OX40L and IL-2 combination strategy for gastric cancer immunotherapy

Introduction

Gastric cancer (GC), also defined as stomach cancer, commonly afflicts adults above 65 years of age. Different risk factors, including persistent mucosal inflammation caused by Helicobacter pylori (H. pylori) (1-3) and Epstein-Barr virus (EBV) (4) are known to be associated with the onset of GC. After breast, lung, colorectum, and prostate cancers, GC is one of the most commonly diagnosed tumors worldwide, while the mortality rate places GC in the fifth position (https://gco.iarc.who.int/today, accessed 20 June 2025) (5). The lack of early symptoms means GC is usually diagnosed late, placing a heavy burden on patients and society. Because GC is often diagnosed late, surgery with adjuvant chemotherapy is the standard treatment for locally advanced disease, while immunotherapy may be used in selected cases. Despite recent advances in immunotherapy for other solid tumors, GC remains a significant challenge. The persistently low remission rates associated with GC urge the development of targeted and effective treatments for GC patients. Under current investigations are costimulatory molecules that play a pivotal role in enhancing the patient’s T cells immune response. Among these, OX40 (TNFRSF4, CD134) have been shown to play important modulatory functions in inducing a robust immune response (6,7).

OX40 is a transmembrane glycoprotein, a member of the tumor necrosis factor receptor (TNFR) superfamily first identified in 1987 on activated rat CD4⁺ T cells (8,9). Effective CD4⁺ and CD8⁺ T cells, after initial T cell receptor (TCR) stimulation, transiently express OX40 after 1 to 2 days upon activation, whereas it is absent from resting lymphocytes (10,11). OX40 is a potent costimulatory molecule that sustains expansion and survival of activated CD4⁺ and CD8⁺ T cells, increases generation of memory T cells, and promotes cytokine production by effector T cells (12-16).

The ligand-receptor interaction is fundamental to immune response enhancement in the cancer microenvironment. OX40L (TNFSF4, CD252) is the only known homologous ligand for OX40, firstly discovered in 1985 as a glycoprotein 34 (gp34) on human T-lymphotropic virus-I (HTLV-I) transformed cells and later found to be the ligand for OX40 (17). OX40L is a type II transmembrane protein principally expressed in antigen presenting cells (APCs), such as dendritic cells (DCs), B cells, and macrophages (15,18-20). Moreover, it can be present in other cell types, such as endothelial cells, mast cells, Langerhans cells, and natural killer (NK) cells (21-24).

The OX40-OX40L interaction, occurring under defined conditions and in response to specific signals and antigens, initiates an intracellular signaling cascade in effector T cells (Teffs), which enhances immune responses and intensively sustains their clonal expansion (11,25). Furthermore, a noticeable increase of important cytokines secretion, such as the T cell growth factor interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), and interferon gamma (IFN-γ), was previously observed in effector T cells (11,26). The role of IL-2 in the activation of immune system, supporting T cells survival and proliferation, has placed this cytokine in a prominent place in immunotherapy since its discovery. IL-2 is a type I glycoprotein secreted by activated T lymphocytes. Originally discovered as a T cell growth factor, further studies have shown that IL-2 participates in wide and indispensable biological activities. IL-2 is accountable for the growth, differentiation, and progression of T lymphocytes and NK cells (27-30). Currently, the administration of high doses of IL-2 is only approved for advanced stage of renal cell carcinoma and metastatic melanoma (https://www.cancer.gov/about-cancer/treatment/drugs/aldesleukin) (31). This limitation is due to the in vivo short half-life time of IL-2, which requires repeated administrations, often leading to systemic adverse events. This limitation is due to the in vivo short half-life of IL-2, which requires repeated administrations, often leading to systemic adverse events, including vascular leak syndrome, edema, anemia, fever, and hypotension (32). On the contrary, low doses of IL-2 support the stimulation and survival of regulatory T cells (T_regs) that, while being beneficial for autoimmune diseases, this subpopulation of T cells is detrimental in the tumor microenvironment (TME) since they compromise the host antitumor response and the immunotherapy treatments (33,34). However, the potential of IL-2 as an attractive therapeutic approach in enhancing antitumor immune response is validated by the steady increase of new strategies that are exploring this potent cytokine in an engineered, modified form for selective binding to specific cell types or employed in combination with checkpoint inhibitor or costimulatory molecules, in order to bypass its limitations (35,36).

