Chemokines and Chemokine Receptors in Lymphoid Tissue Dynamics.

The continuous migration of immune cells between lymphoid and nonlymphoid organs is a key feature of the immune system, facilitating the distribution of effector cells within nearly all compartments of the body. Furthermore, reaching their correct position within primary, secondary, or tertiary lymphoid organs is a prerequisite to ensure immune cells' unimpaired differentiation, maturation, and selection, as well as their activation or functional silencing. The superfamilies of chemokines and chemokine receptors are of major importance in guiding immune cells to and within lymphoid and nonlymphoid tissues. In this review we focus on the role of the chemokine system in the migration dynamics of immune cells within lymphoid organs at the steady state and on how these dynamics are affected by infectious and inflammatory processes.

Chemokines and chemokine receptors: positioning cells for host defense and immunity.

Chemokines are chemotactic cytokines that control the migratory patterns and positioning of all immune cells. Although chemokines were initially appreciated as important mediators of acute inflammation, we now know that this complex system of approximately 50 endogenous chemokine ligands and 20 G protein-coupled seven-transmembrane signaling receptors is also critical for the generation of primary and secondary adaptive cellular and humoral immune responses. Recent studies demonstrate important roles for the chemokine system in the priming of naive T cells, in cell fate decisions such as effector and memory cell differentiation, and in regulatory T cell function. In this review, we focus on recent advances in understanding how the chemokine system orchestrates immune cell migration and positioning at the organismic level in homeostasis, in acute inflammation, and during the generation and regulation of adoptive primary and secondary immune responses in the lymphoid system and peripheral nonlymphoid tissue.

Molecular Pathways of Colon Inflammation Induced by Cancer Immunotherapy.

Checkpoint blockade with antibodies specific for the PD-1 and CTLA-4 inhibitory receptors can induce durable responses in a wide range of human cancers. However, the immunological mechanisms responsible for severe inflammatory side effects remain poorly understood. Here we report a comprehensive single-cell analysis of immune cell populations in colitis, a common and severe side effect of checkpoint blockade. We observed a striking accumulation of CD8 T cells with highly cytotoxic and proliferative states and no evidence of regulatory T cell depletion. T cell receptor (TCR) sequence analysis demonstrated that a substantial fraction of colitis-associated CD8 T cells originated from tissue-resident populations, explaining the frequently early onset of colitis symptoms following treatment initiation. Our analysis also identified cytokines, chemokines, and surface receptors that could serve as therapeutic targets for colitis and potentially other inflammatory side effects of checkpoint blockade.

Unleashing Type-2 Dendritic Cells to Drive Protective Antitumor CD4+ T Cell Immunity.

Differentiation of proinflammatory CD4+ conventional T cells (Tconv) is critical for productive antitumor responses yet their elicitation remains poorly understood. We comprehensively characterized myeloid cells in tumor draining lymph nodes (tdLN) of mice and identified two subsets of conventional type-2 dendritic cells (cDC2) that traffic from tumor to tdLN and present tumor-derived antigens to CD4+ Tconv, but then fail to support antitumor CD4+ Tconv differentiation. Regulatory T cell (Treg) depletion enhanced their capacity to elicit strong CD4+ Tconv responses and ensuing antitumor protection. Analogous cDC2 populations were identified in patients, and as in mice, their abundance relative to Treg predicts protective ICOS+ PD-1lo CD4+ Tconv phenotypes and survival. Further, in melanoma patients with low Treg abundance, intratumoral cDC2 density alone correlates with abundant CD4+ Tconv and with responsiveness to anti-PD-1 therapy. Together, this highlights a pathway that restrains cDC2 and whose reversal enhances CD4+ Tconv abundance and controls tumor growth.

CD10+GPR77+ Cancer-Associated Fibroblasts Promote Cancer Formation and Chemoresistance by Sustaining Cancer Stemness.

