Predominant CD8+ cell infiltration and low accumulation of regulatory T cells in immune checkpoint inhibitor-induced tubulointerstitial nephritis.

Immune checkpoint inhibitors (ICIs) can provide survival benefits to cancer patients; however, they sometimes result in the development of renal immune-related adverse events (irAEs). Tubulointerstitial nephritis (TIN) is the most representative pathological feature of renal irAEs. However, the clinicopathological entity and underlying pathogenesis of ICI-induced TIN are unclear. Therefore, we compared the clinical and histological features of this condition with those of non-ICI drug-induced TIN. Age and C-reactive protein levels were significantly higher in ICI-induced TIN, but there were no significant differences in renal function. Immunophenotyping of ICI-induced TIN showed massive T cell and macrophage infiltration with fewer B cells, plasma cells, neutrophils, and eosinophils. Compared with those in non-ICI drug-induced TIN, CD4+ cell numbers were significantly lower in ICI-induced TIN but CD8+ cell numbers were not significantly different. However, CD8/CD3 and CD8/CD4 ratios were higher in ICI-induced TIN. Moreover, CD25+ and FOXP3+ cells, namely regulatory T cells, were less abundant in ICI-induced TIN. In conclusion, T cell, B cell, plasma cell, neutrophil, and eosinophil numbers proved useful for differentiating ICI-induced and non-ICI drug-induced TIN. Furthermore, the predominant distribution of CD8+ cells and low accumulation of regulatory T cells might be associated with ICI-induced TIN development.

Disulfiram, an Anti-alcoholic Drug, Targets Macrophages and Attenuates Acute Rejection in Rat Lung Allografts.

Macrophages contribute to post-transplant lung rejection. Disulfiram (DSF), an anti-alcoholic drug, has an anti-inflammatory effect and regulates macrophage chemotactic activity. Here, we investigated DSF efficacy in suppressing acute rejection post-lung transplantation. Male Lewis rats (280-300 g) received orthotopic left lung transplants from Fisher 344 rats (minor histocompatibility antigen-mismatched transplantation). DSF (0.75 mg/h) monotherapy or co-solvent only (50% hydroxypropyl-ß-cyclodextrin) as control was subcutaneously administered for 7 days (n = 10/group). No post-transplant immunosuppressant was administered. Grades of acute rejection, infiltration of immune cells positive for CD68, CD3, or CD79a, and gene expression of monocyte chemoattractant protein and pro-inflammatory cytokines in the grafts were assessed 7 days post-transplantation. The DSF-treated group had significantly milder lymphocytic bronchiolitis than the control group. The infiltration levels of CD68+ or CD3+ cells to the peribronchial area were significantly lower in the DSF than in the control groups. The normalized expression of chemokine ligand 2 and interleukin-6 mRNA in allografts was lower in the DSF than in the control groups. Validation assay revealed interleukin-6 expression to be significantly lower in the DSF than in the control groups. DSF can alleviate acute rejection post-lung transplantation by reducing macrophage accumulation around peripheral bronchi and suppressing pro-inflammatory cytokine expression.

Disulfiram treatment suppresses antibody-producing reactions by inhibiting macrophage activation and B cell pyrimidine metabolism.

Antibody responses, involving B cells, CD4 + T cells, and macrophages, are implicated in autoimmune diseases and organ transplant rejection. We have previously shown that inhibiting FROUNT with disulfiram (DSF) suppresses macrophage activation and migration, effectively treating inflammatory diseases. In this study, we investigated the effectiveness of DSF in antibody-producing reactions. Using a heart transplantation mouse model with antibody-mediated rejection, we administered anti-CD8 antibody to exclude cellular rejection. DSF directly inhibited B cell responses in vitro and significantly reduced plasma donor-specific antibodies and graft antibody deposition in vivo, resulting in prolonged survival of the heart graft. DSF also mediated various effects, including decreased macrophage infiltration and increased Foxp3+ regulatory T-cells in the grafts. Additionally, DSF inhibited pyrimidine metabolism-related gene expression induced by B-cell stimulation. These findings demonstrate that DSF modulates antibody production in the immune response complexity by regulating B-cell and macrophage responses.

A Novel Multi-Observation System to Study the Effects of Anterior Ocular Inflammation in Zinn's Zonule Using One Specimen.

