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1.
Proc Natl Acad Sci U S A ; 114(51): E10956-E10964, 2017 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-29158404

RESUMO

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a major human pandemic. Germline-encoded mycolyl lipid-reactive (GEM) T cells are donor-unrestricted and recognize CD1b-presented mycobacterial mycolates. However, the molecular requirements governing mycolate antigenicity for the GEM T cell receptor (TCR) remain poorly understood. Here, we demonstrate CD1b expression in TB granulomas and reveal a central role for meromycolate chains in influencing GEM-TCR activity. Meromycolate fine structure influences T cell responses in TB-exposed individuals, and meromycolate alterations modulate functional responses by GEM-TCRs. Computational simulations suggest that meromycolate chain dynamics regulate mycolate head group movement, thereby modulating GEM-TCR activity. Our findings have significant implications for the design of future vaccines that target GEM T cells.


Assuntos
Antígenos CD1/imunologia , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Tuberculose/imunologia , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Antígenos CD1/química , Antígenos CD1/genética , Expressão Gênica , Granuloma/imunologia , Granuloma/metabolismo , Granuloma/microbiologia , Granuloma/patologia , Humanos , Imuno-Histoquímica , Ativação Linfocitária/imunologia , Modelos Moleculares , Conformação Molecular , Ácidos Micólicos/química , Ácidos Micólicos/metabolismo , Ligação Proteica , Receptores de Antígenos de Linfócitos T/metabolismo , Tuberculose/microbiologia
2.
Proc Natl Acad Sci U S A ; 113(9): E1266-75, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26884207

RESUMO

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.


Assuntos
Antígenos CD1/imunologia , Ésteres do Colesterol/metabolismo , Glicoproteínas/imunologia , Linfócitos T/imunologia , Antígenos CD1/química , Antígenos CD1d , Glicoproteínas/química , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica
3.
Cancer Immunol Immunother ; 62(4): 773-85, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23263452

RESUMO

NY-ESO-1 and LAGE-1 are cancer testis antigens with an ideal profile for tumor immunotherapy, combining up-regulation in many cancer types with highly restricted expression in normal tissues and sharing a common HLA-A*0201 epitope, 157-165. Here, we present data to describe the specificity and anti-tumor activity of a bifunctional ImmTAC, comprising a soluble, high-affinity T-cell receptor (TCR) specific for NY-ESO-1157-165 fused to an anti-CD3 scFv. This reagent, ImmTAC-NYE, is shown to kill HLA-A2, antigen-positive tumor cell lines, and freshly isolated HLA-A2- and LAGE-1-positive NSCLC cells. Employing time-domain optical imaging, we demonstrate in vivo targeting of fluorescently labelled high-affinity NYESO-specific TCRs to HLA-A2-, NY-ESO-1157-165-positive tumors in xenografted mice. In vivo ImmTAC-NYE efficacy was tested in a tumor model in which human lymphocytes were stably co-engrafted into NSG mice harboring tumor xenografts; efficacy was observed in both tumor prevention and established tumor models using a GFP fluorescence readout. Quantitative RT-PCR was used to analyze the expression of both NY-ESO-1 and LAGE-1 antigens in 15 normal tissues, 5 cancer cell lines, 10 NSCLC, and 10 ovarian cancer samples. Overall, LAGE-1 RNA was expressed at a greater frequency and at higher levels than NY-ESO-1 in the tumor samples. These data support the clinical utility of ImmTAC-NYE as an immunotherapeutic agent for a variety of cancers.


Assuntos
Antígenos de Neoplasias/imunologia , Antígenos de Superfície/imunologia , Proteínas de Membrana/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Linfócitos T/imunologia , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Antígenos de Neoplasias/biossíntese , Antígenos de Superfície/biossíntese , Complexo CD3/imunologia , Linhagem Celular Tumoral , Epitopos/imunologia , Feminino , Antígeno HLA-A2/imunologia , Humanos , Fragmentos de Imunoglobulinas/imunologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Melanoma/imunologia , Melanoma/metabolismo , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/metabolismo , Distribuição Aleatória , Proteínas Recombinantes de Fusão/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Nat Med ; 18(6): 980-7, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22561687

