Evaluación de la inmunogenicidad de antígenos formulados en minigenes transfectados a células presentadoras de antígeno

ilustraciones, diagramas

Autores:
Villota Alava, María Alejandra
Tipo de recurso:
Fecha de publicación:
2024
Institución:
Universidad Nacional de Colombia
Repositorio:
Universidad Nacional de Colombia
Idioma:
spa
OAI Identifier:
oai:repositorio.unal.edu.co:unal/86062
Acceso en línea:
https://repositorio.unal.edu.co/handle/unal/86062
https://repositorio.unal.edu.co/
Palabra clave:
610 - Medicina y salud::615 - Farmacología y terapéutica
Antígenos de Neoplasias
Antígenos Virales/análisis
Inmunoterapia/métodos
Antigens, Neoplasm
Antigens, Viral/analysis
Immunotherapy/methods
Minigenes
Neoantígenos
Células presentadoras de antígeno artificiales
Transfección
Transducción
Citometría de Flujo
Inmunoterapia
Minigene
Neoantigens
Artificial Antigen Presenting Cells
Transfection
Flow Cytometry
Transduction
Immunotherapy
Rights
openAccess
License
Reconocimiento 4.0 Internacional
id UNACIONAL2_96394703b3f85e18900399ca42abc643
oai_identifier_str oai:repositorio.unal.edu.co:unal/86062
network_acronym_str UNACIONAL2
network_name_str Universidad Nacional de Colombia
repository_id_str
dc.title.spa.fl_str_mv Evaluación de la inmunogenicidad de antígenos formulados en minigenes transfectados a células presentadoras de antígeno
dc.title.translated.eng.fl_str_mv Assessment of the immunogenicity of antigens formulated in minigenes transfected into antigen-presenting cells
title Evaluación de la inmunogenicidad de antígenos formulados en minigenes transfectados a células presentadoras de antígeno
spellingShingle Evaluación de la inmunogenicidad de antígenos formulados en minigenes transfectados a células presentadoras de antígeno
610 - Medicina y salud::615 - Farmacología y terapéutica
Antígenos de Neoplasias
Antígenos Virales/análisis
Inmunoterapia/métodos
Antigens, Neoplasm
Antigens, Viral/analysis
Immunotherapy/methods
Minigenes
Neoantígenos
Células presentadoras de antígeno artificiales
Transfección
Transducción
Citometría de Flujo
Inmunoterapia
Minigene
Neoantigens
Artificial Antigen Presenting Cells
Transfection
Flow Cytometry
Transduction
Immunotherapy
title_short Evaluación de la inmunogenicidad de antígenos formulados en minigenes transfectados a células presentadoras de antígeno
title_full Evaluación de la inmunogenicidad de antígenos formulados en minigenes transfectados a células presentadoras de antígeno
title_fullStr Evaluación de la inmunogenicidad de antígenos formulados en minigenes transfectados a células presentadoras de antígeno
title_full_unstemmed Evaluación de la inmunogenicidad de antígenos formulados en minigenes transfectados a células presentadoras de antígeno
title_sort Evaluación de la inmunogenicidad de antígenos formulados en minigenes transfectados a células presentadoras de antígeno
dc.creator.fl_str_mv Villota Alava, María Alejandra
dc.contributor.advisor.spa.fl_str_mv Parra López, Carlos Alberto
dc.contributor.author.spa.fl_str_mv Villota Alava, María Alejandra
dc.contributor.educationalvalidator.spa.fl_str_mv Clavijo Ramirez, Carlos Arturo
dc.contributor.researchgroup.spa.fl_str_mv Inmunología y Medicina Traslacional
dc.contributor.subjectmatterexpert.spa.fl_str_mv Patarroyo Gutiérrez, Manuel Alfonso
dc.contributor.cvlac.spa.fl_str_mv Villota Alava, María Alejandra [https://scienti.minciencias.gov.co/cvlac/visualizador/generarCurriculoCv.do?cod_rh=0001786060]
dc.subject.ddc.spa.fl_str_mv 610 - Medicina y salud::615 - Farmacología y terapéutica
topic 610 - Medicina y salud::615 - Farmacología y terapéutica
Antígenos de Neoplasias
Antígenos Virales/análisis
Inmunoterapia/métodos
Antigens, Neoplasm