Carcinoma-associated fibroblasts (CAFs) are abundant and heterogeneous stromal cells in tumor microenvironment that are critically involved in cancer progression. Here, we demonstrate that two cell-surface molecules, CD10 and GPR77, specifically define a CAF subset correlated with chemoresistance and poor survival in multiple cohorts of breast and lung cancer patients. CD10+GPR77+ CAFs promote tumor formation and chemoresistance by providing a survival niche for cancer stem cells (CSCs). Mechanistically, CD10+GPR77+ CAFs are driven by persistent NF-κB activation via p65 phosphorylation and acetylation, which is maintained by complement signaling via GPR77, a C5a receptor. Furthermore, CD10+GPR77+ CAFs promote successful engraftment of patient-derived xenografts (PDXs), and targeting these CAFs with a neutralizing anti-GPR77 antibody abolishes tumor formation and restores tumor chemosensitivity. Our study reveals a functional CAF subset that can be defined and isolated by specific cell-surface markers and suggests that targeting the CD10+GPR77+ CAF subset could be an effective therapeutic strategy against CSC-driven solid tumors.

Graded expression of the chemokine receptor CX3CR1 marks differentiation states of human and murine T cells and enables cross-species interpretation.

T cells differentiate into functionally distinct states upon antigen encounter. These states are delineated by different cell surface markers for murine and human T cells, which hamper cross-species translation of T cell properties. We aimed to identify surface markers that reflect the graded nature of CD8+ T cell differentiation and delineate functionally comparable states in mice and humans. CITEseq analyses revealed that graded expression of CX3CR1, encoding the chemokine receptor CX3CR1, correlated with the CD8+ T cell differentiation gradient. CX3CR1 expression distinguished human and murine CD8+ and CD4+ T cell states, as defined by migratory and functional properties. Graded CX3CR1 expression, refined with CD62L, accurately captured the high-dimensional T cell differentiation continuum. Furthermore, the CX3CR1 expression gradient delineated states with comparable properties in humans and mice in steady state and on longitudinally tracked virus-specific CD8+ T cells in both species. Thus, graded CX3CR1 expression provides a strategy to translate the behavior of distinct T cell differentiation states across species.

Newly recruited intraepithelial Ly6A+CCR9+CD4+ T cells protect against enteric viral infection.

The intestinal epithelium comprises the body's largest surface exposed to viruses. Additionally, the gut epithelium hosts a large population of intraepithelial T lymphocytes, or IELs, although their role in resistance against viral infections remains elusive. By fate-mapping T cells recruited to the murine intestine, we observed an accumulation of newly recruited CD4+ T cells after infection with murine norovirus CR6 and adenovirus type-2 (AdV), but not reovirus. CR6- and AdV-recruited intraepithelial CD4+ T cells co-expressed Ly6A and chemokine receptor CCR9, exhibited T helper 1 and cytotoxic profiles, and conferred protection against AdV in vivo and in an organoid model in an IFN-γ-dependent manner. Ablation of the T cell receptor (TCR) or the transcription factor ThPOK in CD4+ T cells prior to AdV infection prevented viral control, while TCR ablation during infection did not impact viral clearance. These results uncover a protective role for intraepithelial Ly6A+CCR9+CD4+ T cells against enteric adenovirus.

Robust Anti-viral Immunity Requires Multiple Distinct T Cell-Dendritic Cell Interactions.

Host defense against viruses and intracellular parasites depends on effector CD8(+) T cells, whose optimal clonal expansion, differentiation, and memory properties require signals from CD4(+) T cells. Here, we addressed the role of dendritic cell (DC) subsets in initial activation of the two T cell types and their co-operation. Surprisingly, initial priming of CD4(+) and CD8(+) T cells was spatially segregated within the lymph node and occurred on different DCs with temporally distinct patterns of antigen presentation via MHCI versus MHCII molecules. DCs that co-present antigen via both MHC molecules were detected at a later stage; these XCR1(+) DCs are the critical platform involved in CD4(+) T cell augmentation of CD8(+) T cell responses. These findings delineate the complex choreography of cellular interactions underlying effective cell-mediated anti-viral responses, with implications for basic DC subset biology, as well as for translational application to the development of vaccines that evoke optimal T cell immunity.

