Journal: International Journal of Molecular Sciences

Article Title: Roles of OX40 and OX40 Ligand in Mycosis Fungoides and Sézary Syndrome

doi: 10.3390/ijms222212576

Figure Lengend Snippet: OX40 and OX40 ligand (OX40L) expression in patients with cutaneous T-cell lymphoma (CTCL). ( A , B ) Quantitative RT-PCR was performed to measure expression levels of OX40 and OX40L using mRNA extracted from MF/SS lesional skin ( n = 57; 49 MF cases and 8 SS cases) and normal skin ( n = 13). ( C ) Correlations between soluble IL-2 receptor (sIL-2R) and OX40 or OX40L mRNA expression in MF/SS patients. ( D ) Positive correlations between OX40 and OX40L mRNA expression in MF/SS lesional skin. ( E ) Serum soluble OX40 and soluble OX40L levels in CTCL patients. ( F ) MF/SS patients with high OX40 mRNA expression exhibited poor prognosis. Means are presented as bars. ** p < 0.01.

Article Snippet: Anti-human OX40 (2 μg/mL; AF3388; R&D Systems) and anti-human OX40L (2 μg/mL; clone MM0505-8S23; Novus Biologicals, Centennial, CO, USA) were used as neutralizing antibodies.

Techniques: Expressing, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Roles of OX40 and OX40 Ligand in Mycosis Fungoides and Sézary Syndrome

doi: 10.3390/ijms222212576

Figure Lengend Snippet: OX40 and OX40L expression in lesional skin of MF/SS was determined by immunohistochemistry (original magnification ×400, scale bar = 100 µm). ( A ) Representative results are shown. ( B ) Counts of OX40 and OX40L-positive cells. Ten cases of patch/plaque MF, nine cases of tumor MF/SS, and seven cases of healthy subjects. ** p < 0.01.

Article Snippet: Anti-human OX40 (2 μg/mL; AF3388; R&D Systems) and anti-human OX40L (2 μg/mL; clone MM0505-8S23; Novus Biologicals, Centennial, CO, USA) were used as neutralizing antibodies.

Techniques: Expressing, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: Roles of OX40 and OX40 Ligand in Mycosis Fungoides and Sézary Syndrome

doi: 10.3390/ijms222212576

Figure Lengend Snippet: OX40 and OX40L expression was analyzed by flow cytometry in CD4 + CD7 − T cells from 6 Sézary syndrome (SS) patients and CD4 + T cells from 6 healthy controls. Comparison of OX40 and OX40L-positive cell ratios in peripheral blood CD4 + CD7 − T cells in patients with SS or in healthy controls peripheral blood CD4 + T cells. n.s.: not significant. * p < 0.05.

Article Snippet: Anti-human OX40 (2 μg/mL; AF3388; R&D Systems) and anti-human OX40L (2 μg/mL; clone MM0505-8S23; Novus Biologicals, Centennial, CO, USA) were used as neutralizing antibodies.

Techniques: Expressing, Flow Cytometry, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Roles of OX40 and OX40 Ligand in Mycosis Fungoides and Sézary Syndrome

doi: 10.3390/ijms222212576

Figure Lengend Snippet: Involvement of OX40 and OX40L in CTCL cell lines. ( A ) OX40 and OX40L expression was analyzed by flow cytometry in human CTCL cell lines (Hut78, MyLa, and HH cells). ( B ) HH, Hut, and MyLa cells (1.0 × 10 5 /well) were cultured with anti-OX40 (5 μg/mL) and/or anti-OX40L (5 μg/mL) neutralizing antibodies for 48 hours. ( C ) Hut and HH cells (1.0 × 10 5 /well) were cultured with anti-OX40 neutralizing antibody (5 μg/mL) for 24 hours. Apoptosis (Annexin V + and 7-AAD − ) was evaluated with Annexin V and 7-AAD staining for flow cytometric analysis. * p < 0.05, ** p < 0.01.

