A CD22-Shp1 phosphatase axis controls integrin ß7 display and B cell function in mucosal immunity.

The integrin α4ß7 selectively regulates lymphocyte trafficking and adhesion in the gut and gut-associated lymphoid tissue (GALT). Here, we describe unexpected involvement of the tyrosine phosphatase Shp1 and the B cell lectin CD22 (Siglec-2) in the regulation of α4ß7 surface expression and gut immunity. Shp1 selectively inhibited ß7 endocytosis, enhancing surface α4ß7 display and lymphocyte homing to GALT. In B cells, CD22 associated in a sialic acid-dependent manner with integrin ß7 on the cell surface to target intracellular Shp1 to ß7. Shp1 restrained plasma membrane ß7 phosphorylation and inhibited ß7 endocytosis without affecting ß1 integrin. B cells with reduced Shp1 activity, lacking CD22 or expressing CD22 with mutated Shp1-binding or carbohydrate-binding domains displayed parallel reductions in surface α4ß7 and in homing to GALT. Consistent with the specialized role of α4ß7 in intestinal immunity, CD22 deficiency selectively inhibited intestinal antibody and pathogen responses.

Single-Cell Survey of Human Lymphatics Unveils Marked Endothelial Cell Heterogeneity and Mechanisms of Homing for Neutrophils.

Lymphatic vessels form a critical component in the regulation of human health and disease. While their functional significance is increasingly being recognized, the comprehensive heterogeneity of lymphatics remains uncharacterized. Here, we report the profiling of 33,000 lymphatic endothelial cells (LECs) in human lymph nodes (LNs) by single-cell RNA sequencing. Unbiased clustering revealed six major types of human LECs. LECs lining the subcapsular sinus (SCS) of LNs abundantly expressed neutrophil chemoattractants, whereas LECs lining the medullary sinus (MS) expressed a C-type lectin CD209. Binding of a carbohydrate Lewis X (CD15) to CD209 mediated neutrophil binding to the MS. The neutrophil-selective homing by MS LECs may retain neutrophils in the LN medulla and allow lymph-borne pathogens to clear, preventing their spread through LNs in humans. Our study provides a comprehensive characterization of LEC heterogeneity and unveils a previously undefined role for medullary LECs in human immunity.

Role and species-specific expression of colon T cell homing receptor GPR15 in colitis.

Lymphocyte recruitment maintains intestinal immune homeostasis but also contributes to inflammation. The orphan chemoattractant receptor GPR15 mediates regulatory T cell homing and immunosuppression in the mouse colon. We show that GPR15 is also expressed by mouse TH17 and TH1 effector cells and is required for colitis in a model that depends on the trafficking of these cells to the colon. In humans GPR15 is expressed by effector cells, including pathogenic TH2 cells in ulcerative colitis, but is expressed poorly or not at all by colon regulatory T (Treg) cells. The TH2 transcriptional activator GATA-3 and the Treg-associated transcriptional repressor FOXP3 robustly bind human, but not mouse, GPR15 enhancer sequences, correlating with receptor expression. Our results highlight species differences in GPR15 regulation and suggest it as a potential therapeutic target for colitis.

Transcriptional programs of lymphoid tissue capillary and high endothelium reveal control mechanisms for lymphocyte homing.

Lymphocytes are recruited from blood by high-endothelial venules (HEVs). We performed transcriptomic analyses and identified molecular signatures that distinguish HEVs from capillary endothelium and that define tissue-specific HEV specialization. Capillaries expressed gene programs for vascular development. HEV-expressed genes showed enrichment for genes encoding molecules involved in immunological defense and lymphocyte migration. We identify capillary and HEV markers and candidate mechanisms for regulated recruitment of lymphocytes, including a lymph node HEV-selective transmembrane mucin; transcriptional control of functionally specialized carbohydrate ligands for lymphocyte L-selectin; HEV expression of molecules for transendothelial migration; and metabolic programs for lipid mediators of lymphocyte motility and chemotaxis. We also elucidate a carbohydrate-recognition pathway that targets B cells to intestinal lymphoid tissues, defining CD22 as a lectin-homing receptor for mucosal HEVs.

Comparative transcriptional and functional profiling defines conserved programs of intestinal DC differentiation in humans and mice.

