Co-inhibition of TGF-ß and PD-L1 pathways in a metastatic colorectal cancer mouse model triggers interferon responses, innate cells and T cells, alongside metabolic changes and tumor resistance.

Colorectal cancer (CRC) is the third most prevalent cancer worldwide with a high mortality rate (20-30%), especially due to metastasis to adjacent organs. Clinical responses to chemotherapy, radiation, targeted and immunotherapies are limited to a subset of patients making metastatic CRC (mCRC) difficult to treat. To understand the therapeutic modulation of immune response in mCRC, we have used a genetically engineered mouse model (GEMM), "KPN", which resembles the human 'CMS4'-like subtype. We show here that transforming growth factor (TGF-ß1), secreted by KPN organoids, increases cancer cell proliferation, and inhibits splenocyte activation in vitro. TGF-ß1 also inhibits activation of naive but not pre-activated T cells, suggesting differential effects on specific immune cells. In vivo, the inhibition of TGF-ß inflames the KPN tumors, causing infiltration of T cells, monocytes and monocytic intermediates, while reducing neutrophils and epithelial cells. Co-inhibition of TGF-ß and PD-L1 signaling further enhances cytotoxic CD8+T cells and upregulates innate immune response and interferon gene signatures. However, simultaneous upregulation of cancer-related metabolic genes correlated with limited control of tumor burden and/or progression despite combination treatment. Our study illustrates the importance of using GEMMs to predict better immunotherapies for mCRC.

The small and large intestine contain related mesenchymal subsets that derive from embryonic Gli1+ precursors.

The intestinal lamina propria contains a diverse network of fibroblasts that provide key support functions to cells within their local environment. Despite this, our understanding of the diversity, location and ontogeny of fibroblasts within and along the length of the intestine remains incomplete. Here we show that the small and large intestinal lamina propria contain similar fibroblast subsets that locate in specific anatomical niches. Nevertheless, we find that the transcriptional profile of similar fibroblast subsets differs markedly between the small intestine and colon suggesting region specific functions. We perform in vivo transplantation and lineage-tracing experiments to demonstrate that adult intestinal fibroblast subsets, smooth muscle cells and pericytes derive from Gli1-expressing precursors present in embryonic day 12.5 intestine. Trajectory analysis of single cell RNA-seq datasets of E12.5 and adult mesenchymal cells suggest that adult smooth muscle cells and fibroblasts derive from distinct embryonic intermediates and that adult fibroblast subsets develop in a linear trajectory from CD81+ fibroblasts. Finally, we provide evidence that colonic subepithelial PDGFRαhi fibroblasts comprise several functionally distinct populations that originate from an Fgfr2-expressing fibroblast intermediate. Our results provide insights into intestinal stromal cell diversity, location, function, and ontogeny, with implications for intestinal development and homeostasis.

The IκB-protein BCL-3 controls Toll-like receptor-induced MAPK activity by promoting TPL-2 degradation in the nucleus.

Proinflammatory responses induced by Toll-like receptors (TLRs) are dependent on the activation of the NF-ĸB and mitogen-activated protein kinase (MAPK) pathways, which coordinate the transcription and synthesis of proinflammatory cytokines. We demonstrate that BCL-3, a nuclear IĸB protein that regulates NF-ĸB, also controls TLR-induced MAPK activity by regulating the stability of the TPL-2 kinase. TPL-2 is essential for MAPK activation by TLR ligands, and the rapid proteasomal degradation of active TPL-2 is a critical mechanism limiting TLR-induced MAPK activity. We reveal that TPL-2 is a nucleocytoplasmic shuttling protein and identify the nucleus as the primary site for TPL-2 degradation. BCL-3 interacts with TPL-2 and promotes its degradation by promoting its nuclear localization. As a consequence, Bcl3-/- macrophages have increased TPL-2 stability following TLR stimulation, leading to increased MAPK activity and MAPK-dependent responses. Moreover, BCL-3-mediated regulation of TPL-2 stability sets the MAPK activation threshold and determines the amount of TLR ligand required to initiate the production of inflammatory cytokines. Thus, the nucleus is a key site in the regulation of TLR-induced MAPK activity. BCL-3 links control of the MAPK and NF-ĸB pathways in the nucleus, and BCL-3-mediated TPL-2 regulation impacts on the cellular decision to initiate proinflammatory cytokine production in response to TLR activation.

