ABSTRACT
Plasma membrane proteins mediate important aspects of physiology, including nutrient acquisition, cell-cell interactions, and monitoring homeostasis. The trafficking of these proteins, involving internalisation from and/or recycling back to the cell surface, is often critical to their functions. These processes can vary among different proteins and cell types and states and are still being elucidated. Current strategies to measure surface protein internalisation and recycling are typically microscopy or biochemical assays; these are accurate but generally limited to analysing a homogenous cell population and are often low throughput. Here, we present flow cytometry-based methods involving probe-conjugated antibodies that enable quantification of internalisation or recycling rates at the single-cell level in complex samples. To measure internalisation, we detail an assay where the protein of interest is labelled with a specific antibody conjugated to a fluorescent oligonucleotide-labelled probe. To measure recycling, a specific antibody conjugated to a cleavable biotin group is employed. These probes permit the differentiation of molecules that have been internalised or recycled from those that have not. When combined with cell-specific marker panels, these methods allow the quantitative study of plasma membrane protein trafficking dynamics in a heterogenous cell mixture at the single-cell level. Key features ⢠These assays allow sensitive quantification of internalised or recycled surface molecules using oligonucleotide or cleavable biotin-conjugated probes, respectively, and detected by flow cytometry. ⢠They can be adapted to any membrane protein that transits via the cell surface and for which a specific purified antibody is available. ⢠The dynamics of a cell surface protein can be measured in heterogenous cell populations simultaneously, including various cellular activation states. ⢠The internalisation assay builds upon the method developed by Liu et al. [1,2] and extends its application to heterogenous human peripheral blood mononuclear cells. ⢠These assays have been extensively used on suspension cells but have not been tested on adherent cells.
ABSTRACT
Bacterial synthesis of vitamin B2 generates a by-product, 5-(2-oxopropylideneamino)-d-ribityl-aminouracil (5-OP-RU), with potent immunological properties in mammals, but it is rapidly degraded in water. This natural product covalently bonds to the key immunological protein MR1 in the endoplasmic reticulum of antigen presenting cells (APCs), enabling MR1 refolding and trafficking to the cell surface, where it interacts with T cell receptors (TCRs) on mucosal associated invariant T lymphocytes (MAIT cells), activating their immunological and antimicrobial properties. Here, we strategically modify this natural product to understand the molecular basis of its recognition by MR1. This culminated in the discovery of new water-stable compounds with extremely powerful and distinctive immunological functions. We report their capacity to bind MR1 inside APCs, triggering its expression on the cell surface (EC50 17â nM), and their potent activation (EC50 56â pM) or inhibition (IC50 80â nM) of interacting MAIT cells. We further derivatize compounds with diazirine-alkyne, biotin, or fluorophore (Cy5 or AF647) labels for detecting, monitoring, and studying cellular MR1. Computer modeling casts new light on the molecular mechanism of activation, revealing that potent activators are first captured in a tyrosine- and serine-lined cleft in MR1 via specific pi-interactions and H-bonds, before more tightly attaching via a covalent bond to Lys43 in MR1. This chemical study advances our molecular understanding of how bacterial metabolites are captured by MR1, influence cell surface expression of MR1, interact with T cells to induce immunity, and offers novel clues for developing new vaccine adjuvants, immunotherapeutics, and anticancer drugs.
