Journal: bioRxiv

Article Title: Engineered immunomodulatory extracellular vesicles derived from epithelial cells acquire capacity for positive and negative T cell co-stimulation in cancer and autoimmunity

doi: 10.1101/2023.11.02.565371

Figure Lengend Snippet: ( a ) Schematic representation of the experimental design deployed to investigate the functionality of engineered EVs ex vivo using human PBMCs. ( b ) Representative histograms of activation markers (CD25 and CD69), proliferation marker (Ki67), and IFNγ in human CD4 + T cells and CD8 + T cells treated with hCD80 EVs. Accompanying bar graphs show mean +/- s.e.m. of MFI from n=6 PBMC samples. ( c ) Representative histograms of activation markers (CD25 and CD69), proliferation marker (Ki67), and IL-2 in human CD4 + T cells, and activation markers (CD25 and CD69), IFNγ, and cytolytic marker (Granzyme B), in human CD8 + T cells treated with hOX40L EVs. Accompanying bar graphs show mean +/- s.e.m. of MFI from n=3 PBMC samples. ( d ) Representative histograms of activation markers (CD25 and CD69), proliferation marker (Ki67), and IFNγ in human CD8 + T cells treated with hPD-L1 EVs. Accompanying bar graphs show mean +/- s.e.m. of MFI from n=8 PBMC samples. Statistical significance was determined using ordinary one-way ANOVA, p-values are shown. Statistical significance defined as p < 0.05.

Article Snippet: To generate overexpressing cells, plasmids for hCD80 (Sino Biological HG10698-UT)), hOX40L (Sino Biological HG13127-UT), hPD-L1 (Origene RC213071L4), mCD80 (Sino Biological MG50446-UT), mOX40L (Sino Biological MG53582-UT), and mPD-L1 (Origene MR203953L4) were transfected using lipofectamine 3000 (Invitrogen) in HEK293T/17 cells according to manufacturer’s instructions and selected using hygromycin (CD80 and OX40L plasmids) or puromycin (PD-L1 plasmids).

Techniques: Ex Vivo, Activation Assay, Marker

Journal: Immunity

Article Title: Tissue-Restricted Adaptive Type 2 Immunity Is Orchestrated by Expression of the Costimulatory Molecule OX40L on Group 2 Innate Lymphoid Cells

doi: 10.1016/j.immuni.2018.05.003

Figure Lengend Snippet: ILC2s Are the Main OX40L-Expressing Cell in Response to IL-33 (A) TNFSF gene expression was analyzed in the indicated immune cell populations using IMMGEN and naive lung ILC2 microarray data. (B) OX40L expression was measured on lung DCs or ILC2s in WT mice on day 2 after treatment with PBS or IL-33 (i.n., day 0 and 1). (C) The percentage of OX40L + ILC2s (top) and DCs (bottom) was measured on day 2 in response to the indicated stimuli (i.n., day 0 and 1). (D) The percent of Ki67 + ILC2s and Treg cells was measured on day 2 after treatment with PBS or IL-33 (day 0 and 1). (E) OX40L expression was measured on ILC2s and ILC3s in the lung or mLN on day 2 after PBS or IL-33 treatment (i.n., day 0 and 1). Numbers in (B) and (E) indicate percent (mean ± SD) gated populations. (A), two independent datasets per group; (B), three repeat experiments; (C), ANOVA, two repeat experiments; (D), ANOVA, two repeat experiments; (E), two-tailed Student’s t test, two repeat experiments. ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001. See also Figure S4 .

Article Snippet: Anti-human OX40L , Biolegend , 326307; RRID: AB_2207272.

