A novel inhibitor of class IIa histone deacetylases attenuates collagen-induced arthritis.

BACKGROUND AND PURPOSE: Most inhibitors of histone deacetylases (HDACs) are not selective and are cytotoxic. Some have anti-inflammatory activity in disease models, but cytotoxicity prevents long-term uses in non-fatal diseases. Inhibitors selective for class IIa HDACs are much less cytotoxic and may have applications in management of chronic inflammatory diseases. EXPERIMENTAL APPROACH: LL87 is a novel HDAC inhibitor examined here for HDAC enzyme selectivity. It was also investigated in macrophages for cytotoxicity and for inhibition of lipopolysaccharide (LPS)-stimulated cytokine secretion. In a rat model of collagen-induced arthritis, LL87 was investigated for effects on joint inflammation in Dark Agouti rats. Histological, immunohistochemical, micro-computed tomography and molecular analyses characterise developing arthritis and anti-inflammatory efficacy. KEY RESULTS: LL87 was significantly more inhibitory against class IIa than class I or IIb HDAC enzymes. In macrophages, LL87 was not cytotoxic and reduced both LPS-induced secretion of pro-inflammatory cytokines, and IL6-induced class IIa HDAC activity. In rats, LL87 attenuated paw swelling and clinical signs of arthritis, reducing collagen loss and histological damage in ankle joints. LL87 decreased immune cell infiltration, especially pro-inflammatory macrophages and osteoclasts, into synovial joints and significantly reduced expression of pro-inflammatory cytokines and tissue-degrading proteases. CONCLUSION AND IMPLICATIONS: A novel inhibitor of class IIa HDACs has been shown to have an anti-inflammatory and anti-arthritic profile distinct from current therapies. It is efficacious in reducing macrophage infiltration and joint inflammation in a chronic model of rat arthritis.

MAIT cell-MR1 reactivity is highly conserved across multiple divergent species.

Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T cells that recognize small molecule metabolites presented by major histocompatibility complex class I related protein 1 (MR1), via an αß T cell receptor (TCR). MAIT TCRs feature an essentially invariant TCR α-chain, which is highly conserved between mammals. Similarly, MR1 is the most highly conserved major histocompatibility complex-I-like molecule. This extreme conservation, including the mode of interaction between the MAIT TCR and MR1, has been shown to allow for species-mismatched reactivities unique in T cell biology, thereby allowing the use of selected species-mismatched MR1-antigen (MR1-Ag) tetramers in comparative immunology studies. However, the pattern of cross-reactivity of species-mismatched MR1-Ag tetramers in identifying MAIT cells in diverse species has not been formally assessed. We developed novel cattle and pig MR1-Ag tetramers and utilized these alongside previously developed human, mouse, and pig-tailed macaque MR1-Ag tetramers to characterize cross-species tetramer reactivities. MR1-Ag tetramers from each species identified T cell populations in distantly related species with specificity that was comparable to species-matched MR1-Ag tetramers. However, there were subtle differences in staining characteristics with practical implications for the accurate identification of MAIT cells. Pig MR1 is sufficiently conserved across species that pig MR1-Ag tetramers identified MAIT cells from the other species. However, MAIT cells in pigs were at the limits of phenotypic detection. In the absence of sheep MR1-Ag tetramers, a MAIT cell population in sheep blood was identified phenotypically, utilizing species-mismatched MR1-Ag tetramers. Collectively, our results validate the use and define the limitations of species-mismatched MR1-Ag tetramers in comparative immunology studies.

Polyethylene Glycol Priming Enhances the Seed Germination and Seedling Growth of Scutellaria baicalensis Georgi under Salt Stress.

Seed priming has become a practical pre-sowing strategy to deal with abiotic stresses. This study aims to explore the effects of polyethylene glycol (PEG) priming on seed germination and seedling growth of Scutellaria baicalensis Georgi under salt stress. Regardless of seed priming, salt stress significantly inhibited the seed germination and seedling growth of S. baicalensis. PEG priming significantly alleviates the inhibitory effects of salt stress on seed germination and seedling growth when compared to non-priming and water priming. Among all treatments, PEG priming exhibited the highest germination rate, germination potential, seed vigor index, fresh weight, dry weight, and plant length; the highest contents of proline, soluble sugar, and soluble protein; the highest K+/Na+ ratio and relative water content; the highest antioxidant activities and contents; but the lowest H2O2, malondialdehyde (MDA) content, and relative electrical conductivity in response to salt stress. In addition, PEG priming had the highest transcript levels of antioxidant-related genes among all treatments under NaCl stress. Taken together, the results demonstrated that seed priming with PEG could be recommended as an effective practice to enhance the germination and early seedling growth of S. baicalensis under saline conditions.

