Pulmonary Epithelial Cell-Derived Cytokine TGF-ß1 Is a Critical Cofactor for Enhanced Innate Lymphoid Cell Function.

Epithelial cells orchestrate pulmonary homeostasis and pathogen defense and play a crucial role in the initiation of allergic immune responses. Maintaining the balance between homeostasis and inappropriate immune activation and associated pathology is particularly complex at mucosal sites that are exposed to billions of potentially antigenic particles daily. We demonstrated that epithelial cell-derived cytokine TGF-ß had a central role in the generation of the pulmonary immune response. Mice that specifically lacked epithelial cell-derived TGF-ß1 displayed a reduction in type 2 innate lymphoid cells (ILCs), resulting in suppression of interleukin-13 and hallmark features of the allergic response including airway hyperreactivity. ILCs in the airway lumen were primed to respond to TGF-ß by expressing the receptor TGF-ßRII and ILC chemoactivity was enhanced by TGF-ß. These data demonstrate that resident epithelial cells instruct immune cells, highlighting the central role of the local environmental niche in defining the nature and magnitude of immune reactions.

A Requirement for Neutrophil Glycosaminoglycans in Chemokine:Receptor Interactions Is Revealed by the Streptococcal Protease SpyCEP.

To evade the immune system, the lethal human pathogen Streptococcus pyogenes produces SpyCEP, an enzyme that cleaves the C-terminal α-helix of CXCL8, resulting in markedly impaired recruitment of neutrophils to sites of invasive infection. The basis for chemokine inactivation by SpyCEP is, however, poorly understood, as the core domain of CXCL8 known to interact with CXCL8 receptors is unaffected by enzymatic cleavage. We examined the in vitro migration of human neutrophils and observed that their ability to efficiently navigate a CXCL8 gradient was compromised following CXCL8 cleavage by SpyCEP. SpyCEP-mediated cleavage of CXCL8 also impaired CXCL8-induced migration of transfectants expressing the human chemokine receptors CXCR1 or CXCR2. Despite possessing an intact N terminus and preserved disulfide bonds, SpyCEP-cleaved CXCL8 had impaired binding to both CXCR1 and CXCR2, pointing to a requirement for the C-terminal α-helix. SpyCEP-cleaved CXCL8 had similarly impaired binding to the glycosaminoglycan heparin. Enzymatic removal of neutrophil glycosaminoglycans was observed to ablate neutrophil navigation of a CXCL8 gradient, whereas navigation of an fMLF gradient remained largely intact. We conclude, therefore, that SpyCEP cleavage of CXCL8 results in chemokine inactivation because of a requirement for glycosaminoglycan binding in productive chemokine:receptor interactions. This may inform strategies to inhibit the activity of SpyCEP, but may also influence future approaches to inhibit unwanted chemokine-induced inflammation.

Evidence for the Existence of a CXCL17 Receptor Distinct from GPR35.

The chemokine CXCL17 is associated with the innate response in mucosal tissues but is poorly characterized. Similarly, the G protein-coupled receptor GPR35, expressed by monocytes and mast cells, has been implicated in the immune response, although its precise role is ill-defined. A recent manuscript reported that GPR35 was able to signal in response to CXCL17, which we set out to confirm in this study. GPR35 was readily expressed using transfection systems but failed to signal in response to CXCL17 in assays of ß-arrestin recruitment, inositol phosphate production, calcium flux, and receptor endocytosis. Similarly, in chemotaxis assays, GPR35 did not confirm sensitivity to a range of CXCL17 concentrations above that observed in the parental cell line. We subsequently employed a real time chemotaxis assay (TAXIScan) to investigate the migratory responses of human monocytes and the monocytic cell line THP-1 to a gradient of CXCL17. Freshly isolated human monocytes displayed no obvious migration to CXCL17. Resting THP-1 cells showed a trend toward directional migration along a CXCL17 gradient, which was significantly enhanced by overnight incubation with PGE2 However, pretreatment of PGE2-treated THP-1 cells with the well-characterized GPR35 antagonist ML145 did not significantly impair their migratory responses to CXCL17 gradient. CXCL17 was susceptible to cleavage with chymase, although this had little effect its ability to recruit THP-1 cells. We therefore conclude that GPR35 is unlikely to be a bona fide receptor for CXCL17 and that THP-1 cells express an as yet unidentified receptor for CXCL17.

