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1.
J Immunol ; 208(3): 562-570, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-35031578

RESUMO

Aging is associated with functional deficits in the naive T cell compartment, which compromise the generation of de novo immune responses against previously unencountered Ags. The mechanisms that underlie this phenomenon have nonetheless remained unclear. We found that naive CD8+ T cells in elderly humans were prone to apoptosis and proliferated suboptimally in response to stimulation via the TCR. These abnormalities were associated with dysregulated lipid metabolism under homeostatic conditions and enhanced levels of basal activation. Importantly, reversal of the bioenergetic anomalies with lipid-altering drugs, such as rosiglitazone, almost completely restored the Ag responsiveness of naive CD8+ T cells. Interventions that favor lipid catabolism may therefore find utility as adjunctive therapies in the elderly to promote vaccine-induced immunity against targetable cancers and emerging pathogens, such as seasonal influenza viruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).


Assuntos
Envelhecimento/imunologia , Linfócitos T CD8-Positivos/imunologia , Imunocompetência/efeitos dos fármacos , Metabolismo dos Lipídeos , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Linfócitos T CD8-Positivos/metabolismo , COVID-19/imunologia , Vacinas Anticâncer/imunologia , Divisão Celular , Feminino , Fenofibrato/farmacologia , Glucose/metabolismo , Antígeno HLA-A2/imunologia , Humanos , Hipolipemiantes/farmacologia , Hipolipemiantes/uso terapêutico , Influenza Humana/imunologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Ativação Linfocitária , Antígeno MART-1/química , Antígeno MART-1/imunologia , Masculino , Pessoa de Meia-Idade , Neoplasias/imunologia , Fragmentos de Peptídeos/imunologia , Rosiglitazona/farmacologia , Método Simples-Cego , Vacinação , Vacinas Virais/imunologia , Adulto Jovem
2.
ChemMedChem ; 15(9): 799-807, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32162475

RESUMO

A click-chemistry-based approach was implemented to prepare peptidomimetics designed in silico and made from aromatic azides and a propargylated GIGI-mimicking platform derived from the altered Melan-A/MART-126(27L)-35 antigenic peptide ELAGIGILTV. The CuI -catalyzed Huisgen cycloaddition was carried out on solid support to generate rapidly a first series of peptidomimetics, which were evaluated for their capacity to dock at the interface between the major histocompatibility complex class-I (MHC-I) human leucocyte antigen (HLA)-A2 and T-cell receptors (TCRs). Despite being a weak HLA-A2 ligand, one of these 11 first synthetic compounds bearing a p-nitrobenzyl-triazole side chain was recognized by the receptor proteins of Melan-A/MART-1-specific T-cells. After modification of the N and C termini of this agonist, which was intended to enhance HLA-A2 binding, one of the resulting seven additional compounds triggered significant T-cell responses. Thus, these results highlight the capacity of naturally circulating human TCRs that are specific for the native Melan-A/MART-126-35 peptide to cross-react with peptidomimetics bearing organic motifs structurally different from the native central amino acids.


Assuntos
Haptenos/química , Antígeno MART-1/química , Oligopeptídeos/síntese química , Química Click , Antígeno HLA-A2/imunologia , Haptenos/imunologia , Humanos , Antígeno MART-1/imunologia , Estrutura Molecular , Oligopeptídeos/química , Oligopeptídeos/imunologia , Peptidomiméticos , Receptores de Antígenos de Linfócitos T/imunologia
3.
Eur J Immunol ; 49(7): 1052-1066, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31091334

RESUMO

The HLA-A*02:01-restricted decapeptide EAAGIGILTV, derived from melanoma antigen recognized by T-cells-1 (MART-1) protein, represents one of the best-studied tumor associated T-cell epitopes, but clinical results targeting this peptide have been disappointing. This limitation may reflect the dominance of the nonapeptide, AAGIGILTV, at the melanoma cell surface. The decapeptide and nonapeptide are presented in distinct conformations by HLA-A*02:01 and TCRs from clinically relevant T-cell clones recognize the nonapeptide poorly. Here, we studied the MEL5 TCR that potently recognizes the nonapeptide. The structure of the MEL5-HLA-A*02:01-AAGIGILTV complex revealed an induced fit mechanism of antigen recognition involving altered peptide-MHC anchoring. This "flexing" at the TCR-peptide-MHC interface to accommodate the peptide antigen explains previously observed incongruences in this well-studied system and has important implications for future therapeutic approaches. Finally, this study expands upon the mechanisms by which molecular plasticity can influence antigen recognition by T cells.