Zinn's zonule is a fragile and thin tissue, and little is known about its pathogenesis. The aim of this study was to develop an experimental setup for a comprehensive analysis of Zinn's zonule. Rats were divided into two groups: a control group (n = 4) and an alkali injury group (n = 4). Seven days after injury, the eyes were enucleated, the anterior eye was dissected and embedded in gelatin, and macroscopic observations were made. The gelatin specimens were then embedded in paraffin and observed in detail by low-vacuum scanning electron microscopy, immunofluorescence, and quantitative reverse transcription polymerase chain reaction (RT-qPCR). The results show qualitative changes in Zinn's zonules in both macroscopic and microscopic observations. In addition, macrophage infiltration and increased matrix metalloproteinase 2 (MMP2) expression were observed in the injured group, consistent with the RT-qPCR results. The experimental system in this study allowed us to capture the morphological and molecular biological changes of Zinn's zonule and to gain insight into its pathogenesis. In conclusion, this study presents a new experimental setup for the comprehensive analysis of the rat Zinn's zonule. The results suggest that this system can be used in the future to study and analyze a variety of paraffin-embedded tissues and specimens.

Immune checkpoint inhibitors associated granulomatous small vessel vasculitis accompanied with tubulointerstitial nephritis: a case report.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have provided significant benefits in cancer treatment, but they could develop immune-related adverse events (irAE). ICI-associated renal adverse effects are rare and tubulointerstitial nephritis (TIN) is the most common in the renal irAE. However, only a few case reports of renal vasculitis associated with ICI have been reported. In addition, the characteristics of infiltrating inflammatory cells of ICI-associated TIN and renal vasculitis have been uncertain. CASE PRESENTATION: A 65-year-old man received immune checkpoint inhibitors (ICIs), anti-CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) and anti-PD-1 (programmed cell death 1) antibodies for aggravated metastatic malignant melanoma. About 1 week after the second administration of nivolumab and ipilimumab, acute kidney injury developed. A renal biopsy was performed that showed TIN and non-necrotizing granulomatous vasculitis in interlobular arteries. Massive CD3+ T cells and CD163+ macrophages infiltrated both tubulointerstitium and interlobular arteries. Many infiltrating cells tested positive for Ki-67 and PD-1 ligand (PD-L1), but negative for PD-1. In CD3+ T cells, CD8+ T cells were predominantly infiltrated, and these cells were positive for Granzyme B (GrB) and cytotoxic granule TIA-1, but negative for CD25, indicating antigen-independent activated CD8+ T cells. Infiltration of CD4+ T cells was noted without obvious CD4+ CD25+ regulatory T (Treg) cells. His renal dysfunction recovered within 2 months of treatment with prednisolone in addition to discontinuation of nivolumab and ipilimumab. CONCLUSIONS: We herein reported a case of ICI-related TIN and renal granulomatous vasculitis with infiltration of massive antigen-independent activated CD8+ T cells and CD163+ macrophages, and none or few CD4+ CD25+ Treg cells. These infiltrating cells might be a characteristic of the development of renal irAE.

Disulfiram Ophthalmic Solution Inhibited Macrophage Infiltration by Suppressing Macrophage Pseudopodia Formation in a Rat Corneal Alkali Burn Model.

FROUNT is an intracellular protein that promotes pseudopodia formation by binding to the chemokine receptors CCR2 and CCR5 on macrophages. Recently, disulfiram (DSF), a drug treatment for alcoholism, was found to have FROUNT inhibitory activity. In this study, we investigated the effect of DSF eye drops in a rat corneal alkali burn model. After alkali burn, 0.5% DSF eye drops (DSF group) and vehicle eye drops (Vehicle group) were administered twice daily. Immunohistochemical observations and real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed at 6 h and 1, 4, and 7 days after alkali burn. Results showed a significant decrease in macrophage accumulation in the cornea in the DSF group, but no difference in neutrophils. RT-PCR showed decreased expression of macrophage-associated cytokines in the DSF group. Corneal scarring and neovascularization were also suppressed in the DSF group. Low-vacuum scanning electron microscopy imaging showed that macrophage length was significantly shorter in the DSF group, reflecting the reduced extension of pseudopodia. These results suggest that DSF inhibited macrophage infiltration by suppressing macrophage pseudopodia formation.

The reduced number of nephrons with shortening renal tubules in mouse postnatal adverse environment.

