Ação das proteínas LMP1 e RPMS1 do vírus de Epstein- Barr (EBV) na regulação de genes codificadores de proteínas de checkpoint imunológico em células humanas cultivadas in vitro
Carregando...
Arquivos
Data
2021-10-25
Autores
Biggi, Alison Felipe Bordini
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
A maior parcela da população mundial adulta apresenta infecção latente pelo vírus de Epstein-Barr (EBV), reconhecida como potencialmente cancerígena para humanos. Esse vírus é encontrado nas células neoplásicas de virtualmente todos os casos da forma endêmica africana do linfoma de Burkitt e do carcinoma indiferenciado de nasofaringe, além de estar associado ao desenvolvimento de parcela de outros cânceres, especialmente linfomas. A fase latente do ciclo biológico do EBV é caracterizada por restrição na expressão de alguns produtos virais, o que favorece sua imunoevasão e infecção persistente no hospedeiro. Por outro lado, a expressão de alguns genes do EBV durante o ciclo latente viral pode efetuar regulação de checkpoints imunológicos, indicando que o EBV também pode beneficiar a carcinogênese por esse mecanismo. Embora há alguns indícios de ação da oncoproteína viral LMP1 na expressão de PD-L1 e CTLA-4, são escassos os dados sobre outros produtos virais do EBV na regulação de checkpoints imunológicos, incluindo a proteína viral RPMS1. Assim, este estudo avaliou os efeitos da expressão dos genes LMP1 e RPMS1 do EBV na regulação de genes codificadores de proteínas de checkpoints imunológicos empregando linhagens de células neoplásicas derivadas de linfomas EBV-positivos. Para tanto, foram desenhados iniciadores para análise da expressão em nível transcricional de genes codificadores para as seguintes moléculas reguladoras de checkpoints imunológicos: B7-H3, B7-H4, BTLA, CTLA-4, LAG-3, PD-L1, PD-L2, TIM-3 e VISTA. Por problemas na repressão dos produtos virais, vetores com expressão constitucional de LMP1 e RPMS1 foram transfectados transientemente em linhagem Ramos para avaliação dos possíveis efeitos em relação à expressão das moléculas de checkpoint imunológico investigadas. Foi observado que, a nível transcricional, as células que expressavam LMP1 apresentaram diminuição de BTLA e aumento de PD-L1 e TIM-3; enquanto que células que expressavam RPMS1 apresentaram diminuição de PD-L1 e aumento de BTLA e TIM-3. Esses resultados apontam interação de LMP1 e RPMS1, não previamente descrita, com moléculas associadas com checkpoints imunológicos, contribuindo para um melhor entendimento sobre a atuação do vírus na etiopatogenia e progressão de cânceres, além de subsidiar possível desenvolvimento de terapias a base de moduladores de checkpoints imunológicos para portadores de cânceres associados ao EBV.
Most of the world's adult population has latent Epstein-Barr virus (EBV) infection, recognized as potentially carcinogenic to humans. This virus is found in neoplastic cells of virtually all cases of the African endemic form of Burkitt's lymphoma and of undifferentiated nasopharyngeal carcinoma, in addition to being associated with the development of a portion of other cancers, especially lymphomas. The latent phase of EBV biological cycle is characterized by restriction in the expression of some viral products, which favors its immunoevasion and persistent infection in the host. On the other hand, the expression of some EBV genes during the latent viral cycle can regulate immune checkpoints, indicating that EBV can also benefit carcinogenesis through this mechanism. Although there is some evidence for the role of the viral oncoprotein LMP1 on the expression of PD-L1 and CTLA-4, data on other EBV viral products in the regulation of immune checkpoints, including the viral protein RPMS1 are scarce. Thus, this study evaluated the effects of the expression of EBV LMP1 and RPMS1 genes in the regulation of genes encoding immune checkpoint using EBV-positive lymphoma-derived neoplastic cell lines. Therefore, primers were designed to analyze the expression at transcriptional level of genes encoding the following immune checkpoint molecules: B7-H3, B7-H4, BTLA, CTLA-4, LAG-3, PD-L1, PD-L2, TIM-3 and VISTA. Due to problems in the knock-down of viral products, vectors with constitutional expression of LMP1 and RPMS1 were transiently transfected into Ramos cell line to evaluate their possible effects in the expression of the investigated immune checkpoint molecules. It was observed that, at transcriptional level, Ramos cells that expressed LMP1 showed a decrease in BTLA and an increase in PD-L1 and TIM-3; while cells that expressed RPMS1 showed a decrease in PD-L1 and an increase in BTLA and TIM-3. These results point to an interaction, not previously described, between LMP1 and RPMS1 with molecules associated with immune checkpoints, contributing to a better understanding of the role of the virus in the etiopathogenesis and progression of cancers, in addition to supporting the possible development of therapies based on checkpoint inhibitors for patients with EBV-associated cancers.
Most of the world's adult population has latent Epstein-Barr virus (EBV) infection, recognized as potentially carcinogenic to humans. This virus is found in neoplastic cells of virtually all cases of the African endemic form of Burkitt's lymphoma and of undifferentiated nasopharyngeal carcinoma, in addition to being associated with the development of a portion of other cancers, especially lymphomas. The latent phase of EBV biological cycle is characterized by restriction in the expression of some viral products, which favors its immunoevasion and persistent infection in the host. On the other hand, the expression of some EBV genes during the latent viral cycle can regulate immune checkpoints, indicating that EBV can also benefit carcinogenesis through this mechanism. Although there is some evidence for the role of the viral oncoprotein LMP1 on the expression of PD-L1 and CTLA-4, data on other EBV viral products in the regulation of immune checkpoints, including the viral protein RPMS1 are scarce. Thus, this study evaluated the effects of the expression of EBV LMP1 and RPMS1 genes in the regulation of genes encoding immune checkpoint using EBV-positive lymphoma-derived neoplastic cell lines. Therefore, primers were designed to analyze the expression at transcriptional level of genes encoding the following immune checkpoint molecules: B7-H3, B7-H4, BTLA, CTLA-4, LAG-3, PD-L1, PD-L2, TIM-3 and VISTA. Due to problems in the knock-down of viral products, vectors with constitutional expression of LMP1 and RPMS1 were transiently transfected into Ramos cell line to evaluate their possible effects in the expression of the investigated immune checkpoint molecules. It was observed that, at transcriptional level, Ramos cells that expressed LMP1 showed a decrease in BTLA and an increase in PD-L1 and TIM-3; while cells that expressed RPMS1 showed a decrease in PD-L1 and an increase in BTLA and TIM-3. These results point to an interaction, not previously described, between LMP1 and RPMS1 with molecules associated with immune checkpoints, contributing to a better understanding of the role of the virus in the etiopathogenesis and progression of cancers, in addition to supporting the possible development of therapies based on checkpoint inhibitors for patients with EBV-associated cancers.
Descrição
Palavras-chave
Carcinogenese viral, Checkpoints imunologicos, Ensaios in vitro, Vírus de Epstein-Barr, Progressão tumoral