Assuntos
Epitopos Imunodominantes/metabolismo , Imunoterapia Adotiva/métodos , Antígeno MART-1/metabolismo , Melanoma/imunologia , Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Aminoácidos , Apresentação de Antígeno , Sítios de Ligação , Células Cultivadas , Células Clonais , Antígeno HLA-A2/química , Antígeno HLA-A2/metabolismo , Humanos , Ativação Linfocitária , Antígeno MART-1/química , Melanoma/terapia , Peptídeos/química , Ligação Proteica , Conformação Proteica , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/transplante
4.
Biomater Sci ; 7(3): 773-788, 2019 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-30601510

RESUMO

The clinical success of dendritic cell (DC)-based genetic immunization remains critically dependent on the availability of effective and safe nano-carriers for targeting antigen-encoded DNA vaccines to DCs, the most potent antigen-presenting cells in the human body in vivo. Recent studies revealed the efficacies of mannose receptor-mediated in vivo DC-targeted genetic immunization by liposomal DNA vaccine carriers containing both mannose-mimicking shikimoyl and transfection enhancing guanidinyl functionalities. However, to date, the efficacies of this approach have not been examined for metal-based nanoparticle DNA vaccine carriers. Herein, we report for the first time, the design, synthesis, physico-chemical characterization and bioactivities of gold nanoparticles covalently functionalized with a thiol ligand containing both shikimoyl and guanidinyl functionalities (Au-SGSH). We show that Au-SGSH nanoparticles can deliver DNA vaccines to mouse DCs under in vivo conditions. Subcutaneous administration of near infrared (NIR) dye-labeled Au-SGSH showed significant accumulation of the NIR dye in the DCs of the nearby lymph nodes compared to that for the non-targeting NIR-labeled Au-GSH nanoconjugate containing only a covalently tethered guanidinyl group, not the shikimoyl-functionality. Under prophylactic settings, in vivo immunization (s.c.) with the Au-SGSH-pCMV-MART1 nanoplex induced a long-lasting (180 days) immune response against murine melanoma. Notably, mannose receptor-mediated in vivo DC-targeted immunization (s.c.) with the Au-SGSH-MART1 nanoplex significantly inhibited established melanoma growth and increased the overall survivability of melanoma-bearing mice under therapeutic settings. The Au-SGSH nanoparticles reported herein have potential use for in vivo DC-targeted genetic immunization against cancer and infectious diseases.


Assuntos
Células Dendríticas/metabolismo , Ouro/química , Nanopartículas Metálicas/química , Nanoconjugados/química , Vacinas de DNA/imunologia , Animais , Células da Medula Óssea/citologia , Linhagem Celular Tumoral , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/citologia , Células Dendríticas/imunologia , Feminino , Corantes Fluorescentes/química , Imunidade Ativa , Antígeno MART-1/química , Antígeno MART-1/imunologia , Masculino , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Melanoma Experimental/prevenção & controle , Nanopartículas Metálicas/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos/química , Plasmídeos/metabolismo , Compostos de Sulfidrila/química , Vacinas de DNA/química
5.
Sci Immunol ; 3(30)2018 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-30552102

RESUMO

Antigen recognition by T cells bearing αß T cell receptors (TCRs) is restricted by major histocompatibility complex (MHC). However, how antigens are recognized by T cells bearing γδ TCRs remains unclear. Although γδ T cells can recognize nonclassical MHC, it is generally thought that recognition of antigens is not MHC restricted. Here, we took advantage of an in vitro system to generate antigen-specific human T cells and show that melanoma-associated antigens, MART-1 and gp100, can be recognized by γδ T cells in an MHC-restricted fashion. Cloning and transferring of MART-1-specific γδ TCRs restored the specific recognition of the initial antigen MHC/peptide reactivity and conferred antigen-specific functional responses. A crystal structure of a MART-1-specific γδ TCR, together with MHC/peptide, revealed distinctive but similar docking properties to those previously reported for αß TCRs, recognizing MART-1 on HLA-A*0201. Our work shows that antigen-specific and MHC-restricted γδ T cells can be generated in vitro and that MART-1-specific γδ T cells can also be found and cloned from the naïve repertoire. These findings reveal that classical MHC-restricted human γδ TCRs exist in the periphery and have the potential to be used in developing of new TCR-based immunotherapeutic approaches.