BACKGROUND: The intrauterine adverse environment during nephrogenesis reduces the nephron number, probably associates with impaired ureteric bud (UB) branching. METHODS: The kidneys in C57/BL6 mice were irradiated with a single dose of 10 gray (10 Gy) as adverse environment on postnatal day 3 (irradiated PND3 kidneys) after UB branching ceased. The renal functions and pathological findings of irradiated PND3 kidneys were compared with those of non-irradiated control and 10 Gy irradiation on PND14 (irradiated PND14 kidney) from 1 to 18 months. RESULTS: The number and density of glomeruli in irradiated PND3 kidneys were reduced by 1 month with renal dysfunction at 6 months. The morphologically incomplete glomeruli with insufficient capillaries were involuted by 1 month in the superficial cortex. Reduced tubular numbers and developmental disability with shortening renal tubules occurred in irradiated PND3 kidneys with impaired urine concentration at 6 months. Hypertrophy of glomeruli developed, and occasional sclerotic glomeruli appeared in the juxtamedullary cortex with hypertension and albuminuria at 12 to 18 months. CONCLUSIONS: The reduced number of nephrons with shortening renal tubules occurred with impaired renal functions in a postnatal adverse environment after cessation of UB branching, and glomerular hypertrophy with occasional glomerulosclerosis developed accompanied with hypertension and albuminuria in the adulthood. IMPACT: The reduced number of nephrons with shortening renal tubules occurred with impaired renal functions in a postnatal adverse environment after cessation of ureteric bud branching. The reduced number of glomeruli were associated with not only the impaired formation of glomeruli but also involution of morphologically small incomplete glomeruli after an adverse environment. The insufficiently developed nephrons were characterized by the shortening renal tubules with impaired urine concentration. In addition, glomerular hypertrophy and occasional glomerulosclerosis developed with hypertension and albuminuria in adulthood. The present study can help to understand the risk of alternations of premature nephrons in preterm neonates.

Inhibition of the chemokine signal regulator FROUNT by disulfiram ameliorates crescentic glomerulonephritis.

Activated monocytes/macrophages promote glomerular injury, including crescent formation, in anti-glomerular basement membrane (GBM) glomerulonephritis. Disulfiram, an alcohol-aversion drug, inhibits monocyte/macrophage migration by inhibiting FROUNT, a cytosolic protein that enhances chemokine receptor signaling. Our study found that disulfiram at a human equivalent dose successfully blocked albuminuria and crescent formation with podocyte loss, and later stage kidney fibrotic lesions, in a rat model of anti-GBM glomerulonephritis. A disulfiram derivative, DSF-41, with more potent FROUNT inhibition activity, inhibited glomerulonephritis at a lower dose than disulfiram. Disulfiram markedly reduced the number of monocytes or macrophages at the early stage of glomerulonephritis and that of CD3+ and CD8+ lymphocytes at the established stage. Impaired pseudopodia formation was observed in the glomerular monocytes/macrophages of the disulfiram group; consistent with the in vitro observation that disulfiram blocked chemokine-dependent pseudopodia formation and chemotaxis of bone marrow-derived monocytes/macrophages. Furthermore, disulfiram suppressed macrophage activation as revealed by reduced expression of inflammatory cytokines and chemokines (TNF-α, CCL2, and CXCL9) and reduced CD86 and MHC class II expressions in monocytes/macrophages during glomerulonephritis. The dramatic reduction in monocyte/macrophage number might have resulted from disulfiram suppression of both the chemotactic response of monocytes/macrophages and their subsequent activation to produce cytokines and chemokines, which further recruit monocytes. Additionally, FROUNT was expressed in CD68+ monocytes/macrophages infiltrating the crescentic glomeruli in human anti-GBM glomerulonephritis. Thus, disulfiram can be a highly effective and safe drug for the treatment of glomerulonephritis by blocking the chemotactic responses of monocytes/macrophages and their activation status in the glomerulus.

Heavy Metal Enhancement Technique for Diaminobenzidine in Immunohistochemistry Enables Ultrastructural Observation by Low-vacuum Scanning Electron Microscopy.