Tumor-infiltrating lymphocytes (TILs) in the cancer microenvironment play an important role in antitumor activity. Thus, their detection is a useful diagnostic biomarker that has the potential to predict the outcome of immunotherapy and the clinical course of patients with various cancers (37,38). Tumor infiltrating lymphocytes can suppress tumor growth and progression but reversibly can also be hijacked by tumor cells as allies, creating an immunosuppressive environment (38). Regulatory T cells (T_reg cells) identified by the expression of the transcription factor forkhead box P3 (FOXP3) is frequently detected in the TME (39). T_regs have been shown to be responsible for immunological tolerance to self-antigens. Cancer cells are recruiting T_regs to escape the host active response carried on by effector T cells by means of T_regs maintenance functions in homeostasis and self-tolerance. Regulatory T cells promote tumor progression by suppressing effector T-cell proliferation through IL-2 consumption and by releasing inhibitory cytokines such as IL-10 and TGF-β (40,41). In the case of GC, the presence of T_regs in the TME is usually correlated with a poor chance of survival (42-44). Hence, evaluating the heterogeneity and complexity of TILs is a fundamental first approach to predict the progression of cancer growth and prognosis and thus can be used in determining a better immunotherapy strategy.

Taking into the account all these important players and their role in the cancer microenvironment, we developed a new immunotherapy strategy using OX40L combined with IL-2. This new approach is a basis for a new complementary immunotherapy strategy that could provide a robust immune response against tumor growth and progression, leading to a promising and beneficial outcome for GC patients. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-707/rc).

Methods

Bioinformatics analysis

The gastric adenocarcinoma RNA sequencing (RNA-seq) dataset was retrieved from The Cancer Genome Atlas (TCGA) (https://www.cancer.gov/tcga) using the TCGA biolinks R package (45). Differential gene expression analysis was performed using the DESeq2 R package (46), and the results were visualized with the ggplot2 R package. Kaplan-Meier survival analysis and visualization were conducted using the survival and survminer R package. The single-cell RNA-seq dataset was downloaded from the GSA-Human database under access number HR000704 (available at http://bigd.big.ac.cn/gsa-human). After downloading, analysis began from the UMI count matrix, which was imported into R and further processed using the Seurat R package (47).

Gastric tissues samples and peripheral blood acquisition from patients

The samples of para-cancerous, tissue within the range of less than 2 cm from the tumor tissue, and paired cancerous, tumor tissue itself, from 70 GC patients, were obtained from the Department of Gastrointestinal Surgery of Affiliated Hospital of Jiangnan University between June 2019 and December 2020 in the form of paraffin embedded sections. Patients with GC combined with other primary tumors were excluded. Total of 54 peripheral blood samples from the same GC patients were taken preoperatively through deep vein puncture tubes. All tissue samples after acquisition were immediately placed in sterile containers and then transported to the central laboratory for analyses. None of the examined patients were treated with radiotherapy or chemotherapy before surgery and samples collections.

The study protocols involving human samples were approved by the Ethics Committee of affiliated Hospital of Jiangnan University (No. LS2019061). Each patient enrolled in the study signed a consent form approved by the ethics committee. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Histochemical and immunohistochemical analysis

Firstly, hematoxylin & eosin (H&E) staining were used for identification and classification of GC and normal gastric tissues by a pathologist. The stage of cancer malignancy was determined according to TNM classification system. Representative sample blocks previously dehydrated and embedded in paraffin were cut 4 µm thick with microtome, and successively mounted on glass slides for further staining. Immunohistochemistry (IHC) analyses were performed using specific primary rabbit anti-human CD3 monoclonal antibody (Proteintech Group, Wuhan, China), mouse anti-human OX40 and rabbit anti-human OX40L monoclonal antibodies (CST, Danvers, MA, USA), and rabbit anti-human CD4 (Proteintech Group, Wuhan, China), mouse anti-human CD8 (Proteintech Group, Wuhan, China) antibody, rabbit anti-human FOXP3 monoclonal antibodies (CST, Danvers, MA, USA). After washing 3 times with phosphate buffer saline (PBS), these were followed by secondary anti-rabbit or anti-mouse antibodies coupled to horseradish peroxidase (SignalStain^® Boost IHC Detection Reagent). Tissue sections were scanned on a digital section scanning system (3DHISTECH Hungary), and five fields of view were taken at each site to determine the number of positive cells per mm² using Image Pro Plus 6.0.

Immunofluorescence analyses

Paraffin-embedded GC tissue sections (4–5 µm) were deparaffinized, rehydrated, and subjected to antigen retrieval in citrate buffer (pH 6.0). After blocking with 5% BSA for 1 hour, sections were incubated overnight at 4 ℃ with mouse anti-human OX40 and rabbit anti-human CD3 antibodies (CST, Danvers, MA, USA). The next day, Alexa Fluor^® 488 conjugated anti-mouse IgG (H+L) and Alexa Fluor^® 594 Conjugated anti-rabbit IgG (H+L) (CST, Danvers, MA, USA) were applied for 1 hour at room temperature in the dark. Nuclei were counterstained with DAPI (1 µg/mL). Images were acquired using a confocal laser scanning microscope (LSM 880, Zeiss, Germany) and processed with ZEN software.

