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In the absence of the CD4 receptor at the cell surface, primary Envs sample a \u201cclosed\u201d conformation that occludes CD4-induced (CD4i) epitopes. The virus controls CD4 expression through the actions of Nef and Vpu accessory proteins, thus protecting infected cells from ADCC responses. However, gp120 shed from infected cells can bind to CD4 present on uninfected bystander cells, sensitizing them to ADCC mediated by CD4i antibodies (Abs). Therefore, we hypothesized that these bystander cells could impact the interpretation of ADCC measurements. To investigate this, we evaluated the ability of antibodies to CD4i epitopes and broadly neutralizing Abs (bNAbs) to mediate ADCC measured by five ADCC assays commonly used in the field. Our results indicate that the uninfected bystander cells coated with gp120 are efficiently recognized by the CD4i ligands but not the bNabs. Consequently, the uninfected bystander cells substantially affect\n            <jats:italic>in vitro<\/jats:italic>\n            measurements made with ADCC assays that fail to identify responses against infected versus uninfected cells. Moreover, using an mRNA flow technique that detects productively infected cells, we found that the vast majority of HIV-1-infected cells in\n            <jats:italic>in vitro<\/jats:italic>\n            cultures or\n            <jats:italic>ex vivo<\/jats:italic>\n            samples from HIV-1-infected individuals are CD4 negative and therefore do not expose significant levels of CD4i epitopes. Altogether, our results indicate that ADCC assays unable to differentiate responses against infected versus uninfected cells overestimate responses mediated by CD4i ligands.\n          <\/jats:p>\n          <jats:p>\n            <jats:bold>IMPORTANCE<\/jats:bold>\n            Emerging evidence supports a role for antibody-dependent cellular cytotoxicity (ADCC) in protection against HIV-1 transmission and disease progression. However, there are conflicting reports regarding the ability of nonneutralizing antibodies targeting CD4-inducible (CD4i) Env epitopes to mediate ADCC. Here, we performed a side-by-side comparison of different methods currently being used in the field to measure ADCC responses to HIV-1. We found that assays which are unable to differentiate virus-infected from uninfected cells greatly overestimate ADCC responses mediated by antibodies to CD4i epitopes and underestimate responses mediated by broadly neutralizing antibodies (bNAbs). Our results strongly argue for the use of assays that measure ADCC against HIV-1-infected cells expressing physiologically relevant conformations of Env to evaluate correlates of protection in vaccine trials.\n          <\/jats:p>","DOI":"10.1128\/mbio.00358-18","type":"journal-article","created":{"date-parts":[[2018,3,19]],"date-time":"2018-03-19T10:00:36Z","timestamp":1521453636000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":89,"title":["Uninfected Bystander Cells Impact the Measurement of HIV-Specific Antibody-Dependent Cellular Cytotoxicity Responses"],"prefix":"10.1128","volume":"9","author":[{"given":"Jonathan","family":"Richard","sequence":"first","affiliation":[{"name":"Centre de Recherche du CHUM, Montreal, Quebec, Canada"},{"name":"Department of Microbiology, Infectiology and Immunology, Universit\u00e9 de Montr\u00e9al, Montreal, Quebec, 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