Low-vacuum scanning electron microscopy (LV-SEM) is a powerful tool that allows to observe light microscopic specimens with periodic acid-silver methenamine (PAM) staining at a higher magnification, simply by removing the coverslip. However, it is not suitable for observation of immunohistochemistry (IHC) using 3,3'-diaminobenzidine (DAB) due to insufficient backscattered electron image. Traditional heavy metal enhancement techniques for DAB in IHC, (1) osmium tetroxide and iron, (2) cobalt, (3) methenamine silver (Ag), (4) gold chloride (Gold), and (5) both Ag and Gold (Ag + Gold), were examined by LV-SEM. Tissue specimens from Thy1.1 glomerulonephritis rat kidney stained with α-smooth muscle actin and visualized with DAB were enhanced by each of these enhancement methods. We found, in light microscopic and LV-SEM, that the enhancement with Ag, Gold, or Ag + Gold had better intensity and contrast than others. At a higher magnification, Ag + Gold enhancement showed high intensity and low background, although only Ag or Gold enhancement had nonspecific background. Even after observation by LV-SEM, the quality of specimens was maintained after remounting the coverslip. It was also confirmed that Ag + Gold enhancement could be useful for IHC using clinical human renal biopsy. These findings indicate that Ag + Gold provided an adequate enhancement in IHC for both LM and LV SEM observation.

Disulfiram Produces Potent Anxiolytic-Like Effects Without Benzodiazepine Anxiolytics-Related Adverse Effects in Mice.

Disulfiram is an FDA approved drug for the treatment of alcoholism. The drug acts by inhibiting aldehyde dehydrogenase, an enzyme essential to alcohol metabolism. However, a recent study has demonstrated that disulfiram also potently inhibits the cytoplasmic protein FROUNT, a common regulator of chemokine receptor CCR2 and CCR5 signaling. Several studies have reported that chemokine receptors are associated with the regulation of emotional behaviors in rodents, such as anxiety. Therefore, this study was performed to clarify the effect of disulfiram on emotional behavior in rodents. The anxiolytic-like effects of disulfiram were investigated using an elevated plus-maze (EPM) test, a typical screening model for anxiolytics. Disulfiram (40 or 80 mg/kg) significantly increased the amount of time spent in the open arms of the maze and the number of open arm entries without affecting the total open arms entries. Similar results were obtained in mice treated with a selective FROUNT inhibitor, disulfiram-41 (10 mg/kg). These disulfiram-associated behavioral changes were similar to those observed following treatment with the benzodiazepine anxiolytic diazepam (1.5 mg/kg). Moreover, disulfiram (40 mg/kg) significantly and completely attenuated increased extracellular glutamate levels in the prelimbic-prefrontal cortex (PL-PFC) during stress exposure on the elevated open-platform. However, no effect in the EPM test was seen following administration of the selective aldehyde dehydrogenase inhibitor cyanamide (40 mg/kg). In contrast to diazepam, disulfiram caused no sedation effects in the open-field, coordination disorder on a rotarod, or amnesia in a Y-maze. This is the first report suggesting that disulfiram produces anxiolytic-like effects in rodents. We found that the presynaptic inhibitory effects on glutaminergic neurons in the PL-PFC may be involved in its underlying mechanism. Disulfiram could therefore be an effective and novel anxiolytic drug that does not produce benzodiazepine-related adverse effects, such as amnesia, coordination disorder, or sedation, as found with diazepam. We propose that the inhibitory activity of disulfiram against FROUNT function provides an effective therapeutic option in anxiety.

Uterine leiomyosarcomas with osteoclast-like giant cells associated with high expression of RUNX2 and RANKL.

Uterine leiomyosarcoma (ULMS) with osteoclast-like giant cells (OLGCs) has been reported as a rare phenomenon in ULMS, and its clinico-pathological features and tumorigenesis remain unclear. We recently reported high expression of receptor activator of nuclear factor κB ligand (RANKL) in ULMS with OLGCs. As osteoblasts produce RANKL, in this study, we analyzed the expression of Runt-related transcription factor 2 (RUNX2), a critical transcription factor for osteoblasts, and osteoclast-related proteins in three cases of ULMS with OLGCs as well as five conventional ULMSs and nine leiomyomas. Immunohistochemistry and real-time reverse transcription quantitative polymerase chain reaction analyses showed high expression of RUNX2 and RANKL in ULMS with OLGCs. In these cases, macrophages expressed receptor activator of nuclear factor κB (RANK), and OLGCs expressed osteoclast-related proteins (nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), and cathepsin K). Accumulation sites of cathepsin K-positive OLGCs showed hemorrhagic appearance and degraded type IV collagen. We reviewed reported cases of ULMS with OLGCs, including ours, and found that they presented an aggressive course even at stage I. Furthermore, metastatic lesions showed similar histological features to those of OLGC association in ULMS. Here, we show that tumor cells in ULMS with OLGCs highly express RUNX2 and RANKL and that osteoclastic differentiation of macrophages occurs in the tumor tissue.

