Transcription factor networks directing the development, function, and evolution of innate lymphoid effectors.

Mammalian lymphoid immunity is mediated by fast and slow responders to pathogens. Fast innate lymphocytes are active within hours after infections in mucosal tissues. Slow adaptive lymphocytes are conventional T and B cells with clonal antigen receptors that function days after pathogen exposure. A transcription factor (TF) regulatory network guiding early T cell development is at the core of effector function diversification in all innate lymphocytes, and the kinetics of immune responses is set by developmental programming. Operational units within the innate lymphoid system are not classified by the types of pathogen-sensing machineries but rather by discrete effector functions programmed by regulatory TF networks. Based on the evolutionary history of TFs of the regulatory networks, fast effectors likely arose earlier in the evolution of animals to fortify body barriers, and in mammals they often develop in fetal ontogeny prior to the establishment of fully competent adaptive immunity.

T cell antigen receptor recognition of antigen-presenting molecules.

The Major Histocompatibility Complex (MHC) locus encodes classical MHC class I and MHC class II molecules and nonclassical MHC-I molecules. The architecture of these molecules is ideally suited to capture and present an array of peptide antigens (Ags). In addition, the CD1 family members and MR1 are MHC class I-like molecules that bind lipid-based Ags and vitamin B precursors, respectively. These Ag-bound molecules are subsequently recognized by T cell antigen receptors (TCRs) expressed on the surface of T lymphocytes. Structural and associated functional studies have been highly informative in providing insight into these interactions, which are crucial to immunity, and how they can lead to aberrant T cell reactivity. Investigators have determined over thirty unique TCR-peptide-MHC-I complex structures and twenty unique TCR-peptide-MHC-II complex structures. These investigations have shown a broad consensus in docking geometry and provided insight into MHC restriction. Structural studies on TCR-mediated recognition of lipid and metabolite Ags have been mostly confined to TCRs from innate-like natural killer T cells and mucosal-associated invariant T cells, respectively. These studies revealed clear differences between TCR-lipid-CD1, TCR-metabolite-MR1, and TCR-peptide-MHC recognition. Accordingly, TCRs show remarkable structural and biological versatility in engaging different classes of Ag that are presented by polymorphic and monomorphic Ag-presenting molecules of the immune system.

Biology of CD1- and MR1-restricted T cells.

Over the past 15 years, investigators have shown that T lymphocytes can recognize not only peptides in the context of MHC class I and class II molecules but also foreign and self-lipids in association with the nonclassical MHC class I-like molecules, CD1 proteins. In this review, we describe the most recent events in the field, with particular emphasis on (a) structural and functional aspects of lipid presentation by CD1 molecules, (b) the development of CD1d-restricted invariant natural killer T (iNKT) cells and transcription factors required for their differentiation, (c) the ability of iNKT cells to modulate innate and adaptive immune responses through their cross talk with lymphoid and myeloid cells, and (d) MR1-restricted and group I (CD1a, CD1b, and CD1c)-restricted T cells.

An infectivity-enhancing site on the SARS-CoV-2 spike protein targeted by antibodies.

Antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein prevent SARS-CoV-2 infection. However, the effects of antibodies against other spike protein domains are largely unknown. Here, we screened a series of anti-spike monoclonal antibodies from coronavirus disease 2019 (COVID-19) patients and found that some of antibodies against the N-terminal domain (NTD) induced the open conformation of RBD and thus enhanced the binding capacity of the spike protein to ACE2 and infectivity of SARS-CoV-2. Mutational analysis revealed that all of the infectivity-enhancing antibodies recognized a specific site on the NTD. Structural analysis demonstrated that all infectivity-enhancing antibodies bound to NTD in a similar manner. The antibodies against this infectivity-enhancing site were detected at high levels in severe patients. Moreover, we identified antibodies against the infectivity-enhancing site in uninfected donors, albeit at a lower frequency. These findings demonstrate that not only neutralizing antibodies but also enhancing antibodies are produced during SARS-CoV-2 infection.

Pathways of antigen processing.

