The anti-tumor effect and increased tregs infiltration mediated by rAAV-SLC vector.
Bottom Line: To explore the anti-tumor effect and immune mechanism mediated by a new recombinant adeno-associated virus (rAAV) encoding secondary lymphoid tissue chemokine (SLC) mature peptide gene.More importantly, there was higher infiltration of Foxp3+ regulatory T cells (Tregs).Local elaboration of SLC mediated by rAAV-SLC has strong T cell mediated anti-tumor effect.
To explore the anti-tumor effect and immune mechanism mediated by a new recombinant adeno-associated virus (rAAV) encoding secondary lymphoid tissue chemokine (SLC) mature peptide gene. AAV Helper-Free system was used for rAAV-SLC package. The anti-tumor effect of SLC was detected by bearing tumor established from Hepal-6 cells both in C57BL/6J and nude mice. Flow cytometry analysis and IHC for Tumor-infiltrating T cells and CD11c+DCs were also investigated to explore the immunological mechanism. rAAV-SLC was successfully packaged in AAV293 cells and transfected Hepal-6 tumor cells at high efficiency. The anti-tumor effect was demonstrated by less tumor weight and longer survival outcome. Coincident with the anti-tumor response, local elaboration of SLC within the tumor bed elicited a heavy infiltration of CD4+, CD8+T cells and CD11c+ dendritic cells into the tumor sites. More importantly, there was higher infiltration of Foxp3+ regulatory T cells (Tregs). Local elaboration of SLC mediated by rAAV-SLC has strong T cell mediated anti-tumor effect. The study also suggested that Tregs in the tumor microenvironment tampered the anti-tumor effect.
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Mentions: AAV Help-Free System was used for the package of rAAV-SLC. Firstly, the entire nucleotide sequence of SLC was cloned into the MCS of the pAAV-IRES-hrGFP vector. Positive clones was demonstrated by restriction digestion and PCR, as shown in Fig. 1a–c. As followed, a new rAAV 2 encoding SLC peptide (rAAV-SLC) was successfully packaged in AAV293 cells by three-plasmids co-transfection system, as shown in Fig. 1d, 72 h post-transfection, the virus particles was harvested. Virus titer was 1.25 × 1011 analysed by Real-time PCR, which was used to infect Hepal-6 cells later, with GFP as a marker of gene expression. Hepal-6 cells are highly susceptible to rAAV-mediated transduction. 2 h post-transfection the viral particles were observed to shift toward the nucleus and begin to accumulate at the nuclear envelope (Fig. 1e). 72 h post-transfection, based on the expression the marker gene GFP, it was found that about 50 % Hepal-6 tumor cells were positive in showing the transduction efficiency (Fig. 1f). There was no difference in transduction efficiency between the rAAV-SLC and rAAV-GFP. The control groups of single rAAV transduction have black background. The expression of SLC and its bioactivities were demonstrated in intro by ELISA and chemotaxis assay (Data not shown).Fig. 1