Search

Genome-level rearrangements of Ig genes during B cell development are critical for generation of a diverse repertoire of BCRs that bind to a multitude of foreign Ags and some self Ags. Bone marrow B cell development involves a variety of cell–cell interactions, cell migration, and receptor signaling that likely benefit from the activity of membrane-cytoskeletal reorganizing proteins. However, the specific contribution of such proteins toward BCR repertoire diversification is poorly understood. Ezrin is a membrane-cytoskeletal linker protein that regulates mature B cell activation through spatial organization of the BCR. We employed next-generation sequencing to investigate whether Ezrin plays a role in IgH rearrangements and generation of BCR diversity in developing bone marrow B cells. BCR repertoire development occurred stochastically in B cell progenitors from both control and B cell conditional Ezrin-deficient mice. However, the loss of Ezrin resulted in fewer unique CDRs (CDR3s) in the BCRs and reduced Shannon entropy. Ezrin-deficient pre-B cells revealed similar utilization of joining (J) genes but significantly fewer variable (V) genes, thereby decreasing V-J combinatorial diversity. V-J junctional diversity, measured by CDR3 length and nucleotide additions and deletions, was not altered in Ezrin-deficient pre-B cells. Mechanistically, Ezrin-deficient cells showed a marked decrease in RAG1 gene expression, indicating a less efficient DNA recombination machinery. Overall, our results demonstrate that Ezrin shapes the BCR repertoire through combinatorial diversification.

The ability of individual T and B cells to display Ag receptors of unique uniform specificity is the molecular basis of adaptive immunity. Most αβ T cells achieve uniform specificity by assembling in-frame genes on only one allelic copy of TCRβ and TCRα loci, while others prevent incorporation of TCRα protein from both alleles into TCRs. Analysis of mice expressing TCR proteins from a restricted combination of transgenes showed that TCR protein pairing restrictions achieve uniform specificity of cells expressing two types of TCRβ protein. However, whether this mechanism operates in the physiological context where each dual-TCRβ cell expresses one set of a vast number of different TCRβ proteins remains an open question, largely because there is a low, but significant, portion of cells carrying two in-frame TCRβ genes. To resolve this issue, we inactivated one allelic copy of the TCRα locus in a new mouse strain that assembles two in-frame TCRβ genes in an elevated fraction of cells. This genetic manipulation has no effect on the frequency of cells that display multiple types of αβ TCR, yet increases the representation of cells displaying TCRβ proteins that generate more highly expressed TCRs. Our data demonstrate that some TCRβ proteins exhibit differential functional pairing with TCRα proteins, but these restrictions have negligible contribution for ensuring uniform specificity of cells that express two types of TCRβ protein. Therefore, we conclude that mechanisms governing monogenic assembly and expression of TCRβ genes in individual cells are paramount for uniform specificity of αβ T lymphocytes.

CD4

The variable domain of the new antigen receptor (V

Heavy chain only binding proteins, such as variable new antigen receptors (VNARs), have emerged as an alternative to the highly successful therapeutic monoclonal antibodies (mAb). Owing to their small size (∼ 11 kDa) and single chain only architecture, they are amenable to modular reformatting and can be produced using inexpensive expression systems. Furthermore, due to their low molecular weight (MW) and high stability, they may be suitable for alternative delivery strategies, such as microarray array patches (MAPs). In this study, the transdermal delivery of ELN22-104, a multivalent anti-hTNF-α VNAR, was examined using both dissolving and hydrogel-forming MAPs. For dissolving MAPs, the cumulative in vitro permeation of ELN22-104 reached a plateau after 2 h (12.24 ± 0.17 µg). This could be important for bolus dosing. Assessing two hydrogel-forming MAPs in vitro, PVP/PVA hydrogel-forming MAPs delivered significantly higher drug doses when compared to 'super swelling' MAPs, equivalent to 43.13 ± 10.36 µg and 23.13 ± 5.66 µg, respectively (p < 0.05). Consequently, this study has proven that by modifying the MAP system, the transdermal delivery of a VNAR across the skin can be enhanced. Furthermore, this proof-of-concept study has shown that transdermal delivery of 'next generation' biotherapeutics is achievable using MAP technology.