IRF5 controls both acute and chronic inflammation.

Whereas the importance of macrophages in chronic inflammatory diseases is well recognized, there is an increasing awareness that neutrophils may also play an important role. In addition to the well-documented heterogeneity of macrophage phenotypes and functions, neutrophils also show remarkable phenotypic diversity among tissues. Understanding the molecular pathways that control this heterogeneity should provide abundant scope for the generation of more specific and effective therapeutics. We have shown that the transcription factor IFN regulatory factor 5 (IRF5) polarizes macrophages toward an inflammatory phenotype. IRF5 is also expressed in other myeloid cells, including neutrophils, where it was linked to neutrophil function. In this study we explored the role of IRF5 in models of acute inflammation, including antigen-induced inflammatory arthritis and lung injury, both involving an extensive influx of neutrophils. Mice lacking IRF5 accumulate far fewer neutrophils at the site of inflammation due to the reduced levels of chemokines important for neutrophil recruitment, such as the chemokine (C-X-C motif) ligand 1. Furthermore we found that neutrophils express little IRF5 in the joints and that their migratory properties are not affected by the IRF5 deficiency. These studies extend prior ones suggesting that inhibiting IRF5 might be useful for chronic macrophage-induced inflammation and suggest that IRF5 blockade would ameliorate more acute forms of inflammation, including lung injury.

Distinct conformations of the chemokine receptor CCR4 with implications for its targeting in allergy.

CC chemokine receptor 4 (CCR4) is expressed by Th2 and regulatory T cells and directs their migration along gradients of the chemokines CCL17 and CCL22. Both chemokines and receptor are upregulated in allergic disease, making CCR4 a therapeutic target for the treatment of allergy. We set out to assess the mechanisms underlying a previous report that CCL22 is a dominant ligand of CCR4, which may have implications for its therapeutic targeting. Human T cells expressing endogenous CCR4 and transfectants engineered to express CCR4 were assessed for receptor function, using assays of calcium release, chemotaxis, receptor endocytosis, and ligand binding. Despite the two ligands having equal potency in calcium flux and chemotaxis assays, CCL22 showed dominance in both receptor endocytosis assays and heterologous competitive binding assays. Using two different CCR4-specific Abs, we showed that CCR4 exists in at least two distinct conformations, which are differentially activated by ligand. A major population is activated by both CCL17 and CCL22, whereas a minor population is activated only by CCL22. Mutation of a single C-terminal residue K310 within a putative CCR4 antagonist binding site ablated activation of CCR4 by CCL17, but not by CCL22, despite having no effect on the binding of either ligand. We conclude that CCL17 and CCL22 are conformationally selective ligands of CCR4 and interact with the receptor by substantially different mechanisms. This finding suggests that the selective blockade of CCR4 in allergy may be feasible when one CCR4 ligand dominates, allowing the inhibition of Th2 signaling via one ligand while sparing regulatory T cell recruitment via another.

CCL17/thymus and activation-regulated chemokine induces calcitonin gene-related peptide in human airway epithelial cells through CCR4.

