Scalable, multimodal profiling of chromatin accessibility, gene expression and protein levels in single cells.

Recent technological advances have enabled massively parallel chromatin profiling with scATAC-seq (single-cell assay for transposase accessible chromatin by sequencing). Here we present ATAC with select antigen profiling by sequencing (ASAP-seq), a tool to simultaneously profile accessible chromatin and protein levels. Our approach pairs sparse scATAC-seq data with robust detection of hundreds of cell surface and intracellular protein markers and optional capture of mitochondrial DNA for clonal tracking, capturing three distinct modalities in single cells. ASAP-seq uses a bridging approach that repurposes antibody:oligonucleotide conjugates designed for existing technologies that pair protein measurements with single-cell RNA sequencing. Together with DOGMA-seq, an adaptation of CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing) for measuring gene activity across the central dogma of gene regulation, we demonstrate the utility of systematic multi-omic profiling by revealing coordinated and distinct changes in chromatin, RNA and surface proteins during native hematopoietic differentiation and peripheral blood mononuclear cell stimulation and as a combinatorial decoder and reporter of multiplexed perturbations in primary T cells.

The lectin-specific activity of Toxoplasma gondii microneme proteins 1 and 4 binds Toll-like receptor 2 and 4 N-glycans to regulate innate immune priming.

Infection of host cells by Toxoplasma gondii is an active process, which is regulated by secretion of microneme (MICs) and rhoptry proteins (ROPs and RONs) from specialized organelles in the apical pole of the parasite. MIC1, MIC4 and MIC6 assemble into an adhesin complex secreted on the parasite surface that functions to promote infection competency. MIC1 and MIC4 are known to bind terminal sialic acid residues and galactose residues, respectively and to induce IL-12 production from splenocytes. Here we show that rMIC1- and rMIC4-stimulated dendritic cells and macrophages produce proinflammatory cytokines, and they do so by engaging TLR2 and TLR4. This process depends on sugar recognition, since point mutations in the carbohydrate-recognition domains (CRD) of rMIC1 and rMIC4 inhibit innate immune cells activation. HEK cells transfected with TLR2 glycomutants were selectively unresponsive to MICs. Following in vitro infection, parasites lacking MIC1 or MIC4, as well as expressing MIC proteins with point mutations in their CRD, failed to induce wild-type (WT) levels of IL-12 secretion by innate immune cells. However, only MIC1 was shown to impact systemic levels of IL-12 and IFN-γ in vivo. Together, our data show that MIC1 and MIC4 interact physically with TLR2 and TLR4 N-glycans to trigger IL-12 responses, and MIC1 is playing a significant role in vivo by altering T. gondii infection competency and murine pathogenesis.

CD14 is critical for TLR2-mediated M1 macrophage activation triggered by N-glycan recognition.

Agonist interaction with Toll-like receptors (TLRs) induces T cell-mediated immunity, which is effective against intracellular pathogens. Consequently, TLR agonists are being tried as immunomodulatory agents. The lectin ArtinM targets TLR2 N-glycans on macrophages, induces cytokines production, and promotes T helper-1 immunity, a process that culminates in resistance to several parasitic and fungal infections in vivo. Because co-receptors influence agonist binding to TLRs, we investigated whether CD14 is required for macrophage activation induced by ArtinM. Macrophages from wild-type mice stimulated by ArtinM not only produced cytokines but also had the following activation profile: (i) expression of M1 polarization markers; (ii) nitrite oxide production; (iii) cellular migration; (iv) enhanced phagocytic and fungicide activity; (v) modulation of TLR2 expression; and (vi) activation of NF-κB pathway. This activation profile induced by ArtinM was evaluated in macrophages lacking CD14 that showed none of the ArtinM effects. We demonstrated by immunoprecipitation and sugar inhibition assays the physical interaction of ArtinM, TLR2, and CD14, which depends on recognition of the trimannoside that constitutes the core of N-glycans. Thus, our study showed that CD14 is critical for ArtinM-induced macrophage activation, providing fundamental insight into the design of anti-infective therapies based on carbohydrate recognition.