Assuntos
Antígeno MART-1/imunologia , Melanoma/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Adulto , Células Cultivadas , Cristalografia por Raios X , Humanos , Antígeno MART-1/química , Modelos Moleculares , Receptores de Antígenos de Linfócitos T gama-delta/química
6.
J Chem Phys ; 149(7): 072316, 2018 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-30134695

RESUMO

A method for calculating the free energy difference between two structurally defined conformational states of a chemical system is developed. A path is defined using a previously reported collective variable that interpolates between two or more conformations, and a restraint is introduced in order to keep the system close to the path. The evolution of the system along the path, which typically presents a high free energy barrier, is generated using enhanced sampling schemes. Although the formulation of the method in terms of a path is quite general, an important advance in this work is the demonstration that prior knowledge of the path is, in fact, not needed and that the free energy difference can be obtained using a simplified definition of the path collective variable that only involves the endpoints. We first validate this method on cyclohexane isomerization. The method is then tested for an extensive conformational change in a realistic molecular system by calculating the free energy difference between the α-helix and ß-hairpin conformations of deca-alanine in solution. Finally, the method is applied to a biologically relevant system to calculate the free energy difference of an observed and a hypothetical conformation of an antigenic peptide bound to a major histocompatibility complex.


Assuntos
Cicloexanos/química , Antígeno HLA-A2/química , Antígeno MART-1/química , Simulação de Dinâmica Molecular , Peptídeos/química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Termodinâmica
7.
J Biol Chem ; 293(5): 1820-1834, 2018 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-29229779

RESUMO

Most affinity-maturation campaigns for antibodies and T-cell receptors (TCRs) operate on the residues at the binding site, located within the loops known as complementarity-determining regions (CDRs). Accordingly, mutations in contact residues, or so-called "second shell" residues, that increase affinity are typically identified by directed evolution involving combinatorial libraries. To determine the impact of residues located at a distance from the binding site, here we used single-codon libraries of both CDR and non-CDR residues to generate a deep mutational scan of a human TCR against the cancer antigen MART-1·HLA-A2. Non-CDR residues included those at the interface of the TCR variable domains (Vα and Vß) and surface-exposed framework residues. Mutational analyses showed that both Vα/Vß interface and CDR residues were important in maintaining binding to MART-1·HLA-A2, probably due to either structural requirements for proper Vα/Vß association or direct contact with the ligand. More surprisingly, many Vα/Vß interface substitutions yielded improved binding to MART-1·HLA-A2. To further explore this finding, we constructed interface libraries and selected them for improved stability or affinity. Among the variants identified, one conservative substitution (F45ßY) was most prevalent. Further analysis of F45ßY showed that it enhanced thermostability and increased affinity by 60-fold. Thus, introducing a single hydroxyl group at the Vα/Vß interface, at a significant distance from the TCR·peptide·MHC-binding site, remarkably affected ligand binding. The variant retained a high degree of specificity for MART-1·HLA-A2, indicating that our approach provides a general strategy for engineering improvements in either soluble or cell-based TCRs for therapeutic purposes.


Assuntos
Regiões Determinantes de Complementaridade/química , Antígeno HLA-A2/química , Antígeno MART-1/química , Sítios de Ligação , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Humanos , Antígeno MART-1/genética , Antígeno MART-1/imunologia , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Saccharomyces cerevisiae
8.
Immunology ; 152(3): 462-471, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28664991

RESUMO

Peptide recognition through the MHC class I molecule by cytotoxic T lymphocytes (CTLs) leads to the killing of cancer cells. A potential challenge for T-cell immunotherapy is that dendritic cells (DCs) are exposed to the MHC class I-peptide complex for an insufficient amount of time. To improve tumour antigen presentation to T cells and thereby initiate a more effective T-cell response, we generated artificial antigen-presenting cells (aAPCs) by incubating human immature DCs (imDCs) with poly(lactic-co-glycolic) acid nanoparticles (PLGA-NPs) encapsulating tumour antigenic peptides, followed by maturation with lipopolysaccharide. Tumour antigen-specific CTLs were then induced using either peptide-loaded mature DCs (mDCs) or aAPCs, and their activities were analysed using both ELISpot and cytotoxicity assays. We found that the aAPCs induced significantly stronger tumour antigen-specific CTL responses than the controls, which included both mDCs and aAPCs loaded with empty nanoparticles. Moreover, frozen CTLs that were generated by exposure to aAPCs retained the capability to eradicate HLA-A2-positive tumour antigen-bearing cancer cells. These results indicated that aAPCs are superior to DCs when inducing the CTL response because the former are capable of continuously presenting tumour antigens to T cells in a sustained manner. The development of aAPCs with PLGA-NPs encapsulating tumour antigenic peptides is a promising approach for the generation of effective CTL responses in vitro and warrants further assessments in clinical trials.