Plasmid construction

Peripheral blood mononuclear cell (PBMC) cDNA were used as a template with primers OX40L EcoRV 5'-ACAGATATCGCAGGTATCACATCGGTATC-3' and OX40L XhoHis 5'-ACAGATATCGAAAGGACACAGAATTCAC-3'. The PCR product was digested with EcoRV and ligated with the pFuse-hIgG1-Fc2 plasmid, previously digested with EcoRV to generate IL2ss-OX40L-Fc-His. The resulted ligation product was used as the template for secondary amplification with the primers pFuse-hIgG1-Fc2 Sac 5'-ACAGAGCTCGAAGGAGGGCCACCATGTAC-3' and FcHisKpnR 5'-ACAGGTACCTTAGTGGTGGTGATGGTGATGTTTACCCGGAGACAGG-3'. The PCR product was digested with SacI and KpnI and then cloned into pCAGGS. The resultant plasmid was named pCAGGS-IL2ss-OX40L-Fc-His.

Generation of adenoviral recombinant

This was performed as described before (48). First, we constructed pShuttleIL-2-OX40L plasmid as follow procedure. The IL-2 DNA fragment was amplified by PCR using F primers 5'-ACAGGTACCGCCACCATGTACAGGA-3' and 5'-AAGTTAGTAGCTCCGCTTCCAGTCAGTGTTGAGATG-3' from pCDNA3.0 IL-2 NEO. The OX40L DNA fragment was amplified by PCR for four times using primers F1, 5'-CCTGGGGCTGCTCCTGGTGTTGCCTGCTGCCTTCCCTGCCCCACAGGTATCACATCGGT-3', F2, 5'-TGAACTCCTTCTCCACAAGCGCCTTCGGTCCAGTTGCCTTCTCCCTGGGGCTGCTCCTG-3', F3, 5'-CTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTATGAACTCCTTCTCCAC-3', F4, 5'-GGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTG-3' and R prime, 5'-AGCCAGAAGTCAGATGCTCAAG-3' from pCAGGS-OX40L-puro-Flag. The IL2-OX40L DNA fragment was amplified by PCR using primers 5'-ACAGGTACCGCCACCATGTACAGGA-3' and 5'-AGCCAGAAGTCAGATGCTCAAG-3', digested with KpnI restriction enzyme and then cloned into pShuttle plasmid.

pShuttleIL-2-OX40L plasmid was linearized with PmeI, purified and mixed with supercoiled pAdEasy-1, then electroporation was performed in E. coli BJ5183 cells. Those clones that had inserts were further tested by PacI restriction endonuclease digestions.

Recombinant adenoviral vector pAdIL-2-OX40L, digested with PacI and ethanol precipitated, was used for transfection of 293 GNTI^- cells. Transfected cells were collected 7–10 days after transfection. After three cycles of freezing in a dry ice bath and rapid thawing at 37 ℃, viral lysate was used to infect 293 GNTI^- cells (49).

OX40L protein generation

To generate the OX40L protein, the pCAGGS-IL2ss-OX40L-Fc-His plasmid was transformed into 293T cells using polyethyleneimine (PEI; Polysciences) according to the manufacturer’s instructions. Two days after transfection, the OX40L protein in the culture medium was purified using Ni-NTA (Qiagen, Hilden, Germany) affinity chromatography.

Cell isolation and CFSE staining

PBMCs obtained from patients were isolated by density gradient centrifugation using standard Ficoll-Hypaque (tbdscience) procedures (50). TILs were isolated by standard Percoll procedures (51). CFSE dilution assay was used for labeling live cells and cumulative monitoring their division via flow cytometry and fluorescent microscopy.

Flow cytometry

Cells suspension in 200 µL of 1× PBS were firstly incubated with 5 µL PerCP anti-human CD45 (Biolegend, San Diego, CA, USA), FITC-conjugated anti-human CD3 (Biolegend), and APC-conjugated anti-human CD134 (Biolegend) reagents for 20 minutes at 4 ℃ in the dark. After washing each sample with 4 mL with PBS containing 1% penicillin-streptomycin, cells were centrifuged at 2,000 rpm for 5 minutes, resuspended in 200 µL of 1640 medium, and were assessed using a flow cytometer (BD FACSLyric™, San Jose, CA, USA). FlowJo version 10.4 software was used to analyze the flow cytometry data.

