RESUMO
Synthetic α-galactosylceramide (αGalCer) and its analogues as powerful agonists for natural killer T (NKT) cell manipulation have received significant attention in immunotherapy and adjuvant development. However, identifying new potent NKT cell agonists, especially those with Th1 selectivity that promote anticancer effects, remains a challenging task. In this work, we introduced a sulfonamide group into the acyl chain of αGalCer to form additional hydrogen bonds to intensify the glycolipid/CD1d interaction. Two compounds GCS-11 and GCS-12 demonstrated remarkable potency while exhibiting different cytokine induction patterns. Compared to αGalCer, the Th1-biased GCS-11 exhibited a 6-fold increase in IFN-γ but not IL-4, while the Th1/2-balanced GCS-12 elicited 7- and 5-fold increase in IFN-γ and IL-4, respectively, in vivo. These findings place them among the most potent NKT cell agonists, with superior antitumor effects. Therefore, hydrogen-bond-involved derivatization could be a powerful strategy to develop potent and polarized NKT cell agonists for various immunotherapies.
Assuntos
Antígenos CD1d , Citocinas , Desenho de Fármacos , Galactosilceramidas , Ligação de Hidrogênio , Células T Matadoras Naturais , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/efeitos dos fármacos , Células T Matadoras Naturais/metabolismo , Animais , Galactosilceramidas/química , Galactosilceramidas/farmacologia , Galactosilceramidas/síntese química , Camundongos , Citocinas/metabolismo , Antígenos CD1d/metabolismo , Antígenos CD1d/química , Humanos , Interleucina-4/metabolismo , Relação Estrutura-Atividade , Camundongos Endogâmicos C57BL , Interferon gama/metabolismo , Sulfonamidas/química , Sulfonamidas/farmacologia , Sulfonamidas/síntese química , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Linhagem Celular TumoralRESUMO
Microbes produce a rich array of lipidic species that through their location in the cell wall and ability to mingle with host lipids represent a privileged class of immune-active molecules. Lipid-sensing immunity recognizes microbial lipids from pathogens and commensals causing immune responses. Yet microbial lipids are often heterogeneous, in limited supply and in some cases their structures are incompletely defined. Total synthesis can assist in structural determination, overcome supply issues, and provide access to high-purity, homogeneous samples and analogues. This account highlights synthetic approaches to lipidic species from pathogenic and commensal bacteria and fungi that have supported immunological studies involving lipid sensing through the pattern recognition receptor Mincle and cell-mediated immunity through the CD1-T cell axis.
Assuntos
Bactérias/metabolismo , Fungos/metabolismo , Imunidade Celular , Lipídeos/imunologia , Antígenos CD1d/química , Antígenos CD1d/metabolismo , Glicolipídeos/química , Glicolipídeos/imunologia , Humanos , Lipídeos/química , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Natural killer T (NKT) cells constitute a unique subset of T lymphocytes characterized by specifically interacting with antigenic glycolipids conjugated to the CD1d receptor on antigen-presenting cells. Functionally, NKT cells are capable of performing either effector or suppressor immune responses, depending on their production of proinflammatory or anti-inflammatory cytokines, respectively. Effector NKT cells are subdivided into three subsets, termed NKT1, NKT2, and NKT17, based on the cytokines they produce and their similarity to the cytokine profile produced by Th1, Th2, and Th17 lymphocytes, respectively. Recently, a new subgroup of NKT cells termed NKT10 has been described, which cooperates and interacts with other immune cells to promote immunoregulatory responses. Although the tissue-specific functions of NKT cells have not been fully elucidated, their activity has been associated with the pathogenesis of different inflammatory diseases with immunopathogenic similarities to periodontitis, including osteolytic pathologies such as rheumatoid arthritis and osteoporosis. In the present review, we revise and discuss the pathogenic characteristics of NKT cells in these diseases and their role in the pathogenesis of periodontitis; particularly, we analyze the potential regulatory role of the IL-10-producing NKT10 cells.