Dendritic cells (DCs) that orchestrate mucosal immunity have been studied in mice. Here we characterized human gut DC populations and defined their relationship to previously studied human and mouse DCs. CD103(+)Sirpα(-) DCs were related to human blood CD141(+) DCs and to mouse intestinal CD103(+)CD11b(-) DCs and expressed markers of cross-presenting DCs. CD103(+)Sirpα(+) DCs aligned with human blood CD1c(+) DCs and mouse intestinal CD103(+)CD11b(+) DCs and supported the induction of regulatory T cells. Both CD103(+) DC subsets induced the TH17 subset of helper T cells, while CD103(-)Sirpα(+) DCs induced the TH1 subset of helper T cells. Comparative analysis of transcriptomes revealed conserved transcriptional programs among CD103(+) DC subsets and identified a selective role for the transcriptional repressors Bcl-6 and Blimp-1 in the specification of CD103(+)CD11b(-) DCs and intestinal CD103(+)CD11b(+) DCs, respectively. Our results highlight evolutionarily conserved and divergent programming of intestinal DCs.

Development of follicular dendritic cells in lymph nodes depends on retinoic acid-mediated signaling.

Specialized stromal cells occupy and help define B- and T-cell domains, which are crucial for proper functioning of our immune system. Signaling through lymphotoxin and TNF receptors is crucial for the development of different stromal subsets, which are thought to arise from a common precursor. However, mechanisms that control the selective generation of the different stromal phenotypes are not known. Using in vitro cultures of embryonic mouse stromal cells, we show that retinoic acid-mediated signaling is important for the differentiation of precursors towards the Cxcl13pos follicular dendritic cell (FDC) lineage, and also blocks lymphotoxin-mediated Ccl19pos fibroblastic reticular cell lineage differentiation. Accordingly, at the day of birth we observe the presence of Cxcl13posCcl19neg/low and Cxcl13neg/lowCcl19pos cells within neonatal lymph nodes. Furthermore, ablation of retinoic acid receptor signaling in stromal precursors early after birth reduces Cxcl13 expression, and complete blockade of retinoic acid signaling prevents the formation of FDC networks in lymph nodes.

Single-cell analysis of early progenitor cells that build coronary arteries.

Arteries and veins are specified by antagonistic transcriptional programs. However, during development and regeneration, new arteries can arise from pre-existing veins through a poorly understood process of cell fate conversion. Here, using single-cell RNA sequencing and mouse genetics, we show that vein cells of the developing heart undergo an early cell fate switch to create a pre-artery population that subsequently builds coronary arteries. Vein cells underwent a gradual and simultaneous switch from venous to arterial fate before a subset of cells crossed a transcriptional threshold into the pre-artery state. Before the onset of coronary blood flow, pre-artery cells appeared in the immature vessel plexus, expressed mature artery markers, and decreased cell cycling. The vein-specifying transcription factor COUP-TF2 (also known as NR2F2) prevented plexus cells from overcoming the pre-artery threshold by inducing cell cycle genes. Thus, vein-derived coronary arteries are built by pre-artery cells that can differentiate independently of blood flow upon the release of inhibition mediated by COUP-TF2 and cell cycle factors.

Chemerin triggers migration of a CD8 T cell subset with natural killer cell functions.

The recruitment of cells with effector functions into the tumor microenvironment holds potential for delaying cancer progression. We show that subsets of human CD28-effector CD8 T cells, CCR7- CD45RO+ effector memory, and CCR7- CD45RO- effector memory RA phenotypes, express the chemerin receptor CMKLR1 and bind chemerin via the receptor. CMKLR1-expressing human CD8 effector memory T cells present gene, protein, and cytotoxic features of NK cells. Active chemerin promotes chemotaxis of CMKLR1-expressing CD8 effector memory cells and triggers activation of the α4ß1 integrin. In an experimental prostate tumor mouse model, chemerin expression is downregulated in the tumor microenvironment, which is associated with few tumor-infiltrating CD8+ T cells, while forced overexpression of chemerin by mouse prostate cancer cells leads to an accumulation of intra-tumor CD8+ T cells. Furthermore, α4 integrin blockade abrogated the chemerin-dependent recruitment of CD8+ T effector memory cells into implanted prostate tumors in vivo. The results identify a role for chemerin:CMKLR1 in defining a specialized NK-like CD8 T cell, and suggest the use of chemerin-dependent modalities to target effector CMKLR1-expressing T cells to the tumor microenvironment for immunotherapeutic purposes.