BACKGROUND: Calcitonin gene-related peptide (CGRP) is a potent arterial and venous vasodilator. Increased airway epithelial cell expression of CGRP, together with increased CCL17 expression, was previously observed in a model of provoked asthma in atopic human subjects. OBJECTIVE: We sought to determine whether CCL17 induces CCR4-dependent CGRP synthesis and secretion by human airway epithelial cells. METHODS: Human airway epithelial cell lines (BEAS-2B and A549) and human primary airway cells were cultured with CCL17 or various other cytokines, and CGRP expression was measured by using RT-PCR, quantitative immunofluorescence, and enzyme immunoassay. CCR4 expression was determined in cultured cells by using flow cytometry and in bronchial biopsy specimens by using immunohistochemistry. RESULTS: CCL17 induced a several thousand-fold increase in CGRP mRNA expression and released peptide product from BEAS-2B and A549 cells in a time- and concentration-dependent fashion. Concentration-dependent CCL17-induced release of CGRP by primary human airway epithelial cells was also observed. Under comparable conditions, CCL17 induced greater CGRP release from BEAS-2B cells than either IL-13, a cytokine mixture (TNF-α, GM-CSF, and IL-1), or CCL22. CCR4 was expressed by BEAS-2B and A549 cells and internalized after ligation with CCL17. CCL17-induced CGRP release was inhibited by a specific anti-CCR4 blocking antibody. Bronchial biopsy specimens obtained from healthy volunteers and asthmatic patients before and after provoked asthma all exhibited CCR4 staining of equivalent intensity, indicating that the receptor is constitutively expressed. CONCLUSIONS: CCL17-induced, CCR4-dependent release of CGRP by human airway epithelial cells represents a novel inflammatory pathway and a possible target in patients with asthma and allergic disease.

The chemokine receptor CCR8 is not a high-affinity receptor for the human chemokine CCL18.

The primate-specific chemokine CCL18 is a potent chemoattractant for T cells and is expressed at elevated levels in several inflammatory diseases. However, the cognate receptor for CCL18 remains unconfirmed. Here, we describe attempts to validate a previous report that the chemokine receptor CCR8 is the human CCL18 receptor (Islam et al. J Exp Med. 2013, 210:1889-98). Two mouse pre-B cell lines (4DE4 and L1.2) exogenously expressing CCR8 exhibited robust migration in response to the known CCR8 ligand CCL1 but not to CCL18. Similarly, CCL1 but not CCL18 induced internalization of CCR8 on 4DE4 cells. CCR8 expressed on Chinese hamster ovarian (CHO) cells mediated robust G protein activation, inhibition of cAMP synthesis and ß-arrestin2 recruitment in response to CCL1 but not CCL18. Several N- and C-terminal variants of CCL18 also failed to stimulate CCR8 activation. On the other hand, and as previously reported, CCL18 inhibited CCL11-stimulated migration of 4DE4 cells expressing the receptor CCR3. These data suggest that CCR8, at least in the absence of unidentified cofactors, does not function as a high affinity receptor for CCL18.

Human T(H)2 cells respond to cysteinyl leukotrienes through selective expression of cysteinyl leukotriene receptor 1.

BACKGROUND: Allergic asthma is characterized by reversible airway obstruction and bronchial hyperresponsiveness associated with T(H)2 cell-mediated inflammation. Cysteinyl leukotrienes (CysLTs) are potent lipid mediators involved in bronchoconstriction, mucus secretion, and cell trafficking in asthmatic patients. Recent data have implicated CysLTs in the establishment and amplification of T(H)2 responses in murine models, although the precise mechanisms are unresolved. OBJECTIVES: Preliminary microarray studies suggested that human T(H)2 cells might selectively express cysteinyl leukotriene receptor 1 (CYSLTR1) mRNA. We sought to establish whether human T(H)2 cells are indeed a CysLT target cell type. METHODS: We examined the expression of CYSLTR1 using real-time PCR in human T(H)1 and T(H)2 cells. We functionally assessed cysteinyl leukotriene receptor 1 protein (CysLT(1)) expression using calcium flux, cyclic AMP, and chemotaxis assays. RESULTS: We show that human T(H)2 cells selectively express CYSLTR1 mRNA at high levels compared with T(H)1 cells after in vitro differentiation from naive precursors. Human T(H)2 cells are selectively responsive to CysLTs in a calcium flux assay when compared with T(H)1 cells with a rank order of potency similar to that described for CysLT(1) (leukotriene [LT] D(4) > LTC(4) > LTE(4)). We also show that LTD(4)-induced signaling in T(H)2 cells is mediated through CysLT(1) coupled to G(α)q and G(α)i proteins, and both pathways can be completely inhibited by selective CysLT(1) antagonists. LTD(4) is also found to possess potent chemotactic activity for T(H)2 cells at low nanomolar concentrations. CONCLUSIONS: These findings suggest a novel mechanism of action for CysLTs in the pathogenesis of asthma and provide a potential explanation for the anti-inflammatory effects of CysLT(1) antagonists.

