Single Immunoglobulin Research Articles

The hyaluronan-binding activity of aggrecan is important, but not essential, for its specific insertion into perineuronal nets.

Aggrecan (ACAN) is a large, secreted chondroitin sulfate proteoglycan that includes three globular regions named G1, G2, G3, and is decorated with multiple glycosaminoglycan attachments between its G2 and G3 domains. The N-terminal G1 region interacts with the glycosaminoglycan hyaluronan (HA), which is an essential component of the vertebrate extracellular matrix. In the central nervous system, ACAN is found in perineuronal nets (PNNs), honeycomb-like structures that are enriched on parvalbumin-positive neurons in specific neural circuits. PNNs regulate the plasticity of the central nervous system, and it is believed that association between ACAN and HA is a foundational event in the assembly of these reticular structures. Here, we report the co-crystal structure of the G1 region of ACAN in the absence and presence of an HA decasaccharide and analyze the importance of the HA-binding activity of ACAN for its integration into PNNs. We demonstrate that the single immunoglobulin domain and the two Link modules that comprise the G1 region form a single structural unit, and that HA is clamped inside a groove that spans the length of the tandem Link domains. Introduction of point mutations in the glycosaminoglycan-binding site eliminates HA-binding activity in ACAN, but, surprisingly, only decreases the integration of ACAN into PNNs. Thus, these results suggest that the HA-binding activity of ACAN is important for its recruitment to PNNs, but it does not appear to be essential.

Nanobody-based heavy chain antibodies and chimeric antibodies.

Nanobodies are the products of an intriguing invention in the evolution of immunoglobulins. This invention can be traced back approximately 45 million years to the common ancestor of extant dromedaries, camels, llamas, and alpacas. Next to conventional heterotetrameric H2L2 antibodies, these camelids produce homodimeric nanobody-based heavy chain antibodies, composed of shortened heavy chains that a lack the CH1 domain. Nanobodies against human target antigens are derived from immunized animals and/or synthetic nanobody libraries. As a robust, highly soluble, single immunoglobulin domain, a nanobody can easily be fused to another protein, for example to another nanobody and/or the hinge and constant domains of other immunoglobulins. Nanobody-derived heavy chain antibodies hold promise as a new form of immunotherapeutics.

Congophilic fibrils in the glomeruli with polyclonal immunoglobulin gamma staining - another cause for diagnostic overlap: A case report

BACKGROUND Glomerulopathy with fibrillary deposits is not uncommon in routine nephropathology practice, with amyloidosis and fibrillary glomerulonephritis being the two most frequently encountered entities. Renal amyloid heavy and light chain (AHL) is relatively uncommon and its biopsy diagnosis is usually limited to cases that show strong equivalent staining for a single immunoglobulin (Ig) heavy chain and a single light chain, further supported by mass spectrometry (MS) and serum studies for monoclonal protein. But polyclonal light chain staining can pose a challenge. CASE SUMMARY Herein we present a challenging case of renal AHL with polyclonal and polytypic Ig gamma (IgG) staining pattern by immunofluorescence. The patient is a 62-year-old Caucasian male who presented to an outside institution with a serum creatinine of up to 8.1 mg/dL and nephrotic range proteinuria. Despite the finding of a polyclonal and polytypic staining pattern on immunofluorescence, ultrastructural study of the renal biopsy demonstrated the presence of fibrils with a mean diameter of 10 nm. Congo red was positive while DNAJB9 was negative. MS suggested a diagnosis of amyloid AHL type with IgG and lambda, but kappa light chains were also present supporting the immunofluorescence staining results. Serum immunofixation studies demonstrated IgG lambda monoclonal spike. The patient was started on chemotherapy. The chronic renal injury however was quite advanced and he ended up needing dialysis shortly after. CONCLUSION Tissue diagnosis of AHL amyloid can be tricky. Thorough confirmation using other available diagnostic techniques is recommended in such cases.

Near-Field Terahertz Morphological Reconstruction Nanoscopy for Subsurface Imaging of Protein Layers.