In the adaptive immune system, V(D)J recombination initiates the production of a diverse antigen receptor repertoire in developing B and T cells. Recombination activating proteins, RAG1 and RAG2 (RAG1/2), catalyze V(D)J recombination by cleaving adjacent to recombination signal sequences (RSSs) that flank antigen receptor gene segments. Previous studies defined the consensus RSS as containing conserved heptamer and nonamer sequences separated by a less conserved 12 or 23 base-pair spacer sequence. However, many RSSs deviate from the consensus sequence. Here, we developed a cell-based, massively parallel assay to evaluate V(D)J recombination activity on thousands of RSSs where the 12-RSS heptamer and adjoining spacer region contained randomized sequences. While the consensus heptamer sequence (CACAGTG) was marginally preferred, V(D)J recombination was highly active on a wide range of non-consensus sequences. Select purine/pyrimidine motifs that may accommodate heptamer unwinding in the RAG1/2 active site were generally preferred. In addition, while different coding flanks and nonamer sequences affected recombination efficiency, the relative dependency on the purine/pyrimidine motifs in the RSS heptamer remained unchanged. Our results suggest RAG1/2 specificity for RSS heptamers is primarily dictated by DNA structural features dependent on purine/pyrimidine pattern, and to a lesser extent, RAG:RSS base-specific interactions.

Sharks and other cartilaginous fish produce new antigen receptor (IgNAR) antibodies, as key part of their humoral immune response and are the phylogenetically oldest living organisms that possess an immunoglobulin (Ig)-based adaptive immune system. IgNAR antibodies are naturally occurring heavy-chain-only antibodies, that recognize antigens with their single domain variable regions (VNARs). In this study, we structurally and biophysically elucidate the effect of antibody humanization of a previously published spiny dogfish VNAR (parent E06), which binds with high affinity to the human serum albumin (HSA). We analyze different humanization variants together with the parental E06 VNAR and the human Vκ1 light chain germline DPK9 antibody to characterize the influence of point mutations in the framework and the antigen binding site on the specificity of VNARs as reported by Kovalenko et al. We find substantially higher flexibility in the humanized variants, reflected in a broader conformational space and a higher conformational entropy, as well as population shifts of the dominant binding site ensembles in solution. A further variant, in which some mutations are reverted, largely restores the conformational stability and the dominant binding minimum of the parent E06. We also identify differences in surface hydrophobicity between the human Vκ1 light chain germline DPK9 antibody, the parent VNAR E06 and the humanized variants. Additional simulations of VNAR-HSA complexes of the parent E06 VNAR and a humanized variant reveal that the parent VNAR features a substantially stronger network of stabilizing interactions. Thus, we conclude that a structural and dynamic understanding of the VNAR binding site upon humanization is a key aspect in antibody humanization.

Chimeric antigen receptor (CAR) T cell therapy that involves genetic engineering a patient's own immune cells with antigen-specific receptors has shown remarkable efficacy in blood cancer treatment. Numerous clinical studies with CAR T cells targeting the blood cell surface protein CD19 led to the FDA 's first approval of a genetically engineered cell therapy. The process of generating potent CAR T cells involves several carefully performed manufacturing steps. Here, we describe the generation of redirected engineered human CAR T cells for preclinical studies starting with the CAR design, retroviral gene transfer, detection of CAR expression, and expansion of transduced T cells.

Immunoglobulins and T cell receptors (TCR) have obvious structural similarities as well as similar immunogenetic diversification and selection mechanisms. Nevertheless, the two receptor systems and the loci that encode them are distinct in humans and classical murine models, and the gene segments comprising each repertoire are mutually exclusive. Additionally, while both B and T cells employ recombination-activating genes (RAG) for primary diversification, immunoglobulins are afforded a supplementary set of activation-induced cytidine deaminase (AID)-mediated diversification tools. As the oldest-emerging vertebrates sharing the same adaptive B and T cell receptor systems as humans, extant cartilaginous fishes allow a potential view of the ancestral immune system. In this review, we discuss breakthroughs we have made in studies of nurse shark (Ginglymostoma cirratum) T cell receptors demonstrating substantial integration of loci and diversification mechanisms in primordial B and T cell repertoires. We survey these findings in this shark model where they were first described, while noting corroborating examples in other vertebrate groups. We also consider other examples where the gnathostome common ancestry of the B and T cell receptor systems have allowed dovetailing of genomic elements and AID-based diversification approaches for the TCR. The cartilaginous fish seem to have retained this T/B cell plasticity to a greater extent than more derived vertebrate groups, but representatives in all vertebrate taxa except bony fish and placental mammals show such plasticity.

