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1.
J Exp Med ; 218(7)2021 07 05.
Article in English | MEDLINE | ID: mdl-33961028

ABSTRACT

We optimized lipidomics methods to broadly detect endogenous lipids bound to cellular CD1a proteins. Whereas membrane phospholipids dominate in cells, CD1a preferentially captured sphingolipids, especially a C42, doubly unsaturated sphingomyelin (42:2 SM). The natural 42:2 SM but not the more common 34:1 SM blocked CD1a tetramer binding to T cells in all human subjects tested. Thus, cellular CD1a selectively captures a particular endogenous lipid that broadly blocks its binding to TCRs. Crystal structures show that the short cellular SMs stabilized a triad of surface residues to remain flush with CD1a, but the longer lipids forced the phosphocholine group to ride above the display platform to hinder TCR approach. Whereas nearly all models emphasize antigen-mediated T cell activation, we propose that the CD1a system has intrinsic autoreactivity and is negatively regulated by natural endogenous inhibitors selectively bound in its cleft. Further, the detailed chemical structures of natural blockers could guide future design of therapeutic blockers of CD1a response.


Subject(s)
Antigens, CD1/immunology , T-Lymphocytes/immunology , Antigen Presentation/immunology , Cell Line , Cell Membrane/immunology , HEK293 Cells , Humans , K562 Cells , Lymphocyte Activation/immunology , Phospholipids/immunology , Receptors, Antigen, T-Cell/immunology
2.
J Clin Invest ; 131(1)2021 01 04.
Article in English | MEDLINE | ID: mdl-33393500

ABSTRACT

CD1a-autoreactive T cells contribute to skin disease, but the identity of immunodominant self-lipid antigens and their mode of recognition are not yet solved. In most models, MHC and CD1 proteins serve as display platforms for smaller antigens. Here, we showed that CD1a tetramers without added antigen stained large T cell pools in every subject tested, accounting for approximately 1% of skin T cells. The mechanism of tetramer binding to T cells did not require any defined antigen. Binding occurred with approximately 100 lipid ligands carried by CD1a proteins, but could be tuned upward or downward with certain natural self-lipids. TCR recognition mapped to the outer A' roof of CD1a at sites remote from the antigen exit portal, explaining how TCRs can bind CD1a rather than carried lipids. Thus, a major antigenic target of CD1a T cell autoreactivity in vivo is CD1a itself. Based on their high frequency and prevalence among donors, we conclude that CD1a-specific, lipid-independent T cells are a normal component of the human skin T cell repertoire. Bypassing the need to select antigens and effector molecules, CD1a tetramers represent a simple method to track such CD1a-specific T cells from tissues and in any clinical disease.


Subject(s)
Antigens, CD1/immunology , Membrane Lipids/immunology , Receptors, Antigen, T-Cell/immunology , Skin/immunology , T-Lymphocytes/immunology , HEK293 Cells , Humans , K562 Cells
3.
Sci Immunol ; 5(43)2020 01 03.
Article in English | MEDLINE | ID: mdl-31901073

ABSTRACT

During industrialization, humans have been exposed to increasing numbers of foreign chemicals. Failure of the immune system to tolerate drugs, cosmetics, and other skin products causes allergic contact dermatitis, a T cell-mediated disease with rising prevalence. Models of αß T cell response emphasize T cell receptor (TCR) contact with peptide-MHC complexes, but this model cannot readily explain activation by most contact dermatitis allergens, which are nonpeptidic molecules. We tested whether CD1a, an abundant MHC I-like protein in human skin, mediates contact allergen recognition. Using CD1a-autoreactive human αß T cell clones to screen clinically important allergens present in skin patch testing kits, we identified responses to balsam of Peru, a tree oil widely used in cosmetics and toothpaste. Additional purification identified benzyl benzoate and benzyl cinnamate as antigenic compounds within balsam of Peru. Screening of structurally related compounds revealed additional stimulants of CD1a-restricted T cells, including farnesol and coenzyme Q2. Certain general chemical features controlled response: small size, extreme hydrophobicity, and chemical constraint from rings and unsaturations. Unlike lipid antigens that protrude to form epitopes and contact TCRs, the small size of farnesol allows sequestration deeply within CD1a, where it displaces self-lipids and unmasks the CD1a surface. These studies identify molecular connections between CD1a and hypersensitivity to consumer products, defining a mechanism that could plausibly explain the many known T cell responses to oily substances.