Protein layers formed on solid surfaces have important applications in various fields. High-resolution characterization of the morphological structures of protein forms in the process of developing protein layers has significant implications for the control of the layer's quality as well as for the evaluation of the layer's performance. However, it remains challenging to precisely characterize all possible morphological structures of protein in various forms, including individuals, networks, and layers involved in the formation of protein layers with currently available methods. Here, we report a terahertz (THz) morphological reconstruction nanoscopy (THz-MRN), which can reveal the nanoscale three-dimensional structural information on a protein sample from its THz near-field image by exploiting an extended finite dipole model for a thin sample. THz-MRN allows for both surface imaging and subsurface imaging with a vertical resolution of ∼0.5 nm, enabling the characterization of various forms of proteins at the single-molecule level. We demonstrate the imaging and morphological reconstruction of single immunoglobulin G (IgG) molecules, their networks, a monolayer, and a heterogeneous double layer comprising an IgG monolayer and a horseradish peroxidase-conjugated anti-IgG layer. The established THz-MRN presents a useful approach for the label-free and nondestructive study of the formation of protein layers.

Mechanism of Anti-Salmonella Rabbit Immunoglobulin Adsorption on Polymer Particles.

The adsorption of anti-Salmonella rabbit immunoglobulin (IgaR) on negatively charged polymer particles leading to the formation of immunolatex was studied using various techniques comprising atomic force microscopy (AFM) and laser Doppler velocimetry (LDV). Initially, the basic physicochemical properties of IgaR molecules and the particles, inter alia their electrophoretic mobilities, the zeta potentials and hydrodynamic diameters, were determined under different ionic strengths and pHs. Applying AFM, single immunoglobulin molecules adsorbed on mica were also imaged, which allowed to determine their size. The adsorption of the IgaR molecules on the particles leading to changes in their electrophoretic mobility was monitored in situ using the LDV method. The obtained results were interpreted applying a general electrokinetic model which yielded quantitative information about the molecule coverage on the particles. The obtained immunolatex was thoroughly characterized with respect to its acid-base properties and its stability upon storage. Notably, the developed procedure demonstrated better efficiency compared to commercially applied methods, characterized by a higher immunoglobulin consumption.

The role of single immunoglobulin interleukin-1–related receptor in formation of necrotising enterocolitis in infants with intrauterine infection

Necrotizing Enterocolitis is one of the actual problems that primarily affects premature infants, particularly those born before 32 weeks of gestation. The aim of this study is to investigate the role of genetic factors in the development of both necrotizing enterocolitis and intrauterine infection, exploring the possible links between these two conditions. A total of 52 with NEC and 10 healthy, were included in this study. All the examined children with NEC were divided into 2 groups: 19 newborns with intrauterine infection (1st subgroup), 33 infants without intrauterine infection (2nd subgroup). It was determined SIGIRR gene mutations with DNA isolation and standardized PCR reactions. It was found that C58 gene locus changes is a potential predictive marker for differentiating between infants with intrauterine infection and NEC and those without these conditions. Genetic screening may help identify infants at higher risk of developing NEC, allowing for targeted interventions and personalized care.

Deregulated Expression of IL-37 in Patients with Bladder Urothelial Cancer: The Diagnostic Potential of the IL-37e Isoform

Cellular and molecular immune components play a crucial role in the development and perpetuation of human malignancies, shaping anti-tumor responses. A novel immune regulator is interleukin-37 (IL-37), already shown to be involved in the inflammation associated with the pathophysiology of many human disorders, including cancer. The interplay between tumor and immune cells is of great importance, especially for highly immunogenic tumors such as bladder urothelial carcinoma (BLCA). This study aimed to investigate the potential of IL-37 and its receptor SIGIRR (single immunoglobulin IL-1-related receptor) to serve as prognostic and/or diagnostic markers in patients with BLCA. To this end, a series of bioinformatics tools processing -omics datasets and specifically designed qPCR assays on human BLCA tumors and cancer cell lines were utilized. Bioinformatics analysis revealed that IL-37 levels correlate with BLCA tumor development and are higher in patients with longer overall survival. Furthermore, mutations on SIGIRR are associated with enhanced infiltration of the tumor by regulatory T cells and dendritic cells. Based on the qPCR validation experiments, BLCA epithelial cells express the IL-37c and IL-37e isoforms, while the latter is the predominant variant detected in tumor biopsies, also associated with higher grade and the non-muscle-invasive type. This is the first time, to the best of our knowledge, that IL-37 and SIGIRR levels have been assessed in BLCA tumor lesions, and associations with pathological and survival parameters are described, while a transcript variant-specific signature is indicated to have a diagnostic potential. These data strongly indicate the need for further investigation of the involvement of this cytokine and interconnected molecules in the pathophysiology of the disease and its prospective as a therapeutic target and biomarker for BLCA.