CAR (Chimeric Antigen Receptor) T-cell therapy has revolutionized the field of oncology in recent years. This innovative shift in cancer treatment also provides the opportunity to improve therapies for many patients suffering from various autoimmune diseases. Recent studies have confirmed the therapeutic suppressive potential of regulatory T cells (Tregs) to modulate immune response in autoimmune diseases. However, the polyclonal character of regulatory T cells and their unknown TCR specificity impaired their therapeutic potency in clinical implementation. Genetical engineering of these immune modulating cells to express antigen-specific receptors and using them therapeutically is a logical step on the way to overcome present limitations of the Treg strategy for the treatment of autoimmune diseases. Encouraging preclinical studies successfully demonstrated immune modulating properties of CAR Tregs in various mouse models. Still, there are many concerns about targeted Treg therapies relating to CAR target selectivity, suppressive functions, phenotype stability and safety aspects. Here, we summarize recent developments in CAR design, Treg biology and future strategies and perspectives in CAR Treg immunotherapy aiming at clinical translation.

Development of B cells requires the programmed generation and repair of double-stranded DNA breaks in antigen receptor genes. Investigation of the cellular responses to these DNA breaks has established important insights into B cell development and, more broadly, has provided fundamental advances into the molecular mechanisms of DNA damage response pathways. Abelson transformed pre-B cell lines and primary pre-B cell cultures are malleable experimental systems with diverse applications for studying DNA damage responses. This chapter describes methods for generating these cellular systems, inducing and quantifying DSBs, and assessing DNA damage programs.

Whitespotted bamboo shark (Chiloscyllium plagiosum) is a demersal cartilaginous fish with an adaptive immune system founded upon immunoglobulins. In this manuscript, we characterize the IgNAR of the whitespotted bamboo shark. A newly discovered alternative splicing form of IgNAR Sec (IgNARshort (ΔC2-C3) Sec) was identified, in which the C1 domain was spliced directly to the C4 domain, the process resulted in a molecule containing three constant domains. However, a single unpaired cysteine remains in the highly flexible hinge region, contributing in the formation of an interchain disulfide bond. Two types of C1 domain were found, and the one lacking a short α-helix showed lower proportion. This finding suggests that short α-helices might be important to the stability of IgNAR. High-throughput sequencing revealed that the percentage of VNAR types significantly vary between the diverse species of sharks. The variable region of IgNAR (the VNAR) with small size and stabilization is a potential candidate for immunotherapeutic agents. The structure and stability analysis in this manuscript may be useful in future biomedical applications.

Nonclonal innate immune responses mediated by germ line–encoded receptors, such as Toll-like receptors or natural killer receptors, are commonly contrasted with diverse, clonotypic adaptive responses of lymphocyte antigen receptors generated by somatic recombination. However, the Variable (V) regions of antigen receptors include germ line–encoded motifs unaltered by somatic recombination, and theoretically available to mediate nonclonal, innate responses, that are independent of or largely override clonotypic responses. Recent evidence demonstrates that such responses exist, underpinning the associations of particular γδ T cell receptors (TCRs) with specific anatomical sites. Thus, TCRγδ can make innate and adaptive responses with distinct functional outcomes. Given that αβ T cells and B cells can also make nonclonal responses, we consider that innate responses of antigen receptor V-regions may be more widespread, for example, inducing states of preparedness from which adaptive clones are better selected. We likewise consider that potent, nonclonal T cell responses to microbial superantigens may reflect subversion of physiologic innate responses of TCRα/β chains.

A bispecific antibody (bsAb) is an emerging class of next-generation biological therapeutics. BsAbs are engineered antibodies possessing dual antigen-binding paratopes in one molecule. The circular backbone topology has never been demonstrated, although an enormous number of bispecific constructs have been proposed. The circular topology is potentially beneficial for fixing the orientation of two paratopes and protection from exopeptidase digestion. We construct herein a circularly connected bispecific VHH, termed cyclobody, using the split-intein circular ligation of peptides and proteins. The constructed cyclobodies are protected from proteolysis with a retained bispecificity. The anti-EGFR × anti-GFP cyclobody can specifically stain EGFR-positive cells with GFP. The anti-EGFR × anti-CD16 cyclobody shows cytotoxic activity against EGFR-positive cancer cells with comparative activity of a tandem VHH construct. Successful demonstration of a new topology for the bispecific antibody will expand the construction strategy for developing antibody-based drugs and reagents.