Enzyme-linked immunosorbent assay (ELISA)

The TILs isolated from the GC patients were collected for ELISAs. Human TNF-α/IFN-γ/Perforin/Granzyme B ELISA Kits (Biolegend) were used for detection. The specific experimental procedures for each ELISA were performed according to the manufacturer’s instructions.

Statistical analysis

SPSS 26.0 software was used for statistical analysis. Continuous variables were expressed as mean ± standard deviation (SD). Categorical variables were shown as counts and percentages. Descriptive statistics were shown as mean (SD) or median (interquartile spacing), depending on the data distribution. One-way ANOVA was used for comparison of continuous variables between different groups, and S-N-K test was used for post hoc testing. Two-tailed t-test was used for comparison of continuous variables between two groups. P values <0.05 were considered statistically significant (ns: nonsignificant, *P<0.05, **P<0.01, ***P<0.001).

Results

RNA Expression profiling of TNFRSF4 and TNFSF4 in GC

The Cancer Genome Atlas (TCGA) dataset was used to investigate mRNA expression profile in the tumor tissues from stomach adenocarcinoma patients. Differential pairwise analyses of TNFSF4, TNFRSF4, IL-2 and three IL-2 receptors subtypes, IL2RA, ILRB, and ILRG revealed that their expression significantly increased within tumors compared with the normal tissue, except for IL-2 expression, whose expression remained low and showed no significant difference between GC and normal tissues (Figure 1A). Applying Kaplan-Meier plot evaluation we found that TNFSF4 and TNFRSF4 expression did not have a significant impact on the survival period of patients (Figure 1B). To better understand the role of the TNFSF4-TNFRSF4 axis in the microenvironment of GC, we selected a single-cell sequencing cohort composed of normal tissue, primary tumor tissue, and metastatic tumor tissue using Uniform Manifold Approximation and Projection (UMAP) for investigating the heterogeneity of T cells (52). Normal and primary tumor tissues were shown in different colors (Figure 1C) and classified into different clusters (Figure 1D). We further identified cells with high expression of the TNFRSF4 gene, which were mainly located in clusters 4, 5, and 6. We then re-performed UMAP clustering on these three clusters and found that the TNFRSF4-high cell populations (clusters 1, 2, 3, and 7) were co-localized with the FOXP3-high cell populations, whereas TNFSF4 expression displayed a scattered pattern and was not confined to the same clusters (Figure 1E). The T cell cluster was identified with CD3D gene and NK cells were characterized with NKG7 gene (Figure 1F).

Figure 1 Molecular characterization of distinct TME cell population and expression signature in gastric cancer. (A) Box plot showing differences in genes expression between normal tissue and gastric tumor tissue and dot plot showing pair comparison between normal tissue and gastric tumor tissue of TNFRSF4, TNFSF4, IL-2, IL2RA, IL2RB, and IL2RG expression. (B) Survival analysis with Kaplan-Meier method for STAD patients grouped by the expression level of TNFRSF4 and TNFSF4. (C) UMAP representation of tissue; normal tissue (red), primary tumor (light blue), from GSE163558. (D) UMAP plot of single-cell transcriptomic profiles grouped into the major cell types clusters from GSE163558. (E) Expression of marker genes for identification of T cells and NK cells. (F) Expression of TNFRSF4, TNFSF4, and FOXP3 marker genes. ns, not significant; **, P<0.01; ***, P<0.001. CI, confidence interval; HR, hazard ratio; NK, natural killer; STAD, stomach adenocarcinoma; TME, tumor microenvironment; UMAP, Uniform Manifold Approximation and Projection.

Expression profile of CD3, CD4, CD8, FOXP3, OX40, and OX40L in GC tissues

We obtained paraffin sections from a group of 32 untreated patients diagnosed with GC (Table 1, Group 1). The pathological evaluation determined the area of normal gastric tissue, the junction between GC and normal gastric tissue, and the internal boundaries of the cancer tissue using standard H&E staining (Figure 2A). T cells in the TME and within the tumor were evaluated by immunohistochemical staining with anti-CD3, anti-CD4, and anti-CD8 antibodies. Within the TME area, the presence of CD3⁺ and CD4⁺ was elevated compared with their presence in the tumor area. CD8⁺ in TME was less frequent than CD3⁺ and CD4⁺. In the tumor area, all three markers were poorly expressed (Figure 2B).

