Enhanced activation of human dendritic cells by silencing SOCS1 and activating TLRs simultaneously

Abstract

There was established evidence that silencing the attenuator and activating the TLRs could activate the dendritic cells in synergic effects. In this study, we constructed a plasmid, namely pshS1NH, which encodes SOCS1-shRNA, NY-ESO-1-MAGE3 (HLA-A2*0201) fusion antigen and secretory HMGB1, an agent used to modify dendritic cells (DCs), aiming to generate potent DC vaccine against tumors. The SOCS1-shRNA could efficiently downregulate the expression of SOCS1, as indicated by real-time RT-PCR and Western blot. The fusion antigen was detected in the pshS1NH-DCs by PCR and Western blot. Simultaneously, HMGB1 level in the pshS1NH-DCs culture media was significantly higher than that in the control DCs culture media. Levels of Th1 cytokines in pshS1NH-DCs culture media, such as IL-1β, IL-6, TNF-α and IL-12p70, were dramatically higher than those in control DCs culture media. In addition, lymphocytes co-cultured with pshS1NH-DCs secreted dramatically higher level of IFN-γ, whereas no difference was detected in IL-4 levels. Taken together, these data suggest that pshS1NH-DCs may be a potential adjuvant immunotherapy for cancers in clinical applications.

Fig. 1

Schematic representation of pshS1NH

Fig. 2

Verification of the structure of pshS1NH. a Identification by restriction enzyme digestion. Lane 1 DL15000 marker; lane 2 pshS1NH without double enzyme digestion (6,191 bp); lane 3 pshS1NH with double enzyme digestion by BglII and EcoRI (576 bp and 5,614 bp); lane 4 DL2000 marker. b Amplification of NY-ESO-1 and HMGB1 from pshS1NH. Lane 1 DL2000 marker; lane 2 NY-ESO-1 (113 bp); lane 3 HMGB1 (150 bp). All of the fragments exhibited expected sizes

Fig. 3

The expression of transgenes in DCs. a Western blot to verify the downregulation of SOCS1 in pshS1-DCs. SOCS1 could not be detected in pshS1NH–DCs. b Western blot confirmation of NY-ESO-1 in pshS1NH–DCs. c PCR confirmation of the presence of NY-ESO-1 in pshS1NH–DCs. 1 DL2000 marker; 2, 5 non-nucleofected-DCs; 3, 6 pNC-GFP-DCs; 4, 7 pshS1NH–DCs. It was obviously that NY-ESO-1 was only expressed in pshS1NH–DCs. d Secretion of HMGB1 in the supernatants. All transgenes are expressed as expected and exhibited their specific functions in DCs

Fig. 4

Phenotypic analysis of DCs. At 24 h after nucleofection, DCs were harvested and stained with mAbs against DCs surface markers or appropriate isotype control antibody as described in “Materials and methods”. Percentage of positive DCs is indicated. One representative experiment of five is shown

Fig. 5

Th1 cytokines secreted by pshS1NH–DCs were higher than those secreted by non-nucleofected DCs and pNC-GFP-DCs. 1 non-nucleofected DCs; 2 pNC-GFP-DCs; 3 pshS1NH–DCs. At 48 h after nucleofection, supernatants were collected for TNF-α, IL-6, IL-1β and IL-12p70 ELISA analyses. The pshS1NH–DCs secreted significantly higher level of Th1 cytokines than the pNC-GFP-DCs and non-nucleofected DCs (P < 0.05). Data shown are the means ± SEM and representative of six independent experiments. The significant differences between pshS1NH–DCs and control were determined by ANOVA

Fig. 6

The Th1/Th2 cytokine balance in media of syngeneic naive T cells co-cultured with modified DCs. 1 T cells alone; 2 T cells co-cultured with pNC-GFP-DC; 3 T cells co-cultured with pshS1NH–DCs. The IFN-γ secreted by T cells co-cultured with pshS1NH–DCs was significantly higher than that in control groups (P < 0.05). However, there was no significant difference in IL-4 levels between the two groups (P > 0.05). Data shown are the means ± SEM and representative of six independent experiments. The significant differences between T cells co-cultured with pshS1NH–DCs and T cells alone and T cells co-cultured with pNC-GFP-DC were determined by ANOVA