Interleukin-37 promotes DMBA/TPA skin cancer through SIGIRR-mediated inhibition of glycolysis in CD103+DC cells.

Interleukin 37 (IL-37), a member of the IL-1 family, is considered a suppressor of innate and adaptive immunity and, hence is a regulator of tumor immunity. However, the specific molecular mechanism and role of IL-37 in skin cancer remain unclear. Here, we report that IL-37b-transgenic mice (IL-37tg) treated with the carcinogenic 7,12-dimethylbenzoanthracene (DMBA)/12-o-tetradecylphorbol-13-acetate (TPA) exhibited enhanced skin cancer and increased tumor burden in the skin by inhibiting the function of CD103+ dendritic cells (DCs). Notably, IL-37 induced rapid phosphorylation of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), and via single immunoglobulin IL-1-related receptor (SIGIRR), inhibited the long-term Akt activation. Specifically, by affecting the SIGIRR-AMPK-Akt signaling axis, which is related to the regulation of glycolysis in CD103+DCs, IL-37 inhibited their anti-tumor function. Our results show that a marked correlation between the CD103+DC signature (IRF8, FMS-like tyrosine kinase 3 ligand, CLEC9A, CLNK, XCR1, BATF3, and ZBTB46) and chemokines C-X-C motif chemokine ligand 9, CXCL10, and CD8A in a mouse model with DMBA/TPA-induced skin cancer. In a word, our results highlight that IL-37 as an inhibitor of tumor immune surveillance through modulating CD103+DCs and establishing an important link between metabolism and immunity as a therapeutic target for skin cancer.

Differences in the clinical characteristics of Kawasaki disease between older and younger children (2015-2019): A single-center, retrospective study.

The objective of this study was to investigate the differences in the clinical characteristics of Kawasaki disease between older and younger children. This retrospective study examined 405 children with Kawasaki disease admitted to Showa University Northern Yokohama Hospital between 2015 and2019. Eligible patients were classified into the older (≥3.0years of age, n=169) and younger (<3.0years of age, n=236) groups. Skin rash was found in significantly fewer cases (112 [66.3%] vs 229 [97.0%], P<.001 in the younger group). Cervical lymphadenopathy was more common in older children (153 [90.5%] vs 165 [69.9%], P<.001) and in incomplete Kawasaki disease (3 or 4 findings) (34 [20.1%] vs 25 [10.6%], P=.0078). The diagnosis was more delayed in older children (median: 5.0days vs 4.0days, P=.003) than the younger group. Additionally, fever nonresponsive to a single intravenous immunoglobulin was more common, and the duration of fever was significantly longer in the older group (48 [28.4%] vs 47 [19.9%], P=.0479). Kawasaki disease should be suspected in children aged >3.0years with cervical lymphadenopathy and fever, despite the absence of skin rash. Additionally, incomplete Kawasaki disease, fever unresolved by a single intravenous immunoglobulin infusion, and the tendency to delay treatment initiation are more common in children aged >3.0years.

A TMEM16J variant leads to dysregulated cytosolic calcium which may lead to renal disease.