Figure 2 Immunohistochemical and flow cytometric analysis of gastric cancer tissue. (A) H&E histochemical staining of tumor-free area (left), tumor margin (middle), and gastric tumor site (right). (B) Representative IHC images for CD3⁺, CD4⁺, CD8⁺, FOXP3, OX40, and OX40L in tumor-free area (left column), tumor margin (middle column), and gastric tumor site (right column) sections. (C) Immunofluorescence staining for the co-expression of CD3⁺ and OX40 in gastric cancer with anti-CD3 and anti-OX40 antibodies and DAPI for nuclear staining. (D) Flow cytometry analysis of OX40 expression in CD3 cells obtained from PBMC of gastric cancer patients (upper panel) and OX40 expression on CD3 on TILs in gastric cancer patients (low panel). Statistical analysis of all data were performed using one-way ANOVA. Data are shown as mean ± standard deviation. ns, nonsignificant; *, P<0.05; ***, P<0.001. ANOVA, analysis of variance; DAPI, 4',6-diamidino-2-phenylindole; H&E, hematoxylin & eosin; IHC, immunohistochemistry; OX40L, OX40 ligand; PBMC, peripheral blood mononuclear cells; TILs, tumor-infiltrating lymphocytes.

Since the immunosuppression exerted by T_regs in the tumor area led to a poor chance of survival, we performed IHC staining with anti-FOXP3 antibody. The number of cells expressing FOXP3+ inside the GC area was significantly higher than that of normal tissue and the junction of GC tissue (Figure 2B). Our subsequent analysis focused on the expression profile of OX40 and OX40L in these samples. We found that the distribution pattern of OX40⁺ cells predominantly accumulated at the junctions of GC and normal tissue and was highly reduced within the tumor area. OX40L was primarily expressed in gastric smooth muscle cells within the microenvironment of GC (Figure 2B).

Co-expression of OX40 and CD3 in the immune microenvironment of GC

To further profile the OX40-expressing cells, we randomly selected 6 samples from the above paraffin sections obtained from GC patients and performed the IHC staining with anti-OX40 and anti-CD3 antibodies. Laser confocal microscopy imaging results confirmed that the OX40 was mainly co-expressed with the CD3 on the surface of CD3⁺ T cells (Figure 2C). These CD3⁺ T cells accumulated in the tumor margin around the GC tissue and were less frequent within the GC area (Figure 2C). These results are consistent with the immunohistochemical results presented in Figure 2B.

Flow cytometry detection of OX40 expression in PBMCs and TILs of GC patients

We isolated PBMCs from the blood of 32 GC patients and analyzed the expression of OX40 with flow cytometry. The results showed a ratio of 1.67% of CD3⁺ T cells expressing OX40 in patients PBMCs (Figure 2D).

TILs obtained from the normal gastric tissue, inside the area of GC, and at the junction between normal gastric tissue and GC were also analyzed using flow cytometry. The expression of OX40 in CD3⁺ T cells in TILs was significantly high in the tumor margin with a ratio of 4.28% of CD3⁺ T cells co-expressing OX40 (Figure 2D). In contrast, the percentage of CD3⁺ T cells expressing OX40 was less abundant in the tumor-free area and within the tumor, underscoring the spatial variation of OX40 expression (Figure 2D).

OX40L and IL-2 promote T cell proliferation in PBMCs and TILs of GC patients

We used an in vitro culturing assay followed by flow cytometry analysis to explore the combined effect of OX40L and IL-2. Peripheral blood samples were collected from a group of 22 untreated GC patients (Table 1, Group 2). To reach the peak of T cell expression, we tested different concentrations of IL-2 (5, 10, and 20 ng/mL), whereas the concentration of OX40L was fixed at 10 µg/mL (Figure 3A). The suitable concentration of IL-2 and OX40L, 20 ng/mL and 10 µg/mL, respectively, resulted in significant enhancement of the expression of T cells in PBMCs (Figure 3B). Similar results were obtained in TILs where the propagation pattern was confirmed. TILs stimulated with combined OX40L, and IL-2 showed the highest proliferation (51.60%) compared with IL-2 alone (7.81%) or OX40L alone (38.70%) (Figure 3B). Statistical analyses of our presented data stated the significance of our results (Figure 3B).

Figure 3 OX40L/IL-2 stimulation of PBMCs and TILs. (A) Flow cytometry analyses of T cell expression upon stimulation with different concentration of OX40L/IL-2. (B) Flow cytometry analyses of PBMCs and TILs upon stimulation with IL-2, OX40L, and OX40L/IL-2. (C) Expression at mRNA and secreted protein level of TNF-α, IFN-γ, Perforin, and Granzyme B after OX40L/IL-2 stimulation in PBMCs. (D) Expression at mRNA and secreted protein level of TNF-α, IFN-γ, Perforin, and Granzyme B after OX40L/IL-2 stimulation in TILs. Statistical analysis of all data was performed using one-way ANOVA. Data are shown as mean ± standard deviation. ns, nonsignificant; *, P<0.05; **, P<0.01; ***, P<0.001. ANOVA, analysis of variance; IFN-γ, interferon-gamma; IL-2, interleukin-2; OX40L, OX40 ligand; PBMCs, peripheral blood mononuclear cells; TILs, tumor-infiltrating lymphocytes; TNF-α, tumor necrosis factor-alpha.

