Human inhibitory leukocyte Ig-like receptors: from immunotolerance to immunotherapy.

In recent decades, immunotherapeutic strategies have been used to treat a wide range of pathologies, many of which were previously incurable, such as cancer and autoimmune disorders. Despite this unprecedented success, a considerable number of patients fail to respond to currently approved immunotherapies or develop resistance over time. Therefore, there is an urgent need to develop the next generation of immune-targeted therapies. Various members of the Ig superfamily play essential roles in regulating leukocyte functions. One such group, the leukocyte Ig-like receptors (LILRs), have been implicated in both innate and adaptive immune regulation. Human inhibitory LILRs (LILRBs) are primarily expressed on leukocytes and mediate their signaling through multiple cytoplasmic immunoreceptor tyrosine-based inhibitory motifs. Engagement of LILRBs by endogenous and pathogenic ligands can markedly suppress immune responses, leading to tolerance or immunoevasion, whereas blocking these inhibitory receptors can potentiate immune responses. In this Review, we discuss the immunoregulatory functions of human LILRBs and the potential of targeting them to manipulate immune responses in various pathologies.

Novel CD33 antibodies unravel localization, biology and therapeutic implications of CD33 isoforms.

The aim of this study was to establish the therapeutic relevance of the CD33D2 isoform by developing novel antibodies targeting the IgC domain of CD33. Two novel IgC-targeting antibodies, HL2541 and 5C11-2, were developed, and CD33 isoforms were assessed using multiple assays in cells overexpressing either CD33FL or CD33D2 isoforms, unmodified acute myeloid leukemia (AML) cell lines and primary AML specimens representing different genotypes for the CD33 splicing single nucleotide polymorphism. CD33D2 was recognized on cells overexpressing CD33D2 and unmodified AML cell lines; however, minimal/no cell surface detection of CD33D2 was observed in primary AML specimens. Both isoforms were detected intracellularly using novel antibodies. Minimal cell surface expression of CD33D2 on primary AML/progenitor cells warrants further studies on anti-CD33D2 immunotherapeutics.

Crystal Structures of Bat and Human Coronavirus ORF8 Protein Ig-Like Domain Provide Insights Into the Diversity of Immune Responses.

ORF8 is a viral immunoglobulin-like (Ig-like) domain protein encoded by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA genome. It tends to evolve rapidly and interfere with immune responses. However, the structural characteristics of various coronavirus ORF8 proteins and their subsequent effects on biological functions remain unclear. Herein, we determined the crystal structures of SARS-CoV-2 ORF8 (S84) (one of the epidemic isoforms) and the bat coronavirus RaTG13 ORF8 variant at 1.62 Å and 1.76 Å resolution, respectively. Comparison of these ORF8 proteins demonstrates that the 62-77 residues in Ig-like domain of coronavirus ORF8 adopt different conformations. Combined with mutagenesis assays, the residue Cys20 of ORF8 is responsible for forming the covalent disulfide-linked dimer in crystal packing and in vitro biochemical conditions. Furthermore, immune cell-binding assays indicate that various ORF8 (SARS-CoV-2 ORF8 (L84), ORF8 (S84), and RaTG13 ORF8) proteins have different interaction capabilities with human CD14+ monocytes in human peripheral blood. These results provide new insights into the specific characteristics of various coronavirus ORF8 and suggest that ORF8 variants may influence disease-related immune responses.

Association of soluble T cell immunoglobulin domain and mucin-3 (sTIM-3) and mac-2 binding protein glycosylation isomer (M2BPGi) in patients with autoimmune hepatitis.

BACKGROUND: Autoimmune hepatitis (AIH) is a disorder of unknown etiology in which immune-mediated liver injury progress to cirrhosis or hepatocellular carcinoma (HCC). The aim of the present study was to determine whether circulating soluble TIM3 (sTIM3) is elevated in patients with AIH patients and whether sTIM-3 levels are associated with clinical parameters of AIH. METHODS: We enrolled 123 Japanese patients with AIH who were identified from the National Hospital Organization-AIH-liver-network database, as well as 32 patients with chronic hepatitis C (CHC), 30 patients with primary biliary cholangitis (PBC) and healthy control subjects. Serum sTIM-3 concentrations were quantified by ELISA. RESULTS: Serum levels of sTIM-3 were significantly higher in AIH patients (median 4865 pg/ml; [interquartile range (IQR); 3122-7471]) compared to those in CHC (1026 pg/ml [IQR: 806-1283] p<0.001), PBC (2395 pg/ml [IQR: 2012-3422] p<0.001) or healthy controls (1285 pg/ml [IQR: 1098-1812] p<0.001). In AIH group, serum sTIM-3 were correlated with alanine aminotransferase (ALT), or total bilirubin (TB) and negatively correlated with serum levels of albumin (Alb). Serum levels of sTIM-3 were also strongly correlated with Mac-2 binding protein glycosylation isomer (M2BPGi) levels, but did not correlate with the histological grade of liver fibrosis. Steroid treatment of AIH patients significantly reduced serum sTIM-3 levels (2147±623pg/ml versus 1321±378pg/ml, p<0.001). CONCLUSIONS: Circulating sTIM-3 levels were elevated in AIH patients and are associated with AIH disease activity and AIH-related liver damage. These findings indicate that serum sTIM-3 correlated with disease status of AIH and could be useful biomarkers to detect autoimmune-mediated liver injury. Our data suggest a possible link between the TIM-3/GAL-9 pathway and AIH severity or phenotype, and further investigations of the TIM-3 pathway and AIH pathophysiology is warranted.

