The adaptable major histocompatibility complex (MHC) fold: structure and function of nonclassical and MHC class I-like molecules.

The MHC fold is found in proteins that have a range of functions in the maintenance of an organism's health, from immune regulation to fat metabolism. Well adapted for antigen presentation, as seen for peptides in the classical MHC molecules and for lipids in CD1 molecules, the MHC fold has also been modified to perform Fc-receptor activity (e.g., FcRn) and for roles in host homeostasis (e.g., with HFE and ZAG). The more divergent MHC-like molecules, such as some of those that interact with the NKG2D receptor, represent the minimal MHC fold, doing away with the α3 domain and ß2m while maintaining the α1/α2 platform domain for receptor engagement. Viruses have also co-opted the MHC fold for immune-evasive functions. The variations on the theme of a ß-sheet topped by two semiparallel α-helices are discussed in this review, highlighting the fantastic adaptability of this fold for good and for bad.

CRISPR screens decode cancer cell pathways that trigger γδ T cell detection.

γδ T cells are potent anticancer effectors with the potential to target tumours broadly, independent of patient-specific neoantigens or human leukocyte antigen background1-5. γδ T cells can sense conserved cell stress signals prevalent in transformed cells2,3, although the mechanisms behind the targeting of stressed target cells remain poorly characterized. Vγ9Vδ2 T cells-the most abundant subset of human γδ T cells4-recognize a protein complex containing butyrophilin 2A1 (BTN2A1) and BTN3A1 (refs. 6-8), a widely expressed cell surface protein that is activated by phosphoantigens abundantly produced by tumour cells. Here we combined genome-wide CRISPR screens in target cancer cells to identify pathways that regulate γδ T cell killing and BTN3A cell surface expression. The screens showed previously unappreciated multilayered regulation of BTN3A abundance on the cell surface and triggering of γδ T cells through transcription, post-translational modifications and membrane trafficking. In addition, diverse genetic perturbations and inhibitors disrupting metabolic pathways in the cancer cells, particularly ATP-producing processes, were found to alter BTN3A levels. This induction of both BTN3A and BTN2A1 during metabolic crises is dependent on AMP-activated protein kinase (AMPK). Finally, small-molecule activation of AMPK in a cell line model and in patient-derived tumour organoids led to increased expression of the BTN2A1-BTN3A complex and increased Vγ9Vδ2 T cell receptor-mediated killing. This AMPK-dependent mechanism of metabolic stress-induced ligand upregulation deepens our understanding of γδ T cell stress surveillance and suggests new avenues available to enhance γδ T cell anticancer activity.

Identification of Natural Regulatory T Cell Epitopes Reveals Convergence on a Dominant Autoantigen.

Regulatory T (Treg) cells expressing the transcription factor Foxp3 are critical for the prevention of autoimmunity and the suppression of anti-tumor immunity. The major self-antigens recognized by Treg cells remain undefined, representing a substantial barrier to the understanding of immune regulation. Here, we have identified natural Treg cell ligands in mice. We found that two recurrent Treg cell clones, one prevalent in prostate tumors and the other associated with prostatic autoimmune lesions, recognized distinct non-overlapping MHC-class-II-restricted peptides derived from the same prostate-specific protein. Notably, this protein is frequently targeted by autoantibodies in experimental models of prostatic autoimmunity. On the basis of these findings, we propose a model in which Treg cell responses at peripheral sites converge on those self-proteins that are most susceptible to autoimmune attack, and we suggest that this link could be exploited as a generalizable strategy for identifying the Treg cell antigens relevant to human autoimmunity.

Human Leukocyte Antigen F Presents Peptides and Regulates Immunity through Interactions with NK Cell Receptors.

Evidence is mounting that the major histocompatibility complex (MHC) molecule HLA-F (human leukocyte antigen F) regulates the immune system in pregnancy, infection, and autoimmunity by signaling through NK cell receptors (NKRs). We present structural, biochemical, and evolutionary analyses demonstrating that HLA-F presents peptides of unconventional length dictated by a newly arisen mutation (R62W) that has produced an open-ended groove accommodating particularly long peptides. Compared to empty HLA-F open conformers (OCs), HLA-F tetramers bound with human-derived peptides differentially stained leukocytes, suggesting peptide-dependent engagement. Our in vitro studies confirm that NKRs differentiate between peptide-bound and peptide-free HLA-F. The complex structure of peptide-loaded ß2m-HLA-F bound to the inhibitory LIR1 revealed similarities to high-affinity recognition of the viral MHC-I mimic UL18 and a docking strategy that relies on contacts with HLA-F as well as ß2m, thus precluding binding to HLA-F OCs. These findings provide a biochemical framework to understand how HLA-F could regulate immunity via interactions with NKRs.

