Regulatory T-cell sensing of extracellular ATP via P2RX7 promotes their accumulation and suppression and drives lung tumor growth

Abstract

Lung cancer is the leading cause of cancer-related deaths worldwide and, despite advances in treatment, immune suppression remains an obstacle to effective therapy. Effector CD4⁺ T cells (CD4⁺ Teffs) are critical for antitumor immunity, but their function is often inhibited by regulatory T cells (Tregs), which accumulate in lung tumors and mediate suppressive functions through multiple mechanisms. This suppression leads to tumor progression and poor patient outcomes. However, the mechanisms underlying Treg-mediated suppression are not fully understood. Herein, we identify the extracellular ATP receptor P2RX7 as a key regulator of Treg function in lung tumors. In a murine lung cancer model induced by Lewis lung carcinoma cells, we found that P2RX7 enhanced the suppressive capacity of tumor-infiltrating Tregs, promoting tumor growth. In T cell–specific P2RX7-KO mice, reduced Treg infiltration was accompanied by increased CD4⁺ Teff accumulation and improved tumor control. Treg-specific P2RX7-KO mice exhibited reduced tumor growth, confirming a Treg-intrinsic role of P2RX7. Suppression assays revealed that tumor-infiltrating wild-type Tregs had greater suppressive activity compared to P2RX7-KO Tregs, which failed to inhibit type 1 and Tfh-like responses. This was associated with increased tumor-specific IgG production by lung B cells in P2RX7-KO mice. We also observed that wild-type Tregs expressed higher levels of the immunosuppressive molecule CTLA-4 when compared to P2RX7-KO Tregs. Thus, we conclude that P2RX7 expression on Tregs is essential for their suppressive function in lung cancer and targeting of P2RX7 may constitute a strategy to improve lung cancer treatment by alleviating Treg-mediated immune suppression.

INTRODUCTION

Lung cancer remains the leading cause of cancer-related deaths worldwide, accounting for over 1.8 million deaths annually worldwide (1). Despite recent advances in early diagnosis and the development of immunotherapies, the prognosis for patients with lung cancer, especially non-small cell lung cancer (NSCLC), remains poor (2). A major challenge to successful therapy is the immunosuppressive environment established within the tumor, which hampers effective antitumor immune responses (3).

CD4⁺ T cells are key players in the immune response against lung tumors (4). These cells orchestrate antitumor immunity through many mechanisms, including direct cytotoxicity against tumor cells, support for cytotoxic CD8⁺ T cells, and collaboration with B cells to induce the production of tumor-specific antibodies (5,6). In both human and murine lung tumors, effector CD4⁺ T cells (CD4⁺ Teffs) have been shown to contribute to tumor control and to enhance the quality and magnitude of cytotoxic responses (7). Furthermore, CD4⁺ T-cell help is critical for the formation and maintenance of intratumoral tertiary lymphoid structures (TLS), which are associated with improved prognosis and better responses to immunotherapy (8). Despite their importance, the antitumor function of CD4⁺ Teffs is often undermined within the tumor microenvironment (TME) (4).

A major contributor to Teff dysfunction is the accumulation and suppressive activity of FOXP3⁺ CD4⁺ regulatory T cells (Tregs) (9). Tregs are enriched in lung tumors and inhibit Teff proliferation, cytokine production, and cytotoxic activity (10,11). Their presence is frequently associated with worse clinical outcomes and resistance to immune checkpoint blockade (12). Tregs can limit CD4⁺ Teff-mediated help for B cells and CD8⁺ T cells, dampening the ability of the immune system to eliminate tumor cells (13,14). Given their central role in immunosuppression, understanding the mechanisms that govern Treg accumulation and function in the lung TME is essential for developing strategies to restore effective antitumor immunity.

Several factors have been implicated in driving Treg function in tumors, such as cytokines, metabolic cues, and engagement of co-inhibitory receptors (15). However, the role of damage-associated molecular patterns (DAMPs), such as extracellular ATP (eATP), in shaping Treg responses in cancer remains incompletely understood (16). eATP is often released in inflamed or damaged tissues, including tumors, and is sensed by immune cells through purinergic receptors (17,18). Among these receptors, P2RX7 has emerged as a key regulator of immune cell fate and function in inflammatory and infectious settings (19). In Teffs, P2RX7 signaling has been linked to cell survival, tissue residency, and effector differentiation (20–22). However, whether and how P2RX7 contributes to the immunosuppressive activity of Tregs in the context of lung cancer has not been investigated.

In this study, we identified P2RX7 as a regulator of Treg function in lung tumors. Using human NSCLC public data and murine models of lung cancer, we found that P2RX7 promoted the suppressive capacity of tumor-infiltrating Tregs and favored their accumulation in the TME. CD4⁺ T cell–specific P2RX7-KO mice exhibited reduced tumor growth, accompanied by a significant increase in Teff infiltration and antitumor activity. Treg-specific deletion of P2RX7 revealed a Treg-intrinsic role of this receptor in enhancing Treg-mediated immunosuppression. We also found that lung P2RX7-KO Tregs failed to upregulate the key suppressive co-inhibitory receptor CTLA-4 and showed diminished ability to suppress both Th1 and Tfh-like responses. Together, our findings reveal a role for P2RX7 in promoting Treg-mediated suppression in lung cancer and suggest that targeting this pathway may shift the balance between Tregs and Teffs, unlocking antitumor immunity.

MATERIALS AND METHODS

Single-cell RNA-sequencing

Single-cell RNA sequencing (scRNA-seq) data from 42 samples from patients with NSCLC were obtained from the publicly available GEO NCBI repository (GSE148071) (23). Downstream analyses were performed using the Seurat v5 workflow in R. Each sample was individually filtered to exclude cells expressing fewer than 200 genes or less than 350 RNA features, as well as cells exhibiting mitochondrial gene expression above the 95th percentile. Cells with gene counts above the 95th percentile were also removed. Data integration was performed using Harmony, implemented within Seurat. After loading the count matrices into Seurat objects, we applied NormalizeData, FindVariableFeatures, ScaleData, and RunPCA (n_dims = 50), followed by batch correction with RunHarmony. Clustering was performed by running FindNeighbors and FindClusters, and cell types were assigned using the human Lung v2 reference from Azimuth (https://azimuth.hubmapconsortium.org). Cluster annotations were further refined based on canonical marker gene expression. Differential gene expression analysis was conducted using Seurat’s FindAllMarkers function, with significance thresholds set at p-adjusted < 0.1, |log2 fold change| ≥ 1, and expression in at least 30% of cells.

Spatial transcriptomics

10x Genomics Visium spatial transcriptomic data from 8 patients with NSCLC were obtained from the Human Tumor Atlas Network (HTAN) consortium (https://humantumoratlas.org). In summary, publicly available spatial transcriptomics (ST) data were obtained from the study by Zhao et al. (2025) (24) through the HTAN Data Coordinating Center. Results were accessed via the HTAN online portal (https://data.humantumoratlas.org/explore/) by selecting the HTAN HTAPP atlas and lung as the organ of interest. Processed ST data and mutation information can be downloaded from Zenodo (https://doi.org/10.5281/zenodo.16546233). A total of 20 spatial transcriptomic slides were analyzed, with 2 to 4 tissue sections profiled per patient. For each patient, samples were collected from distinct tumor regions within the same lung lobe or from different lobes to capture intratumoral spatial heterogeneity. Raw data processing and downstream analyses were conducted in R using the Seurat package. Spot transcriptomes were filtered to exclude spots with fewer than 300 detected genes. Sampling areas with fewer than 20 high-quality spot transcriptomes were excluded from further analysis. Count matrices were normalized and scaled using NormalizeData and ScaleData functions with default parameters. Deconvolution analysis was performed using RCTD (25), as implemented in the spacexr package. Single-cell RNA-seq data served as the reference. For each sampling area, an RCTD object was created by combining the single-cell reference and the corresponding SpatialRNA object. RCTD analysis was run using the run.RCTD function with the doublet mode parameter set to “full”.

Survival probability analysis

Survival data from over 1,000 NSCLC patients (Affy ID 207091_at; patients split by median; Histology: adenocarcinoma, n=1308) were analyzed using the Kaplan-Meier Plotter tool (https://kmplot.com/analysis). Overall survival was evaluated based on P2RX7 mRNA expression levels. Patients were stratified into “high” and “low” expression groups according to their P2RX7 median value. Kaplan-Meier survival curves were generated, and statistical significance was determined using the long-rank test.

Lung cancer biopsies collection and analysis

Samples (n=5) were collected from patients during biopsy (Mayo Clinic Hospital, Jacksonville; samples collected between September and October of 2025) with different types and stages of lung cancer (adenocarcinoma), rapidly frozen, and sent for analysis. Upon arrival, tissues were processed in RPMI medium (Gibco) with 0.5 mg/mL collagenase type IV (Gibco) and subjected to cell separation using GentleMACS. Cells were washed with FACS buffer (PBS 2% FBS) and stained with the following surface antibodies diluted in FACS buffer: anti-human CD4 (Spark UV 395, clone SK3, BioLegend), anti-human CD8 (PerCP, clone SK1, BioLegend, #344701), anti-mouse/human CD44 (BV786, clone IM7, BioLegend, Cat# 103041), anti-mouse P2RX7 (BV421, clone 1F11, BD Biosciences, Cat# 744779; laboratory-optimized staining), and a Live/Dead viability dye (Ghost Dye 778). Surface staining was performed for 40 min at room temperature. After staining, cells were washed with FACS buffer and fixed and permeabilized using the True-Nuclear^™ Transcription Factor Buffer Set (BioLegend, Cat# 424401) for 30 min at room temperature. Cells were then washed with the kit buffer (PermWash) and stained with anti-human FOXP3 (BB515, clone 236A/E7, eBioscience, #53-4777-42) diluted in PermWash overnight at 4°C. Data were acquired on a Cytek Aurora flow cytometer. For P2RX7 identification, fluorescent minus one (FMO) control samples were used. Samples were collected after written informed consent was obtained from the patients, and the studies were conducted in accordance with the Declaration of Helsinki. These studies were approved under the IRB protocol 14-004094 (Mayo Clinic Florida).

