TREM2 Modulation Remodels the Tumor Myeloid Landscape Enhancing Anti-PD-1 Immunotherapy.

Checkpoint immunotherapy unleashes T cell control of tumors, but is undermined by immunosuppressive myeloid cells. TREM2 is a myeloid receptor that transmits intracellular signals that sustain microglial responses during Alzheimer's disease. TREM2 is also expressed by tumor-infiltrating macrophages. Here, we found that Trem2-/- mice are more resistant to growth of various cancers than wild-type mice and are more responsive to anti-PD-1 immunotherapy. Furthermore, treatment with anti-TREM2 mAb curbed tumor growth and fostered regression when combined with anti-PD-1. scRNA-seq revealed that both TREM2 deletion and anti-TREM2 are associated with scant MRC1+ and CX3CR1+ macrophages in the tumor infiltrate, paralleled by expansion of myeloid subsets expressing immunostimulatory molecules that promote improved T cell responses. TREM2 was expressed in tumor macrophages in over 200 human cancer cases and inversely correlated with prolonged survival for two types of cancer. Thus, TREM2 might be targeted to modify tumor myeloid infiltrates and augment checkpoint immunotherapy.

CNS-Native Myeloid Cells Drive Immune Suppression in the Brain Metastatic Niche through Cxcl10.

Brain metastasis (br-met) develops in an immunologically unique br-met niche. Central nervous system-native myeloid cells (CNS-myeloids) and bone-marrow-derived myeloid cells (BMDMs) cooperatively regulate brain immunity. The phenotypic heterogeneity and specific roles of these myeloid subsets in shaping the br-met niche to regulate br-met outgrowth have not been fully revealed. Applying multimodal single-cell analyses, we elucidated a heterogeneous but spatially defined CNS-myeloid response during br-met outgrowth. We found Ccr2+ BMDMs minimally influenced br-met while CNS-myeloid promoted br-met outgrowth. Additionally, br-met-associated CNS-myeloid exhibited downregulation of Cx3cr1. Cx3cr1 knockout in CNS-myeloid increased br-met incidence, leading to an enriched interferon response signature and Cxcl10 upregulation. Significantly, neutralization of Cxcl10 reduced br-met, while rCxcl10 increased br-met and recruited VISTAHi PD-L1+ CNS-myeloid to br-met lesions. Inhibiting VISTA- and PD-L1-signaling relieved immune suppression and reduced br-met burden. Our results demonstrate that loss of Cx3cr1 in CNS-myeloid triggers a Cxcl10-mediated vicious cycle, cultivating a br-met-promoting, immune-suppressive niche.

Liver Immune Profiling Reveals Pathogenesis and Therapeutics for Biliary Atresia.

Biliary atresia (BA) is a severe cholangiopathy that leads to liver failure in infants, but its pathogenesis remains to be fully characterized. By single-cell RNA profiling, we observed macrophage hypo-inflammation, Kupffer cell scavenger function defects, cytotoxic T cell expansion, and deficiency of CX3CR1+effector T and natural killer (NK) cells in infants with BA. More importantly, we discovered that hepatic B cell lymphopoiesis did not cease after birth and that tolerance defects contributed to immunoglobulin G (IgG)-autoantibody accumulation in BA. In a rhesus-rotavirus induced BA model, depleting B cells or blocking antigen presentation ameliorated liver damage. In a pilot clinical study, we demonstrated that rituximab was effective in depleting hepatic B cells and restoring the functions of macrophages, Kupffer cells, and T cells to levels comparable to those of control subjects. In summary, our comprehensive immune profiling in infants with BA had educed that B-cell-modifying therapies may alleviate liver pathology.

BATF regulates progenitor to cytolytic effector CD8+ T cell transition during chronic viral infection.