Subject(s)
Allergens/immunology , Antigens, CD1/immunology , Antigens, Plant/immunology , Balsams , T-Lymphocytes/immunology , Cell Line , Cosmetics/adverse effects , Dermatitis, Allergic Contact/immunology , Humans , Patch Tests , Plant Extracts/adverse effects , Receptors, Antigen, T-Cell/immunology , Skin Care
4.
Nat Chem Biol ; 15(9): 889-899, 2019 09.
Article in English | MEDLINE | ID: mdl-31427817

ABSTRACT

Mycobacterium tuberculosis (Mtb) is the world's most deadly pathogen. Unlike less virulent mycobacteria, Mtb produces 1-tuberculosinyladenosine (1-TbAd), an unusual terpene nucleoside of unknown function. In the present study 1-TbAd has been shown to be a naturally evolved phagolysosome disruptor. 1-TbAd is highly prevalent among patient-derived Mtb strains, where it is among the most abundant lipids produced. Synthesis of TbAd analogs and their testing in cells demonstrate that their biological action is dependent on lipid linkage to the 1-position of adenosine, which creates a strong conjugate base. Furthermore, C20 lipid moieties confer passage through membranes. 1-TbAd selectively accumulates in acidic compartments, where it neutralizes the pH and swells lysosomes, obliterating their multilamellar structure. During macrophage infection, a 1-TbAd biosynthesis gene (Rv3378c) confers marked phagosomal swelling and intraphagosomal inclusions, demonstrating an essential role in regulating the Mtb cellular microenvironment. Although macrophages kill intracellular bacteria through phagosome acidification, Mtb coats itself abundantly with antacid.


Subject(s)
Antacids/metabolism , Lipids/biosynthesis , Lipids/chemistry , Mycobacterium tuberculosis/metabolism , Phagosomes/metabolism , Animals , Gene Expression Regulation, Bacterial , Humans , Hydrogen-Ion Concentration , Lysosomes , Macrophages/metabolism , Mice , Molecular Structure , Mycobacterium kansasii/genetics , Prevalence
5.
Curr Opin Immunol ; 52: 93-99, 2018 06.
Article in English | MEDLINE | ID: mdl-29738961

ABSTRACT

Peptide and lipid antigens are presented to T cells when bound to MHC or CD1 proteins, respectively. The general paradigm of T cell antigen recognition is that T cell receptors (TCRs) co-recognize an epitope comprised of the antigen and antigen presenting molecule. Here we review the latest studies in which T cells operate outside the co-recognition paradigm: TCRs can broadly contact CD1 itself, but not the carried lipid. The essential structural feature in these new mechanisms is a large 'antigen free' zone on the outer surface of certain antigen presenting molecules. Whereas peptides dominate the exposed surface of MHC-peptide complexes, all human CD1 proteins have a closed, antigen-free surface, which is known as the A' roof. These new structural models help to interpret recent biological studies of CD1 autoreactive T cells in vivo, which have now been broadly observed in studies on TCR-transgenic mice, healthy humans and patients with autoimmune disease.


Subject(s)
Antigens, CD1/immunology , Antigens, CD1/metabolism , Autoimmunity , Lipids/immunology , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Animals , Antigen Presentation , Antigens/chemistry , Antigens/immunology , Antigens, CD1/chemistry , Humans , Ligands , Lipids/chemistry , Lymphocyte Activation , Receptors, Antigen, T-Cell/chemistry
6.
Curr Opin Immunol ; 46: 127-133, 2017 Jun.
Article in English | MEDLINE | ID: mdl-28756303

ABSTRACT

CD1a, CD1b, CD1c and CD1d proteins migrate through distinct subcellular compartments of antigen presenting cells and so can be considered to take four separate pathways leading to display of lipid antigens to T cell receptors. This review discusses the intersection of CD1 trafficking and lipid antigen loading mechanisms in cells, highlighting key controversies relating to CD1 gene expression, size mismatches between antigens and CD1 binding clefts and unexpected mechanisms of T cell receptor-based recognition.


Subject(s)
Antigen Presentation/immunology , Antigens, CD1/immunology , Antigens, CD1/metabolism , Signal Transduction , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Animals , Antigen Presentation/genetics , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , Antigens, CD1/chemistry , Antigens, CD1/genetics , Gene Expression Regulation , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class I/metabolism , Histocompatibility Antigens Class II/immunology , Histocompatibility Antigens Class II/metabolism , Humans , Lipid Metabolism , Lipids/immunology , Protein Binding/immunology , Receptors, Antigen, T-Cell/metabolism
7.
Chemistry ; 23(7): 1694-1701, 2017 Jan 31.
Article in English | MEDLINE | ID: mdl-27925318

ABSTRACT

Mycobacterium tuberculosis produces dideoxymycobactin-838 (DDM-838), a lipopeptide that potently activates T cells upon binding to the MHC-like antigen-presenting molecule CD1a. M. tuberculosis produces DDM-838 in only trace amounts and a previous solid-phase synthesis provided sub-milligram quantities. We describe a high-yielding solution-phase synthesis of DDM-838 that features a Mitsunobu substitution that avoids yield-limiting epimerization at lysine during esterification, and amidation conditions that prevent double-bond isomerization of the Z-C20:1 acyl chain, and provides material with equivalent antigenicity to natural DDM-838. Isomers of DDM-838 that varied in stereochemistry at the central lysine and the C20:1 acyl chain were compared for their ability to be recognised by CD1a-restricted T cell receptors (TCRs). These TCRs, derived from unrelated human donors, exhibited a similar spectrum of reactivity towards the panel of DDM-838 isomers, highlighting the exquisite sensitivity of lipopeptide-reactive T cells for the natural DDM stereochemistry.