Article Snippet: Anti-human OX40 (2 μg/mL; AF3388; R&D Systems) and anti-human OX40L (2 μg/mL; clone MM0505-8S23; Novus Biologicals, Centennial, CO, USA) were used as neutralizing antibodies.

Techniques: Expressing, Flow Cytometry, Cell Culture, Staining

Journal: International Journal of Molecular Sciences

Article Title: Roles of OX40 and OX40 Ligand in Mycosis Fungoides and Sézary Syndrome

doi: 10.3390/ijms222212576

Figure Lengend Snippet: Anti-OX40 and anti-OX40L antibodies suppressed tumor growth. Hut78 cells (5.0 × 10 6 cells) in 100 µL of PBS were injected subcutaneously into the shaved left abdomen of NSG mice. Anti-OX40 or anti-OX40L neutralizing antibodies were injected on days 0, 4, 7, and 11 intraperitoneally. ( A ) Representative images are shown. ( B ) The tumor volume was calculated using the equation: V = π (L1 × L2 2 )/6, where V = volume (mm 3 ), L1 = longest diameter (mm), and L2 = shortest diameter (mm). Values are means ± SEM. *,# p < 0.05 by Mann–Whitney U test compared with control group.

Article Snippet: Anti-human OX40 (2 μg/mL; AF3388; R&D Systems) and anti-human OX40L (2 μg/mL; clone MM0505-8S23; Novus Biologicals, Centennial, CO, USA) were used as neutralizing antibodies.

Techniques: Injection, MANN-WHITNEY, Control

Journal: Retrovirology

Article Title: Increased expression of OX40 is associated with progressive disease in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis

doi: 10.1186/1742-4690-10-51

Figure Lengend Snippet: Tax-dependent constitutive expression of OX40 in HTLV-1-infected T-cell lines and Tax-inducible JPX-9 cell line. A. Representative histograms of OX40 expression in 6 HTLV-1 infected T-cell lines (HUT-102, MT-1, MT-2, MT-4, C5/MJ, SLB-1) and two HTLV-1-uninfected T-cell lines (CEM-OX40L and CEM-OX40). Shaded histograms represent the isotype control. Tax+ or Tax- means whether these cells express Tax (Tax+) or not (Tax-). B. Representative histograms of OX40L expression in 6 HTLV-1 infected T-cell lines (HUT-102, MT-1, MT-2, MT-4, C5/MJ, SLB-1) and two HTLV-1-uninfected T-cell lines (CEM-OX40L and CEM-OX40). Shaded histograms are isotype controls. C. Flow cytometric analysis of expression of OX40 after induction of Tax in JPX-9 cells. D. Flow cytometric analysis of expression of OX40L after induction of Tax in JPX-9 cells. E. Soluble OX40 and OX40L levels in cell culture supernatant and cell lysate from 6 HTLV-1 infected T-cell lines (HUT-102, MT-1, MT-2, MT-4, C5/MJ, SLB-1) and three HTLV-1-uninfected T-cell lines (CEM-mock, CEM-OX40L and CEM-OX40). F. Soluble OX40 and OX40L levels in cell culture supernatant and cell lysate from JPX-9 cell line treated with CdCl 2 along with the induction of viral transactivator Tax.

Article Snippet: Recombinant human OX40 ligand/TNFSF4 and recombinant human OX40/TNFRSF4/Fc Chimera were purchased from R&D Systems (Minneapolis, MN) and used for the standard curve in sOX40L and sOX40 ELISA, respectively.