CXCL17 binds efficaciously to glycosaminoglycans with the potential to modulate chemokine signaling.

Introduction: CXCL17 is a mucosally secreted protein, and the most recently identified human chemokine, an assignment based on protein fold prediction and chemotactic activity for leukocytes. However, these credentials have been the subject of much recent discussion and no experimental evidence has been presented regarding the definitive structure of CXCL17. In this study, we evaluated the structural and chemoattractant credentials of CXCL17 to better characterize this molecule, and gain deeper insights into its functional role as a glycosaminoglycan (GAG) binding protein. Methods: In the absence of structural information, in silico modeling techniques assessed the likelihood of CXCL17 adopting a chemokine fold. Recombinant CXCL17 was synthesized in mammalian and prokaryotic systems. Modified Boyden chamber and real-time chemotaxis assays assessed the ability of CXCL17 to promote chemotaxis of murine splenocytes, human neutrophils, and CXCR1 transfectants. The efficacy of CXCL17 binding to GAGs was quantified with solid-phase assays and bio-layer interferometry techniques. Results: All modeling efforts failed to support classification of CXCL17 as a chemokine based on its predicted conformation. Recombinant CXCL17 was observed to dimerize as a function of concentration, a characteristic of several chemokines. Contrary to a previous report, CXCL17 was not chemotactic for murine splenocytes, although it was a low-potency chemoattractant for human neutrophils at micromolar concentrations, several orders of magnitude higher than those required for CXCL8. As anticipated owing to its highly basic nature, CXCL17 bound to GAGs robustly, with key C-terminal motifs implicated in this process. While inactive via CXCR1, CXCL17 was found to inhibit CXCR1-mediated chemotaxis of transfectants to CXCL8 in a dose-dependent manner. Discussion: In summary, despite finding little evidence for chemokine-like structure and function, CXCL17 readily bound GAGs, and could modulate chemotactic responses to another chemokine in vitro. We postulate that such modulation is a consequence of superior GAG binding, and that C-terminal fragments of CXCL17 may serve as prototypic inhibitors of chemokine function.

The protease associated (PA) domain in ScpA from Streptococcus pyogenes plays a role in substrate recruitment.

Annually, over 18 million disease cases and half a million deaths worldwide are estimated to be caused by Group A Streptococcus. ScpA (or C5a peptidase) is a well characterised member of the cell enveleope protease family, which possess a S8 subtilisin-like catalytic domain and a shared multi-domain architecture. ScpA cleaves complement factors C5a and C3a, impairing the function of these critical anaphylatoxins and disrupts complement-mediated innate immunity. Although the high resolution structure of ScpA is known, the details of how it recognises its substrate are only just emerging. Previous studies have identified a distant exosite on the 2nd fibronectin domain that plays an important role in recruitment via an interaction with the substrate core. Here, using a combination of solution NMR spectroscopy, mutagenesis with functional assays and computational approaches we identify a second exosite within the protease-associated (PA) domain. We propose a model in which the PA domain assists optimal delivery of the substrate's C terminus to the active site for cleavage.

A distinct subset of human NK cells expressing HLA-DR expand in response to IL-2 and can aid immune responses to BCG.