Multiple Isomers of Photolumazine V Bind MR1 and Differentially Activate MAIT Cells.

In response to microbial infection, the nonclassical Ag-presenting molecule MHC class I-related protein 1 (MR1) presents secondary microbial metabolites to mucosal-associated invariant T (MAIT) cells. In this study, we further characterize the repertoire of ligands captured by MR1 produced in Hi5 (Trichoplusia ni) cells from Mycobacterium smegmatis via mass spectrometry. We describe the (to our knowledge) novel MR1 ligand photolumazine (PL)V, a hydroxyindolyl-ribityllumazine with four isomers differing in the positioning of a hydroxyl group. We show that all four isomers are produced by M. smegmatis in culture and that at least three can induce MR1 surface translocation. Furthermore, human MAIT cell clones expressing distinct TCR ß-chains differentially responded to the PLV isomers, demonstrating that the subtle positioning of a single hydroxyl group modulates TCR recognition. This study emphasizes structural microheterogeneity within the MR1 Ag repertoire and the remarkable selectivity of MAIT cell TCRs.

Parkinson's disease-associated mutations in α-synuclein alters its lipid-bound state.

Lipid-binding properties of α-synuclein play a central role in protein aggregation and progression of Parkinson's disease (PD). α-Synuclein, an intrinsically disordered protein, binds to lipid membranes through the formation of two amphipathic helices that insert into the lipid bilayer. All disease-associated single point mutations have been identified to be within these helical regions of α-synuclein: V15A, A30P, E46K, H50Q, G51D, A53T, and A53V. However, the effects of these mutations on the bound states of the two α helices of the protein have yet to be fully characterized. In this report, we use a tryptophan fluorescence assay to measure the binding of the α helices of these PD-associated mutants to lipid membranes within the lipid-depletion regime. We characterize the binding behavior of each helix, revealing that, generally, the PD-associated mutants shift the equilibrium bound state away from the N-terminal helix of the protein toward helix 2 at lower lipid concentrations. Altogether, our results indicate that disruption to the equilibrium binding of the two α helices of α-synuclein could play a role in PD progression.

The molecular characterization of antibody binding to a superantigen-like protein from a commensal microbe.

Microorganisms have coevolved diverse mechanisms to impair host defenses. A major one, superantigens, can result in devastating effects on the immune system. While all known superantigens induce vast immune cell proliferation and come from opportunistic pathogens, recently, proteins with similar broad specificity to antibody variable (V) domain families were identified in a commensal microbiota. These proteins, identified in the human commensal Ruminococcus gnavus, are called immunoglobulin-binding protein (Ibp) A and B and have been shown to activate B cells in vitro expressing either human VH3 or murine VH5/6/7. Here, we provide molecular and functional studies revealing the basis of this Ibp/immunoglobulin (Ig) interaction. The crystal structure and biochemical assays of a truncated IbpA construct in complex with mouse VH5 antigen-binding fragment (Fab) shows a binding of Ig heavy chain framework residues to the Ibp Domain D and the C-terminal heavy chain binding domain (HCBD). We used targeted mutagenesis of contact residues and affinity measurements and performed studies of the Fab-IbpA complex to determine the stoichiometry between Ibp and VH domains, suggesting Ibp may serve to cluster full-length IgA antibodies in vivo. Furthermore, in vitro stimulation experiments indicate that binding of the Ibp HCBD alone is sufficient to activate responsive murine B cell receptors. The presence of these proteins in a commensal microbe suggest that binding a broad repertoire of immunoglobulins, particularly in the gut/microbiome environment, may provide an important function in the maintenance of host/microbiome homeostasis contrasting with the pathogenic role of structurally homologous superantigens expressed by pathogens.

Molecular design of the γδT cell receptor ectodomain encodes biologically fit ligand recognition in the absence of mechanosensing.