Combined OX40L/IL-2 stimulation increases expression of effector molecules at mRNA and secreted protein level in PBMCs and TILs

Since TNF-α, IFN-γ, Perforin, and Granzyme B are important antitumor effector molecules secreted by CD4⁺ T helper and C8⁺ T cells, we further study the effects of combined OX40L and IL-2 on their expression in PBMCs and TILs obtained from GC patients.

At the mRNA level, the expression of TNF-α, IFN-γ, perforin, and granzyme B mRNAs after 72 hours of incubation was significantly highest in PBMCs treated with a combination of OX40L and IL-2, followed by their gradually lowering expressions in PBMCs treated with OX40L alone, and PBMCs treated with IL-2 alone. As expected, their expression was lowest in the control untreated PBMCs (Figure 3C).

ELISA data showed that the amount of secreted TNF-a, IFN-γ, perforin, and granzyme B proteins was significantly highest in PBMCs treated with a combination of OX40L and IL-2 (Figure 3C). The lowest expression was found in the control untreated PBMCs, followed by their gradually increasing secretion in PBMCs treated with IL-2 alone and in PBMCs treated with OX40L alone (Figure 3C).

The expression of four effectors mRNA and their proteins in TILs was qualitatively the same as for PBMCs. The combination of OX40L and IL-2 stimulation resulted in the highest effectors expression compared to the lowest expression in the untreated TILs control, whereas the values for OX40 alone and IL-2 alone were in between (Figure 3D).

The secretion levels of four effectors in the supernatant of TILs were qualitatively the same as for their mRNA. The highest stimulation was achieved by the combination of OX40L and IL-2 (Figure 3D). The lower level was measured after stimulation with OX40L and IL-2 alone, whereas the lowest minimal stimulation was in untreated control cells (Figure 3D). These results show that in PBMCs and TILs, the combination of OX40L/IL-2 exerts a robust effect on increasing the secretion levels of these effector molecules.

The apoptotic effect of OX40L and IL-2 on co-culture of PBMCs and TILs from GC patients and GC primary cells

To explore the effect of OX40L and IL-2 close to an in vivo situation, we used an in vitro system employing GC primary cells obtained from fresh GC specimens.

Obtained primary GC cells from a group of 16 untreated GC patients (Table 1, Group 3) were fixed in alcohol, embedded in paraffin, and sectioned for H&E staining and IHC labeling with a cytokeratin antibody (Figure 4A). After identification by a pathologist, the purity of the isolated GC primary cell was approximately 81.3% (Figure 4A).

Figure 4 Apoptotic effect of OX40L and IL-2 on co-culture of PBMCs or TILs from gastric cancer patients with gastric cancer primary cells. (A) H&E staining and immunohistochemistry labeling with a cytokeratin antibodies of primary gastric cancer cells. (B) Flow cytometry analyses of apoptosis in PBMCs co-cultured with primary gastric cancer cells alone, and after stimulation with either IL-2, or OX40L, or OX40L/IL-2. (C) Flow cytometry analyses of apoptosis in TILs co-cultured with primary gastric cancer cells alone and after stimulation with either IL-2, or OX40L, or OX40L/IL-2. Statistical analysis of all data was performed using one-way ANOVA. Data are shown as mean ± standard deviation. ns, nonsignificant; *, P<0.05; **, P<0.01. ANOVA, analysis of variance; H&E, hematoxylin & eosin; IL-2, interleukin-2; OX40L, OX40 ligand; PBMCs, peripheral blood mononuclear cells; TILs, tumor-infiltrating lymphocytes.

We isolated the primary GC cells and performed flow cytometry using carboxyfluorescein succinimidyl ester (CFSE) staining to detect apoptotic cells. The Ficoll separation method was applied to isolate corresponding peripheral blood PBMCs obtained from GC patients.

The apoptotic effect of OX40L and IL-2 was performed using the cell co-culture of either PBMCs or TILs (10⁶ cells/well) from GC patients and GC primary cells (2×10⁵ cells/well). For PBMCs, the strongest 62.8% of apoptotic cells in the co-culture was measured for the combined OX40L with IL-2 stimulation (Figure 4B). OX40L alone resulted in 48.5% of cell apoptosis, IL-2 induced 40.2% cell apoptosis, and untreated control PBMCs showed 38.7% of apoptotic cells (Figure 4B).

In the case of TILs, the number of apoptotic cells was highest (69.4%) for the combined OX40L and IL-2 stimulation compared to the OX40L stimulation alone (54.3%). In contrast, a minor difference was observed between IL-2 treatment alone (46.3%) and the control untreated TILs (44.2%) (Figure 4C).

