Phagocytosis can be induced via the engagement of Fcγ receptors by antibody-opsonized material. Furthermore, the efficiency of antibody-induced effector functions has been shown to be dramatically modulated by changes in antibody glycosylation. Because infection can modulate antibody glycans, which in turn modulate antibody functions, assays capable of determining the induction of effector functions rather than neutralization or titer provide a valuable opportunity to more fully characterize the quality of the adaptive immune response. Here we describe a robust and high-throughput flow cytometric assay to define the phagocytic activity of antigen-specific antibodies from clinical samples. This assay employs a monocytic cell line that expresses numerous Fc receptors: including inhibitory and activating, and high and low affinity receptorsallowing complex phenotypes to be studied. We demonstrate the adaptability of this highthroughput, flow-based assay to measure antigen-specific antibody-mediated phagocytosis against an array of viruses, including influenza, HIV, and dengue. The phagocytosis assay format further allows for simultaneous analysis of cytokine release, as well as determination of the role of specific Fcγ-receptor subtypes, making it a highly useful system for parsing differences in the ability of clinical and vaccine induced antibody samples to recruit this critical effector function.

No abstract

Natural killer (NK) cells are effector cells of the innate immune system and are important in the control of viral infections. Their relevance is reflected by the multiple mechanisms evolved by viruses to evade NK cell-mediated immune responses. Over recent years, our understanding of the interplay between NK cell immunity and viral pathogenesis has improved significantly. Here, we review the role of NK cells in the control of four important viral infections in humans: cytomegalovirus, influenza virus, HIV-1, and hepatitis C virus.

Natural killer (NK) cells have traditionally been considered nonspecific components of innate immunity, but recent studies have shown features of antigen-specific memory in murine NK cells. However, it has remained unclear whether this phenomenon also exists in primates. Compared to NK cells from uninfected macaques, we found splenic and hepatic NK cells from SHIV-SF162P3- and SIVmac251-infected animals specifically lysed Gag- and Env-pulsed dendritic cells (DCs) in an NKG2-dependent fashion. Moreover, splenic and hepatic NK cells from Ad26-vaccinated macaques efficiently lysed antigen-matched but not antigen-mismatched targets 5 years post-vaccination. These data demonstrate that robust, durable, antigen-specific NK cell memory can be induced in primates following both infection and vaccination, and could be important for vaccines against HIV-1 and other pathogens.

The activating NK-cell receptor KIR3DS1 has been implicated in the outcome of various human diseases, including delayed HIV-1 disease progression, yet a ligand that accounts for its biological effects remained unknown. We screened 100 HLA-I proteins and found that KIR3DS1 binds HLA-F, which was validated biochemically and functionally. Primary human KIR3DS1+ NK cells degranulated and produced antiviral cytokines upon encountering HLA-F, and inhibited HIV-1 replication in vitro. CD4+ T-cell activation triggered HLA-F transcription and expression and induced KIR3DS1 ligand expression. HIV-1 infection further increased HLA-F transcription, but decreased KIR3DS1 ligand expression, indicating an immune-evasion mechanism. Altogether, we established HLA-F as a ligand of KIR3DS1, and demonstrated cell-context-dependent expression of HLA-F that may explain the widespread influence of KIR3DS1 in human diseases.