Antigens, Viral/analysis
Immunotherapy/methods
Minigenes
Neoantígenos
Células presentadoras de antígeno artificiales
Transfección
Transducción
Citometría de Flujo
Inmunoterapia
Minigene
Neoantigens
Artificial Antigen Presenting Cells
Transfection
Flow Cytometry
Transduction
Immunotherapy
dc.subject.decs.spa.fl_str_mv Antígenos de Neoplasias
Antígenos Virales/análisis
Inmunoterapia/métodos
dc.subject.decs.eng.fl_str_mv Antigens, Neoplasm
Antigens, Viral/analysis
Immunotherapy/methods
dc.subject.proposal.spa.fl_str_mv Minigenes
Neoantígenos
Células presentadoras de antígeno artificiales
Transfección
Transducción
Citometría de Flujo
Inmunoterapia
dc.subject.proposal.eng.fl_str_mv Minigene
Neoantigens
Artificial Antigen Presenting Cells
Transfection
Flow Cytometry
Transduction
Immunotherapy
description ilustraciones, diagramas
publishDate 2024
dc.date.accessioned.none.fl_str_mv 2024-05-09T18:42:36Z
dc.date.available.none.fl_str_mv 2024-05-09T18:42:36Z
dc.date.issued.none.fl_str_mv 2024-04-19
dc.type.spa.fl_str_mv Trabajo de grado - Maestría
dc.type.driver.spa.fl_str_mv info:eu-repo/semantics/masterThesis
dc.type.version.spa.fl_str_mv info:eu-repo/semantics/acceptedVersion
dc.type.content.spa.fl_str_mv Text
dc.type.redcol.spa.fl_str_mv http://purl.org/redcol/resource_type/TM
status_str acceptedVersion
dc.identifier.uri.none.fl_str_mv https://repositorio.unal.edu.co/handle/unal/86062
dc.identifier.instname.spa.fl_str_mv Universidad Nacional de Colombia
dc.identifier.reponame.spa.fl_str_mv Repositorio Institucional Universidad Nacional de Colombia
dc.identifier.repourl.spa.fl_str_mv https://repositorio.unal.edu.co/
url https://repositorio.unal.edu.co/handle/unal/86062
https://repositorio.unal.edu.co/
identifier_str_mv Universidad Nacional de Colombia
Repositorio Institucional Universidad Nacional de Colombia
dc.language.iso.spa.fl_str_mv spa
language spa
dc.relation.indexed.spa.fl_str_mv Bireme
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dc.rights.license.spa.fl_str_mv Reconocimiento 4.0 Internacional
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spelling Reconocimiento 4.0 Internacionalhttp://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2Parra López, Carlos Alberto72ac583cfa47cd3a2fb760ecf10befcc600Villota Alava, María Alejandra304cff981c087fba7679071d7b010f61Clavijo Ramirez, Carlos ArturoInmunología y Medicina TraslacionalPatarroyo Gutiérrez, Manuel AlfonsoVillota Alava, María Alejandra [https://scienti.minciencias.gov.co/cvlac/visualizador/generarCurriculoCv.do?cod_rh=0001786060]2024-05-09T18:42:36Z2024-05-09T18:42:36Z2024-04-19https://repositorio.unal.edu.co/handle/unal/86062Universidad Nacional de ColombiaRepositorio Institucional Universidad Nacional de Colombiahttps://repositorio.unal.edu.co/ilustraciones, diagramasLa deficiente presentación del antígeno por parte de las células tumorales juega un papel importante en la evasión de la respuesta inmune antitumoral por parte de los linfocitos T, siendo, de hecho, un factor primordial del origen de los tumores. Por esta razón distintos tipos de células presentadoras de antígeno (APCs) han sido ampliamente utilizadas para la inmunoterapia del cáncer debido a su capacidad de procesar y presentar eficientemente antígenos a los linfocitos T en pacientes con cáncer. Frecuentemente las APCs son pulsadas con el antígeno en forma de péptidos, sin embargo, este enfoque puede resultar en la presentación de epítopes que no son generadas producto del procesamiento natural por las células tumorales, afectando la efectividad de este tipo de inmunoterapia. En este contexto, el uso de minigenes, que corresponden a secuencias de antígenos concatenados, emerge como