Exogenous Sorbitol Application Confers Drought Tolerance to Maize Seedlings through Up-Regulating Antioxidant System and Endogenous Sorbitol Biosynthesis.

This study aims to explore the impacts of exogenous sorbitol on maize seedlings under polyethylene glycol (PEG)-simulated drought stress. Six treatments were set: normal condition (CK), PEG (P), 10 mM sorbitol (10S), PEG plus 10 mM sorbitol (10SP), 100 mM sorbitol (100S) and PEG plus 100 mM sorbitol (100SP). Maize seedlings' growth under PEG-simulated drought stress was significantly inhibited and exogenous sorbitol largely alleviated this growth inhibition. The seedlings under 10SP treatment grew much better than those under P, 100S and 100SP treatments and no significant difference in growth parameters was observed between the control and 10S treatment. The seedlings treated with 10SP had higher contents of soluble sugar, soluble protein, proline, ascorbic acid (AsA), reduced glutathione (GSH), sorbitol and relative water content, higher activities of antioxidant enzymes and aldose reductase, but lower contents of malondialdehyde (MDA), H2O2 and relative electrical conductivity than those treated with P, 100S and 100SP. qRT-PCR analysis showed that the transcript levels of genes encoding putative aldose reductase (AR) under P treatment were significantly up-regulated in sorbitol-applied treatments. Taken together, the results demonstrated that exogenous sorbitol application conferred drought tolerance to maize seedlings by up-regulating the expression levels of AR-related genes to enhance the accumulation of intracellular osmotic substances such as sorbitol and improve antioxidant systems to tone down the damage caused by drought stress.

Quantitative affinity measurement of small molecule ligand binding to major histocompatibility complex class-I-related protein 1 MR1.

The Major Histocompatibility Complex class I-related protein 1 (MR1) presents small molecule metabolites, drugs, and drug-like molecules that are recognized by MR1-reactive T cells. While we have an understanding of how antigens bind to MR1 and upregulate MR1 cell surface expression, a quantitative, cell-free, assessment of MR1 ligand-binding affinity was lacking. Here, we developed a fluorescence polarization-based assay in which fluorescent MR1 ligand was loaded into MR1 protein in vitro and competitively displaced by candidate ligands over a range of concentrations. Using this assay, ligand affinity for MR1 could be differentiated as strong (IC50 < 1 µM), moderate (1 µM < IC50 < 100 µM), and weak (IC50 > 100 µM). We demonstrated a clear correlation between ligand-binding affinity for MR1, the presence of a covalent bond between MR1 and ligand, and the number of salt bridge and hydrogen bonds formed between MR1 and ligand. Using this newly developed fluorescence polarization-based assay to screen for candidate ligands, we identified the dietary molecules vanillin and ethylvanillin as weak bona fide MR1 ligands. Both upregulated MR1 on the surface of C1R.MR1 cells and the crystal structure of a MAIT cell T cell receptor-MR1-ethylvanillin complex revealed that ethylvanillin formed a Schiff base with K43 of MR1 and was buried within the A'-pocket. Collectively, we developed and validated a method to quantitate the binding affinities of ligands for MR1 that will enable an efficient and rapid screening of candidate MR1 ligands.

Helical structure in cyclic peptides: effect of N-methyl amides versus esters.

An alpha helical turn can be reproduced in a cyclic pentapeptide if the first and fifth amino acid sidechains are correctly joined. Here structural studies (CD, NMR, in silico) reveal why N-methylation at positions not involved in hydrogen bonds disrupts helicity whereas ester bonds can maintain helicity and promote greater cell uptake.

CD8 coreceptor engagement of MR1 enhances antigen responsiveness by human MAIT and other MR1-reactive T cells.

Mucosal-associated invariant T (MAIT) cells detect microbial infection via recognition of riboflavin-based antigens presented by the major histocompatibility complex class I (MHC-I)-related protein 1 (MR1). Most MAIT cells in human peripheral blood express CD8αα or CD8αß coreceptors, and the binding site for CD8 on MHC-I molecules is relatively conserved in MR1. Yet, there is no direct evidence of CD8 interacting with MR1 or the functional consequences thereof. Similarly, the role of CD8αα in lymphocyte function remains ill-defined. Here, using newly developed MR1 tetramers, mutated at the CD8 binding site, and by determining the crystal structure of MR1-CD8αα, we show that CD8 engaged MR1, analogous to how it engages MHC-I molecules. CD8αα and CD8αß enhanced MR1 binding and cytokine production by MAIT cells. Moreover, the CD8-MR1 interaction was critical for the recognition of folate-derived antigens by other MR1-reactive T cells. Together, our findings suggest that both CD8αα and CD8αß act as functional coreceptors for MAIT and other MR1-reactive T cells.