Infection of co-cultured GC patients TILs and GC primary cell with adenovirus pAd-IL-2-OX40L leads to tumor cell apoptosis

To test the efficiency of combined OX40L and IL-2 produced internally by co-cultured cells, we constructed a pAd-IL-2-OX40L adenovirus vector carrying OX40L and IL-2 genes (Figure 5A). pAd-IL-2-OX40L, at a concentration of 20MOI, was added to TILs and primary GC co-culture 24 hours after establishing the TILs/GC primary cell at a ratio of 0.2 using 10⁶ GC primary cells per well.

Figure 5 Apoptotic effects of infectious adenovirus pAd-IL-2-OX40L on co-cultured gastric cancer patients TILs and gastric cancer primary cells. (A) Schematic presentation of adenoviral vector construction expressing OX40L/IL-2 and transfection of HEK293K cells with subsequent production of infectious AdV vector particles. (B) Flow cytometry analyses of gastric cancer primary cells, gastric cancer primary cells infected with pAd-IL-2-OX40L, gastric cancer primary cells and TILs, and co-culture of gastric cancer primary cells infected with pAd-IL-2-OX40L and TILs. Statistical analysis of all data was performed using one-way ANOVA. Data are shown as mean ± standard deviation. ns, nonsignificant; *, P<0.05; **, P<0.01. ANOVA, analysis of variance; IL-2, interleukin-2; OX40L, OX40 ligand; TILs, tumor-infiltrating lymphocytes.

We prepared four different groups of cultures: (I) GC primary cells; (II) GC primary cells infected with pAd-IL-2-OX40L; (III) GC primary cells and TILs; (IV) co-culture of GC primary cells infected with pAd-IL-2-OX40L and TILs. Flow cytometry performed after 72 hours in co-culture infected cells with pAd-IL-2-OX40L, showed the highest number of apoptotic cells (64.7%) compared to the other three groups (Figure 5B). As expected, the co-culture of TILs and GC primary cells shown in Figure 5B was almost identical, with 43.1% of apoptosis, compared to results presented in Figure 4C, 44.2% of apoptosis. The GC primary cells group has the lowest apoptosis of 21.6%, whereas the GC primary cells infected with pAd-IL-2-OX40L had 30.3% apoptosis (Figure 5B). These results suggest the possibility of applying this new combinatory OX40L/IL-2 immunotherapy to GC patients.

Discussion

Despite the noticeable immunotherapy improvements reached in different types of tumors, GC persists as a challenge, and the various immunotherapy strategies that have shown positive results in animal models are still facing different problems in enhancing a correct and durable anti-tumor host’s immune response (53). Furthermore, these therapies have limited effects on tumor growth and do not have significant outcomes on GC patients’ survival (54). In the first place it is fundamentally important to comprehensively examine the biological molecular behavior of the cell repertoire present in the TME to improve the design of specific immunotherapy strategies. Moreover, the spatial expression of distinct genes within the tumor and the stroma can be correlated with the specific behavior of the tumor cells and tumor evolution. Recently, an increased number of studies have been based on one hand on the analysis of gene expression at the mRNA level using bioinformatics tools, and on the other hand, the histochemical and biochemical analysis of TILs that reside within the TME in different types of tumors (38,55-59). This phenotypically diverse population affected by different metabolic factors is proven to influences the results of anti-tumor immunotherapies. For this reason, the relationship between the TILs in the TME and the anti-tumor immune response is recognized as an important research subject related to immunotherapeutic treatments in many different tumor types (38). The TILs assessment has shown its value as a valid biomarker in various tumors, such as melanoma, prostate cancer, breast cancer, and non-small cell lung carcinoma, as well as in GC (38,60-62). In GC, the investigation of the different T cell subpopulations within the tumor and its surroundings is an applied tool for the evaluation of the cancer advancement, and in the case of immunotherapy approaches, can potentially predict the prognosis and help to design a specific strategy to elicit a proper host immune response (63,64).

In our study, we have extended the knowledge about the molecular mechanism of TILs potentially applicable in immunotherapies by characterizing cell heterogeneity and further exploring the expression of specific genes that can potentially be used as biomarkers in GC. Our bulk RNA-seq analysis of the genome dataset of GC patients obtained from TCGA showed that the expression of TNFRSF4 is partially detected in the same location of T_reg cells whereas TNFSF4 is poorly expressed. Furthermore, we characterized the cells positive for CD3D gene since expression of CD3D gene and the detection of CD3D protein in intratumor area of GC samples is correlated with better prognosis (65). The obtained results from scRNA-seq led us to further explore the effects of OX40L in combination with IL-2, as an improved and complementary immunotherapy approach.