During chronic viral infection, CD8+ T cells develop into three major phenotypically and functionally distinct subsets: Ly108+TCF-1+ progenitors, Ly108-CX3CR1- terminally exhausted cells and the recently identified CX3CR1+ cytotoxic effector cells. Nevertheless, how CX3CR1+ effector cell differentiation is transcriptionally and epigenetically regulated remains elusive. Here, we identify distinct gene regulatory networks and epigenetic landscapes underpinning the formation of these subsets. Notably, our data demonstrate that CX3CR1+ effector cells bear a striking similarity to short-lived effector cells during acute infection. Genetic deletion of Tbx21 significantly diminished formation of the CX3CR1+ subset. Importantly, we further identify a previously unappreciated role for the transcription factor BATF in maintaining a permissive chromatin structure that allows the transition from TCF-1+ progenitors to CX3CR1+ effector cells. BATF directly bound to regulatory regions near Tbx21 and Klf2, modulating their enhancer accessibility to facilitate the transition. These mechanistic insights can potentially be harnessed to overcome T cell exhaustion during chronic infection and cancer.

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.

Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage.

Tissue fibrosis is a major cause of mortality that results from the deposition of matrix proteins by an activated mesenchyme. Macrophages accumulate in fibrosis, but the role of specific subgroups in supporting fibrogenesis has not been investigated in vivo. Here, we used single-cell RNA sequencing (scRNA-seq) to characterize the heterogeneity of macrophages in bleomycin-induced lung fibrosis in mice. A novel computational framework for the annotation of scRNA-seq by reference to bulk transcriptomes (SingleR) enabled the subclustering of macrophages and revealed a disease-associated subgroup with a transitional gene expression profile intermediate between monocyte-derived and alveolar macrophages. These CX3CR1+SiglecF+ transitional macrophages localized to the fibrotic niche and had a profibrotic effect in vivo. Human orthologs of genes expressed by the transitional macrophages were upregulated in samples from patients with idiopathic pulmonary fibrosis. Thus, we have identified a pathological subgroup of transitional macrophages that are required for the fibrotic response to injury.

Identification of a novel arthritis-associated osteoclast precursor macrophage regulated by FoxM1.

Osteoclasts have a unique bone-destroying capacity, playing key roles in steady-state bone remodeling and arthritic bone erosion. Whether the osteoclasts in these different tissue settings arise from the same precursor states of monocytoid cells is presently unknown. Here, we show that osteoclasts in pannus originate exclusively from circulating bone marrow-derived cells and not from locally resident macrophages. We identify murine CX3CR1hiLy6CintF4/80+I-A+/I-E+ macrophages (termed here arthritis-associated osteoclastogenic macrophages (AtoMs)) as the osteoclast precursor-containing population in the inflamed synovium, comprising a subset distinct from conventional osteoclast precursors in homeostatic bone remodeling. Tamoxifen-inducible Foxm1 deletion suppressed the capacity of AtoMs to differentiate into osteoclasts in vitro and in vivo. Furthermore, synovial samples from human patients with rheumatoid arthritis contained CX3CR1+HLA-DRhiCD11c+CD80-CD86+ cells that corresponded to mouse AtoMs, and human osteoclastogenesis was inhibited by the FoxM1 inhibitor thiostrepton, constituting a potential target for rheumatoid arthritis treatment.

Self-renewing resident cardiac macrophages limit adverse remodeling following myocardial infarction.

Macrophages promote both injury and repair after myocardial infarction, but discriminating functions within mixed populations remains challenging. Here we used fate mapping, parabiosis and single-cell transcriptomics to demonstrate that at steady state, TIMD4+LYVE1+MHC-IIloCCR2- resident cardiac macrophages self-renew with negligible blood monocyte input. Monocytes partially replaced resident TIMD4-LYVE1-MHC-IIhiCCR2- macrophages and fully replaced TIMD4-LYVE1-MHC-IIhiCCR2+ macrophages, revealing a hierarchy of monocyte contribution to functionally distinct macrophage subsets. Ischemic injury reduced TIMD4+ and TIMD4- resident macrophage abundance, whereas CCR2+ monocyte-derived macrophages adopted multiple cell fates within infarcted tissue, including those nearly indistinguishable from resident macrophages. Recruited macrophages did not express TIMD4, highlighting the ability of TIMD4 to track a subset of resident macrophages in the absence of fate mapping. Despite this similarity, inducible depletion of resident macrophages using a Cx3cr1-based system led to impaired cardiac function and promoted adverse remodeling primarily within the peri-infarct zone, revealing a nonredundant, cardioprotective role of resident cardiac macrophages.