Subject(s)
Riboflavin , Humans , Riboflavin/metabolism , Riboflavin/chemistry , Riboflavin/pharmacology , Riboflavin/biosynthesis , Riboflavin/analogs & derivatives , Immunomodulating Agents/chemistry , Immunomodulating Agents/pharmacology , Immunomodulating Agents/metabolism , Histocompatibility Antigens Class I/metabolism , Histocompatibility Antigens Class I/immunology , Minor Histocompatibility Antigens/metabolism , Mucosal-Associated Invariant T Cells/metabolism , Mucosal-Associated Invariant T Cells/immunology , Immunologic Factors/pharmacology , Immunologic Factors/chemistry , Immunologic Factors/metabolism , Antigen-Presenting Cells/metabolism , Antigen-Presenting Cells/immunology , Ribitol/analogs & derivatives , Uracil/analogs & derivativesABSTRACT
Sepsis induces immune alterations, which last for months after the resolution of illness. The effect of this immunological reprogramming on the risk of developing cancer is unclear. Here we use a national claims database to show that sepsis survivors had a lower cumulative incidence of cancers than matched nonsevere infection survivors. We identify a chemokine network released from sepsis-trained resident macrophages that triggers tissue residency of T cells via CCR2 and CXCR6 stimulations as the immune mechanism responsible for this decreased risk of de novo tumor development after sepsis cure. While nonseptic inflammation does not provoke this network, laminarin injection could therapeutically reproduce the protective sepsis effect. This chemokine network and CXCR6 tissue-resident T cell accumulation were detected in humans with sepsis and were associated with prolonged survival in humans with cancer. These findings identify a therapeutically relevant antitumor consequence of sepsis-induced trained immunity.
Subject(s)
Macrophages , Neoplasms , Sepsis , Humans , Sepsis/immunology , Macrophages/immunology , Female , Neoplasms/immunology , Neoplasms/therapy , Male , Receptors, CXCR6/metabolism , Animals , T-Lymphocytes/immunology , Receptors, CCR2/metabolism , Middle Aged , Mice , Aged , Chemokines/metabolism , AdultABSTRACT
Immunity to infectious diseases is predominantly studied by measuring immune responses towards a single pathogen, although co-infections are common. In-depth mechanisms on how co-infections impact anti-viral immunity are lacking, but are highly relevant to treatment and prevention. We established a mouse model of co-infection with unrelated viruses, influenza A (IAV) and Semliki Forest virus (SFV), causing disease in different organ systems. SFV infection eight days before IAV infection results in prolonged IAV replication, elevated cytokine/chemokine levels and exacerbated lung pathology. This is associated with impaired lung IAV-specific CD8+ T cell responses, stemming from suboptimal CD8+ T cell activation and proliferation in draining lymph nodes, and dendritic cell paralysis. Prior SFV infection leads to increased blood brain barrier permeability and presence of IAV RNA in brain, associated with increased trafficking of IAV-specific CD8+ T cells and establishment of long-term tissue-resident memory. Relative to lung IAV-specific CD8+ T cells, brain memory IAV-specific CD8+ T cells have increased TCR repertoire diversity within immunodominant DbNP366+CD8+ and DbPA224+CD8+ responses, featuring suboptimal TCR clonotypes. Overall, our study demonstrates that infection with an unrelated neurotropic virus perturbs IAV-specific immune responses and exacerbates IAV disease. Our work provides key insights into therapy and vaccine regimens directed against unrelated pathogens.
Subject(s)
Coinfection , Influenza Vaccines , Influenza, Human , Orthomyxoviridae Infections , Viruses , Mice , Animals , Humans , Influenza, Human/pathology , CD8-Positive T-Lymphocytes , Coinfection/pathology , Receptors, Antigen, T-Cell , Lung/pathologyABSTRACT
The major histocompatibility complex (MHC), Class-I-related (MR1) molecule presents microbiome-synthesized metabolites to Mucosal-associated invariant T (MAIT) cells, present at sites of herpes simplex virus (HSV) infection. During HSV type 1 (HSV-1) infection there is a profound and rapid loss of MR1, in part due to expression of unique short 3 protein. Here we show that virion host shutoff RNase protein downregulates MR1 protein, through loss of MR1 transcripts. Furthermore, a third viral protein, infected cell protein 22, also downregulates MR1, but not classical MHC-I molecules. This occurs early in the MR1 trafficking pathway through proteasomal degradation. Finally, HSV-2 infection results in the loss of MR1 transcripts, and intracellular and surface MR1 protein, comparable to that seen during HSV-1 infection. Thus HSV coordinates a multifaceted attack on the MR1 antigen presentation pathway, potentially protecting infected cells from MAIT cell T cell receptor-mediated detection at sites of primary infection and reactivation.
