections were incubated with 3% BSA for 1 h at 37◦C. Following this, they were stained with rat anti-human CD39 mAb (1:150) overnight at 4◦C. After washing with PBS, primary antibodies were detected with rabbit anti-rat CD8 mAb (1:300). In some cases, rat anti-human HIF-α mAb (1:150) was stained. After washing, sections were subsequently embedded with DAPI. Sections were scanned using Nikon imaging system (Eclipse Ni-U).To count the number of target cells, six representative HPFS (\u0026times;400 or \u0026times;200 amplification) were selected for each section. The number of target cells in each field of view was calculated, and then statistical analysis was conducted using Graphpad.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 In vitro CD39 inhibition assay\u003c/h2\u003e\u003cp\u003eSingle cells from lung cancer tissues were cultured with CD39 inhibition POM-1 (100 \u0026micro;mol/L, Sigma-Aldrich) or vehicle for 2h at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e[13, 17]\u003c/sup\u003e. After culture, the cells were stained with several antibodies for 30 min at 4\u0026deg;C in the dark: APC-Cyanine7-labeled anti-CD45 mAb (HI30), FITC-labeled anti-CD8α mAb (RPA-T8), and PE-Cyanine7-labeled anti-CD39 mAb (A1). Cells were then fixed (eBioscience\u0026trade; Foxp3/ Transcription Factor Staining Buffer Set, ThermoFisher) for 30 min and stained with PE-labeled anti-granzyme B mAb (QA18A28) in a permeabilization buffer (ThermoFisher) for 30 min\u003csup\u003e[18]\u003c/sup\u003e. Multi-colored flow cytometry was performed using Cytoflex (Beckman) and data were analyzed using FlowJo software (Tree Star).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 In vitro blockade culture\u003c/h2\u003e\u003cp\u003eHuman lung cancer tissues were placed in RPMI 1640 medium containing 5% fetal bovine serum (FBS) and 1% antibiotics, and immediately transported to the laboratory at 4\u0026deg;C. After removing necrotic tissues, blood clots, and other debris, the tumor tissues were washed several times with PBS and cut into 1 mm2 fragments in the culture medium. An appropriate amount of 0.1% collagenase IV was added, and the mixture was incubated in a shaker water bath at 37\u0026deg;C. After 30 min, the digestion was terminated by adding culture medium. The cell suspension was filtered through a 100-\u0026micro;m cell strainer and centrifuged for 5 min, discarding the supernatant. The cells were resuspended in culture medium and randomly divided into two treatment groups: (i) control IgG; and (ii) human anti-PD-1 mAb (10 \u0026micro;g/mL, Biolegend). After incubation at 37\u0026deg;C with 5% CO₂ for 72 h, cells were stained with fluorochrome-conjugated antibodies for CD45, CD8, and CD39, followed by flow cytometric analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Assessment of Immunotherapy\u003c/h2\u003e\u003cp\u003eAntitumor efficacy was assessed by reviewing computed tomography (CT) scans following four treatment courses. The same imaging modality was recommended for all patients. The change from baseline regarding the sum of target lesion diameters was measured according to RECIST 1.1.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Statistical Analysis\u003c/h2\u003e\u003cp\u003eDifferences in mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of mean (SEM) were evaluated with Student\u0026rsquo;s t-test, paired t-test, and Welch\u0026rsquo;s t-test. The correlation analysis was evaluated with Pearson correlation and linear regression. The association between response status and TIL frequency was analyzed using Fisher\u0026rsquo;s exact test. Receiver operating characteristic (ROC) curves for response status were constructed to assess the prognostic ability of TILs. All analyses were two-sided and performed at a significance level of 5% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) using GraphPad 8.0.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Patient characteristics\u003c/h2\u003e\u003cp\u003eThe baseline characteristics of the 53 patients with lung cancer are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The median age was 66 years (range, 37\u0026ndash;87 years), and 83.01% of patients were male. Histological analysis revealed 30 squamous cell carcinomas, 14 adenocarcinomas, and 9 small cell carcinomas. Over half of the patients (50.95%) were in the late pathological stage (IIIB-IV), and 30 (56.6%) patients had a primary lesion larger than 3 cm.\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\u003ePatient clinical characteristics.