Analyzing microglial-associated Aß in Alzheimer's disease transgenic mice with a novel mid-domain Aß-antibody.

The mechanisms of amyloid-ß (Aß)-degradation and clearance in Alzheimer's disease (AD) pathogenesis have been relatively little studied. Short Aß-fragments form by enzymatic cleavage and alternate amyloid-beta precursor protein (APP)-processing. Here we characterized a novel polyclonal Aß-antibody raised against an Aß mid-domain and used it to investigate microglial Aß-uptake in situ by microscopy at the light- and ultrastructural levels. The rabbit Aß-mid-domain antibody (ab338), raised against the mid-domain amino acids 21-34 (Aß21-34), was characterized with biochemical and histological techniques. To identify the epitope in Aß recognized by ab338, solid phase and solution binding data were compared with peptide folding scores as calculated with the Tango software. The ab338 antibody displayed high average affinity (KD: 6.2 × 10-10 M) and showed preference for C-terminal truncated Aß-peptides ending at amino acid 34 and Aß-mid domain peptides with high scores of ß-turn structure. In transgenic APP-mouse brain, ab338 labelled amyloid plaques and detected Aß-fragments in microglia at the ultra- and light microscopic levels. This reinforces a role of microglia/macrophages in Aß-clearance in vivo. The ab338 antibody might be a valuable tool to study Aß-clearance by microglial uptake and Aß-mid-domain peptides generated by enzymatic degradation and alternate production.

Engineering of Group A Streptococcus Isopeptide Bonds into Immunoglobulin-Like Protein Domains.

Intramolecular isopeptide bonds, formed autocatalytically between Lys and Asn/Asp side chains, are widely present in the immunoglobulin-like domains of Gram-positive bacterial adhesins, including Group A Streptococcus, and confer considerable mechanical and chemical stability. These properties make them attractive for applications in biotechnology. Here, we detail the practical considerations that are involved in engineering isopeptide bonds into Ig-like proteins, including the choice of a site where bond-forming residues could be introduced and the appropriate methodology for mutagenesis. We specify how to determine whether an isopeptide bond has formed, what strategies can be adopted to overcome problems, and how to monitor the stability of the engineered protein.

Ancient Use of Ig Variable Domains Contributes Significantly to the TCRδ Repertoire.

The loci encoding B and T cell Ag receptors are generally distinct in commonly studied mammals, with each receptor's gene segments limited to intralocus, cis chromosomal rearrangements. The nurse shark (Ginglymostoma cirratum) represents the oldest vertebrate class, the cartilaginous fish, with adaptive immunity provided via Ig and TCR lineages, and is one species among a growing number of taxa employing Ig-TCRδ rearrangements that blend these distinct lineages. Analysis of the nurse shark Ig-TCRδ repertoire found that these rearrangements possess CDR3 characteristics highly similar to canonical TCRδ rearrangements. Furthermore, the Ig-TCRδ rearrangements are expressed with TCRγ, canonically found in the TCRδ heterodimer. We also quantified BCR and TCR transcripts in the thymus for BCR (IgHV-IgHC), chimeric (IgHV-TCRδC), and canonical (TCRδV-TCRδC) transcripts, finding equivalent expression levels in both thymus and spleen. We also characterized the nurse shark TCRαδ locus with a targeted bacterial artifical chromosome sequencing approach and found that the TCRδ locus houses a complex of V segments from multiple lineages. An IgH-like V segment, nestled within the nurse shark TCRδ translocus, grouped with IgHV-like rearrangements we found expressed with TCRδ (but not IgH) rearrangements in our phylogenetic analysis. This distinct lineage of TCRδ-associated IgH-like V segments was termed "TAILVs." Our data illustrate a dynamic TCRδ repertoire employing TCRδVs, NARTCRVs, bona fide trans-rearrangements from shark IgH clusters, and a novel lineage in the TCRδ-associated Ig-like V segments.

The Fab fragment of anti-IgE Cε2 domain prevents allergic reactions through interacting with IgE-FcεRIα complex on rat mast cells.