Early detection of COPD based on graph convolutional network and small and weakly labeled data.

Chronic obstructive pulmonary disease (COPD) is a common disease with high morbidity and mortality, where early detection benefits the population. However, the early diagnosis rate of COPD is low due to the absence or slight early symptoms. In this paper, a novel method based on graph convolution network (GCN) for early detection of COPD is proposed, which uses small and weakly labeled chest computed tomography image data from the publicly available Danish Lung Cancer Screening Trial database. The key idea is to construct a graph using regions of interest randomly selected from the segmented lung parenchyma and then input it into the GCN model for COPD detection. In this way, the model can not only extract the feature information of each region of interest but also the topological structure information between regions of interest, that is, graph structure information. The proposed GCN model achieves an acceptable performance with an accuracy of 0.77 and an area under a curve of 0.81, which is higher than the previous studies on the same dataset. GCN model also outperforms several state-of-the-art methods trained at the same time. As far as we know, it is also the first time using the GCN model on this dataset for COPD detection.

A Novel CT-Based Radiomics Features Analysis for Identification and Severity Staging of COPD.

RATIONALE AND OBJECTIVES: To evaluate the role of radiomics based on Chest Computed Tomography (CT) in the identification and severity staging of chronic obstructive pulmonary disease (COPD). MATERIALS AND METHODS: This retrospective analysis included 322 participants (249 COPD patients and 73 control subjects). In total, 1395 chest CT-based radiomics features were extracted from each participant's CT images. Three feature selection methods, including variance threshold, Select K Best method, and least absolute shrinkage and selection operator (LASSO), and two classification methods, including support vector machine (SVM) and logistic regression (LR), were used as identification and severity classification of COPD. Performance was compared by AUC, accuracy, sensitivity, specificity, precision, and F1-score. RESULTS: 38 and 10 features were selected to construct radiomics models to detect and stage COPD, respectively. For COPD identification, SVM classifier achieved AUCs of 0.992 and 0.970, while LR classifier achieved AUCs of 0.993 and 0.972 in the training set and test set, respectively. For the severity staging of COPD, the mentioned two machine learning classifiers can better differentiate less severity (GOLD1 + GOLD2) group from greater severity (GOLD3 + GOLD4) group. The AUCs of SVM and LR is 0.907 and 0.903 in the training set, and that of 0.799 and 0.797 in the test set. CONCLUSION: The present study showed that the novel radiomics approach based on chest CT images that can be used for COPD identification and severity classification, and the constructed radiomics model demonstrated acceptable performance.

QSAR Classification Models for Prediction of Hydroxamate Histone Deacetylase Inhibitor Activity against Malaria Parasites.

Malaria, caused by Plasmodium parasites, results in >400,000 deaths annually. There is no effective vaccine, and new drugs with novel modes of action are needed because of increasing parasite resistance to current antimalarials. Histone deacetylases (HDACs) are epigenetic regulatory enzymes that catalyze post-translational protein deacetylation and are promising malaria drug targets. Here, we describe quantitative structure-activity relationship models to predict the antiplasmodial activity of hydroxamate-based HDAC inhibitors. The models incorporate P. falciparum in vitro activity data for 385 compounds containing a hydroxamic acid and were subject to internal and external validation. When used to screen 22 new hydroxamate-based HDAC inhibitors for antiplasmodial activity, model A7 (external accuracy 91%) identified three hits that were subsequently verified as having potent in vitro activity against P. falciparum parasites (IC50 = 6, 71, and 84 nM), with 8 to 51-fold selectivity for P. falciparum versus human cells.

Chemical Modulators of Mucosal Associated Invariant T Cells.