Techniques: Expressing, Infection, Cell Culture

Journal: Retrovirology

Article Title: Increased expression of OX40 is associated with progressive disease in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis

doi: 10.1186/1742-4690-10-51

Figure Lengend Snippet: OX40 is specifically expressed on the surface of T cells naturally infected with HTLV-1 that have the potential to produce pro-inflammatory cytokines. A. OX40 was detected on CD4 + T cells of HAM/TSP patient with anti-OX40 mAb (clones B-7B5) after 16 hours in vitro cultivation in the absence of any growth factors or mitogen. B. OX40L was not detected on CD4 + T cells of HAM/TSP patient with anti-OX40L mAb (clones 5A8) after 16 hours in vitro cultivation in the absence of any growth factors or mitogen. C. Tax protein was detected in CD4 + T cells of HAM/TSP patient after 16 hours in vitro cultivation. D. OX40 was expressed almost exclusively in naturally infected CD4 + T cells that also expressed Tax in HAM/TSP patient. E. Both HTLV-1 tax and OX40 mRNA expression in CD4 + T cells was increased after 16 hours in vitro cultivation. F. The frequency of pro-inflammatory cytokine positive cells within the OX40 + CD4 + and Tax + CD4 + populations from HTLV-1 infected individuals are significantly higher than OX40 - CD4 + and Tax - CD4 + T cells, respectively (p<0.001, Student’s t- test). One representative experiment of HAM/TSP patient (HAM/TSP1) is shown.

Article Snippet: Recombinant human OX40 ligand/TNFSF4 and recombinant human OX40/TNFRSF4/Fc Chimera were purchased from R&D Systems (Minneapolis, MN) and used for the standard curve in sOX40L and sOX40 ELISA, respectively.

Techniques: Infection, Clone Assay, In Vitro, Expressing

Journal: Retrovirology

Article Title: Increased expression of OX40 is associated with progressive disease in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis

doi: 10.1186/1742-4690-10-51

Figure Lengend Snippet: Increased expression of OX40 in vivo in rapidly progressive HAM/TSP patients. A. The plasma levels of soluble OX40 (sOX40) measured by ELISA. The plasma levels of sOX40 in typical HAM/TSP patients (chronic HAM: n=20), asymptomatic carriers (ACs: n=9) and normal uninfected healthy controls (NCs: n=13). B. No correlation between the plasma levels of sOX40 and HTLV-1 proviral load (tax copies/10,000PBMCs) from 29 HTLV-1 infected individuals (20 chronic HAM/TSP patients and 9 ACs). Data were analyzed by Spearman rank correlation. C. The cerebrospinal fluid (CSF) levels of sOX40 in rapidly progressive HAM/TSP patients (n=3), chronic HAM/TSP patients (n=22) and other neurological diseases including multiple sclerosis (MS) (n=12), aseptic meningitis (n=8), systemic lupus erythematosus (SLE) with neurological manifestations (n=5), chronic inflammatory demyelinating polyneuropathy (CIDP) (n=9), Guillain-Barré syndrome (GBS) (n=6), and amyotrophic lateral sclerosis (ALS) (n=9). Chronic HAM/TSP means typical cases fulfilling diagnostic criteria and rapidly progressive HAM/TSP is defined by patients’ incapacity to walk unaided within three months after symptoms’ onset. D. The levels of sOX40 in the CSF from HTLV-1 infected other inflammatory neurological diseases (HTLV-1+ OINDs), i.e. any inflammatory neurological disorders except for HAM/TSP which occurred in HTLV-1 infected individuals, was not significantly different from that of chronic HAM/TSP, whereas the levels of sOX40 from HTLV-1+ OINDs was significantly increased than that of non-infected OINDs (HTLV-1- OINDs). HTLV-1+ OINDs: 1 multiple sclerosis (MS), 1 SLE with neurological manifestations, 4 aseptic meningitis. HTLV-1- OINDs: 9 MS, 5 SLE with neurological manifestations, 7 CIDP, 5 GBS.

Article Snippet: Recombinant human OX40 ligand/TNFSF4 and recombinant human OX40/TNFRSF4/Fc Chimera were purchased from R&D Systems (Minneapolis, MN) and used for the standard curve in sOX40L and sOX40 ELISA, respectively.

