Nucleotide modifications enable rational design of TLR7-selective ligands by blocking RNase cleavage.

Toll-like receptors 7 (TLR7) and 8 (TLR8) each sense single-stranded RNA (ssRNA), but their activation results in different immune activation profiles. Attempts to selectively target either TLR7 or TLR8 have been hindered by their high degree of homology. However, recent studies revealed that TLR7 and TLR8 bind different ligands resulting from the processing of ssRNA by endolysosomal RNases. We demonstrate that by introducing precise 2' sugar-modified bases into oligoribonucleotides (ORNs) containing known TLR7 and TLR8 binding motifs, we could prevent RNase-mediated degradation into the monomeric uridine required for TLR8 activation while preserving TLR7 activation. Furthermore, a novel, optimized protocol for CRISPR-Cas9 knockout in primary human plasmacytoid dendritic cells showed that TLR7 activation is dependent on RNase processing of ORNs and revealed a previously undescribed role for RNase 6 in degrading ORNs into TLR ligands. Finally, 2' sugar-modified ORNs demonstrated robust innate immune activation in mice. Altogether, we identified a strategy for creating tunable TLR7-selective agonists.

Transitional dendritic cells are distinct from conventional DC2 precursors and mediate proinflammatory antiviral responses.

High-dimensional approaches have revealed heterogeneity amongst dendritic cells (DCs), including a population of transitional DCs (tDCs) in mice and humans. However, the origin and relationship of tDCs to other DC subsets has been unclear. Here we show that tDCs are distinct from other well-characterized DCs and conventional DC precursors (pre-cDCs). We demonstrate that tDCs originate from bone marrow progenitors shared with plasmacytoid DCs (pDCs). In the periphery, tDCs contribute to the pool of ESAM+ type 2 DCs (DC2s), and these DC2s have pDC-related developmental features. Different from pre-cDCs, tDCs have less turnover, capture antigen, respond to stimuli and activate antigen-specific naïve T cells, all characteristics of differentiated DCs. Different from pDCs, viral sensing by tDCs results in IL-1ß secretion and fatal immune pathology in a murine coronavirus model. Our findings suggest that tDCs are a distinct pDC-related subset with a DC2 differentiation potential and unique proinflammatory function during viral infections.

Landscape of coordinated immune responses to H1N1 challenge in humans.

Influenza is a significant cause of morbidity and mortality worldwide. Here we show changes in the abundance and activation states of more than 50 immune cell subsets in 35 individuals over 11 time points during human A/California/2009 (H1N1) virus challenge monitored using mass cytometry along with other clinical assessments. Peak change in monocyte, B cell, and T cell subset frequencies coincided with peak virus shedding, followed by marked activation of T and NK cells. Results led to the identification of CD38 as a critical regulator of plasmacytoid dendritic cell function in response to influenza virus. Machine learning using study-derived clinical parameters and single-cell data effectively classified and predicted susceptibility to infection. The coordinated immune cell dynamics defined in this study provide a framework for identifying novel correlates of protection in the evaluation of future influenza therapeutics.

Chromatin Landscape Underpinning Human Dendritic Cell Heterogeneity.

Human dendritic cells (DCs) comprise subsets with distinct phenotypic and functional characteristics, but the transcriptional programs that dictate their identity remain elusive. Here, we analyze global chromatin accessibility profiles across resting and stimulated human DC subsets by means of the assay for transposase-accessible chromatin using sequencing (ATAC-seq). We uncover specific regions of chromatin accessibility for each subset and transcriptional regulators of DC function. By comparing plasmacytoid DC responses to IFN-I-producing and non-IFN-I-producing conditions, we identify genetic programs related to their function. Finally, by intersecting chromatin accessibility with genome-wide association studies, we recognize DC subset-specific enrichment of heritability in autoimmune diseases. Our results unravel the basis of human DC subset heterogeneity and provide a framework for their analysis in disease pathogenesis.

Integrated Cross-Species Analysis Identifies a Conserved Transitional Dendritic Cell Population.

