ILT2 and ILT4 Drive Myeloid Suppression via Both Overlapping and Distinct Mechanisms.

Solid tumors are dense three-dimensional (3D) multicellular structures that enable efficient receptor-ligand trans interactions via close cell-cell contact. Immunoglobulin-like transcript (ILT)2 and ILT4 are related immune-suppressive receptors that play a role in the inhibition of myeloid cells within the tumor microenvironment. The relative contribution of ILT2 and ILT4 to immune inhibition in the context of solid tumor tissue has not been fully explored. We present evidence that both ILT2 and ILT4 contribute to myeloid inhibition. We found that although ILT2 inhibits myeloid cell activation in the context of trans-engagement by MHC-I, ILT4 efficiently inhibits myeloid cells in the presence of either cis- or trans-engagement. In a 3D spheroid tumor model, dual ILT2/ILT4 blockade was required for the optimal activation of myeloid cells, including the secretion of CXCL9 and CCL5, upregulation of CD86 on dendritic cells, and downregulation of CD163 on macrophages. Humanized mouse tumor models showed increased immune activation and cytolytic T-cell activity with combined ILT2 and ILT4 blockade, including evidence of the generation of immune niches, which have been shown to correlate with clinical response to immune-checkpoint blockade. In a human tumor explant histoculture system, dual ILT2/ILT4 blockade increased CXCL9 secretion, downregulated CD163 expression, and increased the expression of M1 macrophage, IFNγ, and cytolytic T-cell gene signatures. Thus, we have revealed distinct contributions of ILT2 and ILT4 to myeloid cell biology and provide proof-of-concept data supporting the combined blockade of ILT2 and ILT4 to therapeutically induce optimal myeloid cell reprogramming in the tumor microenvironment.

The Fibronectin-ILT3 Interaction Functions as a Stromal Checkpoint that Suppresses Myeloid Cells.

Suppressive myeloid cells inhibit antitumor immunity by preventing T-cell responses. Immunoglobulin-like transcript 3 (ILT3; also known as LILRB4) is highly expressed on tumor-associated myeloid cells and promotes their suppressive phenotype. However, the ligand that engages ILT3 within the tumor microenvironment and renders tumor-associated myeloid cells suppressive is unknown. Using a screening approach, we identified fibronectin as a functional ligand for ILT3. The interaction of fibronectin with ILT3 polarized myeloid cells toward a suppressive state, and these effects were reversed with an ILT3-specific antibody that blocked the interaction of ILT3 with fibronectin. Furthermore, ex vivo treatment of human tumor explants with anti-ILT3 reprogrammed tumor-associated myeloid cells toward a stimulatory phenotype. Thus, the ILT3-fibronectin interaction represents a "stromal checkpoint" through which the extracellular matrix actively suppresses myeloid cells. By blocking this interaction, tumor-associated myeloid cells may acquire a stimulatory phenotype, potentially resulting in increased antitumor T-cell responses.

Apelin-36 Modulates Blood Glucose and Body Weight Independently of Canonical APJ Receptor Signaling.

Apelin-36 was discovered as the endogenous ligand for the previously orphan receptor APJ. Apelin-36 has been linked to two major types of biological activities: cardiovascular (stimulation of cardiac contractility and suppression of blood pressure) and metabolic (improving glucose homeostasis and lowering body weight). It has been assumed that both of these activities are modulated through APJ. Here, we demonstrate that the metabolic activity of apelin-36 can be separated from canonical APJ activation. We developed a series of apelin-36 variants in which evolutionarily conserved residues were mutated, and evaluated their ability to modulate glucose homeostasis and body weight in chronic mouse models. We found that apelin-36(L28A) retains full metabolic activity, but is 100-fold impaired in its ability to activate APJ. In contrast to its full metabolic activity, apelin-36(L28A) lost the ability to suppress blood pressure in spontaneously hypertensive rats (SHR). We took advantage of these findings to develop a longer-acting variant of apelin-36 that could modulate glucose homeostasis without impacting blood pressure (or activating APJ). Apelin-36-[L28C(30kDa-PEG)] is 10,000-fold less potent than apelin-36 at activating the APJ receptor but retains its ability to significantly lower blood glucose and improve glucose tolerance in diet-induced obese mice. Apelin-36-[L28C(30kDa-PEG)] provides a starting point for the development of diabetes therapeutics that are devoid of the blood pressure effects associated with canonical APJ activation.

Mutations of GPR126 are responsible for severe arthrogryposis multiplex congenita.