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCharacteristics\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNumber(%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSex\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e44(83.01)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e9 (16.99)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge (years)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e20(37.74)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e33(62.26)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTypes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNSCLC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e44(83.01)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAdenocarcinomas\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e14(26.41)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSquamous carcinomas\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e30(56.60)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSCLC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e9 (16.99)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII\u0026ndash;IIIA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e26(49.05)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIIIB\u0026ndash;IV\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e27(50.95)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSize(cm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e23(43.40)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e30(56.60)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Infiltration of CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cell subsets in human lung cancer\u003c/h2\u003e\u003cp\u003eTo further explore the potential contribution of CD8\u003csup\u003e+\u003c/sup\u003eT cells to the TME, we examined the expression of the ectonucleotidases CD39 on CD8\u003csup\u003e+\u003c/sup\u003eT cell in lung cancer prior to any therapy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). We found that the percentage of infiltrated CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells was comparable between lung cancer patients from stage II-IIIA to IIIB-IV (36.12\u0026thinsp;\u0026plusmn;\u0026thinsp;4.53% vs 28.46\u0026thinsp;\u0026plusmn;\u0026thinsp;4.80%, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In addition, similar percentages of infiltrated CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells were observed between different ages, pathologic types, and different lesion sizes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). While it was noticed that the percentage of CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cell subsets was higher in males than that in females (35.54\u0026thinsp;\u0026plusmn;\u0026thinsp;3.71 vs 15.98\u0026thinsp;\u0026plusmn;\u0026thinsp;4.39%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Decreased infiltration of CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells in center of lung cancer tissue\u003c/h2\u003e\u003cp\u003eTo gain a more in-depth understanding of CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cell infiltration, peripheral and central TME were assessed whether heterogenous reginal distribution was involved in its accumulation. Here, neoplasm-superficial biopsy and intratumoral EBUS-TBNAs were used to obtain peripheral and central lung cancer tissue samples, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells were both found in the peripheral and central sites, whereas peripheral TME was more enriched with this T cell subset than that in the central TME (36.84\u0026thinsp;\u0026plusmn;\u0026thinsp;3.58% vs 16.45\u0026thinsp;\u0026plusmn;\u0026thinsp;6.34%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Heterogeneous infiltration of CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells is dependent on tumor size\u003c/h2\u003e\u003cp\u003eWe next sought to determine whether this heterogenous distribution of CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells was correlated with tumor size. In Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, distinct infiltrated profile of CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells between peripheral and central region was only found in lesions with diameter larger than 3cm (39.98\u0026thinsp;\u0026plusmn;\u0026thinsp;4.68% vs 3.59\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), rather than tumor diameters less than 3cm (31.94\u0026thinsp;\u0026plusmn;\u0026thinsp;5.51% vs 25.64\u0026thinsp;\u0026plusmn;\u0026thinsp;9.56%, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). As supported, immunofluorescence technique was further verified the distribution of tumoral CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells within TME, which was consistent with the findings obtained by flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Reduced CD39 expression on intratumoral CD8\u003csup\u003e+\u003c/sup\u003eT cell under hypoxia\u003c/h2\u003e\u003cp\u003eAs CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells were heterogeneous resident in the context of compartment of TME characterized by location and size, we hypothesized that hypoxia has distinct effects on continuous experience of CD8\u003csup\u003e+\u003c/sup\u003eT cell within TME. Then, immunofluorescence technique was again used to verify the distribution of HIF-α within central and peripheral TME. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, central region exhibited significant enrichment of HIF-α compared to peripheral tumor.\u003c/p\u003e\u003cp\u003eTherefore, we sought to a model mimic this stress in vitro, where fresh lung cancer derived cells were co-cultured under conditions of HIF-α (100ng/ml) for 3 d. Accordingly, it was found that HIF-α significantly decreased the expression of CD39 on CD8\u003csup\u003e+\u003c/sup\u003eT cells, resulting in a significant decrease in the number of CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Thus, these data show that a strong potential for the reginal hypoxia to serve as a mechanistic role in heterogenous CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cell infiltration.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Abundance of peri-neoplastic CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells predicted efficacy of PD-1 blockade\u003c/h2\u003e\u003cp\u003eOf the total cases studied, 17 patients were administered anti-PD-1 therapy. We then investigated whether the quantification of CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells could serve as a predictive biomarker for response to anti-PD-1 therapy. Initial analysis revealed that random CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells displayed similar level between responders and non-responders (45.02\u0026thinsp;\u0026plusmn;\u0026thinsp;19.13% vs 34.80\u0026thinsp;\u0026plusmn;\u0026thinsp;17.83%, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). It only produced 80.00% sensitivity and 71.43% specificity, with a corresponding AUC of 0.6571 to predict therapeutic response to anti-PD-1 mAb (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eConsidering central tumor CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cell vary according to lesion size and hypoxia, peri-neoplastic CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells was selected as candidate biomarker for anti-PD-1 therapy. In this cohort (n\u0026thinsp;=\u0026thinsp;13), it was found that the fraction of CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells in the tumor samples from responders (n\u0026thinsp;=\u0026thinsp;8) was significantly higher compared to non-responders (n\u0026thinsp;=\u0026thinsp;5), (52.05\u0026thinsp;\u0026plusmn;\u0026thinsp;11.81% vs 24.72\u0026thinsp;\u0026plusmn;\u0026thinsp;4.433%, p\u0026thinsp;\u0026le;\u0026thinsp;0.01). An AUC of 1.00 with a PPV of 1.00 and NPV of 1.00 suggests that the peri-neoplastic CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cell fraction can serve as a potential predictive biomarker of therapeutic response to anti-PD-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B).\u003c/p\u003e\u003cp\u003eNext, it is generated by calculating the percentage change in the total diameter of the target lesions compared to the baseline. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and D, unlike random CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells (r=-0.2355, p\u0026thinsp;=\u0026thinsp;0.3628), the higher the degree of peri-neoplastic CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells infiltration correlated with the deeper the clinical remission after receiving anti-PD-1 treatment (r=-0.7793, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.7 Absence of CD39 limited functional restore of CD8\u003csup\u003e+\u003c/sup\u003eT cells upon PD-1 blockade\u003c/h2\u003e\u003cp\u003eTo further confirm that immunotherapy is more effective in patients with higher CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells, we explored the correlation of CD39 expression and PD-1, whom is the target of anti-PD-1 mAb. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells expressed significantly higher levels of PD-1 compared to their CD39\u003csup\u003e\u0026minus;\u003c/sup\u003ecounterparts (54.21\u0026thinsp;\u0026plusmn;\u0026thinsp;4.00% vs 16.18\u0026thinsp;\u0026plusmn;\u0026thinsp;2.77%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Especially, CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells derived from the peri-neoplastic site were more PD-1 enriched than those from the central-neoplastic TME (59.83\u0026thinsp;\u0026plusmn;\u0026thinsp;3.40% vs 44.83\u0026thinsp;\u0026plusmn;\u0026thinsp;7.45%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This pattern indicated that CD39\u003csup\u003e+\u003c/sup\u003e subset might be the dominant CD8\u003csup\u003e+\u003c/sup\u003eT cells population adapted by the PD-1 blockade.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCorrespondingly, we stimulated single cells isolated from human lung cancer tissues with anti-PD-1 mAb in vitro for 72 h to mimic clinical conditions. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, anti-PD-1 treatment significantly reduced CD39 expression on CD8\u003csup\u003e+\u003c/sup\u003eT cells, leading to a marked decrease in the proportion of CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These data further support the notion that CD39\u003csup\u003e+\u003c/sup\u003e subset could be a main targeted CD8\u003csup\u003e+\u003c/sup\u003eT cell regulated by ICI.\u003c/p\u003e\u003cp\u003eNext, we aimed to determine the function of CD39 expressed by CD8\u003csup\u003e+\u003c/sup\u003eT cells by Polyoxometalate 1 (POM-1), which was used to block CD39 eATPase activity. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, the CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells demonstrated increased cytotoxic potential, indicated by a higher frequency of granzyme B positive cells following POM-1 treatment (36.32\u0026thinsp;\u0026plusmn;\u0026thinsp;2.69% vs 29.47\u0026thinsp;\u0026plusmn;\u0026thinsp;3.41%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while this effect was not found in CD39\u003csup\u003e\u0026minus;\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells. Collectively, these data show that CD39 is not merely a marker to define which CD8\u003csup\u003e+\u003c/sup\u003eT cell subset would be renewed capacity after receiving anti-PD-1 treatment, but serves a mechanistic role in rendering CD8\u003csup\u003e+\u003c/sup\u003eT cells dysfunctionality.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study provides the comprehensive characterization of spatial heterogeneity in CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells distribution within NSNLC. It was demonstrated that CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells are significantly enriched in peripheral tumor regions compared to central areas, and worked as a biomarker for anti-PD-1 therapy. Importantly, regional hypoxia was identified as a mechanism reducing CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells. Furthermore, our data reveal that the loss of CD39 limits functional restoration of CD8\u003csup\u003e+\u003c/sup\u003eT cells following PD-1 blockade, together with downregulated CD39 expression driving by anti-PD-1 therapy itself, these findings not only advance our understanding of spatial immune architecture in tumors but also uncover a novel mechanism of immunotherapy resistance.\u003c/p\u003e\u003cp\u003eRecent developments in studies of tumor heterogeneity have led to new insights into cancer management. For example, a higher level of intratumoral but not peritumoral CD8\u003csup\u003e+\u003c/sup\u003eT cells in conjunction with a low level of myeloid cells was reported to be associated with favorable outcomes in melanoma patients treated with mitogen-activated protein kinase inhibitors\u003csup\u003e[19]\u003c/sup\u003e. In estrogen receptor (ER)-negative/human epidermal growth factor receptor 2 (Her2)-negative\u003csup\u003e[20]\u003c/sup\u003eand Her2-positive\u003csup\u003e[21]\u003c/sup\u003e breast cancer patients, a high degree of immune infiltration in tumor stroma was associated with increased survival and complete response rates. In lung cancer, recent evidence shows that PD-1\u003csup\u003e+\u003c/sup\u003eTim-3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e tissue-resident memory T cells preferentially localized at invasive margins compared to tumor centers, with decreased spatial interactions independently associated with recurrence\u003csup\u003e[22]\u003c/sup\u003e. Tian et al. showed that CD8\u003csup\u003e+\u003c/sup\u003eT cell infiltration markedly reduced in larger tumors\u003csup\u003e[23]\u003c/sup\u003e. These observations align with our findings, implying heterogeneity of CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cell distribution within the TME of NSCLC extend concepts in spatial tumor immunology, establishing tumor size and location as a critical determinant of immune spatial organization.\u003c/p\u003e\u003cp\u003eThen, we hypothesized that regional hypoxia correlates strongly infiltrated degree of CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells. As reported, hypoxic conditions promoted exhausted-like states through reactive oxygen species generation\u003csup\u003e[24]\u003c/sup\u003e. Scharping et al. elucidated the continuous TCR stimulation combined with hypoxia rapidly induces T cell exhaustion\u003csup\u003e[25]\u003c/sup\u003e. In NSCLC specifically, Giatromanolaki et al. confirmed that CD73 and CD39 expression correlates with HIF-α, creating an immunosuppressive feedback loop\u003csup\u003e[26]\u003c/sup\u003e. In our study, HIF-α treatment significantly reduces CD39 expression on CD8\u003csup\u003e+\u003c/sup\u003eT cells, indicating hypoxia as a driver of decreased CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cell frequency in tumor. And presumably, the more severe hypoxic conditions in larger tumor centers explain why spatial heterogeneity manifests only in tumors exceeding 3 cm.