Can molecular stratification improve the treatment of inflammatory bowel disease?

Inflammatory bowel disease (IBD) is a debilitating chronic inflammatory disease of the gastrointestinal (GI) tract. It affects more than 3.5 million people in the western world and places a huge financial burden on healthcare systems. IBD is highly heterogeneous; disease severity and outcomes in IBD are highly variable, and patients may experience episodes of relapse and remission. However, treatment often follows a step-up model whereby the patients start with anti-inflammatory agents (corticosteroids or immunosuppressants) and step-up to monoclonal anti-tumour necrosis factor-α (TNFα) antibodies and then other biologics if the initial drugs cannot control disease. Unfortunately, many patients do not respond to the costly biologics, and thus often still require gut-resective surgery, which decreases quality of life. In order to decrease rates of surgery and ineffective treatments, it is important to identify markers that accurately predict disease progression and treatment responses, to inform decisions about the best choice of therapeutics. Here we examine molecular approaches to patient stratification that aim to increase the effectiveness of treatments and potentially reduce healthcare costs. In the future, it may become possible to stratify patients based on their suitability for specific molecular-targeted therapeutic agents, and eventually use molecular stratification for personalised medicine in IBD.

Understanding and overcoming the resistance of cancer to PD-1/PD-L1 blockade.

Greater understanding of tumour immunobiology has led to a new era of cancer treatment in which immuno-oncology (IO) therapies are used to boost anti-cancer immune responses. Prominent among these therapies are immune checkpoint inhibitors (ICIs), antibody-based drugs that can unleash the power of tumour-specific CD8 + T-cells. ICIs targeting the Programmed cell death protein 1 (PD-1) cell surface receptor or its ligand PD-L1 are particularly effective, with clinical studies reporting powerful and durable therapeutic impact against many cancer types, including melanoma and non-small cell lung cancer. ICIs have the potential to transform the landscape of cancer treatment, and their development was recognised by the award of the 2018 Nobel Prize in Physiology or Medicine to James Allison and Tasuku Honjo. However, the proportion of patients responding to anti-PD-1/PD-L1 monotherapy can be low. The next major challenge involves understanding and overcoming the innate and acquired resistance that prevents most patients from responding to PD-1/PD-L1 blockade. In this review, we outline the physiological function of PD-1 and its exploitation by developing tumours. We give an overview of current FDA-approved drugs targeting PD-1 or PD-L1 and summarise clinical progress so far. We then discuss key mechanisms thought to underpin resistance to PD-1/PD-L1 blockade, describing biomarkers that could allow patient responses to be predicted before treatment, and tracked once treatment has started. We also present clinical and pre-clinical combination therapies that have been developed to overcome resistance and which have the potential to substantially extend the therapeutic reach of these revolutionary drugs.

The Atypical Chemokine Receptor Ackr2 Constrains NK Cell Migratory Activity and Promotes Metastasis.

Chemokines have been shown to be essential players in a range of cancer contexts. In this study, we demonstrate that mice deficient in the atypical chemokine receptor Ackr2 display impaired development of metastasis in vivo in both cell line and spontaneous models. Further analysis reveals that this relates to increased expression of the chemokine receptor CCR2, specifically by KLRG1+ NK cells from the Ackr2-/- mice. This leads to increased recruitment of KLRG1+ NK cells to CCL2-expressing tumors and enhanced tumor killing. Together, these data indicate that Ackr2 limits the expression of CCR2 on NK cells and restricts their tumoricidal activity. Our data have important implications for our understanding of the roles for chemokines in the metastatic process and highlight Ackr2 and CCR2 as potentially manipulable therapeutic targets in metastasis.