Assuntos
Células T Matadoras Naturais/fisiologia , Periodontite/etiologia , Animais , Antígenos CD1d/química , Citocinas/fisiologia , Glicolipídeos/química , Humanos , Ativação Linfocitária , Células T Matadoras Naturais/citologia , Periodontite/imunologiaRESUMO
CD1d-restricted invariant natural killer T cells (iNKT cells) mediate strong antitumor immunity when stimulated by glycolipid agonists. However, attempts to develop effective iNKT cell agonists for clinical applications have been thwarted by potential problems with dose-limiting toxicity and by activation-induced iNKT cell anergy, which limits the efficacy of repeated administration. To overcome these issues, we developed a unique bispecific T-cell engager (BiTE) based on covalent conjugates of soluble CD1d with photoreactive analogues of the glycolipid α-galactosylceramide. Here we characterize the in vivo activities of iNKT cell-specific BiTEs and assess their efficacy for cancer immunotherapy in mouse models using transplantable colorectal cancer or melanoma tumor lines engineered to express human Her2 as a tumor-associated antigen. Systemic administration of conjugated BiTEs stimulated multiple iNKT cell effector functions including cytokine release, secondary activation of NK cells, and induction of dendritic cell maturation and also initiated epitope spreading for tumor-specific CD8+ cytolytic T-cell responses. The antitumor effects of iNKT-cell activation with conjugated BiTEs were further enhanced by simultaneous checkpoint blockade with antibodies to CTLA-4, providing a potential approach for combination immunotherapy. Multiple injections of covalently stabilized iNKT cell-specific BiTEs activated iNKT cells without causing iNKT cell anergy or exhaustion, thus enabling repeated administration for effective and nontoxic cancer immunotherapy regimens. SIGNIFICANCE: Covalently stabilized conjugates that engage the antigen receptors of iNKT cells and target a tumor antigen activate potent antitumor immunity without induction of anergy or depletion of the responding iNKT cells.
Assuntos
Antígenos CD1d/farmacologia , Anergia Clonal/efeitos dos fármacos , Galactosilceramidas/farmacologia , Imunoterapia/métodos , Células T Matadoras Naturais/efeitos dos fármacos , Animais , Antígenos CD1d/química , Antígenos CD1d/imunologia , Anergia Clonal/imunologia , Feminino , Galactosilceramidas/química , Humanos , Imunoconjugados/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células T Matadoras Naturais/imunologia , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/terapia , Células Tumorais CultivadasRESUMO
CD1 molecules present lipid antigens to T-cells in early stages of immune responses. Whereas CD1âlipidâT-cell receptors interactions are reasonably understood, molecular details on initial trafficking and loading of lipids onto CD1 proteins are less complete. We present a molecular dynamics (MD) study of human CD1d, the isotype that activates iNKT cells. MD simulations and calculations of properties and Poisson-Boltzmann electrostatic potentials were used to explore the dynamics of the antigen-binding domain of the apo-form, CD1d complexes with three lipid-antigens that activate iNKT cells and CD1d complex with GM2AP, a protein that assists lipid loading onto CD1 molecules in endosomes/lysosomes. The study was done at pH 7 and 4.5, values representative of strongly acidic environments in endosomal compartments. Our findings revealed dynamic features of the entrance to the hydrophobic channels of CD1d modulated by two α helices with sensitivity to the type of lipid. We also found lipid- and pH-dependent dynamic changes in three exposed tryptophans unique to CD1d among the five human CD1 isotypes. On the basis of modelled structures, our data also revealed external effects produced by the helper protein GM2AP only when it interacts in its open form, thus suggesting that the own assistant protein also adapts conformation to association with CD1d.
Assuntos
Antígenos CD1d/química , Antígenos CD1d/metabolismo , Antígenos/metabolismo , Metabolismo dos Lipídeos , Simulação de Dinâmica Molecular , Sítios de Ligação , Humanos , Concentração de Íons de Hidrogênio , Ligação Proteica , Domínios Proteicos , Eletricidade EstáticaRESUMO
CD1d is a major histocompatibility complex (MHC) class I-like glycoprotein and binds to glycolipid antigens that are recognized by natural killer T (NKT) cells. To date, our understanding of the structural basis for glycolipid binding and receptor recognition of CD1d is still limited. Here, we established a preparation method for the ectodomain of human and mouse CD1d using a silkworm-baculovirus expression system. The co-expression of human and mouse CD1d and ß2-microglobulin (ß2m) in the silkworm-baculovirus system was successful, but the yield of human CD1d was low. A construct of human CD1d fused with ß2m via a flexible GS linker as a single polypeptide was prepared to improve protein yield. The production of this single-chained complex was higher (50 µg/larva) than that of the co-expression complex. Furthermore, differential scanning calorimetry revealed that the linker made the CD1d complex more stable and homogenous. These results suggest that the silkworm-baculovirus expression system is useful for structural and biophysical studies of CD1d in several aspects including low cost, easy handling, biohazard-free, rapid, and high yielding.