Conventional monoclonal antibodies (mAbs) have been widely used in research and diagnostic applications due to their high affinity and specificity. However, multiple limitations, such as large size, complex structure and sensitivity to extreme ambient temperature potentially weaken the performance of mAbs in certain applications. To address this problem, the exploration of new antigen binders is extensively required in relation to improve the quality of current diagnostic platforms. In recent years, a new immunoglobulin-based protein, namely variable domain of new antigen receptor (VNAR) was discovered in sharks. Unlike conventional mAbs, several advantages of VNARs, include small size, better thermostability and peculiar paratope structure have attracted interest of researchers to further explore on it. This article aims to first present an overview of the shark VNARs and outline the characteristics as an outstanding new reagent for diagnostic and therapeutic applications., Highlights • Antibodies are powerful tools due to their high affinity and specificity. • In tropical regions degradation of mAbs in test kits due to high temperatures is problematic. • Shark single domain antibody (VNAR) can have better thermostability than mAbs. • VNAR potentially provides new reagents for diagnostic and therapeutic applications.

The approval of the first VHH-based drug caplacizumab (anti-von Willebrand factor) has validated a two-decade long commitment in time and research effort to realize the clinical potential of single-domain antibodies. The variable domain (VNAR) of the immunoglobulin new antigen receptor (IgNAR) found in sharks provides an alternative small binding domain to conventional monoclonal antibodies and their fragments and heavy-chain antibody-derived VHHs. Evolutionarily distinct from mammalian antibody variable domains, VNARs have enhanced thermostability and unusual convex paratopes. This predisposition to bind cryptic and recessed epitopes has facilitated both the targeting of new antigens and new (neutralizing) epitopes on existing antigens. Together these unique properties position the VNAR platform as an alternative non-antibody binding domain for therapeutic drug, diagnostic and reagent development. In this introductory chapter, we highlight recent VNAR advancements that further underline the exciting potential of this discovery platform.

In chronic lymphocytic leukemia (CLL), the B cell receptor (BCR) plays a critical role in disease development and progression, as indicated by the therapeutic efficacy of drugs blocking BCR signaling. However, the mechanism(s) underlying BCR responsiveness are not completely defined. Selective engagement of membrane IgM or IgD on CLL cells, each coexpressed by more than 90% of cases, leads to distinct signaling events. Since both IgM and IgD carry the same antigen-binding domains, the divergent actions of the receptors are attributed to differences in immunoglobulin (Ig) structure or the outcome of signal transduction. We showed that IgM, not IgD, level and organization associated with CLL-cell birth rate and the type and consequences of BCR signaling in humans and mice. The latter IgM-driven effects were abrogated when BCR signaling was inhibited. Collectively, these studies demonstrated a critical, selective role for IgM in BCR signaling and B cell fate decisions, possibly opening new avenues for CLL therapy., publishedVersion

The jawless vertebrates (lamprey and hagfish) evolved a novel adaptive immune system with many similarities to that found in the jawed vertebrates, including the production of antigen-specific circulating antibodies in response to immunization. However, the jawless vertebrates use leucine-rich repeat (LRR)-based antigen receptors termed variable lymphocyte receptors (VLRs) for immune recognition, instead of immunoglobulin (Ig)-based receptors. VLR genes are assembled in developing lymphocytes through a gene conversion-like process, in which hundreds of LRR gene segments are randomly selected as template donors to generate a large repertoire of distinct antigen receptors, similar to that found within the mammalian adaptive immune system. Here we describe the development of a robust platform using immunized lampreys (Petromyzon marinus) for generating libraries of anti-carbohydrate (anti-glycan) variable lymphocyte receptor B, or VLRBs. The anti-carbohydrate VLRBs are isolated using a yeast surface display (YSD) expression platform and enriched by binding to glycan microarrays through the anti-glycan VLRB. This enables both the initial identification and enrichment of individual yeast clones against hundreds of glycans simultaneously. Through this enrichment strategy a broad array of glycan-specific VLRs can be isolated from the YSD library. Subsequently, the bound yeast cells are directly removed from the microarray, the VLR antibody clone is sequenced, and the end product is expressed as a VLR-IgG-Fc fusion protein that can be used for ELISA, Western blotting, flow cytometry, and immunomicroscopy. Thus, by combining yeast surface display with glycan microarray technology, we have developed a rapid, efficient, and novel method for generating chimeric VLR-IgG-Fc proteins that recognize a broad array of unique glycan structures with exquisite specificity.