Over the past decade, we have contributed to the chemistry of microbial natural products and synthetic ligands, related to riboflavin and uracils, that modulate immune cells called mucosal associated invariant T cells (MAIT cells). These highly abundant T lymphocytes were only discovered in 2003 and have become recognized for their importance in mammalian immunology. Unlike other T cells, MAIT cells are not activated by peptide or lipid antigens. In collaboration with immunology and structural biology research groups, we discovered that they are instead activated by unstable nitrogen-containing heterocycles synthesized by bacteria. The most potent naturally occurring activating compound (antigen) is 5-(2-oxopropylideneamino)-d-ribitylaminouracil (5-OP-RU). This compound is an imine (Schiff base) formed through condensation between an intermediate in the biosynthesis of riboflavin (vitamin B2) and a metabolic byproduct of mammalian and microbial glycolysis. Although it is very unstable in water due to intramolecular ring closure or hydrolysis, we were able to develop a non-enzymatic synthesis that yields a pure kinetically stable compound in a nonaqueous solvent. This compound has revolutionized the study of MAIT cell immunology due to its potent activation (EC50 = 2 pM) of MAIT cells and its development into immunological reagents for detecting and characterizing MAIT cells in tissues. MAIT cells are now linked to key physiological processes and disease, including antibacterial defense, tissue repair, regulation of graft-vs-host disease, gastritis, inflammatory bowel diseases, and cancer. 5-OP-RU activates MAIT cells and, like a vaccine, has been shown to protect mice from bacterial infections and cancers. Mechanistic studies on the binding of 5-OP-RU to its dual protein targets, the major histocompatibility complex class I related protein (MR1) and the MAIT cell receptor (MAIT TCR), have involved synthetic chemistry, 2D 1H NMR spectroscopy, mass spectrometry, computer modeling and molecular dynamics simulations, biochemical, cellular, and immunological assays, and protein structural biology. These combined studies have revealed structural influences for 5-OP-RU in solution on protein binding and antigen presentation and potency; informed the development of potent (EC50 = 2 nM) and water stable analogues; led to fluorescent analogues for detecting and tracking binding proteins in and on cells; and enabled discovery of drugs and drug-like molecules that bind MR1 and modulate MAIT cell function. MAIT cells offer new opportunities for chemical synthesis to enhance the stability, potency, selectivity, and bioavailability of small molecule ligands for MR1 or MAIT TCR proteins, and to contribute to the understanding of T cell immunity and the development of prospective new immunomodulating medicines.

Targeting Class IIa HDACs: Insights from Phenotypes and Inhibitors.

This review summarizes key literature defining the phenotypes of individual class IIa HDAC proteins and compounds that selectively target their enzymatic catalytic domain (CD). The focus is on the effects of class IIa HDACs in physiological and pathological conditions, both in vitro and in vivo, and on their mode of action in regulating genes, upstream proteins and signaling pathways. Phenotype studies further demonstrate either beneficial or detrimental effects of silencing selected class IIa HDACs or their enzymatic properties. We also summarize the knowledge gained from structure-activity relationships of CD inhibitors as well as molecular mechanisms underpinning isozyme selectivity where crystal structures or modelling studies are available. Given that the number of genes affected by silencing class IIa HDACs is much smaller than class I, the role of gene regulation of class IIa HDACs could be much more selective. Since class IIa HDACs have restricted tissue distributions and multiple functions independent of their CD, targeting the CD of class IIa HDACs could lead to more selective therapeutic agents with significantly fewer side-effects than other HDAC ligands.

Identification and Phenotype of MAIT Cells in Cattle and Their Response to Bacterial Infections.

Mucosal-associated invariant T (MAIT) cells are a population of innate-like T cells that utilize a semi-invariant T cell receptor (TCR) α chain and are restricted by the highly conserved antigen presenting molecule MR1. MR1 presents microbial riboflavin biosynthesis derived metabolites produced by bacteria and fungi. Consistent with their ability to sense ligands derived from bacterial sources, MAIT cells have been associated with the immune response to a variety of bacterial infections, such as Mycobacterium spp., Salmonella spp. and Escherichia coli. To date, MAIT cells have been studied in humans, non-human primates and mice. However, they have only been putatively identified in cattle by PCR based methods; no phenotypic or functional analyses have been performed. Here, we identified a MAIT cell population in cattle utilizing MR1 tetramers and high-throughput TCR sequencing. Phenotypic analysis of cattle MAIT cells revealed features highly analogous to those of MAIT cells in humans and mice, including expression of an orthologous TRAV1-TRAJ33 TCR α chain, an effector memory phenotype irrespective of tissue localization, and expression of the transcription factors PLZF and EOMES. We determined the frequency of MAIT cells in peripheral blood and multiple tissues, finding that cattle MAIT cells are enriched in mucosal tissues as well as in the mesenteric lymph node. Cattle MAIT cells were responsive to stimulation by 5-OP-RU and riboflavin biosynthesis competent bacteria in vitro. Furthermore, MAIT cells in milk increased in frequency in cows with mastitis. Following challenge with virulent Mycobacterium bovis, a causative agent of bovine tuberculosis and a zoonosis, peripheral blood MAIT cells expressed higher levels of perforin. Thus, MAIT cells are implicated in the immune response to two major bacterial infections in cattle. These data suggest that MAIT cells are functionally highly conserved and that cattle are an excellent large animal model to study the role of MAIT cells in important zoonotic infections.