Assuntos
Antígenos CD1d , Baculoviridae , Expressão Gênica , Animais , Antígenos CD1d/biossíntese , Antígenos CD1d/química , Antígenos CD1d/genética , Antígenos CD1d/isolamento & purificação , Bombyx , Humanos , Camundongos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
α-Galactosylceramide (α-GalCer) is recognized by the CD1d proteins on antigen-presenting cells at the ceramide moiety and the galactose moiety is presented to iNKT cells, which stimulates the immune responses. However, the immune suppression by repeated injections of α-GalCer has discouraged its development as an anti-cancer agent. To overcome the shortcoming by spatiotemporal restriction of its exposure, we synthesized the photochromic azobenzene-incorporated analogues and tested the photo-immunoregulation effect in its binding to CD1d. FACS analyses indicated that some of these analogues enhanced the affinity to CD1d on photo-irradiation by about 20%. A docking simulation suggests that the photochromic molecule should be bulkier for a clearer discrimination between on and off states.
Assuntos
Antígenos CD1d/metabolismo , Compostos Azo/metabolismo , Galactosilceramidas/metabolismo , Animais , Antígenos CD1d/química , Compostos Azo/síntese química , Compostos Azo/efeitos da radiação , Galactosilceramidas/síntese química , Galactosilceramidas/efeitos da radiação , Humanos , Camundongos , Simulação de Acoplamento Molecular , Células T Matadoras Naturais/metabolismo , Ligação Proteica/efeitos da radiaçãoRESUMO
CD1d is a nonpolymorphic antigen-presenting protein responsible for the regulation of natural killer T (NKT) cell activation. α-Galactosyl ceramide (α-GalCer, KRN7000) is the representative CD1d ligand that can bind to the CD1d protein. The resulting complex is recognized by the T cell receptors of the NKT cell, inducing various immune responses. Previous structure-activity relationship studies of α-GalCer have revealed that the ability of NKT cells to induce cytokines depends on the ligand structure, and in particular, ligands that form more stable complexes with CD1d display potent activity. We focused on the Cys residue of the large hydrophobic pockets of CD1d (A' pocket) and developed α-GalCer derivatives containing groups that can form covalent bonds. The assay results revealed that these ligands displayed higher levels of cytokine production and Th2 cell-type cytokine polarization response. Furthermore, the LC-MS/MS analysis indicated that the chloroacetylamide-containing ligand was covalently bound to Cys12 of CD1d, which suggests that the enhanced activities result from the formation of a stable CD1d-ligand complex. To our knowledge, this is the first ligand that allows covalent bond formation to CD1d under physiological conditions.
Assuntos
Antígenos CD1d/metabolismo , Galactosilceramidas/farmacologia , Acetamidas/síntese química , Acetamidas/metabolismo , Acetamidas/farmacologia , Acrilamidas/síntese química , Acrilamidas/metabolismo , Acrilamidas/farmacologia , Animais , Antígenos CD1d/química , Cisteína/química , Desenho de Fármacos , Descoberta de Drogas , Galactosilceramidas/síntese química , Galactosilceramidas/metabolismo , Interferon gama/metabolismo , Interleucina-4/metabolismo , Ligantes , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Simulação de Acoplamento Molecular , Células T Matadoras Naturais/efeitos dos fármacos , Células T Matadoras Naturais/metabolismo , Ligação ProteicaRESUMO
Antibody-mediated modulation of major histocompatibility complex (MHC) molecules, or MHC class I-like molecules, could constitute an effective immunotherapeutic approach. We describe how single-domain antibodies (VHH), specific for the human MHC class I-like molecule CD1d, can modulate the function of CD1d-restricted T cells and how one VHH (1D12) specifically induced strong type I natural killer T (NKT) cell activation. The crystal structure of the VHH1D12-CD1d(α-GalCer)-NKT T-cell receptor (TCR) complex revealed that VHH1D12 simultaneously contacted CD1d and the type I NKT TCR, thereby stabilizing this interaction through intrinsic bispecificity. This led to greatly enhanced type I NKT cell-mediated antitumor activity in in vitro, including multiple myeloma and acute myeloid leukemia patient-derived bone marrow samples, and in vivo models. Our findings underscore the versatility of VHH molecules in targeting composite epitopes, in this case consisting of a complexed monomorphic antigen-presenting molecule and an invariant TCR, and represent a generalizable antitumor approach.