una alternativa propicia para la selección de antígenos procesados naturalmente por las APCs. Se espera que esta estrategia simule el procesamiento y presentación natural de los antígenos a los linfocitos T y aumente la probabilidad de identificar antígenos inmunogénicos, evitando así la selección de epítopes que no son procesados y presentados naturalmente como candidatos a vacuna para la inmunoterapia. Para hacer entrega de estos minigenes a las APCs, se han explorado distintos métodos, siendo la electroporación, la administración mediante liposomas catiónicos y la transducción por vectores virales los más comunes. Aunque se reconocen las ventajas y desventajas inherentes a cada método, pocos estudios han comparado el rendimiento en la estimulación de los linfocitos T por parte de APCs transfectadas con diferentes sistemas. En este sentido, el presente trabajo propuso la evaluación de la capacidad que tienen las APCs transfectadas con un constructo de minigen que codifica para antígenos inmunogénicos restringidos al haplotipo HLA-A*0201, de estimular precursores de linfocitos T-CD8+ (LT-CD8+) antígeno-específicos in vitro. Además de la transfección con el minigen, también se diseñaron APCs artificiales (en adelante denominaremos aAPCs), cotransfectando las líneas celulares HEK293 y K562 con plásmidos que codifican para moléculas co-estimuladoras (CD80, CD83, CD137L) y en el caso de las K562 también con un plásmido que codifica para la molécula HLA-A*0201, con el fin de evaluar la eficiencia de activación de LT-CD8+ por estas aAPCs, en términos de la producción de citoquinas intracelulares, la actividad citotóxica, la expresión de marcadores de activación y agotamiento; y el perfil de las subpoblaciones de memoria de los LT-CD8+ estimulados. Los resultados de este trabajo permitieron implementar una metodología de transfección con el uso de lipofectamina y electroporación en células HEK293 de un minigen codificante para epítopes HLA-A*0201. Estas células se emplearon como aAPCs para analizar el fenotipo de poblaciones de LT-CD8+ antígeno-específicas. Considerando que el uso de las células HEK293 como aAPCs no ha sido explorado, y debido a su alta eficiencia de transfección y transducción con minigenes, la metodología implementada en este trabajo posibilita su uso para la identificación de neoantígenos inmunogénicos naturalmente procesados. Consideramos que nuestros hallazgos pueden contribuir con la selección y el diseño de vacunas personalizadas basadas en neoantígenos tumorales útiles para la inmunoterapia del cáncer. (Texto tomado de la fuente).The deficient antigen presentation by tumor cells plays a significant role in the evasion of the anti-tumor immune response by T lymphocytes, being, in fact, a primordial factor in tumor genesis. For this reason, various types of antigen-presenting cells (APCs) have been widely used for cancer immunotherapy due to their ability to efficiently process and present antigens to T lymphocytes in cancer patients. APCs are often pulsed with antigenic peptides; however, this approach may result in the presentation of epitopes that are not naturally processed by tumor cells, affecting the effectiveness of this type of immunotherapy. In this context, the use of minigenes, which correspond to concatenated antigen sequences, emerges as a promising alternative for the selection of antigens naturally processed by APCs. It is expected that this strategy mimics the natural processing and presentation of antigens to T lymphocytes, increasing the probability of identifying immunogenic antigens, thus avoiding the selection of epitopes that are not naturally processed and presented as vaccine