ABSTRACT
MHC antigen presentation plays a fundamental role in adaptive and semi-invariant T cell immunity. Distinct MHC molecules bind antigens that differ in chemical structure, origin and location and present them to specialized T cells. MHC class I-related protein 1 (MR1) presents a range of small molecule antigens to MR1-restricted T (MR1T) lymphocytes. The best studied MR1 ligands are derived from microbial metabolism and are recognized by a major class of MR1T cells known as mucosal-associated invariant T (MAIT) cells. Here, we describe the MR1 antigen presentation pathway: the known types of antigens presented by MR1, the location where MR1-antigen complexes form, the route followed by the complexes to the cell surface, the mechanisms involved in termination of MR1 antigen presentation and the accessory cellular proteins that comprise the MR1 antigen presentation machinery. The current road map of the MR1 antigen presentation pathway reveals potential strategies for therapeutic manipulation of MR1T cell function and provides a foundation for further studies that will lead to a deeper understanding of MR1-mediated immunity.
Subject(s)
Antigen Presentation , Mucosal-Associated Invariant T Cells , Humans , Minor Histocompatibility Antigens , Histocompatibility Antigens Class I , AntigensABSTRACT
Among the 16 two-component systems in the opportunistic human pathogen Staphylococcus aureus, only WalKR is essential. Like the orthologous systems in other Bacillota, S. aureus WalKR controls autolysins involved in peptidoglycan remodeling and is therefore intimately involved in cell division. However, despite the importance of WalKR in S. aureus, the basis for its essentiality is not understood and the regulon is poorly defined. Here, we defined a consensus WalR DNA-binding motif and the direct WalKR regulon by using functional genomics, including chromatin immunoprecipitation sequencing, with a panel of isogenic walKR mutants that had a spectrum of altered activities. Consistent with prior findings, the direct regulon includes multiple autolysin genes. However, this work also revealed that WalR directly regulates at least five essential genes involved in lipoteichoic acid synthesis (ltaS): translation (rplK), DNA compaction (hup), initiation of DNA replication (dnaA, hup) and purine nucleotide metabolism (prs). Thus, WalKR in S. aureus serves as a polyfunctional regulator that contributes to fundamental control over critical cell processes by coordinately linking cell wall homeostasis with purine biosynthesis, protein biosynthesis, and DNA replication. Our findings further address the essentiality of this locus and highlight the importance of WalKR as a bona fide target for novel anti-staphylococcal therapeutics. IMPORTANCE The opportunistic human pathogen Staphylococcus aureus uses an array of protein sensing systems called two-component systems (TCS) to sense environmental signals and adapt its physiology in response by regulating different genes. This sensory network is key to S. aureus versatility and success as a pathogen. Here, we reveal for the first time the full extent of the regulatory network of WalKR, the only staphylococcal TCS that is indispensable for survival under laboratory conditions. We found that WalKR is a master regulator of cell growth, coordinating the expression of genes from multiple, fundamental S. aureus cellular processes, including those involved in maintaining cell wall metabolism, protein biosynthesis, nucleotide metabolism, and the initiation of DNA replication.
ABSTRACT
Introduction: The antigen presentation molecule MHC class I related protein-1 (MR1) is best characterized by its ability to present bacterially derived metabolites of vitamin B2 biosynthesis to mucosal-associated invariant T-cells (MAIT cells). Methods: Through in vitro human cytomegalovirus (HCMV) infection in the presence of MR1 ligand we investigate the modulation of MR1 expression. Using coimmunoprecipitation, mass spectrometry, expression by recombinant adenovirus and HCMV deletion mutants we investigate HCMV gpUS9 and its family members as potential regulators of MR1 expression. The functional consequences of MR1 modulation by HCMV infection are explored in coculture activation assays with either Jurkat cells engineered to express the MAIT cell TCR or primary MAIT cells. MR1 dependence in these activation assays is established by addition of MR1 neutralizing antibody and CRISPR/Cas-9 mediated MR1 knockout. Results: Here we demonstrate that HCMV infection efficiently suppresses MR1 surface expression and reduces total MR1 protein levels. Expression of the viral glycoprotein gpUS9 in isolation could reduce both cell surface and total MR1 levels, with analysis of a specific US9 HCMV deletion mutant suggesting that the virus can target MR1 using multiple mechanisms. Functional assays with primary MAIT cells demonstrated the ability of HCMV infection to inhibit bacterially driven, MR1-dependent activation using both neutralizing antibodies and engineered MR1 knockout cells. Discussion: This study identifies a strategy encoded by HCMV to disrupt the MR1:MAIT cell axis. This immune axis is less well characterized in the context of viral infection. HCMV encodes hundreds of proteins, some of which regulate the expression of antigen presentation molecules. However the ability of this virus to regulate the MR1:MAIT TCR axis has not been studied in detail.