Subject(s)
Antigens, CD1/metabolism , Lipopeptides/chemistry , Mycobacterium tuberculosis/metabolism , Oxazoles/chemistry , T-Lymphocytes/metabolism , Antigens, CD1/genetics , Cell Line , Humans , Interferon-gamma/metabolism , Jurkat Cells , Lipopeptides/chemical synthesis , Lipopeptides/metabolism , Oxazoles/chemical synthesis , Oxazoles/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Stereoisomerism , T-Lymphocytes/cytology , T-Lymphocytes/immunology
8.
Infect Immun ; 84(9): 2463-72, 2016 09.
Article in English | MEDLINE | ID: mdl-27297394

ABSTRACT

Immune modulation is a hallmark of patent filarial infection, including suppression of antigen-presenting cell function and downmodulation of filarial antigen-specific T cell responses. The mammalian target of rapamycin (mTOR) signaling pathway has been implicated in immune regulation, not only by suppressing T cell responses but also by regulating autophagy (through mTOR sensing amino acid availability). Global proteomic analysis (liquid chromatography-tandem mass spectrometry) of microfilaria (mf)-exposed monocyte-derived dendritic cells (DC) indicated that multiple components of the mTOR signaling pathway, including mTOR, eIF4A, and eIF4E, are downregulated by mf, suggesting that mf target this pathway for immune modulation in DC. Utilizing Western blot analysis, we demonstrate that similar to rapamycin (a known mTOR inhibitor), mf downregulate the phosphorylation of mTOR and its regulatory proteins, p70S6K1 and 4E-BP1, a process essential for DC protein synthesis. As active mTOR signaling regulates autophagy, we examined whether mf exposure alters autophagy-associated processes. mf-induced autophagy was reflected in marked upregulation of phosphorylated Beclin 1, known to play an important role in both autophagosome formation and autolysosome fusion, in induction of LC3II, a marker of autophagosome formation, and in induced degradation of p62, a ubiquitin-binding protein that aggregates protein in autophagosomes and is degraded upon autophagy that was reduced significantly by mf exposure and by rapamycin. Together, these results suggest that Brugia malayi mf employ mechanisms of metabolic modulation in DC to influence the regulation of the host immune response by downregulating mTOR signaling, resulting in increased autophagy. Whether this is a result of the parasite-secreted rapamycin homolog is currently under study.


Subject(s)
Autophagy/physiology , Brugia malayi/parasitology , Dendritic Cells/parasitology , Microfilariae/physiology , TOR Serine-Threonine Kinases/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , Apoptosis Regulatory Proteins/metabolism , Autophagosomes/metabolism , Autophagosomes/parasitology , Beclin-1/metabolism , Cell Cycle Proteins , Dendritic Cells/metabolism , Down-Regulation/physiology , Eukaryotic Initiation Factor-4A/metabolism , Eukaryotic Initiation Factor-4E/metabolism , Humans , Lysosomes/metabolism , Lysosomes/parasitology , Monocytes/metabolism , Monocytes/parasitology , Phosphoproteins/metabolism , Phosphorylation/physiology , Proteomics/methods , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Signal Transduction/physiology , Ubiquitin/metabolism , Up-Regulation/physiology
9.
J Immunol ; 192(12): 5863-72, 2014 Jun 15.
Article in English | MEDLINE | ID: mdl-24808365

ABSTRACT

Leishmania major-infected human dendritic cells (DCs) exhibit a marked induction of IL-12, ultimately promoting a robust Th1-mediated response associated with parasite killing and protective immunity. The host cell transcription machinery associated with the specific IL-12 induction observed during L. major infection remains to be thoroughly elucidated. In this study, we used Affymetrix GeneChip (Affymetrix) to globally assess the host cell genes and pathways associated with early L. major infection in human myeloid-derived DCs. Our data revealed 728 genes were significantly differentially expressed and molecular signaling pathway revealed that the type I IFN pathway was significantly enriched. Addition of a neutralizing type I IFN decoy receptor blocked the expression of IRF7 and IL-12p40 during DC infection, indicating the L. major-induced expression of IL-12p40 is dependent upon the type I IFN signaling pathway. In stark contrast, IL-12p40 expression is not elicited by L. donovani, the etiological agent of deadly visceral leishmaniasis. Therefore, we examined the gene expression profile for several IFN response genes in L. major versus L. donovani DC infections. Our data revealed that L. major, but not L. donovani, induces expression of IRF2, IRF7, and IFIT5, implicating the regulation of type I IFN-associated signaling pathways as mediating factors toward the production of IL-12.


Subject(s)
Dendritic Cells/immunology , Gene Expression Regulation/immunology , Interferon Type I/immunology , Interleukin-12 Subunit p40/immunology , Leishmania major/immunology , Leishmaniasis, Cutaneous/immunology , Dendritic Cells/pathology , Female , Humans , Interferon Regulatory Factor-2/immunology , Interferon Regulatory Factor-7/immunology , Leishmania donovani/immunology , Leishmaniasis, Cutaneous/pathology , Leishmaniasis, Visceral/immunology , Male , Neoplasm Proteins/immunology , Signal Transduction/immunology
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