ChIP-Seq analysis

Raw FASTQ files were downloaded from the Gene Expression Omnibus under accession codes GSE139960 (25), GSE40684 (26), and DRA003955 (27) and processed using the Galaxy platform (https://usegalaxy.org/). Read quality control and adapter trimming were performed using Trim Galore and/or Trimmomatic. Trimmed reads were aligned to the reference genome using Bowtie, generating BAM files. Aligned reads were filtered, and PCR duplicates were removed using rmDuplicate. Peak calling was performed with MACS2, and the resulting peak files were exported in BED format for downstream analyses.

Mice

Male and female mice, aged 6–8 weeks, with a specific-pathogen-free status, were used in this study. Mice were housed in the animal facilities of The Department of Immunology at Mayo Clinic Arizona. All mice were randomly assigned to experimental groups. The following mouse strains were utilized: C57BL/6, RAG2-KO, CD4-cre, CD4-cre P2rx7^flox/flox, Foxp3 eGFP-Cre-ERT2, and Foxp3 eGFP-Cre-ERT2 P2rx7^flox/flox (all obtained from The Jackson Laboratory except the P2rx7^flox/flox, which was obtained from Gyorgy Hasko, Rutgers University). C57BL/6 Kras^LSL-G12D/+;Tp53^fl/fl (KP) mice were developed by Dr. Verline Justilien and previously published (26). All experimental procedures were conducted in accordance with institutional guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC – A00005172-20-R23, A00005542-20-R23, A99997437-R23 and A00005139-20-R23).

Cell lines, tumor injection and Ad-Cre intratracheal instillation

The Lewis lung carcinoma (LLC) cell line, both GFP-expressing (LLC-GFP) and non-GFP (LLC) variants, was used for murine lung cancer experiments. LLC cells were cultured in complete Dulbecco’s Modified Eagle Medium (DMEM - Gibco) supplemented with 10% fetal bovine serum (FBS – Cardinal Healthcare), 1% penicillin-streptomycin (Corning), and 1% L-glutamine (Gibco) at 37°C in a 5% CO₂ atmosphere. Cells were maintained in an exponential growth phase and were used for injections after confirming proper growth and morphology. LLC-GFP cells (5x10⁵) were subcutaneously or intravenously injected into experimental mice to establish ectopic primary tumors or lung metastases (27), respectively, while LLC cells were used in some experiments to assess tumor growth without GFP labeling. MC38 (colon) and R1WT (liver) tumor cells were injected subcutaneously, and tumor growth was monitored by measuring tumor volume (calculated as length × width² / 2). SB28 (brain) tumors were monitored by bioluminescence imaging due to their expression of luciferase. Lung tumors were monitored based on body weight stabilization or weight loss.

For experiments with cell lines representative of other tumor types, SB28 (brain), R1WT (liver), and MC38 (colon) cell lines were cultured using the same protocol described for the LLC cell line. SB28 cells (3 × 10⁴) were injected directly into the brain of C57BL/6 mice by surgical procedure, and tumor growth was monitored for 10 days before harvest. R1WT (5 × 10⁵) and MC38 (5 × 10⁵) cells were injected subcutaneously into the flank, and tumor growth was monitored for 21 and 14 days, respectively, followed by flow cytometry analyses. Lung tumors were initiated in KP mice (6-10 weeks old) through intratracheal instillation of Adenovirus expressing Cre recombinase (1.5 x 10⁸ PFU/ml) (Vector Laboratories, Malvern, PA, USA).

The LLC cell line was obtained from ATCC (2023). MC38 was obtained from Kerafast (2023). R1WT was obtained from Dr. Mitesh Borad (Mayo Clinic Arizona, 2024). SB28 was obtained from the Leibniz Institut-DSMZ (2024). LLC-GFP was engineered in house (2023). All lines were tested negative for Mycoplasma (IDEXX): LLC, LLC-GFP, R1WT and MC38 were last tested in June/2025, and SB28 was last tested in August/2025. LLC, LLC-GFP, R1WT are male cell lines, while SB28 and MC38 are female cell lines. In all experiments, thawed vials of tumor cells were passed twice (with a 2-to-3-day interval between passages) prior to inoculation into mice.

CD4⁺ T-cell purification and adoptive transfer experiments

For single adoptive transfer experiments, splenocytes were isolated from C57BL/6 (CD45.1, WT) and T cell–specific P2RX7-KO (CD45.2) mice. Naive CD4⁺ T cells were purified from splenocytes by negative selection using the EasySep^™ Mouse Naïve CD4⁺ T Cell Isolation Kit (STEMCELL Technologies), following the manufacturer’s instructions. The negative fraction, containing splenocytes depleted of CD4⁺ T cells, was washed with 1x PBS and transferred into RAG2-KO mice. Mice were randomly assigned into two groups, receiving either WT or P2RX7-KO naive CD4⁺ T cells. After 24 hours, both groups were injected intravenously with 5x10⁵ LLC-GFP tumor cells. Lungs were harvested on day 30 post-LLC injection (p.i.) for histological and flow cytometry analyses.

For co-adoptive transfer experiments, splenocytes from WT (CD45.1) and P2RX7-KO (CD45.2) mice were isolated and co-transferred into RAG2-KO mice. At 24 hours post-transfer, recipients were injected with 5x10⁵ LLC-GFP cells. Lungs were collected on day 30 p.i. for flow cytometry analysis.

Administration of tamoxifen in mice

On day 5, 7, 9 and 11 p.i., Foxp3 eGFP-Cre-ERT2 (WT) and Foxp3 eGFP-Cre-ERT2 P2rx7^flox/flox mice were treated intraperitoneally with 1 mg of tamoxifen (Sigma-Aldrich) diluted in sunflower oil (vehicle) (28).

Tissue processing for flow cytometry and ELISA

On day 30 p.i., lungs were harvested, and the lobes were processed and digested with 0.5 mg/mL (diluted in RPMI) collagenase type IV (Gibco) at 37°C for 40 minutes under agitation (200 rpm) (22). Mediastinal lymph nodes (medLNs) and spleens were mechanically processed using cell strainers (Corning). Lung homogenates were separated for IgG measurement by ELISA (see Antitumor IgG quantification). The cell suspension obtained from the tissues was homogenized, filtered through cell filters, and incubated with ACK lysis buffer (made in house) at room temperature for 2 minutes to deplete erythrocytes. The cell suspensions were washed with PBS containing 10% FBS. Afterward, the cell suspension was centrifuged at 1,200 rpm for 5 minutes and resuspended in FACS buffer (PBS + 2% FBS) until the time of staining.

Flow cytometry analysis

Cell suspensions from the lung, medLN, and spleen were stained with fluorochrome-conjugated monoclonal antibodies (see Supplementary Table S1 for details of the antibodies and other reagents used in this study) diluted in FACS buffer and incubated at room temperature (RT) for 40 minutes. After staining, cells were washed with FACS buffer and then prepared for intracellular IFN-γ staining (22). For this, lung cells were incubated with eBioscience^™ Cell Stimulation Cocktail plus protein transport inhibitors (Invitrogen, Cat# 00-4970-03 - 2 μM) for 4 hours at 37°C in 5% CO₂. After incubation, cells were fixed and permeabilized using the BD Cytofix/Cytoperm^™ Fixation/Permeabilization Kit (BD Biosciences) and then washed with the buffer provided in the kit. ViaDye^™ Red Fixable Viability Dye Kit (Cytek Biosciences) was used to identify dead cells. Samples were analyzed using multiparametric flow cytometry with a 27-parameter panel on Cytek Aurora (Cytek Biosciences) flow cytometer. Data was processed for conventional analysis and cluster distribution through t-distributed stochastic neighbor embedding (tSNE) using FlowJo software v10.8.2 (BD Biosciences).

Histological analysis

The upper left lung lobes were collected, rinsed in PBS, and fixed in 4% paraformaldehyde (PFA) for 24 hours at 4°C. Fixed tissues were then embedded in paraffin, sectioned into 4-5 μm slices, and stained with hematoxylin and eosin (H&E) to evaluate tissue pathology. Stained sections were visualized under a light microscope (Olympus IX71 Inverted microscope, 20X Objective, CCD cameras and using the Infinity Capture Software). Representative images were acquired for analysis.