T cell recognition of antigen-presenting cells depends on their expression of a spectrum of peptides bound to major histocompatibility complex class I (MHC-I) and class II (MHC-II) molecules. Conversion of antigens from pathogens or transformed cells into MHC-I- and MHC-II-bound peptides is critical for mounting protective T cell responses, and similar processing of self proteins is necessary to establish and maintain tolerance. Cells use a variety of mechanisms to acquire protein antigens, from translation in the cytosol to variations on the theme of endocytosis, and to degrade them once acquired. In this review, we highlight the aspects of MHC-I and MHC-II biosynthesis and assembly that have evolved to intersect these pathways and sample the peptides that are produced.

Restriction of HIV-1 Escape by a Highly Broad and Potent Neutralizing Antibody.

Broadly neutralizing antibodies (bNAbs) represent a promising approach to prevent and treat HIV-1 infection. However, viral escape through mutation of the HIV-1 envelope glycoprotein (Env) limits clinical applications. Here we describe 1-18, a new VH1-46-encoded CD4 binding site (CD4bs) bNAb with outstanding breadth (97%) and potency (GeoMean IC50 = 0.048 µg/mL). Notably, 1-18 is not susceptible to typical CD4bs escape mutations and effectively overcomes HIV-1 resistance to other CD4bs bNAbs. Moreover, mutational antigenic profiling uncovered restricted pathways of HIV-1 escape. Of most promise for therapeutic use, even 1-18 alone fully suppressed viremia in HIV-1-infected humanized mice without selecting for resistant viral variants. A 2.5-Å cryo-EM structure of a 1-18-BG505SOSIP.664 Env complex revealed that these characteristics are likely facilitated by a heavy-chain insertion and increased inter-protomer contacts. The ability of 1-18 to effectively restrict HIV-1 escape pathways provides a new option to successfully prevent and treat HIV-1 infection.

VLR-based adaptive immunity.

Lampreys and hagfish are primitive jawless vertebrates capable of mounting specific immune responses. Lampreys possess different types of lymphocytes, akin to T and B cells of jawed vertebrates, that clonally express somatically diversified antigen receptors termed variable lymphocyte receptors (VLRs), which are composed of tandem arrays of leucine-rich repeats. The VLRs appear to be diversified by a gene conversion mechanism involving lineage-specific cytosine deaminases. VLRA is expressed on the surface of T-like lymphocytes; B-like lymphocytes express and secrete VLRB as a multivalent protein. VLRC is expressed by a distinct lymphocyte lineage. VLRA-expressing cells appear to develop in a thymus-like tissue at the tip of gill filaments, and VLRB-expressing cells develop in hematopoietic tissues. Reciprocal expression patterns of evolutionarily conserved interleukins and chemokines possibly underlie cell-cell interactions during an immune response. The discovery of VLRs in agnathans illuminates the origins of adaptive immunity in early vertebrates.

A Public BCR Present in a Unique Dual-Receptor-Expressing Lymphocyte from Type 1 Diabetes Patients Encodes a Potent T Cell Autoantigen.

T and B cells are the two known lineages of adaptive immune cells. Here, we describe a previously unknown lymphocyte that is a dual expresser (DE) of TCR and BCR and key lineage markers of both B and T cells. In type 1 diabetes (T1D), DEs are predominated by one clonotype that encodes a potent CD4 T cell autoantigen in its antigen binding site. Molecular dynamics simulations revealed that this peptide has an optimal binding register for diabetogenic HLA-DQ8. In concordance, a synthetic version of the peptide forms stable DQ8 complexes and potently stimulates autoreactive CD4 T cells from T1D patients, but not healthy controls. Moreover, mAbs bearing this clonotype are autoreactive against CD4 T cells and inhibit insulin tetramer binding to CD4 T cells. Thus, compartmentalization of adaptive immune cells into T and B cells is not absolute, and violators of this paradigm are likely key drivers of autoimmune diseases.

Deubiquitination of NLRP6 inflammasome by Cyld critically regulates intestinal inflammation.

