Invariant natural killer T cells recognize lipid self antigen induced by microbial danger signals.

Invariant natural killer T cells (iNKT cells) have a prominent role during infection and other inflammatory processes, and these cells can be activated through their T cell antigen receptors by microbial lipid antigens. However, increasing evidence shows that they are also activated in situations in which foreign lipid antigens would not be present, which suggests a role for lipid self antigen. We found that an abundant endogenous lipid, ß-D-glucopyranosylceramide (ß-GlcCer), was a potent iNKT cell self antigen in mouse and human and that its activity depended on the composition of the N-acyl chain. Furthermore, ß-GlcCer accumulated during infection and in response to Toll-like receptor agonists, contributing to iNKT cell activation. Thus, we propose that recognition of ß-GlcCer by the invariant T cell antigen receptor translates innate danger signals into iNKT cell activation.

The Clonal Invariant NKT Cell Repertoire in People with Type 1 Diabetes Is Characterized by a Loss of Clones Expressing High-Affinity TCRs.

Invariant NKT (iNKT) cells in healthy people express iNKT-TCRs with widely varying affinities for CD1d, suggesting different roles for high- and low-affinity iNKT clones in immune regulation. However, the functional implications of this heterogeneity have not yet been determined. Functionally aberrant iNKT responses have been previously demonstrated in different autoimmune diseases, including human type 1 diabetes, but their relationship to changes in the iNKT clonal repertoire have not been addressed. In this study, we directly compared the clonal iNKT repertoire of people with recent onset type 1 diabetes and age- and gender-matched healthy controls with regard to iNKT-TCR affinity and cytokine production. Our results demonstrate a selective loss of clones expressing high-affinity iNKT-TCRs from the iNKT repertoire of people with type 1 diabetes. Furthermore, this bias in the clonal iNKT repertoire in type 1 diabetes was associated with increased GM-CSF, IL-4, and IL-13 cytokine secretion among Ag-stimulated low-affinity iNKT clones. Thus, qualitative changes of the clonal iNKT repertoire with the potential to affect the regulatory function of this highly conserved T cell population are already established at the early stages in type 1 diabetes. These findings may inform future rationales for the development of iNKT-based therapies aiming to restore immune tolerance in type 1 diabetes.

Cholesteryl esters stabilize human CD1c conformations for recognition by self-reactive T cells.

Cluster of differentiation 1c (CD1c)-dependent self-reactive T cells are abundant in human blood, but self-antigens presented by CD1c to the T-cell receptors of these cells are poorly understood. Here we present a crystal structure of CD1c determined at 2.4 Å revealing an extended ligand binding potential of the antigen groove and a substantially different conformation compared with known CD1c structures. Computational simulations exploring different occupancy states of the groove reenacted these different CD1c conformations and suggested cholesteryl esters (CE) and acylated steryl glycosides (ASG) as new ligand classes for CD1c. Confirming this, we show that binding of CE and ASG to CD1c enables the binding of human CD1c self-reactive T-cell receptors. Hence, human CD1c adopts different conformations dependent on ligand occupancy of its groove, with CE and ASG stabilizing CD1c conformations that provide a footprint for binding of CD1c self-reactive T-cell receptors.

Multitissue Transcriptomics Delineates the Diversity of Airway T Cell Functions in Asthma.