Assuntos
Receptores de Antígenos de Linfócitos T , Antígenos CD1d/química , Humanos , Receptores de Antígenos de Linfócitos T/químicaRESUMO
α-Galactosylceramides are glycosphingolipids that show promise in cancer immunotherapy. After presentation by CD1d, they activate natural killer T cells (NKT), which results in the production of a variety of pro-inflammatory and immunomodulatory cytokines. Herein, we report the synthesis and biological evaluation of photochromic derivatives of KRN-7000, the activity of which can be modulated with light. Based on established structure-activity relationships, we designed photoswitchable analogues of this glycolipid that control the production of pro-inflammatory cytokines, such as IFN-γ. The azobenzene derivative α-GalACer-4 proved to be more potent than KRN-7000 itself when activated with 370â nm light. Photolipids of this type could improve our mechanistic understanding of cytokine production and could open new directions in photoimmunotherapy.
Assuntos
Antígenos CD1d/metabolismo , Citocinas/química , Galactosilceramidas/farmacologia , Glicolipídeos/química , Células Matadoras Naturais/efeitos dos fármacos , Antígenos CD1d/química , Citocinas/metabolismo , Galactosilceramidas/química , Células Matadoras Naturais/química , Células T Matadoras Naturais , Relação Estrutura-AtividadeRESUMO
High-throughput TCR sequencing allows interrogation of the human TCR repertoire, potentially connecting TCR sequences to antigenic targets. Unlike the highly polymorphic MHC proteins, monomorphic Ag-presenting molecules such as MR1, CD1d, and CD1b present Ags to T cells with species-wide TCR motifs. CD1b tetramer studies and a survey of the 27 published CD1b-restricted TCRs demonstrated a TCR motif in humans defined by the TCR ß-chain variable gene 4-1 (TRBV4-1) region. Unexpectedly, TRBV4-1 was involved in recognition of CD1b regardless of the chemical class of the carried lipid. Crystal structures of two CD1b-specific TRBV4-1+ TCRs show that germline-encoded residues in CDR1 and CDR3 regions of TRBV4-1-encoded sequences interact with each other and consolidate the surface of the TCR. Mutational studies identified a key positively charged residue in TRBV4-1 and a key negatively charged residue in CD1b that is shared with CD1c, which is also recognized by TRBV4-1 TCRs. These data show that one TCR V region can mediate a mechanism of recognition of two related monomorphic Ag-presenting molecules that does not rely on a defined lipid Ag.