Techniques: Expressing, Microarray, Two Tailed Test

Journal: Immunity

Article Title: Tissue-Restricted Adaptive Type 2 Immunity Is Orchestrated by Expression of the Costimulatory Molecule OX40L on Group 2 Innate Lymphoid Cells

doi: 10.1016/j.immuni.2018.05.003

Figure Lengend Snippet: OX40L Expression by ILC2s Is Essential for Tissue-Specific Adaptive Immune Response to IL-33 (A) OX40L expression on lung ILC2s in the specified genotypes on day 2 after IL-33 administration (i.n., day 0 and 1). (B–D) Lung GATA3 + and GATA3 − Treg and Th2 cell numbers were quantified on day 5 after treatment with PBS or IL-33 (i.n., days 0 and 1). (E–G) OX40L expression on ILC2s was measured in the indicated tissues (L. int., large intestine) after treatment with PBS or IL-33 (days 0 and 1) on day 2 (E), while the number of ILC2s (F) and GATA3 + Treg cells was quantified on day 5 (G). Numbers in (A) and (E) indicate percent (mean ± SD) gated populations. Bar graphs indicate mean (±SEM). (A), three repeat experiments; (B)–(D), ANOVA, two repeat experiments; (E)–(G), ANOVA, two repeat experiments. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗∗ p ≤ 0.0001. See also Figure S5 .

Article Snippet: Anti-human OX40L , Biolegend , 326307; RRID: AB_2207272.

Techniques: Expressing

Journal: Immunity

Article Title: Tissue-Restricted Adaptive Type 2 Immunity Is Orchestrated by Expression of the Costimulatory Molecule OX40L on Group 2 Innate Lymphoid Cells

doi: 10.1016/j.immuni.2018.05.003

Figure Lengend Snippet: OX40L-Driven Response to IL-33 Is Restricted to ILC2s (A–C) Lung GATA3 + and GATA3 − Treg cells and Th2 cells were quantified on day 5 in the specified genotypes, treated with PBS or IL-33 (i.n., days 0 and 1). (D and E) Bone marrow and mixed-bone marrow chimeric mice were created with the indicated genotypes. 6 to 7 months after bone marrow transfer, mice received IL-33 (i.n., days 0 and 1) followed by quantification of lung GATA3 + Treg (D) and GATA3 − Treg (E) cells on day 5. Bar graphs indicate mean (±SEM). ANOVA, two repeat experiments. ns = not significant, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗ p ≤ 0.0001. See also Figure S6 .

Article Snippet: Anti-human OX40L , Biolegend , 326307; RRID: AB_2207272.

Techniques:

Journal: Immunity

Article Title: Tissue-Restricted Adaptive Type 2 Immunity Is Orchestrated by Expression of the Costimulatory Molecule OX40L on Group 2 Innate Lymphoid Cells

doi: 10.1016/j.immuni.2018.05.003

Figure Lengend Snippet: OX40L on ILC2s Orchestrates Adaptive Type 2 Immunity to Allergens (A–D) Mice of the specified genotypes were treated with PBS or papain (Pap) (i.n., day 0 and 1), followed by quantification of lung Th2, GATA3 + Treg, and GATA3 − Treg cells on day 5 (A–C). Whole lung cell suspensions were re-stimulated with PMA and ionomycin, followed by quantification of IL-13 + Th2 cells by intracellular staining (D). (E–G) Mice were treated with papain on days 0, 1, 14, and 21, followed by quantification on day 24 of lung eosinophils (E), and detection (F) and quantification (G) of RELMα + M2 alveolar (CD45 + SiglecF + CD11c + CD11b − F4/80 + ) macrophages (MΦ). (H and I) Mice were treated with A. alternata ( A.alt ) (i.n., days 0 and 1), followed by quantification on day 9 of lung Th2, GATA3 + Treg, and GATA3 − Treg cells (H) and serum IgE concentration (I). Bar graphs indicate mean (±SEM). (A)–(D), ANOVA, two repeat experiments; (E), ANOVA, three repeat experiments; (F) and (G), ANOVA, two repeat experiments, representative gate shown in (F); (H) and (I), ANOVA, two repeat experiments. ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001. See also Figure S7 .

