Antibody density on bacteria regulates C1q recruitment by monoclonal IgG but not IgM.

Antibodies that trigger the complement system play a pivotal role in the immune defense against pathogenic bacteria and offer potential therapeutic avenues for combating antibiotic-resistant bacterial infections, a rising global concern. To gain a deeper understanding of the key parameters regulating complement activation by monoclonal antibodies, we developed a novel bioassay for quantifying classical complement activation at the monoclonal antibody level, and employed this assay to characterize rare complement-activating antibacterial antibodies on the single-antibody level in postimmunization murine antibody repertoires. We characterized monoclonal antibodies from various antibody isotypes against specific pathogenic bacteria (Bordetella pertussis and Neisseria meningitidis) to broaden the scope of our findings. We demonstrated activation of the classical pathway by individual IgM- and IgG-secreting cells, that is, monoclonal IgM and IgG2a/2b/3 subclasses. Additionally, we could observe different epitope density requirements for efficient C1q binding depending on antibody isotype, which is in agreement with previously proposed molecular mechanisms. In short, we found that antibody density most crucially regulated C1q recruitment by monoclonal IgG isotypes, but not IgM isotypes. This study provides additional insights into important parameters for classical complement initiation by monoclonal antibodies, a knowledge that might inform antibody screening and vaccination efforts.

Measuring single-cell protein secretion in immunology: Technologies, advances, and applications.

The dynamics, nature, strength, and ultimately protective capabilities of an active immune response are determined by the extracellular constitution and concentration of various soluble factors. Generated effector cells secrete such mediators, including antibodies, chemo- and cytokines to achieve functionality. These secreted factors organize the individual immune cells into functional tissues, initiate, orchestrate, and regulate the immune response. Therefore, a single-cell resolved analysis of protein secretion is a valuable tool for studying the heterogeneity and functionality of immune cells. This review aims to provide a comparative overview of various methods to characterize immune reactions by measuring single-cell protein secretion. Spot-based and cytometry-based assays, such as ELISpot and flow cytometry, respectively, are well-established methods applied in basic research and clinical settings. Emerging novel technologies, such as microfluidic platforms, offer new ways to measure and exploit protein secretion in immune reactions. Further technological advances will allow the deciphering of protein secretion in immunological responses with unprecedented detail, linking secretion to functionality. Here, we summarize the development and recent advances of tools that allow the analysis of protein secretion at the single-cell level, and discuss and contrast their applications within immunology.

A Guide to the Quantitation of Protein Secretion Dynamics at the Single-Cell Level.

Protein secretion is a key cellular functionality, particularly in immunology, where cells can display large heterogeneity in this crucial activity in addition to binary secretion behavior. However, few methods enable quantitative secretion rate measurements at the single-cell level, and these methods are mostly based on microfluidics systems. Here, we describe such a microfluidic single-cell method for precisely measuring protein secretion rates in detail, building on the published droplet-based microfluidic platform DropMap. We give an updated, detailed guide toward quantifying protein secretion rates, discussing its setup and limitations. We illustrate the protocol on two key immunological analytes, immunoglobulin G, and interferon-γ.

Single-B cell analysis correlates high-lactate secretion with stress and increased apoptosis.

While cellular metabolism was proposed to be a driving factor of the activation and differentiation of B cells and the function of the resulting antibody-secreting cells (ASCs), the study of correlations between cellular metabolism and functionalities has been difficult due to the absence of technologies enabling the parallel measurement. Herein, we performed single-cell transcriptomics and introduced a direct concurrent functional and metabolic flux quantitation of individual murine B cells. Our transcriptomic data identified lactate metabolism as dynamic in ASCs, but antibody secretion did not correlate with lactate secretion rates (LSRs). Instead, our study of all splenic B cells during an immune response linked increased lactate metabolism with acidic intracellular pH and the upregulation of apoptosis. T cell-dependent responses increased LSRs, and added TLR4 agonists affected the magnitude and boosted LSRhigh B cells in vivo, while resulting in only a few immunoglobulin-G secreting cells (IgG-SCs). Therefore, our observations indicated that LSRhigh cells were not differentiating into IgG-SCs, and were rather removed due to apoptosis.

Insights into the relationship between persistent antibody secretion and metabolic programming - A question for single-cell analysis.

Vaccination aims to generate a protective and persisting antibody response. Indeed, humoral vaccine-mediated protection depends on the quality and quantity of the produced antigen-specific antibodies for its initial magnitude and the persistence of the plasma cells for its duration. Therefore, understanding the mechanisms behind the generation, selection and maintenance of long-lived plasma cells secreting protective antibodies is of fundamental importance for understanding long-term immunity, vaccine responses, therapeutical approaches for autoimmune disease and multiple myeloma. Recent studies have observed correlations between the generation, function and lifespan of plasma cells and their metabolism, with metabolism being both a main driver and primary consequence of changes in cellular behavior. This review introduces how metabolic programs influence and drive immune cell functions in general and plasma cell differentiation and longevity more specifically, summarizing the current knowledge on metabolic pathways and their influences on cellular fate. In addition, available technologies to profile metabolism and their limitations are discussed, leading to the unique and open technological challenges for further advancement of this research field.