Sunday, May 5, 2024
5/5/2024
Project Summary/Abstract Multiple studies have demonstrated that Natural Killer (NK) cell activity, including antibody dependent cellular cytotoxicity (ADCC), is correlated with delayed HIV disease progression. Furthermore, protein engineering has revealed that glycosylation of the antibody Fc region, particularly for IgG1, affects Fc gamma receptor IIIa (CD16) binding affinity and ADCC activity. However, the mechanism by which Fc glycosylation alters ADCC activity remains incompletely understood, specifically regarding the spatiotemporal localization of CD16 and associated signaling molecules. The long-term goal of this proposal is to obtain a more complete understanding of how antibody glycobiology interplays with the innate immune response to generate guiding principles for more effective vaccines and therapeutics. The objective of this proposal is to determine how antibody glycosylation influences CD16 mediated NK cell activation at single cell resolution in HIV infected individuals and to define the spaciotemporal dynamics of CD16-based signaling. The rationale for this work is that the combination of cutting- edge microscopy observations and temporal protein analysis will provide new insights into ADCC function with immediate impacts on antibody therapeutic design. Our central hypothesis is that specific antibody glycosylation profiles will differentially modulate CD16 interactions and ADCC activity through changes in CD16 signaling. Our central hypothesis will be tested in two specific aims: 1) Determine how narrow glycosylation profiles of monoclonal antibodies (mAbs) directed to key sites of vulnerability on the HIV Envelope (Env) affect phosphorylation states of signaling proteins during NK cell ADCC, comparing NK cells from healthy and HIV+ donors; 2) Determine how antibody glycosylation alters the subcellular localization of CD16 and signaling proteins within the NK cell immunological synapse (NKIS) throughout each stage of ADCC. We will pursue these aims with innovative techniques in multi-color quantitative fluorescent microscopy and spectral flow cytometry. Detailed single cell studies using MINFLUX nanoscopy, a super-resolution fluorescent microscopy technique capable of 2 nm or better spatial resolution in 3D, will complement our proteomic analysis. The expected outcome of our studies is that marrying our quantitative proteomic analysis with more detailed mechanistic studies of ADCC will rationalize previously observed changes in cellular activity associated with differential antibody glycosylation. Our results will impact human health by providing a molecular rational for the effect of antibody glycosylation on ADCC activity, thus improving antibody and cellular therapeutic development for HIV as well as a broad range of other diseases.
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