Phagocytosis is a process in which target cells or particles are engulfed and taken up by other cells, typically professional phagocytes; this process is crucial in many physiological processes and disease states. The detection of targets for phagocytosis is directed by a complex repertoire of cell surface receptors. Pattern recognition receptors directly detect targets for binding and uptake, while opsonic and complement receptors detect objects coated by soluble factors. However, the importance of single and combinatorial surface marker expression across different phenotypes of professional phagocytes is not known. Here we developed a novel mass cytometry-based phagocytosis assay that enables the simultaneous detection of phagocytic events in combination with up to 40 other protein markers. We applied this assay to distinct monocyte derived macrophage (MDM) populations and found that prototypic M2-like MDMs phagocytose more E. coli than M1-like MDMs. Surface markers such as CD14, CD206, and CD163 rendered macrophages phagocytosis competent, but only CD209 directly correlated with the amount of particle uptake. Similarly, M2-like MDMs also phagocytosed more cancer cells than M1-like MDMs but, unlike M1-like MDMs, were insensitive to anti-CD47 opsonization. Our approach facilitates the simultaneous study of single-cell phenotypes, phagocytic activity, signaling and transcriptional events in complex cell mixtures.

Nasal colonization is a major risk factor for S. aureus infections. The mechanisms responsible for colonization are still not well understood and involve several factors on the host and the bacterial side. One key factor is the cell wall teichoic acid (WTA) of S. aureus, which governs direct interactions with nasal epithelial surfaces. We report here the first receptor for the cell wall glycopolymer WTA on nasal epithelial cells. In several assay systems this type F-scavenger receptor, termed SREC-I, bound WTA in a charge dependent manner and mediated adhesion to nasal epithelial cells in vitro. The impact of WTA and SREC-I interaction on epithelial adhesion was especially pronounced under shear stress, which resembles the conditions found in the nasal cavity. Most importantly, we demonstrate here a key role of the WTA-receptor interaction in a cotton rat model of nasal colonization. When we inhibited WTA mediated adhesion with a SREC-I antibody, nasal colonization in the animal model was strongly reduced at the early onset of colonization. More importantly, colonization stayed low over an extended period of 6 days. Therefore we propose targeting of this glycopolymer-receptor interaction as a novel strategy to prevent or control S. aureus nasal colonization.

The human immune system is composed of a distributed network of cells circulating throughout the body, which must dynamically form physical associations and communicate using interactions between their cell-surface proteomes1. Despite their therapeutic potential2, our map of these surface interactions remains incomplete3,4. Here, using a high-throughput surface receptor screening method, we systematically mapped the direct protein interactions across a recombinant library that encompasses most of the surface proteins that are detectable on human leukocytes. We independently validated and determined the biophysical parameters of each novel interaction, resulting in a high-confidence and quantitative view of the receptor wiring that connects human immune cells. By integrating our interactome with expression data, we identified trends in the dynamics of immune interactions and constructed a reductionist mathematical model that predicts cellular connectivity from basic principles. We also developed an interactive multi-tissue single-cell atlas that infers immune interactions throughout the body, revealing potential functional contexts for new interactions and hubs in multicellular networks. Finally, we combined targeted protein stimulation of human leukocytes with multiplex high-content microscopy to link our receptor interactions to functional roles, in terms of both modulating immune responses and maintaining normal patterns of intercellular associations. Together, our work provides a systematic perspective on the intercellular wiring of the human immune system that extends from systems-level principles of immune cell connectivity down to mechanistic characterization of individual receptors, which could offer opportunities for therapeutic intervention.

Phenotypic plasticity is essential to the immune system, yet the factors that shape it are not fully understood. Here, we comprehensively analyze immune cell phenotypes including morphology across human cohorts by single-round multiplexed immunofluorescence, automated microscopy, and deep learning. Using the uncertainty of convolutional neural networks to cluster the phenotypes of eight distinct immune cell subsets, we find that the resulting maps are influenced by donor age, gender, and blood pressure, revealing distinct polarization and activation-associated phenotypes across immune cell classes. We further associate T cell morphology to transcriptional state based on their joint donor variability and validate an inflammation-associated polarized T cell morphology and an age-associated loss of mitochondria in CD4 + T cells. Together, we show that immune cell phenotypes reflect both molecular and personal health information, opening new perspectives into the deep immune phenotyping of individual people in health and disease.