Human igg4 isotype control

Cp20 (Ac-I[CV-1MeW-QDW-Sar-AHRC]mI-NH₂), a potent compstatin analog, was used to inhibit convertase-mediated C3 activation, and was produced by solid phase synthesis (29 (link)). PMX53 (30 (link)) was produced as described. SALO (31 (link)) was a generous donation from Dr. Jesus Valenzuela (National Institutes of Health). Eculizumab was purchased from Creative Biolabs, and OmCI (32 (link)) was a generous donation from Dr. Susan Lea (University of Oxford). Human IgG4 isotype control (BioLegend) was used as a control for Eculizumab.

To set up the TICS assay, human cancer lines or primary ascetic cells were seeded at indicated concentrations in 100 μl assay medium in a 96-well flat bottom plate and incubated at 37° overnight to allow cell adherence. On the same day, ‘assay-ready’ primary human lymphocytes were quickly thawed at 37 °C in a water bath, washed twice in 15 ml PBS, resuspended in assay medium and incubated at 37 °C with 5% CO₂ overnight. Next, lymphocytes were resuspended at 3 million cells per ml and added to the wells containing tumour cells in 100 μl assay medium. To block the human PD-1/PD-L1 pathway, a human IgG4 isotype control (BioLegend, California, USA) or nivolumab (Bristol-Myers Squibb, New York, USA), a human IgG1 isotype control (NIP228, AstraZeneca, Cambridge, UK), a PD-1 blocking antibody (clone LO115, AstraZeneca, Cambridge, UK) or durvalumab (MEDI4736, AstraZeneca, Cambridge, UK) were added at 10 μg/ml. Activation of different immune cell subsets was analysed by flow cytometry on day 6, and soluble IFNγ levels in the supernatants were analysed by Human IFNγ MAX Deluxe ELISA kit (BioLegend, California, USA).
For real-time monitoring of tumour cell killing, TICS assay was set up in the presence of 1 μM green caspase-3/7 cell apoptosis reagent (Essen Bioscience/Sartorius, Göttingen, Germany) and imaged using an IncuCyte ZOOM instrument.