RESUMO

T cell immunity can potentially eradicate malignant cells and lead to clinical remission in a minority of patients with cancer. In the majority of these individuals, however, there is a failure of the specific T cell receptor (TCR)­mediated immune recognition and activation process. Here we describe the engineering and characterization of new reagents termed immune-mobilizing monoclonal TCRs against cancer (ImmTACs). Four such ImmTACs, each comprising a distinct tumor-associated epitope-specific monoclonal TCR with picomolar affinity fused to a humanized cluster of differentiation 3 (CD3)-specific single-chain antibody fragment (scFv), effectively redirected T cells to kill cancer cells expressing extremely low surface epitope densities. Furthermore, these reagents potently suppressed tumor growth in vivo. Thus, ImmTACs overcome immune tolerance to cancer and represent a new approach to tumor immunotherapy.


Assuntos
Citotoxicidade Imunológica , Neoplasias Experimentais/terapia , Receptores de Antígenos de Linfócitos T/fisiologia , Animais , Linfócitos T CD8-Positivos/imunologia , Humanos , Memória Imunológica , Imunoterapia , Interferon gama/biossíntese , Ativação Linfocitária , Camundongos , Camundongos SCID , Neoplasias Experimentais/imunologia
5.
PLoS Biol ; 8(6): e1000402, 2010 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-20585371

RESUMO

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.


Assuntos
Regiões Determinantes de Complementaridade , Imunidade Inata , Células Matadoras Naturais/imunologia , Sequência de Aminoácidos , Animais , Antígenos CD1/imunologia , Galactosilceramidas , Humanos , Ligantes , Camundongos , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos T/química , Ressonância de Plasmônio de Superfície
6.
Protein Cell ; 1(12): 1118-27, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21213105

RESUMO

Using directed mutagenesis and phage display on a soluble fragment of the human immunoglobulin super-family receptor ILT2 (synonyms: LIR1, MIR7, CD85j), we have selected a range of mutants with binding affinities enhanced by up to 168,000-fold towards the conserved region of major histocompatibility complex (MHC) class I molecules. Produced in a dimeric form, either by chemical cross-linking with bivalent polyethylene glycol (PEG) derivatives or as a genetic fusion with human IgG Fc-fragment, the mutants exhibited a further increase in ligand-binding strength due to the avidity effect, with resident half-times (t(1/2)) on the surface of MHC I-positive cells of many hours. The novel compounds antagonized the interaction of CD8 co-receptor with MHC I in vitro without affecting the peptide-specific binding of T-cell receptors (TCRs). In both cytokine-release assays and cell-killing experiments the engineered receptors inhibited the activation of CD8(+) cytotoxic T lymphocytes (CTLs) in the presence of their target cells, with subnanomolar potency and in a dose-dependent manner. As a selective inhibitor of CD8(+) CTL responses, the engineered high affinity ILT2 receptor presents a new tool for studying the activation mechanism of different subsets of CTLs and could have potential for the development of novel autoimmunity therapies.


Assuntos
Antígenos CD/genética , Antígenos CD/farmacologia , Fatores Imunológicos/genética , Fatores Imunológicos/farmacologia , Ativação Linfocitária/imunologia , Receptores Imunológicos/genética , Sequência de Aminoácidos , Antígenos CD/química , Autoimunidade , Bioensaio , Linhagem Celular , Citotoxicidade Imunológica/genética , Citotoxicidade Imunológica/imunologia , Relação Dose-Resposta Imunológica , Humanos , Imunoglobulinas/imunologia , Imunoglobulinas/metabolismo , Fatores Imunológicos/química , Cinética , Receptor B1 de Leucócitos Semelhante a Imunoglobulina , Ativação Linfocitária/genética , Complexo Principal de Histocompatibilidade/genética , Complexo Principal de Histocompatibilidade/imunologia , Dados de Sequência Molecular , Terapia de Alvo Molecular , Mutagênese Sítio-Dirigida , Biblioteca de Peptídeos , Polietilenoglicóis , Ligação Proteica/genética , Ligação Proteica/imunologia , Receptores Imunológicos/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo
7.
J Exp Med ; 203(3): 699-710, 2006 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-16520393

RESUMO

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.