Fluorescence microscopy

PFA-fixed paraffin-embedded samples were deparaffinized and subjected to antigen retrieval by steaming for 20 minutes in 1X Citrate Buffer (Diagnostic Biosystems). Next, sections were permeabilized with 2% Triton-X 100 in 1% bovine serum albumin (BSA) in PBS for 30 minutes, followed by blocking in 5% BSA in PBS for 30 minutes at RT. Sections were stained for 48 hours at 4°C in a humidity chamber in the dark with the following primary antibodies diluted in BSA blocking buffer: rabbit polyclonal anti-CD4 (1:50, Abcam, Cat#Ab288724), rat anti-B220 (1:200, Biolegend, Cat#103201) and mouse anti-Ep-CAM (1:100, Invitrogen, Cat#MA5-12436). Sections were washed three times with 0.1% Tween-20 in PBS before incubation for 2 hours at RT in the dark with goat anti-rabbit IgG AF546 (Invitrogen A11035) and goat anti-mouse IgG AF488 (Invitrogen A11001) secondary antibodies diluted (1:200) in BSA blocking buffer. Slides were counterstained with DAPI (Invitrogen, Cat#D1306) for 15 minutes, washed thrice, and then mounted with ProLong^™ Gold Antifade Mountant (Thermo Fisher Scientific) and secured with a coverslip. Sections were imaged using LSM 800 confocal microscope (Zeiss) equipped with Plan-Neofluar 20× objective lens (Zeiss, NA 0.4). Images were processed and analyzed using the Zeiss ZEN software, where background signals were adjusted based on unstained controls.

Quantitative PCR analyses

Tregs were isolated from lungs using StemCell columns (Supplementary Table S1). RNA was extracted with the RNeasy kit (QIAGEN, Cat#74134) and reverse-transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher, Cat#4368813). For qPCR reactions, 5 ng of template cDNA was used per reaction (three replicates per sample). Amplification was performed with ROX SYBR Green Master Mix (Applied Biosystems) on a QuantStudio 7 Pro system. Relative expression was calculated by the ΔΔCt method, normalizing Ct values of Ctla4 to the housekeeping gene Actb. The following primers were used: forward 5′-GTACCTCTGCAAGGTGGAACTC-3′ and reverse 5′-CCAAAGGAGGAAGTCAGAATCCG-3′ (Ctla4; ThermoFisher); forward 5’-TGAGCTGCGTTTTACACCCT-3’ and reverse 5’-TTTGGGGGATGTTTGCTCCA-3’ (Actb; ThermoFisher).

Suppression assays

Tregs (Foxp3/GFP⁺CD25⁺CD4⁺ T cells) were sorted from the lung of WT and Treg-specific P2RX7-KO mice at day 30 p.i. using a MA900 Multi-Application Cell Sorter (Sony Biotechnology). Sorted lung Tregs were plated with pre-activated WT CD4⁺ or CD8⁺ T cells isolated from the spleen with appropriate columns at 0:1, 1:5 and 1:20 and Treg/Teff ratios (STEMCELL Technologies) and stimulated in vitro with anti-CD3/CD28. Information about anti-CD3/CD28 and the kits used for cell isolation are shown in Supplementary Table S1. Teffs were labeled with CellTrace^™ Violet (Thermo Fisher Scientific) and Tregs were labeled with CFSE (Carboxyfluorescein succinimidyl ester), to allow tracking of proliferation and identification of distinct populations within the same well. After 4 days in culture, cells were collected, washed with FACS buffer, and stained for flow cytometry analysis. The percentage of suppression was calculated using the following formula:

$$\text{\%}\mathrm{s}\mathrm{u}\mathrm{p}\mathrm{p}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{s}\mathrm{i}\mathrm{o}\mathrm{n}=\frac{\text{\%}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}{{\mathrm{C}\mathrm{F}\mathrm{S}\mathrm{E}}^{diluted}}_{0:1}-\text{\%}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}{{\mathrm{C}\mathrm{F}\mathrm{S}\mathrm{E}}^{diluted}}_{1:n}}{\text{\%}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}{{\mathrm{C}\mathrm{F}\mathrm{S}\mathrm{E}}^{diluted}}_{0:1}}\left(29\right).$$

In vitro Treg activation and treatments

Splenic Tregs were isolated using the EasySep^™ Mouse CD4⁺CD25⁺ Regulatory T Cell Isolation Kit II (STEMCELL Technologies), following the manufacturer’s protocol. Cells were resuspended in RPMI medium supplemented with 10% of FBS, 1% L-glutamine, 1% sodium pyruvate (Sigma-Aldrich, Cat#58636), and 1% penicillin-streptomycin, then plated (5x10⁴) in 96-well plates pre-coated with anti-CD3 (10 μg/ml) and anti-CD28 (20 μg/ml) for in vitro activation. Interleukin-2 (mouse IL-2, Abcam, 10 ng/ml) was added daily to the culture. Some wells received the P2RX7 agonist HEI3090 (Axon Medchem, 50μM), A438079 (MedChemExpress – 100 μM) and Adenosine 5-triphosphate (ATP, Sigma-Aldrich, 50 μM) at the time of plating (30). After 72 hours, cells were washed with FACS buffer and stained for flow cytometry analysis.

CD8⁺ T-cell cytotoxicity assay

CD44⁺CD8⁺ T cells were sorted from the lungs of WT and P2RX7-KO tumor-bearing mice at day 30 p.i. and cultured with LLC-GFP cells (5 × 10⁴) at a 1:10 LLC:CD8 ratio. 24h after the start of co-culture, cells were collected from the wells and analyzed by flow cytometry to assess LLC cell viability.

P2RX7 inhibition in vivo

Lung tumor–bearing C57BL/6 mice were treated daily intranasally (35 μL) with vehicle (PBS) or the P2RX7 inhibitor A438079 (MedChemExpress; 80 mg/kg) from day 15 until day 30 p.i. On day 30, lungs and medLNs were collected for analysis.

Calcium influx assays

For assessment of calcium influx, cells were stained with surface markers, then incubated for 1 h at 37 °C with 1 μM Fluo-4-AM-FITC (ION Biosciences) and then washed with HEPES buffered saline (made in house) (31). Cells were analyzed by kinetic (Time vs Fluo-4-AM-FITC) flow cytometry, with a baseline reading being determined for 5 min followed by ionomycin (1 μg/ml) (STEMCELL Technologies) addition and continued analysis for the remaining 20 min. Subsequently, the P2RX7 agonist HEI3090 (Axon Medchem – 50 μM) was added, and cells were acquired for an additional 10 minutes. After this period, the P2RX7 inhibitor A438079 (MedChemExpress – 100 μM) was added (32), and cells were analyzed for another 10 minutes. The entire acquisition was performed at low speed and continuously, with the sample tube being removed for cell treatments without stopping acquisition. Flow cytometric analysis was performed using a FACSymphony^™ (BD Biosciences) and data were analyzed with FlowJo software v10.8.2 (BD Biosciences).

Antitumor IgG quantification

Tumor-specific IgG antibodies were quantified using an indirect ELISA approach. High-binding ELISA plates (ELISA MAX^™, BioLegend, Cat# 423501) were coated with LLC cell lysates overnight at 4°C. After 24 h, plates were washed three times with PBS containing 0.05% Tween-20 (washing buffer) and blocked with PBS supplemented with 3% BSA for 1 h at room temperature. Plates were then washed three times with washing buffer and incubated with lung homogenates from tumor-bearing WT and P2RX7-KO mice for 2 h at room temperature. Following incubation, plates were washed four times with washing buffer and incubated for 1 h with an HRP-conjugated anti-mouse IgG antibody (Cat# W402B). Plates were subsequently washed four times, and signal was developed using the TMB Substrate Kit (Thermo Scientific, Cat# 34021) (33). Optical density was measured at 450 nm using a Synergy HTX Multi-Mode plate reader (BioTek).

Statistical analysis

Specific statistical tests applied to each experiment are detailed in the respective figure legends. All analyses were conducted using GraphPad Prism 10.4.0 software. Data are presented as Mean values with standard deviation (SD) shown as error bars. For comparisons between two groups, Paired or Unpaired T-tests were used. Differences between groups were considered significant when p < 0.05 (*), p < 0.01 (**) or p < 0.001 (***).

RESULTS

P2RX7 is highly expressed in tumor-infiltrating T cells of lung cancer patients

To investigate the association between P2RX7 expression and lung cancer outcomes, we analyzed clinical datasets publicly available (see the Methods subsection “Survival probability analysis” for more information about the analyses). Patients with higher P2RX7 mRNA expression exhibited significantly poorer overall survival compared to those with lower expression levels (Fig. 1A). Next, P2RX7 expression at the cellular level was evaluated using scRNA-seq data from the TME of NSCLC patients. After quality control, 90,406 high-quality cells were retained from 42 advanced-stage NSCLC samples. These cells were clustered into nine major groups, comprising structural cells (epithelial, endothelial, and fibroblasts) and immune cells (macrophages, dendritic cells, B cells, and T cells) (Fig. 1B). P2RX7 expression was detected across all clusters, with the highest levels found in T cells (Fig. 1C). Further analysis of T-cell clusters revealed that among these cells, Tregs and Teff CD8⁺ expressed the highest level of P2RX7 (Fig. 1D–E). We validated this expression at the protein level in T cells isolated from biopsies of five lung cancer patients (Supplementary Table S2), and the data confirmed that Tregs expressed significantly higher levels of P2RX7 compared to CD4⁺ Teff cells (Fig. 1F; gating strategy in Supplementary Fig. S1D).

Fig. 1. P2RX7 expression on immune cells in lung cancer patients.