High-acuity αßT cell receptor (TCR) recognition of peptides bound to major histocompatibility complex molecules (pMHCs) requires mechanosensing, a process whereby piconewton (pN) bioforces exert physical load on αßTCR-pMHC bonds to dynamically alter their lifetimes and foster digital sensitivity cellular signaling. While mechanotransduction is operative for both αßTCRs and pre-TCRs within the αßT lineage, its role in γδT cells is unknown. Here, we show that the human DP10.7 γδTCR specific for the sulfoglycolipid sulfatide bound to CD1d only sustains a significant load and undergoes force-induced structural transitions when the binding interface-distal γδ constant domain (C) module is replaced with that of αß. The chimeric γδ-αßTCR also signals more robustly than does the wild-type (WT) γδTCR, as revealed by RNA-sequencing (RNA-seq) analysis of TCR-transduced Rag2-/- thymocytes, consistent with structural, single-molecule, and molecular dynamics studies reflective of γδTCRs as mediating recognition via a more canonical immunoglobulin-like receptor interaction. Absence of robust, force-related catch bonds, as well as γδTCR structural transitions, implies that γδT cells do not use mechanosensing for ligand recognition. This distinction is consonant with the fact that their innate-type ligands, including markers of cellular stress, are expressed at a high copy number relative to the sparse pMHC ligands of αßT cells arrayed on activating target cells. We posit that mechanosensing emerged over ∼200 million years of vertebrate evolution to fulfill indispensable adaptive immune recognition requirements for pMHC in the αßT cell lineage that are unnecessary for the γδT cell lineage mechanism of non-pMHC ligand detection.

Diversity in recognition and function of human γδ T cells.

As interest increases in harnessing the potential power of tissue-resident cells for human health and disease, γδ T cells have been thrust into the limelight due to their prevalence in peripheral tissues, their sentinel-like phenotypes, and their unique antigen recognition capabilities. This review focuses primarily on human γδ T cells, highlighting their distinctive characteristics including antigen recognition, function, and development, with an emphasis on where they differ from their αß T cell comparators, as well as from γδ T cell populations in the mouse. We review the antigens that have been identified thus far to regulate members of the human Vδ1 population and discuss what players are involved in transducing phosphoantigen-mediated signals to human Vγ9Vδ2 T cells. We also briefly review distinguishing features of these cells in terms of TCR signaling, use of coreceptor and costimulatory molecules and their development. These cells have great potential to be harnessed in a clinical setting, but caution must be taken to understand their unique capabilities and how they differ from the populations to which they are commonly compared.

The intracellular B30.2 domain of butyrophilin 3A1 binds phosphoantigens to mediate activation of human Vγ9Vδ2 T cells.

In humans, Vγ9Vδ2 T cells detect tumor cells and microbial infections, including Mycobacterium tuberculosis, through recognition of small pyrophosphate containing organic molecules known as phosphoantigens (pAgs). Key to pAg-mediated activation of Vγ9Vδ2 T cells is the butyrophilin 3A1 (BTN3A1) protein that contains an intracellular B30.2 domain critical to pAg reactivity. Here, we have demonstrated through structural, biophysical, and functional approaches that the intracellular B30.2 domain of BTN3A1 directly binds pAg through a positively charged surface pocket. Charge reversal of pocket residues abrogates binding and Vγ9Vδ2 T cell activation. We have also identified a gain-of-function mutation within this pocket that, when introduced into the B30.2 domain of the nonstimulatory BTN3A3 isoform, transfers pAg binding ability and Vγ9Vδ2 T cell activation. These studies demonstrate that internal sensing of changes in pAg metabolite concentrations by BTN3A1 molecules is a critical step in Vγ9Vδ2 T cell detection of infection and tumorigenesis.

MFG-E8: a model of multiple binding modes associated with ps-binding proteins.

Membrane-binding proteins often associate with lipid membranes through a singular binding interface which is generally modeled as a two-state system: bound or unbound. However, even a single interface can engage with more than one mode of binding since a variety of interactions can contribute to the binding event. Unfortunately, the ability to clearly delineate the different binding modes of a singular binding interface has been elusive with existing models. Here, we present a study on milk fat globule EGF factor 8 (MFG-E8), which belongs to a class of proteins that identifies and binds phosphatidylserine (PS). These proteins detect membrane dysregulation implicated in exposed PS in apoptosis and malignant cells. In order to elucidate the factors affecting the binding of MFG-E8, we used a model system consisting of a series of lipid vesicles with varying PS mole fraction to identify the sensitivity of MFG-E8's binding affinity to changes in electrostatics using a tryptophan fluorescence spectral shift assay. Using a newly developed model, we experimentally identified three binding modes, each associated with a different number of PS lipids, with its cooperativity for binding being enhanced by the availability of negatively charged lipids. X-ray reflectivity experiments additionally suggest that MFG-E8's binding modes are influenced by membrane packing. The protocols established for elucidating MFG-E8's interaction with lipid membranes under different membrane conditions can be applied to the study of other membrane-binding proteins that target specific membrane attributes, such as fluidity and electrostatics, and help elucidate these membrane targeting mechanisms and their subsequent binding events.