Asthma arises from the complex interplay of inflammatory pathways in diverse cell types and tissues. We sought to undertake a comprehensive transcriptomic assessment of the epithelium and airway T cells that remain understudied in asthma and investigate interactions between multiple cells and tissues. Epithelial brushings and flow-sorted CD3+ T cells from sputum and BAL were obtained from healthy subjects (n = 19) and patients with asthma (mild, moderate, and severe asthma; n = 46). Gene expression was assessed using Affymetrix HT HG-U133+ PM GeneChips, and results were validated by real-time quantitative PCR. In the epithelium, IL-13 response genes (POSTN, SERPINB2, and CLCA1), mast cell mediators (CPA3 and TPSAB1), inducible nitric oxide synthase, and cystatins (CST1, CST2, and CST4) were upregulated in mild asthma, but, except for cystatins, were suppressed by corticosteroids in moderate asthma. In severe asthma-with predominantly neutrophilic phenotype-several distinct processes were upregulated, including neutrophilia (TCN1 and MMP9), mucins, and oxidative stress responses. The majority of the disease signature was evident in sputum T cells in severe asthma, where 267 genes were differentially regulated compared with health, highlighting compartmentalization of inflammation. This signature included IL-17-inducible chemokines (CXCL1, CXCL2, CXCL3, IL8, and CSF3) and chemoattractants for neutrophils (IL8, CCL3, and LGALS3), T cells, and monocytes. A protein interaction network in severe asthma highlighted signatures of responses to bacterial infections across tissues (CEACAM5, CD14, and TLR2), including Toll-like receptor signaling. In conclusion, the activation of innate immune pathways in the airways suggests that activated T cells may be driving neutrophilic inflammation and steroid-insensitive IL-17 response in severe asthma.

The versatility of the CD1 lipid antigen presentation pathway.

The family of non-classical major histocompatibility complex (MHC) class-I like CD1 molecules has an emerging role in human disease. Group 1 CD1 includes CD1a, CD1b and CD1c, which function to display lipids on the cell surface of antigen-presenting cells for direct recognition by T-cells. The recent advent of CD1 tetramers and the identification of novel lipid ligands has contributed towards the increasing number of CD1-restricted T-cell clones captured. These advances have helped to identify novel donor unrestricted and semi-invariant T-cell populations in humans and new mechanisms of T-cell recognition. However, although there is an opportunity to design broadly acting lipids and harness the therapeutic potential of conserved T-cells, knowledge of their role in health and disease is lacking. We briefly summarize the current evidence implicating group 1 CD1 molecules in infection, cancer and autoimmunity and show that although CD1 are not as diverse as MHC, recent discoveries highlight their versatility as they exhibit intricate mechanisms of antigen presentation.

Structural and Functional Changes of the Invariant NKT Clonal Repertoire in Early Rheumatoid Arthritis.

Invariant NKT cells (iNKT) are potent immunoregulatory T cells that recognize CD1d via a semi-invariant TCR (iNKT-TCR). Despite the knowledge of a defective iNKT pool in several autoimmune conditions, including rheumatoid arthritis (RA), a clear understanding of the intrinsic mechanisms, including qualitative and structural changes of the human iNKT repertoire at the earlier stages of autoimmune disease, is lacking. In this study, we compared the structure and function of the iNKT repertoire in early RA patients with age- and gender-matched controls. We analyzed the phenotype and function of the ex vivo iNKT repertoire as well as CD1d Ag presentation, combined with analyses of a large panel of ex vivo sorted iNKT clones. We show that circulating iNKTs were reduced in early RA, and their frequency was inversely correlated to disease activity score 28. Proliferative iNKT responses were defective in early RA, independent of CD1d function. Functional iNKT alterations were associated with a skewed iNKT-TCR repertoire with a selective reduction of high-affinity iNKT clones in early RA. Furthermore, high-affinity iNKTs in early RA exhibited an altered functional Th profile with Th1- or Th2-like phenotype, in treatment-naive and treated patients, respectively, compared with Th0-like Th profiles exhibited by high-affinity iNKTs in controls. To our knowledge, this is the first study to provide a mechanism for the intrinsic qualitative defects of the circulating iNKT clonal repertoire in early RA, demonstrating defects of iNKTs bearing high-affinity TCRs. These defects may contribute to immune dysregulation, and our findings could be exploited for future therapeutic intervention.

Multidimensional endotypes of asthma: topological data analysis of cross-sectional clinical, pathological, and immunological data.