(A) Kaplan-Meier curves showing the overall survival values related to P2RX7 expression in TCGA database. (B) UMAP projection of 9 clusters of immune and non-immune cells in human lungs with cancer. (C) Boxplot showing the average expression (gMFI + percentiles) of P2RX7 in immune and non-immune cell clusters in human lungs with cancer. (D) UMAP showing the subclusters of CD4⁺ and CD8⁺ T cells in human lungs with cancer. (E) Boxplot (gMFI + percentiles) showing the average expression of P2RX7 in CD4⁺ and CD8⁺ T cells subclusters in human lungs with cancer. (F) Left: histograms of P2RX7 expression on CD44⁺CD4⁺ Tregs, Teff CD4⁺ and CD8⁺. Right: average frequencies of P2RX7⁺ on CD44⁺CD4⁺ Tregs, Teff CD4⁺ and CD8⁺. (G) Spatial images showing the spots expressing P2RX7, CD4 and FOXP3 in lung cancer grade 2 (G2) and grade 3 (G3) patients. (H) Violin plots showing the average expression of P2RX7, CD4 and FOXP3 in lung cancer G2 and G3 patients. (I) Spatial images showing the location of spots co-expressing CD4 and P2RX7 G2 and G3 lung cancer patients. (J) Spatial images showing the location of spots co-expressing FOXP3 and P2RX7 in G2 and G3 lung cancer patients. (K) Spatial images showing the location of spots co-expressing CD4 and FOXP3 in G2 and G3 lung cancer patients. Spatial images are representative sections from two patients. (L) Boxplot showing the average coexpression of CD4 and P2RX7, FOXP3 and P2RX7, and CD4 and FOXP3 in G2 and G3 lung cancer patients. Differences in P2RX7 expression between tumor grades were assessed using the Wilcoxon rank-sum test. p-values were calculated and displayed on the plot. Error bars represent standard deviation (SD) within each experimental group. (E) Data from n = 5 human samples/experimental group per experiment. Data shown as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Statistical significance was determined by one-way ANOVA (E-F; Tukey post tests) or unpaired t-tests (H, L).

We further analyzed spatial transcriptomics data from NSCLC patients to assess the expression patterns of P2RX7, CD4, and FOXP3 across distinct regions of tumor tissue (Supplementary Table S3) (25). A spatial trend in P2RX7 expression was observed in relation to tumor grade. Multiple P2RX7-positive spots were detected in both grade 2 (G2) and grade 3 (G3) tumors, with higher expression levels observed in G3 samples (Fig. 1G–H). CD4 expression was detected in both tumor grades; however, no significant differences in overall CD4 expression were observed between G2 and G3 tumors. Similar to P2RX7, FOXP3 signal was higher in G3 compared to G2 tumors. Both P2RX7 and FOXP3 showed increased expression levels in samples from patients with higher tumor grade. Finally, we analyzed the co-expression of these genes and observed a greater number of regions co-expressing CD4 - P2RX7, FOXP3 - P2RX7, and CD4 - FOXP3 in G3 patients when compared to G2 patients (Fig. 1I–K). This is consistent with our statistical analysis of the fraction of co-expression spots (Fig. 1L). Together, these data support the notion that CD4⁺ T cell–intrinsic P2RX7 expression in Tregs may serve as a negative prognostic biomarker in lung cancer.

Tregs exhibit higher P2RX7 expression than CD4⁺ Teffs in murine lung cancers

To investigate the in vivo role of P2RX7 in T-cell responses to lung cancer, we used an experimental murine model induced by the LLC cell line (Fig. 2A). To facilitate tumor visualization in the lung, we used GFP-expressing LLC cells (LLC-GFP). Cancer cells were injected intravenously, and 30 days p.i., we harvested the lungs and medLNs for analysis. GFP⁺ tumor nodules were macroscopically visible through the lung tissue (Fig. 2B, left). Histological analysis showed widespread tumor development beyond the visible nodules, with areas of immune-cell infiltration (Fig. 2B, right), indicating a robust ongoing antitumor immune response by day 30 p.i.

Fig. 2. P2RX7 expression on CD4⁺ T cells in lung cancer metastasis experimental model.

C57BL/6 (B6) mice were injected i.v. with LLC-GFP cells and lungs and medLNs were collected 30 days p.i. (A) Schematic illustration showing the lung cancer experimental protocol (Created in BioRender [Santiago de Carvalho, I]. [2026] [https://BioRender.com/uptb9q6]). (B) Left: Representative fluorescence macroscopy of the left upper lung lobes with tumors (20 μm). Right: Representative section of the left upper lung lobes stained with H&E (200 μm). (C) Left: histograms of P2RX7 expression on lung activated Tregs and Teffs. Right: average geometric mean fluorescence intensity (gMFI) of P2RX7 on lung activated Tregs and Teffs. (D) Left: histograms of P2RX7 expression on medLN activated Tregs and Teffs. Right: average gMFI of P2RX7 on medLN activated Tregs and Teffs. (E) Schematic illustration showing the lung cancer experimental protocol (Created in BioRender [Santiago de Carvalho, I]. [2026] [https://BioRender.com/ulf63sd]). (F) Representative section of the left upper lung lobe from KP (Kras^LSL-G12D Trp53^fl/fl) mice instilled intratracheally with Adeno-Cre virus, collected at 16 weeks post-instillation, and stained with H&E (1 mm). (G) Top: histograms of P2RX7 expression on lung-activated Tregs and Teff cells from KP mice. Bottom: average gMFI of P2RX7 on lung-activated Tregs and Teff cells from KP mice. (H) B6 mice were inoculated with brain (SB28), liver (R1WT), or colon (MC38) tumors, which were collected at different time points for analysis. Top: histograms of P2RX7 expression on activated Tregs and Teff cells from SB28, R1WT, and MC38 tumors. Bottom: average gMFI of P2RX7 on activated Tregs and Teff cells from SB28, R1WT, and MC38 tumors. (I) Schematic illustration of the lung cancer experimental protocol (Created in BioRender [Santiago de Carvalho, I]. [2026] [https://BioRender.com/a7obfux]). C57BL/6 mice were injected i.v. or in the flank with LLC-GFP cells, and lungs or flank tumors were collected at 30 and 14 days p.i., respectively. (J) Average gMFI of P2RX7 on activated Tregs and Teff cells from SB28, R1WT, and MC38 tumors; average gMFI of P2RX7 on activated Tregs from naive lungs, LLC lung tumors, and LLC flank tumors. (K) Average numbers of activated Foxp3⁺ Tregs from naive lungs, LLC lung tumors, and LLC flank tumors. (L) Public ChIP-seq data analysis from B6 mouse Tregs showing FOXP3 and TCF-1 binding at the P2rx7 active enhancer. Data from 2-3 independent experiments, n = 3–4/experimental group per experiment. Data shown as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Statistical significance was determined by one-way ANOVA (Tukey post tests).

We next analyzed activated CD4⁺ T cells (CD44⁺) in the lungs and medLNs of tumor-bearing mice (gating strategy in Supplementary Fig. S1A). We separated T cells into Teffs CD4⁺, CD8⁺, and Tregs and compared P2RX7 expression between these populations in both organs. Our data showed that among the T cells, Tregs expressed higher levels of P2RX7 than CD4⁺ and CD8⁺ Teffs (Fig. 2C–D). We also observed that CD8⁺ Teff cells expressed lower levels of P2RX7 compared to CD4⁺ Teff cells. We next validated these findings in a genetic model of lung cancer using KP mice (Fig. 2E). Sixteen weeks after intratracheal instillation of Adeno-Cre virus, these mice developed tumors in the lung tissue (Fig. 2F). In this model, we observed higher expression of P2RX7 in Tregs compared to both CD4⁺ and CD8⁺ Teff cells (Fig. 2G). Together, these findings indicate that lung tumors induce the accumulation of P2RX7-expressing T cells, with Tregs expressing higher levels of this receptor.

To assess whether the higher expression of P2RX7 in Tregs is specific to lung tumors, we analyzed T cells from brain (SB28), liver (R1WT), and colon (MC38) tumors (Supplementary Fig. S2A–C). In SB28 tumors, both Tregs and CD4⁺ Teff cells expressed higher levels of P2RX7 than CD8⁺ Teff cells (Fig. 2H). In contrast, T cells from R1WT liver tumors followed the same pattern observed in LLC lung tumors, with Tregs expressing higher levels of P2RX7 compared to both CD4⁺ and CD8⁺ Teff cells. In MC38 colon tumors, all T-cell subsets expressed comparable levels of P2RX7. These findings demonstrate that P2RX7 expression in T cells is heterogeneous across different tumor types, suggesting that the elevated P2RX7 levels in Tregs in lung cancer may be driven by tumor-derived signals or by the lung TME.

To determine whether the elevated expression of P2RX7 in Tregs was dictated by the lung tissue or by the lung tumor type itself, we compared P2RX7 expression in these cells using an ectopic (flank injected) tumor model, LLC-induced lung tumors, and naïve lung tissue (Fig. 2I and Supplementary Fig. S2D). The data showed that P2RX7 expression in Tregs was upregulated in the presence of cancer, as indicated by its low expression in naïve lung (Fig. 2J). However, no significant differences were observed between Tregs from ectopic tumors and those from LLC-induced lung tumors, indicating that the increased expression of P2RX7 in Tregs was induced by the tumor type rather than the lung tissue. Nevertheless, LLC-induced lung tumors promoted a higher accumulation of Tregs compared to the ectopic model (Fig. 2K).

