Journal: bioRxiv

Article Title: Novel APC-like properties of human NK cells directly regulate T cell activation

doi: 10.1101/016816

Figure Lengend Snippet: (A) ANK cells (1 × 10 6 ) were incubated with recombinant HA or BSA proteins (0.5 μg/ml) and pooled anti- HA or control sera (1 μl per well), for 180 minutes at 37C. The cells were subsequently stained for surface expression of CD16. MFI levels are indicated for CD16 − (upper left corner) and CD16 + NK cells (upper right corner). (B) The proliferative response of 5 × 10 4 OFR3 clone T cells to different concentrations of HA or BSA proteins, in the presence of a fixed concentration of pooled anti-HA or control sera (1 μl per well). Irradiated ANK cells (1 × 10 5 ) were used as APCs. The cells were cultured for 48 hours and pulsed with [ 3 H]thymidine for the last 24 hours. Values are mean ± SD for triplicate samples. Prior to harvesting, 100 μl of supernatant was taken up for ELISA measurement of IL-2 and IFN-γ. Data are representative of 3 separate experiments. In vivo expression of MHC class II and costimulatory molecules on human NK cells. We next sought to characterize the in vivo regulation of MHC class II and costimulatory molecules on human NK cells. We isolated NK cells from inflamed tonsils obtained from 4 donors who underwent elective tonsillectomies. Interestingly, NK cells derived from all samples displayed significant levels of HLA-DR,DP,DQ, CD86, CD70, OX40 ligand, and, less prominently, CD80 . These observations indicate that human NK cells acquire APC-like phenotype in vivo in inflamed lymphoid organs without any external manipulation.

Article Snippet: Anti- CD56 and anti-CD4 were obtained from DAKO Corp. Anti-CXCR3, anti-NKp30, and anti-NKG2D were obtained from R&D Systems Inc. Biotinylated anti–OX40 ligand mAb was obtained from MBL International Corp.

Techniques: Incubation, Recombinant, Staining, Expressing, Concentration Assay, Irradiation, Cell Culture, Enzyme-linked Immunosorbent Assay, In Vivo, Isolation, Derivative Assay

Journal: Journal of Ophthalmic Inflammation and Infection

Article Title: OX40 ligand expression abrogates the immunosuppressive function of retinal pigment epithelium

doi: 10.1186/1869-5760-3-12

Figure Lengend Snippet: OX40L gene identification by PCR. PCR performed on purified plasmid with and without OX40L insert. Note the presence of OX40L in lanes 7 and 9. (Lane 6, low range DNA ladder; lane 7, eYFP-C1 + OX40L insert; lane 8, eYFP-C1 plasmid; lane 9, eYFP-N1 +OX40L insert; lane 10, eYFP-N1 plasmid).

Article Snippet: Biotinylated polyclonal goat anti-human OX40L antibody (Catalog # BAF1054) was obtained from R & D systems (Minneapolis, MN, USA).

Techniques: Purification, Plasmid Preparation

Journal: Journal of Ophthalmic Inflammation and Infection

Article Title: OX40 ligand expression abrogates the immunosuppressive function of retinal pigment epithelium

doi: 10.1186/1869-5760-3-12

Figure Lengend Snippet: Time trial after vector transfection. (a) Bar graph representing the expression pattern of the fluorescent protein (YFP) and OX40L after transfection of eYFP-C1-OX40L into RPE at five time points (time point 1 = 30 min, time point 2 = 1 h, time point 3 = 4 h, time point 4 = 6 h, and time point 5 = 24 h). Time-dependent increased expression is observed. (b) Flow cytometric analysis at 4 h after transfection of RPE cells with eYFP-C1-OX40L, with YFP expression (bold) in the left panel and OX40L expression (bold) in the right panel (the thinner lines represent RPE that was not transfected). (c) Bar graph expression pattern of YFP and OX40L after transfection of eYFP-N1-OX40L into RPE cells at the same time points. (d) Flow cytometric analysis at 4 h after transfection of RPE cells with eYFP-N1-OX40L.