BACKGROUND: Incomplete understanding of mechanisms and clinicopathobiological heterogeneity in asthma hinders research progress. Pathogenic roles for T-helper-type 17 (Th17) cells and invariant T cells implied by murine data have yet to be assessed in man. We aimed to investigate the role of Th17 and mucosal associated invariant T (MAIT) cells in airway inflammation; to characterise associations between diverse clinical and immunological features of asthma; and to identify novel multidimensional asthma endotypes. METHODS: In this single-centre, cross-sectional observational study in the UK, we assessed volunteers with mild-to-severe asthma and healthy non-atopic controls using clinical and physiological assessment and immunological sampling of blood, induced sputum, endobronchial biopsy, and bronchoalveolar lavage for flow cytometry and multiplex-electrochemiluminescence assays. Primary outcomes were changes in frequencies of Th17 and MAIT cells between health and asthma using Mann-Whitney U tests and the Jonckheere-Terpstra test (linear trend across ranked groups). The study had 80% power to detect 60% differences in T-cell frequencies at p<0·05. Bayesian Network Analysis (BNA) was used to explore associations between parameters. Topological Data Analysis (TDA) was used to identify multidimensional endotypes. The study had local research ethics approval. All participants provided informed consent. FINDINGS: Participants were 84 male and female volunteers (60 with mild-to-severe asthma and 24 healthy, non-atopic controls) aged 18-70 years recruited from clinics and research cohorts. Th17 cells and γδ17 cells were not associated with asthma, even in severe neutrophilic forms. MAIT-cell frequencies were strikingly reduced in asthma compared with health (median frequency in blood 0·9% of CD3+ cells [IQR 0·3-1·8] in asthma vs 1·6 [1·2-2·6] in health, p=0·005; in sputum 1·1 [0·7-2·0] vs 1·8 [1·6-2·3], p=0·002; and in biopsy samples 1·3 [0·7-2·3] vs 3·9% [1·3-5·3%], p=0·02), especially in severe asthma where BAL regulatory T cells were also reduced compared with those in health (4·4, 3·1-6·1, vs 8·1, 5·6-10; p=0·02). BNA and TDA identified six novel clinicopathobiological clusters of underlying disease mechanisms, with elevated mast cell mediators tryptase (p<0·0001), chymase (p=0·02), and carboxypeptidase A3 (p=0·02) in severe asthma. INTERPRETATION: This study suggests that Th17 cells do not have a major pathogenic role in human asthma. We describe a novel deficiency of MAIT cells in severe asthma. We also provide proof of concept for application of TDA to identification of multidimensional clinicopathobiological endotypes. Endotypes will require validation in further cohorts. FUNDING: Wellcome Trust.

NK cell killer Ig-like receptor repertoire acquisition and maturation are strongly modulated by HLA class I molecules.

The interaction between clonally distributed inhibitory receptors and their activating counterparts on NK cells and HLA class I molecules defines NK cell functions, but the role of HLA class I ligands in the acquisition of their receptors during NK development is still unclear. Although some studies demonstrated that HLA-C affects the expression of killer Ig-like receptors (KIR), other studies showed that NK cells acquire their KIR repertoire in a stochastic manner. Only when infected with human CMV is an expansion of self-specific KIR(+) NKG2C(+) NK cells detected. To gain more insight into this question, we compared the coexpression of different KIR molecules, NKG2A, CD8, and CD57, on NK cells in healthy donors and seven patients with deficient HLA class I expression due to mutations in one of the TAP genes. Our results show a correlation between the presence/absence of HLA class I molecules and the coexpression of their receptors. In an HLA class I low-expression context, an increase in KIR molecules' coexpression is detected on the NKG2A(+) CD8(+) subset. In functional assays, hyporesponsiveness was observed for TAP-deficient NK cells derived from four patients. In contrast, NK cells from patient five were functional, whereas CD107a(+) and IFN-γ(+) CD56(dim) NK cells presented a different pattern of HLA class I receptors compared with healthy donors. Taken together, our results provide strong evidence for the role of HLA class I molecules in NK cell maturation and KIR repertoire acquisition.

Innate and adaptive T cells in asthmatic patients: Relationship to severity and disease mechanisms.