Atypical chemokine receptor 1 on nucleated erythroid cells regulates hematopoiesis.

Healthy individuals of African ancestry have neutropenia that has been linked with the variant rs2814778(G) of the gene encoding atypical chemokine receptor 1 (ACKR1). This polymorphism selectively abolishes the expression of ACKR1 in erythroid cells, causing a Duffy-negative phenotype. Here we describe an unexpected fundamental role for ACKR1 in hematopoiesis and provide the mechanism that links its absence with neutropenia. Nucleated erythroid cells had high expression of ACKR1, which facilitated their direct contact with hematopoietic stem cells. The absence of erythroid ACKR1 altered mouse hematopoiesis including stem and progenitor cells, which ultimately gave rise to phenotypically distinct neutrophils that readily left the circulation, causing neutropenia. Individuals with a Duffy-negative phenotype developed a distinct profile of neutrophil effector molecules that closely reflected the one observed in the ACKR1-deficient mice. Thus, alternative physiological patterns of hematopoiesis and bone marrow cell outputs depend on the expression of ACKR1 in the erythroid lineage, findings with major implications for the selection advantages that have resulted in the paramount fixation of the ACKR1 rs2814778(G) polymorphism in Africa.

Brown-adipose-tissue macrophages control tissue innervation and homeostatic energy expenditure.

Tissue macrophages provide immunological defense and contribute to the establishment and maintenance of tissue homeostasis. Here we used constitutive and inducible mutagenesis to delete the nuclear transcription regulator Mecp2 in macrophages. Mice that lacked the gene encoding Mecp2, which is associated with Rett syndrome, in macrophages did not show signs of neurodevelopmental disorder but displayed spontaneous obesity, which was linked to impaired function of brown adipose tissue (BAT). Specifically, mutagenesis of a BAT-resident Cx3Cr1+ macrophage subpopulation compromised homeostatic thermogenesis but not acute, cold-induced thermogenesis. Mechanistically, malfunction of BAT in pre-obese mice with mutant macrophages was associated with diminished sympathetic innervation and local titers of norepinephrine, which resulted in lower expression of thermogenic factors by adipocytes. Mutant macrophages overexpressed the signaling receptor and ligand PlexinA4, which might contribute to the phenotype by repulsion of sympathetic axons expressing the transmembrane semaphorin Sema6A. Collectively, we report a previously unappreciated homeostatic role for macrophages in the control of tissue innervation. Disruption of this circuit in BAT resulted in metabolic imbalance.

CXCR1 and CXCR2 Chemokine Receptor Agonists Produced by Tumors Induce Neutrophil Extracellular Traps that Interfere with Immune Cytotoxicity.

Neutrophils are expanded and abundant in cancer-bearing hosts. Under the influence of CXCR1 and CXCR2 chemokine receptor agonists and other chemotactic factors produced by tumors, neutrophils, and granulocytic myeloid-derived suppressor cells (MDSCs) from cancer patients extrude their neutrophil extracellular traps (NETs). In our hands, CXCR1 and CXCR2 agonists proved to be the major mediators of cancer-promoted NETosis. NETs wrap and coat tumor cells and shield them from cytotoxicity, as mediated by CD8+ T cells and natural killer (NK) cells, by obstructing contact between immune cells and the surrounding target cells. Tumor cells protected from cytotoxicity by NETs underlie successful cancer metastases in mice and the immunotherapeutic synergy of protein arginine deiminase 4 (PAD4) inhibitors, which curtail NETosis with immune checkpoint inhibitors. Intravital microscopy provides evidence of neutrophil NETs interfering cytolytic cytotoxic T lymphocytes (CTLs) and NK cell contacts with tumor cells.

High Amount of Transcription Factor IRF8 Engages AP1-IRF Composite Elements in Enhancers to Direct Type 1 Conventional Dendritic Cell Identity.

