Anti-S-layer monoclonal antibodies impact Clostridioides difficile physiology.

Clostridioides difficile (C. difficile), a gram-positive anaerobic and spore-forming bacterium, is the leading cause of nosocomial antibiotic-associated diarrhea in adults which is characterized by high levels of recurrence and mortality. Surface (S)-layer Protein A (SlpA), the most abundantly expressed protein on the bacterial surface, plays a crucial role in the early stages of infection although the nature of its involvement in C. difficile physiology is yet to be fully understood. Anti-S-layer antibodies have been identified in the sera of convalescent patients and have been correlated with improved outcomes of C. difficile infection (CDI). However, the precise mechanisms by which anti-S-layer antibodies confer protection to the host remain unknown. In this study, we report the first monoclonal antibodies (mAbs) targeting the S-layer of reference strain 630. Characterization of these mAbs unraveled important roles for the S-layer protein in growth, toxin secretion, and biofilm formation by C. difficile, with differential and even opposite effects of various anti-SlpA mAbs on these functions. Moreover, one anti-SlpA mAb impaired C. difficile growth and conferred sensitivity to lysozyme-induced lysis. The results of this study show that anti-S-layer antibody responses can be beneficial or harmful for the course of CDI and provide important insights for the development of adequate S-layer-targeting therapeutics.

Isolation methods determine human neutrophil responses after stimulation.

Studying neutrophils is challenging due to their limited lifespan, inability to proliferate, and resistance to genetic manipulation. Neutrophils can sense various cues, making them susceptible to activation by blood collection techniques, storage conditions, RBC lysis, and the isolation procedure itself. Here we assessed the impact of the five most used methods for neutrophil isolation on neutrophil yield, purity, activation status and responsiveness. We monitored surface markers, reactive oxygen species production, and DNA release as a surrogate for neutrophil extracellular trap (NET) formation. Our results show that neutrophils isolated by negative immunomagnetic selection and density gradient methods, without RBC lysis, resembled untouched neutrophils in whole blood. They were also less activated and more responsive to milder stimuli in functional assays compared to neutrophils obtained using density gradients requiring RBC lysis. Our study highlights the importance of selecting the appropriate method for studying neutrophils, and underscores the need for standardizing isolation protocols to facilitate neutrophil subset characterization and inter-study comparisons.

The role of neutrophils in antibody-driven autoimmune cytopenias.

Autoimmune cytopenias are a consequence of autoantibodies that target blood cell lineages and mark them for their accelerated destruction, mostly through phagocytosis by monocytes and macrophages and complement activation. Neutrophils, although equipped with Fc and complement receptors and effector mechanisms that are critical in other autoimmune conditions, remained long overlooked. Recent reports, however, propose a new and possibly critical role of neutrophils. In this review, we gathered available evidence on the contribution of neutrophils to the development, onset, and consequences of autoantibody-dependent cytopenias.

Specificity of mouse and human Fcgamma receptors and their polymorphic variants for IgG subclasses of different species.

IgG is the predominant antibody class generated during infections and used for the generation of therapeutic antibodies. Antibodies are mainly characterized in or generated from animal models that support particular infections, respond to particular antigens or allow the generation of hybridomas. Due to the availability of numerous transgenic mouse models and the ease of performing bioassays with human blood cells in vitro, most antibodies from species other than mice and humans are tested in vitro using human cells and/or in vivo using mice. In this process, it is expected, but not yet systematically documented, that IgG from these species interact with human or mouse IgG receptors (FcγRs). In this study, we undertook a systematic assessment of binding specificities of IgG from various species to the families of mouse and human FcγRs including their polymorphic variants. Our results document the specific binding patterns for each of these IgG (sub)classes, reveal possible caveats of antibody-based immunoassays, and will be a useful reference for the transition from one animal model to preclinical mouse models or human cell-based bioassays.

IgG Fc sialylation is regulated during the germinal center reaction following immunization with different adjuvants.

BACKGROUND: Effector functions of IgG Abs are regulated by their Fc N-glycosylation pattern. IgG Fc glycans that lack galactose and terminal sialic acid residues correlate with the severity of inflammatory (auto)immune disorders and have also been linked to protection against viral infection and discussed in the context of vaccine-induced protection. In contrast, sialylated IgG Abs have shown immunosuppressive effects. OBJECTIVE: We sought to investigate IgG glycosylation programming during the germinal center (GC) reaction following immunization of mice with a foreign protein antigen and different adjuvants. METHODS: Mice were analyzed for GC T-cell, B-cell, and plasma cell responses, as well as for antigen-specific serum IgG subclass titers and Fc glycosylation patterns. RESULTS: Different adjuvants induce distinct IgG+ GC B-cell responses with specific transcriptomes and expression levels of the α2,6-sialyltransferase responsible for IgG sialylation that correspond to distinct serum IgG Fc glycosylation patterns. Low IgG Fc sialylation programming in GC B cells was overall highly dependent on the Foxp3- follicular helper T (TFH) cell-inducing cytokine IL-6, here in particular induced by water-in-oil adjuvants and Mycobacterium tuberculosis. Furthermore, low IgG Fc sialylation programming was dependent on adjuvants that induced IL-27 receptor-dependent IFN-γ+ TFH1 cells, IL-6/IL-23-dependent IL-17A+ TFH17 cells, and high ratios of TFH cells to Foxp3+ follicular regulatory T cells. Here, the 2 latter were dependent on M tuberculosis and its cord factor. CONCLUSION: This study's findings regarding adjuvant-dependent GC responses and IgG glycosylation programming may aid in the development of novel vaccination strategies to induce IgG Abs with both high affinity and defined Fc glycosylation patterns in the GC.