Prognostic immunological implications of OX40L expression in the tumor microenvironment of melanoma

Immunotherapy targeting immune checkpoint proteins (ICPs) has transformed cancer care, yet current treatments focus on a narrow set of inhibitory ICPs and benefit only a subset of patients. The co-stimulatory pair OX40-OX40L, implicated in inflammation and autoimmunity, also plays roles in cancer immunity. We previously showed that high OX40L mRNA expression in melanoma correlates with favorable prognosis and improved responses to PD-1 blockade. However, the protein-level expression and functions of OX40L in melanoma remain poorly defined.

Formalin-fixed paraffin-embedded primary tumor samples from 30 patients with stage II-III melanoma were analyzed by multiplex immunofluorescence combined with quantitative image analysis. OX40L and OX40 expression were evaluated alongside immune cell phenotyping markers. Regulatory T cells (Tregs) isolated from human peripheral blood were examined by flow cytometry and RT-qPCR. Associations with recurrence were assessed in depth-matched subsets (n=22) using Kaplan-Meier analysis.

OX40L was detected across tumor, immune, and stromal compartments, with marked intertumoral heterogeneity. OX40⁺ cells were less frequent but were often found in spatial proximity to OX40L⁺ cells. OX40L was infrequently detected in melanoma cancer cells, and was more prevalent in antigen-presenting cells, CD4⁺/CD8⁺ T cells, and regulatory T cells. Strikingly, intratumoral Tregs expressed OX40L more frequently than OX40 or other ICPs, whereas blood-derived Tregs showed the opposite pattern, with OX40 predominating over OX40L. Disease recurrence following resection of the primary tumor was associated with lower proportions of OX40L-expressing myeloid cells, providing preliminary evidence for a potential link between myeloid OX40L expression and recurrence risk.

OX40L protein expression is a heterogeneous but prominent feature of the melanoma microenvironment, with cell type-specific expression patterns that include regulatory T cells. An exploratory association was seen between myeloid OX40L expression and clinical outcome, warranting further investigation.