Single-Cell Analysis of Blood-Brain Barrier Response to Pericyte Loss.

RATIONALE: Pericytes are capillary mural cells playing a role in stabilizing newly formed blood vessels during development and tissue repair. Loss of pericytes has been described in several brain disorders, and genetically induced pericyte deficiency in the brain leads to increased macromolecular leakage across the blood-brain barrier (BBB). However, the molecular details of the endothelial response to pericyte deficiency remain elusive. OBJECTIVE: To map the transcriptional changes in brain endothelial cells resulting from lack of pericyte contact at single-cell level and to correlate them with regional heterogeneities in BBB function and vascular phenotype. METHODS AND RESULTS: We reveal transcriptional, morphological, and functional consequences of pericyte absence for brain endothelial cells using a combination of methodologies, including single-cell RNA sequencing, tracer analyses, and immunofluorescent detection of protein expression in pericyte-deficient adult Pdgfbret/ret mice. We find that endothelial cells without pericyte contact retain a general BBB-specific gene expression profile, however, they acquire a venous-shifted molecular pattern and become transformed regarding the expression of numerous growth factors and regulatory proteins. Adult Pdgfbret/ret brains display ongoing angiogenic sprouting without concomitant cell proliferation providing unique insights into the endothelial tip cell transcriptome. We also reveal heterogeneous modes of pericyte-deficient BBB impairment, where hotspot leakage sites display arteriolar-shifted identity and pinpoint putative BBB regulators. By testing the causal involvement of some of these using reverse genetics, we uncover a reinforcing role for angiopoietin 2 at the BBB. CONCLUSIONS: By elucidating the complexity of endothelial response to pericyte deficiency at cellular resolution, our study provides insight into the importance of brain pericytes for endothelial arterio-venous zonation, angiogenic quiescence, and a limited set of BBB functions. The BBB-reinforcing role of ANGPT2 (angiopoietin 2) is paradoxical given its wider role as TIE2 (TEK receptor tyrosine kinase) receptor antagonist and may suggest a unique and context-dependent function of ANGPT2 in the brain.

Neutrophils Recirculate through Lymph Nodes to Survey Tissues for Pathogens.

The adaptive immune function of lymph nodes is dependent on constant recirculation of lymphocytes. In this article, we identify neutrophils present in the lymph node at steady state, exhibiting the same capacity for recirculation. In germ-free mice, neutrophils still recirculate through lymph nodes, and in mice cohoused with wild microbiome mice, the level of neutrophils in lymph nodes increases significantly. We found that at steady state, neutrophils enter the lymph node entirely via L-selectin and actively exit via efferent lymphatics via an S1P dependent mechanism. The small population of neutrophils in the lymph node can act as reconnaissance cells to recruit additional neutrophils in the event of bacterial dissemination to the lymph node. Without these reconnaissance cells, there is a delay in neutrophil recruitment to the lymph node and a reduction in swarm formation following Staphylococcus aureus infection. This ability to recruit additional neutrophils by lymph node neutrophils is initiated by LTB4. This study establishes the capacity of neutrophils to recirculate, much like lymphocytes via L-selectin and high endothelial venules in lymph nodes and demonstrates how the presence of neutrophils at steady state fortifies the lymph node in case of an infection disseminating through lymphatics.

DGAT1 inhibits retinol-dependent regulatory T cell formation and mediates autoimmune encephalomyelitis.

The balance of effector versus regulatory T cells (Tregs) controls inflammation in numerous settings, including multiple sclerosis (MS). Here we show that memory phenotype CD4+ T cells infiltrating the central nervous system during experimental autoimmune encephalomyelitis (EAE), a widely studied animal model of MS, expressed high levels of mRNA for Dgat1 encoding diacylglycerol-O-acyltransferase-1 (DGAT1), an enzyme that catalyzes triglyceride synthesis and retinyl ester formation. DGAT1 inhibition or deficiency attenuated EAE, with associated enhanced Treg frequency; and encephalitogenic, DGAT1-/- in vitro-polarized Th17 cells were poor inducers of EAE in adoptive recipients. DGAT1 acyltransferase activity sequesters retinol in ester form, preventing synthesis of retinoic acid, a cofactor for Treg generation. In cultures with T cell-depleted lymphoid tissues, retinol enhanced Treg induction from DGAT1-/- but not from WT T cells. The WT Treg induction defect was reversed by DGAT1 inhibition. These results demonstrate that DGAT1 suppresses retinol-dependent Treg formation and suggest its potential as a therapeutic target for autoimmune inflammation.