Rational Design of Monodispersed Mutants of Proteins by Identifying Aggregation Contact Sites Using Solubilizing Agents.

Suppression of protein aggregation is a subject of growing importance in the treatment of protein aggregation diseases, an urgent worldwide human health problem, and the production of therapeutic proteins, such as antibody drugs. We previously reported a method to identify compounds that suppress aggregation, based on screening using multiple terminal deletion mutants. We now present a method to determine the aggregation contact sites of proteins, using such solubilizing compounds, to design monodispersed mutants. We applied this strategy to the chemokine receptor-binding domain (CRBD) of FROUNT, which binds to the membrane-proximal C-terminal intracellular region of CCR2. Initially, the backbone NMR signals were assigned to a certain extent by available methods, and the putative locations of five α-helices were identified. Based on NMR chemical shift perturbations upon varying the protein concentrations, the first and third helices were found to contain the aggregation contact sites. The two helices are amphiphilic, and based on an NMR titration with 1,6-hexanediol, a CRBD solubilizing compound, the contact sites were identified as the hydrophobic patches located on the hydrophilic sides of the two helices. Subsequently, we designed multiple mutants targeting amino acid residues on the contact sites. Based on their NMR spectra, a doubly mutated CRBD (L538E/P612S) was selected from the designed mutants, and its monodispersed nature was confirmed by other biophysical methods. We then assessed the CCR2-binding activities of the mutants. Our method is useful for the protein structural analyses, the treatment of protein aggregation diseases, and the improvement of therapeutic proteins.

Targeting FROUNT with disulfiram suppresses macrophage accumulation and its tumor-promoting properties.

Tumor-associated macrophages affect tumor progression and resistance to immune checkpoint therapy. Here, we identify the chemokine signal regulator FROUNT as a target to control tumor-associated macrophages. The low level FROUNT expression in patients with cancer correlates with better clinical outcomes. Frount-deficiency markedly reduces tumor progression and decreases macrophage tumor-promoting activity. FROUNT is highly expressed in macrophages, and its myeloid-specific deletion impairs tumor growth. Further, the anti-alcoholism drug disulfiram (DSF) acts as a potent inhibitor of FROUNT. DSF interferes with FROUNT-chemokine receptor interactions via direct binding to a specific site of the chemokine receptor-binding domain of FROUNT, leading to inhibition of macrophage responses. DSF monotherapy reduces tumor progression and decreases macrophage tumor-promoting activity, as seen in the case of Frount-deficiency. Moreover, co-treatment with DSF and an immune checkpoint antibody synergistically inhibits tumor growth. Thus, inhibition of FROUNT by DSF represents a promising strategy for macrophage-targeted cancer therapy.

1H, 13C and 15N resonance assignments for a chemokine receptor-binding domain of FROUNT, a cytoplasmic regulator of chemotaxis.

FROUNT is a cytoplasmic protein that interacts with the membrane-proximal C-terminal regions (Pro-Cs) of the CCR2 and CCR5 chemokine receptors. The interactions between FROUNT and the chemokine receptors play an important role in the migration of inflammatory immune cells. Therefore, FROUNT is a potential drug target for inflammatory diseases. However, the structural basis of the interactions between FROUNT and the chemokine receptors remains to be elucidated. We previously identified the C-terminal region (residues 532-656) of FROUNT as the structural domain responsible for the Pro-C binding, referred to as the chemokine receptor-binding domain (CRBD), and then constructed its mutant, bearing L538E/P612S mutations, with improved NMR spectral quality, referred to as CRBD_LEPS. We now report the main-chain and side-chain 1H, 13C, and 15N resonance assignments of CRBD_LEPS. The NMR signals of CRBD_LEPS were well dispersed and their intensities were uniform on the 1H-15N HSQC spectrum, and thus almost all of the main-chain and side-chain resonances were assigned. This assignment information provides the foundation for NMR studies of the three-dimensional structure of CRBD_LEPS in solution and its interactions with chemokine receptors.

Efficient identification of compounds suppressing protein precipitation via solvent screening using serial deletion mutants of the target protein.

