Human igg1 isotype control

For CDC assay of the LALAPG variant, Huh-7 cells were used as target cells. Pooled normal human serum (NHS) was purchased from Innovative Research, Inc. and with or without heat-inactivation at 56°C for 30 min. Cell death was determined by using the LDH cytotoxicity colorimetric assay kit (BioVision). Experiments were based on 96-well assay. In brief, Huh-7 cells were seeded into wells of a 96-well culture plate at day 1 and infected at day 2. After 48 h, Huh-7 cells were replaced with 100 μl fresh medium (DMEM without phenol red; Thermo Fisher Scientific) with indicated human IgG1 isotype control (BioXCell), h33D2, and h33D2-LALAPG followed by the addition of 100 μl 10% NHS contained DMEM. Cell viability was analyzed after 4 h. To prepare positive control, 20 μl lysis buffer was added into a non-treated well. A non-infected well was used as negative control. Positive controls were calculated as mean values relative to the maximum LDH release. For the LDH assay, samples (20 μl) were mixed with 100 μl LDH reaction mix and incubated for 30 min at room temperature. Absorbance at 450 nm was recorded and the cytotoxicity percentage was calculated as follows:
% cytotoxicity = (Experimental value—NHS/Heat-inactivated NHS cell control—Negative control)
/ (Positive control—Negative control) x 100.

Anti-VISTA antibodies and isotype control (Human IgG1 Isotype control, Bio X Cell BP0297) were labeled with Alexa Fluor 647 using the protocol in the conjugation kit (Biotium). Monocytes, T-cells, and Neutrophils were labeled in a human blood sample (BIOIVT) by flow cytometry using CD45-VioGreen (130-110-638), CD3-PE-Vio 770 (130-113-140), CD16-Vio Bright B515 (130-119-616) and CD14-PE (130-113-147) in the presence of FcR Blocking Reagent (130-059-901; all reagents from Miltenyi Biotec and antibodies diluted 50-fold as per manufacturers recommendation). Samples with appropriate FMO (Fluorescence Minus One) controls for each antibody were analyzed in parallel. RBC were lysed using Lysis Buffer (BD Biosciences 555899). Samples were washed in 1x PBS pH7.4 containing 1% heat inactivated fetal bovine serum, and propidium iodide (PI) staining (Miltenyi 130-093-233) for Live/dead cell discrimination done immediately before analysis using a MACS Quant Analyzer (Miltenyi). For NK cells, isolated human NK cells (Hemacare/Charles River Labs) were stained with labeled anti-VISTA or isotype control mAbs and CD56-FITC (Miltenyi 130-114-740; 50-fold diluted) in MACSQuant Running Buffer (Miltenyi 130-092-747), and Sytox Blue (Thermo Fisher Scientific S34862) was used for gating for live cells.

University of Cincinnati’s Institutional Animal Care and Use Committee approved all animal studies. Antibody co-implantation studies: Pt45.P1 cells were preincubated with the indicated antibody diluted in PBS to a concentration of 5 mg/mL for 30 minutes prior to surgery. Human IgG1 isotype control (BioXCell, BE0297) was utilized as a negative control. 1x10⁶ cells were implanted with antibody into the pancreata of athymic nude mice. Pre-formed orthotopic tumor studies: 1x10⁶ Pt45.P1 cells were implanted into the pancreata of athymic nude mice (Fox1^nu/nu; Jackson 002019). Mice were randomized into control and treatment groups. Tumor growth was followed for 7 weeks.