Subject(s)
Mucosal-Associated Invariant T Cells , Humans , Histocompatibility Antigens Class I , Cytomegalovirus/metabolism , Minor Histocompatibility Antigens , Receptors, Antigen, T-Cell/metabolismABSTRACT
Hexanucleotide expansion mutations in C9ORF72 are a frequent cause of amyotrophic lateral sclerosis. We previously reported that long arginine-rich dipeptide repeats (DPRs), mimicking abnormal proteins expressed from the hexanucleotide expansion, caused translation stalling when expressed in cell culture models. Whether this stalling provides a mechanism of pathogenicity remains to be determined. Here, we explored the molecular features of DPR-induced stalling and examined whether known mechanisms such as ribosome quality control (RQC) regulate translation elongation on sequences that encode arginine-rich DPRs. We demonstrate that arginine-rich DPRs lead to stalling in a length-dependent manner, with lengths longer than 40 repeats invoking severe translation arrest. Mutational screening of 40×Gly-Xxx DPRs shows that stalling is most pronounced when Xxx is a charged amino acid (Arg, Lys, Glu, or Asp). Through a genome-wide knockout screen, we find that genes regulating stalling on polyadenosine mRNA coding for poly-Lys, a canonical RQC substrate, act differently in the case of arginine-rich DPRs. Indeed, these findings point to a limited scope for natural regulatory responses to resolve the arginine-rich DPR stalls, even though the stalls may be sensed, as evidenced by an upregulation of RQC gene expression. These findings therefore implicate arginine-rich DPR-mediated stalled ribosomes as a source of stress and toxicity and may be a crucial component in pathomechanisms.
Subject(s)
Amyotrophic Lateral Sclerosis , Humans , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/physiopathology , Arginine/metabolism , C9orf72 Protein/genetics , C9orf72 Protein/metabolism , Dipeptides/chemistry , Ribosomes/genetics , Ribosomes/metabolism , Gene Knockout Techniques , Mutation , Up-RegulationABSTRACT
The antigen presentation molecule MR1 (major histocompatibility complex, class I-related) presents ligands derived from the riboflavin (vitamin B) synthesis pathway, which is not present in mammalian species or viruses, to mucosal-associated invariant T (MAIT) cells. In this study, we demonstrate that varicella zoster virus (VZV) profoundly suppresses MR1 expression. We show that VZV targets the intracellular reservoir of immature MR1 for degradation, while preexisting, ligand-bound cell surface MR1 is protected from such targeting, thereby highlighting an intricate temporal relationship between infection and ligand availability. We also identify VZV open reading frame (ORF) 66 as functioning to suppress MR1 expression when this viral protein is expressed during transient transfection, but this is not apparent during infection with a VZV mutant virus lacking ORF66 expression. This indicates that VZV is likely to encode multiple viral genes that target MR1. Overall, we identify an immunomodulatory function of VZV whereby infection suppresses the MR1 biosynthesis pathway.
Subject(s)
Herpesvirus 3, Human , Histocompatibility Antigens Class I , Animals , Herpesvirus 3, Human/genetics , Ligands , Minor Histocompatibility Antigens , Major Histocompatibility Complex , MammalsABSTRACT
MR1 is a highly conserved microbial immune-detection system in mammals. It captures vitamin B-related metabolite antigens from diverse microbes and presents them at the cell surface to stimulate MR1-restricted lymphocytes including mucosal-associated invariant T (MAIT) cells. MR1 presentation and MAIT cell recognition mediate homeostasis through host defense and tissue repair. The cellular mechanisms regulating MR1 cell surface expression are critical to its function and MAIT cell recognition, yet they are poorly defined. Here, we report that human MR1 is equipped with a tyrosine-based motif in its cytoplasmic domain that mediates low affinity binding with the endocytic adaptor protein 2 (AP2) complex. This interaction controls the kinetics of MR1 internalization from the cell surface and minimizes recycling. We propose MR1 uses AP2 endocytosis to define the duration of antigen presentation to MAIT cells and the detection of a microbial metabolic signature by the immune system.