HDAC7 Inhibition by Phenacetyl and Phenylbenzoyl Hydroxamates.

The zinc-containing histone deacetylase enzyme HDAC7 is emerging as an important regulator of immunometabolism and cancer. Here, we exploit a cavity in HDAC7, filled by Tyr303 in HDAC1, to derive new inhibitors. Phenacetyl hydroxamates and 2-phenylbenzoyl hydroxamates bind to Zn2+ and are 50-2700-fold more selective inhibitors of HDAC7 than HDAC1. Phenylbenzoyl hydroxamates are 30-70-fold more potent HDAC7 inhibitors than phenacetyl hydroxamates, which is attributed to the benzoyl aromatic group interacting with Phe679 and Phe738. Phthalimide capping groups, including a saccharin analogue, decrease rotational freedom and provide hydrogen bond acceptor carbonyl/sulfonamide oxygens that increase inhibitor potency, liver microsome stability, solubility, and cell activity. Despite being the most potent HDAC7 inhibitors to date, they are not selective among class IIa enzymes. These strategies may help to produce tools for interrogating HDAC7 biology related to its catalytic site.

MiR-138-5p knockdown promotes osteogenic differentiation through FOXC1 up-regulation in human bone mesenchymal stem cells.

This study examined the hypothesis that the microRNA miR-138-5p reduces the osteodifferentiation of human bone mesenchymal stem cells (hBMSCs) by downregulating the expression of forkhead box C1 (FOXC1). For this, hBMSCs were separated from bone marrow and osteogenic induction medium was added to stimulate osteogenic differentiation. Flow cytometric analysis was applied to evaluate the expression of cell-surface antigens associated with hBMSCs, including CD29, CD44, CD90, CD45, and CD34. qRT-PCR assays and Western blot assays were used to measure the mRNA and protein expression of miR-138-5p, osteocalcin, runt-related transcription factor 2, bone sialoprotein, alkaline phosphatase (ALP), and FOXC1. ALP staining assays and Alizarin Red staining (ARS) assays were used to confirm osteogenic differentiation. We used a luciferase assay to test the interaction between miR-138-5p and FOXC1. We demonstrated that miR-138-5p is downregulated in osteogenic differentiated hBMSCs. Further, overexpression of miR-138-5p reduced the expression of markers for osteodifferentiation, ALP activity, and ARS activity. Furthermore, we showed that FOXC1 is a downstream target gene of miR-138-5p, and that knockdown of miR-138-5p improves the osteogenesis differentiation of hBMSCs by upregulating FOXC1. The results from this study indicate miR-138-5p as a new target for osteogenic differentiation of hBMSCs and the treatment of bone defects.

Endoplasmic reticulum chaperones stabilize ligand-receptive MR1 molecules for efficient presentation of metabolite antigens.

The antigen-presenting molecule MR1 (MHC class I-related protein 1) presents metabolite antigens derived from microbial vitamin B2 synthesis to activate mucosal-associated invariant T (MAIT) cells. Key aspects of this evolutionarily conserved pathway remain uncharacterized, including where MR1 acquires ligands and what accessory proteins assist ligand binding. We answer these questions by using a fluorophore-labeled stable MR1 antigen analog, a conformation-specific MR1 mAb, proteomic analysis, and a genome-wide CRISPR/Cas9 library screen. We show that the endoplasmic reticulum (ER) contains a pool of two unliganded MR1 conformers stabilized via interactions with chaperones tapasin and tapasin-related protein. This pool is the primary source of MR1 molecules for the presentation of exogenous metabolite antigens to MAIT cells. Deletion of these chaperones reduces the ER-resident MR1 pool and hampers antigen presentation and MAIT cell activation. The MR1 antigen-presentation pathway thus co-opts ER chaperones to fulfill its unique ability to present exogenous metabolite antigens captured within the ER.

