Bound fusion protein was detected using goat anti-human IgG HRP. same cell but not in between two cells, which suggests a model in which PD-L1 can bend via its 11-amino acid, flexible stalk to bind to B7C1 in between two cells. However, when PD-L1 was presented in a more accessible and flexible form, a strong interaction between PD-L1 and B7C1 was observed. This was confirmed using two separate approaches of ELISA and flow cytometry. These results failed to confirm a interaction but suggested a interaction. We cotransfected PD-L1 and B7C1 in the same cell and, using a proximity assay (NanoBiT), confirmed a binding interaction. Our results indicate binding between PD-L1 and B7C1 in on the same cell surface but not in between two cells. We further distinguish the binding region of PD-L1 to B7C1 as being overlapping but higher on the GFCCC face of the PD-L1 IgV domain than the binding surface for PD-1. Together, our study offers molecular insight into the PD-L1 pathway. Materials and Methods Cells and culture media Mouse 300.19 cells are an Abelson mouse leukemia virus transformed pre-B cell line from Swiss Webster mice that grows as a nonadherent, single-cell suspension. The mouse EL4 T-cell line was obtained from American Type Culture Collection (ATCC). The 300.19 cells, 300.19 PD-L1 transfected cells, 300.19 PD-L1-IgV-Tim-3 mucin domain transfected cells, and EL4 cells were transfected by electroporation Nedisertib with mouse or human PD-L1, B7C1, PD-1, CD28, CTLA-4, or other appropriate construct cDNA in the pEF-Puro Nedisertib or pEF6-Blasticidin expression vectors in our laboratory. Cells were selected in media containing puromycin or blasticidin, sorted with specific monoclonal antibodies (mAbs), and subcloned. Cell-surface expression of the indicated molecules was verified by flow cytometry using specific mAbs. Cells were cultured no more than 4 months before new thaws, but have not been reauthenticated within the last year. Cells were cultured at 37C with 5% CO2 in RPMI-1640 (Mediatech) supplemented with 10% heat-inactivated FBS (Invitrogen), 1% streptomycin/penicillin, 15g/ml gentamicin (Invitrogen), 1% glutamax (Invitrogen), 50M -mercaptoethanol (Sigma-Aldrich), and 5g/ml puromycin or blasticidin. The same media minus -mercaptoethanol was used for EL4 cells. COS cells were cultured at 37C with 10% CO2 in DMEM (Mediatech) supplemented with 10% heat-inactivated FBS (Invitrogen), 1% streptomycin/penicillin, 15g/ml gentamicin (Invitrogen), 1% glutamax (Invitrogen). COS Cell Transfection COS cells were plated on day 1 to reach 40C60% confluency on the day of transfection. Transfection was Nedisertib Rabbit Polyclonal to P2RY5 performed on day 2 using a 3:1 ratio of GeneJuice (Novagen) to plasmid. Cells were harvested 48C60 hr after transfection and analyzed by flow cytometry. Fusion Proteins Recombinant proteins human B7C1-hIgG1 and human PD-1-hIgG1 were purchased from R & D systems. Human IgG was purchased from Jackson and BioXcell. Mouse IgG1 isotype control antibody (clone MOPC21) was purchased from BioXcell. Mouse IgG2b and Mouse IgG2a were purchased from Southern Biotech. hPD-1-mIgG2a and hB7C1-mIgG2a were purchased from Chimerigen. hPD-L1-mIgG2a was made in our laboratory (14). Antibodies Secondary antibodies absorbed against the other species (mouse or human) were used. Goat F(ab)2 anti-mouse IgG2a, PE-conjugated goat F(ab)2 anti-human IgG (absorbed against mouse Ig), PE-conjugated goat anti-human IgG (absorbed against mouse Ig) and HRP-conjugated goat anti-human IgG (absorbed against mouse Ig) were purchased from Southern Biotech. Antibodies specific for human PD-L1, mouse PD-L1, and human TIM-3 were made in our laboratory (15). Flow Cytometry Cells were incubated with the indicated primary antibody or fusion protein, washed, and incubated with 10 g/ml of the appropriate secondary antibody, washed and analyzed by flow cytometry on a BD FACS Canto II. Data were analyzed using FlowJo 10 software. Half-maximal effective Nedisertib concentration (EC-50) values were calculated using 4 parameter variable slope regression curve (Prism 7, GraphPad Software). ELISA ELISA plates were coated with 2 g/ml primary protein in PBS overnight at 4o, then blocked in 1% BSA and washed in ELISA washing buffer (Phosphate buffered saline pH 7.4 with 0.05% Tween 20). Fusion proteins were diluted in PBS Nedisertib plus 1% BSA at the indicated concentrations and incubated with plate-bound ligand for an hour. Bound fusion protein was detected using goat anti-human IgG HRP. EC-50 values were calculated using 4 parameter variable slope regression curve (Prism 7, GraphPad Software). Cell conjugation assay A cell conjugation assay for cell surface receptor-ligand binding was developed in our previous study (13). Briefly, cells transfected with cell surface gene 1 were labeled with the red fluorescent dye PKH26 (Sigma), and cells transfected with cell.