Given that our data indicated distinct expression of P2RX7 in T-cell subsets, we investigated whether transcription factors associated with Treg specification (FOXP3) and Treg functional capacity (TCF-1) (34) directly regulated the P2rx7 gene locus. Our ChIP-Seq analysis revealed strong bindings of both TCF-1 and FOXP3 at the P2rx7 active enhancer region in mice (Fig. 2L). These peaks colocalized with histone modifications associated with active enhancer elements (H3K27Ac and H3K4me1), supporting a possible functional relevance for TCF-1 and/or FOXP3 binding of this region. Future studies will be needed to define if these ChIP-Seq results are also true for lung tumor–infiltrating Tregs – for instance, other transcription factors may be involved. It will be also important, in the future, to determine if FOXP3 overexpression can directly drive P2RX7 expression in intratumoral Tregs. Overall, our data show that P2RX7 expression is consistently higher in Tregs than in Teff cells in murine lung cancer, is shaped by tumor type rather than tissue context, and can be directly regulated by FOXP3.

T cell–specific P2RX7 expression permits tumor growth in the lungs

To investigate the T cell–specific role of P2RX7 in lung cancer, we injected LLC-GFP cells into WT (CD4-cre) and T cell–specific P2RX7-KO mice (CD4-cre P2rx7^fl/fl) mice (Fig. 3A). Over a 30-day period, we monitored body weight and observed that, while WT mice experienced weight loss or lack of weight gain, T cell–specific P2RX7-KO mice continued to gain weight over time (Fig. 3B). In line with apparent increased disease development, WT mice exhibited heavier lungs compared to P2RX7-KO mice (Fig. 3C). Macroscopic fluorescence imaging revealed larger and more disseminated GFP⁺ tumor nodules in WT lungs than in P2RX7-KO lungs (Fig. 3D and Supplementary Fig. S3A). Flow cytometry confirmed a higher number of LLC-GFP cells in WT lungs (Fig. 3E and Supplementary Fig. S3B). Histological analysis showed extensive tumor areas in WT lungs, whereas P2RX7-KO lungs displayed smaller tumor regions, contrasting with a pronounced immune-cell infiltrate in P2RX7-KO LLC-inoculated lungs (Fig. 3F).

Fig. 3. Effects of T-cell intrinsic P2RX7 in lung cancer experimental model.

WT (CD4-cre) and T cell-P2RX7-KO (CD4-cre P2rx7^fl/fl) mice were injected i.v. with LLC-GFP cells and lungs were collected on day 30 p.i. (A) Schematic illustration showing the lung cancer experimental protocol (Created in BioRender [Santiago de Carvalho, I]. [2026] [https://BioRender.com/ethjmqb]). (B) Average body weights (percentages related to day 0). (C) Lung weight values on day 30 p.i. (D) Representative fluorescence macroscopy of the left upper lung lobes with tumors (20 μm). (E) Average numbers of GFP⁺ LLC cells in the lungs. (F) Representative section of the left upper lung lobes stained with H&E (200 μm). (G) Average numbers of CD44⁺CD8⁺ T cells in the lungs. (H) Schematic illustration of the lung cancer experimental protocol (Created in BioRender [Santiago de Carvalho, I]. [2026] [https://BioRender.com/35gqj4e]). (I) Average frequencies of GFP⁺ LLC cells after 24 h of co-culture with lung tumor–infiltrating WT and P2RX7-KO CD8⁺ T cells. (J) Average frequencies of WT and P2RX7-KO CD44⁺CD8⁺ T cells after 24 h of co-culture with LLC-GFP cells. (K) Average numbers of CD44⁺CD4⁺ T cells in the lungs. (L) Representative confocal images of DAPI (blue), EpCAM (green), and CD4 (orange) staining in lung tissues (scale bar, 50 μm). (M) t-SNE plot showing cell density and Foxp3 expression on CD44⁺CD4⁺ T cells in lung cancer. (N) Average numbers of CD44⁺CD4⁺ Tregs (Foxp3⁺) and Teffs (Foxp3⁻) in the lungs and medLNs. (O) Schematic illustration of the lung cancer experimental protocol (Created in BioRender [Santiago de Carvalho, I]. [2026] [https://BioRender.com/1aezlbz]). RAG-KO mice were reconstituted with splenocytes from WT or T cell–specific P2RX7-KO mice and injected i.v. with LLC-GFP cells. At day 30 p.i., lungs, medLNs, and spleens were collected. (P) Left: flow cytometry plots of CD45.1 and CD45.2 expression on CD44⁺CD4⁺ T cells. Right: average frequencies of CD45.1⁺ and CD45.2⁺ CD44⁺CD4⁺ T cells. Data from 2-3 independent experiments, n = 3–5/experimental group per experiment. Data shown as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Statistical significance was determined by paired/unpaired t tests and one-way ANOVA (Tukey post tests).

Next, we analyzed the phenotypic characteristics of lung-infiltrating T cells. The number of CD44⁺CD8⁺ T cells was comparable between WT and P2RX7-KO mice (Fig. 3G). We also isolated tumor-infiltrating CD44⁺CD8⁺ T cells and performed cytotoxicity assays (Fig. 3H). The data showed that both WT and P2RX7-KO CD8⁺ T cells exhibited the same capacity to kill LLC-GFP cells ex vivo (Fig. 3I–J). On the other hand, P2RX7-KO lungs contained higher numbers of CD44⁺CD4⁺ T cells (Fig. 3K and Supplementary Fig. S1A for the gating strategy). This was further confirmed using confocal microscopy, showing that while in WT mice CD4⁺ T cells were less abundant and sparsely distributed, P2RX7-KO mice exhibited increased CD4⁺ T-cell numbers across the lung tissue (Fig. 3L). Collectively, our findings suggested that T-cell intrinsic P2RX7 allowed lung tumor growth while limiting the accumulation of CD4⁺ Teffs within the lung tissue. Among the CD4⁺ T cells, T cell–specific P2RX7-KO mice exhibited an increased number of CD4⁺ Teffs in the lungs compared to WT, while Tregs were more abundant in WT lungs (Fig. 3M–N). In the medLNs of P2RX7-KO mice, both Treg and CD4⁺ Teff numbers were increased (Fig. 3N). In the spleens, only P2RX7-KO Teff numbers were higher than WT ones (Supplementary Fig. S3C).

To assess the T cell–intrinsic impact of P2RX7 expression on T cells within a competitive setting, we transferred splenocytes from WT and T cell–specific P2RX7-KO mice into RAG2-KO recipients, followed by LLC-GFP tumor injection (Fig. 3O). At day 30 p.i., we analyzed host T-cell numbers and found a higher frequency of activated P2RX7-KO CD4⁺ T cells (CD45.2⁺) compared to WT (CD45.1⁺) in the lung, as well as in the medLNs and spleens (Fig. 3P and Supplementary Fig. S3D). CD8⁺ P2RX7-KO T cells also accumulated in greater numbers than WT counterparts in the lung (Supplementary Fig. S3E).

Next, we evaluated the phenotype of lung-infiltrating T cells. While CD44⁺CD8⁺ T cells showed no phenotypic differences (Supplementary Fig. S3F), WT CD44⁺CD4⁺ T cells exhibited higher expression of CXCR6 than P2RX7-KO cells in the lungs (Supplementary Fig. S3G). Conversely, P2RX7-KO CD4⁺ T cells expressed higher levels of PD-1, CD69, CXCR5, ICOS, and CD39, with FR4 (Folate Receptor 4) being significantly increased across all organs analyzed (Supplementary Fig. S3G). Collectively, our findings suggest that T cell–intrinsic P2RX7 allows lung tumor growth while limiting the accumulation of CD4⁺ Teffs within the lung tissue.

eATP–P2RX7 axis on CD4⁺ T cells promotes Treg accumulation and allows tumor growth in the lungs

To evaluate whether P2RX7 expression in CD4⁺ T cells affected tumor growth, we used a single transfer model into RAG2-KO mice (Supplementary Fig. S4A). In this single transfer model, P2RX7-KO CD4⁺ T cells prevented tumor growth or disease, differently from WT CD4⁺ T cells which allowed substantial weight loss and tumor development (Supplementary Fig. S4B–E). We analyzed T cells and found no differences in the numbers of bulk CD4⁺ T cells in the lungs; however, their numbers were increased in the medLNs of mice that received P2RX7-KO CD4⁺ T cells (Supplementary Fig. S4F–G). We observed higher numbers of WT Tregs compared to P2RX7-KO Tregs in the lungs, while the opposite was true in the medLNs (Supplementary Fig. S4H–J). In contrast, P2RX7-KO CD4⁺ Teffs increased only in the medLNs. While no differences were observed in the numbers of bulk or activated CD8⁺ T cells in the lungs, these populations were increased in the medLNs of mice transferred with P2RX7-KO CD4⁺ T cells (Supplementary Fig. S4K–L).

Treg-intrinsic P2RX7 promotes tumor growth in the lung

To evaluate the intrinsic effects of P2RX7 in Tregs, we employed a Foxp3 Cre tamoxifen-inducible P2RX7 ablation system (Fig. 4A) Tamoxifen-induced ablation of P2RX7 in Tregs led to weight gain in tumor-bearing mice, whereas WT mice exhibited weight stability or loss (Fig. 4B). Treg-specific P2RX7-KO mice also survived longer than WT mice over time (Supplementary Fig. S5A). Additionally, lighter lungs were observed in Treg-specific P2RX7-KO mice at day 30 p.i. (Fig. 4C), accompanied by reduced numbers of LLC-GFP cells in the lungs, which was confirmed by lung fluorescence imaging showing larger and more extensive GFP⁺ nodules in WT compared to P2RX7-KO mice and flow cytometry (Fig. 4D–E and Supplementary Fig. S5B). Histology confirmed that the tumor area was reduced in the Treg-specific P2RX7-KO mice compared to WT mice (Fig. 4F).

