Effect of exosome-derived lncRNA on MAL ⁺ CTL apoptosis in remodeling of the immune microenvironment in HPV ⁺ penile squamous cell carcinoma.

e17022 Background: Patients with HPV + PSCC have a poor prognosis, and the immune escape mechanisms remain a major challenge in improving clinical outcomes. This study aims to investigate the role of exosome-derived long non-coding RNAs (lncRNAs) in modulating the immune microenvironment, particularly through their interaction with MAL + CTLs in HPV + PSCC. Methods: We collected tumor samples from HPV + PSCC patients and performed scRNA-seq to analyze the distribution and functional state of immune cells within the tumor microenvironment. Spatial transcriptomics were employed to explore the spatial localization of Tregs and CTLs within the tumor. Exosomes were isolated from the culture media of Treg cells and analyzed for the presence of lncRNAs. Additionally, the interaction between exosome-derived lncRNAs and miRNAs, particularly hsa-miR-619-3p, was examined in vitro. The impact of exosome-derived lncRNAs on MAL + CTL apoptosis was assessed using flow cytometry and apoptosis assays. Results: Our clinical data and scRNA-seq analysis revealed that HPV + PSCC tumors are characterized by an increased proportion of Treg cells and a decreased presence of CD8 + CTLs compared to HPV- PSCC or normal tissues. Importantly, we observed a significant correlation between the number of Tregs and the severity of immune suppression in HPV + PSCC. Spatial transcriptomics further revealed that Tregs were predominantly located in close proximity to CD8 + CTLs within the tumor microenvironment, suggesting potential interactions between these cell populations. Interestingly, exosome isolation from Tregs confirmed the presence of exosome-derived lncRNAs, which were shown to significantly alter the miRNA profile in surrounding immune cells. Specifically, we identified that exosome-derived lncRNAs from Tregs bound to hsa-miR-619-3p, a miRNA previously linked to T-cell dysfunction and immune evasion. We further demonstrated that this interaction led to an increase in MAL expression in CTLs, which in turn triggered their apoptosis. Flow cytometry analysis revealed that exposure to exosome-derived lncRNAs from Tregs resulted in a significant increase in MAL + CTLs and a concomitant increase in CTL apoptosis. Besides, the in vivo experiments using HPV + PSCC xenografts in mice showed that Treg-derived exosomes significantly impaired anti-tumor immunity, leading to increased tumor growth and reduced CTL infiltration in the tumor. Importantly, inhibition of MAL expression in CTLs by RNA interference rescued CTL function and partially restored anti-tumor immunity, confirming the pivotal role of MAL + CTLs in immune suppression. Conclusions: Our findings reveal a novel mechanism of immune evasion in HPV + PSCC, where Tregs secrete exosome-derived lncRNAs that interact with miR-619-3p to upregulate MAL expression in CTLs, leading to their apoptosis.