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

Journal: Frontiers in Immunology

Article Title: Loss of ATG5 impairs CD4+ T cell activation and promotes anti-tumor responses

doi: 10.3389/fimmu.2025.1284391

Figure Lengend Snippet: Characterization of the OX40-Cre deleter line. (A) Scheme describing the generation of Rosa26-mT/mG fl/wt OX40-Cre tg/wt mice. (B) Dot plots of tdTomato and EGFP expression within unstimulated or anti-CD3-stimulated CD3 + , CD19 + and GR-1 + cells from Rosa26-mT/mG fl/wt , OX40-Cre tg/wt and Rosa26-mT/mG fl/wt OX40-Cre tg/wt mice. (C) Dot plots of tdTomato and EGFP expression within unstimulated or anti-CD3-stimulated CD4 + , CD8 + and CD4 + Foxp3 + cells from Rosa26-mT/mG fl/wt OX40-Cre tg/wt mice.

Article Snippet: OX40- and OX40+ cells were purified using a CD4+ CD25+ regulatory T cell isolation kit for magnetic separation (130-091-041; Miltenyi Biotec) according to the manufacturer’s protocol, with the modification that the anti-OX40-PE (#119410, BioLegend) was used instead of the anti-CD25-PE provided in the kit.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Loss of ATG5 impairs CD4+ T cell activation and promotes anti-tumor responses

doi: 10.3389/fimmu.2025.1284391

Figure Lengend Snippet: No gross alterations of T cell development in Atg5 ΔOX40 mice. (A) Scheme describing the generation of Atg5 ΔOX40 mice. (B, C) Immunoblot of ATG5 and LC3 expression in OX40-negative and OX40-positive cells from Atg5 ΔOX40 mice. Actin was used as a loading control. (D) Dot plots of thymic CD4- and CD8-expressing cells in Atg5 fl/fl , OX40-Cre and Atg5 ΔOX40 mice. Mean frequency +/-SD are given; n=10-12. The one-way ANOVA and Tukey’s multiple comparison tests were used for statistical analysis. (E-G) Absolute cell numbers of thymi (E) , spleen (F) and pLN (G) from the indicated mice. Mean and SD are shown; n=12-16. (H) Representative examples of lymph nodes from Atg5 ΔOX40 and control mice. **p < 0.01 and ***p < 0.001.

Article Snippet: OX40- and OX40+ cells were purified using a CD4+ CD25+ regulatory T cell isolation kit for magnetic separation (130-091-041; Miltenyi Biotec) according to the manufacturer’s protocol, with the modification that the anti-OX40-PE (#119410, BioLegend) was used instead of the anti-CD25-PE provided in the kit.

Techniques: Western Blot, Expressing, Control, Comparison

Journal: Frontiers in Immunology

Article Title: Loss of ATG5 impairs CD4+ T cell activation and promotes anti-tumor responses

doi: 10.3389/fimmu.2025.1284391

Figure Lengend Snippet: Characterization of the CD4+ and CD8+ T cell compartment in Atg5 ΔOX40 mice. (A) Dot plots of CD4 + and CD8 + cell populations from pLN of the indicated mice. (B) Graphical presentation of frequencies of CD4 + and CD8 + cells. Mean frequency +/-SD are given; n=10-12. (C) Graphical presentation of absolute CD4 + and CD8 + cell numbers in pLN of the indicated mice. Mean and SD are shown; n=9-11. (D) Dot plots of CD3 + T cells vs. CD19 + B cells in pLN of Atg5 fl/fl and Atg5 ΔOX40 mice. **p < 0.01 and ***p <0.001.

Article Snippet: OX40- and OX40+ cells were purified using a CD4+ CD25+ regulatory T cell isolation kit for magnetic separation (130-091-041; Miltenyi Biotec) according to the manufacturer’s protocol, with the modification that the anti-OX40-PE (#119410, BioLegend) was used instead of the anti-CD25-PE provided in the kit.

Techniques:

Journal: Frontiers in Immunology

Article Title: Loss of ATG5 impairs CD4+ T cell activation and promotes anti-tumor responses

doi: 10.3389/fimmu.2025.1284391

Figure Lengend Snippet: Lymphadenopathy and activation defect in peripheral T cells of Atg5 ΔOX40 mice. (A) Absolute cell numbers of pLN from anti-CD3 injected mice. Mean and SD are indicated; n=12-16. (B) Absolute cell numbers of CD4 + cells and CD8 + cells from pLN of anti-CD3 injected mice. Mean and SD are shown; n=12-16. (C) Histograms of CD69 expression by CD4 + T cells in pLN, as well as graphs showing the corresponding frequency and geomean. Mean and SD are shown; n=2-3. (D) Histograms of CD25 expression by CD4 + T cells in pLN, as well as graphs showing the corresponding frequency and geomean. Mean and SD are shown; n=9-11. (E) Histograms of OX40 expression by CD4 + T cells in pLN, as well as graphs showing the corresponding frequency and geomean. Mean and SD are shown; n=7-9. *p < 0.05; **p < 0.01 and ***p < 0.001.