TGFß inhibition restores a regenerative response in acute liver injury by suppressing paracrine senescence.

Liver injury results in rapid regeneration through hepatocyte proliferation and hypertrophy. However, after acute severe injury, such as acetaminophen poisoning, effective regeneration may fail. We investigated how senescence may underlie this regenerative failure. In human acute liver disease, and murine models, p21-dependent hepatocellular senescence was proportionate to disease severity and was associated with impaired regeneration. In an acetaminophen injury mouse model, a transcriptional signature associated with the induction of paracrine senescence was observed within 24 hours and was followed by one of impaired proliferation. In mouse genetic models of hepatocyte injury and senescence, we observed transmission of senescence to local uninjured hepatocytes. Spread of senescence depended on macrophage-derived transforming growth factor-ß1 (TGFß1) ligand. In acetaminophen poisoning, inhibition of TGFß receptor 1 (TGFßR1) improved mouse survival. TGFßR1 inhibition reduced senescence and enhanced liver regeneration even when delivered beyond the therapeutic window for treating acetaminophen poisoning. This mechanism, in which injury-induced senescence impairs liver regeneration, is an attractive therapeutic target for developing treatments for acute liver failure.

CXCR2 Inhibition Profoundly Suppresses Metastases and Augments Immunotherapy in Pancreatic Ductal Adenocarcinoma.

CXCR2 has been suggested to have both tumor-promoting and tumor-suppressive properties. Here we show that CXCR2 signaling is upregulated in human pancreatic cancer, predominantly in neutrophil/myeloid-derived suppressor cells, but rarely in tumor cells. Genetic ablation or inhibition of CXCR2 abrogated metastasis, but only inhibition slowed tumorigenesis. Depletion of neutrophils/myeloid-derived suppressor cells also suppressed metastasis suggesting a key role for CXCR2 in establishing and maintaining the metastatic niche. Importantly, loss or inhibition of CXCR2 improved T cell entry, and combined inhibition of CXCR2 and PD1 in mice with established disease significantly extended survival. We show that CXCR2 signaling in the myeloid compartment can promote pancreatic tumorigenesis and is required for pancreatic cancer metastasis, making it an excellent therapeutic target.

Nuclear FAK controls chemokine transcription, Tregs, and evasion of anti-tumor immunity.

Focal adhesion kinase (FAK) promotes anti-tumor immune evasion. Specifically, the kinase activity of nuclear-targeted FAK in squamous cell carcinoma (SCC) cells drives exhaustion of CD8(+) T cells and recruitment of regulatory T cells (Tregs) in the tumor microenvironment by regulating chemokine/cytokine and ligand-receptor networks, including via transcription of Ccl5, which is crucial. These changes inhibit antigen-primed cytotoxic CD8(+) T cell activity, permitting growth of FAK-expressing tumors. Mechanistically, nuclear FAK is associated with chromatin and exists in complex with transcription factors and their upstream regulators that control Ccl5 expression. Furthermore, FAK's immuno-modulatory nuclear activities may be specific to cancerous squamous epithelial cells, as normal keratinocytes do not have nuclear FAK. Finally, we show that a small-molecule FAK kinase inhibitor, VS-4718, which is currently in clinical development, also drives depletion of Tregs and promotes a CD8(+) T cell-mediated anti-tumor response. Therefore, FAK inhibitors may trigger immune-mediated tumor regression, providing previously unrecognized therapeutic opportunities.

CXCR2 inhibition suppresses acute and chronic pancreatic inflammation.