The control of protein solubility is a subject of broad interest. Although several solvent screening methods are available to search for compounds that enhance protein solubilization, their performance is influenced by the intrinsic solubility of the tested protein. We now present a method for screening solubilizing compounds, using an array of N- or C-terminal deletion mutants of the protein. A key behind this approach is that such terminal deletions of the protein affect its aggregation propensity. The solubilization activities of trial solvents are individually assessed, based on the number of solubilized mutants. The solubilizing compounds are then identified from the screened solvents. In this study, the C-terminal chemokine receptor-binding region of the cytoplasmic protein, FROUNT (FNT-C), which mediates intracellular signals leading to leukocyte migration, was subjected to the multicomponent screening. In total, 192 solution conditions were tested, using eight terminal deletion mutants of FNT-C. We identified five solvent conditions that solubilized four or five mutants of FNT-C, and the compounds in the screened solvents were then, respectively, assessed in terms of their solubilization ability. The best compound for solubilizing FNT-C was 1,6-hexanediol. Indeed, 1,6-hexanediol bound to FNT-C and suppressed its precipitation, as showed by NMR and dynamic light scattering analyses.

Identification and Preparation of a Novel Chemokine Receptor-Binding Domain in the Cytoplasmic Regulator FROUNT.

FROUNT is a cytoplasmic protein that binds to the membrane-proximal C-terminal regions (Pro-Cs) of chemokine receptors, CCR2 and CCR5. The FROUNT-chemokine receptor interactions play a pivotal role in the migration of inflammatory immune cells, indicating the potential of FROUNT as a drug target for inflammatory diseases. To provide the foundation for drug development, structural information of the Pro-C binding region of FROUNT is desired. Here, we defined the novel structural domain (FNT-CB), which mediates the interaction with the chemokine receptors. A recombinant GST-tag-fused FNT-CB protein expression system was constructed. The protein was purified by affinity chromatography and then subjected to in-gel protease digestion of the GST-tag. The released FNT-CB was further purified by anion-exchange and size-exclusion chromatography. Purified FNT-CB adopts a helical structure, as indicated by CD. NMR line-broadening indicated that weak aggregation occurred at sub-millimolar concentrations, but the line-broadening was mitigated by using a deuterated sample in concert with transverse relaxation-optimized spectroscopy. The specific binding of FNT-CB to CCR2 Pro-C was confirmed by the fluorescence-based assay. The improved NMR spectral quality and the retained functional activity of FNT-CB support the feasibility of further structural and functional studies targeted at the anti-inflammatory drug development.

Robust Antitumor Effects of Combined Anti-CD4-Depleting Antibody and Anti-PD-1/PD-L1 Immune Checkpoint Antibody Treatment in Mice.

Depletion of CD4(+) cells in tumor-bearing mice has strong antitumor effects. However, the mechanisms underlying these effects and the therapeutic benefits of CD4(+) cell depletion relative to other immunotherapies have not been fully evaluated. Here, we investigated the antitumor effects of an anti-CD4-depleting mAb as a monotherapy or in combination with immune checkpoint mAbs. In B16F10, Colon 26, or Lewis lung carcinoma subcutaneous tumor models, administration of the anti-CD4 mAb alone had strong antitumor effects that were superior to those elicited by CD25(+) Treg depletion or other immune checkpoint mAbs, and which were completely reversed by CD8(+) cell depletion. CD4(+) cell depletion led to the proliferation of tumor-specific CD8(+) T cells in the draining lymph node and increased infiltration of PD-1(+)CD8(+) T cells into the tumor, with a shift toward type I immunity within the tumor. Combination treatment with the anti-CD4 mAb and immune checkpoint mAbs, particularly anti-PD-1 or anti-PD-L1 mAbs, synergistically suppressed tumor growth and greatly prolonged survival. To our knowledge, this work represents the first report of robust synergy between anti-CD4 and anti-PD-1 or anti-PD-L1 mAb therapies.

Structural basis for the binding of the membrane-proximal C-terminal region of chemokine receptor CCR2 with the cytosolic regulator FROUNT.

