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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 proteomes 1 . Despite their therapeutic potential 2 , our map of these surface interactions remains incomplete 3 , 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. Systematic measurements of the interactions between proteins found on the surfaces of human leukocytes provides a global view of the way that immune cells are dynamically connected by receptors.

The progenitor stage of commitment toward the conventional dendritic cell subsets and the transcriptional networks that control it remain poorly understood. Two articles from Ginhoux and colleagues and Murphy and colleagues offer insight into these processes. Mouse conventional dendritic cells (cDCs) can be classified into two functionally distinct lineages: the CD8α + (CD103 + ) cDC1 lineage, and the CD11b + cDC2 lineage. cDCs arise from a cascade of bone marrow (BM) DC-committed progenitor cells that include the common DC progenitors (CDPs) and pre-DCs, which exit the BM and seed peripheral tissues before differentiating locally into mature cDCs. Where and when commitment to the cDC1 or cDC2 lineage occurs remains poorly understood. Here we found that transcriptional signatures of the cDC1 and cDC2 lineages became evident at the single-cell level from the CDP stage. We also identified Siglec-H and Ly6C as lineage markers that distinguished pre-DC subpopulations committed to the cDC1 lineage (Siglec-H − Ly6C − pre-DCs) or cDC2 lineage (Siglec-H − Ly6C + pre-DCs). Our results indicate that commitment to the cDC1 or cDC2 lineage occurs in the BM and not in the periphery.

An adeno-associated virus (AAV) receptor protein essential for AAV2 entry into cells is identified; AAV receptor binds directly to the virus, and its ablation renders a diverse range of mammalian cell types and mice resistant to infection by AAV of multiple serotypes. A receptor for adeno-associated virus infection The recent revival of interest in gene therapy has been fueled by the availability of safer and more effective viral gene delivery methods, most notably adeno-associated virus (AAV) vectors. Jan Carette and colleagues now identify a protein that is essential for AAV entry into cells, subsequent to cell attachment. This protein, which they call AAVR, rapidly traffics from the plasma membrane to the trans -Golgi network. The authors show that the virus directly binds to AAVR and that genetic ablation of AAVR renders a diverse range of mammalian cell types and mice resistant to AAV infection. Adeno-associated virus (AAV) vectors are currently the leading candidates for virus-based gene therapies because of their broad tissue tropism, non-pathogenic nature and low immunogenicity 1 . They have been successfully used in clinical trials to treat hereditary diseases such as haemophilia B (ref. 2 ), and have been approved for treatment of lipoprotein lipase deficiency in Europe 3 . Considerable efforts have been made to engineer AAV variants with novel and biomedically valuable cell tropisms to allow efficacious systemic administration 1 , 4 , yet basic aspects of AAV cellular entry are still poorly understood. In particular, the protein receptor(s) required for AAV entry after cell attachment remains unknown. Here we use an unbiased genetic screen to identify proteins essential for AAV serotype 2 (AAV2) infection in a haploid human cell line. The most significantly enriched gene of the screen encodes a previously uncharacterized type I transmembrane protein, KIAA0319L (denoted hereafter as AAV receptor (AAVR)). We characterize AAVR as a protein capable of rapid endocytosis from the plasma membrane and trafficking to the trans -Golgi network. We show that AAVR directly binds to AAV2 particles, and that anti-AAVR antibodies efficiently block AAV2 infection. Moreover, genetic ablation of AAVR renders a wide range of mammalian cell types highly resistant to AAV2 infection. Notably, AAVR serves as a critical host factor for all tested AAV serotypes. The importance of AAVR for in vivo gene delivery is further highlighted by the robust resistance of Aavr −/− (also known as Au040320 −/− and Kiaa0319l −/− ) mice to AAV infection. Collectively, our data indicate that AAVR is a universal receptor involved in AAV infection.

Mesenchymal stem cells (MSC) hold great potential for regenerative medicine because of their ability for self‐renewal and differentiation into tissue‐specific cells such as osteoblasts, chondrocytes, and adipocytes. MSCs orchestrate tissue development, maintenance and repair, and are useful for musculoskeletal regenerative therapies to treat age‐related orthopedic degenerative diseases and other clinical conditions. Importantly, MSCs produce secretory factors that play critical roles in tissue repair that support both engraftment and trophic functions (autocrine and paracrine). The development of uniform protocols for both preparation and characterization of MSCs, including standardized functional assays for evaluation of their biological potential, are critical factors contributing to their clinical utility. Quality control and release criteria for MSCs should include cell surface markers, differentiation potential, and other essential cell parameters. For example, cell surface marker profiles (surfactome), bone‐forming capacities in ectopic and orthotopic models, as well as cell size and granularity, telomere length, senescence status, trophic factor secretion (secretome), and immunomodulation, should be thoroughly assessed to predict MSC utility for regenerative medicine. We propose that these and other functionalities of MSCs should be characterized prior to use in clinical applications as part of comprehensive and uniform guidelines and release criteria for their clinical‐grade production to achieve predictably favorable treatment outcomes for stem cell therapy. Stem Cells Translational Medicine 2017;6:2173–2185 Adopting a multifaceted approach for characterizing mesenchymal stem cells (MSCs) is critical for the selection of best‐in‐class cells for therapeutic use. Assaying for surfactome, secretome, self‐renewal, colony formation, trophic factor secretion, and multilineage differentiation, all form part of an assessment of cellular naivety or “stemness.” When this status is combined with functional assays such as immunomodulation, and other key parameters of cellular health that include telomere length, they collectively help provide a robust assessment of characteristics that are increasingly becoming important indicators of clinical efficacy.