Previous studies have established that OX40/OX40L axis plays an important role in regulating effective anti-tumor response (5,63,64). OX40 and OX40L since the beginning were investigated for their costimulatory properties on effector T cells in different tumor immunotherapies (66-69). Several mouse models using OX40 and OX40L alone or in combination with other molecules have shown strong efficacy, while different clinical trials are still underway to test the increasing therapeutic potency of OX40 either as agonist antibodies monotherapy or in combination with other molecules. A variety of agonistic antibodies targeting OX40 have been developed. Among them, BAT6026 is a new anti-OX40 agonist antibody currently under phase I clinical evaluation (70). Another example is ivuxolimab, which, in combination with the 4-1BB agonist antibody utomilumab, has shown encouraging efficacy with manageable adverse events in patients with solid tumors, although further studies are required due to the limited number of treated cases (71). The strong immunostimulatory properties of OX40L were shown in combination with nanoparticle-mediated mRNa-based immunotherapy in mice, where the delivery of OX40L mRNA alone resulted in a strong response against solid tumors (72). The gene therapy was also applied by anchoring OX40L to tumor cells to induce tumor “self-killing” in combination with anti-PD1 therapy (73). An oncolytic virus bearing OX40L was constructed in a mice model and tested against pancreatic ductal adenocarcinoma (PDAC). This system was shown to decrease T_regs, increase the T cell’s anti-tumor response, and prolong the survival in PDCA-bearing mice (74). All these strategies that have validated the efficacy of these molecules in anti-tumor activity require novel or complementary approaches to extend the benefits to all types of tumors in a broader cohort.

To further validate the potential of the costimulatory molecule OX40, herein we employed a new approach to test OX40L in combination with IL-2 on GC samples of non-treated patients. The importance of IL-2 has been proven since its discovery. It was the first approved immunotherapy in metastatic renal cell carcinoma and melanoma, validating its potential to stimulate a robust immune response (75-77). However, due to the short half-life of this cytokine and its rapid clearance, the systemic administration of IL-2 in cancer patients requires high-dose administration with repeating cycles that often results in AE. Instead, low doses primarily activate and promote the survival of T_reg cells that compete with effector T cells in IL-2 absorption, causing their apoptosis due to cytokine deprivation (78). Moreover, in GC patients, the presence of T_reg cells in TME is associated with poor prognosis since they limit the efficacy of current immunotherapies. To circumvent these obstacles, we tested a possible suitable immunotherapy that applied OX40L in combination with IL-2 as costimulatory factors for enhancing the immune response elicited by T cells. Specifically, we first tested whether OX40L and IL-2 promote T cell proliferation in PBMCs and TILs of GC patients. Indeed, we found that combining OX40L/IL-2 greatly improved the proliferation of TILs and T cells in PMBC compared with IL-2 alone and OX40L alone. Moreover, to complement the knowledge about the molecular mechanism, we tested the effect on T cells by measuring the expression levels of TNF-a, IFN-γ, perforin, and granzyme B, which are active during the anti-tumor immune response. Quantitative PCR (qPCR) measurements for mRNA and ELISA for secreted proteins revealed the highest expression of all these molecules when OX40L and IL-2 were used in combination. These findings strengthen the notion that our novel combinatorial approach can be applied as an emerging complementary strategy for GC immunotherapy. The confirmed usefulness of the combinatory OX40L/IL-2 was also shown by the high number of apoptotic tumor cells in the treated primary culture cells obtained from GC patients. Thus, this new approach can be considered as an anti-GC immunotherapy. The final step in showing the efficacy of our approach and its possible application as immunotherapy in GC was the implementation of the delivery of a recombinant adenoviral vector containing OX40L and IL-2 to patients’ TILs.

Conclusions

Our data revealed that IL-2 combined with OX40L, delivered either in the medium or in a form of cellular therapy involving patients TILs transfected with a recombinant adenoviral vector, greatly influenced the activation of TILs, resulting in tumor cells apoptosis. Thus, the combination of IL-2 and OX40L could be considered as a new immunotherapeutic strategy complementing other costimulatory modulators as enhancement of a proper host immune response against tumor growth and progression in GC.

Acknowledgments

We thank Erguang Li (Nanjing University, China) for providing adenovirus vectors.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-707/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-707/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-707/prf

Funding: This research was funded by the Nanjing Special Fund for Health science and Technology Development Program of China (No. YKK21154).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-707/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study protocols involving human samples were approved by the Ethics Committee of affiliated Hospital of Jiangnan University (No.LS2019061). Each patient enrolled in the study signed a consent form approved by the ethics committee.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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