BACKGROUND: Asthma is a chronic inflammatory disease involving diverse cells and mediators whose interconnectivity and relationships to asthma severity are unclear. OBJECTIVE: We performed a comprehensive assessment of TH17 cells, regulatory T cells, mucosal-associated invariant T (MAIT) cells, other T-cell subsets, and granulocyte mediators in asthmatic patients. METHODS: Sixty patients with mild-to-severe asthma and 24 control subjects underwent detailed clinical assessment and provided induced sputum, endobronchial biopsy, bronchoalveolar lavage, and blood samples. Adaptive and invariant T-cell subsets, cytokines, mast cells, and basophil mediators were analyzed. RESULTS: Significant heterogeneity of T-cell phenotypes was observed, with levels of IL-13-secreting T cells and type 2 cytokines increased at some, but not all, asthma severities. TH17 cells and γδ-17 cells, proposed drivers of neutrophilic inflammation, were not strongly associated with asthma, even in severe neutrophilic forms. MAIT cell frequencies were strikingly reduced in both blood and lung tissue in relation to corticosteroid therapy and vitamin D levels, especially in patients with severe asthma in whom bronchoalveolar lavage regulatory T-cell numbers were also reduced. Bayesian network analysis identified complex relationships between pathobiologic and clinical parameters. Topological data analysis identified 6 novel clusters that are associated with diverse underlying disease mechanisms, with increased mast cell mediator levels in patients with severe asthma both in its atopic (type 2 cytokine-high) and nonatopic forms. CONCLUSION: The evidence for a role for TH17 cells in patients with severe asthma is limited. Severe asthma is associated with a striking deficiency of MAIT cells and high mast cell mediator levels. This study provides proof of concept for disease mechanistic networks in asthmatic patients with clusters that could inform the development of new therapies.

CD1d protein structure determines species-selective antigenicity of isoglobotrihexosylceramide (iGb3) to invariant NKT cells.

Isoglobotrihexosylceramide (iGb3) has been identified as a potent CD1d-presented self-antigen for mouse invariant natural killer T (iNKT) cells. The role of iGb3 in humans remains unresolved, however, as there have been conflicting reports about iGb3-dependent human iNKT-cell activation, and humans lack iGb3 synthase, a key enzyme for iGb3 synthesis. Given the importance of human immune responses, we conducted a human-mouse cross-species analysis of iNKT-cell activation by iGb3-CD1d. Here we show that human and mouse iNKT cells were both able to recognise iGb3 presented by mouse CD1d (mCD1d), but not human CD1d (hCD1d), as iGb3-hCD1d was unable to support cognate interactions with the iNKT-cell TCRs tested in this study. The structural basis for this discrepancy was identified as a single amino acid variation between hCD1d and mCD1d, a glycine-to-tryptophan modification within the α2-helix that prevents flattening of the iGb3 headgroup upon TCR ligation. Mutation of the human residue, Trp153, to the mouse ortholog, Gly155, therefore allowed iGb3-hCD1d to stimulate human iNKT cells. In conclusion, our data indicate that iGb3 is unlikely to be a major antigen in human iNKT-cell biology.

Natural variations at position 93 of the invariant Vα24-Jα18 α chain of human iNKT-cell TCRs strongly impact on CD1d binding.

Human invariant natural killer T (NKT) cell TCRs bind to CD1d via an "invariant" Vα24-Jα18 chain (iNKTα) paired to semi-invariant Vß11 chains (iNKTß). Single-amino acid variations at position 93 (p93) of iNKTα, immediately upstream of the "invariant" CDR3α region, have been reported in a substantial proportion of human iNKT-cell clones (4-30%). Although p93, a serine in most human iNKT-cell TCRs, makes no contact with CD1d, it could affect CD1d binding by altering the conformation of the crucial CDR3α loop. By generating recombinant refolded iNKT-cell TCRs, we show that natural single-nucleotide variations in iNKTα, translating to serine, threonine, asparagine or isoleucine at p93, exert a powerful effect on CD1d binding, with up to 28-fold differences in affinity between these variants. This effect was observed with CD1d loaded with either the artificial α-galactosylceramide antigens KRN7000 or OCH, or the endogenous glycolipid ß-galactosylceramide, and its importance for autoreactive recognition of endogenous lipids was demonstrated by the binding of variant iNKT-cell TCR tetramers to cell surface expressed CD1d. The serine-containing variant showed the strongest CD1d binding, offering an explanation for its predominance in vivo. Complementary molecular dynamics modeling studies were consistent with an impact of p93 on the conformation of the CDR3α loop.