Retinoic Acid and Lymphotoxin Signaling Promote Differentiation of Human Intestinal M Cells.

BACKGROUND & AIMS: Intestinal microfold (M) cells are a unique subset of intestinal epithelial cells in the Peyer's patches that regulate mucosal immunity, serving as portals for sampling and uptake of luminal antigens. The inability to efficiently develop human M cells in cell culture has impeded studies of the intestinal immune system. We aimed to identify signaling pathways required for differentiation of human M cells and establish a robust culture system using human ileum enteroids. METHODS: We analyzed transcriptome data from mouse Peyer's patches to identify cell populations in close proximity to M cells. We used the human enteroid system to determine which cytokines were required to induce M-cell differentiation. We performed transcriptome, immunofluorescence, scanning electron microscope, and transcytosis experiments to validate the development of phenotypic and functional human M cells. RESULTS: A combination of retinoic acid and lymphotoxin induced differentiation of glycoprotein 2-positive human M cells, which lack apical microvilli structure. Upregulated expression of innate immune-related genes within M cells correlated with a lack of viral antigens after rotavirus infection. Human M cells, developed in the enteroid system, internalized and transported enteric viruses, such as rotavirus and reovirus, across the intestinal epithelium barrier in the enteroids. CONCLUSIONS: We identified signaling pathways required for differentiation of intestinal M cells, and used this information to create a robust culture method to develop human M cells with capacity for internalization and transport of viruses. Studies of this model might increase our understanding of antigen presentation and the systemic entry of enteric pathogens in the human intestine.

Plasmacytoid dendritic cells transport peripheral antigens to the thymus to promote central tolerance.

Central tolerance can be mediated by peripheral dendritic cells (DCs) that transport innocuous antigens (Ags) to the thymus for presentation to developing T cells, but the responsible DC subsets remained poorly defined. Immature plasmacytoid DCs (pDCs) express CCR9, a chemokine receptor involved in migration of T cell precursors to the thymus. We show here that CCR9 mediated efficient thymic entry of endogenous or i.v. transfused pDCs. pDCs activated by Toll-like receptor (TLR) ligands downregulated CCR9 and lost their ability to home to the thymus. Moreover, endogenous pDCs took up subcutaneously injected fluorescent Ag and, in the absence of TLR signals, transported Ag to the thymus in a CCR9-dependent fashion. Injected, Ag-loaded pDCs effectively deleted Ag-specific thymocytes, and this thymic clonal deletion required CCR9-mediated homing and was prevented by infectious signals. Thus, peripheral pDCs can contribute to immune tolerance through CCR9-dependent transport of peripheral Ags and subsequent deletion of Ag-reactive thymocytes.

Endothelial HIF-2α as a Key Endogenous Mediator Preventing Emphysema.

Rationale: Endothelial injury may provoke emphysema, but molecular pathways of disease development require further discernment. Emphysematous lungs exhibit decreased expression of HIF-2α (hypoxia-inducible factor-2α)-regulated genes, and tobacco smoke decreases pulmonary HIF-2α concentrations. These findings suggest that decreased HIF-2α expression is important in the development of emphysema.Objectives: The objective of this study was to evaluate the roles of endothelial-cell (EC) HIF-2α in the pathogenesis of emphysema in mice.Methods: Mouse lungs were examined for emphysema after either the loss or the overexpression of EC Hif-2α. In addition, SU5416, a VEGFR2 inhibitor, was used to induce emphysema. Lungs were evaluated for HGF (hepatocyte growth factor), a protein involved in alveolar development and homeostasis. Lungs from patients with emphysema were measured for endothelial HIF-2α expression.Measurements and Main Results: EC Hif-2α deletion resulted in emphysema in association with fewer ECs and pericytes. After SU5416 exposure, EC Hif-2α-knockout mice developed more severe emphysema, whereas EC Hif-2α-overexpressing mice were protected. EC Hif-2α-knockout mice demonstrated lower levels of HGF. Human emphysema lung samples exhibited reduced EC HIF-2α expression.Conclusions: Here, we demonstrate a unique protective role for pulmonary endothelial HIF-2α and how decreased expression of this endogenous factor causes emphysema; its pivotal protective function is suggested by its ability to overcome VEGF antagonism. HIF-2α may maintain alveolar architecture by promoting vascular survival and associated HGF production. In summary, HIF-2α may be a key endogenous factor that prevents the development of emphysema, and its upregulation has the potential to foster lung health in at-risk patients.