Pancreatitis is a significant clinical problem and the lack of effective therapeutic options means that treatment is often palliative rather than curative. A deeper understanding of the pathogenesis of both acute and chronic pancreatitis is necessary to develop new therapies. Pathological changes in pancreatitis are dependent on innate immune cell recruitment to the site of initial tissue damage, and on the coordination of downstream inflammatory pathways. The chemokine receptor CXCR2 drives neutrophil recruitment during inflammation, and to investigate its role in pancreatic inflammation, we induced acute and chronic pancreatitis in wild-type and Cxcr2(-/-) mice. Strikingly, Cxcr2(-/-) mice were strongly protected from tissue damage in models of acute pancreatitis, and this could be recapitulated by neutrophil depletion or by the specific deletion of Cxcr2 from myeloid cells. The pancreata of Cxcr2(-/-) mice were also substantially protected from damage during chronic pancreatitis. Neutrophil depletion was less effective in this model, suggesting that CXCR2 on non-neutrophils contributes to the development of chronic pancreatitis. Importantly, pharmacological inhibition of CXCR2 in wild-type mice replicated the protection seen in Cxcr2(-/-) mice in acute and chronic models of pancreatitis. Moreover, acute pancreatic inflammation was reversible by inhibition of CXCR2. Thus, CXCR2 is critically involved in the development of acute and chronic pancreatitis in mice, and its inhibition or loss protects against pancreatic damage. CXCR2 may therefore be a viable therapeutic target in the treatment of pancreatitis.

The atypical chemokine receptor ACKR2 suppresses Th17 responses to protein autoantigens.

Chemokine-directed leukocyte migration is a critical component of all innate and adaptive immune responses. The atypical chemokine receptor ACKR2 is expressed by lymphatic endothelial cells and scavenges pro-inflammatory CC chemokines to indirectly subdue leukocyte migration. This contributes to the resolution of acute inflammatory responses in vivo. ACKR2 is also universally expressed by innate-like B cells, suppressing their responsiveness to the non-ACKR2 ligand CXCL13, and controlling their distribution in vivo. The role of ACKR2 in autoimmunity remains relatively unexplored, although Ackr2 deficiency reportedly lessens the clinical symptoms of experimental autoimmune encephalomyelitis induced by immunization with encephalogenic peptide (MOG(35-55)). This was attributed to poor T-cell priming stemming from the defective departure of dendritic cells from the site of immunization. However, we report here that Ackr2-deficient mice, on two separate genetic backgrounds, are not less susceptible to autoimmunity induced by immunization, and in some cases develop enhanced clinical symptoms. Moreover, ACKR2 deficiency does not suppress T-cell priming in response to encephalogenic peptide (MOG(35-55)), and responses to protein antigen (collagen or MOG(1-125)) are characterized by elevated interleukin-17 production. Interestingly, after immunization with protein, but not peptide, antigen, Ackr2 deficiency was also associated with an increase in lymph node B cells expressing granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that enhances T helper type 17 (Th17) cell development and survival. Thus, Ackr2 deficiency does not suppress autoreactive T-cell priming and autoimmune pathology, but can enhance T-cell polarization toward Th17 cells and increase the abundance of GM-CSF(+) B cells in lymph nodes draining the site of immunization.

CCRL1/ACKR4 is expressed in key thymic microenvironments but is dispensable for T lymphopoiesis at steady state in adult mice.

Thymus colonisation and thymocyte positioning are regulated by interactions between CCR7 and CCR9, and their respective ligands, CCL19/CCL21 and CCL25. The ligands of CCR7 and CCR9 also interact with the atypical receptor CCRL1 (also known as ACKR4), which is expressed in the thymus and has recently been reported to play an important role in normal αßT-cell development. Here, we show that CCRL1 is expressed within the thymic cortex, predominantly by MHC-II(low) CD40(-) cortical thymic epithelial cells and at the subcapsular zone by a population of podoplanin(+) thymic epithelial cells in mice. Interestingly, CCRL1 is also expressed by stromal cells which surround the pericytes of vessels at the corticomedullary junction, the site for progenitor cell entry and mature thymocyte egress from the thymus. We show that CCRL1 suppresses thymocyte progenitor entry into the thymus, however, the thymus size and cellularity are the same in adult WT and CCRL1(-/-) mice. Moreover, CCRL1(-/-) mice have no major perturbations in T-cell populations at different stages of thymic differentiation and development, and have a similar rate of thymocyte migration into the blood. Collectively, our findings argue against a major role for CCRL1 in normal thymus development and function.