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.

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.

Proliferating Transitory T Cells with an Effector-like Transcriptional Signature Emerge from PD-1+ Stem-like CD8+ T Cells during Chronic Infection.

T cell dysfunction is a characteristic feature of chronic viral infection and cancer. Recent studies in chronic lymphocytic choriomeningitis virus (LCMV) infection have defined a PD-1+ Tcf-1+ CD8+ T cell subset capable of self-renewal and differentiation into more terminally differentiated cells that downregulate Tcf-1 and express additional inhibitory molecules such as Tim3. Here, we demonstrated that expression of the glycoprotein CD101 divides this terminally differentiated population into two subsets. Stem-like Tcf-1+ CD8+ T cells initially differentiated into a transitory population of CD101-Tim3+ cells that later converted into CD101+ Tim3+ cells. Recently generated CD101-Tim3+ cells proliferated in vivo, contributed to viral control, and were marked by an effector-like transcriptional signature including expression of the chemokine receptor CX3CR1, pro-inflammatory cytokines, and granzyme B. PD-1 pathway blockade increased the numbers of CD101-Tim3+ CD8+ T cells, suggesting that these newly generated transitional cells play a critical role in PD-1-based immunotherapy.

A Subset of Skin Macrophages Contributes to the Surveillance and Regeneration of Local Nerves.

The skin comprises tissue macrophages as the most abundant resident immune cell type. Their diverse tasks including resistance against invading pathogens, attraction of bypassing immune cells from vessels, and tissue repair require dynamic specification. Here, we delineated the postnatal development of dermal macrophages and their differentiation into subsets by adapting single-cell transcriptomics, fate mapping, and imaging. Thereby we identified a phenotypically and transcriptionally distinct subset of prenatally seeded dermal macrophages that self-maintained with very low postnatal exchange by hematopoietic stem cells. These macrophages specifically interacted with sensory nerves and surveilled and trimmed the myelin sheath. Overall, resident dermal macrophages contributed to axon sprouting after mechanical injury. In summary, our data show long-lasting functional specification of macrophages in the dermis that is driven by stepwise adaptation to guiding structures and ensures codevelopment of ontogenetically distinct cells within the same compartment.

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.

Macrophages of multiple hematopoietic origins reside in the developing prostate.

Tissue-resident macrophages contribute to the organogenesis of many tissues. Growth of the prostate is regulated by androgens during puberty, yet androgens are considered immune suppressive. In this study, we characterized the localization, androgen receptor expression and hematopoietic origin of prostate macrophages, and transiently ablated macrophages during postnatal prostate organogenesis in the mouse. We show that myeloid cells were abundant in the prostate during puberty. However, nuclear androgen receptor expression was not detected in most macrophages. We found Cx3cr1, a marker for macrophages, monocytes and dendritic cells, expressed in interstitial macrophages surrounding the prostate and associated with nerve fibers. Furthermore, we provide evidence for the co-existence of embryonic origin, self-renewing, tissue-resident macrophages and recruited macrophages of bone-marrow monocyte origin in the prostate during puberty. Our findings suggest that prostate macrophages promote neural patterning and may shed further light on our understanding of the role of the innate immune system in prostate pathology in response to inflammation and in cancer.

Treg Cells: A LAGging Hand Holds the Double-Edged Sword of the IL-23 Axis.

Intestinal Treg cells suppress colitis; yet the mechanisms behind the intricate pathways involved in this process remain largely unknown. In this issue of Immunity,Bauché et al. (2018) show that Treg cells engage MHCII on CX3CR1+ macrophages via LAG3. This indirectly reduces IL-22 mediated colonic inflammation.

Critical Role for the Microbiota in CX3CR1+ Intestinal Mononuclear Phagocyte Regulation of Intestinal T Cell Responses.