SIGIRR (single immunoglobulin IL-1 related receptor), PKP3 (plakophilin 3), and TMEM16J (anoctamin 9), a putative calcium-activated ion channel and phospholipid scramblase, control the immune response and the extent of inflammation. Variants of SIGIRR/PKP3/TMEM16J lead to severe inflammatory diseases such as pneumonia, enterocolitis, and kidney graft rejection. Meta-analysis of genome-wide association studies identified TMEM16J-T604A as a promotor for chronic kidney disease (CKD), but the disease mechanism and function of TMEM16J remain unknown. Here, we demonstrate TMEM16J as a calcium-activated calcium-permeable channel, which is expressed in the endoplasmic reticulum (ER). TMEM16J controls the intracellular distribution of calcium, and inhibits intracellular receptor-mediated Ca2+ signals and Ca2+ -dependent activation of ion channels, but augments transcription and release of pro-inflammatory cytokines. Renal epithelial cells expressing the variant TMEM16J-T604A show enhanced calcium signals when compared to cells expressing wt-TMEM16J, and demonstrate spontaneous transcription and release of cytokines. This study identifies TMEM16J as an important regulator of intracellular Ca2+ signals, ion channel activity, and cytokine release. TMEM16J may therefore affect immune response in renal tissue and immune cells.

Cardiac sodium channel complexes and arrhythmia: structural and functional roles of the β1 and β3 subunits.

In cardiac myocytes, the voltage-gated sodium channel NaV 1.5 opens in response to membrane depolarisation and initiates the action potential. The NaV 1.5 channel is typically associated with regulatory β-subunits that modify gating and trafficking behaviour. These β-subunits contain a single extracellular immunoglobulin (Ig) domain, a single transmembrane α-helix and an intracellular region. Here we focus on the role of the β1 and β3 subunits in regulating NaV 1.5. We catalogue β1 and β3 domain specific mutations that have been associated with inherited cardiac arrhythmia, including Brugada syndrome, long QT syndrome, atrial fibrillation and sudden death. We discuss how new structural insights into these proteins raises new questions about physiological function.

Aberrant Expression and Prognostic Potential of IL-37 in Human Lung Adenocarcinoma.

Interleukin-37 (IL-37) is a relatively new IL-1 family cytokine that, due to its immunoregulatory properties, has lately gained increasing attention in basic and translational biomedical research. Emerging evidence supports the implication of this protein in any human disorder in which immune homeostasis is compromised, including cancer. The aim of this study was to explore the prognostic and/or diagnostic potential of IL-37 and its receptor SIGIRR (single immunoglobulin IL-1-related receptor) in human tumors. We utilized a series of bioinformatics tools and -omics datasets to unravel possible associations of IL-37 and SIGIRR expression levels and genetic aberrations with tumor development, histopathological parameters, distribution of tumor-infiltrating immune cells, and survival rates of patients. Our data revealed that amongst the 17 human malignancies investigated, IL-37 exhibits higher expression levels in tumors of lung adenocarcinoma (LUAD). Moreover, the expression profiles of IL-37 and SIGIRR are associated with LUAD development and tumor stage, whereas their high mRNA levels are favorable prognostic factors for the overall survival of patients. What is more, IL-37 correlates positively with a LUAD-associated transcriptomic signature, and its nucleotide changes and expression levels are linked with distinct infiltration patterns of certain cell subsets known to control LUAD anti-tumor immune responses. Our data indicate the potential value of IL-37 and its receptor SIGIRR to serve as biomarkers and/or immune-checkpoint therapeutic targets for LUAD patients. Further, the data highlight the urgent need for further exploration of this cytokine and the underlying pathogenetic mechanisms to fully elucidate its implication in LUAD development and progression.

Short-chain fatty acids ameliorate necrotizing enterocolitis-like intestinal injury through enhancing Notch1-mediated single immunoglobulin interleukin-1-related receptor, toll-interacting protein, and A20 induction.