The inflammasome NLRP6 plays a crucial role in regulating inflammation and host defense against microorganisms in the intestine. However, the molecular mechanisms by which NLRP6 function is inhibited to prevent excessive inflammation remain unclear. Here, we demonstrate that the deubiquitinase Cyld prevents excessive interleukin 18 (IL-18) production in the colonic mucosa by deubiquitinating NLRP6. We show that deubiquitination inhibited the NLRP6-ASC inflammasome complex and regulated the maturation of IL-18. Cyld deficiency in mice resulted in elevated levels of active IL-18 and severe colonic inflammation following Citrobacter rodentium infection. Further, in patients with ulcerative colitis, the concentration of active IL-18 was inversely correlated with CYLD expression. Thus, we have identified a novel regulatory mechanism that inhibits the NLRP6-IL-18 pathway in intestinal inflammation.

Structural basis for the constitutive activity and immunomodulatory properties of the Epstein-Barr virus-encoded G protein-coupled receptor BILF1.

Epstein-Barr virus (EBV) encodes a G protein-coupled receptor (GPCR) termed BILF1 that is essential for EBV-mediated immunosuppression and oncogenesis. BILF1 couples with inhibitory G protein (Gi), the major intracellular signaling effector for human chemokine receptors, and exhibits constitutive signaling activity; the ligand(s) for BILF1 are unknown. We studied the origins of BILF1's constitutive activity through structure determination of BILF1 bound to the inhibitory G protein (Gi) heterotrimer. The 3.2-Å resolution cryo-electron microscopy structure revealed an extracellular loop within BILF1 that blocked the typical chemokine binding site, suggesting ligand-autonomous receptor activation. Rather, amino acid substitutions within BILF1 transmembrane regions at hallmark ligand-activated class A GPCR "microswitches" stabilized a constitutively active BILF1 conformation for Gi coupling in a ligand-independent fashion. Thus, the constitutive activity of BILF1 promotes immunosuppression and virulence independent of ligand availability, with implications for the function of GPCRs encoded by related viruses and for therapeutic targeting of EBV.

Pleiotropic consequences of metabolic stress for the major histocompatibility complex class II molecule antigen processing and presentation machinery.

Hyperglycemia and hyperlipidemia are often observed in individuals with type II diabetes (T2D) and related mouse models. One dysmetabolic biochemical consequence is the non-enzymatic reaction between sugars, lipids, and proteins, favoring protein glycation, glycoxidation, and lipoxidation. Here, we identified oxidative alterations in key components of the major histocompatibility complex (MHC) class II molecule antigen processing and presentation machinery in vivo under conditions of hyperglycemia-induced metabolic stress. These modifications were linked to epitope-specific changes in endosomal processing efficiency, MHC class II-peptide binding, and DM editing activity. Moreover, we observed some quantitative and qualitative changes in the MHC class II immunopeptidome of Ob/Ob mice on a high-fat diet compared with controls, including changes in the presentation of an apolipoprotein B100 peptide associated previously with T2D and metabolic syndrome-related clinical complications. These findings highlight a link between glycation reactions and altered MHC class II antigen presentation that may contribute to T2D complications.

Neutralization potency of monoclonal antibodies recognizing dominant and subdominant epitopes on SARS-CoV-2 Spike is impacted by the B.1.1.7 variant.

Interaction of the SARS-CoV-2 Spike receptor binding domain (RBD) with the receptor ACE2 on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies, and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS-CoV-2 variants has revealed mutations arising in the RBD, N-terminal domain (NTD) and S2 subunits of Spike. To understand how these mutations affect Spike antigenicity, we isolated and characterized >100 monoclonal antibodies targeting epitopes on RBD, NTD, and S2 from SARS-CoV-2-infected individuals. Approximately 45% showed neutralizing activity, of which ∼20% were NTD specific. NTD-specific antibodies formed two distinct groups: the first was highly potent against infectious virus, whereas the second was less potent and displayed glycan-dependant neutralization activity. Mutations present in B.1.1.7 Spike frequently conferred neutralization resistance to NTD-specific antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes should be considered when investigating antigenic drift in emerging variants.

The CD47-SIRPα Immune Checkpoint.