Arthrogryposis multiplex congenita is defined by the presence of contractures across two or more major joints and results from reduced or absent fetal movement. Here, we present three consanguineous families affected by lethal arthrogryposis multiplex congenita. By whole-exome or targeted exome sequencing, it was shown that the probands each harbored a different homozygous mutation (one missense, one nonsense, and one frameshift mutation) in GPR126. GPR126 encodes G-protein-coupled receptor 126, which has been shown to be essential for myelination of axons in the peripheral nervous system in fish and mice. A previous study reported that Gpr126(-/-) mice have a lethal arthrogryposis phenotype. We have shown that the peripheral nerves in affected individuals from one family lack myelin basic protein, suggesting that this disease in affected individuals is due to defective myelination of the peripheral axons during fetal development. Previous work has suggested that autoproteolytic cleavage is important for activating GPR126 signaling, and our biochemical assays indicated that the missense substitution (p.Val769Glu [c.2306T>A]) impairs autoproteolytic cleavage of GPR126. Our data indicate that GPR126 is critical for myelination of peripheral nerves in humans. This study adds to the literature implicating defective axoglial function as a key cause of severe arthrogryposis multiplex congenita and suggests that GPR126 mutations should be investigated in individuals affected by this disorder.

Type IV collagen is an activating ligand for the adhesion G protein-coupled receptor GPR126.

GPR126 is an orphan heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) that is essential for the development of diverse organs. We found that type IV collagen, a major constituent of the basement membrane, binds to Gpr126 and activates its signaling function. Type IV collagen stimulated the production of cyclic adenosine monophosphate in rodent Schwann cells, which require Gpr126 activity to differentiate, and in human embryonic kidney (HEK) 293 cells expressing exogenous Gpr126. Type IV collagen specifically bound to the extracellular amino-terminal region of Gpr126 containing the CUB (complement, Uegf, Bmp1) and pentraxin domains. Gpr126 derivatives lacking the entire amino-terminal region were constitutively active, suggesting that this region inhibits signaling and that ligand binding relieves this inhibition to stimulate receptor activity. A new zebrafish mutation that truncates Gpr126 after the CUB and pentraxin domains disrupted development of peripheral nerves and the inner ear. Thus, our findings identify type IV collagen as an activating ligand for GPR126, define its mechanism of activation, and highlight a previously unrecognized signaling function of type IV collagen in basement membranes.

Brain-specific angiogenesis inhibitor-1 signaling, regulation, and enrichment in the postsynaptic density.

Brain-specific angiogenesis inhibitor-1 (BAI1) is an adhesion G protein-coupled receptor that has been studied primarily for its anti-angiogenic and anti-tumorigenic properties. We found that overexpression of BAI1 results in activation of the Rho pathway via a Gα(12/13)-dependent mechanism, with truncation of the BAI1 N terminus resulting in a dramatic enhancement in receptor signaling. This constitutive activity of the truncated BAI1 mutant also resulted in enhanced downstream phosphorylation of ERK as well as increased receptor association with ß-arrestin2 and increased ubiquitination of the receptor. To gain insights into the regulation of BAI1 signaling, we screened the C terminus of BAI1 against a proteomic array of PDZ domains to identify novel interacting partners. These screens revealed that the BAI1 C terminus interacts with a variety of PDZ domains from synaptic proteins, including MAGI-3. Removal of the BAI1 PDZ-binding motif resulted in attenuation of receptor signaling to Rho but had no effect on ERK activation. Conversely, co-expression with MAGI-3 was found to potentiate signaling to ERK by constitutively active BAI1 in a manner that was dependent on the PDZ-binding motif of the receptor. Biochemical fractionation studies revealed that BAI1 is highly enriched in post-synaptic density fractions, a finding consistent with our observations that BAI1 can interact with PDZ proteins known to be concentrated in the post-synaptic density. These findings demonstrate that BAI1 is a synaptic receptor that can activate both the Rho and ERK pathways, with the N-terminal and C-terminal regions of the receptor playing key roles in the regulation of BAI1 signaling activity.

Adhesion G protein-coupled receptors: signaling, pharmacology, and mechanisms of activation.

The adhesion G protein-coupled receptors (GPCRs) are a distinct family of more than 30 receptors in vertebrate genomes. These receptors have been shown to play pivotal roles in a diverse range of biological functions and are characterized by extremely large N termini featuring various adhesion domains capable of mediating cell-cell and cell-matrix interactions. The adhesion GPCR N termini also contain GPCR proteolytic site motifs that undergo autocatalytic cleavage during receptor processing to create mature GPCRs existing as noncovalently attached complexes between the N terminus and transmembrane regions. There is mounting evidence that adhesion GPCRs can couple to G proteins to activate a variety of different downstream signaling pathways. Furthermore, recent studies have demonstrated that adhesion GPCR N termini can bind to multiple ligands, which may differentially activate receptor signaling and/or mediate cell adhesion. In addition, studies on several distinct adhesion GPCRs have revealed that truncations of the N termini result in constitutively active receptors, suggesting a model of receptor activation in which removal of the N terminus may be a key event in stimulating receptor signaling. Because mutations to certain adhesion GPCRs cause human disease and because many members of this receptor family exhibit highly discrete distribution patterns in different tissues, the adhesion GPCRs represent a class of potentially important drug targets that have not yet been exploited. For this reason, understanding the mechanisms of activation for these receptors and elucidating their downstream signaling pathways can provide insights with the potential to lead to novel therapeutic agents.

