The research on the mechanism of CD40 RNAi mouse dendritic cells inhibits T lymphocyte proliferation / 中华微生物学和免疫学杂志

ABSTRACT

Objective To construct the mouse CD40 RNA interfering(RNAi) lentiviral vector and prepare dendritic cells (DCs) with low expression of CD40. And to explore the mechanism of inducing T lymphocyte incompetence by blocking CD40/CD40L costimulatory pathway. Methods Mouse myeloid DCs were cultured in selective medium containing necessary cytokines for DC growth in vitro. CD40 RNAi gene was transfected into DCs with lentiviral vector. The expression levels of CD40 mRNA and protein were assayed by real-time quantitative PCR and flow cytometry respectively. The influences on DCs stimulating the proliferation ability of T lymphocyte were observed through mixed lymphocyte culture(MLC). Results Myeloid DCs have been harvested from mouse through cell culture in vitro. A mouse CD40 RNAi ientiviral vector was built successfully. The lentiviral titer was 8×10~9 TU/ml. The CD40 mRNA inhibition rate after infection was significantly higher(P<0.05). The CD40 protein expression of DCs was significandy lower(P<0.05). The result of MLC demonstrated that the index of stimulation to T lymphocyte of CD40 RNAi transfected DC significantly decreased compared with non-transfected DC and empty plasmid transfected DC(P< 0.01). Conclusion Large quantity of myeloid DCs with typical histological configuration are obtained through in vitro culture in selective medium which may activate naive T lymphocyte to generate immune response. While DCs were infected by CD40 RNAi lentiviral vector, CD40 protein expression was inhibited significantly. The DCs could hamper the activation of allogeneic T lymphocyte by blocking CD40/CD40L cestimulatory pathway and induce T cell allergy. This will make the good foundation for studying the immune tolerance of CD40 RNAi.