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: 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

Journal: Journal of Hepatocellular Carcinoma

Article Title: The Combination Immunotherapy of TLR9 Agonist and OX40 Agonist via Intratumoural Injection for Hepatocellular Carcinoma

doi: 10.2147/jhc.s301375

Figure Lengend Snippet: Figure 1 The stimulatory effect of CpG on the OX40 expression of intratumoural CD4+ T cells verified using an ectopic liver tumour model. (A) OX40 expression in subcutaneous hepatoma tissues intratumourally injected with different doses of CpG (12.5 µg, 25 µg, 50 µg) was assessed by Western blot analysis (n=3; ***P<0.001, ****P<0.0001). (B) Forty-eight hours after the intratumoural injection of CpG, single cell suspension from the subcutaneous hepatoma tissues was prepared, and the OX40 expression of the CD3+CD4+ T cell subgroup was analyzed by flow cytometry (n=5; *P<0.05). (C) OX40 expression on effector CD4+ T cells (Foxp3-negative) was revealed by flow cytometry (n=5; **P<0.01).

Article Snippet: Anti-OX40 (CD134) monoclonal antibody (clone OX86) and isotype control were purchased from BioXCell (Lebanon, NH, USA).

Techniques: Expressing, Injection, Western Blot, Suspension, Flow Cytometry

Journal: Journal of Hepatocellular Carcinoma

Article Title: The Combination Immunotherapy of TLR9 Agonist and OX40 Agonist via Intratumoural Injection for Hepatocellular Carcinoma

doi: 10.2147/jhc.s301375

Figure Lengend Snippet: Figure 2 The combination of CpG and anti-OX40 mAb via intratumoural injection induces improved immunotherapy against both local and distal Hepa1-6 hepatoma. (A) Schematic diagram of immunotherapy for the ectopic HCC model. C57BL/6 mice were subcutaneously inoculated with Hepa1-6 cells on both sides of the abdomen. When the tumour diameter reached 5–7 mm, the left tumour was injected with different immune drugs (CpG, αOX40 or CpG plus αOX40) every other day for a total of 4 times. The bilateral tumour volumes were continuously measured. (B) Tumour growth of the subcutaneous tumour model. Left: noninjected (NI) tumour volume, right: injected (In) tumour volume; the arrow indicates the beginning of the treatment (n=8; ***P<0.001, ****P<0.0001). (C) Survival curve of the subcutaneous HCC model (n=8; ****P<0.0001). (D) Schematic diagram of immunotherapy for the orthotopic HCC model. C57BL/6 mice were inoculated with Hepa1-6 cells in the subcapsular region of the liver and the subcutaneous site of the left abdomen. The treatment scheme was consistent with (A). (E) Tumour growth of the orthotopic HCC model. Left: the volume of the noninjected (NI) intrahepatic tumour was measured by MRI scan before treatment and on day 16 after the first treatment. Right: the volume of the injected (In) left subcutaneous tumour was continuously measured (n=8; ****P<0.0001). (F) Representative MRI images of the intrahepatic tumours. (G) Survival curve of the orthotopic HCC model (n=8; ****P<0.0001).

Article Snippet: Anti-OX40 (CD134) monoclonal antibody (clone OX86) and isotype control were purchased from BioXCell (Lebanon, NH, USA).

Techniques: Injection

Journal: Journal of Hepatocellular Carcinoma

Article Title: The Combination Immunotherapy of TLR9 Agonist and OX40 Agonist via Intratumoural Injection for Hepatocellular Carcinoma

doi: 10.2147/jhc.s301375

Figure Lengend Snippet: Figure 3 The combined CpG and anti-OX40 mAb via intratumoural injection triggers a tumour-specific immune response. (A) Schematic diagram of immunotherapy for the ectopic HCC model inoculated with Hepa1-6 cells. (B) Tumour growth of the subcutaneous HCC model receiving combined CpG and anti-OX40 mAb via different methods of drug delivery. Growth of the right noninjected tumours after treatment was monitored (n=8; ****P<0.0001). (C) On day 7 after treatment, splenocytes derived from ectopic HCC mice receiving different treatments were cocultured with media, B16-F10 cells or Hepa1-6 cells for 24 hours, and the percentage of IFN-γ+CD44+CD8+ T cells was analyzed by flow cytometry (n=5; ****P<0.0001). (D) Tumour growth of the mixed tumour model, with two Hepa1-6 tumours on bilateral abdomen and a B16- F10 tumour on the right armpit (n=8; *P<0.05). (E) Tumour growth of the mixed tumour model, with two B16-F10 tumours on bilateral abdomen and a Hepa1-6 tumour on the right armpit (n=8; *P<0.05, **P<0.01). (F) Tumour growth of the mixed tumour model, with a mixed tumour (Hepa1-6 and B16-F10) on the left abdomen, B16-F10 tumour on the right abdomen and Hepa1-6 tumour on the right armpit.

Article Snippet: Anti-OX40 (CD134) monoclonal antibody (clone OX86) and isotype control were purchased from BioXCell (Lebanon, NH, USA).

Techniques: Injection, Derivative Assay, Flow Cytometry

Journal: Journal of Hepatocellular Carcinoma

Article Title: The Combination Immunotherapy of TLR9 Agonist and OX40 Agonist via Intratumoural Injection for Hepatocellular Carcinoma

doi: 10.2147/jhc.s301375

Figure Lengend Snippet: Figure 5 The combination immunotherapy exerts T cell-dependent anti-tumour effect and induces effective immune memory response to prevent tumour recurrence. (A) To perform immunotherapy for the ectopic HCC model, Hepa1-6 cells were subcutaneously inoculated into the bilateral abdomen of nude mice, and the left tumours were injected with CpG and anti-OX40 mAb. The tumour growth curves of the nude mice were recorded (NI: non-injected, In: injected; n=8). (B) Schematic diagram of the adoptive transfer experiment. (C-F) Different T cell subsets derived from mice receiving different treatments were mixed with Hepa1-6 cells and subcutaneously implanted into naive C57BL/6 mice. The tumour growth curves of the hosts were recorded (n=6; ****P<0.0001). (G) Schematic diagram of the in vivo immune memory response assay. On day 90 after the primary tumour disappeared, the mice treated with combination immunotherapy were rechallenged with Hepa1-6 cells. (H) The tumour growth curves of the control mice and the mice rechallenged with Hepa1-6 cells. Age-matched mice without any treatment but only transplanted subcutaneously with Hepa1-6 cells were used as controls (n=8; ****P<0.0001).

Article Snippet: Anti-OX40 (CD134) monoclonal antibody (clone OX86) and isotype control were purchased from BioXCell (Lebanon, NH, USA).

Techniques: Injection, Adoptive Transfer Assay, Derivative Assay, In Vivo, Control