The cytotoxic activity of myeloid cells is regulated by a balance of signals that are transmitted through inhibitory and activating receptors. The Cluster of Differentiation 47 (CD47) protein, expressed on both healthy and cancer cells, plays a pivotal role in this balance by delivering a "don't eat me signal" upon binding to the Signal-regulatory protein alpha (SIRPα) receptor on myeloid cells. Here, we review the current understanding of the role of the CD47-SIRPα axis in physiological tissue homeostasis and as a promising therapeutic target in, among others, oncology, fibrotic diseases, atherosclerosis, and stem cell therapies. We discuss gaps in understanding and highlight where additional insight will be beneficial to allow optimal exploitation of this myeloid cell checkpoint as a target in human disease.

CRISPR-based peptide library display and programmable microarray self-assembly for rapid quantitative protein binding assays.

CRISPR-inspired systems have been extensively developed for applications in genome editing and nucleic acid detection. Here, we introduce a CRISPR-based peptide display technology to facilitate customized, high-throughput in vitro protein interaction studies. We show that bespoke peptide libraries fused to catalytically inactive Cas9 (dCas9) and barcoded with unique single guide RNA (sgRNA) molecules self-assemble from a single mixed pool to programmable positions on a DNA microarray surface for rapid, multiplexed binding assays. We develop dCas9-displayed saturation mutagenesis libraries to characterize antibody-epitope binding for a commercial anti-FLAG monoclonal antibody and human serum antibodies. We also show that our platform can be used for viral epitope mapping and exhibits promise as a multiplexed diagnostics tool. Our CRISPR-based peptide display platform and the principles of complex library self-assembly using dCas9 could be adapted for rapid interrogation of varied customized protein libraries or biological materials assembly using DNA scaffolding.

Rapid characterization of spike variants via mammalian cell surface display.

The SARS-CoV-2 spike protein is a critical component of vaccines and a target for neutralizing monoclonal antibodies (nAbs). Spike is also undergoing immunogenic selection with variants that increase infectivity and partially escape convalescent plasma. Here, we describe Spike Display, a high-throughput platform to rapidly characterize glycosylated spike ectodomains across multiple coronavirus-family proteins. We assayed ∼200 variant SARS-CoV-2 spikes for their expression, ACE2 binding, and recognition by 13 nAbs. An alanine scan of all five N-terminal domain (NTD) loops highlights a public epitope in the N1, N3, and N5 loops recognized by most NTD-binding nAbs. NTD mutations in variants of concern B.1.1.7 (alpha), B.1.351 (beta), B.1.1.28 (gamma), B.1.427/B.1.429 (epsilon), and B.1.617.2 (delta) impact spike expression and escape most NTD-targeting nAbs. Finally, B.1.351 and B.1.1.28 completely escape a potent ACE2 mimic. We anticipate that Spike Display will accelerate antigen design, deep scanning mutagenesis, and antibody epitope mapping for SARS-CoV-2 and other emerging viral threats.

Structural interplay between germline interactions and adaptive recognition determines the bandwidth of TCR-peptide-MHC cross-reactivity.

The T cell antigen receptor (TCR)-peptide-major histocompatibility complex (MHC) interface is composed of conserved and diverse regions, yet the relative contribution of each in shaping recognition by T cells remains unclear. Here we isolated cross-reactive peptides with limited homology, which allowed us to compare the structural properties of nine peptides for a single TCR-MHC pair. The TCR's cross-reactivity was rooted in highly similar recognition of an apical 'hot-spot' position in the peptide with tolerance of sequence variation at ancillary positions. Furthermore, we found a striking structural convergence onto a germline-mediated interaction between the TCR CDR1α region and the MHC α2 helix in twelve TCR-peptide-MHC complexes. Our studies suggest that TCR-MHC germline-mediated constraints, together with a focus on a small peptide hot spot, might place limits on peptide antigen cross-reactivity.

The intracellular pathway for the presentation of vitamin B-related antigens by the antigen-presenting molecule MR1.