Development and function of conventional dendritic cell (cDC) subsets, cDC1 and cDC2, depend on transcription factors (TFs) IRF8 and IRF4, respectively. Since IRF8 and IRF4 can each interact with TF BATF3 at AP1-IRF composite elements (AICEs) and with TF PU.1 at Ets-IRF composite elements (EICEs), it is unclear how these factors exert divergent actions. Here, we determined the basis for distinct effects of IRF8 and IRF4 in cDC development. Genes expressed commonly by cDC1 and cDC2 used EICE-dependent enhancers that were redundantly activated by low amounts of either IRF4 or IRF8. By contrast, cDC1-specific genes relied on AICE-dependent enhancers, which required high IRF concentrations, but were activated by either IRF4 or IRF8. IRF8 was specifically required only by a minority of cDC1-specific genes, such as Xcr1, which could distinguish between IRF8 and IRF4 DNA-binding domains. Thus, these results explain how BATF3-dependent Irf8 autoactivation underlies emergence of the cDC1-specific transcriptional program.

Self-renewing resident arterial macrophages arise from embryonic CX3CR1(+) precursors and circulating monocytes immediately after birth.

Resident macrophages densely populate the normal arterial wall, yet their origins and the mechanisms that sustain them are poorly understood. Here we use gene-expression profiling to show that arterial macrophages constitute a distinct population among macrophages. Using multiple fate-mapping approaches, we show that arterial macrophages arise embryonically from CX3CR1(+) precursors and postnatally from bone marrow-derived monocytes that colonize the tissue immediately after birth. In adulthood, proliferation (rather than monocyte recruitment) sustains arterial macrophages in the steady state and after severe depletion following sepsis. After infection, arterial macrophages return rapidly to functional homeostasis. Finally, survival of resident arterial macrophages depends on a CX3CR1-CX3CL1 axis within the vascular niche.

Microglia promote learning-dependent synapse formation through brain-derived neurotrophic factor.

Microglia are the resident macrophages of the CNS, and their functions have been extensively studied in various brain pathologies. The physiological roles of microglia in brain plasticity and function, however, remain unclear. To address this question, we generated CX3CR1(CreER) mice expressing tamoxifen-inducible Cre recombinase that allow for specific manipulation of gene function in microglia. Using CX3CR1(CreER) to drive diphtheria toxin receptor expression in microglia, we found that microglia could be specifically depleted from the brain upon diphtheria toxin administration. Mice depleted of microglia showed deficits in multiple learning tasks and a significant reduction in motor-learning-dependent synapse formation. Furthermore, Cre-dependent removal of brain-derived neurotrophic factor (BDNF) from microglia largely recapitulated the effects of microglia depletion. Microglial BDNF increases neuronal tropomyosin-related kinase receptor B phosphorylation, a key mediator of synaptic plasticity. Together, our findings reveal that microglia serve important physiological functions in learning and memory by promoting learning-related synapse formation through BDNF signaling.

The fractalkine/CX3CR1 system regulates ß cell function and insulin secretion.

Here, we demonstrate that the fractalkine (FKN)/CX3CR1 system represents a regulatory mechanism for pancreatic islet ß cell function and insulin secretion. CX3CR1 knockout (KO) mice exhibited a marked defect in glucose and GLP1-stimulated insulin secretion, and this defect was also observed in vitro in isolated islets from CX3CR1 KO mice. In vivo administration of FKN improved glucose tolerance with an increase in insulin secretion. In vitro treatment of islets with FKN increased intracellular Ca(2+) and potentiated insulin secretion in both mouse and human islets. The KO islets exhibited reduced expression of a set of genes necessary for the fully functional, differentiated ß cell state, whereas treatment of wild-type (WT) islets with FKN led to increased expression of these genes. Lastly, expression of FKN in islets was decreased by aging and high-fat diet/obesity, suggesting that decreased FKN/CX3CR1 signaling could be a mechanism underlying ß cell dysfunction in type 2 diabetes.

Nr4a1-dependent Ly6C(low) monocytes monitor endothelial cells and orchestrate their disposal.