Fig. 4. Effects of Treg-intrinsic P2RX7 on tumor growth in lung cancer experimental model.

WT (Foxp3 Cre-ERT2 P2rx7^fl/fl - Vehicle) and Treg-specific P2RX7-KO (Foxp3 Cre-ERT2 P2rx7 ^fl/fl – tamoxifen) mice were injected i.v. with LLC-GFP cells and lung were collected on day 30 p.i. (A) Schematic illustration showing the lung cancer experimental protocol (Created in BioRender [Santiago de Carvalho, I]. [2026] [https://BioRender.com/rq8djbm]). (B) Average body weights (percentages related to day 0). (C) Lung weight values on day 30 p.i. (D) Average numbers of GFP⁺ LLC cells in the lungs. (E) Representative fluorescence macroscopy of the left upper lung lobes with tumors (20 μm). (F) Representative section of the left upper lung lobes stained with H&E (200 μm). (G) Flow-cytometry plots of CD44 and Foxp3 expression in the lungs. (H) Average numbers of Tregs (Foxp3⁺) and Teff (Foxp3⁻) CD44⁺CD4⁺ T cells in the lungs. (I) Average numbers of Tregs (Foxp3⁺) and Teff (Foxp3⁻) CD44⁺CD4⁺ T cells in the medLNs. (J) Average numbers of CD44⁺CD8⁺ T cells in the lungs and medLNs. RAG-KO mice were reconstituted with WT B cells, CD8⁺ T cells, and Tregs; one group received WT CD4⁺ Teffs while the other received P2RX7-KO CD4⁺ Teffs. Mice were injected i.v. with LLC-GFP cells, and lungs and medLNs were collected at day 30 p.i. (K) Schematic illustration showing the lung cancer experimental protocol (Created in BioRender [Santiago de Carvalho, I]. [2026] [https://BioRender.com/di965yy]). (L) Average body weights (percentages related to day 0). (M) Lung weight values on day 30 p.i. (N) Average numbers of GFP⁺ LLC cells in the lungs. (O) Representative section of the left upper lung lobes stained with H&E (200 μm). (P) Flow-cytometry plots of CD44 and Foxp3 expression in the lungs. (Q) Average numbers of Tregs (Foxp3⁺) and Teff (Foxp3⁻) CD44⁺CD4⁺ T cells in the lungs. (R) Average numbers of Tregs (Foxp3⁺) and Teff (Foxp3⁻) CD44⁺CD4⁺ T cells in the medLNs. Data from 2-3 independent experiments, n = 3–5/experimental group per experiment. Data shown as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Statistical significance was determined by unpaired t test.

Subsequent analysis of T-cell infiltration in the lungs and medLNs revealed an increase in the numbers of bulk CD4⁺ T cells (Supplementary Fig. S5C), but no numerical differences in Tregs between WT and Treg-specific P2RX7-KO mice (Fig. 4G–H). However, an increased number of Live/dead⁺Foxp3⁺ Tregs was detected in both lungs and medLNs, suggesting higher cell death rates among P2RX7-KO Tregs (Supplementary Fig. S5D). In addition, higher numbers of CD4⁺ Teffs were present in the Treg-specific P2RX7-KO mice (Fig. 4G–H). In the medLNs, we observed increased numbers of bulk CD4⁺ T cells and when we gated on Tregs and Teff cells, both populations increased in Treg-specific P2RX7-KO mice compared to WT counterparts. (Fig. 4I and Supplementary Fig. S5C). Additionally, we observed increased numbers of bulk and CD44⁺CD8⁺ T cells in both lungs and medLNs (Fig. 4J and Supplementary Fig. S5E).

We next evaluated the effects of P2RX7 specifically in CD4⁺ Teffs. For this, we transferred WT or P2RX7-KO CD4⁺ Teffs into tumor-bearing RAG-KO mice reconstituted with WT B cells, CD8⁺ T cells, and Tregs (Fig. 4K). No differences in body weight were observed between mice that received WT or P2RX7-KO CD4⁺ Teffs, and this was also the case for lung weight and numbers of LLC-GFP⁺ cells in the lungs (Fig. 4L–N and Supplementary Fig. S5F). Histology showed extensive tumor areas in both groups, with no significant differences in tumor control (Fig. 4O). Similarly, there were no differences in the numbers of bulk CD4⁺ or CD8⁺ T cells (Supplementary Fig. S5G–H). While the numbers of infiltrating Tregs in the lungs and medLNs did not differ between groups, a modest increase in WT CD4⁺ Teffs was observed in the lungs, but not in the medLNs (Fig. 4P–R). These data indicate that in an experimental model of lung cancer, Treg-intrinsic P2RX7, but not Teff-intrinsic P2RX7, promotes lung tumor growth in vivo.

Treg-intrinsic P2RX7 promotes immunosuppression in the lungs favoring tumor growth

To assess the suppressive capacity of tumor-infiltrating WT versus P2RX7-KO Tregs ex vivo, we sorted these cells from lung tumors and conducted suppression assays (Fig. 5A and Supplementary Fig. S1C for gating strategy). Our data showed that WT lung Tregs were more potent in inhibiting the proliferation of in vitro activated CD4⁺ and CD8⁺ T cells compared to P2RX7-KO Tregs at 1:5 and 1:20 Treg:Teff ratios (Fig. 5B–C). WT lung Tregs were also better at suppression of CD8⁺ T-cell cytotoxic function, as evidenced by co-cultures with Tregs, CD8⁺ T cells and LLC tumors (Fig. 5D–E). This enhanced suppressive function correlated with higher expression of Ki-67, CD25, OX-40 and CD39, but not TCF-1 and Foxp3 in WT Tregs (Fig. 5F). Conversely, in the medLNs, no differences were observed in Ki-67, OX-40 and CD39 expression, while CD25 was upregulated in P2RX7-KO Tregs and TCF-1 expression was increased in WT Tregs (Fig. 5G). We also evaluated CTLA-4 expression at the intracellular and extracellular levels, and we found that both extracellular and intracellular CTLA-4 expression was higher in lung WT Tregs than in P2RX7-KO Tregs (Fig. 5H). In the medLNs, we observed only higher intracellular CTLA-4 expression in WT Tregs (Fig. 5I). To assess whether the reduction of CTLA-4 occured at the pre- or post-transcriptional level, we measured Ctla4 mRNA levels in Tregs isolated from lung tumors. The data showed lower Ctla4 mRNA expression in P2RX7-KO Tregs compared to WT (Fig. 5J). This data indicated a tissue-specific role for P2RX7 in promoting Treg function within the TME and decreased expression of CTLA-4 at the transcriptional and protein levels.

Fig. 5. Effects of P2RX7 on Treg suppressive activity in lung cancer experimental model.

WT (Foxp3 Cre-ERT2 P2rx7^fl/fl - Vehicle) and Treg-specific P2RX7-KO (Foxp3 Cre-ERT2 P2rx7 ^fl/fl – tamoxifen) mice were injected i.v. with LLC-GFP cells and lung were collected on day 30 p.i. (A) Schematic illustration showing the suppression assay using WT and P2RX7-KO lung Tregs lung experimental protocol (Created in BioRender [Santiago de Carvalho, I]. [2026] [https://BioRender.com/vdn5hwy]). (B) Left: Histograms showing the Cell Trace staining on CD44⁺CD4⁺ T cells. Right: Average percentage of Treg suppression on CD44⁺CD4⁺ T cell proliferation. (C) Left: Histograms showing the Cell Trace staining on CD44⁺CD8⁺ T cells. Right: Average percentage of Treg suppression on CD44⁺CD8⁺ T cell proliferation. (D) Schematic illustration showing the in vitro suppression of cytotoxicity assay, using WT or P2RX7-KO lung Tregs in co-culture with lung CD8⁺ T cells and LLC-GFP tumors (Created in BioRender [Santiago de Carvalho, I]. [2026] [https://BioRender.com/c7n4uer]). (E) Average numbers of LLC-GFP tumor cells in the indicated experimental groups. (F) Average geometric mean fluorescence intensity (gMFI) of Ki-67, CD25, OX-40, CD39, TCF-1 and Foxp3 on lung Tregs (Foxp3⁺). (G) Average gMFI of Ki-67, CD25, OX-40, CD39, TCF-1 and Foxp3 on medLN Tregs (Foxp3⁺). (H) Left: flow-cytometry plots of CTLA-4 intracellular and extracellular expression on lung Tregs. Right: average gMFI of CTLA-4 intracellular and extracellular on lung Tregs. (I) Left: flow-cytometry plots of CTLA-4 intracellular and extracellular expression on medLN Tregs. Right: average gMFI of CTLA-4 intracellular and extracellular on medLN Tregs. (J) Relative Ctla4 mRNA expression (fold change normalized to WT) in lung-isolated WT and P2RX7-KO Tregs. (K) Schematic illustration showing the in vitro activation of Tregs using P2RX7 agonist (HEI3090) or not (Created in BioRender [Santiago de Carvalho, I]. [2026] [https://BioRender.com/g91bxs3]). (L) Left: flow-cytometry plots of CD25 and Foxp3 expression in vitro activated Tregs. Right: average numbers CD25⁺Foxp3⁺ in vitro activated Tregs. (M) Line graph of calcium influx per time (s) analyzed by FlowJo Kinetics. Data from 2-3 independent experiments, n = 3–5/experimental group per experiment. Data shown as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Statistical significance was determined by unpaired t tests and one-way ANOVA (Tukey post tests).