Article Snippet: Biotinylated polyclonal goat anti-human OX40L antibody (Catalog # BAF1054) was obtained from R & D systems (Minneapolis, MN, USA).

Techniques: Plasmid Preparation, Transfection, Expressing

Journal: Journal of Ophthalmic Inflammation and Infection

Article Title: OX40 ligand expression abrogates the immunosuppressive function of retinal pigment epithelium

doi: 10.1186/1869-5760-3-12

Figure Lengend Snippet: RT-PCR demonstrated expression of OX40L in the RNA of ARPE cells stimulated by pro-inflammatory cytokines. Lanes 1 and 2 are two different DNA ladders; lanes 3 and 4 represent two different concentrations of the same RNA from stimulated RPE cells.

Article Snippet: Biotinylated polyclonal goat anti-human OX40L antibody (Catalog # BAF1054) was obtained from R & D systems (Minneapolis, MN, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Journal of Ophthalmic Inflammation and Infection

Article Title: OX40 ligand expression abrogates the immunosuppressive function of retinal pigment epithelium

doi: 10.1186/1869-5760-3-12

Figure Lengend Snippet: ARPE-19 and PBMC co-culture study results. (a) OX40L-C + PBMC abrogates the immunosuppressive effect of RPE alone. No difference was observed between OX40L (C- or N- vectors) or GITR ligand. An approximately 20% to 30% reversal of RPE-mediated immunosuppression is seen when stimulated pBMCs were co-cultured with RPE-OX40L vector. (b) A repeat experiment shows that concomitant transfection of both eYFP-C1-OX40L and eYFP-C1-GITRL vectors into RPE cells resulted into an additive reversal of immunosuppression of stimulated pBMCs, when comparing it to either RPE transfected with OX40L-C or GITRL alone. (pBMC unstim, unstimulated peripheral blood mononuclear cells; pBMC stim, stimulated pBMC with anti-CD3 and anti-CD28; RPE, retinal pigment epithelial cells without transfection; OX40L-C, RPE cells transfected with eYFP-C1-OX40L; OX40L-N, RPE cells transfected with eYFP-N1-OX40L; GITRL, RPE cells transfected with eYFP-C1-GITRL).

Article Snippet: Biotinylated polyclonal goat anti-human OX40L antibody (Catalog # BAF1054) was obtained from R & D systems (Minneapolis, MN, USA).

Techniques: Co-Culture Assay, Cell Culture, Plasmid Preparation, Transfection

Journal: Journal for Immunotherapy of Cancer

Article Title: Agonist redirected checkpoint, PD1-Fc-OX40L, for cancer immunotherapy

doi: 10.1186/s40425-018-0454-3

Figure Lengend Snippet: Mouse PD1-Fc-OX40L ARC is efficacious at treating CT26 colon cancer tumors and increasing survival. a Mice were inoculated subcutaneously on the rear flank with 5 × 10 5 CT26 cells on day 0, and then treated with 2 doses (all by IP injection on days 5 & 7 once the tumors established and were ~ 4–6 mm in diameter), consisting of 100 μg for each antibody dose and either 100 μg, 150 μg or 300 μg for each dose of the murine PD1-Fc-OX40L ARC. Each line represents the tumor growth from an individual mouse. The first dotted line (~day 18) represents the mean when all untreated mice reached tumor burden. On day 30, surviving mice were challenged with a secondary tumor consisting of 3 × 10 5 cells on the opposing rear flank. Thirteen new untreated mice were also inoculated on day 30 as a control for tumor re-challenge growth. All ARC treated mice that survived until the day 30 challenge, were combined when plotting the ‘re-challenge’ tumor growth curves. b Kaplan-Meier curves were generated for each treatment group, and are plotted identically in order to directly compare sample groups. c A cohort of mice was euthanized 13 days following tumor inoculation, and tumors were excised, dissociated, and subjected to immune phenotyping by flow cytometry. Total populations of CD4 + and CD8 + T cells were assessed, as well as CD4 + CD25 − effector and CD8 + AH1 + antigen-specific cytotoxic cells. d Some mice were treated on days − 1, 1 and 10 with CD4, CD8, or a combination of the two, depleting antibodies. CT26 tumors were again inoculated on day 0, and mice were treated with two IP treatments of 300 μg of the mPD1-Fc-OX40L ARC on days 5 and 7. The percent change in tumor growth between the first treatment day (day 5) and day 17 is shown

Article Snippet: Primary antibodies used for probing hPD1-Fc-OX40L and mPD1-Fc-OX40L were obtained from Cell Signaling Technology, Jackson ImmunoResearch Laboratories, Inc. and R&D Systems.