Enhancers are defined as regulatory elements that control transcription in a cell-type and developmental stage-specific manner. They achieve this by physically interacting with their cognate gene promoters. Significantly, these interactions can occur through long genomic distances since enhancers may not be near their cognate promoters. The optimal coordination of enhancer-regulated transcription is essential for the function and identity of the cell. Although great efforts to fully understand the principles of this type of regulation are ongoing, other potential functions of the long-range chromatin interactions (LRCIs) involving enhancers are largely unexplored. We recently uncovered a new role for enhancer elements in determining the three-dimensional (3D) structure of the immunoglobulin kappa (Igκ) light chain receptor locus suggesting a structural function for these DNA elements. This enhancer-mediated locus configuration shapes the resulting Igκ repertoire. We also propose a role for enhancers as critical components of sub-topologically associating domain (subTAD) formation and nuclear spatial localization.

In the two decades since the invention of laser-based super resolution microscopy this family of technologies has revolutionised the way life is viewed and understood. Its unparalleled resolution, speed, and accessibility makes super resolution imaging particularly useful in examining the highly complex and dynamic immune system. Here we introduce the super resolution technologies and studies that have already fundamentally changed our understanding of a number of central immunological processes and highlight other immunological puzzles only addressable in super resolution.

In response to the invasion of exogenous microorganisms, one of the defence strategies of the immune system is to produce antibodies. Cartilaginous fish is among those who evolved the earliest humoral immune system that utilizes immunoglobulin-type antibodies. The cartilaginous fish antibodies fall into three categories: IgW, IgM, and IgNAR. The shark Immunoglobulin Novel Antigen Receptor (IgNAR) constitutes disulfide-bonded dimers of two protein chains, similar to the heavy chain of mammalian IgGs. Shark IgNAR is the primary antibody of a shark's adaptive immune system with a serum concentration of 0.1-1.0 mg/mL. Its structure comprises of one variable (V) domain (VNAR) and five constant (C1 -C5) domains in the secretory form. VNARs are classified into several subclasses based on specific properties such as the quantity and position of additional non-canonical cysteine (Cys) residues in the VNAR. The VDJ recombination in IgNAR comprises various fragments; one variable component, three diverse sections, one joining portion, and a solitary arrangement of constant fragments framed in each IgNAR gene cluster. The re-arrangement happens just inside this gene cluster bringing about a VD1D2D3J segment. Therefore, four re-arrangement procedures create the entire VNAR space. IgNAR antibody can serve as an excellent diagnostic, therapeutic, and research tool because it has a smaller size, high specificity for antigen-binding, and perfect stability. The domain characterization, structural features, types, diversity and therapeutic applications of IgNAR molecules are highlighted in this review. It would be helpful for further research on IgNAR antibodies acting as an essential constituent of the adaptive immune system and a potential therapeutic agent.

Single-domain Variable New Antigen Receptors (VNARs) from the immune system of sharks are the smallest naturally occurring binding domains found in nature. Possessing flexible paratopes that can recognize protein motifs inaccessible to classical antibodies, VNARs have yet to be exploited for the development of SARS-CoV-2 therapeutics. Here, we detail the identification of a series of VNARs from a VNAR phage display library screened against the SARS-CoV-2 receptor binding domain (RBD). The ability of the VNARs to neutralize pseudotype and authentic live SARS-CoV-2 virus rivalled or exceeded that of full-length immunoglobulins and other single-domain antibodies. Crystallographic analysis of two VNARs found that they recognized separate epitopes on the RBD and had distinctly different mechanisms of virus neutralization unique to VNARs. Structural and biochemical data suggest that VNARs would be effective therapeutic agents against emerging SARS-CoV-2 mutants, including the Delta variant, and coronaviruses across multiple phylogenetic lineages. This study highlights the utility of VNARs as effective therapeutics against coronaviruses and may serve as a critical milestone for nearing a paradigm shift of the greater biologic landscape., Shark antibodies (Variable New Antigen Receptors, VNARs) are the smallest naturally occurring antibody fragments. Here, the authors screen a VNAR phage display library against the SARS-CoV2 receptor binding domain (RBD) and identify VNARs that neutralize the SARSCoV-2 virus and discuss their mechanisms of viral neutralization.