Artificially induced MAIT cells inhibit M. bovis BCG but not M. tuberculosis during in vivo pulmonary infection.

There is significant interest in targeting MAIT cells with immunostimulatory agents to enhance immune responses. Mycobacterium tuberculosis (M. tb.) is a pervasive respiratory disease that could benefit from treatments that augment immunity. Here we investigate the role of MAIT cells in M. tb. infection and the potential for MAIT cell-targeted immunotherapy to control bacterial burdens. We find that MAIT cells fail to substantially accumulate in the lungs during murine pulmonary M. bovis BCG and M. tb. infections but this defect is overcome by intranasal installation of a TLR2/6 agonist and a MAIT cell antigen. Although artificially induced MAIT cells produce important cytokines in both infections, they control BCG but not M. tb. growth in the lungs. Correspondingly, M. tb.-infected mouse macrophages are relatively resistant to MAIT cell antimicrobial activities in vitro. Thus, MAIT cell antigen-mediated immunotherapy for M. tb. presents a complex challenge.

CXCL16 Stimulates Antigen-Induced MAIT Cell Accumulation but Trafficking During Lung Infection Is CXCR6-Independent.

Mucosa-associated invariant T (MAIT) cells are a unique T cell subset that contributes to protective immunity against microbial pathogens, but little is known about the role of chemokines in recruiting MAIT cells to the site of infection. Pulmonary infection with Francisella tularensis live vaccine strain (LVS) stimulates the accrual of large numbers of MAIT cells in the lungs of mice. Using this infection model, we find that MAIT cells are predominantly CXCR6+ but do not require CXCR6 for accumulation in the lungs. However, CXCR6 does contribute to long-term retention of MAIT cells in the airway lumen after clearance of the infection. We also find that MAIT cells are not recruited from secondary lymphoid organs and largely proliferate in situ in the lungs after infection. Nevertheless, the only known ligand for CXCR6, CXCL16, is sufficient to drive MAIT cell accumulation in the lungs in the absence of infection when administered in combination with the MAIT cell antigen 5-OP-RU. Overall, this new data advances the understanding of mechanisms that facilitate MAIT cell accumulation and retention in the lungs.

Atypical TRAV1-2- T cell receptor recognition of the antigen-presenting molecule MR1.

MR1 presents vitamin B-related metabolites to mucosal associated invariant T (MAIT) cells, which are characterized, in part, by the TRAV1-2+ αß T cell receptor (TCR). In addition, a more diverse TRAV1-2- MR1-restricted T cell repertoire exists that can possess altered specificity for MR1 antigens. However, the molecular basis of how such TRAV1-2- TCRs interact with MR1-antigen complexes remains unclear. Here, we describe how a TRAV12-2+ TCR (termed D462-E4) recognizes an MR1-antigen complex. We report the crystal structures of the unliganded D462-E4 TCR and its complex with MR1 presenting the riboflavin-based antigen 5-OP-RU. Here, the TRBV29-1 ß-chain of the D462-E4 TCR binds over the F'-pocket of MR1, whereby the complementarity-determining region (CDR) 3ß loop surrounded and projected into the F'-pocket. Nevertheless, the CDR3ß loop anchored proximal to the MR1 A'-pocket and mediated direct contact with the 5-OP-RU antigen. The D462-E4 TCR footprint on MR1 contrasted that of the TRAV1-2+ and TRAV36+ TCRs' docking topologies on MR1. Accordingly, diverse MR1-restricted T cell repertoire reveals differential docking modalities on MR1, thus providing greater scope for differing antigen specificities.

Absence of mucosal-associated invariant T cells in a person with a homozygous point mutation in MR1.

The role unconventional T cells play in protective immunity in humans is unclear. Mucosal-associated invariant T (MAIT) cells are an unconventional T cell subset restricted to the antigen-presenting molecule MR1. Here, we report the discovery of a patient homozygous for a rare Arg31His (R9H in the mature protein) mutation in MR1 who has a history of difficult-to-treat viral and bacterial infections. MR1R9H was unable to present the potent microbially derived MAIT cell stimulatory ligand. The MR1R9H crystal structure revealed that the stimulatory ligand cannot bind due to the mutation lying within, and causing structural perturbation to, the ligand-binding domain of MR1. While MR1R9H could bind and be up-regulated by a MAIT cell inhibitory ligand, the patient lacked circulating MAIT cells. This shows the importance of the stimulatory ligand for MAIT cell selection in humans. The patient had an expanded γδ T cell population, indicating a compensatory interplay between these unconventional T cell subsets.