Assuntos
Apresentação de Antígeno , Vacinas Anticâncer/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Proteínas Inibidoras de Apoptose/farmacologia , Ácido Láctico/química , Lipopolissacarídeos/farmacologia , Antígeno MART-1/farmacologia , Nanopartículas , Neoplasias/terapia , Fragmentos de Peptídeos/farmacologia , Ácido Poliglicólico/química , Linfócitos T Citotóxicos/efeitos dos fármacos , Vacinas Anticâncer/química , Vacinas Anticâncer/imunologia , Sobrevivência Celular/efeitos dos fármacos , Preparações de Ação Retardada , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Composição de Medicamentos , Liberação Controlada de Fármacos , Humanos , Proteínas Inibidoras de Apoptose/química , Proteínas Inibidoras de Apoptose/imunologia , Cinética , Lipopolissacarídeos/imunologia , Antígeno MART-1/química , Antígeno MART-1/imunologia , Células MCF-7 , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Solubilidade , Survivina , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo
9.
Sci Rep ; 6: 36360, 2016 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-27821860

RESUMO

Generalized vitiligo is an autoimmune disease characterized by melanocyte loss, which results in patchy depigmentation of skin and hair. Recent studies suggested the key role of CD8+T lymphocytes for mediating immune response in vitiligo through melanocyte differentiation antigens, including tyrosinase, gp100 and MelanA/Mart-1. However, the specific epitopes of these auto-antigens are still unknown. In our study, we predicted the possible HLA-A*0201-restricted nonapeptides overlaying the full-length amino acid sequences of these three known antigens and investigated the lymphocytes reactivity to these nonapeptides by Elispot assay. In addition, we evaluated the abilities of these nonapeptides to activate CD8+T cells. We screened out 5 possible epitopes originated from tyrosinase and gp100, numbered P28, P41, P112, P118 and P119. Among these 5 epitopes, notably, P28 and P119 played the dominant role in activating CTLs, with a significant increase in proliferation rate and Interferon-γ (IFN-γ) production of CD8+T cells. Nevertheless, antigen-specific T cell reactivity was not detected in MelanA/Mart-1 peptides. Our studies identified two novel epitopes originated from proteins of gp100 and tyrosinase, which may have implications for the development of immunotherapies for vitiligo.


Assuntos
Autoantígenos/química , Epitopos/genética , Antígeno HLA-A2/genética , Linfócitos T Citotóxicos/imunologia , Vitiligo/genética , Adolescente , Adulto , Sequência de Aminoácidos , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Criança , China , Epitopos/imunologia , Feminino , Humanos , Interferon gama/metabolismo , Antígeno MART-1/química , Masculino , Pessoa de Meia-Idade , Monofenol Mono-Oxigenase/química , Vitiligo/imunologia , Adulto Jovem , Antígeno gp100 de Melanoma/química
10.
Appl Immunohistochem Mol Morphol ; 24(7): 531-4, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-26317308

RESUMO

INTRODUCTION: Heat-induced epitope retrieval (HIER) of formalin-fixed paraffin-embedded tissues is now a standard practice in immunohistochemistry (IHC). In this study, we aimed to test the effect of altering HIER temperature on IHC staining quality at high altitude, the hypothesis being that lower HIER temperatures would result in improved staining patterns. MATERIALS AND METHODS: In a laboratory at high altitude (Aurora, CO), we used a platform with automated onboard epitope retrieval, and systematically tested 3 different HIER temperatures (100°C, 95°C, 90°C) with 4 IHC stains that are commonly used in routine practice: CD3, Ki67, CK20, and Melan A (n=10 for each antibody/epitope retrieval temperature combination). A scoring system was devised, the slides were scored in a blinded manner, and statistical analysis was performed. For comparison, the same study was performed in a laboratory near sea level (Atlanta, GA). RESULTS: At high altitude, lower HIER temperatures resulted in improved staining patterns, as quantified by stronger staining intensity and greater area of the slides stained. The scores obtained with HIER temperatures of 95°C and 90°C were higher than those obtained with HIER of 100°C, and the difference was found to be statistically significantly for some antibody/epitope retrieval temperature combinations (P<0.05). This effect was not seen in the laboratory near sea level. CONCLUSIONS: We show that alternate epitope retrieval recommendations are warranted for laboratories at high altitude. Furthermore, we suggest that manufactures should consider how their instruments will perform at high altitude as they further automate the process of IHC.