Techniques: Expressing, In Vivo, Enzyme-linked Immunosorbent Assay, Infection, Diagnostic Assay

Journal: Retrovirology

Article Title: Increased expression of OX40 is associated with progressive disease in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis

doi: 10.1186/1742-4690-10-51

Figure Lengend Snippet: The expression of pro-inflammatory cytokines in peripheral blood mononuclear cells of HTLV-1 infected individuals

Article Snippet: Recombinant human OX40 ligand/TNFSF4 and recombinant human OX40/TNFRSF4/Fc Chimera were purchased from R&D Systems (Minneapolis, MN) and used for the standard curve in sOX40L and sOX40 ELISA, respectively.

Techniques: Expressing, Infection

Journal: Retrovirology

Article Title: Increased expression of OX40 is associated with progressive disease in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis

doi: 10.1186/1742-4690-10-51

Figure Lengend Snippet: Clinical and laboratory findings of HAM/TSP patients for whom paired CSF and plasma samples were tested for soluble OX40 (sOX40)

Article Snippet: Recombinant human OX40 ligand/TNFSF4 and recombinant human OX40/TNFRSF4/Fc Chimera were purchased from R&D Systems (Minneapolis, MN) and used for the standard curve in sOX40L and sOX40 ELISA, respectively.

Techniques:

Journal: Retrovirology

Article Title: Increased expression of OX40 is associated with progressive disease in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis

doi: 10.1186/1742-4690-10-51

Figure Lengend Snippet: Expression of OX40 in inflammatory mononuclear cells in spinal cord lesions of HAM/TSP patient with short disease duration and progressive symptoms. We studied autopsy specimens from 9 HAM/TSP patients by immunohistochemical staining. A. No OX40 positive cells are detected in the spinal cord lesion without active inflammation of a HAM/TSP patient with a long duration of illness. Magnification: ×40. B. Many infiltrating mononuclear cells are positively stained by anti-OX40 mAb in the spinal cord lesion with active inflammation of HAM/TSP patient with 2.5 years of illness. Magnification: ×40. C. There was reduced or no OX40L protein expression in spinal cord tissues of HAM/TSP patients. OX40L showed only low background staining and there was no OX40L positive staining on inflammatory mononuclear cells in the spinal cord lesions. Magnification: ×20. D. Positive control staining for OX40L positive CEM-OX40L cells. Magnification: ×20. Bar: 50 μm.

Article Snippet: Recombinant human OX40 ligand/TNFSF4 and recombinant human OX40/TNFRSF4/Fc Chimera were purchased from R&D Systems (Minneapolis, MN) and used for the standard curve in sOX40L and sOX40 ELISA, respectively.

Techniques: Expressing, Immunohistochemical staining, Staining, Positive Control

Journal: Retrovirology

Article Title: Increased expression of OX40 is associated with progressive disease in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis

doi: 10.1186/1742-4690-10-51

Figure Lengend Snippet: Anti-OX40 monoclonal antibody specifically eliminated naturally infected CD4 + T cells via antibody-dependent cell-mediated cytotoxicity (ADCC) in cultured PBMCs. Anti-OX40 mAb (clone B-7B5) reduces the percentage of Tax-positive cells, whereas the isotype control mAb (clone 2C2: anti-HIV-1 gp21, mouse IgG1) has no effect on Tax expression (1st, 2nd, and 3rd panels from left). Culture of PBMCs with anti-CD16/CD32 (Fc receptor) antibody to block Fc receptors abolishes Tax suppression by anti-OX40 mAb (4th panels from left). F(ab’) 2 fragment do not suppress Tax exppression (right panels).

Article Snippet: Recombinant human OX40 ligand/TNFSF4 and recombinant human OX40/TNFRSF4/Fc Chimera were purchased from R&D Systems (Minneapolis, MN) and used for the standard curve in sOX40L and sOX40 ELISA, respectively.

