Signal Regulatory Protein A Sets A Threshold For Dendritic Cell Activation And The Preparation And Application Of Its Monoclonal Antibody

Part I Signal Regulatory Protein a Sets a Threshold for Dendritic Cell ActivationPurpose:Signal regulatory protein (SIRP)a, also known as SHPS-1 or SIRPa, is a transmembrane protein that binds to the protein tyrosine phosphatases SHP-1 and SHP-2 through its cytoplasmic region. Due to its high expression in dendritic cells and macrophages, SIRPa was often thought to have an important role in the immune system. In innate immune responses, SIRPa had been proved to play a critically negative role in macrophage activation. However, whether or how SIRPa regulate the adaptive immune response is not very clear. Here we firstly purposed to delineate potential regulations of SIRPa in DCs, and then discussed its potential functions in adaptive immunity against tumor.Experimental Design:To examine SIRPa regulation of the adaptive immune responses, lentiviral-mediated, microRNA-based RNA interference of SIRPa in DC was designed. After infected by lentivirus, in vitro and in vivo growth and survival of DCs were evaluated firstly. Secondly, maturation and cytokine production of DCs regulated by SIRPa were detected. Thirdly, T cell responses and anti-tumor immune effects were evaluated. Finally, the molecular mechanisms.of SIRPa in regulation of DC function were discussed.Results:SIRPa was found to suppress the survival, phenotypic and functional maturation of immature dendritic cells (DCs) and to inhibit cytokine production by mature DCs. Moreover, SIRPa silenced-DCs showed potent antitumor effects by activation of antigen-specific cytotoxic T lymphocytes.Conclusions:This study indicates that SIRPa is a critical regulator of DC function, and inhibiting SIRPa is necessary to evoke potent adaptive immunity and induce effective antitumor responses in an antigen-specific manner. Partâ…¡Preparation and Application of a Novel Monoclonal Antibody against Human SIRPaPurpose:To generate monoclonal antibody against human SIRPa by using synthetic peptide.Experimental Design:The synthetic peptide of SIRPa linked with KLH was used as antigen for immunization. The mAb anti-SIRPa was obtained by hybridoma technique. The flow cytometry and Western Blot assay were carrying out by the mAb. TNF-a secreted by PMA-treated THP-1 with antibodies was also tested.Results:Hybridoma cell clone was developed which could secret mAb. The results showed that the mAb can be used in flow cytometry and Western Blot assay. And the mAb can stimulate the PMA-treated THP-1 to secret TNF-a and IL-6.Conclusions:The mAb against SIRPa is prepared successfully.