Plants deploy cell-surface and intracellular leucine rich-repeat domain (LRR) immune receptors to detect pathogens 1 . LRR receptor kinases and LRR receptor proteins at the plasma membrane recognize microorganism-derived molecules to elicit pattern-triggered immunity (PTI), whereas nucleotide-binding LRR proteins detect microbial effectors inside cells to confer effector-triggered immunity (ETI). Although PTI and ETI are initiated in different host cell compartments, they rely on the transcriptional activation of similar sets of genes 2 , suggesting pathway convergence upstream of nuclear events. Here we report that PTI triggered by the Arabidopsis LRR receptor protein RLP23 requires signalling-competent dimers of the lipase-like proteins EDS1 and PAD4, and of ADR1 family helper nucleotide-binding LRRs, which are all components of ETI. The cell-surface LRR receptor kinase SOBIR1 links RLP23 with EDS1, PAD4 and ADR1 proteins, suggesting the formation of supramolecular complexes containing PTI receptors and transducers at the inner side of the plasma membrane. We detected similar evolutionary patterns in LRR receptor protein and nucleotide-binding LRR genes across Arabidopsis accessions; overall higher levels of variation in LRR receptor proteins than in LRR receptor kinases are consistent with distinct roles of these two receptor families in plant immunity. We propose that the EDS1–PAD4–ADR1 node is a convergence point for defence signalling cascades, activated by both surface-resident and intracellular LRR receptors, in conferring pathogen immunity. The authors provide mechanistic insights into the crosstalk between signalling components of pattern-triggered immunity and effector-triggered immunity and their molecular linkers.

Adiponectin plays a central role as an antidiabetic and antiatherogenic adipokine. AdipoR1 and AdipoR2 serve as receptors for adiponectin in vitro , and their reduction in obesity seems to be correlated with reduced adiponectin sensitivity. Here we show that adenovirus-mediated expression of AdipoR1 and R2 in the liver of Lepr −/− mice increased AMP-activated protein kinase (AMPK) activation and peroxisome proliferator-activated receptor (PPAR)-α signaling pathways, respectively. Activation of AMPK reduced gluconeogenesis, whereas expression of the receptors in both cases increased fatty acid oxidation and lead to an amelioration of diabetes. Alternatively, targeted disruption of AdipoR1 resulted in the abrogation of adiponectin-induced AMPK activation, whereas that of AdipoR2 resulted in decreased activity of PPAR-α signaling pathways. Simultaneous disruption of both AdipoR1 and R2 abolished adiponectin binding and actions, resulting in increased tissue triglyceride content, inflammation and oxidative stress, and thus leading to insulin resistance and marked glucose intolerance. Therefore, AdipoR1 and R2 serve as the predominant receptors for adiponectin in vivo and play important roles in the regulation of glucose and lipid metabolism, inflammation and oxidative stress in vivo .

Ovarian cancer and triple-negative breast cancer are among the most lethal diseases affecting women, with few targeted therapies and high rates of metastasis. Cancer cells are capable of evading clearance by macrophages through the overexpression of anti-phagocytic surface proteins called ‘don’t eat me’ signals—including CD47 1 , programmed cell death ligand 1 (PD-L1) 2 and the beta-2 microglobulin subunit of the major histocompatibility class I complex (B2M) 3 . Monoclonal antibodies that antagonize the interaction of ‘don’t eat me’ signals with their macrophage-expressed receptors have demonstrated therapeutic potential in several cancers 4 , 5 . However, variability in the magnitude and durability of the response to these agents has suggested the presence of additional, as yet unknown ‘don’t eat me’ signals. Here we show that CD24 can be the dominant innate immune checkpoint in ovarian cancer and breast cancer, and is a promising target for cancer immunotherapy. We demonstrate a role for tumour-expressed CD24 in promoting immune evasion through its interaction with the inhibitory receptor sialic-acid-binding Ig-like lectin 10 (Siglec-10), which is expressed by tumour-associated macrophages. We find that many tumours overexpress CD24 and that tumour-associated macrophages express high levels of Siglec-10. Genetic ablation of either CD24 or Siglec-10, as well as blockade of the CD24–Siglec-10 interaction using monoclonal antibodies, robustly augment the phagocytosis of all CD24-expressing human tumours that we tested. Genetic ablation and therapeutic blockade of CD24 resulted in a macrophage-dependent reduction of tumour growth in vivo and an increase in survival time. These data reveal CD24 as a highly expressed, anti-phagocytic signal in several cancers and demonstrate the therapeutic potential for CD24 blockade in cancer immunotherapy. CD24 interacts with the tumour-associated-macrophage receptor Siglec-10 to inhibit the macrophage-mediated clearance of cancer cells, revealing a new ‘don’t eat me’ signal as a potential target for cancer immunotherapy.