Assuntos
Altitude , Complexo CD3 , Temperatura Alta , Imuno-Histoquímica/métodos , Complexo CD3/química , Humanos , Imuno-Histoquímica/normas , Queratina-20/química , Antígeno Ki-67/química , Antígeno MART-1/química , Inclusão em Parafina , Controle de Qualidade
11.
J Immunol ; 194(7): 3487-500, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25710913

RESUMO

TCRα- and ß-chains cooperatively recognize peptide-MHC complexes. It has been shown that a "chain-centric" TCR hemichain can, by itself, dictate MHC-restricted Ag specificity without requiring major contributions from the paired TCR counterchain. Little is known, however, regarding the relative contributions and roles of chain-centric and its counter, non-chain-centric, hemichains in determining T cell avidity. We comprehensively analyzed a thymically unselected T cell repertoire generated by transducing the α-chain-centric HLA-A*02:01(A2)/MART127-35 TCRα, clone SIG35α, into A2-matched and unmatched postthymic T cells. Regardless of their HLA-A2 positivity, a substantial subset of peripheral T cells transduced with SIG35α gained reactivity for A2/MART127-35. Although the generated A2/MART127-35-specific T cells used various TRBV genes, TRBV27 predominated with >10(2) highly diverse and unique clonotypic CDR3ß sequences. T cells individually reconstituted with various A2/MART127-35 TRBV27 TCRß genes along with SIG35α possessed a wide range (>2 log orders) of avidity. Approximately half possessed avidity higher than T cells expressing clone DMF5, a naturally occurring A2/MART127-35 TCR with one of the highest affinities. Importantly, similar findings were recapitulated with other self-Ags. Our results indicate that, although a chain-centric TCR hemichain determines Ag specificity, the paired counterchain can regulate avidity over a broad range (>2 log orders) without compromising Ag specificity. TCR chain centricity can be exploited to generate a thymically unselected Ag-specific T cell repertoire, which can be used to isolate high-avidity antitumor T cells and their uniquely encoded TCRs rarely found in the periphery because of tolerance.


Assuntos
Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Linhagem Celular Tumoral , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/metabolismo , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Antígeno HLA-A2/química , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Humanos , Imunofenotipagem , Antígeno MART-1/química , Antígeno MART-1/genética , Antígeno MART-1/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Multimerização Proteica , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Transdução Genética
12.
Eur J Immunol ; 45(2): 380-2, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25581444

RESUMO

Although it has been shown that human tumor-associated, HLA anchor residue modified "heteroclitic" peptides may induce stronger immune responses than wild-type peptides in cancer vaccine trials, it has also been shown that some T cells primed with these heteroclitic peptides subsequently fail to recognize the natural, tumor-expressed peptide efficiently. This may provide a molecular reason for why clinical trials of these peptides have been thus far unsuccessful. In this issue of the European Journal of Immunology, Madura et al. [Eur. J. Immunol. 2015. 45: 584-591] highlight a novel twist on T-cell receptor (TCR) recognition of HLA-peptide complexes. Tumor-associated peptides often lack canonical anchor residues, which can be substituted for the optimal residue to improve their antigenicity. T-cell cross-reactivity between the natural and modified (heteroclitic) peptides is essential for this approach to work and depends on whether the anchor residue substitution influences peptide conformation. The Melan-A/MART-126-35 peptide epitope is an example where T cells can make this distinction, with the natural peptide stimulating higher affinity CD8(+) T cells than the heteroclitic peptide, despite the heteroclitic peptide's more stable association with HLA-A2. The molecular basis for peptide discrimination is identified through the structure of the TCR bound to the natural peptide; TCR engagement of the natural peptide "lifts" its amino-terminus partly away from the HLA peptide binding groove, forming a higher affinity interface with the TCR than is formed with the anchor residue "optimized" heteroclitic peptide, which cannot be "pulled" from the HLA groove.


Assuntos
Alanina/química , Epitopos de Linfócito T/metabolismo , Antígeno HLA-A2/química , Leucina/química , Antígeno MART-1/química , Peptídeos/química , Receptores de Antígenos de Linfócitos T alfa-beta/química , Humanos
13.
J Control Release ; 198: 91-103, 2015 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-25483429

RESUMO

We hypothesized that the co-entrapment of melanoma-associated antigens and the Toll-like receptor (TLR) ligands Poly(I:C) and CpG, known to be Th1-immunopotentiators, in mannose-functionalized aliphatic polyester-based nanoparticles (NPs) could be targeted to mannose receptors on antigen-presenting cells and induce anti-tumor immune responses. High entrapment efficiencies of antigens and immunopotentiators in 150nm NPs were obtained. The co-entrapment of the model antigen ovalbumin and the TLR ligands was crucial to induce high IgG2c/IgG1 ratios and high levels of IFN-γ and IL-2. Mannose-functionalization of NPs potentiated the Th1 immune response. The nanoparticulate vaccines decreased the growth rate of murine B16F10 melanoma tumors in therapeutic and prophylatic settings. The combination of mannose-functionalized NPs containing MHC class I- or class II-restricted melanoma antigens and the TLR ligands induced the highest tumor growth delay. Overall, we demonstrate that the multifunctional properties of NPs in terms of targeting and antigen/adjuvant delivery have high cancer immunotherapeutic potential.