Subsets of NK cells can have distinct functions. Here, we report that >25% of human peripheral blood NK cells express HLA-DR after culture with IL-2. This can be driven by an expansion of a small subset of NK cells expressing HLA-DR, in contrast to previous assumptions that HLA-DR is upregulated on previously negative cells. HLA-DR-expressing NK cells showed enhanced degranulation to susceptible target cells and expressed chemokine receptor CXCR3, which facilitated their enrichment following exposure to CXCL11/I-TAC. Suggesting HLA-DR-expressing NK cells have an important role in an immune response, stimulation of PBMCs with Mycobacterium bovis BCG (BCG) triggered expansion of this subset. Importantly, the magnitude of an individual's NK cell IFN-γ response triggered by BCG was associated with the initial frequency of HLA-DR-expressing NK cells in PBMCs. More directly indicating the importance of HLA-DR-expressing NK cells, enriching the frequency of this subset in PBMCs substantially augmented the IFN-γ response to BCG. Thus, HLA-DR expression marks a distinct subset of NK cells, present at low frequency in circulating blood but readily expanded by IL-2, that can play an important role during immune responses to BCG.

The CXCL16 A181V mutation selectively inhibits monocyte adhesion to CXCR6 but is not associated with human coronary heart disease.

OBJECTIVE: The chemokine CXCL16 serves as a scavenger receptor for oxidized low-density lipoprotein and as an adhesion molecule and chemoattractant for cells expressing the receptor CXCR6. A commonly occurring CXCL16 allele has been described containing 2 nonsynonymous single-nucleotide polymorphisms in complete linkage disequilibrium, although the effects on CXCL16 function are unknown. Here, we examined the effect of the single-nucleotide polymorphisms on CXCL16 function and assessed the association of the mutant allele with coronary heart disease (CHD). METHODS AND RESULTS: Both wild-type and mutant T123V181-CXCL16 were readily expressed in vitro and were similarly functional in assays of oxidized low-density lipoprotein scavenging and chemotaxis. However, unlike wild-type CXCL16, T123V181-CXCL16 was unable to promote adhesion of CXCR6(+) cells. Findings were confirmed ex vivo, with monocytes from donors homozygous for the T123V181 allele unable to facilitate adhesion of CXCR6 transfectants. In the London Life Sciences Prospective Population cohort (n = 2797), we found that the T123V181 allele was not associated with protection or susceptibility to CHD (adjusted odds ratio, 1.01; 95% CI, 0.95 to 1.10; P = 0.74). CONCLUSIONS: CXCL16-mediated cell adhesion plays at best a modest role in CHD, and the scavenging and chemotactic properties of the chemokine are more likely to be more important in disease pathogenesis.

The role of the CCL2/CCR2 axis in mouse mast cell migration in vitro and in vivo.

Tissue-resident mast cells (MCs) are important in allergic diseases. In a mouse model of allergic airways inflammation, an increase in peribronchiolar MCs was associated with increased concentrations of the chemokine CCL2 in lung lavage. MC progenitors (MCps) arising in bone marrow (BM) are recruited to tissues by transendothelial migration, and we found that CCL2 is chemotactic for MCps in freshly isolated BM in vitro. Immature, but not mature, BM-derived MCs migrated in response to CCL2 when cultured in IL-3+stem cell factor (SCF) but not when cultured in IL-3 alone. However, the cells under both culture conditions expressed mRNA for CCR2, the receptor for CCL2, and bound the radiolabeled chemokine with similar affinities, highlighting SCF as a key mediator in coupling CCR2 to downstream events, culminating in chemotaxis. Immature BM-derived MCs from IL-3 +SCF cultures, when administered i.v., accumulated at skin sites injected with CCL2 in vivo. MCp recruitment to the allergen-sensitized/challenged lung was significantly reduced in CCR2(-/-) and CCL2(-/-) mouse strains. However, reconstitution studies of sublethally irradiated and BM-reconstituted mice indicated that BM cells and stromal elements could provide CCL2, whereas the CCR2 function resided with stromal elements rather than BM cells. These experiments revealed a new function of SCF in chemokine receptor coupling, but they suggest a complex role of the CCL2/CCR2 axis in recruiting MCps during pulmonary inflammation.