Single immunoglobulin interleukin-1-related receptor (SIGIRR), toll-interacting protein (TOLLIP), and A20 are major inhibitors of toll-like receptor (TLR) signaling induced postnatally in the neonatal intestine. Short-chain fatty acids (SCFAs), fermentation products of indigestible carbohydrates produced by symbiotic bacteria, inhibit intestinal inflammation. Herein, we investigated the mechanisms by which SCFAs regulate SIGIRR, A20, and TOLLIP expression and mitigate experimental necrotizing enterocolitis (NEC). Butyrate induced NOTCH activation by repressing sirtuin 1 (SIRT1)-mediated deacetylation of the Notch intracellular domain (NICD) in human intestinal epithelial cells (HIECs). Overexpression of NICD induced SIGIRR, A20, and TOLLIP expression. Chromatin immunoprecipitation revealed that butyrate-induced NICD binds to the SIGIRR, A20, and TOLLIP gene promoters. Notch1-shRNA suppressed butyrate-induced SIGIRR/A20 upregulation in mouse enteroids and HIEC. Flagellin (TLR5 agonist)-induced inflammation in HIEC was inhibited by butyrate in a SIGIRR-dependent manner. Neonatal mice fed butyrate had increased NICD, A20, SIGIRR, and TOLLIP expression in the ileal epithelium. Butyrate inhibited experimental NEC-induced intestinal apoptosis, cytokine expression, and histological injury. Our data suggest that SCFAs can regulate the expression of the major negative regulators of TLR signaling in the neonatal intestine through Notch1 and ameliorate experimental NEC. Enteral SCFAs supplementation in preterm infants provides a promising bacteria-free, therapeutic option for NEC.NEW & NOTEWORTHY Short-chain fatty acids (SCFAs), such as propionate and butyrate, metabolites produced by symbiotic gut bacteria are known to be anti-inflammatory, but the mechanisms by which they protect against NEC are not fully understood. In this study, we reveal that SCFAs regulate intestinal inflammation by inducing the key TLR and IL1R inhibitors, SIGIRR and A20, through activation of the pluripotent transcriptional factor NOTCH1. Butyrate-mediated SIGIRR and A20 induction represses experimental NEC in the neonatal intestine.

SIGIRR deficiency contributes to CD4 T cell abnormalities by facilitating the IL1/C/EBPβ/TNF-α signaling axis in rheumatoid arthritis

BackgroundRheumatoid arthritis (RA) is a complex autoimmune disease with multiple etiological factors, among which aberrant memory CD4 T cells activation plays a key role in the initiation and perpetuation of the disease. SIGIRR (single immunoglobulin IL-1R-related receptor), a member of the IL-1 receptor (ILR) family, acts as a negative regulator of ILR and Toll-like receptor (TLR) downstream signaling pathways and inflammation. The aim of this study was to investigate the potential roles of SIGIRR on memory CD4 T cells in RA and the underlying cellular and molecular mechanisms.MethodsSingle-cell transcriptomics and bulk RNA sequencing data were integrated to predict SIGIRR gene distribution on different immune cell types of human PBMCs. Flow cytometry was employed to determine the differential expression of SIGIRR on memory CD4 T cells between the healthy and RA cohorts. A Spearman correlation study was used to determine the relationship between the percentage of SIGIRR+ memory CD4 T cells and RA disease activity. An AIA mouse model (antigen-induced arthritis) and CD4 T cells transfer experiments were performed to investigate the effect of SIGIRR deficiency on the development of arthritis in vivo. Overexpression of SIGIRR in memory CD4 T cells derived from human PBMCs or mouse spleens was utilized to confirm the roles of SIGIRR in the intracellular cytokine production of memory CD4 T cells. Immunoblots and RNA interference were employed to understand the molecular mechanism by which SIGIRR regulates TNF-α production in CD4 T cells.ResultsSIGIRR was preferentially distributed by human memory CD4 T cells, as revealed by single-cell RNA sequencing. SIGIRR expression was substantially reduced in RA patient-derived memory CD4 T cells, which was inversely associated with RA disease activity and related to enhanced TNF-α production. SIGIRR-deficient mice were more susceptible to antigen-induced arthritis (AIA), which was attributed to unleashed TNF-α production in memory CD4 T cells, confirmed by decreased TNF-α production resulting from ectopic expression of SIGIRR. Mechanistically, SIGIRR regulates the IL-1/C/EBPβ/TNF-α signaling axis, as established by experimental evidence and cis-acting factor bioinformatics analysis.ConclusionTaken together, SIGIRR deficiency in memory CD4 T cells in RA raises the possibility that receptor induction can target key abnormalities in T cells and represents a potentially novel strategy for immunomodulatory therapy.