Subject(s)
Antigen Presentation , Endocytosis , Histocompatibility Antigens Class I , Minor Histocompatibility Antigens , Mucosal-Associated Invariant T Cells , Adaptor Protein Complex 2/genetics , Adaptor Protein Complex 2/metabolism , Histocompatibility Antigens Class I/genetics , Humans , Lymphocyte Activation , Minor Histocompatibility Antigens/genetics , Minor Histocompatibility Antigens/metabolism , Mucosal-Associated Invariant T Cells/metabolism , Tyrosine , VitaminsABSTRACT
Transfusion is a specific cause of acute kidney injury (AKI) after cardiac surgery. Whether there is an association between the composition of blood products and the onset of AKI is unknown. The present study suggests that the transfusion of packed red blood cells containing a high amount of myeloid-related protein 14 (MRP_14) could increase the incidence of AKI after cardiac surgery. In a mouse model, MRP_14 increased the influx of neutrophils in the kidney after ischemia-reperfusion and their ability to damage tubular cells. Higher concentrations of MRP_14 were found in packed red blood cells from female donors or prepared by whole blood filtration.
ABSTRACT
The MARCH E3 ubiquitin (Ub) ligase MARCH1 regulates trafficking of major histocompatibility complex class II (MHC II) and CD86, molecules of critical importance to immunity. Here we show, using a genome-wide CRISPR knockout screen, that ubiquitin-like protein 3 (UBL3) is a necessary component of ubiquitination-mediated trafficking of these molecules in mice and in humans. Ubl3-deficient mice have elevated MHC II and CD86 expression on the surface of professional and atypical antigen presenting cells. UBL3 also regulates MHC II and CD86 in human dendritic cells (DCs) and macrophages. UBL3 impacts ubiquitination of MARCH1 substrates, a mechanism that requires UBL3 plasma membrane anchoring via prenylation. Loss of UBL3 alters adaptive immunity with impaired development of thymic regulatory T cells, loss of conventional type 1 DCs, increased number of trogocytic marginal zone B cells, and defective in vivo MHC II and MHC I antigen presentation. In summary, we identify UBL3 as a conserved, critical factor in MARCH1-mediated ubiquitination with important roles in immune responses.
Subject(s)
Histocompatibility Antigens Class II , Ubiquitins , Animals , B7-2 Antigen/metabolism , Dendritic Cells , Histocompatibility Antigens Class II/metabolism , Major Histocompatibility Complex , Mice , Mice, Inbred C57BL , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , Ubiquitins/metabolismABSTRACT
Rationale: Brain injury induces systemic immunosuppression, increasing the risk of viral reactivations and altering neurological recovery. Objectives: To determine if systemic immune alterations and lung replication of herpesviridae are associated and can help predict outcomes after brain injury. Methods: We collected peripheral blood mononuclear cells in patients with severe brain injury requiring invasive mechanical ventilation. We systematically searched for respiratory herpes simplex virus (HSV) replications in tracheal aspirates. We also performed chromatin immunoprecipitation sequencing, RNA-sequencing, and in vitro functional assays of monocytes and CD4 T cells collected on Day 1 to characterize the immune response to severe acute brain injury. The primary outcome was the Glasgow Outcome Scale Extended at 6 months. Measurements and Main Results: In 344 patients with severe brain injury, lung HSV reactivations were observed in 39% of the 232 patients seropositive for HSV and independently associated with poor neurological recovery at 6 months (hazard ratio, 1.90; 95% confidence interval, 1.08-3.57). Weighted gene coexpression network analyses of the transcriptomic response of monocytes to brain injury defined a module of 721 genes, including PD-L1 and CD80, enriched for the binding DNA motif of the transcriptional factor Zeb2 and whose ontogenic analyses revealed decreased IFN-γ-mediated and antiviral response signaling pathways. This monocyte signature was preserved in a validation cohort and predicted the neurological outcome at 6 months with good accuracy (area under the curve, 0.786; 95% confidence interval, 0.593-0.978). Conclusions: A specific monocyte signature is associated with HSV reactivation and predicts poor recovery after brain injury. The alterations of the immune control of herpesviridae replication are understudied and represent a novel therapeutic target.