Assuntos
Antígenos CD1/química , Regiões Determinantes de Complementaridade/química , Galactosilceramidas/química , Células Matadoras Naturais , Receptores de Antígenos de Linfócitos T alfa-beta/química , Linfócitos T , Animais , Apresentação de Antígeno/imunologia , Antígenos CD1/imunologia , Antígenos CD1d , Regiões Determinantes de Complementaridade/imunologia , Cristalografia por Raios X , Galactosilceramidas/imunologia , Humanos , Células Matadoras Naturais/imunologia , Camundongos , Modelos Moleculares , Ligação Proteica/imunologia , Estrutura Quaternária de Proteína , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Relação Estrutura-Atividade , Linfócitos T/imunologia
8.
J Exp Med ; 201(8): 1243-55, 2005 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-15837811

RESUMO

Analogue peptides with enhanced binding affinity to major histocompatibility class (MHC) I molecules are currently being used in cancer patients to elicit stronger T cell responses. However, it remains unclear as to how alterations of anchor residues may affect T cell receptor (TCR) recognition. We correlate functional, thermodynamic, and structural parameters of TCR-peptide-MHC binding and demonstrate the effect of anchor residue modifications of the human histocompatibility leukocyte antigens (HLA)-A2 tumor epitope NY-ESO-1(157-165)-SLLMWITQC on TCR recognition. The crystal structure of the wild-type peptide complexed with a specific TCR shows that TCR binding centers on two prominent, sequential, peptide sidechains, methionine-tryptophan. Cysteine-to-valine substitution at peptide position 9, while optimizing peptide binding to the MHC, repositions the peptide main chain and generates subtly enhanced interactions between the analogue peptide and the TCR. Binding analyses confirm tighter binding of the analogue peptide to HLA-A2 and improved soluble TCR binding. Recognition of analogue peptide stimulates faster polarization of lytic granules to the immunological synapse, reduces dependence on CD8 binding, and induces greater numbers of cross-reactive cytotoxic T lymphocyte to SLLMWITQC. These results provide important insights into heightened immunogenicity of analogue peptides and highlight the importance of incorporating structural data into the process of rational optimization of superagonist peptides for clinical trials.


Assuntos
Antígenos de Neoplasias/química , Vacinas Anticâncer/farmacologia , Epitopos de Linfócito T/química , Proteínas de Membrana/química , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas Sintéticas/farmacologia , Animais , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/química , Linhagem Celular Tumoral , Quimiocina CCL4 , Cristalografia por Raios X , Epitopos de Linfócito T/imunologia , Antígeno HLA-A2/química , Antígeno HLA-A2/imunologia , Imunização , Interferon gama/análise , Proteínas Inflamatórias de Macrófagos/análise , Complexo Principal de Histocompatibilidade/imunologia , Proteínas de Membrana/imunologia , Camundongos , Camundongos Transgênicos , Peptídeos/química , Peptídeos/imunologia , Ligação Proteica/imunologia , Conformação Proteica , Receptores de Antígenos de Linfócitos T/química , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologia , Transfecção , Vacinas Sintéticas/química
9.
Artigo em Inglês | MEDLINE | ID: mdl-16511019

RESUMO

The class I CD8 positive T-cell response is involved in a number of conditions in which artificial down-regulation and control would be therapeutically beneficial. Such conditions include a number of autoimmune diseases and graft rejection in transplant patients. Although the CD8 T-cell response is dominated by the TCR-pMHC interaction, activation of T cells is in most cases also dependent on a number of associated signalling molecules. Previous work has demonstrated the ability of one such molecule (CD8) to act as an antagonist to T-cell activation if added in soluble form. Therefore, a high-affinity mutant CD8 (haCD8) has been developed with the aim of developing a therapeutic immunosuppressor. In order to fully understand the nature of the haCD8 interaction, this protein was crystallized using the sitting-drop vapour-diffusion method. Single haCD8 crystals were cryocooled and used for data collection. These crystals belonged to space group P6(4)22 (assumed by similarity to the wild type), with unit-cell parameters a = 101.08, c = 56.54 A. VM calculations indicated one molecule per asymmetric unit. A 2 A data set was collected and the structure is currently being determined using molecular replacement.


Assuntos
Complexo Principal de Histocompatibilidade , Receptores de Antígenos de Linfócitos T/química , Linfócitos T CD8-Positivos/imunologia , Clonagem Molecular , Cristalização , Humanos , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Receptores de Antígenos de Linfócitos T/isolamento & purificação , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
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