While apoptosis plays a role in B-cell self-tolerance, its significance in preventing autoimmunity remains unclear. Here, we report that dysregulated B cell apoptosis leads to delayed onset autoimmune phenotype in mice. Our longitudinal studies revealed that mice with B cell-specific deletion of pro-apoptotic Bim (BBimfl/fl) have an expanded B cell compartment with a notable increase in transitional, antibody secreting and recently described double negative (DN) B cells. They develop greater hypergammaglobulinemia than mice lacking Bim in all cells and accumulate several autoantibodies characteristic of Systemic Lupus Erythematosus (SLE) and related Sjögren’s Syndrome (SS) including anti-nuclear, anti-Ro/SSA and anti-La/SSB at a level comparable to NODH2h4 autoimmune mouse model. Furthermore, lymphocytes infiltrated the tissues including submandibular glands and formed follicle-like structures populated with B cells, plasma cells and T follicular helper cells indicative of ongoing immune reaction. This autoimmunity was ameliorated upon deletion of Bruton’s tyrosine kinase (Btk) gene, which encodes a key B cell signaling protein. These studies suggest that Bim-mediated apoptosis suppresses and B cell tyrosine kinase signaling promotes B cell-mediated autoimmunity.

Background and objectives High-intensity chemotherapy and advances in novel immunotherapies have seen the emergence of cytomegalovirus (CMV) infections in cancer patients other than allogeneic haematopoietic cell transplantation (HCT). We therefore aimed to evaluate the epidemiology, clinical characteristics, and outcomes of CMV infection in this population. Method A retrospective review of cancer patients other than allogeneic HCT who had CMV DNAemia and/or disease from July 2013 till May 2020 at a quaternary cancer center was performed. Results Of 11,485 cancer patients who underwent treatment during this period, 953 patients had CMV DNA testing performed and 238 of them had CMV DNAemia. After excluding patients with allogeneic HCT, 62 patients with CMV DNAemia were identified of which 10 had concurrent CMV disease. The most frequent underlying malignancies were B-cell lymphoproliferative disease (LPD) (31%, 19/62), T-cell LPD (21%, 13/62), chronic lymphocytic leukemia (11%, 7/62), and multiple myeloma (10%, 6/62). Most patients had lymphopenia (77%, 48/62), multiple cancer therapies (63%, 39/62 received ≥2 previous therapies), co-infection (56%, 35/62 had ≥1 co-infection) and corticosteroid therapy (48%, 30/62) within one month before CMV diagnosis. CMV DNAemia and disease were observed in patients receiving novel immunotherapies including bispecific antibody therapy, chimeric-antigen receptor T-cell therapy and immune checkpoint inhibitors. Conclusion Patients with haematological malignancy, particularly B-cell LPD, T-cell LPD, chronic lymphocytic leukemia and multiple myeloma were frequently identified to have CMV DNAemia and disease. Lymphopenia, multiple cancer therapies, co-infection, and recent receipt of systemic corticosteroids were also commonly observed. Future studies are necessary to determine optimal identification and management of CMV in these patients. This article is protected by copyright. All rights reserved.

Ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus strains is posing new COVID-19 diagnosis and treatment challenges. To help efforts to meet these challenges we examined data acquired from proteomic analyses of human SARS-CoV-2-infected cell lines and samples from COVID-19 patients. Initially, 129 unique peptides were identified, which were rigorously evaluated for repeats, disorders, polymorphisms, antigenicity, immunogenicity, toxicity, allergens, sequence similarity to human proteins, and contributions from other potential cross-reacting pathogenic species or the human saliva microbiome. We also screened SARS-CoV-2-infected NBHE and A549 cell lines for presence of antigenic peptides, and identified paratope peptides from crystal structures of SARS-CoV-2 antigen-antibody complexes. We then selected four antigen peptides for docking with known viral unbound T-cell receptor (TCR), class I and II peptide major histocompatibility complex (pMHC), and identified paratope sequences. We also tested the paratope binding affinity of SARS-CoV T- and B-cell peptides that had been previously experimentally validated. The resultant antigenic peptides have high potential for generating SARS-CoV-2-specific antibodies, and the paratope peptides can be directly used to develop a COVID-19 diagnostics assay. The presented genomics and proteomics-based in-silico approaches have apparent utility for identifying new diagnostic peptides that could be used to fight SARS-CoV-2.