The chemokine receptors ACKR2 and CCR2 reciprocally regulate lymphatic vessel density.

Macrophages regulate lymphatic vasculature development; however, the molecular mechanisms regulating their recruitment to developing, and adult, lymphatic vascular sites are not known. Here, we report that resting mice deficient for the inflammatory chemokine-scavenging receptor, ACKR2, display increased lymphatic vessel density in a range of tissues under resting and regenerating conditions. This appears not to alter dendritic cell migration to draining lymph nodes but is associated with enhanced fluid drainage from peripheral tissues and thus with a hypotensive phenotype. Examination of embryonic skin revealed that this lymphatic vessel density phenotype is developmentally established. Further studies indicated that macrophages and the inflammatory CC-chemokine CCL2, which is scavenged by ACKR2, are associated with this phenotype. Accordingly, mice deficient for the CCL2 signalling receptor, CCR2, displayed a reciprocal phenotype of reduced lymphatic vessel density. Further examination revealed that proximity of pro-lymphangiogenic macrophages to developing lymphatic vessel surfaces is increased in ACKR2-deficient mice and reduced in CCR2-deficient mice. Therefore, these receptors regulate vessel density by reciprocally modulating pro-lymphangiogenic macrophage recruitment, and proximity, to developing, resting and regenerating lymphatic vessels.

The N-terminal region of the atypical chemokine receptor ACKR2 is a key determinant of ligand binding.

The atypical chemokine receptor, ACKR2 is a pivotal regulator of chemokine-driven inflammatory responses and works by binding, internalizing, and degrading inflammatory CC-chemokines. ACKR2 displays promiscuity of ligand binding and is capable of interacting with up to 14 different inflammatory CC-chemokines. Despite its prominent biological role, little is known about the structure/function relationship within ACKR2, which regulates ligand binding. Here we demonstrate that a conserved tyrosine motif at the N terminus of ACKR2 is essential for ligand binding, internalization, and scavenging. In addition we demonstrate that sulfation of this motif contributes to ligand internalization. Furthermore, a peptide derived from this region is capable of binding inflammatory chemokines and inhibits their interaction with their cognate signaling receptors. Importantly, the peptide is only active in the sulfated form, further confirming the importance of the sulfated tyrosines for function. Finally, we demonstrate that the bacterial protease, staphopain A, can cleave the N terminus of ACKR2 and suppress its ligand internalization activity. Overall, these results shed new light on the nature of the structural motifs in ACKR2 that are responsible for ligand binding. The study also highlights ACKR2-derived N-terminal peptides as being of potential therapeutic significance.

Immune regulation by atypical chemokine receptors.

Chemokines have fundamental roles in regulating immune and inflammatory responses, primarily through their control of leukocyte migration and localization. The biological functions of chemokines are typically mediated by signalling through G protein-coupled chemokine receptors, but chemokines are also bound by a small family of atypical chemokine receptors (ACKRs), the members of which are unified by their inability to initiate classical signalling pathways after ligand binding. These ACKRs are emerging as crucial regulatory components of chemokine networks in a wide range of developmental, physiological and pathological contexts. In this Review, we discuss the biochemical and immunological properties of ACKRs and the potential unifying themes in this family, and we highlight recent studies that identify novel roles for these molecules in development , homeostasis, inflammatory disease, infection and cancer.