Assuntos
Vacinas Anticâncer , Antígeno MART-1/administração & dosagem , Melanoma/tratamento farmacológico , Oligodesoxirribonucleotídeos/administração & dosagem , Ovalbumina/administração & dosagem , Receptores Toll-Like/imunologia , Antígeno gp100 de Melanoma/administração & dosagem , Animais , Linhagem Celular Tumoral , Citocinas/imunologia , Modelos Animais de Doenças , Feminino , Granzimas/metabolismo , Imunoglobulina G/sangue , Ligantes , Antígeno MART-1/química , Antígeno MART-1/imunologia , Masculino , Manose/química , Melanoma/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nanopartículas/administração & dosagem , Nanopartículas/química , Oligodesoxirribonucleotídeos/química , Ovalbumina/química , Ovalbumina/imunologia , Peptídeos/administração & dosagem , Peptídeos/química , Poli I-C/administração & dosagem , Poli I-C/química , Polímeros/química , Carga Tumoral/efeitos dos fármacos , Antígeno gp100 de Melanoma/química , Antígeno gp100 de Melanoma/imunologia
14.
Eur J Immunol ; 45(2): 584-91, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25471691

RESUMO

MHC anchor residue-modified "heteroclitic" peptides have been used in many cancer vaccine trials and often induce greater immune responses than the wild-type peptide. The best-studied system to date is the decamer MART-1/Melan-A26-35 peptide, EAAGIGILTV, where the natural alanine at position 2 has been modified to leucine to improve human leukocyte antigen (HLA)-A*0201 anchoring. The resulting ELAGIGILTV peptide has been used in many studies. We recently showed that T cells primed with the ELAGIGILTV peptide can fail to recognize the natural tumor-expressed peptide efficiently, thereby providing a potential molecular reason for why clinical trials of this peptide have been unsuccessful. Here, we solved the structure of a TCR in complex with HLA-A*0201-EAAGIGILTV peptide and compared it with its heteroclitic counterpart , HLA-A*0201-ELAGIGILTV. The data demonstrate that a suboptimal anchor residue at position 2 enables the TCR to "pull" the peptide away from the MHC binding groove, facilitating extra contacts with both the peptide and MHC surface. These data explain how a TCR can distinguish between two epitopes that differ by only a single MHC anchor residue and demonstrate how weak MHC anchoring can enable an induced-fit interaction with the TCR. Our findings constitute a novel demonstration of the extreme sensitivity of the TCR to minor alterations in peptide conformation.


Assuntos
Alanina/química , Epitopos de Linfócito T/metabolismo , Antígeno HLA-A2/química , Leucina/química , Antígeno MART-1/química , Peptídeos/química , Receptores de Antígenos de Linfócitos T alfa-beta/química , Alanina/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Cristalografia por Raios X , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Humanos , Leucina/genética , Antígeno MART-1/genética , Antígeno MART-1/imunologia , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/imunologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo
15.
Nat Commun ; 5: 5223, 2014 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-25376839

RESUMO

Binding of a T-cell receptor (TCR) to a peptide/major histocompatibility complex is the key interaction involved in antigen specificity of T cells. The recognition involves up to six complementarity determining regions (CDR) of the TCR. Efforts to examine the structural basis of these interactions and to exploit them in adoptive T-cell therapies has required the isolation of specific T-cell clones and their clonotypic TCRs. Here we describe a strategy using in vitro-directed evolution of a single TCR to change its peptide specificity, thereby avoiding the need to isolate T-cell clones. The human TCR A6, which recognizes the viral peptide Tax/HLA-A2, was converted to TCR variants that recognized the cancer peptide MART1/HLA-A2. Mutational studies and molecular dynamics simulations identified CDR residues that were predicted to be important in the specificity switch. Thus, in vitro engineering strategies alone can be used to discover TCRs with desired specificities.


