Associations of OX40 and Tumor Microenvironment with Outcomes in Diffuse Large B-Cell Lymphoma

Background: OX40, also termed CD134 and TNFRSF4, is a novel costimulatory molecule expressed on activated T cells. After receiving stimulatory signals from ligands, OX40 initiates downstream signals and promotes T-cell functions, playing an important role in enhancing the anti-tumor immune response. Many preclinical studies, whether for the individual application of OX40 agonists or in combination strategies, have achieved encouraging results, and large number of OX40 agonists have undergone clinical test in solid tumors and been verified to have excellent safety and tolerability, so that their redeployment into B-cell non-Hodgkin’s lymphoma is just occurring now. However, little is known about how OX40 functions in microenvironment in diffuse large B-cell lymphoma (DLBCL). The aims of this study were to determine the OX40 expression in DLBCL microenvironment and the relationship with the prognosis of patients.

Methods: The OX40 mRNA expression between DLBCL tissues and normal tissues, and its associated with survival were analyzed through Oncomine databases, including the Basso lymphoma dataset (n=57), the Compagno lymphoma dataset (n=64) and the Dave lymphoma dataset (n=233). The GEO2R online analysis was utilized to acquire the differentially expressed genes (DEGs) in the OX40 mRNA high and low expression groups, and GO and KEGG functional enrichment analysis on these DEGs using Omicshare tool were further performed. The CD4, CD8, Pax-5, OX40 and Foxp3 multiplexed immunofluorescence staining, and quantitative analyses of OX40+ tumor-infiltrating T-lymphocytes, including CD4+ T-cells, CD8+ T-cells, and Tregs, were performed in DLBCL biopsy samples.

Results: We found that the OX40 mRNA expression was significantly upregulated in DLBCL patients compared to that of normal lymphoid tissues through Oncomine (p<0.001), and high OX40 mRNA expression was significantly correlated with the favorable prognosis (p=0.018). The 45 up-regulated and 4 down-regulated genes were identified as to be significantly associated with OX40 expression (p<0.05). GO analysis results showed that changes in biological processes (BP) of DEGs were significantly enriched in the activation and regulation of T cells and other lymphocytes. Changes in molecular function (MF) were mainly enriched in tumor necrosis factor-activated receptor activity and chemokine receptor binding. Changes in cell component (CC) of DEGs were mainly enriched in the external side of membrane and immunological synapse. KEGG pathway analysis revealed that the DEGs were mainly enriched in the cytokine-cytokine receptor interaction, T cell receptor signaling pathway, chemokine signaling pathway, and NF-κB signaling pathway. The total OX40+ cells expression level in patients was significantly associated with Ann Arbor stage (p=0.039) and IPI score (p=0.047). The expression of OX40, compared with that in stable disease (SD)/progressive disease (PD) patients, was significantly increased in complete response (CR) or partial response (PR) patients (p=0.002; p=0.002, respectively). A higher total expression of OX40 and a greater number of CD8+/OX40+ T-cells were significantly correlated with longer overall survival (OS) (p=0.008; p=0.004, respectively). The expression of CD8+/OX40+ T-cells maintained prognostic value for OS in multivariate analysis (p=0.030; p=0.024, respectively).

Conclusions: Here, for the first time, we describes the expression characteristics of OX40 and tumor microenvironment in DLBCL and demonstrates that the infiltration strength of OX40+ T-cells—particularly the infiltration strength of OX40-expressing CD8+ T-cells on DLBCL—has a significant impact on survival. DLBCL patients may benefit from the OX40 agonist antibodies and may enhancement of the efficacy of existing immune checkpoint inhibition therapies, providing a theoretical foundation for the potential clinical transformational application value of OX40 agonists for DLBCL therapy.