key: cord-0720550-ip5u2lgm authors: Beaudoin-Bussières, Guillaume; Richard, Jonathan; Prévost, Jérémie; Goyette, Guillaume; Finzi, Andrés title: A new flow cytometry assay to measure antibody dependent cellular cytotoxicity against SARS-CoV-2 Spike expressing cells date: 2021-09-13 journal: STAR Protoc DOI: 10.1016/j.xpro.2021.100851 sha: 9dbd5af8370318ce5f7a6385114f9b383dd09070 doc_id: 720550 cord_uid: ip5u2lgm Antibodies can engage specific receptors at the surface of effector cells and mediate several functions beyond viral neutralization. Increasing evidence suggest that Fc-mediated effector functions, such as antibody dependent cellular cytotoxicity (ADCC), have an important role in protection against SARS-CoV-2 infection. We engineered a cell line stably expressing a GFP-tagged SARS-CoV-2 Spike to measure ADCC. This protocol provides an optimized way of measuring ADCC activity mediated by anti-SARS-CoV-2 Spike monoclonal antibodies or plasma from previously infected or vaccinated individuals. For complete details on the use and execution of this protocol, please refer to (Anand et al., 2021b We recently developed a new method to measure antibody dependent cellular cytotoxicity (ADCC) 36 against SARS-CoV-2 Spike expressing cells. In this flow cytometry-based assay, a human T-lymphoid individuals as previously published (Anand et al., 2021b , Tauzin et al., 2021 . Preparation of the media for target and effector cells To study the ADCC response against SARS-CoV-2, we generated a human T-lymphoid cell line resistant 68 to NK cell lysis and stably expressing a full length GFP-tagged SARS-CoV-2 Spike (CEM.NKr.Spike) 69 (Anand et al., 2021b) . As presented in Figure In this experiment, we used the anti-Spike antibody CV3-25. This monoclonal antibody targets the S2 178 subunit of SARS-CoV-2 Spike (Jennewein et al., 2021) and shows ADCC activity against Spike-179 expressing cells ( Figure 6A ). We generated Leucine to Alanine (L234A/L235A, LALA) mutant version of 180 CV3-25 to impair interaction with Fc receptors (Saunders, 2019) and reduce its ADCC activity ( Figure 181 6A). To enhance the ADCC activity of CV3-25, we generated a Glycine to Alanine, Serine to Aspartic 182 Acid, Alanine to Leucine and Isoleucine to Glutamic Acid (G236A/S239D/A330L/I332E, GASDALIE) 183 mutant version of CV3-25 ( Figure 6A ) which strengthen the interaction between the Fc portion of an 184 antibody and Fc receptors (Bournazos et al., 2014a , DiLillo and Ravetch, 2015 , Lazar et al., 2006 185 Richards et al., 2008 , Smith et al., 2012 . Importantly, the differences in ADCC were not the result of 186 differential binding of the different monoclonal antibodies to the CEM.NKr.Spike cells as they all bind 187 the CEM.NKr.Spike cells similarly ( Figure 6B ). Briefly, for the staining on the CEM.NKr.Spike cell line, 188 approximately 300 000 cells were stained in 100 µL of an antibody solution at a concentration of 2,5 189 ng/mL, 10 ng/mL, 50 ng/mL, 250 ng/mL, 1000 ng/mL and 5000 ng/mL. These cells were incubated at Note : As soon as the solution is prepared, protect it from light by covering it with aluminum foil. Note : As soon as the solution is prepared, protect it from light by covering it with aluminum foil. Step-by-step method details The PBMCs should be at a concentration of 5 x 10 6 cells/mL. This section describes how to prepare and organize the cells for the experiment and how to add the 335 antibodies in order to start the ADCC assay. 12. Take the cells out of the incubator. The proportion of effector cells to target cells must also be the same in every well (10:1). To make Pause point: Between step 59 and step 60 you can wait up to 1 week. Analyzing the cells on the cytometer Analyzing the data 516 517 Timing: 1 h This section describes how to analyze the data obtained from the samples that were used in flow As previously reported, this assay can also be used to measure the ADCC mediating capacity of plasma Quantification and statistical analysis 562 563 In this assay, the cells expressing the SARS-CoV-2 Spike are GFP High . A loss of the GFP High