Assuntos
Regiões Determinantes de Complementaridade/genética , Epitopos/genética , Evolução Molecular , Receptores de Antígenos de Linfócitos T/genética , Sequência de Aminoácidos , Células Clonais , Regiões Determinantes de Complementaridade/química , Epitopos/química , Antígeno HLA-A2/química , Antígeno HLA-A2/genética , Humanos , Técnicas In Vitro , Antígeno MART-1/química , Antígeno MART-1/genética , Complexo Principal de Histocompatibilidade , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Oligopeptídeos/química , Oligopeptídeos/genética , Ligação Proteica , Conformação Proteica , Receptores de Antígenos de Linfócitos T/química
16.
J Immunol ; 193(5): 2587-99, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-25070852

RESUMO

Adoptive immunotherapy with Ag-specific T lymphocytes is a powerful strategy for cancer treatment. However, most tumor Ags are nonreactive "self" proteins, which presents an immunotherapy design challenge. Recent studies have shown that tumor-specific TCRs can be transduced into normal PBLs, which persist after transfer in ∼30% of patients and effectively destroy tumor cells in vivo. Although encouraging, the limited clinical responses underscore the need for enrichment of T cells with desirable antitumor capabilities prior to patient transfer. In this study, we used structure-based design to predict point mutations of a TCR (DMF5) that enhance its binding affinity for an agonist tumor Ag-MHC (peptide-MHC [pMHC]), Mart-1 (27L)-HLA-A2, which elicits full T cell activation to trigger immune responses. We analyzed the effects of selected TCR point mutations on T cell activation potency and analyzed cross-reactivity with related Ags. Our results showed that the mutated TCRs had improved T cell activation potency while retaining a high degree of specificity. Such affinity-optimized TCRs have demonstrated to be very specific for Mart-1 (27L), the epitope for which they were structurally designed. Although of somewhat limited clinical relevance, these studies open the possibility for future structural-based studies that could potentially be used in adoptive immunotherapy to treat melanoma while avoiding adverse autoimmunity-derived effects.


Assuntos
Epitopos de Linfócito T , Antígeno MART-1 , Peptídeos , Engenharia de Proteínas , Receptores de Antígenos de Linfócitos T , Animais , Linhagem Celular Tumoral , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Humanos , Ativação Linfocitária , Antígeno MART-1/química , Antígeno MART-1/genética , Antígeno MART-1/imunologia , Peptídeos/química , Peptídeos/imunologia , Mutação Puntual , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Relação Estrutura-Atividade
17.
PLoS One ; 9(2): e89897, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24587108

RESUMO

Antitumor vaccination using synthetic long peptides (SLP) is an additional therapeutic strategy currently under development. It aims to activate tumor-specific CD8(+) CTL by professional APCs such as DCs. DCs can activate T lymphocytes by MHC class I presentation of exogenous antigens - a process referred to as "cross-presentation". Until recently, the intracellular mechanisms involved in cross-presentation of soluble antigens have been unclear. Here, we characterize the cross-presentation pathway of SLP Melan-A16-40 containing the HLA-A2-restricted epitope26-35 (A27L) in human DCs. Using confocal microscopy and specific inhibitors, we show that SLP16-40 is rapidly taken up by DC and follows a classical TAP- and proteasome-dependent cross-presentation pathway. Our data support a role for the ER-associated degradation machinery (ERAD)-related protein p97/VCP in the transport of SLP16-40 from early endosomes to the cytoplasm but formally exclude both sec61 and Derlin-1 as possible retro-translocation channels for cross-presentation. In addition, we show that generation of the Melan-A26-35 peptide from the SLP16-40 was absolutely not influenced by the proteasome subunit composition in DC. Altogether, our findings propose a model for cross-presentation of SLP which tends to enlarge the repertoire of potential candidates for retro-translocation of exogenous antigens to the cytosol.


Assuntos
Apresentação de Antígeno/imunologia , Antígenos/imunologia , Apresentação Cruzada/imunologia , Células Dendríticas/imunologia , Peptídeos/imunologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Antígenos/química , Antígenos de Neoplasias/química , Antígenos de Neoplasias/imunologia , Proteínas de Ciclo Celular/metabolismo , Células Dendríticas/metabolismo , Endocitose/imunologia , Degradação Associada com o Retículo Endoplasmático , Endossomos/metabolismo , Humanos , Cinética , Lisossomos/metabolismo , Antígeno MART-1/química , Antígeno MART-1/imunologia , Proteínas de Membrana/metabolismo , Modelos Biológicos , Peptídeos/síntese química , Peptídeos/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte Proteico , Canais de Translocação SEC , Proteína com Valosina
18.
PLoS Comput Biol ; 10(2): e1003478, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24550723