Targeting chemokine receptors in allergic disease.

The directed migration of cells in response to chemical cues is known as chemoattraction, and plays a key role in the temporal and spatial positioning of cells in lower- and higher-order life forms. Key molecules in this process are the chemotactic cytokines, or chemokines, which, in humans, constitute a family of approx. 40 molecules. Chemokines exert their effects by binding to specific GPCRs (G-protein-coupled receptors) which are present on a wide variety of mature cells and their progenitors, notably leucocytes. The inappropriate or excessive generation of chemokines is a key component of the inflammatory response observed in several clinically important diseases, notably allergic diseases such as asthma. Consequently, much time and effort has been directed towards understanding which chemokine receptors and ligands are important in the allergic response with a view to therapeutic intervention. Such strategies can take several forms, although, as the superfamily of GPCRs has historically proved amenable to blockade by small molecules, the development of specific antagonists has been has been a major focus of several groups. In the present review, I detail the roles of chemokines and their receptors in allergic disease and also highlight current progress in the development of relevant chemokine receptor antagonists.

A single nucleotide polymorphism in the CCR3 gene ablates receptor export to the plasma membrane.

BACKGROUND: The chemokine receptor CCR3 orchestrates the migration of eosinophils, basophils, T(H)2 lymphocytes, and mast cells during the allergic response, with CCR3 blockade a potential means of therapeutic intervention. Non-synonymous single nucleotide polymorphisms (SNPs) within the ccr3 gene have previously been described, with little information regarding their effects on CCR3 function. OBJECTIVE: To characterize the effects of nonsynonymous SNPs within the ccr3 gene. METHODS: Site-directed mutagenesis was used to generate N-terminally tagged mutant CCR3 constructs corresponding to reported SNPs. Cell transfectants expressing either wild-type or mutant CCR3 were studied by flow cytometry, Western blotting, and confocal microscopy and examined for their ability to migrate to the CC chemokine ligand CCL11/eotaxin. RESULTS: An L324P mutant CCR3 protein corresponding to the previously identified T971C SNP was not expressed at the cell surface, and cells remained unresponsive to CCL11 in chemotaxis assays. Confocal microscopy confirmed that L324P-CCR3 had a predominantly intracellular distribution compared with wild-type CCR3. A L324A variant of CCR3 had an identical phenotype to the L324P mutant, suggesting that L324 per se is critical for successful trafficking of nascent CCR3 to the cell membrane. The processes involved appear to be specific for CCR3, because an identical mutation in the homologous receptor CCR1 had minor effects. CONCLUSION: Trafficking to the cell surface of nascent CCR3 is critically dependent on a C-terminal leucine residue, suggestive of specific mechanisms for CCR3 export. Manipulation of these mechanisms may suggest novel means of antagonizing CCR3 function in the treatment of allergy.

Structure and function of A41, a vaccinia virus chemokine binding protein.

The vaccinia virus (VACV) A41L gene encodes a secreted 30 kDa glycoprotein that is nonessential for virus replication but affects the host response to infection. The A41 protein shares sequence similarity with another VACV protein that binds CC chemokines (called vCKBP, or viral CC chemokine inhibitor, vCCI), and strains of VACV lacking the A41L gene induced stronger CD8+ T-cell responses than control viruses expressing A41. Using surface plasmon resonance, we screened 39 human and murine chemokines and identified CCL21, CCL25, CCL26 and CCL28 as A41 ligands, with Kds of between 8 nM and 118 nM. Nonetheless, A41 was ineffective at inhibiting chemotaxis induced by these chemokines, indicating it did not block the interaction of these chemokines with their receptors. However the interaction of A41 and chemokines was inhibited in a dose-dependent manner by heparin, suggesting that A41 and heparin bind to overlapping sites on these chemokines. To better understand the mechanism of action of A41 its crystal structure was solved to 1.9 A resolution. The protein has a globular beta sandwich structure similar to that of the poxvirus vCCI family of proteins, but there are notable structural differences, particularly in surface loops and electrostatic charge distribution. Structural modelling suggests that the binding paradigm as defined for the vCCI-chemokine interaction is likely to be conserved between A41 and its chemokine partners. Additionally, sequence analysis of chemokines binding to A41 identified a signature for A41 binding. The biological and structural data suggest that A41 functions by forming moderately strong (nM) interactions with certain chemokines, sufficient to interfere with chemokine-glycosaminoglycan interactions at the cell surface (microM-nM) and thereby to destroy the chemokine concentration gradient, but not strong enough to disrupt the (pM) chemokine-chemokine receptor interactions.