Techniques: Infection, Cell Culture, Expressing, Blocking Assay

Journal: Pharmaceutics

Article Title: Curcumin Enhances the Abscopal Effect in Mice with Colorectal Cancer by Acting as an Immunomodulator

doi: 10.3390/pharmaceutics15051519

Figure Lengend Snippet: Biodistribution results of 111 In-anti-OX40 mAb in mice receiving different treatments. Mice were euthanized 48 h after being injected with 111 In-anti-OX40 mAb, designated organs/tissues were collected, and their radioactivity was counted using a gamma counter. Except for blood, the liver, spleen, and tumors showed higher relative radioactivity than other organs. The COMB group had the highest spleen and tumor uptakes among all the groups. a, compared with the CTRL group; b, compared with the Curcumin group; c, compared with the RT group; 1, p < 0.05; 2, p < 0.01.

Article Snippet: Prior to the indium-111 radiolabeling, anti-OX40 mAb (#BE0031, clone OX86, BioXCell, Lebanon, NH, USA) was resuspended in PBS at 0.5 mg/mL and then incubated with fivefold excess DOTA-NHS ester (#HY-128890, MedChemExpress, Monmouth Junction, NJ, USA) at 4 °C overnight.

Techniques: Injection, Radioactivity

Journal: Biomedical research (Tokyo, Japan)

Article Title: Blockade of CD80/CD86-CD28 co-stimulation augments the inhibitory function of peptide antigen-specific regulatory T cells.

doi: 10.2220/biomedres.45.115

Figure Lengend Snippet: Fig. 3 Expression of CD27, OX40, ICOS and 4-1BB on Foxp3 + CD4 + or Foxp3 − CD4 + DO 11.10 T cells. Expression of in- dicated co-stimulatory signal receptors on Foxp3 + CD4 + Treg cells and Foxp3 − CD4 + Tconv cells included in freshly isolat- ed DO 11.10, primed DO 11.10, and DO 11.10 iTS cells was examined by flow cytometry. The positive staining gate was set based on isotype-matched control antibody staining. Representative histograms from three independent experiments are presented.

Article Snippet: To assess the iTS population, the following mAbs were used: peridinin chlorophyll protein (PerCP)/Cyanine (Cy) 5.5-conjugated anti-mouse CD3 mAb (17A2, BioLegend), phycoerythrin (PE)/Cy7-conjugated anti-mouse CD4 mAb (GK1.5, BioLegend), fluorescein isothiocyanate (FITC)-conjugated anti-mouse TCR DO 11.10 mAb (KJ1-26, BioLegend), PE-conjugated anti-human CD2 mAb (RPA-2.10, Bio Legend), PE-conjugated anti-mouse CD28 mAb (37.51, Bio Legend), allophycocyanin (APC)-conjugated anti-mouse CD27 mAb (LG.3A10, BioLegend), APC-conjugated anti-mouse OX40 (CD134) mAb (OX-86; Proteintech, Rosemont, IL, USA), PE-conjugated anti-mouse ICOS (CD278) mAb (7E.17G9, Bio Legend), and APC-conjugated anti-mouse 4-1BB (CD137) mAb (17B5, Bio Legend).

Techniques: Expressing, Flow Cytometry, Staining, Control

Journal: Journal of Immunology Research

Article Title: Increased Expressions of OX40 and OX40 Ligand in Patients with Primary Immune Thrombocytopenia

doi: 10.1155/2019/6804806

Figure Lengend Snippet: Detection of antiplatelet autoantibodies in ITP patients. Twenty-two (41%) of 54 patients with primary ITP were positive for antiplatelet autoantibodies detected by ELISA. The peripheral platelet counts in patients with positive antiplatelet autoantibodies (ITP(+)) and negative antiplatelet autoantibodies (ITP(-)) and in HCs. ∗∗∗ p < 0.001; ∗∗ p < 0.01; ∗ p < 0.05.