Single cell clonal analysis identifies an AID‐dependent pathway of plasma cell differentiation

Germinal centers (GC) are microstructures where B cells that have been activated by antigen can improve the affinity of their B cell receptors and differentiate into memory B cells (MBCs) or antibody‐secreting plasma cells. Here, we have addressed the role of activation‐induced deaminase (AID), which initiates somatic hypermutation and class switch recombination, in the terminal differentiation of GC B cells. By combining single cell transcriptome and immunoglobulin clonal analysis in a mouse model that traces AID‐experienced cells, we have identified a novel subset of late‐prePB cells (L‐prePB), which shares the strongest clonal relationships with plasmablasts (PBs). Mice lacking AID have various alterations in the size and expression profiles of transcriptional clusters. We find that AID deficiency leads to a reduced proportion of L‐prePB cells and severely impairs transitions between the L‐prePB and the PB subsets. Thus, AID shapes the differentiation fate of GC B cells by enabling PB generation from a prePB state.

Molecular characterization and functional analysis of DIGIRR from golden pompano (Trachinotus ovatus).

Mammalian single immunoglobulin (Ig) interleukin-1 receptor related molecule (SIGIRR), an important member of the Toll/interleukin-1 receptor (TIR) family, plays important balancing roles in the inflammatory responses. In the present study, the double Ig interleukin-1 receptor related molecule (DIGIRR), the homologous of SIGIRR, was characterized in golden pompano (Trachinotus ovatus) (termed as trDIGIRR). The full-length cDNA of trDIGIRR was 2,167 bp with an open reading frame (ORF) of 1,572 bp encoding 523 amino acids. The trDIGIRR contained several conserved domains including a signal peptide, two Ig domains, a transmembrane domain and a TIR domain, and shared high sequence identities with its teleost counterparts. Realtime qPCR analysis revealed that the trDIGIRR was distributed in all tissues examined, with high expressions in intestine, liver and head kidney. The expressions of trDIGIRR were induced by Vibrio alginolyticus, lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (poly I:C) challenge. Further analysis revealed that trDIGIRR was mainly located in the cytoplasm. In addition, the co-immunoprecipitation (co-IP) assay identified that trDIGIRR could interact with myeloid differentiation factor 88 (MyD88), but not interact with TIR domain containing adaptor protein inducing interferon-β (TRIF). Our results provide basis for studying the immune role of fish DIGIRR.

Dominant-Negative Form of SIGIRR: SIGIRRΔE8 Promotes Tumor Growth Through Regulation of Metabolic Pathways

Colorectal carcinoma is the leading cause of cancer-related death. Previously we have shown that tumor suppressor single immunoglobulin interleukin-1-related receptor (SIGIRR) is frequently inactivated in human colorectal cancer by the increased expression of a novel SIGIRR isoform (SIGIRRΔE8). SIGIRRΔE8 showed increased retention in the cytoplasm and loss of complex glycan modification compared to the full-length SIGIRR. Now we found that the arginine residues located in the C-terminus of SIGIRRΔE8 serve as an endoplasmic reticulum retention signal and are required for resident protein ribophorin 1 (RPN1) interaction. In addition, we found that SIGIRRΔE8 exerts a direct impact on cell metabolism through interaction with the adenosine triphosphate synthase in the colorectal cancer cells. SIGIRRΔE8 expression promoted the metabolic shift through upregulation of mammalian target of rapamycin signaling pathway and dysregulation of mitochondrial function to promote survival and proliferation of colon cancer cells in xenograft model.

A Splice Switch in SIGIRR Causes a Defect of IL-37-Dependent Anti-Inflammatory Activity in Cystic Fibrosis Airway Epithelial Cells.