Subject(s)
Brain Injuries , Herpes Simplex , Herpesvirus 1, Human , Herpesvirus 1, Human/genetics , Humans , Leukocytes, Mononuclear , MonocytesABSTRACT
Understanding the complex elements affecting signal resolution in cytometry is key for quality experimental design and data. In this study, we incorporate autofluorescence as a contributing factor to our understanding of resolution in cytometry and corroborate its impact in fluorescence signal detection through mathematical predictions supported by empirical evidence. Our findings illustrate the critical importance of autofluorescence extraction via full spectrum unmixing in unmasking dim signals and delineating the expression and subset distribution of low abundance markers in discovery projects. We apply our findings to the precise definition of the tissue and cellular distribution of a weakly expressed fluorescent protein that reports on a low-abundance immunological gene. Exploiting the full spectrum coverage enabled by Aurora 5L, we describe a novel approach to the isolation of pure cell subset-specific autofluorescence profiles based on high dimensionality reduction algorithms. This method can also be used to unveil differences in the autofluorescent fingerprints of tissues in homeostasis and after immunological challenges.
Subject(s)
Algorithms , Coloring Agents , ImmunophenotypingABSTRACT
The low mutational burden of epithelial ovarian cancer (EOC) is an impediment to immunotherapies that rely on conventional MHC-restricted, neoantigen-reactive T lymphocytes. Mucosa-associated invariant T (MAIT) cells are MR1-restricted T cells with remarkable immunomodulatory properties. We sought to characterize intratumoral and ascitic MAIT cells in EOC. Single-cell RNA sequencing of six primary human tumor specimens demonstrated that MAIT cells were present at low frequencies within several tumors. When detectable, these cells highly expressed CD69 and VSIR, but otherwise exhibited a transcriptomic signature inconsistent with overt cellular activation and/or exhaustion. Unlike mainstream CD8+ T cells, CD8+ MAIT cells harbored high transcript levels of TNF, PRF1, GZMM and GNLY, suggesting their arming and cytotoxic potentials. In a congenic, MAIT cell-sufficient mouse model of EOC, MAIT and invariant natural killer T cells amassed in the peritoneal cavity where they showed robust IL-17A and IFN-γ production capacities, respectively. However, they gradually lost these functions with tumor progression. In a cohort of 23 EOC patients, MAIT cells were readily detectable in all ascitic fluids examined. In a sub-cohort in which we interrogated ascitic MAIT cells for functional impairments, several exhaustion markers, most notably VISTA, were present on the surface. However, ascitic MAIT cells were capable of producing IFN-γ, TNF-α and granzyme B, but neither IL-17A nor IL-10, in response to an MR1 ligand, bacterial lysates containing MR1 ligands, or a combination of IL-12 and IL-18. In conclusion, ascitic MAIT cells in EOC possess inducible effector functions that may be modified in future immunotherapeutic strategies.