Innate-like control of human iNKT cell autoreactivity via the hypervariable CDR3beta loop.

Invariant Natural Killer T cells (iNKT) are a versatile lymphocyte subset with important roles in both host defense and immunological tolerance. They express a highly conserved TCR which mediates recognition of the non-polymorphic, lipid-binding molecule CD1d. The structure of human iNKT TCRs is unique in that only one of the six complementarity determining region (CDR) loops, CDR3beta, is hypervariable. The role of this loop for iNKT biology has been controversial, and it is unresolved whether it contributes to iNKT TCR:CD1d binding or antigen selectivity. On the one hand, the CDR3beta loop is dispensable for iNKT TCR binding to CD1d molecules presenting the xenobiotic alpha-galactosylceramide ligand KRN7000, which elicits a strong functional response from mouse and human iNKT cells. However, a role for CDR3beta in the recognition of CD1d molecules presenting less potent ligands, such as self-lipids, is suggested by the clonal distribution of iNKT autoreactivity. We demonstrate that the human iNKT repertoire comprises subsets of greatly differing TCR affinity to CD1d, and that these differences relate to their autoreactive functions. These functionally different iNKT subsets segregate in their ability to bind CD1d-tetramers loaded with the partial agonist alpha-linked glycolipid antigen OCH and structurally different endogenous beta-glycosylceramides. Using surface plasmon resonance with recombinant iNKT TCRs and different ligand-CD1d complexes, we demonstrate that the CDR3beta sequence strongly impacts on the iNKT TCR affinity to CD1d, independent of the loaded CD1d ligand. Collectively our data reveal a crucial role for CDR3beta for the function of human iNKT cells by tuning the overall affinity of the iNKT TCR to CD1d. This mechanism is relatively independent of the bound CD1d ligand and thus forms the basis of an inherent, CDR3beta dependent functional hierarchy of human iNKT cells.

Doxycycline for the prevention of progression of COVID-19 to severe disease requiring intensive care unit (ICU) admission: A randomized, controlled, open-label, parallel group trial (DOXPREVENT.ICU).

BACKGROUND: After admission to hospital, COVID-19 progresses in a substantial proportion of patients to critical disease that requires intensive care unit (ICU) admission. METHODS: In a pragmatic, non-blinded trial, 387 patients aged 40-90 years were randomised to receive treatment with SoC plus doxycycline (n = 192) or SoC only (n = 195). The primary outcome was the need for ICU admission as judged by the attending physicians. Three types of analyses were carried out for the primary outcome: "Intention to treat" (ITT) based on randomisation; "Per protocol" (PP), excluding patients not treated according to randomisation; and "As treated" (AT), based on actual treatment received. The trial was undertaken in six hospitals in India with high-quality ICU facilities. An online application serving as the electronic case report form was developed to enable screening, randomisation and collection of outcomes data. RESULTS: Adherence to treatment per protocol was 95.1%. Among all 387 participants, 77 (19.9%) developed critical disease needing ICU admission. In all three primary outcome analyses, doxycycline was associated with a relative risk reduction (RRR) and absolute risk reduction (ARR): ITT 31.6% RRR, 7.4% ARR (P = 0.063); PP 40.7% RRR, 9.6% ARR (P = 0.017); AT 43.2% RRR, 10.8% ARR (P = 0.007), with numbers needed to treat (NTT) of 13.4 (ITT), 10.4 (PP), and 9.3 (AT), respectively. Doxycycline was well tolerated with not a single patient stopping treatment due to adverse events. CONCLUSIONS: In hospitalized COVID-19 patients, doxycycline, a safe, inexpensive, and widely available antibiotic with anti-inflammatory properties, reduces the need for ICU admission when added to SoC.