Cystic fibrosis (CF) is a hereditary disease typically characterized by infection-associated chronic lung inflammation. The persistent activation of toll-like receptor (TLR) signals is considered one of the mechanisms for the CF hyperinflammatory phenotype; however, how negative regulatory signals of TLRs associate with CF inflammation is still elusive. Here, we showed that the cell surface expression of a single immunoglobulin interleukin-1 receptor (IL-1R)-related molecule (SIGIRR), a membrane protein essential for suppressing TLRs- and IL-1R-dependent signals, was remarkably decreased in CF airway epithelial cells compared to non-CF cells. Notably, CF airway epithelial cells specifically and highly expressed a unique, alternative splice isoform of the SIGIRR that lacks exon 8 (Δ8-SIGIRR), which results in the production of a C-terminal truncated form of the SIGIRR. Δ8-SIGIRR was expressed intracellularly, and its over-expression abolished the cell surface expression and function of the full-length SIGIRR (WT-SIGIRR), indicating its dominant-negative effect leading to the deficiency of anti-inflammatory activity in CF cells. Consistently, IL-37, a ligand for the SIGIRR, failed to suppress viral dsRNA analogue poly(I:C)-dependent JNK activation and IL-8 production, confirming the reduction in the functional WT-SIGIRR expression in the CF cells. Together, our studies reveal that SIGIRR-dependent anti-inflammatory activity is defective in CF airway epithelial cells due to the unique splicing switch of the SIGIRR gene and provides the first evidence of IL-37-SIGIRR signaling as a target of CF airway inflammation.

Interleukin-37 promotes colitis-associated carcinogenesis via SIGIRR-mediated cytotoxic T cells dysfunction

Interleukin-37b (hereafter called IL-37) was identified as fundamental inhibitor of natural and acquired immunity. The molecular mechanism and function of IL-37 in colorectal cancer (CRC) has been elusive. Here, we found that IL-37 transgenic (IL-37tg) mice were highly susceptible to colitis-associated colorectal cancer (CAC) and suffered from dramatically increased tumor burdens in colon. Nevertheless, IL-37 is dispensable for intestinal mutagenesis, and CRC cell proliferation, apoptosis, and migration. Notably, IL-37 dampened protective cytotoxic T cell-mediated immunity in CAC and B16-OVA models. CD8+ T cell dysfunction is defined by reduced retention and activation as well as failure to proliferate and produce cytotoxic cytokines in IL-37tg mice, enabling tumor evasion of immune surveillance. The dysfunction led by IL-37 antagonizes IL-18–induced proliferation and effector function of CD8+ T cells, which was dependent on SIGIRR (single immunoglobulin interleukin-1 receptor-related protein). Finally, we observed that IL-37 levels were significantly increased in CRC patients, and positively correlated with serum CRC biomarker CEA levels, but negatively correlated with the CD8+ T cell infiltration in CRC patients. Our findings highlight the role of IL-37 in harnessing antitumor immunity by inactivation of cytotoxic T cells and establish a new defined inhibitory factor IL-37/SIGIRR in cancer-immunity cycle as therapeutic targets in CRC.

Production of Single-Domain Antibodies in Pichia pastoris.

Single-domain antibodies (sdAbs) are binders that consist of a single immunoglobulin domain. SdAbs have gained importance as therapeutics, diagnostic reagents, and research tools. Functional sdAbs are commonly produced in Escherichia coli, which is a simple and widely used host for production of recombinant proteins. However, there are drawbacks of the E. coli expression system, including the potential for misfolded recombinant proteins and pyrogeniccontamination with toxic lipopolysaccharides. Pichia pastoris is an alternative host for the production of heterologous proteins because of its high recombinant protein yields and the ability to produce soluble, properly folded proteins without lipopolysaccharide contamination. Here, we describe a method to produce sdAbs in P. pastoris. We present methods for the cloning of sdAb-encoding genes into a P. pastoris expression vector, production and purification of sdAbs, and measurement of sdAb-binding kinetics. Functional sdAbs are easily and routinely obtained using these methods.