candidates for immunotherapy. To deliver these minigenes to APCs, various methods have been explored, with electroporation, administration via cationic liposomes, and transduction by viral vectors being the most common. Although the advantages and disadvantages of each method are recognized, few studies have compared the performance in T lymphocyte stimulation by APCs transfected with different systems. In this regard, the present work proposed the evaluation of the capacity of APCs transfected with a minigen construct encoding for HLA-A0201-restricted immunogenic antigens to stimulate antigen-specific CD8+ T lymphocyte (CD8+ LT) precursors in vitro. In addition to transfection with the minigen, artificial APCs (hereinafter referred to as aAPCs) were also designed, co-transfecting the HEK293 and K562 cell lines with plasmids encoding co-stimulatory molecules (CD80, CD83, CD137L), and in the case of K562 also with a plasmid encoding the HLA-A0201 molecule, in order to evaluate the efficiency of CD8+ LT activation by these aAPCs, in terms of intracellular cytokine production, cytotoxic activity, expression of activation and exhaustion markers, and the profile of stimulated CD8+ LT memory subpopulations. The results of this work allowed the implementation of a transfection methodology using lipofectamine and electroporation in HEK293 cells of a minigen encoding HLA-A*0201 epitopes. These cells were used as aAPCs to analyze the phenotype of antigen specific CD8+ LT populations. Considering that the use of HEK293 cells as aAPCs has not been explored, and due to their high transfection and transduction efficiency with minigenes, the methodology implemented in this work enables their use for the identification of naturally processed immunogenic neoantigens. We believe that our findings can contribute to the selection and design of personalized vaccines based on tumor neoantigens useful for cancer immunotherapy.MaestríaMagíster en InmunologíaMedicina Traslacional164 páginasapplication/pdfspaUniversidad Nacional de ColombiaBogotá - Medicina - Maestría en InmunologíaFacultad de MedicinaBogotá, ColombiaUniversidad Nacional de Colombia - Sede Bogotá610 - Medicina y salud::615 - Farmacología y terapéuticaAntígenos de NeoplasiasAntígenos Virales/análisisInmunoterapia/métodosAntigens, NeoplasmAntigens, Viral/analysisImmunotherapy/methodsMinigenesNeoantígenosCélulas presentadoras de antígeno artificialesTransfecciónTransducciónCitometría de FlujoInmunoterapiaMinigeneNeoantigensArtificial Antigen Presenting CellsTransfectionFlow CytometryTransductionImmunotherapyEvaluación de la inmunogenicidad de antígenos formulados en minigenes transfectados a células presentadoras de antígenoAssessment of the immunogenicity of antigens formulated in minigenes transfected into antigen-presenting cellsTrabajo de grado - Maestríainfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/acceptedVersionTexthttp://purl.org/redcol/resource_type/TMBiremeTan, S., D. 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Clin Cancer Res, 2007. 13(6): p. 1857-67.Universidad Nacional de ColombiaMincienciasEstudiantesInvestigadoresMaestrosLICENSElicense.txtlicense.txttext/plain; charset=utf-85879https://repositorio.unal.edu.co/bitstream/unal/86062/3/license.txteb34b1cf90b7e1103fc9dfd26be24b4aMD53ORIGINAL1032476383.2024.pdf1032476383.2024.pdfTesis de Maestría en Inmunologíaapplication/pdf8404843https://repositorio.unal.edu.co/bitstream/unal/86062/4/1032476383.2024.pdffa96bd2ecde0b472a2b2f3037202885cMD54unal/86062oai:repositorio.unal.edu.co:unal/860622024-05-09 13:44:24.161Repositorio Institucional Universidad Nacional de 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