Article Snippet: OX40- and OX40+ cells were purified using a CD4+ CD25+ regulatory T cell isolation kit for magnetic separation (130-091-041; Miltenyi Biotec) according to the manufacturer’s protocol, with the modification that the anti-OX40-PE (#119410, BioLegend) was used instead of the anti-CD25-PE provided in the kit.

Techniques: Activation Assay, Injection, Expressing

Journal: Frontiers in Immunology

Article Title: Loss of ATG5 impairs CD4+ T cell activation and promotes anti-tumor responses

doi: 10.3389/fimmu.2025.1284391

Figure Lengend Snippet: Effect of autophagy-deficiency on Treg cells in Atg5 ΔOX40 mice. (A) CD25 and OX40 expression within EGFP - (grey) and EGFP + (black) cells from peripheral lymph nodes of Rosa26-mT/mG fl/wt OX40-Cre tg/wt mice stimulated with anti-CD3 or left untreated. (B) Frequencies and (C) absolute cell numbers of CD4 + Foxp3 + Treg cells in the indicated organs from unstimulated and anti-CD3 stimulated mice. Mean and SD are shown; (A) n=9–13 and (B) n=6-9. Statistical analysis was performed with the one-way ANOVA and Tukey’s multiple comparison tests. (D) Histograms and graphs depicting CTLA-4- and Nrp-1-expression by Treg cells. Unstimulated mice are shown in the left panels and anti-CD3 treated animals are depicted in the right panels. Mean and SD are shown; CTLA-4: n=6-9, Nrp-1: n=5-9. *p < 0.05; **p < 0.01 and ***p < 0.001.

Article Snippet: OX40- and OX40+ cells were purified using a CD4+ CD25+ regulatory T cell isolation kit for magnetic separation (130-091-041; Miltenyi Biotec) according to the manufacturer’s protocol, with the modification that the anti-OX40-PE (#119410, BioLegend) was used instead of the anti-CD25-PE provided in the kit.

Techniques: Expressing, Comparison

Journal: Frontiers in Immunology

Article Title: Loss of ATG5 impairs CD4+ T cell activation and promotes anti-tumor responses

doi: 10.3389/fimmu.2025.1284391

Figure Lengend Snippet: Unaltered T cell death and Treg suppressive capacity. (A) CD4+ T cells were activated with concanavalin A and IL-2 for 3 days and subsequently stimulated with anti-CD3, PMA/ionomycin, or CD95L. Cell death was measured with 7AAD and flow cytometry. The data shown is from three independent experiments. The two-way ANOVA Dunnett´s multiple comparison test was used for statistical analysis. (B) In vitro suppression assay comparing Treg cells from Atg5 ΔOX40 and control mice. Wild-type conventional T cells were labeled with cell trace violet (CTV) and incubated at the indicated ratios with Treg cells of the respective genotype. Cells were analyzed by flow cytometry. Representative dot plots are shown on the left; the right panel shows the summary of three independent experiments. *p < 0.05.

Article Snippet: OX40- and OX40+ cells were purified using a CD4+ CD25+ regulatory T cell isolation kit for magnetic separation (130-091-041; Miltenyi Biotec) according to the manufacturer’s protocol, with the modification that the anti-OX40-PE (#119410, BioLegend) was used instead of the anti-CD25-PE provided in the kit.

Techniques: Flow Cytometry, Comparison, In Vitro, Suppression Assay, Control, Labeling, Incubation

Journal: Frontiers in Immunology

Article Title: Loss of ATG5 impairs CD4+ T cell activation and promotes anti-tumor responses

doi: 10.3389/fimmu.2025.1284391

Figure Lengend Snippet: Enhanced anti-tumor response in Atg5 ΔOX40 mice. (A) B16 tumor growth curves in individual mice. Atg5 ΔOX40 mice are indicated as black triangles and OX40-Cre control mice are depicted as violet squares. (B-D) Frequency of CD4 + Foxp3 + (B) and CD4 + Foxp3 - (C) and CD4 - CD8 + (D) cell populations in the tumor, draining lymph nodes (dLN), control lymph node (cLN) and spleen. (E) Dot plots of CD62L and CD44 expressing CD4 + Foxp3 - and CD4 + Foxp3 + cell populations from the tumor and dLN, mean frequency +/- SD are shown. One of two experiments is depicted where n=7-8. *p < 0.05; **p < 0.01 and ***p < 0.001.

Article Snippet: OX40- and OX40+ cells were purified using a CD4+ CD25+ regulatory T cell isolation kit for magnetic separation (130-091-041; Miltenyi Biotec) according to the manufacturer’s protocol, with the modification that the anti-OX40-PE (#119410, BioLegend) was used instead of the anti-CD25-PE provided in the kit.

Techniques: Control, Expressing