Plant endogenous peptides are crucial for diverse aspects of plant physiology. Among them, C-TERMINALLY ENCODED PEPTIDEs (CEPs) have recently emerged as important regulators of plant growth and stress responses. CEPs are divided into two major subgroups: group I CEPs and the less studied group II CEPs. We recently demonstrated that group I CEPs coordinate cell surface receptor-mediated immunity with nitrogen status in Arabidopsis thaliana (hereafter Arabidopsis). To mount full group I CEP responsiveness, the three phylogenetically related CEP RECEPTOR 1 (CEPR1), CEPR2 and RECEPTOR-LIKE KINASE 7 (RLK7) are required. Here, we provide evidence that biotic stress induces expression of the group II CEP peptide CEP14 . CEP14 and the related CEP13 and CEP15 trigger hallmark immune signalling outputs in a proline hydroxylation pattern-dependent manner in Arabidopsis. Genetic data indicate that group II CEP members contribute to cell surface receptor-mediated immunity against bacterial infection. We further show that group II CEP perception primarily depends on CEPR2. Our work provides new insights into CEP function during biotic stress and sheds new light on the complexity of sequence-divergent CEP signalling mediated by specific endogenous receptors.

Immune checkpoint blockade therapy has been successful in treating some types of cancer but has not shown clinical benefits for treating leukaemia 1 . This result suggests that leukaemia uses unique mechanisms to evade this therapy. Certain immune inhibitory receptors that are expressed by normal immune cells are also present on leukaemia cells. Whether these receptors can initiate immune-related primary signalling in tumour cells remains unknown. Here we use mouse models and human cells to show that LILRB4, an immunoreceptor tyrosine-based inhibition motif-containing receptor and a marker of monocytic leukaemia, supports tumour cell infiltration into tissues and suppresses T cell activity via a signalling pathway that involves APOE, LILRB4, SHP-2, uPAR and ARG1 in acute myeloid leukaemia (AML) cells. Deletion of LILRB4 or the use of antibodies to block LILRB4 signalling impeded AML development. Thus, LILRB4 orchestrates tumour invasion pathways in monocytic leukaemia cells by creating an immunosuppressive microenvironment. LILRB4 represents a compelling target for the treatment of monocytic AML. The receptor LILRB4 on monocytic leukaemia cells suppresses T cell activity and support the infiltration of tumour cells into tissues.

Cell–cell communication mediated by ligand–receptor complexes is critical to coordinating diverse biological processes, such as development, differentiation and inflammation. To investigate how the context-dependent crosstalk of different cell types enables physiological processes to proceed, we developed CellPhoneDB, a novel repository of ligands, receptors and their interactions. In contrast to other repositories, our database takes into account the subunit architecture of both ligands and receptors, representing heteromeric complexes accurately. We integrated our resource with a statistical framework that predicts enriched cellular interactions between two cell types from single-cell transcriptomics data. Here, we outline the structure and content of our repository, provide procedures for inferring cell–cell communication networks from single-cell RNA sequencing data and present a practical step-by-step guide to help implement the protocol. CellPhoneDB v.2.0 is an updated version of our resource that incorporates additional functionalities to enable users to introduce new interacting molecules and reduces the time and resources needed to interrogate large datasets. CellPhoneDB v.2.0 is publicly available, both as code and as a user-friendly web interface; it can be used by both experts and researchers with little experience in computational genomics. In our protocol, we demonstrate how to evaluate meaningful biological interactions with CellPhoneDB v.2.0 using published datasets. This protocol typically takes ~2 h to complete, from installation to statistical analysis and visualization, for a dataset of ~10 GB, 10,000 cells and 19 cell types, and using five threads. CellPhoneDB combines an interactive database and a statistical framework for the exploration of ligand–receptor interactions inferred from single-cell transcriptomics measurements.