Article Snippet: Anti-human OX40L , Biolegend , 326307; RRID: AB_2207272.

Techniques: Staining, Concentration Assay

Journal: Immunity

Article Title: Tissue-Restricted Adaptive Type 2 Immunity Is Orchestrated by Expression of the Costimulatory Molecule OX40L on Group 2 Innate Lymphoid Cells

doi: 10.1016/j.immuni.2018.05.003

Figure Lengend Snippet: ILC2-Expressed OX40L Is Essential for Airway Adaptive Type 2 Immune Response to Helminth Infection Mice of the specified genotypes were infected with Nippostrongylus brasiliensis ( N.b. ) on day 0, followed by analysis on day 28 (or day 5) of: (A–C) Lung Th2, GATA3 + Treg, and GATA3 − Treg cell numbers. (D) Representative lung histology (Mason’s trichrome). (E and F) Lung (E) and bronchoalveolar lavage (F) eosinophil numbers. (G) Lung RELMα + M2 macrophage (MΦ) numbers. (H) Bronchoalveolar lavage IL-4 and IL-5 cytokine concentrations. (I) Whole lung cell suspensions were re-stimulated with PMA and ionomycin, followed by quantification of lung IL-13 + Th2 cell numbers by intracellular staining. (J–L) Mediastinal lymph node Th2, GATA3 + Treg, and GATA3 − Treg cell numbers. (M) Concentration of IgE present in lung homogenate, normalized for total protein content. (N) Intestinal worm burden of indicated mouse genotypes 5 days post infection. Bar graphs indicate mean (±SEM). (A)–(C), ANOVA, three repeat experiments; (D), two repeat experiments; (E)–(I), ANOVA, two repeat experiments; (J)–(L), ANOVA, three repeat experiments; (M), ANOVA, two repeat experiments; (N), two-tailed Student’s t test, two pooled experiments. ns = not significant, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001. See also Figure S7 .

Article Snippet: Anti-human OX40L , Biolegend , 326307; RRID: AB_2207272.

Techniques: Infection, Staining, Concentration Assay, Two Tailed Test

Journal: Immunity

Article Title: Tissue-Restricted Adaptive Type 2 Immunity Is Orchestrated by Expression of the Costimulatory Molecule OX40L on Group 2 Innate Lymphoid Cells

doi: 10.1016/j.immuni.2018.05.003

Figure Lengend Snippet:

Article Snippet: Anti-human OX40L , Biolegend , 326307; RRID: AB_2207272.

Techniques: Recombinant, Staining, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Microarray, Software

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: OX40L-JAG1 induced expansion of lineage-stable regulatory T-cells involves non-canonical NF-kB signaling

doi: 10.4049/jimmunol.1900530

Figure Lengend Snippet: A) CD4+ T-cells from Foxp3.GFP mice were treated with OX40L-JAG1 or α-CD3/CD28 for 3 days. CD4+Foxp3-GFP- Teff cells and CD4+Foxp3+GFP+ Tregs were sorted and subjected to microarray analysis. Unstimulated fresh Teff cells and Tregs used as a control. Heat map shows differential mRNA expression of TRAFs, NF-kB and DNMT signaling related genes between control, OX40L-JAG1 and α-CD3/CD28 treated Tregs. B) CD4+CD25- Tconv cells and CD4+CD25+ Treg cells were pre-treated with indicated NIK inhibitor (10μM/ml) for 2h and co-treated with IL-2 (25U/ml) and OX40L (5 μg/ml) + JAG1 (1 μg/ml) and αCD3+ αCD28 monoclonal antibodies in the presence of IL-2 for 4 days. Cell proliferation was measured by cell trace violet dilution assay. C) Bar graphs show division index calculated from cell trace violet dilution in each culture conditions (Values represent Mean ± SEM, n=3, ****p<0.001). D) Western blots show TRAF1 expression, p100 to p52 processing and total and phospho p65 levels in OX40L-JAG1 treated Tregs compared to fresh and α-CD3/CD28 treated Tregs after 24hr. E) Western blots show p100 to p52 processing in Tregs treated with IL-2 alone, JAG1 alone, OX40L-alone and OX40L+JAG1+IL-2. F) Western blots show TRAF1 and p100 to p52 processing in WT and OX40−/− Tregs treated with or without OX40L.