Structure and binding kinetics of three different human CD1d-alpha-galactosylceramide-specific T cell receptors.

Invariant human TCR Valpha24-Jalpha18+/Vbeta11+ NKT cells (iNKT) are restricted by CD1d-alpha-glycosylceramides. We analyzed crystal structures and binding characteristics for an iNKT TCR plus two CD1d-alpha-GalCer-specific Vbeta11+ TCRs that use different TCR Valpha chains. The results were similar to those previously reported for MHC-peptide-specific TCRs, illustrating the versatility of the TCR platform. Docking TCR and CD1d-alpha-GalCer structures provided plausible insights into their interaction. The model supports a diagonal orientation of TCR on CD1d and suggests that complementarity determining region (CDR)3alpha, CDR3beta, and CDR1beta interact with ligands presented by CD1d, whereas CDR2beta binds to the CD1d alpha1 helix. This docking provides an explanation for the dominant usage of Vbeta11 and Vbeta8.2 chains by human and mouse iNKT cells, respectively, for recognition of CD1d-alpha-GalCer.

Impaired selection of invariant natural killer T cells in diverse mouse models of glycosphingolipid lysosomal storage diseases.

Glycolipid ligands for invariant natural killer T cells (iNKT cells) are loaded onto CD1d molecules in the late endosome/lysosome. Accumulation of glycosphingolipids (GSLs) in lysosomal storage diseases could potentially influence endogenous and exogenous lipid loading and/or presentation and, thus, affect iNKT cell selection or function. The percentages and frequency of iNKT cells were reduced in multiple mouse models of lysosomal GSL storage disease, irrespective of the specific genetic defect or lipid species stored. Reduced numbers of iNKT cells resulted in the absence of cytokine production in response to alpha-galactosylceramide (alpha-GalCer) and reduced iNKT cell-mediated lysis of wild-type targets loaded with alpha-GalCer. The reduction in iNKT cells did not result from defective expression of CD1d or a lack of antigen-presenting cells. Although H-2 restricted CD4(+) T cell responses were generally unaffected, processing of a lysosome-dependent analogue of alpha-GalCer was impaired in all the strains of mice tested. These data suggest that GSL storage may result in alterations in thymic selection of iNKT cells caused by impaired presentation of selecting ligands.

Invariant natural killer T cells in asthma and chronic obstructive pulmonary disease.

BACKGROUND: The number of type 2 helper CD4+ T cells is increased in the airways of persons with asthma. Whether the majority of these cells are class II major-histocompatibility-complex-restricted cells or are among the recently identified CD1d-restricted invariant natural killer T cells is a matter of controversy. We studied the frequency of invariant natural killer T cells in the airways of subjects with mild or moderately severe asthma to investigate the possibility of an association between the number of invariant natural killer T cells in the airway and disease severity. We also studied whether an increased number of these cells is a feature of chronic obstructive pulmonary disease (COPD). METHODS: We enumerated invariant natural killer T cells by flow cytometry with the use of CD1d tetramers loaded with alpha-galactosylceramide and antibodies specific to the invariant natural killer T-cell receptor in samples of bronchoalveolar-lavage fluid, induced sputum, and bronchial-biopsy specimens obtained from subjects with mild or moderately severe asthma, subjects with COPD, and healthy control subjects. Real-time polymerase-chain-reaction analysis was performed on bronchoalveolar-lavage cells for evidence of gene expression of the invariant natural killer T-cell receptor. RESULTS: Fewer than 2% of the T cells obtained from all subjects on airway biopsy, bronchoalveolar lavage, and sputum induction were invariant natural killer T cells, with no significant differences among the three groups of subjects. No expression of messenger RNA for the invariant natural killer T-cell-receptor domains Valpha24 and Vbeta11 was detected in bronchoalveolar-lavage cells from subjects with asthma. CONCLUSIONS: Invariant natural killer T cells are found in low numbers in the airways of subjects with asthma, subjects with COPD, and controls.