Assuntos
Motivos de Aminoácidos , Antígenos CD1d/química , Antígenos CD1d/metabolismo , Sítios de Ligação , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Apresentação de Antígeno , Sequência Conservada , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Rearranjo Gênico , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunofenotipagem , Lipídeos/química , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Relação Estrutura-Atividade , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
iNKT cells are CD1d-restricted T cells recognizing lipid antigens. The prototypic iNKT cell-agonist α-galactosylceramide (α-GalCer) alongside compounds with similar structures induces robust proliferation and cytokine production of iNKT cells and protects against cancer in vivo. Monoclonal antibodies (mAbs) that detect CD1d-α-GalCer complexes have provided critical information for understanding of antigen presentation of iNKT cell agonists. Although most iNKT cell agonists with antitumor properties are α-linked glycosphingolipids that can be detected by anti-CD1d-α-GalCer mAbs, ß-ManCer, a glycolipid with a ß-linkage, induces strong antitumor immunity via mechanisms distinct from those of α-GalCer. In this study, we unexpectedly discovered that anti-CD1d-α-GalCer mAbs directly recognized ß-ManCer-CD1d complexes and could inhibit ß-ManCer stimulation of iNKT cells. The binding of anti-CD1d-α-GalCer mAb with ß-ManCer-CD1d complexes was also confirmed by plasmon resonance and could not be explained by α-anomer contamination. The binding of anti-CD1d-α-GalCer mAb was also observed with CD1d loaded with another ß-linked glycosylceramide, ß-GalCer (C26:0). Detection with anti-CD1d-α-GalCer mAbs indicates that the interface of the ß-ManCer-CD1d complex exposed to the iNKT cell TCR can assume a structure like that of CD1d-α-GalCer, despite its disparate carbohydrate structure. These results suggest that certain ß-linked monoglycosylceramides can assume a structural display similar to that of CD1d-α-GalCer and that the data based on anti-CD1d-α-GalCer binding should be interpreted with caution.
Assuntos
Anticorpos Monoclonais Murinos/imunologia , Apresentação de Antígeno/imunologia , Antígenos CD1d/imunologia , Galactosilceramidas , Células T Matadoras Naturais/imunologia , Animais , Antígenos CD1d/química , Galactosilceramidas/química , Galactosilceramidas/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Células T Matadoras Naturais/patologia , Relação Estrutura-AtividadeRESUMO
Type I natural killer T (NKT) cells are a population of innate like T lymphocytes that rapidly respond to α-GalCer presented by CD1d via the production of both pro- and anti-inflammatory cytokines. While developing novel α-GalCer analogs that were meant to be utilized as potential adjuvants because of their production of pro-inflammatory cytokines (Th1 skewers), we generated α-galactosylsphingamides (αGSA). Surprisingly, αGSAs are not potent antigens in vivo despite their strong T-cell receptor (TCR)-binding affinities. Here, using surface plasmon resonance (SPR), antigen presentation assays, and X-ray crystallography (yielding crystal structures of 19 different binary (CD1d-glycolipid) or ternary (CD1d-glycolipid-TCR) complexes at resolutions between 1.67 and 2.85 Å), we characterized the biochemical and structural details of αGSA recognition by murine NKT cells. We identified a molecular switch within murine (m)CD1d that modulates NKT cell activation by αGSAs. We found that the molecular switch involves a hydrogen bond interaction between Tyr-73 of mCD1d and the amide group oxygen of αGSAs. We further established that the length of the acyl chain controls the positioning of the amide group with respect to the molecular switch and works synergistically with Tyr-73 to control NKT cell activity. In conclusion, our findings reveal important mechanistic insights into the presentation and recognition of glycolipids with polar moieties in an otherwise apolar milieu. These observations may inform the development αGSAs as specific NKT cell antagonists to modulate immune responses.
Assuntos
Antígenos CD1d/química , Glicoesfingolipídeos/química , Células Matadoras Naturais/imunologia , Simulação de Dinâmica Molecular , Animais , Antígenos CD1d/metabolismo , Sítios de Ligação , Glicoesfingolipídeos/metabolismo , Ligação de Hidrogênio , Ativação Linfocitária , Camundongos , Oxigênio/química , Ligação Proteica , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/metabolismo , Células Sf9 , SpodopteraRESUMO
iNKT cells recognize CD1d/α-galactosylceramide (α-GalCer) complexes via their invariant TCR receptor and stimulate the immune response. Many α-GalCer analogues have been investigated to interrogate this interaction. Following our previous work related to the modification of the hydrogen bond network between α-GalCer and CD1d, we have now focused our attention on the synthesis of 3-deoxy-3,3-difluoro- and 3,4-dideoxy-3,3,4,4-tetrafluoro-α-GalCer analogues, and studied their ability to stimulate human iNKT cells. In each case, deoxygenation at the indicated positions was accompanied by difluoro introduction in order to evaluate the resulting electronic effect on the stability of the ternary CD1d/Galcer/TCR complex which has been rationalized by modeling study. With deoxy-difluorination at the 3-position, the two epimeric 4-OH analogues were investigated to establish their capacity to compensate for the lack of the hydrogen bond donating group at the 3-position. The 3,4-dideoxytetrafluoro analogue was of interest to highlight the amide NH-bond hydrogen bond properties.
