Adoptive transfer of IL-4 reprogrammed Tc17 cells elicits anti-tumour immunity through functional plasticity.

Ability of IL-17-producing CD8+ T cells (Tc17) to transform into cytotoxic anti-tumour effectors makes them a promising candidate for immune effector cell (IEC) therapy. However, key factors regulating Tc17 reprogramming remain poorly defined, hindering translation of Tc17-based IEC use from bench to bedside. We probed the effects of multiple cytokines and underlying signalling pathways on Tc17 cells and identified pivotal role for IL-4 and PI3K/AKT in promoting Tc17 transformation into cytotoxic IFN-γ-producing IECs, an effect dependent on Eomes expression. IL-4 not only triggered Tc17 cytotoxicity, but also induced cell expansion, which significantly improved the antitumour potential of Tc17 cells compared to that of IFN-γ-producing CD8+ T cells (Tc1) in a murine model. Furthermore, IL-4/AKT signalling drove the upregulation of the T-cell receptor-associated transmembrane adaptor 1 (Trat1) in Tc17 cells to promote IL-4-induced T-cell receptor stabilization and Tc17 cytotoxicity. Finally, we proposed a possible procedure to expand human Tc17 from peripheral blood of cancer patients, and confirmed the function of IL-4 in Tc17 reprogramming. Collectively, these results document a novel IL-4/AKT/Eomes/Trat1 axis that promotes expansion and transformation of Tc17 cells into cytotoxic effectors with a therapeutic potential. IL-4 priming of Tc17 cells should be further explored as a cell therapy engineering strategy to generate IECs to augment anti-tumour responses.

SUMOylation attenuates c-Maf-dependent IL-4 expression.

The function of transcription factors can be critically regulated by SUMOylation. c-Maf, the cellular counterpart of v-maf oncogene, is a potent transactivator of the IL-4 gene in Th2 cells. We found in a yeast two-hybrid screen that c-Maf can interact with Ubc9 and PIAS1, two key enzymes of the SUMOylation pathway. In this study, we report that c-Maf co-localized with these two SUMO (small ubiquitin-like modifier) ligases in the nucleus and that c-Maf can be SUMOylated in vitro and also in primary Th2 cells. We also demonstrated that lysine-33 is the dominant, if not the only, SUMO acceptor site of c-Maf. SUMOylation of c-Maf attenuated its transcriptional activity. Reciprocally, a SUMOylation resistant c-Maf was more potent than WT-c-Maf in driving IL-4 production in c-Maf-deficient Th2 cells. Furthermore, we showed that ablation of the SUMO site did not alter the subcellular localization or the stability of c-Maf protein but instead enhanced its recruitment to the Il4-promoter. We conclude that SUMOylation at lysine-33 is a functionally critical post-translational modification event of c-Maf in Th cells.

Effective gene expression in the rat dorsal root ganglia with a non-viral vector delivered via spinal nerve injection.

Delivering gene constructs into the dorsal root ganglia (DRG) is a powerful but challenging therapeutic strategy for sensory disorders affecting the DRG and their peripheral processes. The current delivery methods of direct intra-DRG injection and intrathecal injection have several disadvantages, including potential injury to DRG neurons and low transfection efficiency, respectively. This study aimed to develop a spinal nerve injection strategy to deliver polyethylenimine mixed with plasmid (PEI/DNA polyplexes) containing green fluorescent protein (GFP). Using this spinal nerve injection approach, PEI/DNA polyplexes were delivered to DRG neurons without nerve injury. Within one week of the delivery, GFP expression was detected in 82.8% ± 1.70% of DRG neurons, comparable to the levels obtained by intra-DRG injection (81.3% ± 5.1%, p = 0.82) but much higher than those obtained by intrathecal injection. The degree of GFP expression by neurofilament(+) and peripherin(+) DRG neurons was similar. The safety of this approach was documented by the absence of injury marker expression, including activation transcription factor 3 and ionized calcium binding adaptor molecule 1 for neurons and glia, respectively, as well as the absence of behavioral changes. These results demonstrated the efficacy and safety of delivering PEI/DNA polyplexes to DRG neurons via spinal nerve injection.

Dose-response relationship of specific allergen exposure-induced immunological tolerance: a mouse model.

BACKGROUND: It is believed that adequate allergen preimmunization exposure could induce immunologic tolerance. The purpose of this study was to investigate the dose-dependent mechanisms related to antigen-specific tolerance induction in a mouse model. METHODS: Mice were assigned to 5 groups: the control (Cont) group received phosphate-buffered saline (PBS) preimmunization exposure and PBS sham immunization; the other 4 groups were exposed preimmunization to PBS (PBS group) or ovalbumin (OVA) (first mucosal doses: 1.25%, 2.5%, or 5% wt/vol aerosol from days -3 to -1) prior to OVA immunization. The OVA-immunized mice received intraperitoneal doses of 20 µg OVA (on days 1, 7, and 14), and then a second set of mucosal doses with 0.5% wt/vol OVA aerosol (on days 18 to 20). After assessment of airway hyperresponsiveness (AHR), the mice were euthanized and their blood, bronchoalveolar lavage fluids (BALFs), and lung tissues were collected for further analyses. RESULTS: OVA-immunized mice exposed to OVA preimmunization had reduced AHR and immunoglobulin E production when compared to the PBS group. OVA preimmunization exposure inhibited eosinophilic inflammation in lung tissues. The proportions of BALF eosinophil counts from the groups exposed to OVA preimmunization were significantly decreased when compared with those exposed to PBS preimmunization. The balance of T helper 2 (Th2) and T regulatory (Treg) cytokines in BALFs were additionally observed in this mouse model. CONCLUSION: Our results suggest that preimmunization exposure to an appropriate dose of a specific antigen could suppress allergic airway inflammation by induction of immunological tolerance.

N-Acetylcysteine, vitamin C and vitamin E diminish homocysteine thiolactone-induced apoptosis in human promyeloid HL-60 cells.

We showed previously that homocysteine thiolactone (HcyT) is a potent inducer of apoptosis in HL-60 cells. In the present study, the role of some radical scavengers (N-acetylcysteine, vitamin C, vitamin E and folate) on the reduction of HcyT-induced apoptosis was investigated. Preincubation of HcyT-treated HL-60 cells with vitamin C (Vit C; 100 micro mol/L) or vitamin E (Vit E; 100 micro mol/L) for 2 h significantly reduced the proportion of apoptotic cells with hypodiploid DNA contents or with membrane phosphatidylserine exposure, and attenuated the apoptotic DNA fragmentation. Preincubation of cells with N-acetylcysteine (NAC; 5 mmol/L) for 2 h significantly reduced HcyT-promoted apoptosis measured by membrane phosphatidylserine exposure only. The reduction of HcyT-induced apoptosis by NAC, Vit C or Vit E occurred simultaneously with a significant decrease in intracellular H(2)O(2) levels and reduced caspase-3 enzymatic activity. In contrast, folate had no H(2)O(2) scavenging capacity and did not suppress caspase-3 activity 6 h after HcyT treatment, although folate exhibited antioxidant behavior toward superoxide anions, hydroxyl radicals and peroxynitrite. Preincubation of cells with folate (10 micro mol/L) for 3 d did not affect the extent of HcyT-promoted apoptotic damage. Taken together, our findings suggest that antioxidant pretreatment with NAC, Vit C or Vit E exerts more beneficial effects than folate on reducing apoptotic cell damage induced by homocysteine thiolactone.