Publication: Papel de las interacciones KIR/NKG2A con HLA en la expresión génica, la función antitumoral de linfocitos T y NK y la respuesta al tratamiento de pacientes con cáncer
Authors
Ruiz Lorente, Inmaculada
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Escuela Internacional de Doctorado
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Minguela Puras, Alfredo ; Gimeno Arias, Lourdes
Publisher
Universidad de Murcia
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DOI
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info:eu-repo/semantics/doctoralThesis
Description
Abstract
Introducción: El cáncer constituye uno de los principales desafíos de salud a nivel mundial, caracterizado por su elevada incidencia, mortalidad y complejidad biológica. Comprender sus bases moleculares y la interacción con el sistema inmunitario es clave para desarrollar nuevas estrategias terapéuticas.
Hipótesis: La presente tesis doctoral parte de la hipótesis de que la variabilidad genética individual en las interacciones de receptores inmunitarios es un factor determinante en la susceptibilidad y la evolución clínica de los pacientes con cáncer.
Objetivos: Los objetivos generales fueron, por un lado, investigar el papel de las interacciones de los receptores KIR con sus ligandos HLA clase I y la de NKG2A con HLA-E, en la susceptibilidad genética al cáncer y en la capacidad de predicción de la respuesta al tratamiento, en especial a la respuesta a la inmunoterapia con el Bacilo de Calmette-Guérin (BCG). Y por otro, descifrar los mecanismos moleculares que gobiernan la función de las células NK y T, mediante análisis funcionales y de expresión génica en célula única.
Metodología: Se combinaron análisis inmunofenotípicos, genéticos, funcionales y transcriptómicos en una cohorte de pacientes con cáncer: cáncer sólido como cáncer de vejiga (n=328), melanoma (n=311) y cáncer de ovario (n=89), y cáncer hematológico como neoplasias de células plasmáticas (n=335) y leucemia aguda infantil (n=128). Como control, se estudió una cohorte de donantes sanos (n=23.791). En los participantes se realizó la tipificación de los genes KIR y HLA clase I. El impacto pronóstico de estas variables se evaluó mediante análisis de progresión de la enfermedad y supervivencia de los pacientes con el método de Kaplan-Meier. Se realizaron estudios de expresión de receptores de células NK por citometría de flujo y ensayos funcionales in vitro en subgrupos seleccionados de donantes (n=24), donde se midió la respuesta de los linfocitos a la estimulación con BCG y anti-CD3/CD28, evaluando la capacidad proliferativa, citotóxica, y de producción de citoquinas y óxido nítrico intracelular. Para descifrar los programas moleculares subyacentes, se empleó la secuenciación de ARN de célula única en células NK y linfocitos T en donantes seleccionados por sus perfiles genéticos y de supervivencia (n=6).
Conclusiones: Los resultados de esta investigación establecen que el perfil inmunogenético del huésped es un factor crítico en el desarrollo y la evolución clínica del cáncer. Se identificó el gen KIR2DL5 como un factor de riesgo independiente para la susceptibilidad al cáncer de vejiga, cuyo mecanismo se asocia a la desregulación de la vía del óxido nítrico. En el ámbito terapéutico, se validaron biomarcadores predictivos para la inmunoterapia con BCG, demostrando que mientras la interacción inhibidora KIR3DL1/Bw4 predice el fracaso terapéutico al suprimir la respuesta de las células NK, el genotipo MM del dimorfismo HLA B 21M/T predice una respuesta favorable. La investigación culminó al demostrar que el repertorio de receptores KIR activadores es un importante factor pronóstico. La acumulación de estas interacciones es perjudicial, mientras que la interacción aislada KIR2DS1/C2 se asocia a una supervivencia prolongada. La base de este fenómeno no es cuantitativa, sino una reprogramación funcional de las células NK adaptativas (NK3). El perfil genético favorable induce en ellas un programa transcripcional de alta potencia efectora, mientras que el perfil desfavorable las conduce a un fenotipo de agotamiento celular, disfunción metabólica e inmunosupresión. Por tanto, los hallazgos de esta investigación abren una vía hacia una inmunoterapia personalizada, donde la firma inmunogenética del paciente se convierte en la herramienta fundamental para guiar la estrategia terapéutica.