Adoptive transfer of allogeneic and genetically modified T cells, such as CAR-T and TCR-T cells, can induce profound immune reactivity against cancer tissue. At the same time, these therapies are associated with severe off-target and on-target toxicities. For this reason, the development of genetic safety switches that can be used to control the activity of T cells in vivo has become an active field of research. With the spectrum of technologies developed, reversible control of cell products either by supply or removal of small molecules, by supply of protein-based regulators, or by physical stimuli such as light, ultrasound or heat, has become feasible. In this review, we describe the mechanistic classes of genetic safety switches, such as transcription-based or protein-based control of antigen receptors, split receptors, small molecule responsive antibodies, as well as universal remote controls, and discuss their advantages and limitations.

Enteroviruses are potentially linked to the emergence of Acute Flaccid Myelitis (AFM), a rare but very serious condition that affects the nervous system. AFM has been associated with coxsackievirus A16, enterovirus A71 (EVA71) and enterovirus D68 (EVD68). Little is known about host-pathogen interactions for these viruses, and whether immune responses may have a protective or immunopathological role in disease presentations. Towards addressing this issue, we used the Immune Epitope Database to assess the known inventory of B and T cell epitopes from enteroviruses, focusing on data related to human hosts. The extent of conservation in areas that are targets of B and T cell immune responses were examined. This analysis sheds light on regions of the enterovirus polypeptide that can be probed to induce a specific or cross-reactive B or T cell the immune response to enteroviruses, with a particular focus on coxsackievirus A16, EVA71 and EVD68. In addition, these analyses reveal the current gap-of-knowledge in the T and B cell immune responses that future studies should aim to address.

The activation of key signaling pathways downstream of antigen receptor engagement is critically required for normal lymphocyte activation during the adaptive immune response. CARD11 is a multidomain signaling scaffold protein required for antigen receptor signaling to NF-κB, c-Jun N-terminal kinase, and mTOR. Germline mutations in the CARD11 gene result in at least four types of primary immunodeficiency, and somatic CARD11 gain-of-function mutations drive constitutive NF-κB activity in diffuse large B cell lymphoma and other lymphoid cancers. In response to antigen receptor triggering, CARD11 transitions from a closed, inactive state to an open, active scaffold that recruits multiple signaling partners into a complex to relay downstream signaling. However, how this signal-induced CARD11 conversion occurs remains poorly understood. Here we investigate the role of Inducible Element 1 (IE1), a short regulatory element in the CARD11 Inhibitory Domain, in the CARD11 signaling cycle. We find that IE1 controls the signal-dependent Opening Step that makes CARD11 accessible to the binding of cofactors, including Bcl10, MALT1, and the HOIP catalytic subunit of the linear ubiquitin chain assembly complex. Surprisingly, we find that IE1 is also required at an independent step for the maximal activation of HOIP and MALT1 enzymatic activity after cofactor recruitment to CARD11. This role of IE1 reveals that there is an Enzymatic Activation Step in the CARD11 signaling cycle that is distinct from the Cofactor Association Step. Our results indicate that CARD11 has evolved to actively coordinate scaffold opening and the induction of enzymatic activity among recruited cofactors during antigen receptor signaling.

Domestication of a transposon (a DNA sequence that can change its position in a genome) to give rise to the RAG1–RAG2 recombinase (RAG) and V(D)J recombination, which produces the diverse repertoire of antibodies and T cell receptors, was a pivotal event in the evolution of the adaptive immune system of jawed vertebrates. The evolutionary adaptations that transformed the ancestral RAG transposase into a RAG recombinase with appropriately regulated DNA cleavage and transposition activities are not understood. Here, beginning with cryo-electron microscopy structures of the amphioxus ProtoRAG transposase (an evolutionary relative of RAG), we identify amino acid residues and domains the acquisition or loss of which underpins the propensity of RAG for coupled cleavage, its preference for asymmetric DNA substrates and its inability to perform transposition in cells. In particular, we identify two adaptations specific to jawed-vertebrates—arginine 848 in RAG1 and an acidic region in RAG2—that together suppress RAG-mediated transposition more than 1,000-fold. Our findings reveal a two-tiered mechanism for the suppression of RAG-mediated transposition, illuminate the evolution of V(D)J recombination and provide insight into the principles that govern the molecular domestication of transposons. Identification of the changes that converted a transposase to a recombinase sheds light on the evolution of the vertebrate adaptive immune system.

