ox40  (Miltenyi Biotec)

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: Spatial distribution of OX40L + cells in the melanoma TME. (a) Representative mIF image showing OX40L (green), SOX10 (melanoma nuclei; orange when alone, magenta when overlaid with DAPI), and DAPI (blue). OX40L + cells localize within intratumoral (IT), tumor–stroma margin (TM), and tumor-peripheral (TP) regions. (b) Representative image including OX40 (red), illustrating OX40L + and OX40 + cells in spatial proximity suggestive of receptor–ligand co-localization (white-dashed squares). Right: higher-magnification view of the star-marked area.

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:

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: Quantitative analysis of OX40L + and OX40 + cells and their spatial co-localization in melanoma. Multiplex immunofluorescence analysis of OX40L (green), OX40 (red), and DAPI (blue) was performed across 30 melanoma tumors. Quantification was based on 5–10 regions of interest (ROIs; 3 mm² each) per tumor. Co-localization was defined as a distance of <20 μm between OX40 + and OX40L + cells. (a) Total nucleated cell counts (left), OX40L + and OX40 + subpopulation counts (middle), and the corresponding percentages of each subpopulation relative to total cell counts (right) across tumors. Each dot represents the mean value across all ROIs within a single tumor. (b) Percentages of OX40L + cells (green), OX40 + cells (red), and the number of co-localization events normalized to all DAPI + nuclei for individual tumors, ordered by increasing OX40L + frequency. Each dot represents a single ROI. (c) Box plots showing the proportion of OX40L + cells (green) and OX40 + cells (red) within their respective populations that co-localize with the corresponding counterpart. The mean is indicated by a “X” mark and the median by a horizontal line. ***p < 0.001, Student’s t-test.

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: 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

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: Co-expression of OX40 and OX40L with other immune checkpoint proteins (ICPs) in tumor-resident Tregs. Exploratory quantitative mIF analysis of three melanoma tumors (8–15 ROIs per tumor). (a) Box plots showing the proportions of Tregs expressing each ICP (OX40L, OX40, GITR, LAG3, TIM3, and PD1) among all Tregs. Each dot represents one ROI. Statistical comparisons between all groups are summarized in the accompanying table below. (b) Box plots showing the proportions of OX40L + and OX40 + cells within All Tregs, and within GITR + , LAG3 + , TIM3 + , and PD1 + Treg subsets. Each dot represents one ROI. Statistical significance p-values are shown *p<0.05, **p<0.01, ***p<0.001.

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

Journal: Translational Cancer Research

Article Title: OX40L and IL-2 combination strategy for gastric cancer immunotherapy

doi: 10.21037/tcr-2025-707

Figure Lengend Snippet: 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.

Article Snippet: 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).

Techniques: Immunohistochemical staining, Staining, Immunofluorescence, Expressing, Flow Cytometry, Standard Deviation, Immunohistochemistry

Journal: Translational Cancer Research

Article Title: OX40L and IL-2 combination strategy for gastric cancer immunotherapy

doi: 10.21037/tcr-2025-707

Figure Lengend Snippet: 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.

Article Snippet: 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).

Techniques: Flow Cytometry, Expressing, Concentration Assay, Standard Deviation

Journal: Translational Cancer Research

Article Title: OX40L and IL-2 combination strategy for gastric cancer immunotherapy

doi: 10.21037/tcr-2025-707

Figure Lengend Snippet: 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.

Article Snippet: 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).

Techniques: Co-Culture Assay, Staining, Immunohistochemistry, Labeling, Flow Cytometry, Cell Culture, Standard Deviation

Journal: Translational Cancer Research

Article Title: OX40L and IL-2 combination strategy for gastric cancer immunotherapy

doi: 10.21037/tcr-2025-707

Figure Lengend Snippet: 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.

Article Snippet: 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).

Techniques: Cell Culture, Plasmid Preparation, Expressing, Transfection, Flow Cytometry, Infection, Co-Culture Assay, Standard Deviation