The antigen-presenting molecule MR1 presents vitamin B-related antigens (VitB antigens) to mucosal-associated invariant T (MAIT) cells through an uncharacterized pathway. We show that MR1, unlike other antigen-presenting molecules, does not constitutively present self-ligands. In the steady state it accumulates in a ligand-receptive conformation within the endoplasmic reticulum. VitB antigens reach this location and form a Schiff base with MR1, triggering a 'molecular switch' that allows MR1-VitB antigen complexes to traffic to the plasma membrane. These complexes are endocytosed with kinetics independent of the affinity of the MR1-ligand interaction and are degraded intracellularly, although some MR1 molecules acquire new ligands during passage through endosomes and recycle back to the surface. MR1 antigen presentation is characterized by a rapid 'off-on-off' mechanism that is strictly dependent on antigen availability.

Viral DNA Binding to NLRC3, an Inhibitory Nucleic Acid Sensor, Unleashes STING, a Cyclic Dinucleotide Receptor that Activates Type I Interferon.

Immune suppression is a crucial component of immunoregulation and a subgroup of nucleotide-binding domain (NBD), leucine-rich repeat (LRR)-containing proteins (NLRs) attenuate innate immunity. How this inhibitory function is controlled is unknown. A key question is whether microbial ligands can regulate this inhibition. NLRC3 is a negative regulator that attenuates type I interferon (IFN-I) response by sequestering and attenuating stimulator of interferon genes (STING) activation. Here, we report that NLRC3 binds viral DNA and other nucleic acids through its LRR domain. DNA binding to NLRC3 increases its ATPase activity, and ATP-binding by NLRC3 diminishes its interaction with STING, thus licensing an IFN-I response. This work uncovers a mechanism wherein viral nucleic acid binding releases an inhibitory innate receptor from its target.

Butyrophilin-like 3 Directly Binds a Human Vγ4+ T Cell Receptor Using a Modality Distinct from Clonally-Restricted Antigen.

Butyrophilin (BTN) and butyrophilin-like (BTNL/Btnl) heteromers are major regulators of human and mouse γδ T cell subsets, but considerable contention surrounds whether they represent direct γδ T cell receptor (TCR) ligands. We demonstrate that the BTNL3 IgV domain binds directly and specifically to a human Vγ4+ TCR, "LES" with an affinity (∼15-25 µM) comparable to many αß TCR-peptide major histocompatibility complex interactions. Mutations in germline-encoded Vγ4 CDR2 and HV4 loops, but not in somatically recombined CDR3 loops, drastically diminished binding and T cell responsiveness to BTNL3-BTNL8-expressing cells. Conversely, CDR3γ and CDR3δ loops mediated LES TCR binding to endothelial protein C receptor, a clonally restricted autoantigen, with minimal CDR1, CDR2, or HV4 contributions. Thus, the γδ TCR can employ two discrete binding modalities: a non-clonotypic, superantigen-like interaction mediating subset-specific regulation by BTNL/BTN molecules and CDR3-dependent, antibody-like interactions mediating adaptive γδ T cell biology. How these findings might broadly apply to γδ T cell regulation is also examined.

Broad and Potent Neutralizing Antibodies Recognize the Silent Face of the HIV Envelope.

Broadly neutralizing antibodies (bNAbs) against HIV-1 envelope (Env) inform vaccine design and are potential therapeutic agents. We identified SF12 and related bNAbs with up to 62% neutralization breadth from an HIV-infected donor. SF12 recognized a glycan-dominated epitope on Env's silent face and was potent against clade AE viruses, which are poorly covered by V3-glycan bNAbs. A 3.3Å cryo-EM structure of a SF12-Env trimer complex showed additional contacts to Env protein residues by SF12 compared with VRC-PG05, the only other known donor-derived silentface antibody, explaining SF12's increased neutralization breadth, potency, and resistance to Env mutation routes. Asymmetric binding of SF12 was associated with distinct N-glycan conformations across Env protomers, demonstrating intra-Env glycan heterogeneity. Administrating SF12 to HIV-1-infected humanized mice suppressed viremia and selected for viruses lacking the N448gp120 glycan. Effective bNAbs can therefore be raised against HIV-1 Env's silent face, suggesting their potential for HIV-1 prevention, therapy, and vaccine development.