\u003c/p\u003e\u003cp\u003eDespite available biomarker stratification, clinical responses vary\u003csup\u003e[27]\u003c/sup\u003e. Thus, the search for novel biomarkers with improved response predictions is ongoing. Previous study demonstrated that CD39 is highly expressed on neoantigen-reactive CD8\u003csup\u003e+\u003c/sup\u003eT cells\u003csup\u003e[28]\u003c/sup\u003e, supporting CD39 as a marker of tumor-reactive T cells. Duhen et al. found that CD103\u003csup\u003e+\u003c/sup\u003eCD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells are enriched for tumor-specific TCR clones across multiple solid tumor types, while maintaining cytotoxic capacity through granzyme B and perforin\u003csup\u003e[14]\u003c/sup\u003e. Responsively, CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cell proportion can serve as a potential biomarker that predicts response to PD-1 or PD-L1 blockade\u003csup\u003e[29]\u003c/sup\u003e. For example, Chow et al. used mass cytometry to analyze 440 lung cancer specimens, confirming that CD39 identifies CD8\u003csup\u003e+\u003c/sup\u003eT cells as predictive of checkpoint blockade efficacy\u003csup\u003e[16]\u003c/sup\u003e. In our study, we suggested that peripheral CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells might be a better biomarker to predict response to anti-PD-1 therapy. Therefore, evaluating the proportion of regional CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells in TMEs may be a complementary approach to PD-L1 staining for patient stratification prior to immunotherapy.\u003c/p\u003e\u003cp\u003eGiven the higher prevalence of EGFR mutations in female NSCLC patients\u003csup\u003e[30]\u003c/sup\u003e, the observed gender difference likely reflects underlying molecular subtype distributions. Previous reports showing higher frequencies of CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells in EGFR wild-type tumors compared to EGFR-mutant tumors\u003csup\u003e[31]\u003c/sup\u003e. Our recent works further supports this, demonstrating that EGFR mutations suppress CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cell infiltration in malignant pleura effusion\u003csup\u003e[32]\u003c/sup\u003e. However, the data on whether the accumulation of CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cell was associated with the status of EGFR in the TME of lung cancer are not presented in this paper. However, as shown earlier, while CD39 expression reflects T cell reactivity to antigen exposure, we believe that modest response rates for anti-PD-1 therapy in EGFR-mutant NSCLC was associated to lack of these CD8\u003csup\u003e+\u003c/sup\u003eT cells.\u003c/p\u003e\u003cp\u003eIt has been shown that anti-PD-1 can reinvigorate exhausted CD8\u003csup\u003e+\u003c/sup\u003eT cells. However, this might be dependent on the initial cell activation status where tumor-reactive effector cells could be targeted by anti-PD-1 mAb. We found that CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells exhibit higher PD-1 compared to CD39\u003csup\u003e\u0026minus;\u003c/sup\u003e counterpart. This CD39/PD-1 co-expression pattern carries important biological implications, indicated that CD39\u003csup\u003e+\u003c/sup\u003e tumor-reactive T cell subset might be the dominant CD8\u003csup\u003e+\u003c/sup\u003eT cells population adapted by the PD-1 blockade. Alternatively, CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells were depleted by anti-PD-1 mAb. This, in turn, leads to lack of pool in a substantial fraction of CD8\u003csup\u003e+\u003c/sup\u003eT cells to response to PD-1 blockade, further support the notion that CD39\u003csup\u003e+\u003c/sup\u003e subset could be a main targeted CD8\u003csup\u003e+\u003c/sup\u003eT cell regulated by anti-PD-1 mAb. Actually, functional CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cell is generated when CD39 is blocked by POM-1, inducing an effector phenotype. Through these experiments, it was found that CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells are not merely an indicator for anti-PD-1 therapeutic efficacy, but delineated a complex resistance network. This reveals a paradox: while CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells represent the primary targets of anti-PD-1 therapy, the treatment itself may deplete this population, absence of CD39 limited functional restore of CD8\u003csup\u003e+\u003c/sup\u003eT cells upon PD-1 blockade. The clinical implications are profound, suggesting that PD-1 blockade alone may be insufficient to fully activate anti-tumor immunity, particularly in large or severely hypoxic tumors, suggesting that ameliorating hypoxia enhances immunotherapy responses.