Article Snippet: Plasma levels of sOX40L were analyzed by a Quantikine Human OX40 Ligand ELISA Kit (R&D Systems, Minneapolis, MN, USA) in accordance with the manufacturer's protocol.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of Immunology Research

Article Title: Increased Expressions of OX40 and OX40 Ligand in Patients with Primary Immune Thrombocytopenia

doi: 10.1155/2019/6804806

Figure Lengend Snippet: The expression of OX40 on CD4 + T cells in ITP patients. Peripheral blood mononuclear cells (PBMCs) from 54 patients with ITP and 24 healthy controls (HCs) were isolated and stained with labeled antibodies and analyzed by flow cytometry, as described in . (a) The cells were gated initially on lymphocytes and then on CD4 + T cells. (b) The frequency of OX40 + CD4 + T cells among the total CD4 + T cells from HCs and ITP patients. (c) The relationship between the frequency of OX40 + CD4 + T cells and peripheral platelet counts in ITP patients with positive antiplatelet autoantibodies. (d) The relationship between the frequency of OX40 + CD4 + T cells and peripheral platelet counts in ITP patients with negative antiplatelet autoantibodies. ITP(+): ITP patients with positive antiplatelet autoantibodies; ITP(-): ITP patients with negative antiplatelet autoantibodies. ∗∗∗ p < 0.001; ∗∗ p < 0.01; ∗ p < 0.05.

Article Snippet: Plasma levels of sOX40L were analyzed by a Quantikine Human OX40 Ligand ELISA Kit (R&D Systems, Minneapolis, MN, USA) in accordance with the manufacturer's protocol.

Techniques: Expressing, Isolation, Staining, Labeling, Flow Cytometry

Journal: Journal of Immunology Research

Article Title: Increased Expressions of OX40 and OX40 Ligand in Patients with Primary Immune Thrombocytopenia

doi: 10.1155/2019/6804806

Figure Lengend Snippet: Plasma concentrations of soluble OX40L in patients. (a) Concentrations of plasma sOX40L in HCs and in ITP patients with negative and positive antiplatelet autoantibodies. (b) The relationship between the levels of plasma sOX40L and peripheral platelet counts in ITP patients with positive antiplatelet autoantibodies. (c) The relationship between the levels of plasma sOX40L and peripheral platelet counts in ITP patients with negative antiplatelet autoantibodies. ITP(+): ITP patients with positive antiplatelet autoantibodies; ITP(-): ITP patients with negative antiplatelet autoantibodies. ∗∗∗ p < 0.001; ∗∗ p < 0.01; ∗ p < 0.05.

Article Snippet: Plasma levels of sOX40L were analyzed by a Quantikine Human OX40 Ligand ELISA Kit (R&D Systems, Minneapolis, MN, USA) in accordance with the manufacturer's protocol.

Techniques: Clinical Proteomics

Journal: Journal of Immunology Research

Article Title: Increased Expressions of OX40 and OX40 Ligand in Patients with Primary Immune Thrombocytopenia

doi: 10.1155/2019/6804806

Figure Lengend Snippet: The expression of OX40 and OX40L mRNA in PBMCs of patients. (a) The expression levels of OX40 mRNA in PBMCs from 10 ITP patients with positive antiplatelet autoantibodies, 12 ITP patients with negative antiplatelet autoantibodies, and 12 HCs. (b) The expression levels of OX40L mRNA in PBMCs from 10 ITP patients with positive antiplatelet autoantibodies, 12 ITP patients with negative antiplatelet autoantibodies, and 12 HCs. ITP(+): ITP patients with positive antiplatelet autoantibodies; ITP(-): ITP patients with negative antiplatelet autoantibodies. ∗∗∗ p < 0.001; ∗∗ p < 0.01; ∗ p < 0.05.

Article Snippet: Plasma levels of sOX40L were analyzed by a Quantikine Human OX40 Ligand ELISA Kit (R&D Systems, Minneapolis, MN, USA) in accordance with the manufacturer's protocol.