Techniques: Injection, Generated, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: Agonist redirected checkpoint, PD1-Fc-OX40L, for cancer immunotherapy

doi: 10.1186/s40425-018-0454-3

Figure Lengend Snippet: PD1-Fc-OX40L Stimulates Tumor Cell Killing In Vitro. a Human NCI-H2023 cells were co-cultured with human T cells stimulated for 2 days with sub-optimal CD3/CD28/IL2. Tumor cells were labeled with a cell tracker (yellow) and nuclear stain (blue), and then cultured with 150 nM of the human ARC, labeled with AF647 (Red). A cleaved caspase-3/7 reagent (green) was added to cells and then the same field was imaged over a time-course. T cells also fluoresced in the green channel and can be differentiated from tumor cells in the phase images (top images) by morphology. Similar to the red channel, the phase images also read out ARC staining (white). Mouse T cells were isolated from C57BL/6 mice (adoptively transferred with OT-I/OT-II cells and vaccinated with Ova/Alum) and co-cultured with B16.F10-ova melanoma cells in the presence or absence of OX40 agonist antibody (OX86), PD-1 blocking antibody (RMP1–14), the combination of those two antibodies or the PD1-Fc-OX40L ARC. b Following 24 h of culture, T cell degranulation was measured by analyzing the proportion of OT-I+/CD8+ T cells expressing CD107a on the cell surface, and IFNγ intracellularly; in both OT-I+/CD107a + cells and also in OT-II+ cells. c Induction of caspase 3/7 cleavage was also evaluated in B16.F10-ova cells in each condition following 6 h of co-culture. d As a late-stage indicator of tumor cell death, a TUNEL assay was performed on days 1, 2 and 3 of co-culture. Results indicate mean ± SD for ≥2 replicates per condition for each of 2 separate experiments

Article Snippet: Primary antibodies used for probing hPD1-Fc-OX40L and mPD1-Fc-OX40L were obtained from Cell Signaling Technology, Jackson ImmunoResearch Laboratories, Inc. and R&D Systems.

Techniques: In Vitro, Cell Culture, Labeling, Staining, Isolation, Blocking Assay, Expressing, Co-Culture Assay, TUNEL Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Agonist redirected checkpoint, PD1-Fc-OX40L, for cancer immunotherapy

doi: 10.1186/s40425-018-0454-3

Figure Lengend Snippet: PD1-Fc-OX40L ARC retains proper folding and binding to PD-L1 and OX40. a Predicted tertiary structure of human PD1-Fc-OX40L. b Western blot analysis of PD1-Fc-OX40L performed by probing purified protein with human anti-PD1, anti-Fc, and anti-OX40L, under non-reducing and reducing conditions, and ± the deglycosylase PNGase F. c Functional ELISA using capture with recombinant hPD-L1 followed by detection with recombinant hOX40-His and then anti-His-HRP. HVEM-His serves as a negative control. d Functional PD-L1 blocking assay testing the ability of hPD1-Fc-OX40L to outcompete PD1-Biotin for binding to plate bound PD-L1 in an ELISA format. Avidin-HRP was used for signal detection

Article Snippet: Primary antibodies used for probing hPD1-Fc-OX40L and mPD1-Fc-OX40L were obtained from Cell Signaling Technology, Jackson ImmunoResearch Laboratories, Inc. and R&D Systems.