Introduction: Cancer is one of the main health challenges worldwide, characterized by its high incidence, mortality, and biological complexity. Understanding its molecular basis and interaction with the immune system is key to developing new therapeutic strategies. Hypothesis: This doctoral thesis is based on the hypothesis that individual genetic variability in immune receptor interactions is a determining factor in the susceptibility and clinical progression of cancer patients. Objectives: The general objectives were, on the one hand, to investigate the role of KIR receptor interactions with their HLA class I ligands, as well as NKG2A with HLA-E, in genetic susceptibility to cancer and in predicting treatment response, particularly to Bacillus Calmette-Guérin (BCG) immunotherapy. On the other hand, to elucidate the molecular mechanisms that govern the function of NK and T cells through functional and single-cell gene expression analyses. Methodology: Immunophenotypic, genetic, functional, and transcriptomic analyses were conducted in a cohort of cancer patients: solid tumors such as bladder cancer (n=328), melanoma (n=311), and ovarian cancer (n=89), as well as hematological cancers such as plasma cell neoplasms (n=335) and acute childhood leukemia (n=128). A cohort of healthy donors (n = 23,791) was used as a control group. KIR and HLA class I genes were genotyped in all participants. The prognostic impact of these variables was assessed through Kaplan-Meier analyses of disease progression and overall survival. NK cell receptor expression studies were performed by flow cytometry, and in vitro functional assays were performed in selected donor subgroups (n=24) to measure lymphocyte responses to stimulation with BCG and anti-CD3/CD28, evaluating proliferative capacity, cytotoxicity, and intracellular cytokine and nitric oxide production. To elucidate the underlying molecular programs, single-cell RNA sequencing was performed on NK cells and T lymphocytes from donors selected according to their genetic background and survival profiles (n=6). Conclusions: The results of this research establish that the host immunogenetic profile is a critical factor in the development and clinical progression of cancer. The KIR2DL5 gene was identified as an independent risk factor for susceptibility to bladder cancer, whose mechanism is associated with the dysregulation of the nitric oxide pathway. In the therapeutic field, predictive biomarkers for BCG immunotherapy were validated, demonstrating that while the inhibitory KIR3DL1/Bw4 interaction predicts therapeutic failure by suppressing the NK cell response, the MM genotype of HLA B 21M/T dimorphism predicts a favorable response. The research culminated in demonstrating that the repertoire of activating KIR receptors is an important prognostic factor. The accumulation of these interactions is detrimental, while the isolated KIR2DS1/C2 interaction is associated with prolonged survival. The basis of this phenomenon is not quantitative, but reflects a functional reprogramming of adaptive NK cells (NK3). Favorable genetic profiles drive a high-potency effector transcriptional program, whereas unfavorable profiles result in a phenotype of exhaustion, metabolic dysfunction, and immunosuppression. Therefore, the findings of this research open the way to personalized immunotherapy, where the patient's immunogenetic signature becomes the fundamental tool for guiding the therapeutic strategy.
Introduction: Cancer is one of the main health challenges worldwide, characterized by its high incidence, mortality, and biological complexity. Understanding its molecular basis and interaction with the immune system is key to developing new therapeutic strategies. Hypothesis: This doctoral thesis is based on the hypothesis that individual genetic variability in immune receptor interactions is a determining factor in the susceptibility and clinical progression of cancer patients. Objectives: The general objectives were, on the one hand, to investigate the role of KIR receptor interactions with their HLA class I ligands, as well as NKG2A with HLA-E, in genetic susceptibility to cancer and in predicting treatment response, particularly to Bacillus Calmette-Guérin (BCG) immunotherapy. On the other hand, to elucidate the molecular mechanisms that govern the function of NK and T cells through functional and single-cell gene expression analyses. Methodology: Immunophenotypic, genetic, functional, and transcriptomic analyses were conducted in a cohort of cancer patients: solid tumors such as bladder cancer (n=328), melanoma (n=311), and ovarian cancer (n=89), as well as hematological cancers such as plasma cell neoplasms (n=335) and acute childhood leukemia (n=128). A cohort of healthy donors (n = 23,791) was used as a control group. KIR and HLA class I genes were genotyped in all participants. The prognostic impact of these variables was assessed through Kaplan-Meier analyses of disease progression and overall survival. NK cell receptor expression studies were performed by flow cytometry, and in vitro functional assays were performed in selected donor subgroups (n=24) to measure lymphocyte responses to stimulation with BCG and anti-CD3/CD28, evaluating proliferative capacity, cytotoxicity, and intracellular cytokine and nitric oxide production. To elucidate the underlying molecular programs, single-cell RNA sequencing was performed on NK cells and T lymphocytes from donors selected according to their genetic background and survival profiles (n=6). Conclusions: The results of this research establish that the host immunogenetic profile is a critical factor in the development and clinical progression of cancer. The KIR2DL5 gene was identified as an independent risk factor for susceptibility to bladder cancer, whose mechanism is associated with the dysregulation of the nitric oxide pathway. In the therapeutic field, predictive biomarkers for BCG immunotherapy were validated, demonstrating that while the inhibitory KIR3DL1/Bw4 interaction predicts therapeutic failure by suppressing the NK cell response, the MM genotype of HLA B 21M/T dimorphism predicts a favorable response. The research culminated in demonstrating that the repertoire of activating KIR receptors is an important prognostic factor. The accumulation of these interactions is detrimental, while the isolated KIR2DS1/C2 interaction is associated with prolonged survival. The basis of this phenomenon is not quantitative, but reflects a functional reprogramming of adaptive NK cells (NK3). Favorable genetic profiles drive a high-potency effector transcriptional program, whereas unfavorable profiles result in a phenotype of exhaustion, metabolic dysfunction, and immunosuppression. Therefore, the findings of this research open the way to personalized immunotherapy, where the patient's immunogenetic signature becomes the fundamental tool for guiding the therapeutic strategy.
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