The membrane-proximal C-terminal region (Pro-C) is important for the regulation of G-protein-coupled receptors (GPCRs), but the binding of the Pro-C region to a cytosolic regulator has not been structurally analyzed. The chemokine receptor CCR2 is a member of the GPCR superfamily, and the Pro-C region of CCR2 binds to the cytosolic regulator FROUNT. Studying the interaction between CCR2 Pro-C and FROUNT at an atomic level provides a basis for understanding the signal transduction mechanism via GPCRs. NOE-based NMR experiments showed that, when bound to FROUNT, CCR2 Pro-C adopted a helical conformation, as well as when embedded in dodecylphosphocholine micelles. A comparison of two types of cross-saturation-based NMR experiments, applied to a three-component mixture of Pro-C, FROUNT and micelles or a two-component mixture of Pro-C and micelles, revealed that the hydrophobic binding surface on Pro-C for FROUNT mostly overlapped with the binding site for micelles, suggesting competitive binding of Pro-C between FROUNT and micelles. Leu316 was important for both FROUNT and micelle binding. Phe319 was newly identified to be crucial for FROUNT binding, by NMR and mutational analyses. The association and dissociation rates of CCR2 Pro-C for lipid bilayer biomembranes were faster than those for FROUNT. We previously reported that FROUNT binding to CCR2 is detectable even in unstimulated cells and increases in response to chemokine stimulation. Taken together, these results support a model of CCR2 equilibrium: chemokine binding changes the conformational equilibrium of CCR2 toward the active state, and Pro-C switches its binding partner from the membrane to FROUNT.

Identification of a binding element for the cytoplasmic regulator FROUNT in the membrane-proximal C-terminal region of chemokine receptors CCR2 and CCR5.

Chemokine receptors mediate the migration of leucocytes during inflammation. The cytoplasmic protein FROUNT binds to chemokine receptors CCR2 [chemokine (C-C motif) receptor 2] and CCR5, and amplifies chemotactic signals in leucocytes. Although the interaction between FROUNT and chemokine receptors is important for accurate chemotaxis, the interaction mechanism has not been elucidated. In the present study we identified a 16-amino-acid sequence responsible for high-affinity binding of FROUNT at the membrane-proximal C-terminal intracellular region of CCR2 (CCR2 Pro-C) by yeast two-hybrid analysis. Synthesized peptides corresponding to the CCR2 Pro-C sequence directly interacted with FROUNT in vitro. CCR2 Pro-C was predicted to form an amphipathic helix structure. Residues on the hydrophobic side are completely conserved among FROUNT-binding receptors, suggesting that the hydrophobic side is the responsible element for FROUNT binding. The L316T mutation to the hydrophobic side of the predicted helix decreased the affinity for FROUNT. Co-immunoprecipitation assays revealed that the CCR2 L316T mutation diminished the interaction between FROUNT and full-length CCR2 in cells. Furthermore, this mutation impaired the ability of the receptor to mediate chemotaxis. These findings provide the first description of the functional binding element in helix 8 of CCR2 for the cytosolic regulator FROUNT that mediates chemotactic signalling.

Am80, a retinoic acid receptor agonist, ameliorates murine vasculitis through the suppression of neutrophil migration and activation.

OBJECTIVE: Vasculitis is characterized by leukocyte infiltration in the vessel walls, with destructive damage to mural structures. Retinoids are compounds that bind to retinoic acid receptors and exert biologic activities similar to those of vitamin A, including modulatory effects on cell proliferation and differentiation. This study was undertaken to examine the therapeutic effects of a synthetic retinoid, Am80, in a murine model of vasculitis induced by Candida albicans water-soluble fraction (CAWS). METHODS: Vasculitis was induced in BALB/c mice by intraperitoneal injection of CAWS. Neutrophils were depleted by injection of antineutrophil antibody-positive serum. Am80 was administered orally once daily. Vasculitis was evaluated histologically. Migration of labeled adoptively transferred cells was quantified. Chemotaxis was assessed by cell mobility analysis. Production of reactive oxygen species (ROS) and phosphorylation of MAPKs were measured by flow cytometry. Concentrations of elastase were measured by enzyme-linked immunosorbent assay. RESULTS: Administration of CAWS induced vasculitis in the coronary arteries and aortic root, with abundant neutrophil infiltration. Depletion of neutrophils reduced CAWS-induced vasculitis. Treatment with Am80 led to a significant attenuation of the vasculitis score and inhibition of the migration of transferred neutrophils into the site of vasculitis. In vitro, Am80 suppressed fMLP-induced chemotaxis of human peripheral blood neutrophils. ROS production and elastase release by stimulated neutrophils were reduced by AM80 treatment, and Am80 also inhibited phosphorylation of ERK-1/2 and p38 in neutrophils stimulated with fMLP plus lipopolysaccharide. CONCLUSION: Am80 significantly suppressed CAWS-induced vasculitis. This effect was presumably exerted via inhibition of neutrophil migration and activation.