Following removal of PBMCs and RBC lysis from buffy coats, neutrophils (n = 6 donors) were cocultured with DiI-labeled HSV-infected Vero cells pretreated with serum from HSV+ donor containing ADCC-mediating antibodies or HSV– donors. (A) Representative flow plots of target cell DiI cell membrane detection in CD11b⁺CD66b⁺ neutrophils. (B) Percentage or (C) fluorescent intensity of neutrophils positive for target cell membrane detection with either HSV+ or HSV– serum. %DiI⁺ neutrophils and GeoMFI were compared by unpaired t test (**P < 0.01, ****P < 0.0001). (D) Neutrophils were pretreated with DPI, ML-7, or DMSO control before incubation with human serum opsonized HSV infected cell targets in the calcein release assay. Cell-free calcein was measured and converted to percentage cytotoxicity. (E) Representative flow plots of CD11b⁺CD66b⁺ neutrophils for expression HVEM, LIGHT, and CD16a in either FMO (black) or a donor sample (red). (F) Expression of HVEM, LIGHT, and CD16a as measured by flow cytometry from human donor neutrophils (n = 6). (G) Enriched neutrophils (n = 6 different donors) were incubated with antibodies blocking HVEM, LIGHT, or CD16a prior to incubation with human serum-treated (HSV+ or HSV– donors) HSV-infected calcein-labeled Vero cells. (H) Human donor neutrophils (n = 3) were incubated with calcein-labeled SKBR3 cells opsonized with increasing doses of a Trastuzumab biosimilar anti-HER2 mAb or an isotype control for 4 hours in the calcein release assay. (I) Calcein-labeled SKBR3 cells were treated with 10 μg of the anti-HER2 mAb and then cocultured with human neutrophils that had been pretreated with blocking antibodies against HVEM, LIGHT, or an isotype control and cytotoxicity monitored by measuring calcein release and presented as percentage cytotoxicity. Cytotoxicity was compared by 1 way ANOVA with Holm-Šidák’s multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001).