An analysis of the function and expression of D6 on lymphatic endothelial cells.

The mechanisms by which CC chemokine receptor (CCR)7 ligands are selectively presented on lymphatic endothelium in the presence of inflammatory chemokines are poorly understood. The chemokine-scavenging receptor D6 is expressed on lymphatic endothelial cells (LEC) and contributes to selective presentation of CCR7 ligands by suppressing inflammatory chemokine binding to LEC surfaces. As well as preventing inappropriate inflammatory cell attachment to LECs, D6 is specifically involved in regulating the ability of LEC to discriminate between mature and immature dendritic cells (DCs). D6 overexpression reduces immature DC (iDC) adhesion to LECs, whereas D6 knockdown increases adhesion of iDCs that displace mature DCs. LEC D6 expression is regulated by growth factors, cytokines, and tumor microenvironments. In particular, interleukin-6 and interferon-γ are potent inducers, indicating a preferential role for D6 in inflamed contexts. Expression of the viral interleukin-6 homolog from Kaposi sarcoma-associated herpesvirus is also sufficient to induce significant D6 upregulation both in vitro and in vivo, and Kaposi sarcoma and primary effusion lymphoma cells demonstrate high levels of D6 expression. We therefore propose that D6, which is upregulated in both inflammatory and tumor contexts, is an essential regulator of inflammatory leukocyte interactions with LECs and is required for immature/mature DC discrimination by LECs.

CCX-CKR deficiency alters thymic stroma impairing thymocyte development and promoting autoimmunity.

The atypical chemokine receptor CCX-CKR regulates bioavailability of CCL19, CCL21, and CCL25, homeostatic chemokines that play crucial roles in thymic lymphopoiesis. Deletion of CCX-CKR results in accelerated experimental autoimmunity induced by immunization. Here we show that CCX-CKR deletion also increases incidence of a spontaneous Sjögren's syndrome-like pathology, characterized by lymphocytic infiltrates in salivary glands and liver of CCX-CKR(-/-) mice, suggestive of a defect in self-tolerance when CCX-CKR is deleted. This prompted detailed examination of the thymus in CCX-CKR(-/-) mice. Negatively selected mature SP cells were less abundant in CCX-CKR(-/-) thymi, yet expansion of both DP and immature SP cells was apparent. Deletion of CCX-CKR also profoundly reduced proportions of DN3 thymocyte precursors and caused DN2 cells to accumulate within the medulla. These effects are likely driven by alterations in thymic stroma as CCX-CKR(-/-) mice have fewer cTECs per thymocyte, and cTECs express the highest level of CCX-CKR in the thymus. A profound decrease in CCL25 within the thymic cortex was observed in CCX-CKR(-/-) thymi, likely accounting for their defects in thymocyte distribution and frequency. These findings identify a novel role for CCX-CKR in regulating cTEC biology, which promotes optimal thymocyte development and selection important for self-tolerant adaptive immunity.

Inhibition of CXCR2 profoundly suppresses inflammation-driven and spontaneous tumorigenesis.

The chemokine receptor CXCR2 is a key mediator of neutrophil migration that also plays a role in tumor development. However, CXCR2 influences tumors through multiple mechanisms and might promote or inhibit tumor development depending on context. Here, we used several mouse models of spontaneous and inflammation-driven neoplasia to define indispensable roles for CXCR2 in benign and malignant tumors. CXCR2-activating chemokines were part of the secretome of cultured primary benign intestinal adenomas (ApcMin/+) and highly expressed by all tumors in all models. CXCR2 deficiency profoundly suppressed inflammation-driven tumorigenesis in skin and intestine as well as spontaneous adenocarcinoma formation in a model of invasive intestinal adenocarcinoma (AhCreER;Apcfl/+;Ptenfl/fl mice). Pepducin-mediated CXCR2 inhibition reduced tumorigenesis in ApcMin/+ mice. Ly6G+ neutrophils were the dominant source of CXCR2 in blood, and CXCR2 deficiency attenuated neutrophil recruitment. Moreover, systemic Ly6G+ cell depletion purged CXCR2-dependent tumor-associated leukocytes, suppressed established skin tumor growth and colitis-associated tumorigenesis, and reduced ApcMin/+ adenoma formation. CXCR2 is thus a potent protumorigenic chemokine receptor that directs recruitment of tumor-promoting leukocytes into tissues during tumor-inducing and tumor-driven inflammation. Similar leukocyte populations were also found in human intestinal adenomas, which suggests that CXCR2 antagonists may have therapeutic and prophylactic potential in the treatment of cancer.