Techniques: Binding Assay, Western Blot, Purification, Functional Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Negative Control, Blocking Assay, Avidin-Biotin Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Agonist redirected checkpoint, PD1-Fc-OX40L, for cancer immunotherapy

doi: 10.1186/s40425-018-0454-3

Figure Lengend Snippet: Human PD1-Fc-OX40L ARC binds with high affinity to PD-L1, PD-L2, and OX40. On-rate (Ka), off-rate (Kd) and binding affinity (KD) were determined by surface plasmon resonance for PD1-Fc-OX40L binding to chip immobilized ( a ) PD-L1-His, ( b ) PD-L2-His, and ( c ) OX40-His. Recombinant human PD1-Fc and OX40L-Fc were used as controls for binding. ( d ) Summary of binding results depicted in the graphs ( a-c )

Article Snippet: Primary antibodies used for probing hPD1-Fc-OX40L and mPD1-Fc-OX40L were obtained from Cell Signaling Technology, Jackson ImmunoResearch Laboratories, Inc. and R&D Systems.

Techniques: Binding Assay, SPR Assay, Recombinant

Journal: Journal for Immunotherapy of Cancer

Article Title: Agonist redirected checkpoint, PD1-Fc-OX40L, for cancer immunotherapy

doi: 10.1186/s40425-018-0454-3

Figure Lengend Snippet: Human PD1-Fc-OX40L ARC binds efficiently to cells expressing PD-L1, PD-L2, and OX40 in vitro and ex vivo. The in vitro cell binding affinity of the human PD1-Fc-OX40L ARC was assessed using stable cell lines engineered to express high-levels of human PD-L1 (CHOK1/hPD-L1), human PD-L2 (CHOK1/hPD-L2), and human OX40 (Jurkat/hOX40 and HeLa/hOX40). a Representative immunofluorescence and ( b ) flow cytometry binding analysis to PD-L1, (top) PD-L2 and (middle) OX40 (bottom) expressing cells, generating dose-response plots to derive EC50 values

Article Snippet: Primary antibodies used for probing hPD1-Fc-OX40L and mPD1-Fc-OX40L were obtained from Cell Signaling Technology, Jackson ImmunoResearch Laboratories, Inc. and R&D Systems.

Techniques: Expressing, In Vitro, Ex Vivo, Binding Assay, Stable Transfection, Immunofluorescence, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: Agonist redirected checkpoint, PD1-Fc-OX40L, for cancer immunotherapy

doi: 10.1186/s40425-018-0454-3

Figure Lengend Snippet: The human PD1-Fc-OX40L ARC has functional activity in vitro and ex vivo in both tumor co-culture and SEB super-antigen assays. a Schematic of the tumor/T cell co-culture assay. CD3+ human T cells stimulated for 48 h with suboptimal levels of CD3/CD28/IL-2, were plated on mitomycin-c treated PD-L1 low (PC3) and PD-L1 high (HCC827) tumor cells ± the PD1-Fc-OX40L ARC, for an additional 3–5 days (days 5–7 of the entire time-course). b On day 6 of the assay, culture supernatant was collected and analyzed by human IL-2 ELISA. c On days 5 (top) and 7 (bottom) of the assay, floating T cells were collected and subjected to extra- and intra-cellular flow cytometry in order to assess proliferation (Ki67) and markers of T cell activation (IFNγ & TNFα). d Workflow of the SEB super-antigen assay. Total primary human PBMCs were harvested and treated with Staphylococcal enterotoxin B ± the PD1-Fc-OX40L ARC and benchmark antibody controls. Culture supernatants were collected 3 days later and assessed for secreted levels of IL-2 by ELISA

Article Snippet: Primary antibodies used for probing hPD1-Fc-OX40L and mPD1-Fc-OX40L were obtained from Cell Signaling Technology, Jackson ImmunoResearch Laboratories, Inc. and R&D Systems.

Techniques: Functional Assay, Activity Assay, In Vitro, Ex Vivo, Co-Culture Assay, Co-culture Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Activation Assay, Antigen Assay

Journal: Cell reports

Article Title: Persistence of Integrase-Deficient Lentiviral Vectors correlates with the induction of STING-independent CD8 + T cell responses

doi: 10.1016/j.celrep.2019.01.025

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: anti-mouse OX40-L/Biotin (RM134L) , eBioscience , Cat#13-5905-85.

Techniques: Recombinant, Transfection, Isolation, Modification, Software