The N terminus of the adhesion G protein-coupled receptor GPR56 controls receptor signaling activity.

GPR56 is an adhesion G protein-coupled receptor that plays a key role in cortical development. Mutations to GPR56 in humans cause malformations of the cerebral cortex, but little is known about the normal function of the receptor. We found that the large N terminus (NT) of GPR56 is cleaved from the rest of the receptor during processing but remains non-covalently associated with the seven-transmembrane region of the receptor, as indicated by coimmunoprecipitation of the two GPR56 fragments from both transfected cells and native tissue. We also found that truncation of the GPR56 NT results in constitutive activation of receptor signaling, as revealed by increased GPR56-stimulated signaling upon transfection of HEK-293 cells with truncated GPR56, greatly enhanced binding of ß-arrestins by truncated GPR56 relative to the full-length receptor, extensive ubiquitination of truncated GPR56, and cytotoxicity induced by truncated GPR56 that could be rescued by cotransfection of cells with ß-arrestin 2. Furthermore, we found that the GPR56 NT is capable of homophilic trans-trans interactions that enhance receptor signaling activity. On the basis of these findings, we suggest a model of receptor activation in which the large N terminus of GPR56 constrains receptor activity but N-terminal interactions (GPR56 NT with an extracellular ligand and/or GPR56 NT homophilic trans-trans associations) can remove this inhibitory influence of the N terminus to activate receptor signaling.

GLAST stability and activity are enhanced by interaction with the PDZ scaffold NHERF-2.

The astrocytic glutamate transporter GLAST (also known as EAAT1) is a key regulator of extracellular glutamate levels in many regions of vertebrate brains. To identify novel interacting partners that might regulate the localization and function of GLAST in astrocytes, we screened the transporter's C-terminus (GLAST-CT) against a proteomic array of 96 different PDZ domains. The GLAST-CT robustly and specifically interacted with PDZ domains from two related scaffolding proteins, the Na(+)/H(+) exchanger regulatory factors 1 and 2 (NHERF-1 and NHERF-2). Studies on cultured rat cortical astrocytes revealed that these cells are highly enriched in NHERF-2 relative to NHERF-1. Endogenous GLAST and NHERF-2 from cultured astrocytes were found to robustly co-immunoprecipitate, and further co-immunoprecipitation studies on mutant versions of GLAST expressed in transfected cells revealed the GLAST/NHERF-2 interaction to be dependent on the last amino acid of the GLAST-CT. Knockdown of endogenous NHERF-2 in astrocytes via siRNA treatment resulted in a significant reduction in GLAST activity, which corresponded to significantly reduced total expression of GLAST protein and reduced half-life of GLAST, as assessed in pulse-chase metabolic labeling studies. These findings reveal that NHERF-2 can interact with GLAST in astrocytes to enhance GLAST stability and activity.

CCSP regulates cross talk between secretory cells and both ciliated cells and macrophages of the conducting airway.

Pulmonary host defense employs a combination of biochemical and biophysical activities to recognize, inactivate, and mediate clearance of environmental agents as well as modulate the overall response to such challenge. Dysregulation of the inflammatory arm of this response is associated with chronic lung diseases (CLD) including cystic fibrosis and chronic obstructive lung disease. Although mechanisms mediating immunoregulation are incompletely characterized, decrements in levels of the nonciliated secretory cell product Clara cell secretory protein (CCSP) in numerous CLD and identification of proinflammatory state in mice homozygous for a null allele of the CCSP gene (CCSP-/-) suggest a central role for the nonciliated secretory cell in this process. In an effort to determine the molecular basis for immunoregulatory defects associated with CCSP deficiency, we utilized difference gel electrophoresis in combination with matrix-assisted laser desorption ionization time-of-flight to compare the proteomes of wild-type and CCSP-/- mice. We demonstrate a shift in the isoelectric point of the immunomodulatory protein annexin A1 (ANXA1) to more acidic isoforms in CCSP-/- mice. Similar ANXA1 mRNA and protein abundance in wild-type and CCSP-/- tissue and identical localization of ANXA1 protein to alveolar macrophages and the ciliary bed of ciliated cells demonstrated that CCSP deficiency was associated exclusively with altered posttranslational modification of ANXA1. These results suggest that both long- and short-range paracrine signaling between nonciliated secretory cells and cells of the immune system and epithelium impact modification of cell type-specific proteins and implicate nonciliated secretory cells in a regulatory axis that might integrate critical aspects of host defense.