Co-opting endogenous immunoglobulin for the regulation of inflammation and osteoclastogenesis in humans and mice.

OBJECTIVE: Cells of the monocytic lineage play fundamental roles in the regulation of health, ranging from the initiation and resolution of inflammation to bone homeostasis. In rheumatoid arthritis (RA), the inflamed synovium exhibits characteristic infiltration of macrophages along with local osteoclast maturation, which, together, drive chronic inflammation and downstream articular destruction. The aim of this study was to explore an entirely novel route of immunoglobulin-mediated regulation, involving simultaneous suppression of the inflammatory and erosive processes in the synovium. METHODS: Using in vivo and in vitro studies of human cells and a murine model of RA, the ability of staphylococcal protein A (SPA) to interact with and modulate cells of the monocytic lineage was tested. In addition, the efficacy of SPA as a therapeutic agent was evaluated in murine collagen-induced arthritis (CIA). RESULTS: SPA showed a capacity to appropriate circulating IgG, by generating small immunoglobulin complexes that interacted with monocytes, macrophages, and preosteoclasts. Formation of these complexes resulted in Fcγ receptor type I-dependent polarization of macrophages to a regulatory phenotype, rendering them unresponsive to activators such as interferon-γ. The antiinflammatory complexes also had the capacity to directly inhibit differentiation of preosteoclasts into osteoclasts in humans. Moreover, administration of SPA in the early stages of disease substantially alleviated the clinical and histologic erosive features of CIA in mice. CONCLUSION: These findings demonstrate the overarching utility of immunoglobulin complexes for the prevention and treatment of inflammatory diseases. The results shed light on the interface between immunoglobulin complex-mediated pathways, osteoclastogenesis, and associated pathologic processes. Thus, therapeutic agents designed to harness all of these properties may be an effective treatment for arthritis, by targeting both the innate inflammatory response and prodestructive pathways.

Universal expression and dual function of the atypical chemokine receptor D6 on innate-like B cells in mice.

Mouse innate-like B cells are a heterogeneous collection of multifunctional cells that control infection, play housekeeping roles, contribute to adaptive immunity, and suppress inflammation. We show that, among leukocytes, chemokine internalization by the D6 receptor is a unique and universal feature of all known innate-like B-cell populations and, to our knowledge, the most effective unifying marker of these cells. Moreover, we identify novel D6(active) B1-cell subsets, including those we term B1d, which lack CD5 and CD11b but exhibit typical B1-cell properties, including spontaneous ex vivo production of IgM, IL-10, and anti-phosphorylcholine antibody. The unprecedented opportunity to examine D6 on primary cells has allowed us to clarify its ligand specificity and show that, consistent with a scavenging role, D6 internalizes chemokines but cannot induce Ca(2+) fluxes or chemotaxis. Unexpectedly, however, D6 can also suppress the function of CXCR5, a critical chemokine receptor in innate-like B-cell biology. This is associated with a reduction in B1 cells and circulating class-switched anti-phosphorylcholine antibody in D6-deficient mice. Therefore, in the present study, we identify a unifying marker of innate-like B cells, describe novel B1-cell subsets, reveal a dual role for D6, and provide the first evidence of defects in resting D6-deficient mice.