Alpaca (Vicugna pacos), the first nonprimate species with a phosphoantigen-reactive Vγ9Vδ2 T cell subset.

Vγ9Vδ2 T cells are a major γδ T cell population in the human blood expressing a characteristic Vγ9JP rearrangement paired with Vδ2. This cell subset is activated in a TCR-dependent and MHC-unrestricted fashion by so-called phosphoantigens (PAgs). PAgs can be microbial [(E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate, HMBPP] or endogenous (isopentenyl pyrophosphate, IPP) and PAg sensing depends on the expression of B7-like butyrophilin (BTN3A, CD277) molecules. IPP increases in some transformed or aminobisphosphonate-treated cells, rendering those cells a target for Vγ9Vδ2 T cells in immunotherapy. Yet, functional Vγ9Vδ2 T cells have only been described in humans and higher primates. Using a genome-based study, we showed in silico translatable genes encoding Vγ9, Vδ2, and BTN3 in a few nonprimate mammalian species. Here, with the help of new monoclonal antibodies, we directly identified a T cell population in the alpaca (Vicugna pacos), which responds to PAgs in a BTN3-dependent fashion and shows typical TRGV9- and TRDV2-like rearrangements. T cell receptor (TCR) transductants and BTN3-deficient human 293T cells reconstituted with alpaca or human BTN3 or alpaca/human BTN3 chimeras showed that alpaca Vγ9Vδ2 TCRs recognize PAg in the context of human and alpaca BTN3. Furthermore, alpaca BTN3 mediates PAg recognition much better than human BTN3A1 alone and this improved functionality mapped to the transmembrane/cytoplasmic part of alpaca BTN3. In summary, we found remarkable similarities but also instructive differences of PAg-recognition by human and alpaca, which help in better understanding the molecular mechanisms controlling the activation of this prominent population of γδ T cells.

Crystal structure of Vδ1 T cell receptor in complex with CD1d-sulfatide shows MHC-like recognition of a self-lipid by human γδ T cells.

The nature of the antigens recognized by γδ T cells and their potential recognition of major histocompatibility complex (MHC)-like molecules has remained unclear. Members of the CD1 family of lipid-presenting molecules are suggested ligands for Vδ1 TCR-expressing γδ T cells, the major γδ lymphocyte population in epithelial tissues. We crystallized a Vδ1 TCR in complex with CD1d and the self-lipid sulfatide, revealing the unusual recognition of CD1d by germline Vδ1 residues spanning all complementarity-determining region (CDR) loops, as well as sulfatide recognition separately encoded by nongermline CDR3δ residues. Binding and functional analysis showed that CD1d presenting self-lipids, including sulfatide, was widely recognized by gut Vδ1+ γδ T cells. These findings provide structural demonstration of MHC-like recognition of a self-lipid by γδ T cells and reveal the prevalence of lipid recognition by innate-like T cell populations.

How Tim proteins differentially exploit membrane features to attain robust target sensitivity.

Immune surveillance cells such as T cells and phagocytes utilize integral plasma membrane receptors to recognize surface signatures on triggered and activated cells such as those in apoptosis. One such family of plasma membrane sensors, the transmembrane immunoglobulin and mucin domain (Tim) proteins, specifically recognize phosphatidylserine (PS) but elicit distinct immunological responses. The molecular basis for the recognition of lipid signals on target cell surfaces is not well understood. Previous results suggest that basic side chains present at the membrane interface on the Tim proteins might facilitate association with additional anionic lipids including but not necessarily limited to PS. We, therefore, performed a comparative quantitative analysis of the binding of the murine Tim1, Tim3, and Tim4, to synthetic anionic phospholipid membranes under physiologically relevant conditions. X-ray reflectivity and vesicle binding studies were used to compare the water-soluble domain of Tim3 with results previously obtained for Tim1 and Tim4. Although a calcium link was essential for all three proteins, the three homologs differed in how they balance the hydrophobic and electrostatic interactions driving membrane association. The proteins also varied in their sensing of phospholipid chain unsaturation and showed different degrees of cooperativity in their dependence on bilayer PS concentration. Surprisingly, trace amounts of anionic phosphatidic acid greatly strengthened the bilayer association of Tim3 and Tim4, but not Tim1. A novel mathematical model provided values for the binding parameters and illuminated the complex interplay among ligands. In conclusion, our results provide a quantitative description of the contrasting selectivity used by three Tim proteins in the recognition of phospholipids presented on target cell surfaces. This paradigm is generally applicable to the analysis of the binding of peripheral proteins to target membranes through the heterotropic cooperative interactions of multiple ligands.