population 564 indicates that the cells expressing the Spike were specifically killed. The more the cells are killed, the 565 J o u r n a l P r e -p r o o f more there will be a loss in the GFP High population. In order to calculate the % ADCC mediated by CV3-566 25 WT, LALA and GASDALIE (or any other antibody or plasma), the following formula was used : The following tables (table 1 and table 2) show the % GFP High cells that was used to calculate the % ADCC of the three monoclonal antibodies tested in this experiment. We encourage the reader to 571 calculate for themselves the % ADCC of the 3 monoclonal antibodies with the provided To test donor to donor variability in mediating ADCC, we compared 5 donors against the recently It is important to note that for large experiments it is important to limit to a minimum the time an irregular shape they might be contaminated. In that case, throw them away and thaw new cells. CEM.NKr parental and CEM.NKr.Spike cells can also be in an irregular shape when they are too 607 concentrated. In this seems to be the case, dilute them at a concentration of 0.25 x 10 6 cells/mL. Problem 2 The CEM.NKr parental and/or CEM.NKr.Spike cells don't grow. Potential solution If the cells do not multiply, they might be contaminated. If this is the case, throw them and thaw new 616 cells. If you diluted your cells in a concentration less than 0.15 x 10 6 cells/mL, they might take more 617 time to grow and give the impression that they are not multiplying. If this is the case, count them 618 again after 2-3 more days. Problem 3 There are not enough effector cells (PBMCs) to perform the experiment. Potential solution 625 626 PBMCs will not multiply and you can only use them the day after you thaw them. In order to not 627 waste them, perform the experiment with less conditions than you initially prepared. To make sure 628 this does not happen you can count the PBMCs immediately after thawing them. If you do not have 629 enough, thaw one or more vials of PBMCs. Problem 4 There are very few cells in the samples. Potential solution Problem 5 Variation in the calculated percentage of ADCC across different experiments. Potential solution Having some variation between experiments is normal. However, having variation above 30% is not. Use PBMCs from the same donor as not all PBMCs are good at mediating ADCC as shown in figure 8 . C V 3 -2 5 W T C V 3 -2 5 L A L A C V 3 -2 5 G A S D A L I E 5 This cell line is susceptible to antibody mediated ADCC in a dose-dependent manner  This assay can measure ADCC activity of plasma from infected/vaccinated individuals Enhanced ability of plant-derived 702 PGT121 glycovariants to eliminate HIV-1-infected cells Longitudinal analysis of humoral immunity against SARS-CoV-2 Spike in convalescent 708 individuals up to 8 months post-symptom onset Broadly neutralizing anti-HIV-1 antibodies require Fc effector functions for in vivo activity Broadly neutralizing anti-HIV-1 antibodies require Fc effector functions for in vivo activity Integrated immunovirological profiling validates plasma SARS-CoV-2 RNA as an early 723 predictor of COVID-19 mortality Differential Fc-Receptor Engagement Drives an Anti-tumor Vaccinal 725 Effect COVID-19 and the effect of plasma antibodies: a randomized controlled, open-label trial. medRxiv Isolation and characterization of cross-neutralizing 738 coronavirus antibodies from COVID-19+ subjects Engineered antibody 741 Fc variants with enhanced effector function Optimization of 743 antibody binding to FcgammaRIIa enhances macrophage phagocytosis of tumor cells Conceptual Approaches to Modulating Antibody Effector Functions and Circulation 746 Half-Life Mouse model recapitulating human Fcgamma receptor structural and functional diversity A single dose of the SARS-CoV-2 vaccine BNT162b2 elicits Fc-755 mediated antibody effector functions and T cell responses Live imaging of SARS-CoV-2 infection in 761 mice reveals neutralizing antibodies require Fc function for optimal efficacy The authors declare no competing interests. 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