Neutrophils recruited through high endothelial venules of the lymph nodes via PNAd intercept disseminating Staphylococcus aureus.

Staphylococcus aureus is a skin- and respiratory tract-colonizing bacterium and is the leading cause of community-acquired skin infections. Dissemination of these bacteria into systemic circulation causes bacteremia, which has a high mortality rate. Therefore, understanding the immunologic barriers that prevent dissemination is critical to developing novel treatments. In this study, we demonstrate that an S. aureus breach across skin leads to some migration of the pathogen to the draining lymph node, but no further. While subcapsular sinus (SCS) macrophage in lymph nodes were important in detaining S. aureus, a rapid complement-dependent neutrophil recruitment (independent of the SCS macrophage) via high endothelial venules (HEVs) resulted in high numbers of neutrophils that intercepted the bacteria in the lymph nodes. Peripheral Node Addressin together with its two ligands, L-selectin and platelet P-selectin, are critical for recruiting neutrophils via the HEVs. Almost no neutrophils entered the lymph nodes via lymphatics. Neutrophils actively phagocytosed S. aureus and helped sterilize the lymph nodes and prevent dissemination to blood and other organs.

Endothelial Hypoxia-Inducible Factor-2α Is Required for the Maintenance of Airway Microvasculature.

BACKGROUND: Hypoxia-inducible factors (HIFs), especially HIF-1α and HIF-2α, are key mediators of the adaptive response to hypoxic stress and play essential roles in maintaining lung homeostasis. Human and animal genetics studies confirm that abnormal HIF correlates with pulmonary vascular pathology and chronic lung diseases, but it remains unclear whether endothelial cell HIF production is essential for microvascular health. The large airway has an ideal circulatory bed for evaluating histological changes and physiology in genetically modified rodents. METHODS: The tracheal microvasculature of mice, with conditionally deleted or overexpressed HIF-1α or HIF-2α, was evaluated for anatomy, perfusion, and permeability. Angiogenic signaling studies assessed vascular changes attributable to dysregulated HIF expression. An orthotopic tracheal transplantation model further evaluated the contribution of individual HIF isoforms in airway endothelial cells. RESULTS: The genetic deletion of Hif-2α but not Hif-1α caused tracheal endothelial cell apoptosis, diminished pericyte coverage, reduced vascular perfusion, defective barrier function, overlying epithelial abnormalities, and subepithelial fibrotic remodeling. HIF-2α promoted microvascular integrity in airways through endothelial angiopoietin-1/TIE2 signaling and Notch activity. In functional tracheal transplants, HIF-2α deficiency in airway donors accelerated graft microvascular loss, whereas HIF-2α or angiopoietin-1 overexpression prolonged transplant microvascular perfusion. Augmented endothelial HIF-2α in transplant donors promoted airway microvascular integrity and diminished alloimmune inflammation. CONCLUSIONS: Our findings reveal that the constitutive expression of endothelial HIF-2α is required for airway microvascular health.

Environmental cues, dendritic cells and the programming of tissue-selective lymphocyte trafficking.

Lymphocytes are imprinted during activation with trafficking programs (combinations of adhesion and chemoattractant receptors) that target their migration to specific tissues and microenvironments. Cytokines contribute, but, for gut and skin, evolution has cleverly adapted external cues from food (vitamin A) and sunlight (ultraviolet-induced vitamin D3) to imprint lymphocyte homing to the small intestines and T cell migration into the epidermis. Dendritic cells are essential: they process the vitamins to their active metabolites (retinoic acid and 1,25(OH)(2)D3) for presentation with antigen to lymphocytes, and they help export environmental cues through lymphatics to draining lymph nodes, to program the trafficking and effector functions of naive T and B cells.