We next conducted in vitro experiments where Tregs were activated with anti-CD3/CD28 and either treated or not treated with the P2RX7-specific agonist HEI (HEI3090) (Fig. 5K) (30). Activation in the presence of HEI increased Treg numbers in culture (Fig. 5L). Given that P2RX7 functions as an ion channel (35), we performed a calcium influx assay, revealing that HEI-treated Tregs exhibited a higher basal calcium influx activity compared to control Tregs, which further increased upon ionomycin addition (Fig. 5M). Additionally, HEI did not affect previously treated cells but enhanced calcium uptake in control Tregs. The P2RX7 antagonist A4 (A438079) reduced calcium influx only in control Tregs. Collectively, these findings suggest that ablation of P2RX7 diminishes the suppressive capacity of Tregs within lung tumors, possibly because of the reduction in the P2RX7 action on calcium influx leading to a reduction in important suppressive molecules such as CTLA-4.

Treg-intrinsic P2RX7 expression limits type 1 and Tfh-like CD4⁺ T-cell responses in lung cancer

Consistent with the lung tumor suppressive function of Tregs via P2RX7, we observed that tamoxifen-induced ablation of P2RX7 in Tregs enhanced type 1 and cytotoxic responses in the lungs of tumor-bearing mice. Specifically, there was an increased accumulation of IFN-γ-producing CD4⁺ and CD8⁺ T cells (Fig. 6A–B), as well granzyme B–producing CD4⁺ and CD8⁺ T cells (Fig. 6C–D) in the lungs of Treg-specific P2RX7-KO mice compared to WT. Further phenotypic analysis of Teffs in WT and Treg-specific P2RX7-KO mice revealed a significant upregulation of markers characteristic of follicular helper T (Tfh) cells in the P2RX7-KO group. Specifically, geometric mean fluorescence intensity (gMFI) analyses demonstrated higher expression of Bcl6, PD-1, CXCR5, and ICOS in CD4⁺ Teff cells from both lungs and medLNs of Treg-specific P2RX7-KO mice compared to WT mice (Supplementary Fig. S6A–B). Additionally, increased expression of the lung parenchyma homing marker CD69 (36) was noted in Treg-specific P2RX7-KO mice, while the lung vasculature-associated marker KLRG1 (36) was upregulated in CD4⁺ Teffs from WT mice, suggesting enhanced tissue localization of Teff cells within the lung parenchyma in the absence of P2RX7 in Tregs. Indeed, we observed an increase in a resident Tfh-like population (CXCR5⁺CD69⁺) among CD4⁺ Teff cells in the lungs of P2RX7-KO mice (Fig. 6E). This was associated with increased numbers of PD-1⁺ICOS⁺ classical Tfh cells in the medLNs of the P2RX7-KO mice (Fig. 6F).

Fig. 6. Effects of Treg-intrinsic P2RX7 on CD4⁺ T cell-mediated type 1 and Tfh-like responses in lung cancer experimental model.

(A-I) WT (Foxp3 Cre-ERT2 P2rx7^fl/fl - Vehicle) and Treg-specific P2RX7-KO (Foxp3 Cre-ERT2 P2rx7 ^fl/fl – tamoxifen) mice were injected i.v. with LLC-GFP cells, and lung were collected on day 30 p.i. (A) Left: flow-cytometry plots of CD44 and IFN-γ expression on CD44⁺CD4⁺ RT cells in the lungs. Right: average numbers of IFN-γ-producing CD44⁺CD4⁺ T cells in the lungs. (B) Left: flow-cytometry plots of CD44 and IFN-γ expression on CD44⁺CD8⁺ T cells in the lungs. Right: average numbers of IFN-γ-producing CD44⁺CD8⁺ T cells in the lungs. (C) Left: flow-cytometry plots of CD44 and granzyme B (GzmB) expression on CD44⁺CD4⁺ RT cells in the lungs. Right: average numbers of granzyme B-producing CD44⁺CD4⁺ T cells in the lungs. (D) Left: flow-cytometry plots of CD44 and granzyme B expression on CD44⁺CD8⁺ T cells in the lungs. Right: average numbers of granzyme B-producing CD44⁺CD8⁺ T cells in the lungs. (E) Left: flow-cytometry plots of CXCR5 and CD69 expression on CD44⁺CD4⁺ Teff cells in the lungs. Right: average numbers of CXCR5 and CD69 expression on CD44⁺CD4⁺ Teff cells in the lungs. (F) Left: flow-cytometry plots of PD-1 and ICOS expression on CD44⁺CD4⁺ Teff cells in the lungs. Right: average numbers of CXCR5 and CD69 expression on CD44⁺CD4⁺ Teff cells in the lungs. (G) Left: Average numbers of CD19⁺ B cells in the lungs. Right: Average numbers of GL7⁺Fas⁺ GC B cells in the lungs. (H) Left: Average numbers of CD19⁺ B cells in the medLNs. Right: Average numbers of GL7⁺Fas⁺ GC B cells in the medLNs (I) Average optical density (O.D. 450 nm) of LLC-specific IgG in the lung homogenate. (J) Representative confocal images of DAPI (blue), CD4 (red), and B220 (green) staining in lung tissues (scale bar, 50 μm). BV = Blood Vessel and BA = Bronchial Area (K) Left: Spatial images showing the location of spots co-expressing CD4 and MS4A1 (CD20) in G2 and G3 lung cancer patients. Right: Boxplot showing the coexpression of CD4 and MS4A1 (CD20) in G2 and G3 lung cancer patients. Data from 2-3 independent experiments, n = 3–5/experimental group per experiment. Data shown as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Statistical significance was determined by unpaired t tests.

Given the observed increase in Tfh-like cells in the lungs and medLNs of Treg-specific P2RX7-KO mice, we assessed the presence of germinal center (GC) B cells (GL7⁺Fas⁺CD19⁺) in these tumor-bearing mice (gating strategy in Supplementary Fig. S1B). A notable increase in GC B cells was detected in both lungs and medLNs of P2RX7-KO mice compared to WT mice (Fig. 6G–H). Considering that interactions between Tfh and GC B cells in the lungs and lymph nodes lead to the production of antitumor antibodies (21,33), we quantified LLC-specific IgG levels in the lung homogenates of tumor-bearing mice, and elevated levels of LLC-specific IgG were observed in P2RX7-KO mice (Fig. 6I).

The interaction between GC B cells and Tfh-like cells in the lungs contributes to the formation of TLS within tumors (8), facilitating antibody-mediated antitumor protection. Our confocal analyses demonstrated higher numbers of TLS in the lungs of tumor-bearing Treg-specific P2RX7-KO mice compared to WT mice (Fig. 6J). To validate the mouse data, we also analyzed spatial transcriptomics data from patients with grade 2 and grade 3 lung cancer, focusing on spots co-expressing CD4 and MS4A1 (CD20, a B-cell marker). While numerous spots were present in both groups, a higher number was observed in G2 patients (Fig. 6K). These findings suggested that interactions between CD4⁺ Teffs and B cells within lung tumors may be associated with better disease prognosis, mirroring observations from our lung cancer experimental model. Overall, our data supports the conclusion that P2RX7 enhances the suppressive activity of Tregs on type 1 and Tfh-like responses, thereby promoting tumor growth in the lung.

Pharmacological blockade of P2RX7 controls tumor growth and reduces Treg accumulation in the lungs

To assess therapeutic strategies against lung cancer targeting P2RX7, we treated C57BL/6 mice with the P2RX7 inhibitor A438079 (A4; Fig. 7A) and compared their lung cancer progression to vehicle-treated controls. Although A4-treated mice tended to lose less weight compared to the vehicle group, no significant differences were observed between groups (Fig. 7B). No differences were observed for lung weight at day 30 p.i. (Fig. 7C). Nevertheless, histological analyses indicated greater tumor growth in the lungs of vehicle-treated mice compared to those treated with the P2RX7 inhibitor (Fig. 7D). Consistently, flow cytometry showed higher numbers of LLC-GFP⁺ cells in the lungs of vehicle-treated mice than in A4-treated mice (Fig. 7E–F). Further analyses of tumor-infiltrating T cells revealed reduced numbers of Tregs in A4-treated mice compared to vehicle (Fig. 7G), whereas no differences were observed in the numbers of CD4⁺ and CD8⁺ Teffs (Fig. 7H–I).

Fig. 7. Effects of pharmacological inhibition of P2RX7 on tumor growth and Treg responses in the lung.

