Neutrophils, the most abundant white blood cell, play a critical role in anti-pathogen immunity via phagocytic clearance, secretion of enzymes and immunomodulators, and the release of extracellular traps. Neutrophils non-specifically sense infection through an array of innate immune receptors and inflammatory sensors, but are also able to respond in a pathogen/antigen-specific manner when leveraged by antibodies via Fc-receptors. Among neutrophil functions, antibody-dependent neutrophil phagocytosis (ADNP) results in antibody-mediated opsonization, enabling neutrophils to sense and respond to infection in a pathogen-appropriate manner. Here, we describe a high-throughput flow cytometric approach to effectively visualize and quantify ADNP and its downstream consequences. The assay is easily adaptable, supporting both the use of purified neutrophils or white blood cells, the use of purified Ig or serum, and the broad utility of any target antigen. Thus, this ADNP assay represents a high-throughput platform for the in-depth characterization of neutrophil function.

Abundant evidence points to a critical role for antibodies in protection and pathology across infectious diseases. While the antibody variable domain facilitates antibody binding and the blockade of infection, the constant domain (Fc) mediates cross talk with the innate immune system. The biological activity of the Fc region is controlled genetically via class switch recombination, resulting in the selection of distinct antibody isotypes and subclasses. However, a second modification is made to all antibodies, via post-translational changes in antibody glycosylation. Studies from autoimmunity and oncology have established the role of immunoglobulin G (IgG) Fc glycosylation as a key regulator of humoral immune activity. However, a growing body of literature, exploring IgG Fc glycosylation through the lens of infectious diseases, points to the role of inflammation in shaping Fc-glycan profiles, the remarkable immune plasticity in antibody glycosylation across pathogen-exposed populations, the canonical and noncanonical functions of glycans and the existence of antigen-specific control over antibody Fc glycosylation. Ultimately, this work provides critical new insights into the functional roles for antibody glycosylation as well as lays the foundation for leveraging antibody glycosylation to drive prevention or control across diseases.

Development of an effective tuberculosis (TB) vaccine has suffered from an incomplete understanding of the correlates of protection against Mycobacterium tuberculosis (Mtb). Intravenous (i.v.) vaccination with Bacille Calmette–Guérin (BCG) provides nearly complete protection against TB in rhesus macaques, but the antibody response it elicits remains incompletely defined. Here we show that i.v. BCG drives superior antibody responses in the plasma and the lungs of rhesus macaques compared to traditional intradermal BCG administration. While i.v. BCG broadly expands antibody titers and functions, IgM titers in the plasma and lungs of immunized macaques are among the strongest markers of reduced bacterial burden. IgM was also enriched in macaques that received protective vaccination with an attenuated strain of Mtb. Finally, an Mtb-specific IgM monoclonal antibody reduced Mtb survival in vitro. Collectively, these data highlight the potential importance of IgM responses as a marker and mediator of protection against TB.