RESUMO

T cell receptors (TCRs) are key to antigen-specific immunity and are increasingly being explored as therapeutics, most visibly in cancer immunotherapy. As TCRs typically possess only low-to-moderate affinity for their peptide/MHC (pMHC) ligands, there is a recognized need to develop affinity-enhanced TCR variants. Previous in vitro engineering efforts have yielded remarkable improvements in TCR affinity, yet concerns exist about the maintenance of peptide specificity and the biological impacts of ultra-high affinity. As opposed to in vitro engineering, computational design can directly address these issues, in theory permitting the rational control of peptide specificity together with relatively controlled increments in affinity. Here we explored the efficacy of computational design with the clinically relevant TCR DMF5, which recognizes nonameric and decameric epitopes from the melanoma-associated Melan-A/MART-1 protein presented by the class I MHC HLA-A2. We tested multiple mutations selected by flexible and rigid modeling protocols, assessed impacts on affinity and specificity, and utilized the data to examine and improve algorithmic performance. We identified multiple mutations that improved binding affinity, and characterized the structure, affinity, and binding kinetics of a previously reported double mutant that exhibits an impressive 400-fold affinity improvement for the decameric pMHC ligand without detectable binding to non-cognate ligands. The structure of this high affinity mutant indicated very little conformational consequences and emphasized the high fidelity of our modeling procedure. Overall, our work showcases the capability of computational design to generate TCRs with improved pMHC affinities while explicitly accounting for peptide specificity, as well as its potential for generating TCRs with customized antigen targeting capabilities.


Assuntos
Receptores de Antígenos de Linfócitos T/imunologia , Apresentação de Antígeno , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Biologia Computacional , Simulação por Computador , Cristalografia por Raios X , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Antígeno HLA-A2/imunologia , Humanos , Ligantes , Antígeno MART-1/química , Antígeno MART-1/genética , Antígeno MART-1/imunologia , Modelos Moleculares , Peptídeos/química , Peptídeos/genética , Peptídeos/imunologia , Mutação Puntual , Engenharia de Proteínas , Receptores de Antígenos de Linfócitos T/genética , Termodinâmica
19.
Clin Dev Immunol ; 2013: 932318, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24194775

RESUMO

A number of trials of adoptive transfer of tumor-specific T lymphocytes have been performed in the last 20 years in metastatic melanoma, with increasingly encouraging results as the relevant melanoma antigens were identified and the purity/specificity of injected T cells improved. We have previously described a sorting method of epitope-specific T lymphocytes that uses magnetic beads coated with HLA/peptide complexes and we suggested that this method could be applied to a clinical setting. In the present work, we provide a detailed description of the whole GMP process of sorting and amplification of clinical grade T cells specific for the melanoma antigens Melan-A and MELOE-1. All the reagents used in this process including the sorting reagent were produced in GMP conditions and we document the optimization of the different steps of the process such as peptide stimulation, sorting, and amplification. The optimized procedure, validated in 3 blank runs in a clinical setting, allowed the production of at least 108 pure (>90%) Melan-A- and MELOE-1-specific T cells within 28 days starting with 100 mL of blood from metastatic melanoma patients. This GMP process is thus ready to be used in an upcoming phase I/II clinical trial on metastatic melanoma patients.


Assuntos
Epitopos de Linfócito T/imunologia , Imunoterapia Adotiva/métodos , Imunoterapia Adotiva/normas , Melanoma/imunologia , Melanoma/terapia , Linfócitos T/imunologia , Linhagem Celular Tumoral , Antígeno HLA-A2/imunologia , Humanos , Ativação Linfocitária , Antígeno MART-1/química , Antígeno MART-1/imunologia , Melanoma/patologia , Metástase Neoplásica , Peptídeos/imunologia
20.
Protein Expr Purif ; 92(2): 171-82, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24045055

RESUMO

Melan-A is a cancer testis antigen commonly found in melanoma, and has been shown to stimulate the body's immune response against cancerous cells. We have developed and executed a process utilizing current good manufacturing practices (cGMP) to produce the 6 times-His tagged protein in C41DE3 Escherichia coli for use in Phase I clinical trials. Approximately 11 g of purified Melan-A were produced from a 20 L fed-batch fermentation. Purification was achieved through a three column process utilizing immobilized metal affinity, anion exchange, and cation exchange chromatography with a buffer system optimized for low-solubility, high LPS binding capacity proteins. The host cell proteins, residual DNA, and endotoxin concentration were well below limits for a prescribed dose with a final purity level of 91%.


Assuntos
Vacinas Anticâncer , Histidina/metabolismo , Antígeno MART-1/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Pesquisa Biomédica , Química Farmacêutica , Cromatografia por Troca Iônica , Fermentação , Histidina/química , Histidina/genética , Antígeno MART-1/química , Antígeno MART-1/genética , Estabilidade Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Reprodutibilidade dos Testes
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