B6 mice were injected i.v. with LLC-GFP cells and treated with either P2RX7 inhibitor (A438079) or a vehicle control from day 15 to day 30 p.i. On day 30 p.i. the lungs were collected for analysis. (A) Schematic illustration showing the lung cancer and P2RX7 inhibition experimental protocol (Created in BioRender [Santiago de Carvalho, I]. [2026] [https://BioRender.com/4arqgyn]). (B) Average body weights (percentages related to day 0). (C) Lung weight values on day 30 p.i. (D) Representative section of the left upper lung lobes stained with H&E (200 μm). (E) Flow-cytometry plots of GFP expression on LLC cells in the lungs of B6 mice. (F) Average numbers of GFP⁺ LLC cells in the lungs. (G) Average numbers of Tregs (Foxp3⁺) CD44⁺CD4⁺ T cells in the lungs. (H) Average numbers of Teff (Foxp3⁻) CD44⁺CD4⁺ T cells in the lungs. (I) Average numbers of CD44⁺CD8⁺ T cells in the lungs. (J) Schematic illustration showing the suppression assay using untreated and treated (HEI3090, A438079 and ATP) Tregs and CD4⁺ Teffs experimental protocol (Created in BioRender [Santiago de Carvalho, I]. [2026] [https://BioRender.com/5ajk1x7]). Control (CTL) refers to untreated activated Tregs. (K) Left: Histograms showing the Cell Trace staining on CD44⁺CD4⁺ T cells. Right: Average percentage of Treg suppression on CD44⁺CD4⁺ T cell proliferation. (L) Average numbers of Tregs (Foxp3⁺) CD44⁺CD4⁺ T cells in the suppression assay. Data from 2-3 independent experiments, n = 3–5/experimental group per experiment. Data shown as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Statistical significance was determined by unpaired t tests and one-way ANOVA (Tukey post tests).

To evaluate the effects of pharmacological treatments on Treg-mediated suppression, we performed new suppression assays coculturing splenic CD4⁺ Teffs with Tregs that were pretreated with HEI, A4, or a combination of both with ATP (Fig. 7J). As a result, we observed that HEI-treated Tregs exhibited a higher percentage of suppression compared to the control and the other treatments (Fig. 7K). A4-treated Tregs displayed suppression levels similar to the control. When HEI, A4, and ATP were combined, the effects of A4 appeared to be more prominent, as suppression remained comparable to the control and A4 group. Additionally, A4 treatment reduced the numbers of Tregs in the culture during the suppression assay (Fig. 7L). Together, these data indicate that pharmacological inhibition of P2RX7 may represent a therapeutic strategy targeting Tregs to reduce lung tumor growth.

DISCUSSION

eATP serves as a danger signal within the TME, signaling through purinergic receptors such as P2RX7 (17,18,37). While the eATP–P2RX7 axis has been studied across various cancers, including lung cancer, its specific role in T cells during lung tumor progression remained unclear (38). Our work demonstrated that P2RX7 expression in CD4⁺ T cells modulates the balance between Tregs and Teffs, thereby influencing tumor growth in the lung. Single-cell RNA sequencing analyses of advanced-stage NSCLC patients revealed that P2RX7 was highly expressed in T cells within the TME. Spatial transcriptomics further indicated that P2RX7 expression was increased in G3 tumors compared to G2, suggesting its potential as a biomarker for advanced lung cancer stages and as a therapeutic target (39).

Previous studies have shown that pharmacological inhibition or genetic ablation of P2RX7 can prevent tumor growth, potentially by promoting M1 macrophage polarization and enhancing tumor-infiltrating T-cell responses (40). However, the specific impact of P2RX7 on T cells in lung cancer has not been fully elucidated. In melanoma models, P2RX7 limits the accumulation of CD4⁺ and CD8⁺ T cells within tumors by inducing their senescence, without affecting Treg accumulation (41). In our study, Tregs from advanced-stage NSCLC patients exhibited higher P2RX7 expression compared to other CD4⁺ T cells. Spatial transcriptomics confirmed increased co-expression of P2RX7 and FOXP3 in G3 tumors. Similarly, in a murine lung cancer metastasis model, tumor-infiltrating Tregs expressed higher levels of P2RX7 than Teffs. Using various experimental models, we demonstrated that P2RX7 expression in CD4⁺ T cells promoted lung tumor growth, whereas its ablation enhanced Teff accumulation and reduced tumor burden. Previous studies from our group have demonstrated that the eATP–P2RX7 axis is crucial for the accumulation and localization of CD4⁺ T cells in response to severe tuberculosis and influenza infections (22,42). Therefore, the impact of P2RX7 on T-cell responses to tumors may vary depending on the tumor site and/or the type of malignancy. In our present work, we provide additional evidence for this potential context-dependent role of P2RX7, showing that the expression of P2RX7 by Tregs and Teffs, as well as the number of Tregs, varied depending on the type of tumor studied and on the site of injection of LLC tumors. The latter point is important when comparing our findings with a recent report (30), where P2RX7 signaling seems to promote better control of ectopic LLC tumors. It is possible that these discrepant outcomes are linked to differences in the presence of Tregs, which is much lower in the ectopic LLC tumor model.

In T cell–specific P2RX7-KO mice, we observed no differences in LLC-associated Teff numbers, but reduced numbers of Tregs. This decrease, together with their predicted decreased suppressive capacity, may ndicate that effector antitumor immune responses in these mice result from a decrease in suppressive Tregs. In addition, we observed increased numbers of Teff cells in tumor-draining lymph nodes, which may suggest a better ability of P2RX7-KO host mice to develop a reservoir of Teff cells that can replenish the tumor sites over time. Indeed, in vitro activation of Tregs in the presence of a P2RX7 agonist (HEI3090) mimicked the Treg expansion observed in WT mice. In contrast, previous studies using BzATP, a direct P2RX7 agonist, reported Treg apoptosis (43). The use of HEI3090, which sensitizes P2RX7 to ATP without directly activating it, may explain these differing outcomes by enhancing receptor sensitivity to autocrine ATP release (30). The increased Teff numbers in Treg cell–specific P2RX7-KO mice, in contrast, may be mostly associated with reduced Treg suppressive function in the lungs – rather than an absolute loss in Treg numbers.

Treg accumulation in lung cancer impairs effective CD4⁺ and CD8⁺ Teff responses and compromises immunotherapy efficacy (11). In Treg-specific P2RX7-KO mice, we observed that while Treg numbers remain unchanged, their suppressive functions were diminished. Tamoxifen-induced P2RX7 ablation in Tregs led to decreased expression of Ki-67, CTLA-4, and CD25, markers associated with Treg proliferation and suppressive capacity (44,45). Given that T-cell receptor (TCR) activation and calcium influx are crucial for upregulating these molecules (46) and considering the role of P2RX7 as an ion channel facilitating calcium entry (35), our findings suggest that P2RX7 signaling may enhance Treg suppressive function via calcium-mediated pathways. In particular, the P2RX7-induced upregulation of CTLA-4, which our results suggest occurs at the transcriptional level, may happen due to calcium-controlled pathways such as NFAT. NFAT, indeed, is known to bind to the promoter of the CTLA-4 gene in human T cells (47). Future research will be necessary to formally establish this connection between P2RX7, calcium influx, and Treg suppression. We also found that, while Treg numbers increased in the medLNs of P2RX7-KO mice, the suppressive markers reduced in lung Tregs were not similarly affected in medLN Tregs. This indicated that the influence of P2RX7 on Treg function was tissue-specific, primarily affecting Tregs upon their arrival in the lung tissue. This may also help explain why, in contrast with our observations of P2RX7-induced increase in lung tumor Tregs suppressive capacity, previous reports have suggested a P2RX7-mediated decrease in lymph node Treg suppression (43). The possible molecular cues driving this tissue-specific regulation warrant further investigation. In addition, it is possible that intrinsic differences in lung tumor versus circulating Tregs may dictate these differences in function – for example, the expression of CTLA-4, which was noticeably higher in lung tumor-associated Tregs. It is possible that both Treg-intrinsic and tissue instruction cues work together to drive the P2RX7-associated Treg suppressive capacity in the context of lung tumors.

In the absence of Treg-mediated suppression through cell-specific P2RX7 ablation, the protective functions of CD4⁺ and CD8⁺ Teffs were increased. Reduced Treg suppression in P2RX7-KO mice enhanced type 1 Teff responses, characterized by increased IFN-γ and granzyme B production, key antitumor mediators (48,49). The most pronounced effects were observed in Tfh-like cells, which are primary Treg targets due to their high PD-1 expression (50,51). P2RX7-KO Tregs failed to regulate Teffs effectively, leading to Tfh-like cell accumulation in lung tumors and medLNs. This accumulation promotes TLS formation within tumors and germinal centers development in lymph nodes, enhancing antibody-mediated tumor cell elimination (8). Consequently, we observed increased production of tumor-specific IgG and reduced tumor growth in the presence of P2RX7-KO Tregs. The presence of TLS in lung cancer patients correlates with favorable prognosis (8), and our spatial transcriptomics data showed higher CD4⁺ and B cell co-localization in G2 patients. A possible mechanism by which P2RX7-expressing Tregs can suppress TLS responses in lung cancer may be due to an increased ability to infiltrate lung tumor TLS structures, as has been suggested to occur in NSCLC patients (52). Future studies will be necessary to define whether P2RX7 expression favors the ability of Tregs to infiltrate lung tumor TLS. In summary, our findings suggest that in response to lung cancer, P2RX7 promoted Treg infiltration and immunosuppression, limiting Teff accumulation and both type 1 and humoral immune responses. Targeting P2RX7 may offer a therapeutic strategy to mitigate Treg-mediated suppression and enhance Teff-mediated protection against lung cancer.

Synopsis:

Lung tumor–infiltrating Tregs rely on the extracellular ATP receptor P2RX7 for their ability to suppress antitumor immunity. P2RX7 inhibition reduces intratumoral Treg numbers and ameliorates tumor control, suggesting the potential for P2RX7 antagonism for lung cancer treatment.

Data Availability Statement

The data generated in this study are available within the article and its supplementary data files or from the corresponding author upon request.