The functions of Nr4a1-dependent Ly6C(low) monocytes remain enigmatic. We show that they are enriched within capillaries and scavenge microparticles from their lumenal side in a steady state. In the kidney cortex, perturbation of homeostasis by a TLR7-dependent nucleic acid "danger" signal, which may signify viral infection or local cell death, triggers Gαi-dependent intravascular retention of Ly6C(low) monocytes by the endothelium. Then, monocytes recruit neutrophils in a TLR7-dependent manner to mediate focal necrosis of endothelial cells, whereas the monocytes remove cellular debris. Prevention of Ly6C(low) monocyte development, crawling, or retention in Nr4a1(-/-), Itgal(-/-), and Tlr7(host-/-BM+/+) and Cx3cr1(-/-) mice, respectively, abolished neutrophil recruitment and endothelial killing. Prevention of neutrophil recruitment in Tlr7(host+/+BM-/-) mice or by neutrophil depletion also abolished endothelial cell necrosis. Therefore, Ly6C(low) monocytes are intravascular housekeepers that orchestrate the necrosis by neutrophils of endothelial cells that signal a local threat sensed via TLR7 followed by the in situ phagocytosis of cellular debris.

The people behind the papers - Ayumi Fukuoka and Gerry Graham.

Chemokines are perhaps best known for their role in immune responses, but they can also regulate cell migration during embryonic development. A new paper in Development shows that an atypical chemokine receptor sequesters maternal CXCL12 in the placenta to prevent it from entering the embryonic bloodstream, thus ensuring proper haematopoiesis in the embryo. To learn more about the story behind the paper, we caught up with first author Ayumi Fukuoka and corresponding author Gerry Graham, the Gardiner Chair of Immunology at the University of Glasgow, UK.

Two-Factor Authentication for Type 2 Immunity.

The initiation of type 2 responses is tightly regulated. In this issue of Immunity, Sokol et al. (2018) demonstrate that CCL8 is a critical signal that licenses dendritic cells to enter the lymph node parenchyma and induce Th2 differentiation after allergen exposure.

Structural insights into KSHV-GPCR constitutive activation and CXCL1 chemokine recognition.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a viral G protein-coupled receptor, KSHV-GPCR, that contributes to KSHV immune evasion and pathogenesis of Kaposi's sarcoma. KSHV-GPCR shares a high similarity with CXC chemokine receptors CXCR2 and can be activated by selected chemokine ligands. Like other herpesvirus-encoded GPCRs, KSHV-GPCR is characterized by its constitutive activity by coupling to various G proteins. We investigated the structural basis of ligand-dependent and constitutive activation of KSHV-GPCR, obtaining high-resolution cryo-EM structures of KSHV-GPCR-Gi complexes with and without the bound CXCL1 chemokine. Analysis of the apo-KSHV-GPCR-Gi structure (2.81 Å) unraveled the involvement of extracellular loop 2 in constitutive activation of the receptor. In comparison, the CXCL1-bound KSHV-GPCR-Gi structure (3.01 Å) showed a two-site binding mode and provided detailed information of CXCL1 binding to a chemokine receptor. The dual activation mechanism employed by KSHV-GPCR represents an evolutionary adaptation for immune evasion and contributes to the pathogenesis of Kaposi's sarcoma. Together with results from functional assays that confirmed the structural models, these findings may help to develop therapeutic strategies for KSHV infection.

Constant replenishment from circulating monocytes maintains the macrophage pool in the intestine of adult mice.

The paradigm that macrophages that reside in steady-state tissues are derived from embryonic precursors has never been investigated in the intestine, which contains the largest pool of macrophages. Using fate-mapping models and monocytopenic mice, together with bone marrow chimera and parabiotic models, we found that embryonic precursor cells seeded the intestinal mucosa and demonstrated extensive in situ proliferation during the neonatal period. However, these cells did not persist in the intestine of adult mice. Instead, they were replaced around the time of weaning by the chemokine receptor CCR2-dependent influx of Ly6C(hi) monocytes that differentiated locally into mature, anti-inflammatory macrophages. This process was driven largely by the microbiota and had to be continued throughout adult life to maintain a normal intestinal macrophage pool.