Journal: Cell reports

Article Title: Selective decoupling of IgG1 binding to viral Fc receptors restores antibody-mediated NK cell activation against HCMV

doi: 10.1016/j.celrep.2025.116593

Figure Lengend Snippet: (A) BW-CD16A-ζ reporter cells were incubated with SM5–1 antibody, 100 μg/mL isotype, or 1:100 dilution Cytotect and AD169- or Δ3-infected MRC5 fibroblasts (MOI = 5 and 72 hpi), with CD16A activation quantified by mouse IL-2. (B) Fluorescent images of AD169-infected MRC5 cells (MOI = 2 and 96 hpi) incubated with AF647-labeled SM5–1 or SM5–1-mFc (20 μg/mL) for 2 h at 37°C. Cells fixed with 4% paraformaldehyde and scanned using Zeiss LSM 710/Elyra S.1 at 63×. Scale bars, 21 μm. (C and D) Flow cytometry measured the percent of cells positive for (C) extracellular or (D) internalized antibody after incubating 67 nM antibody with infected cells (as above) or mock-infected cells for 2 h at 37°C. Extracellular antibody detected with goat anti-human-Fcy AF647; intracellular with pHrodo-Red-labeled primary antibody. Data are mean ± SD, for n = 2 with * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns: non-significant as determined by two-way ANOVA followed by Tukey’s multiple comparisons test in GraphPad. Data are representative of one experiment; each experiment was repeated twice.

Article Snippet: Bound hu4D5 antibodies were detected by incubation with goat anti-human kappa light chain antibody-HRP (Southern Biotech Cat #2060–05, 1:2000 dilution).

Techniques: Incubation, Infection, Activation Assay, Labeling, Flow Cytometry

Journal: Cell reports

Article Title: Selective decoupling of IgG1 binding to viral Fc receptors restores antibody-mediated NK cell activation against HCMV

doi: 10.1016/j.celrep.2025.116593

Figure Lengend Snippet: (A) Binding of host Fc receptors (FLAG-tagged CD16A-GST, FcRn-GST, or gp34-M) to immobilized human Fc was measured in the presence of competitor (gp34-M, t-gp68, and hu-Fc) by ELISA. Assay area under the curve (AUC) was normalized to controls lacking a competitor and presented as a heatmap. (B and C) Binding of antibody Fc variants to gp34-M, t-gp68, and (B) FcRn or (C) CD16A assessed by ELISA with normalized inverse 50% effective concentration (EC 50 ) values shown as a heatmap with more color indicating better binding. (D and E) The nsEM 3D reconstruction and 2D class averages for particles containing (D) one Fc with one gp34-M bound near the CH 2 tip and (E) one Fc with one t-gp68 bound to the CH 2 -CH 3 interface. RoseTTAFold-based gp34 (green) and t-gp68 (blue) models fit the 3D reconstruction with the Fc crystal structure (PDB: 2GJ7). Scale bars, 5 nm. (F and G) SPR measured (F) t-gp68 binding to immobilized Fc and (G) binding of human IgG1 antibody to immobilized gp34-M with K D values determined using BIAevaluation X100 software. (H and I) Flow cytometry measured (H) binding to and (I) internalization of pHrodo-Red-labeled antibody (67 nM) by AD169-infected MRC5 cells (MOI = 2 and 96 hpi) in the presence of t-gp68 (2 μM) and/or gp34-M (0.1 μM). Extracellular antibody detected with goat-anti-human-Fcy AF647; in (I), the dashed line represents the threshold for detection. Data shown are mean ± SD, n = 2, with * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns, non-significant. All analyses are performed in GraphPad using two-way ANOVA with Tukey’s multiple comparisons test. Representative data of one experiment is shown; each experiment is repeated at least twice.

Article Snippet: Bound hu4D5 antibodies were detected by incubation with goat anti-human kappa light chain antibody-HRP (Southern Biotech Cat #2060–05, 1:2000 dilution).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Software, Flow Cytometry, Labeling, Infection