Plasmacytoid dendritic cells (pDCs) are sensor cells with diverse immune functions, from type I interferon (IFN-I) production to antigen presentation, T cell activation, and tolerance. Regulation of these functions remains poorly understood but could be mediated by functionally specialized pDC subpopulations. We address pDC diversity using a high-dimensional single-cell approach: mass cytometry (CyTOF). Our analysis uncovers a murine pDC-like population that specializes in antigen presentation with limited capacity for IFN-I production. Using a multifaceted cross-species comparison, we show that this pDC-like population is the definitive murine equivalent of the recently described human AXL+ DCs, which we unify under the name transitional DCs (tDCs) given their continuum of pDC and cDC2 characteristics. tDCs share developmental traits with pDCs, as well as recruitment dynamics during viral infection. Altogether, we provide a framework for deciphering the function of pDCs and tDCs during diseases, which has the potential to open new avenues for therapeutic design.

The versatile plasmacytoid dendritic cell: Function, heterogeneity, and plasticity.

Since their identification as the natural interferon-producing cell two decades ago, plasmacytoid dendritic cells (pDCs) have been attributed diverse functions in the immune response. Their most well characterized function is innate, i.e., their rapid and robust production of type-I interferon (IFN-I) in response to viruses. However, pDCs have also been implicated in antigen presentation, activation of adaptive immune responses and immunoregulation. The mechanisms by which pDCs enact these diverse functions are poorly understood. One central debate is whether these functions are carried out by different pDC subpopulations or by plasticity in the pDC compartment. This chapter summarizes the latest reports regarding pDC function, heterogeneity, cell conversion and environmentally influenced plasticity, as well as the role of pDCs in infection, autoimmunity and cancer.

High-Dimensional Phenotypic Mapping of Human Dendritic Cells Reveals Interindividual Variation and Tissue Specialization.

Given the limited efficacy of clinical approaches that rely on ex vivo generated dendritic cells (DCs), it is imperative to design strategies that harness specialized DC subsets in situ. This requires delineating the expression of surface markers by DC subsets among individuals and tissues. Here, we performed a multiparametric phenotypic characterization and unbiased analysis of human DC subsets in blood, tonsil, spleen, and skin. We uncovered previously unreported phenotypic heterogeneity of human cDC2s among individuals, including variable expression of functional receptors such as CD172a. We found marked differences in DC subsets localized in blood and lymphoid tissues versus skin, and a striking absence of the newly discovered Axl+ DCs in the skin. Finally, we evaluated the capacity of anti-receptor monoclonal antibodies to deliver vaccine components to skin DC subsets. These results offer a promising path for developing DC subset-specific immunotherapies that cannot be provided by transcriptomic analysis alone.

Actin-related protein2/3 complex regulates tight junctions and terminal differentiation to promote epidermal barrier formation.

The epidermis provides an essential seal from the external environment and retains fluids within the body. To form an effective barrier, cells in the epidermis must form tight junctions and terminally differentiate into cornified envelopes. Here, we demonstrate that the branched actin nucleator, the actin-related protein (Arp)2/3 complex, is unexpectedly required for both these activities. Loss of the ArpC3 subunit of the Arp2/3 complex resulted in minimal changes in the morphogenesis and architecture of this stratified squamous epithelium, but resulted in profound defects in its physiology. Mutant embryos did not develop an effective barrier to the external environment and died within hours of birth. We discovered two underlying causes for these effects. First, ArpC3 was essential for robust assembly and function of tight junctions, specialized cell-cell adhesions that restrict water loss in the epidermis. Second, there were defects in differentiation of the epidermis and the production of cornified envelopes, structures essential for barrier activity. Underlying this defect, we found that YAP was inappropriately active not only in the ArpC3 mutant tissue, but also in cultured cells. Inhibition of YAP activity rescued the differentiation and barrier defects caused by loss of ArpC3. These results demonstrate previously unappreciated roles for the Arp2/3 complex and highlight the functions of branched actin networks in a complex tissue.