Assuntos
Antígenos CD1d/metabolismo , Galactosilceramidas/farmacologia , Células T Matadoras Naturais/efeitos dos fármacos , Receptores de Antígenos de Linfócitos T/metabolismo , Antígenos CD1d/química , Galactosilceramidas/síntese química , Galactosilceramidas/química , Galactosilceramidas/metabolismo , Células HeLa , Humanos , Ligação de Hidrogênio , Interferon gama/metabolismo , Interleucina-13/metabolismo , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Receptores de Antígenos de Linfócitos T/química , EstereoisomerismoRESUMO
CD1d is a non-polymorphic antigen-presenting glycoprotein that recognizes glycolipids as ligands. Ligands bind to the hydrophobic grooves of CD1d, and the resulting ligand-CD1d complexes activate natural killer T (NKT) cells by means of T cell receptor recognition, leading to the secretion of various cytokines. However, details of the ligand recognition mechanism of a large hydrophobic ligand binding pocket and the relationship between cytokine induction and ligand structure are unclear. We report the synthesis of α-GalCer derivatives containing a Bz amide group having various substituting groups in the ceramide moiety, and the analysis of the structure-activity relationships. The assays reveal that the Bz amide-containing CD1d ligands function as NKT cell modulators displaying Th2 cytokine biasing responses. Furthermore, molecular dynamics simulation studies suggest that the phenyl groups can interact with the aromatic amino acid residues in the lipid binding pocket of CD1d.
Assuntos
Amidas/química , Benzeno/química , Galactosilceramidas/química , Células T Matadoras Naturais/metabolismo , Animais , Antígenos CD1d/química , Antígenos CD1d/metabolismo , Sítios de Ligação , Células Cultivadas , Citocinas/metabolismo , Galactosilceramidas/metabolismo , Galactosilceramidas/farmacologia , Ligantes , Camundongos , Simulação de Dinâmica Molecular , Células T Matadoras Naturais/citologia , Células T Matadoras Naturais/efeitos dos fármacos , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
Activated NKT cells can stimulate antigen-presenting cells leading to enhanced peptide antigen-specific immunity. However, administration of potent NKT cell agonists like α-galactosylceramide (α-GalCer) can be associated with release of high levels of cytokines, and in some situations, hepatotoxicity. Here we show that it is possible to provoke sufficient NKT cell activity to stimulate strong antigen-specific T cell responses without these unwanted effects. This was achieved by chemically conjugating antigenic peptides to α-galactosylphytosphingosine (α-GalPhs), an NKT cell agonist with very weak activity based on structural characterisation and biological assays. Conjugation improved delivery to antigen-presenting cells in vivo, while use of a cathepsin-sensitive linker to release the α-GalPhs and peptide within the same cell promoted strong T cell activation and therapeutic anti-tumour responses in mice. The conjugates activated human NKT cells and enhanced human T cell responses to a viral peptide in vitro. Accordingly, we have demonstrated a means to safely exploit the immunostimulatory properties of NKT cells to enhance T cell activation for virus- and tumour-specific immunity.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Vacinas Anticâncer/administração & dosagem , Células T Matadoras Naturais/efeitos dos fármacos , Células T Matadoras Naturais/imunologia , Neoplasias Experimentais/imunologia , Peptídeos/administração & dosagem , Adjuvantes Imunológicos , Animais , Antígenos CD1d/química , Vacinas Anticâncer/imunologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Epitopos/química , Glicolipídeos/química , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/tratamento farmacológico , Peptídeos/química , Peptídeos/imunologiaRESUMO
Glycosphingolipids and glycerophospholipids bind CD1d. Glycosphingolipid-reactive invariant NKT-cells (iNKT) exhibit myriad immune effects, however, little is known about the functions of phospholipid-reactive T cells (PLT). We report that the normal mouse immune repertoire contains αß T cells, which recognize self-glycerophospholipids such as phosphatidic acid (PA) in a CD1d-restricted manner and don't cross-react with iNKT-cell ligands. PA bound to CD1d in the absence of lipid transfer proteins. Upon in vivo priming, PA induced an expansion and activation of T cells in Ag-specific manner. Crystal structure of the CD1d:PA complex revealed that the ligand is centrally located in the CD1d-binding groove opening for TCR recognition. Moreover, the increased flexibility of the two acyl chains in diacylglycerol ligands and a less stringent-binding orientation for glycerophospholipids as compared with the bindings of glycosphingolipids may allow glycerophospholipids to readily occupy CD1d. Indeed, PA competed with α-galactosylceramide to load onto CD1d, leading to reduced expression of CD1d:α-galactosylceramide complexes on the surface of dendritic cells. Consistently, glycerophospholipids reduced iNKT-cell proliferation, expansion, and cytokine production in vitro and in vivo. Such superior ability of self-glycerophospholipids to compete with iNKT-cell ligands to occupy CD1d may help maintain homeostasis between the diverse subsets of lipid-reactive T cells, with important pathogenetic and therapeutic implications.