In this study, we identified a pair of nonrearranging VJ-joined Ig superfamily genes, termed putative remnants of an Ag receptor precursor (PRARP) genes, in chicken. Both genes encode a single V-set Ig domain consisting of a canonical J-like segment and a potential immunoreceptor tyrosine-based inhibitory or switch motif in the cytoplasmic region. In vitro experiments showed that both genes were expressed at the cell surface as membrane proteins, and their recombinant products formed a monomer and a disulfide-linked homodimer or a heterodimer. These two genes were mainly expressed in B and T cells and were upregulated in response to stimulation with poly(I:C) in vitro and vaccination in vivo. Orthologs of PRARP have been identified in bony fish, amphibians, reptiles, and other birds, and a V-C1 structure similar to that of Ig or TCR chains was found in all these genes, with the exception of those in avian species, which appear to contain degenerated C1 domains or divergent Ig domains. Phylogenetic analyses suggested that the newly discovered genes do not belong to any known immune receptor family and appear to be a novel gene family. Further elucidation of the functions of PRARP and their origin might provide significant insights into the evolution of the immune system of jawed vertebrates.

Immune-cell engineering opens new capabilities for fundamental immunology research and immunotherapy. We developed a system for efficient generation of chimeric antigen receptor (CAR)-engineered T cells (CAR-T cells) with considerably enhanced features by streamlined genome engineering. By leveraging trans-activating CRISPR (clustered regularly interspaced short palindromic repeats) RNA (tracrRNA)-independent CRISPR-Cpf1 systems with adeno-associated virus (AAV), we were able to build a stable CAR-T cell with homology-directed-repair knock-in and immune-checkpoint knockout (KIKO CAR-T cell) at high efficiency in one step. The modularity of the AAV-Cpf1 KIKO system enables flexible and highly efficient generation of double knock-in of two different CARs in the same T cell. Compared with Cas9-based methods, the AAV-Cpf1 system generates double-knock-in CAR-T cells more efficiently. CD22-specific AAV-Cpf1 KIKO CAR-T cells have potency comparable to that of Cas9 CAR-T cells in cytokine production and cancer cell killing, while expressing lower levels of exhaustion markers. This versatile system opens new capabilities of T-cell engineering with simplicity and precision.

Cartilaginous fish are the evolutionarily oldest group of animals which possess antibodies, T cell receptors and major histocompatibility complex (MHC). The immunoglobulin novel antigen receptor (IgNAR) found in cartilaginous fish is a heavy chain homodimer which lacks light chain. The presence of non-canonical cysteine molecules and lack of CDR2 region make it more significant. To synthesize active binding domains based on variable region of IgNAR (VNAR), knowledge on the constant region dynamics play a significant role. The IgNAR exhibit species variations in its primary sequence features; hence, this study was conducted to determine the IgNAR heavy chain constant domain of the brownbanded bamboo shark (Chiloscyllium punctatum). Peripheral blood leukocytes (PBL) isolated from adult bamboo sharks were used to synthesize a cDNA library. A total of four billion residues of two million sequences (average length 218.41 bp) were obtained. Assembled sequences were aligned with published cartilaginous fish IgNAR constant region sequences. Transcriptome analysis revealed two distinct types of IgNAR in the brownbanded bamboo shark. Also, constant-1 domain sequences displayed 13 unique sequences which may reflect the least number of IgNAR gene clusters. The phylogenetic analysis revealed the closest relationship with the nurse shark (Ginglymostoma cirratum) followed by the wobbegong shark (Orectolobus maculatus) which belong to the same order Orectolobiformes. Analysis of the constant domains of the brownbanded bamboo shark IgNAR revealed an evolutionarily conserved nature and this knowledge can be used to design primers for VNAR cloning. Furthermore, knowledge on the structural features in IgNAR constant domains that increase the stability could be useful in the process of stabilizing human immunoglobulins.