Butyrophilin3A proteins and Vγ9Vδ2 T cell activation.

Despite playing critical roles in the immune response and having significant potential in immunotherapy, γδ T cells have garnered little of the limelight. One major reason for this paradox is that their antigen recognition mechanisms are largely unknown, limiting our understanding of their biology and our potential to modulate their activity. One of the best-studied γδ subsets is the human Vγ9Vδ2T cell population, which predominates in peripheral blood and can combat both microbial infections and cancers. Although it has been known for decades that Vγ9Vδ2T cells respond to the presence of small pyrophosphate-based metabolites, collectively named phosphoantigens (pAgs), derived from microbial sources or malignant cells, the molecular basis for this response has been unclear. A major breakthrough in this area came with the identification of the Butyrophilin 3A (BTN3A) proteins, members of the Butyrophilin/Butyrophilin-like protein family, as mediators between pAgs and Vγ9Vδ2T cells. In this article, we review the most recent studies regarding pAg activation of human Vγ9Vδ2T cells, mainly focusing on the role of BTN3A as the pAg sensing molecule, as well as its potential impact on downstream events of the activation process.

An autonomous CDR3delta is sufficient for recognition of the nonclassical MHC class I molecules T10 and T22 by gammadelta T cells.

It remains unclear whether gammadelta T cell antigen receptors (TCRs) detect antigens in a way similar to antibodies or alphabeta TCRs. Here we show that reactivity between the G8 and KN6 gammadelta TCRs and the major histocompatibility complex class Ib molecule T22 could be recapitulated, with retention of wild-type ligand affinity, in an alphabeta TCR after grafting of a G8 or KN6 complementarity-determining region 3-delta (CDR3delta) loop in place of the CDR3alpha loop of an alphabeta TCR. We also found that a shared sequence motif in CDR3delta loops of all T22-reactive gammadelta TCRs bound T22 in energetically distinct ways, and that T10(d), which bound G8 with weak affinity, was converted into a high-affinity ligand by a single point mutation. Our results demonstrate unprecedented autonomy of a single CDR3 loop in antigen recognition.

The immutable recognition of CD1d.

CD1d presents lipid antigens to natural killer T cells. In this issue of Immunity, Wun et al. (2011) and Mallevaey et al. (2011) explore the molecular details of nonself lipid discrimination and self-recognition.

Phosphoantigen-induced conformational change of butyrophilin 3A1 (BTN3A1) and its implication on Vγ9Vδ2 T cell activation.

Human Vγ9Vδ2 T cells respond to microbial infections as well as certain types of tumors. The key initiators of Vγ9Vδ2 activation are small, pyrophosphate-containing molecules called phosphoantigens (pAgs) that are present in infected cells or accumulate intracellularly in certain tumor cells. Recent studies demonstrate that initiation of the Vγ9Vδ2 T cell response begins with sensing of pAg via the intracellular domain of the butyrophilin 3A1 (BTN3A1) molecule. However, it is unknown how downstream events can ultimately lead to T cell activation. Here, using NMR spectrometry and molecular dynamics (MD) simulations, we characterize a global conformational change in the B30.2 intracellular domain of BTN3A1 induced by pAg binding. We also reveal by crystallography two distinct dimer interfaces in the BTN3A1 full-length intracellular domain, which are stable in MD simulations. These interfaces lie in close proximity to the pAg-binding pocket and contain clusters of residues that experience major changes of chemical environment upon pAg binding. This suggests that pAg binding disrupts a preexisting conformation of the BTN3A1 intracellular domain. Using a combination of biochemical, structural, and cellular approaches we demonstrate that the extracellular domains of BTN3A1 adopt a V-shaped conformation at rest, and that locking them in this resting conformation without perturbing their membrane reorganization properties diminishes pAg-induced T cell activation. Based on these results, we propose a model in which a conformational change in BTN3A1 is a key event of pAg sensing that ultimately leads to T cell activation.