Journal: Frontiers in Immunology

Article Title: Prognostic immunological implications of OX40L expression in the tumor microenvironment of melanoma

doi: 10.3389/fimmu.2026.1745742

Figure Lengend Snippet: Cellular phenotyping of OX40L + cells in melanoma tumors. (a) OX40L expression on SOX10 + melanoma cells was manually assessed in 30 tumors (5–10 ROIs per tumor; 3 mm² per ROI). Representative images of membrane-associated OX40L expression and a summary of prevalence across tumors are shown. Rare OX40L + melanoma cells (<10 cells per ROI) were classified as “scattered” or “focally clustered,” whereas “widespread” distribution was defined as OX40L expression in ~10% of SOX10 + melanoma cells per ROI. (b) Exploratory OX40L immune cell phenotyping was performed on serial tumor sections from three melanoma cases. Representative multiplex immunofluorescence images illustrate OX40L expression in macrophages (CD68 + , CD163 + ), dendritic cells (CD11c + ), CD4 + T cells, CD8 + T cells, and Foxp3 + regulatory T cells. (c) Proportions of immune cell subsets within the TME across three tumors (8–15 ROIs per tumor). (d) Violin plots depicting the proportion of OX40L + cells within each immune subset. Mac, macrophages; DC, dendritic cells; T, T cells. Each dot represents one ROI, dot colors represent tumor sample, and mean and median values are shown in red squares and black diamonds.

Article Snippet: Cells were stained with fluorophore-conjugated antibodies against CD4, CD25, OX40, OX40L, and intracellular Foxp3 using the Foxp3 Buffer Set (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Expressing, Membrane, Multiplex Assay, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: Prognostic immunological implications of OX40L expression in the tumor microenvironment of melanoma

doi: 10.3389/fimmu.2026.1745742

Figure Lengend Snippet: Expression of OX40L and OX40 on regulatory T cells in melanoma tumors and peripheral blood of a healthy donor. OX40L and OX40 expression were assessed on Foxp3 + regulatory T cells (Tregs) across 30 melanoma tumors using quantitative multiplex immunofluorescence analysis (10–20 ROIs per tumor; 3 mm² per ROI). (a) Representative images showing Foxp3 + Tregs within a melanoma tumor (Foxp3 + nuclei shown in orange when alone or magenta when overlaid with DAPI) co-expressing OX40L (green), OX40 (red), or both. Right panels show higher-magnification views of circled cells with individual color channels; all panels share the same scale bar, indicated in the DAPI image. (b) Box plots depicting the proportions of OX40L + , OX40 + , and double-positive Tregs across 30 tumors. The mean is indicated by a “X” mark and the median by a horizontal line (***p < 0.001, Student’s t-test). Jittered dots represent the mean value per tumor across ROIs. (c) Box plots showing the prevalence of OX40L + (green), OX40 + (red), and OX40L + /OX40L + double-positive (blue) Tregs per tumor, ordered by OX40L + frequency. Jittered dots represent individual ROIs. (d) Tregs isolated from peripheral blood of a healthy donor were enriched and expanded in vitro under IL-2/CD3/CD28 stimulation. The proportions of OX40L + and OX40 + cells within live, singlet Foxp3 + regulatory T cells, as defined in the Methods, are shown over 13 days of culture.

Article Snippet: Cells were stained with fluorophore-conjugated antibodies against CD4, CD25, OX40, OX40L, and intracellular Foxp3 using the Foxp3 Buffer Set (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Expressing, Multiplex Assay, Immunofluorescence, Isolation, In Vitro