CCR9 expression defines tolerogenic plasmacytoid dendritic cells able to suppress acute graft-versus-host disease.

Dendritic cells (DCs) are 'professional' antigen-presenting cells that are key in the regulation of immune responses. Here we characterize a unique subset of tolerogenic DCs that expressed the chemokine receptor CCR9 and migrated to the CCR9 ligand CCL25, a chemokine linked to the homing of T cells and DCs to the gut. CCR9(+) DCs were of the plasmacytoid DC (pDC) lineage, had an immature phenotype and rapidly downregulated CCR9 in response to maturation-inducing pDC-restricted Toll-like receptor ligands. CCR9(+) pDCs were potent inducers of regulatory T cell function and suppressed antigen-specific immune responses both in vitro and in vivo, including inhibiting acute graft-versus-host disease induced by allogeneic CD4(+) donor T cells in irradiated recipients. Our results identify a highly immunosuppressive population of pDCs present in lymphoid tissues.

Human CD22 Inhibits Murine B Cell Receptor Activation in a Human CD22 Transgenic Mouse Model.

CD22, a sialic acid-binding Ig-type lectin (Siglec) family member, is an inhibitory coreceptor of the BCR with established roles in health and disease. The restricted expression pattern of CD22 on B cells and most B cell lymphomas has made CD22 a therapeutic target for B cell-mediated diseases. Models to better understand how in vivo targeting of CD22 translates to human disease are needed. In this article, we report the development of a transgenic mouse expressing human CD22 (hCD22) in B cells and assess its ability to functionally substitute for murine CD22 (mCD22) for regulation of BCR signaling, Ab responses, homing, and tolerance. Expression of hCD22 on transgenic murine B cells is comparable to expression on human primary B cells, and it colocalizes with mCD22 on the cell surface. Murine B cells expressing only hCD22 have identical calcium (Ca2+) flux responses to anti-IgM as mCD22-expressing wild-type B cells. Furthermore, hCD22 transgenic mice on an mCD22-/- background have restored levels of marginal zone B cells and Ab responses compared with deficiencies observed in CD22-/- mice. Consistent with these observations, hCD22 transgenic mice develop normal humoral responses in a peanut allergy oral sensitization model. Homing of B cells to Peyer's patches was partially rescued by expression of hCD22 compared with CD22-/- B cells, although not to wild-type levels. Notably, Siglec-engaging antigenic liposomes formulated with an hCD22 ligand were shown to prevent B cell activation, increase cell death, and induce tolerance in vivo. This hCD22 transgenic mouse will be a valuable model for investigating the function of hCD22 and preclinical studies targeting hCD22.

A Chimeric Antibody against ACKR3/CXCR7 in Combination with TMZ Activates Immune Responses and Extends Survival in Mouse GBM Models.

Glioblastoma (GBM) is the least treatable type of brain tumor, afflicting over 15,000 people per year in the United States. Patients have a median survival of 16 months, and over 95% die within 5 years. The chemokine receptor ACKR3 is selectively expressed on both GBM cells and tumor-associated blood vessels. High tumor expression of ACKR3 correlates with poor prognosis and potential treatment resistance, making it an attractive therapeutic target. We engineered a single chain FV-human FC-immunoglobulin G1 (IgG1) antibody, X7Ab, to target ACKR3 in human and mouse GBM cells. We used hydrodynamic gene transfer to overexpress the antibody, with efficacy in vivo. X7Ab kills GBM tumor cells and ACKR3-expressing vascular endothelial cells by engaging the cytotoxic activity of natural killer (NK) cells and complement and the phagocytic activity of macrophages. Combining X7Ab with TMZ allows the TMZ dosage to be lowered, without compromising therapeutic efficacy. Mice treated with X7Ab and in combination with TMZ showed significant tumor reduction by MRI and longer survival overall. Brain-tumor-infiltrating leukocyte analysis revealed that X7Ab enhances the activation of M1 macrophages to support anti-tumor immune response in vivo. Targeting ACKR3 with immunotherapeutic monoclonal antibodies (mAbs) in combination with standard of care therapies may prove effective in treating GBM.