Article Snippet: Human recombinant OX40L-Fc and Jagged-1-Fc were from Sino Biologicals.

Techniques: Microarray, Expressing, Dilution Assay, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: OX40L-JAG1 induced expansion of lineage-stable regulatory T-cells involves non-canonical NF-kB signaling

doi: 10.4049/jimmunol.1900530

Figure Lengend Snippet: A-B) CD4+CD25- Tconv cells (A) and CD4+CD25+ Treg (B) cells were pre-treated with indicated kinase inhibitors (10μM/ml) for 2h and co-treated with IL-2 (25U/ml) and OX40L (5 μg/ml) + JAG1 (1 μg/ml) and αCD3+ αCD28 monoclonal antibodies in the presence of IL-2 for 4 days. Cell proliferation was measured by cell trace violet dilution assay. Bar graphs show division index calculated from cell trace violet dilution in each culture conditions (Values represent Mean ± SEM, n=3, ****p<0.001, ***p<0.05). C) Overlay histograms show CD69 and CD25 expression in gated CD4+Foxp3- Teff cells (top panel) and CD4+Foxp3+ Tregs (bottom panel) from the above experiments. Bar graphs show MFI values of CD69 (D) and CD25 expression (E) in Teff and Treg cells summarized from C. Values are expressed as Mean ± SEM (n=3 independent experiments; ***p<0.005, ****p<0.001).

Article Snippet: Human recombinant OX40L-Fc and Jagged-1-Fc were from Sino Biologicals.

Techniques: Dilution Assay, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: OX40L-JAG1 induced expansion of lineage-stable regulatory T-cells involves non-canonical NF-kB signaling

doi: 10.4049/jimmunol.1900530

Figure Lengend Snippet: A) Human thymic fragments were cultured in 3D thymic organ cultures with human IL-2 (50U/ml) or human JAG1-Fc (1μg/ml), human OX40L-Fc (5μg/ml) and JAG1+OX40L in the presence of IL-2 for 5 days and frequencies of CD4+CD25+FOXP3+ Tregs were analyzed. B) Bar graph summarizes the frequencies of CD25+FOXP3+ Tregs. (Values represent Mean ± SEM, n=8 independent experiments, **p<0.01, ***p<0.005 vs IL-2). C) Human thymocytes were cultured with either IL-2 alone or OX40L+JAG1+IL-2 for 5 days and the proliferation of CD4+Foxp3+ and CD4+Foxp3- cells was assayed by cell trace violet dilution. D) The bar graph shows percentages of resting and proliferating CD4+Foxp3+ Tregs (Values represent Mean ± SEM, n=5, *p<0.05 Vs IL-2). E) Representative dot plot from the above experiments show the frequencies of CD45RA+Foxp3low (naïve Tregs), CD45RA-Foxp3hi (effector Tregs) and CD45RA-Foxp3low (effector T-cells) in the culture. F) Overlay histograms show the expression levels of Treg functional markers CTLA4, CD39, Helios and TIGIT in CD4+Foxp3hi Tregs expanded from IL-2 (Blue) Vs OX40L+JAG1 (Red) treated cultures. G) In vitro Treg suppression assay was performed using autologous CD4+CD25-CD127high Teff cells and fresh/OX40L-JAG1 expanded CD4+CD25hiCD127low Tregs in the presence of anti-CD3 stimulation for 4 days. The extent of Teff cell proliferation was measured by cell trace violet dilution assay. H) Bar graphs show division index of Teff cells calculated from each culture conditions I) NSG mice engrafted with human CD34 precursor cells were treated with soluble OX40L and JAG1. Representative dot plots show the development of human CD45+CD4+ T-cells in humanized NSG mice. Representative dot plots and bar graphs show the frequencies of Foxp3hi human Tregs within gated human CD45+CD4+ T-cells in spleen (top panel) and liver of PBS vs OX40L-JAG1 treated mice. (Values represent Mean ± SEM, n=4, *p<0.05).J) Bar graph shows the frequencies of CD45RA+Foxp3low (naïve Tregs), CD45RA-Foxp3hi (effector Tregs) and CD45RA-Foxp3low (effector T-cells) within CD45+CD4+Foxp3+ cells in PBS vs OX40L-JAG1 treated NSG mice. K) Overlay histogram analysis show CTLA4 and TIGIT expression in CD45RA-Foxp3hi effector Tregs from PBS vs OX40L-JAG1 treated NSG mice.