\u003c/p\u003e\u003cp\u003eSeveral limitations warrant consideration when interpreting our results. First, the relatively small sample size, particularly the 13 patients receiving anti-PD-1 therapy, may limit statistical power and generalizability. Second, since most patients undergoing intratumoral EBUS-TBNA sampling did not receive subsequent anti-PD-1 therapy, we could not directly assess the relationship between central CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cell levels and treatment response. Finally, our relatively brief in vitro experiments may not fully recapitulate long-term in vivo treatment effects. Therefore, our findings suggest several important avenues for future investigation. Large-scale prospective cohort studies should validate CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells as immunotherapy predictive biomarkers, particularly incorporating spatial distribution information into predictive models. Mechanistic studies dissecting the hypoxia-HIF-α-CD39 axis may reveal novel therapeutic targets and resistance mechanism.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics Approval and Consent to Participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Institutional Review Boards of The First Affiliated Hospital of Soochow University (2019-070). The processing of clinical tissue samples is in strict compliance with the ethical standards of the Declaration of Helsinki. Informed consent was obtained from all participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (NSFC) grant 81874110 (to QQ), 81672280 (to CC), Natural Science Foundation of Jiangsu Province grant BK20181174 (to QQ), and Gusu Talent Project GSWS2019031(to QQ).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCC and QQX conceived the idea and designed and supervised the study. WJJ, ZJW and LLL performed the experiment and drafted the manuscript. ZYX, WJJ, ZJW, LLL and SY collected data. WJJ, ZJW and LLL analyzed data and performed statistical analysis. All authors reviewed and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thanked Jingyu Mao and Meiqin Su (The First Affiliated Hospital of Soochow University) for the sample collecting.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCEREZO-WALLIS D, CONTRERAS-ALCALDE M, TROUL\u0026eacute; K, et al. Midkine rewires the melanoma microenvironment toward a tolerogenic and immune-resistant state [J]. Nat Med, 2020, 26(12): 1865-77.\u003c/li\u003e\n\u003cli\u003eSUN R, ZHANG Z, BAO R, et al. Loss of SIRT5 promotes bile acid-induced immunosuppressive microenvironment and hepatocarcinogenesis [J]. 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J Transl Med, 2024, 22(1): 653.\u003c/li\u003e\n\u003c/ol\u003e"}],"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":"CD39, PD-1, CD8+T cell, HIF-α, Tumor microenvironment, lung cancer, Immunotherapy","lastPublishedDoi":"10.21203/rs.3.rs-7102657/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7102657/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUnderstanding resistance to anti-PD-1 is crucial for the development of reversal strategies. Here, we analyzed a subset of tumor-infiltrating CD8\u003csup\u003e+\u003c/sup\u003eT cells based on the expression of the ATP ectonucleotidase CD39, further explored its spatial distribution being priming as a contributor to anti-PD-1 therapeutic response or resistance. It is interesting that CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells were more enriched in peritumor compared to center, as well as being severed as a potential immuno-responsive biomarker for anti-PD-1 therapy. Correspondingly, it revealed more hypoxia actually existed in central TME, thereby reducing CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells infiltration and dampening the efficacy of anti-PD-1. As supported, multiple in vitro assays demonstrated that absence of CD39 limited functional restore of CD8\u003csup\u003e+\u003c/sup\u003eT cells upon PD-1 blockade. Collectively, we illustrated the hypoxia was involved in establishing an immunosuppressive TME defined by spatial distribution of CD39\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells and consequentially implied a novel approach of resistance to immunotherapy.\u003c/p\u003e","manuscriptTitle":"Decrease of tumoral CD39+CD8+T cells promoted by hypoxia confers resistance to immunotherapy in NSCLC","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-16 12:18:00","doi":"10.21203/rs.3.rs-7102657/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":"25c3818f-28ab-433f-977d-3eb52996b580","owner":[],"postedDate":"July 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-25T16:28:36+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-16 12:18:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7102657","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7102657","identity":"rs-7102657","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}