Challenges and Opportunities in the Clinical Development of STING Agonists for Cancer Immunotherapy.

Immune checkpoint inhibitors (ICI) have revolutionised cancer therapy. However, they have been effective in only a small subset of patients and a principal mechanism underlying immune-refractoriness is a 'cold' tumour microenvironment, that is, lack of a T-cell-rich, spontaneously inflamed phenotype. As such, there is a demand to develop strategies to transform the tumour milieu of non-responsive patients to one supporting T-cell-based inflammation. The cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes (cGAS-STING) pathway is a fundamental regulator of innate immune sensing of cancer, with potential to enhance tumour rejection through the induction of a pro-inflammatory response dominated by Type I interferons. Recognition of these positive immune-modulatory properties has rapidly elevated the STING pathway as a putative target for immunotherapy, leading to a myriad of preclinical and clinical studies assessing natural and synthetic cyclic dinucleotides and non-nucleotidyl STING agonists. Despite pre-clinical evidence of efficacy, clinical translation has resulted into disappointingly modest efficacy. Poor pharmacokinetic and physiochemical properties of cyclic dinucleotides are key barriers to the development of STING agonists, most of which require intra-tumoral dosing. Development of systemically administered non-nucleotidyl STING agonists, or conjugation with liposomes, polymers and hydrogels may overcome pharmacokinetic limitations and improve drug delivery. In this review, we summarise the body of evidence supporting a synergistic role of STING agonists with currently approved ICI therapies and discuss whether, despite the numerous obstacles encountered to date, the clinical development of STING agonist as novel anti-cancer therapeutics may still hold the promise of broadening the reach of cancer immunotherapy.

A degradatory fate for CCR4 suggests a primary role in Th2 inflammation.

CCR4 is the sole receptor for the chemokines CCL22 and CCL17. Clinical studies of asthmatic airways have shown levels of both ligands and CCR4+ Th2 cells to be elevated, suggestive of a role in disease. Consequently, CCR4 has aroused much interest as a potential therapeutic target and an understanding of how its cell surface expression is regulated is highly desirable. To this end, receptor expression, receptor endocytosis, and chemotaxis were assessed using transfectants expressing CCR4, CCR4+ human T cell lines, and human Th2 cells polarized in vitro. CCL17 and CCL22 drove rapid endocytosis of CCR4 in a dose-dependent manner. Replenishment at the cell surface was slow and sensitive to cycloheximide, suggestive of de novo synthesis of CCR4. Constitutive CCR4 endocytosis was also observed, with the internalized CCR4 found to be significantly degraded over a 6-h incubation. Truncation of the CCR4 C-terminus by 40 amino acids had no effect on cell surface expression, but resulted in significant impairment of ligand-induced endocytosis. Consequently, migration to both CCL17 and CCL22 was significantly enhanced. In contrast, truncation of CCR4 did not impair constitutive endocytosis or degradation, suggesting the use of alternative receptor motifs in these processes. We conclude that CCR4 cell surface levels are tightly regulated, with a degradative fate for endocytosed receptor. We postulate that this strict control is desirable, given that Th2 cells recruited by CCR4 can induce the further expression of CCR4 ligands in a positive feedback loop, thereby enhancing allergic inflammation.