Article Snippet: Human recombinant OX40L-Fc and Jagged-1-Fc were from Sino Biologicals.

Techniques: Cell Culture, Expressing, Functional Assay, In Vitro, Suppression Assay, Dilution Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: OX40L-JAG1 induced expansion of lineage-stable regulatory T-cells involves non-canonical NF-kB signaling

doi: 10.4049/jimmunol.1900530

Figure Lengend Snippet: A) 6–8week-old female NOD mice were injected with PBS or soluble OX40L and JAG1 (100μg each) once a week for 3 consecutive weeks and percentages of CD4+Foxp3+ Tregs were analyzed in the thymus, spleen and peripheral lymph nodes. B) Bar graph show frequency of and number of Tregs (Values represent Mean ± SEM, n=6, *p<0.05, **p<0.01). C) CD4+Foxp3+ from the thymus and spleen were gated and contour plots show Ki67+BCL2- (proliferating) and Ki67-BCL2+ (resting) cells. D) Bar graphs summarize frequencies of Ki67+BCL2- (proliferating) and Ki67-BCL2+ (resting) Tregs in the thymus and spleen calculated from Fig-3C. (Values represent Mean ± SEM, n=6, **p<0.01, ***p<0.005, ****p<0.001). E) Overlay histograms show the expression levels of Treg signature markers CTLA4, GITR, CD25, Foxp3 and Helios expression in CD4+Foxp3+ cells in the thymus, spleen and pancreatic lymph nodes of PBS (Blue) Vs OX40L (Red) treated mice. F) Heat maps show methylation index of individual CpG islands between control and OX40l-JAG1 treated Tregs compared to control Teff cells. G) Percentage methylation levels in the promoter and intronic regions of Treg signature genes Ctla4, Ikzf4, Ikzf2, Il-2ra and Foxp3 in sorted splenic CD4+CD25+ Tregs and CD4+CD25- Teff cells from PBS and OX40L-JAG1 treated NOD mice as analyzed by WGBS.

Article Snippet: Human recombinant OX40L-Fc and Jagged-1-Fc were from Sino Biologicals.

Techniques: Injection, Expressing, Methylation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: OX40L-JAG1 induced expansion of lineage-stable regulatory T-cells involves non-canonical NF-kB signaling