Techniques: Expressing

Journal: Oncoimmunology

Article Title: OX40 and 4-1BB delineate distinct immune profiles in sarcoma

doi: 10.1080/2162402X.2022.2066050

Figure Lengend Snippet: OX40 and 4-1BB delineate distinct immune profiles . ( a ) Correlation analysis was performed between OX40 ( TNFRSF4 ) and 4-1BB ( TNFRSF9 ) transcripts in TCGA data restricted to UPS, MFS and DDLS sarcoma subtypes indicating no clear correlation. ( b ) K-means clustering was performed using TCGA data restricted to UPS, MFS and DDLS sarcoma subtypes for OX40 transcripts, 4-1BB transcripts and other transcripts associated with tumour-resident Tregs. The four clusters identified were labelled OX40-high, 4-1BB-high, double negative, and double positive. ( c ) 4-1BB and OX40 mRNA expression is shown for each of the four clusters shown in b. Abbreviations: DP, double positive for 4-1BB and OX40; DN, double negative for 4-1BB and OX40. ( d ) MethylCIBERSORT-derived immune population estimates corresponding to each of the four clusters in panel b. ( e ) Survival probability for each cluster identified in panel b plotted individually with grey areas indicating 95% confidence intervals. Statistical analysis shown in all panels is by Wilcoxon test between the groups indicated, *p<0.05, **p<0.01, ***0.001, ****p<0.0001.

Article Snippet: Primary antibodies used were: mouse anti-human CD8 (Dako, clone M351501, 1:800, pH 9 retrieval), mouse anti-human CD68 (Dako, M087629, 1:750, pH 6 retrieval), mouse anti-human CD20 (Dako, M075529, 1:1000, pH 6 retrieval), mouse anti-human FOXP3 (Abcam, ab20034, 1:600, pH 6 retrieval), rabbit anti-human CD4 (Abcam, ab133616, 1:1000, pH 9 retrieval) and rabbit anti-human OX40 (Cell Signaling, 98785, 1:400, pH 6 retrieval).

Techniques: Expressing, Derivative Assay

Journal: Oncoimmunology

Article Title: OX40 and 4-1BB delineate distinct immune profiles in sarcoma

doi: 10.1080/2162402X.2022.2066050

Figure Lengend Snippet: Analysis of co-stimulatory receptors indicates OX40 mRNA expression in UPS, DDLS and MFS is amongst the highest in the TCGA dataset . ( a-d ) 4-1BB ( TNFRSF9 ), OX40 ( TNFRSF4 ), ICOS and GITR ( TNFRSF18 ) mRNA expression, split by sarcoma subtype, compared to a range of cancers in the TCGA dataset. ( e ) MethylCIBERSORT-derived binary hot/cold immune classification status comparing sarcoma subtypes to known immune-hot or immunotherapy-responsive cancers in the TCGA dataset. ( f ) A comparison of MethylCIBERSORT-derived CD8 estimates for each sarcoma subtype, split by hot/ cold classification, versus tumours from cancers high in immune-infiltrates or immunotherapy-responsive. ( g-h ) An analysis of the mRNA expression of 4-1BB ( TNFRSF9 ) and OX40 ( TNFRSF4 ) in sarcoma subtypes and tumours from cancers high in immune-infiltrates or immunotherapy responsive, split by hot/cold classification. All statistical comparisons are between immune-hot and immune-cold as shown, using pairwise wilcox test with Bonferroni correction, *p<0.05, **p<0.01, ***0.001, ****p<0.0001. Abbreviations: BLCA, Bladder Urothelial Carcinoma; BRCA, Breast carcinoma; CESC, Cervical squamous cell carcinoma; CHOL, Cholangiocarcinoma; ESCA, Esophageal carcinoma; GBM, Glioblastoma multiforme; HNSC, Head and Neck squamous cell carcinoma; HPV, Human Papiloma Virus; KIRC, Kidney renal clear cell carcinoma; KIRP, Kidney renal papillary cell carcinoma; LGG, Brain Lower Grade Glioma; LIHC, Liver hepatocellular carcinoma; LUAD, Lung adenocarcinoma; LUSC, Lung squamous cell carcinoma; MESO, Mesothelioma; PAAD, Pancreatic adenocarcinoma; PRAD, Prostate adenocarcinoma; SARC, Sarcoma; SKCM, Skin Cutaneous Melanoma; STAD, Stomach adenocarcinoma; THCA, Thyroid carcinoma; UCEC, Uterine Corpus Endometrial Carcinoma.

