Fig 1: Validation of Temporal Profiles by Flow Cytometry and Immunoblot and Investigation of Novel Immune Ligands, Related to Figure 4(A) PM and WCL profiles of MHC-I (HLA-A11) and CD99, validated by flow cytometry and immunoblot. = 95% of HFFF were routinely infected by HCMV, assessed by flow cytometry for cell surface MHC-I.(B) Representative intracellular flow cytometry of 24h-infected HFFF with anti-IE1 confirms > 95% infection efficiency.(C) Flow cytometry of HFFF infected with HCMV confirms proteomic profiles for five additional cell surface proteins.(D and E) NK degranulation assays suggest that CLEC1A and FAT1 are novel activating NK ligands. Top panels – validation of temporal PM and WCL profiles by flow cytometry and immunoblot. CLEC1A was not quantified in any WCL QTV experiments but accumulated by immunoblot of whole-cell lysates, while depleting from the PM. Bottom panels – target cells underwent siRNA knockdown of CLEC1A (D) or FAT1 (E) and were then incubated with stimulated polyclonal NK cells from each of three donors. Degranulation of NK cells in response to both CLEC1A and FAT1 knockdown targets was significantly reduced compared to control. Error bars: +/- SEM (donors A, B), +/- range (donor C). * two-tailed p-value < 0.05, ** two-tailed p-value < 0.005. Cell surface MHC-I was unaffected by siRNA (right bottom panels, staining with W6-32 antibody or control Ig).(F) CD8+ T cell degranulation assay suggests that CEACAM1 is a novel inhibitory ligand for CMV-specific cytotoxic T cells. Top panel – validation of temporal PM profile by flow cytometry. Bottom panels (left) – flow cytometry of a CD8+ T cell line specific to the HCMV HLA-A2 restricted IE1 peptide VLEETSVML confirmed CEACAM1 surface expression. (right) HCMV peptide-specific CD8+ effector cells were incubated with autologous fibroblasts that had been infected with HCMV for 72h then pulsed with peptide or left unpulsed. Effectors and targets were treated with control Ig or anti-CEACAM1. HCMV-specific T cell degranulation was significantly increased with CEACAM1 block. Error bars ± SEM. * two-tailed p-value < 0.0001.y-axes of QTV plots represent relative abundance, and y-axes of flow cytometry plots represent % of max.