Immunoglobulin E (IgE) plays a central role in the pathogenesis of Type I hypersensitivity through interaction with a high-affinity receptor (FcεRIα). For therapeutic applications, substantial attention has been focused recently on the blockade of the IgE interaction with FcεRIα. While exploring better options for preventing allergic diseases, we found that the Fab fragment of the rat anti-murine IgE antibody (Fab-6HD5) strongly inhibited passive cutaneous anaphylaxis (PCA) in vivo, as well as spleen tyrosine kinase (Syk) activity and ß-hexosaminidase release from basophilic leukemia cells in vitro. The in vivo effects of Fab-6HD5 pre-administration were maintained over a long period of time for at least 10 days. Using flow cytometry analysis, we also found that Fab-6HD5 did not recognize the IgE Cε3 domain containing specific binding sites for FcεRIα. Furthermore, deletion-mapping studies revealed that Fab-6HD5 recognized conformational epitopes on the Cε2 domain of IgE. Given that the Cε2 domain plays a key role in stabilizing the interaction of IgE with FcRIα, our results suggest that the specific binding of Fab-6HD5 to the Cε2 domain prevents allergic reactions through destabilizing the preformed IgE-FcεRIα complex on rat mast cells. Although the present study was performed using animal models, these findings support the idea that a certain antibody directed against IgE CH domains may contribute to preventing allergic diseases through interacting with IgE-FcεRIα complex.

ILDR2 Is a Novel B7-like Protein That Negatively Regulates T Cell Responses.

The B7-like protein family members play critical immunomodulatory roles and constitute attractive targets for the development of novel therapies for human diseases. We identified Ig-like domain-containing receptor (ILDR)2 as a novel B7-like protein with robust T cell inhibitory activity, expressed in immune cells and in immune-privileged and inflamed tissues. A fusion protein, consisting of ILDR2 extracellular domain with an Fc fragment, that binds to a putative counterpart on activated T cells showed a beneficial effect in the collagen-induced arthritis model and abrogated the production of proinflammatory cytokines and chemokines in autologous synovial-like cocultures of macrophages and cytokine-stimulated T cells. Collectively, these findings point to ILDR2 as a novel negative regulator for T cells, with potential roles in the development of immune-related diseases, including autoimmunity and cancer.

Sequence composition predicts immunoglobulin superfamily members that could share the intrinsically disordered properties of antibody CH1 domains.

Antibodies are central to the growing sector of protein therapeutics, and increasingly they are being manipulated as fragments and combinations. An improved understanding of the properties of antibody domains in isolation would aid in their engineering. We have conducted an analysis of sequence and domain interactions for IgG antibodies and Fab fragments in the structural database. Of sequence-related properties studied, relative lysine to arginine content was found to be higher in CH1 and CL than in variable domains. As earlier work shows that lysine is favoured over arginine in more soluble proteins, this suggests that individual domains may not be optimised for greater solubility, giving scope for fragment engineering. Across other sequence-based features, CH1 is anomalous. A sequence-based scheme predicts CH1 to be folded, although it is known that CH1 folding is linked to IgG assembly and secretion. Calculations indicate that charge interactions in CH1 domains contribute less to folded state stability than in other Fab domains. Expanding to the immunoglobulin superfamily reveals that a subset of non-antibody domains shares sequence composition properties with CH1, leading us to suggest that some of these may also couple folding, assembly and secretion.

SH3 dependent cell death signaling of the avian chB6 alloantigen.

In chickens, B cells develop in the bursa of Fabricius, a unique organ for B cell development. Most B cells will die within the bursa, mirroring cell losses seen in mammalian bone marrow as central tolerance is enforced at the transition to mature cells. B cell responses are shaped by a complex interplay of signals. Signals in addition to BCR that impact central tolerance have recently been described. We have been interested in chB6, a novel alloantigen on B cells in the chicken. chB6 is found in close proximity to the BCR and can trigger apoptosis after cross-linking by antibody. chB6 has two Ig domains, placing it within the CD2/SLAM family of molecules, but its cytoplasmic domain is unique. We have used a site-specific mutagenesis approach to show that an SH3 binding site in chB6 is required for the induction of apoptosis, suggesting parallels to CD2 signaling.

Elasticity of the Transition State Leading to an Unexpected Mechanical Stabilization of Titin Immunoglobulin Domains.

The giant protein titin plays a critical role in regulating the passive elasticity of muscles, mainly through the stochastic unfolding and refolding of its numerous immunoglobulin domains in the I-band of sarcomeres. The unfolding dynamics of titin immunoglobulin domains at a force range greater than 100 pN has been studied by atomic force microscopy, while that at smaller physiological forces has not been measured before. By using magnetic tweezers, it is found that the titin I27 domain unfolds in a surprising non-monotonic force-dependent manner at forces smaller than 100 pN, with the slowest unfolding rate occurring around 22 pN. We further demonstrate that a model with single unfolding pathway taking into account the elasticity of the transition state can reproduce the experimental results. These results provide important novel insights into the regulation mechanism of the passive elasticity of muscle tissues.