Assuntos
Antígenos CD1d , Células Dendríticas , Ativação Linfocitária , Células T Matadoras Naturais , Ácidos Fosfatídicos , Animais , Antígenos CD1d/química , Antígenos CD1d/genética , Antígenos CD1d/imunologia , Cristalografia por Raios X , Células Dendríticas/química , Células Dendríticas/imunologia , Galactosilceramidas/química , Galactosilceramidas/imunologia , Camundongos , Camundongos Knockout , Células T Matadoras Naturais/química , Células T Matadoras Naturais/imunologia , Ácidos Fosfatídicos/química , Ácidos Fosfatídicos/imunologiaRESUMO
Th2-biasing CD1d ligands are attractive potential candidates for adjuvants and therapeutic drugs. However, the number of potent ligands is limited, and their biasing mechanism remain unclear. Herein, a series of novel Th2-biasing CD1d glycolipid ligands, based on modification of their lipid part, have been identified. These have shown high binding affinities and efficient Th2 cytokine production. Importantly, the truncated acyl chain containing variants still retain their binding affinities and agonistic activities, which can be associated with an "anchoring effect," that is, formation of a buried hydrogen bond between a polar group on the acyl chain and the CD1d lipid-binding pocket. The analysis indicated that the appearance rates of ligand-CD1d complexes on the cell surface were involved in Th2-biasing responses. The designed ligands, having the anchor in the shorter lipid part, are one of the most potent Th2-biasing ligands among the known ligands.
Assuntos
Antígenos CD1d/química , Antígenos CD1d/metabolismo , Citocinas/biossíntese , Lipídeos/química , Células T Matadoras Naturais/metabolismo , Citocinas/química , Humanos , Ligação de Hidrogênio , Ligantes , Estrutura Molecular , Células Th2RESUMO
Invariant Natural Killer T-cells (iNKT-cells) are an attractive target for immune response modulation, as upon CD1d-mediated stimulation with KRN7000, a synthetic α-galactosylceramide, they produce a vast amount of cytokines. Here we present a synthesis that allows swift modification of the phytosphingosine side chain by amidation of an advanced methyl ester precursor. The resulting KRN7000 derivatives, termed α-galactosylsphingamides, were evaluated for their capacity to stimulate iNKT-cells. While introduction of the amide-motif in the phytosphingosine chain is tolerated for CD1d binding and TCR recognition, the studied α-galactosylsphingamides showed compromised antigenic properties.
Assuntos
Antígenos CD1d/metabolismo , Galactosilceramidas/metabolismo , Sondas Moleculares/metabolismo , Animais , Antígenos CD1d/química , Galactosilceramidas/química , Imageamento por Ressonância Magnética , Camundongos , Modelos Moleculares , Conformação Molecular , Sondas Moleculares/química , Estrutura Molecular , Células T Matadoras Naturais/metabolismo , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
In this review, we discuss small-molecule, carbohydrate-based immunostimulants that target Toll-like receptor 4 (TLR-4) and cluster of differentiation 1D (CD1d) receptors. The design and use of these molecules in immunotherapy as well as results from their use in clinical trials are described. How these molecules work and their utilization as vaccine adjuvants are also discussed. Future applications and extensions for the use of these analogues as therapeutic agents will be outlined.