Article Snippet: Primary antibodies used were: mouse anti-human CD8 (Dako, clone M351501, 1:800, pH 9 retrieval), mouse anti-human CD68 (Dako, M087629, 1:750, pH 6 retrieval), mouse anti-human CD20 (Dako, M075529, 1:1000, pH 6 retrieval), mouse anti-human FOXP3 (Abcam, ab20034, 1:600, pH 6 retrieval), rabbit anti-human CD4 (Abcam, ab133616, 1:1000, pH 9 retrieval) and rabbit anti-human OX40 (Cell Signaling, 98785, 1:400, pH 6 retrieval).

Techniques: Expressing, Derivative Assay, Comparison, Virus

Journal: Oncoimmunology

Article Title: OX40 and 4-1BB delineate distinct immune profiles in sarcoma

doi: 10.1080/2162402X.2022.2066050

Figure Lengend Snippet: OX40+ Tregs exhibit both a TLS-associated and TLS-independent phenotype in UPS . Pre-treatment UPS and MFS biopsies were analyzed by multiplex immunohistochemistry. ( a ) Slides were stained for CD4, FOXP3, OX40, CD8, CD20, and CD68, with DAPI as nuclear stain. Example images and phenotype classification are shown. ( b ) Immune cell populations were quantified using QuPath. Log2 values for each cell population per mm 2 are shown corresponding to UPS and MFS biopsies as indicated. ( c ) OX40 staining on each cell was classified as positive or negative. The number of OX40-positive cells is expressed as a percentage of each parent population. Grey indicates less than ten OX40-positive cells were identified for a given parent population, and were excluded due to low numbers. As OX40 strongly associated with Tregs, the number of OX40-positive Tregs per mm 2 is also shown. ( d-e ) Neighborhood analysis was performed on OX40-positive Tregs and OX40- negative Tregs (referred to as Treg only). An overview of the image pipeline is described. Immune cells quantified to be approximately two cell widths from OX40-positive Tregs or OX40-negative Tregs are shown. Grey indicates insufficient cell numbers to perform neighborhood analysis. ( f-i ) OX40 expression on Tregs and CD4s was performed assessing differences between immune cells that are TLS-associated, or cells in surrounding tissue areas classified as TLS-independent. Example images of TLSs identified are shown (f) along with the percentage area identified as a TLS relative to the total biopsy tissue area (g). (h-i) OX40 expression on CD4+ FOXP3+ Tregs and CD4+ FOXP3- CD4s was assessed based on their location outside TLSs (TLS-independent) or inside TLSs (TLS-associated).

Article Snippet: Primary antibodies used were: mouse anti-human CD8 (Dako, clone M351501, 1:800, pH 9 retrieval), mouse anti-human CD68 (Dako, M087629, 1:750, pH 6 retrieval), mouse anti-human CD20 (Dako, M075529, 1:1000, pH 6 retrieval), mouse anti-human FOXP3 (Abcam, ab20034, 1:600, pH 6 retrieval), rabbit anti-human CD4 (Abcam, ab133616, 1:1000, pH 9 retrieval) and rabbit anti-human OX40 (Cell Signaling, 98785, 1:400, pH 6 retrieval).

Techniques: Multiplex Assay, Immunohistochemistry, Staining, Expressing