RESUMO
Little is known about the consequences of immune recognition of mutated gene products, despite their potential relevance to autoimmunity and tumor immunity. To identify mutations that induce immunity, here we have developed a systematic approach in which combinatorial DNA libraries encoding large numbers of random mutations in two syngeneic tyrosinase-related proteins are used to immunize black mice. We show that the libraries of mutated DNA induce autoimmune hypopigmentation and tumor immunity through cross-recognition of nonmutated gene products. Truncations are present in all immunogenic clones and are sufficient to elicit immunity to self, triggering recognition of normally silent epitopes. Immunity is further enhanced by specific amino acid substitutions that promote T helper cell responses. Thus, presentation of a vast repertoire of antigen variants to the immune system can enhance the generation of adaptive immune responses to self.
Assuntos
Autoantígenos/genética , Autoimunidade/genética , Mutação , Neoplasias Experimentais/genética , Neoplasias Experimentais/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/genética , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células COS , Chlorocebus aethiops , Reações Cruzadas , DNA Complementar/genética , Biblioteca Gênica , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Tolerância a Antígenos Próprios/genética , TransfecçãoRESUMO
Among the many promising cancer immunotherapeutic strategies, dendritic cells (DC) have become of particular interest. This study aims to optimize a clinical grade protocol for culture and transfection of human DC. Monocytes and CD34(+) hematopoietic stem cells (HSC) from same donor were differentiated under serum-free conditions and analyzed for their susceptibility to several recently described nonviral transfection methods as compared with established virally mediated gene transfer. Nonviral gene transfer methods studied were square-wave electroporation, lipofection, and particle-mediated transfer of plasmid DNA or in vitro transcribed mRNA. We conclude that DNA is not suitable for transduction of DC using nonviral methods. In contrast, mRNA and square-wave electroporation reproducibly yields 60% and 50% transfected monocyte- and CD34(+)-derived DC, respectively, measured at protein level, without affecting the cell viability. Thus, the transfection efficiency of this method is comparable with the 40-90% transgene expression obtained using retroviral (RV) or adenoviral (AdV) vectors in CD34(+)- and monocyte-derived DC, respectively. In monocyte-derived DC, however, the amount of protein expressed per-cell basis was higher after AdV (MOI = 1000) compared with mRNA electroporation-mediated transfer. This is the first study directly demonstrating side-by-side that mRNA electroporation into DC of different origin indeed results in a comparable number of transduced cells as when using virus-mediated gene transfer.
Assuntos
Adenoviridae/genética , Células Dendríticas/metabolismo , Transdução Genética , Transfecção , Biolística , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas/metabolismo , Células Cultivadas/virologia , DNA Recombinante/administração & dosagem , DNA Recombinante/genética , Vírus Defeituosos/genética , Células Dendríticas/classificação , Células Dendríticas/virologia , Eletroporação , Genes Reporter , Proteínas de Fluorescência Verde , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Lipossomos , Proteínas Luminescentes/genética , Teste de Cultura Mista de Linfócitos , Monócitos/citologia , Monócitos/efeitos dos fármacos , Plasmídeos/genética , RNA Mensageiro/genética , Reprodutibilidade dos TestesRESUMO
Cancer poses a difficult problem for immunotherapy because it arises from the host's own tissues. Many of the target antigens are tissue-specific molecules shared by cancer cells and normal cells. Thus, these are weak antigens that do not typically elicit immunity. In addition, tumors have several features that make their recognition and destruction by the immune system difficult. Despite these obstacles, several strategies for developing effective tumor immunity have been developed. Crucial to these approaches is the discovery and understanding of the molecular identity of antigens and the mechanisms involved in tumor immunity. In this review, strategies to overcome immune ignorance and tolerance are discussed.