Subject(s)
Mucosal-Associated Invariant T Cells , Ovarian Neoplasms , Animals , Ascites , CD8-Positive T-Lymphocytes , Carcinoma, Ovarian Epithelial , Cues , Cytokines , Female , Histocompatibility Antigens Class I/genetics , Humans , Interleukin-17 , Ligands , Mice , Minor Histocompatibility AntigensABSTRACT
MHC-related protein 1 (MR1) presents microbial riboflavin metabolites to mucosal-associated invariant T (MAIT) cells for surveillance of microbial presence. MAIT cells express a semi-invariant T-cell receptor (TCR), which recognizes MR1-antigen complexes in a pattern-recognition-like manner. Recently, diverse populations of MR1-restricted T cells have been described that exhibit broad recognition of tumor cells and appear to recognize MR1 in association with tumor-derived self-antigens, though the identity of these antigens remains unclear. Here, we have used TCR gene transfer and engineered MR1-expressing antigen-presenting cells to probe the MR1 restriction and antigen reactivity of a range of MR1-restricted TCRs, including model tumor-reactive TCRs. We confirm MR1 reactivity by these TCRs, show differential dependence on lysine at position 43 of MR1 (K43) and demonstrate competitive inhibition by the MR1 ligand 6-formylpterin. TCR-expressing reporter lines, however, failed to recapitulate the robust tumor specificity previously reported, suggesting an importance of accessory molecules for MR1-dependent tumor reactivity. Finally, MR1-mutant cell lines showed that distinct residues on the α1/α2 helices were required for TCR binding by different MR1-restricted T cells and suggested central but distinct docking modes by the broad family of MR1-restricted αß TCRs. Collectively, these data are consistent with recognition of distinct antigens by diverse MR1-restricted T cells.
Subject(s)
Mucosal-Associated Invariant T Cells , Receptors, Antigen, T-Cell, alpha-beta , Histocompatibility Antigens Class I/metabolism , Minor Histocompatibility Antigens/genetics , Receptors, Antigen, T-Cell/metabolism , Receptors, Antigen, T-Cell, alpha-beta/geneticsABSTRACT
CD1c presents lipid-based antigens to CD1c-restricted T cells, which are thought to be a major component of the human T cell pool. However, the study of CD1c-restricted T cells is hampered by the presence of an abundantly expressed, non-T cell receptor (TCR) ligand for CD1c on blood cells, confounding analysis of TCR-mediated CD1c tetramer staining. Here, we identified the CD36 family (CD36, SR-B1, and LIMP-2) as ligands for CD1c, CD1b, and CD1d proteins and showed that CD36 is the receptor responsible for non-TCR-mediated CD1c tetramer staining of blood cells. Moreover, CD36 blockade clarified tetramer-based identification of CD1c-restricted T cells and improved identification of CD1b- and CD1d-restricted T cells. We used this technique to characterize CD1c-restricted T cells ex vivo and showed diverse phenotypic features, TCR repertoire, and antigen-specific subsets. Accordingly, this work will enable further studies into the biology of CD1 and human CD1-restricted T cells.
Subject(s)
Antigen Presentation , Antigens, CD1/metabolism , CD36 Antigens/metabolism , Glycoproteins/metabolism , T-Lymphocyte Subsets/immunology , Blood Buffy Coat , CD36 Antigens/antagonists & inhibitors , Healthy Volunteers , Humans , Jurkat Cells , Ligands , Lipids/immunology , Primary Cell Culture , Protein Multimerization , Receptors, Antigen, T-Cell/metabolism , T-Lymphocyte Subsets/metabolismABSTRACT
Mucosal-associated invariant T (MAIT) cells are abundant innate-like T cells important in antimicrobial immunity. These cells express a semi-invariant T cell receptor that recognizes the Major Histocompatibility Complex (MHC) class I-related protein 1 (MR1) in complex with small metabolite antigens derived from a range of commensal and pathogenic bacteria and yeasts, but not other pathogens such as viruses. Thus, MR1 stimulation of MAIT cells was thought to act as a sensor of bacterial infection and was not directly involved in anti-viral immunity. Surprisingly, viruses have recently been shown to directly impair MR1 antigen presentation by targeting the intracellular pool of MR1 for degradation. In this review, we summarize our current understanding of viral evasion of MR1 presentation pathway, and contrast this to evasion of other related MHC molecules. We examine MAIT cell activity in viral infection with a focus on the role of TCR-mediated activation of these innate-like cells and speculate on the selective pressure for viral evasion of MR1 antigen presentation. Overall, viral evasion of MR1 presentation uncovers a new avenue of research and implies that the MR1-MAIT cell axis is more important in viral immunity than was previously appreciated.