The intestinal barrier is vulnerable to damage by microbiota-induced inflammation that is normally restrained through mechanisms promoting homeostasis. Such disruptions contribute to autoimmune and inflammatory diseases including inflammatory bowel disease. We identified a regulatory loop whereby, in the presence of the normal microbiota, intestinal antigen-presenting cells (APCs) expressing the chemokine receptor CX3CR1 reduced expansion of intestinal microbe-specific T helper 1 (Th1) cells and promoted generation of regulatory T cells responsive to food antigens and the microbiota itself. We identified that disruption of the microbiota resulted in CX3CR1+ APC-dependent inflammatory Th1 cell responses with increased pathology after pathogen infection. Colonization with microbes that can adhere to the epithelium was able to compensate for intestinal microbiota loss, indicating that although microbial interactions with the epithelium can be pathogenic, they can also activate homeostatic regulatory mechanisms. Our results identify a cellular mechanism by which the microbiota limits intestinal inflammation and promotes tissue homeostasis.

LAG3+ Regulatory T Cells Restrain Interleukin-23-Producing CX3CR1+ Gut-Resident Macrophages during Group 3 Innate Lymphoid Cell-Driven Colitis.

Interleukin-22 (IL-22)-producing group 3 innate lymphoid cells (ILC3) maintains gut homeostasis but can also promote inflammatory bowel disease (IBD). The regulation of ILC3-dependent colitis remains to be elucidated. Here we show that Foxp3+ regulatory T cells (Treg cells) prevented ILC3-mediated colitis in an IL-10-independent manner. Treg cells inhibited IL-23 and IL-1ß production from intestinal-resident CX3CR1+ macrophages but not CD103+ dendritic cells. Moreover, Treg cells restrained ILC3 production of IL-22 through suppression of CX3CR1+ macrophage production of IL-23 and IL-1ß. This suppression was contact dependent and was mediated by latent activation gene-3 (LAG-3)-an immune checkpoint receptor-expressed on Treg cells. Engagement of LAG-3 on MHC class II drove profound immunosuppression of CX3CR1+ tissue-resident macrophages. Our study reveals that the health of the intestinal mucosa is maintained by an axis driven by Treg cells communication with resident macrophages that withhold inflammatory stimuli required for ILC3 function.

TRPV4 Channel Signaling in Macrophages Promotes Gastrointestinal Motility via Direct Effects on Smooth Muscle Cells.

Intestinal macrophages are critical for gastrointestinal (GI) homeostasis, but our understanding of their role in regulating intestinal motility is incomplete. Here, we report that CX3C chemokine receptor 1-expressing muscularis macrophages (MMs) were required to maintain normal GI motility. MMs expressed the transient receptor potential vanilloid 4 (TRPV4) channel, which senses thermal, mechanical, and chemical cues. Selective pharmacologic inhibition of TRPV4 or conditional deletion of TRPV4 from macrophages decreased intestinal motility and was sufficient to reverse the GI hypermotility that is associated with chemotherapy treatment. Mechanistically, stimulation of MMs via TRPV4 promoted the release of prostaglandin E2 and elicited colon contraction in a paracrine manner via prostaglandin E receptor signaling in intestinal smooth muscle cells without input from the enteric nervous system. Collectively, our data identify TRPV4-expressing MMs as an essential component required for maintaining normal GI motility and provide potential drug targets for GI motility disorders.

Thymic development of gut-microbiota-specific T cells.

Humans and their microbiota have coevolved a mutually beneficial relationship in which the human host provides a hospitable environment for the microorganisms and the microbiota provides many advantages for the host, including nutritional benefits and protection from pathogen infection1. Maintaining this relationship requires a careful immune balance to contain commensal microorganisms within the lumen while limiting inflammatory anti-commensal responses1,2. Antigen-specific recognition of intestinal microorganisms by T cells has previously been described3,4. Although the local environment shapes the differentiation of effector cells3-5 it is unclear how microbiota-specific T cells are educated in the thymus. Here we show that intestinal colonization in early life leads to the trafficking of microbial antigens from the intestine to the thymus by intestinal dendritic cells, which then induce the expansion of microbiota-specific T cells. Once in the periphery, microbiota-specific T cells have pathogenic potential or can protect against related pathogens. In this way, the developing microbiota shapes and expands the thymic and peripheral T cell repertoire, allowing for enhanced recognition of intestinal microorganisms and pathogens.