Discovery of viral and host pathways that protect HIV-infected macrophages from NK cell ADCC

PROJECT SUMMARY Characterization of HIV reservoirs and the immunoevasion mechanisms permitting persistence has largely focused on CD4+ T cells. Yet macrophages represent a physiologically relevant reservoir, with characteristics that may uniquely contribute to viral persistence. They can bud and store virus within virus-containing compartments, which largely exclude neutralizing antibodies, and resist the cytopathic effects of infection. Furthermore, en masse, macrophages vs CD4+ T cells are more resistant to killing by CTL and NK cells. Our published work shows that macrophages resist both NK cell innate cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC), but the mechanisms of this evasion are unknown. As passive transfer of broadly neutralizing antibodies has shown much promise in the cART-free treatment of infection, understanding how macrophages respond to HIV antibodies and how they might succumb to ADCC would aid the design of inclusive HIV cure strategies. Interestingly, while HIV antibody-enhanced NK cell signaling/activation and release of perforin/granzymes towards infected macrophages vs CD4+ T cells is significantly lower, HIV-specific CAR T cells activate in response to both infected cell types equally well. The overall objective of this research project is to determine the mechanisms of how infected macrophages antagonize NK cell signaling that induces ADCC. Given that NK cells signal in response to multiple target cell surface ligands, which are likely different on CD4+ T cells vs macrophages, the central hypothesis of this project is that infected macrophages present an inhibitory signal that is not on CD4+ T cells, which counteracts NK cell ADCC. Specific Aim #1 will characterize the effects of HIV accessory proteins on ADCC-triggered NK cell signaling towards CD4+ T cells vs macrophages. Previous studies suggest that NK ligands can affect ADCC. Since the HIV accessory proteins Nef and Vpu differentially affect CD4+ T cells and macrophages, they may also elicit differential regulation of NK cell ligands in these cell types, leading to differences in ADCC-triggered NK cell signaling. Using HIV with genetically ablated accessory proteins, we propose to assess how each protein regulates the NK cell ligand repertoire and ADCC-triggered NK cell signaling. While these viral proteins may explain the ADCC differences towards the targets, Specific Aim #2 will define how Fc𝛾 receptor (Fc𝛾R) binding to HIV antibody, which also binds to the surface Env on macrophages (in cis) affects ADCC-triggered NK cell signaling (in trans). Fc𝛾R expression on targets cells, which inhibits ADCC towards transplanted cell therapeutics, may be the dominant factor that protects macrophages. We propose to use CRISPR KO of Fc𝛾Rs in macrophages and lentiviral overexpression of Fc𝛾Rs in CD4+ T cells to determine the effects of Fc𝛾Rs on ADCC towards infected cells and uninfected targets that overexpress HIV Env. Together, these studies will provide insight into the mechanisms of macrophage resistance to ADCC, which may help in the design of more effective and inclusive HIV cure strategies, such as blocking accessory protein function with small molecules or design of bNAbs that limit macrophage Fc𝛾R reactivity.