doi: 10.4049/jimmunol.1900530

Figure Lengend Snippet: A) 6–8week-old female CBA/j mice were injected with PBS or soluble OX40L and JAG1 (100μg each) and immunized with porcine thyroglobulin antigen emulsified with CFA. A subset of mice from PBS/OX40L-JAG1 treated mice were sacrificed immediately after 1st immunization and last OX40L-JAG1 treatment as described in materials and methods. Percentages of CD4+Foxp3+ Tregs were analyzed in the thymus, spleen and peripheral lymph nodes. (Values represent Mean ± SEM, n=6, **p<0.01, ***p<0.005 vs PBS). B) Frequencies of CTLA4+, GITR+, CCR4+, Helios+, PD1+, TIGIT+, TIM3+ and LAG3+ Tregs in the spleen (top panel) and draining lymph nodes (bottom panel) were analyzed by flow cytometry. C) MFI values of the above mentioned suppressive markers are summarized. (Values represent Mean ± SEM, n=6, **p<0.01, ***p<0.005 vs PBS). D) Representative dot plots and overlay histograms show increased CTLA4+/TIGIT+ Foxp3+ Tregs and CTLA4/TIGIT expression per se in OX40L-JAG1 treated mice. E-F) PBS and OX40L-JAG1 expanded CD4+CD25+ Treg cells from CBA/j mice were co-cultured with cell trace violet labeled CD4+CD25- Teff cells from PBS and OX40L-JAG1 treated mice in depicted combinations at indicated ratios and stimulated with anti-CD3 in the presence of thyroglobulin antigen (20μg/ml) loaded APCs for 3 days. The extent of Teff cell proliferation was measured by cell trace violet dilution assay. G) Bar graphs show division index of Teff cells calculated from each culture conditions (Values represent Mean ± SEM, n=3, *p<0.05).

Article Snippet: Human recombinant OX40L-Fc and Jagged-1-Fc were from Sino Biologicals.

Techniques: Injection, Flow Cytometry, Expressing, Cell Culture, Labeling, Dilution Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: OX40L-JAG1 induced expansion of lineage-stable regulatory T-cells involves non-canonical NF-kB signaling

doi: 10.4049/jimmunol.1900530

Figure Lengend Snippet: A) All mice from thyroiditis experiments were sacrificed at the end of the experiment 14 days after the second immunization. Images of the spleen and draining lymph nodes are shown. B) Bar graph summarizing spleen weights. C) H & E staining analysis of thyroid sections from CFA control, Tg immunized and treated with PBS, or OX40L-JAG1 (n=6). D) Thyroiditis scoring was done as described in materials and methods. (Values represent Mean ± SEM, n=6, *p<0.05, **p<0.01, ***p<0.005). E) Serum anti-thyroglobulin levels were measured by ELISA and bar graph shows absorbance at 450 nm measured at different dilutions (Values represent Mean ± SEM, n=6). F) Representative dot plots showing frequency of CD4+Foxp3-IFN-γ+ Th1, CD4+Foxp3-IL-4+ Th2 and CD4+Foxp3-IL-17+ Th17 cells in the draining lymph node cells before and after stimulation with PMA/ionomycin. G) Bar graph summarizing results of Fig-5F (Values represent Mean ± SEM, n=6, *p<0.05, **p<0.01 vs PBS).

Article Snippet: Human recombinant OX40L-Fc and Jagged-1-Fc were from Sino Biologicals.

Techniques: Staining, Enzyme-linked Immunosorbent Assay

Journal: Molecular Therapy Oncolytics

Article Title: Characterization of a novel OX40 ligand and CD40 ligand-expressing oncolytic adenovirus used in the PeptiCRAd cancer vaccine platform

doi: 10.1016/j.omto.2021.02.006

Figure Lengend Snippet: Novel VALO-D102 oncolytic adenovirus produces high levels of biologically active human CD40 ligand (CD40L) and OX40 ligand (OX40L) (A) Schematic representation of genetic modifications in VALO-D102. The virus has a 24-base pair deletion in E1A; the CR1-alpha and gp19K genes in the E3A region have been replaced with human OX40L and CD40L genes; the 14.7K gene in the E3B region has been deleted; and finally, the adenovirus 5 knob domain has been replaced with the knob domain from adenovirus serotype 3. (B) A549 cells were infected with VALO-D102 at a MOI of 10. 72 h postinfection, supernatant was collected and added to a culture of Ramos-Blue reporter cells, and CD40 receptor activation by functional virus-produced CD40L was measured. (C) A549 cells were infected with VALO-D102 at a MOI of 10. 48 h postinfection, HEK293-OX40/NF-κB reporter cells were added to the infected A549 cells for 6 h, and OX40 activation by virus-expressed, functional membrane-bound OX40L was measured.

Article Snippet: Recombinant human OX40L (BPS Bioscience) was used as a standard.

Techniques: Infection, Activation Assay, Functional Assay, Produced

Journal: Molecular Therapy Oncolytics

Article Title: Characterization of a novel OX40 ligand and CD40 ligand-expressing oncolytic adenovirus used in the PeptiCRAd cancer vaccine platform

doi: 10.1016/j.omto.2021.02.006

Figure Lengend Snippet: Virus-encoded OX40L and CD40L improve anti-tumor efficacy and induce robust infiltration of tumor-specific CD8 + T cells into the tumor in a syngeneic mouse model of B16.OVA melanoma (A) 1 × 10 9 VP of PeptiCRAd Ad5/3-D24-OVA or PeptiCRAd VALO-mD901-OVA was given intratumorally 6, 8, and 20 days post-tumor implantation. Average tumor growth curves for all treatment groups are shown. (B) Immunological analysis of tumors and tumor-draining lymph nodes of treated mice. Lymph nodes from all mice from each treatment group were pooled in order to get enough cells for the flow cytometric analysis. The number of mice in the mock group was 7 and in both PeptiCRAd groups, was 10. Statistical analysis was performed with one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Recombinant human OX40L (BPS Bioscience) was used as a standard.

Techniques: Tumor Implantation

Journal: Journal for Immunotherapy of Cancer

Article Title: FcγRIIB engagement drives agonistic activity of Fc-engineered αOX40 antibody to stimulate human tumor-infiltrating T cells

doi: 10.1136/jitc-2020-000816

Figure Lengend Snippet: Multimeric OX40 ligation enhances TIL expansion, and activation. Tumor tissues from patients with LM-CRC and HCC were collected; immune cells were isolated and cultured in vitro in the presence of αCD3/CD28 activation beads (ctrl) and additionally (B, C, E) 20 µg/mL or (C) 2 µg/mL OX40L or (B, D, E) 10 µg/mL monomeric αOX40 IgG2 or (F) multimeric αOX40 IgG2 for a period of 8–10 days. (A) Schematic outline of hexameric OX40L, monomeric αOX40 IgG2 antibody and αOX40 IgG2 antibodies conjugated to magnetic beads. (B, C, F) TIL numbers after in vitro cultures were acquired by flow cytometry and normalized to counting beads. (B, C) Relative changes over cell numbers in ctrl are shown (n=11). (D, E) Cytokine levels in culture supernatants were acquired by cytokine multiplex assay; (D) n=12, (E) n=15). (F) Cell numbers are depicted as relative to soluble and bead-conjugated IgG2 isotypes, respectively (n=9). (B) One-way non-parametric Kruskal-Wallis, including Dunn’s post-testing and (C–F) non-parametric Wilcoxon test, was performed. *P≤0.05, **P≤0.01, ***P≤0.001. HCC, hepatocellular carcinoma; IFN, interferon; LM-CRC, liver metastasis colorectal cancer; ns, not significant; OX40L, OX40 ligand; pCRC, primary colorectal cancer; TIL, tumor-infiltrating lymphocyte; TNF-α, tumor necrosis factor alpha.

Article Snippet: Cells were treated with 2–20 μg/mL hexameric human OX40L (Pfizer), 10 μg/mL soluble human αOX40 IgG1, human isotype HA-IgG1, human αOX40 IgG2 (PF-04518600), human isotype HA-IgG2 or human αOX40IgG1_v12 (all Pfizer). αOX40 IgG2 and isotype IgG2 antibody coupling to Dynabeads was performed by using Dynabeads Antibody coupling Kit according to the manufacturer’s instructions (14 311D), whereby 6 μg antibody was coupled per 1 mg of beads.

Techniques: Ligation, Activation Assay, Isolation, Cell Culture, In Vitro, Magnetic Beads, Flow Cytometry, Multiplex Assay