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The anti-tumor effect and increased tregs infiltration mediated by rAAV-SLC vector.

Li R, Hu H, Ma H, Chen L, Zhou B, Liu Y, Liang C - Mol. Biol. Rep. (2013)

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.

View Article: PubMed Central - PubMed

ABSTRACT
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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rAAV-SLC was constructed and infected Hepal-6 cells at high efficiency. a The entire nucleotide sequence of SLC was cloned into the MCS of the pAAV-IRES-hrGFP vector; Positive clones were demonstrated by restriction digestion (b) and by PCR (c); (d) rAAV-SLC was packaged in AAV293 cells by using the three-plasmid co-transfection system. GFP expression was served as a useful expression marker; e distribution of Cy3-rAAV-SLC particles (red) in Hepal-6 cells 2 h post-transduction were observed to shift toward the nucleus and begin to accumulate at the nuclear envelope, detected by laser scanning confocal microscope. The position of the cell nucleus was assessed by DAPI (blue) staining. A representative image is shown, consisting of a single plane of focus through the center of a cell; f 72 h post-transduction, SLC expression in Hepal-6 cells were analyzed by fluorescence microscopy for GFP detection
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Fig1: rAAV-SLC was constructed and infected Hepal-6 cells at high efficiency. a The entire nucleotide sequence of SLC was cloned into the MCS of the pAAV-IRES-hrGFP vector; Positive clones were demonstrated by restriction digestion (b) and by PCR (c); (d) rAAV-SLC was packaged in AAV293 cells by using the three-plasmid co-transfection system. GFP expression was served as a useful expression marker; e distribution of Cy3-rAAV-SLC particles (red) in Hepal-6 cells 2 h post-transduction were observed to shift toward the nucleus and begin to accumulate at the nuclear envelope, detected by laser scanning confocal microscope. The position of the cell nucleus was assessed by DAPI (blue) staining. A representative image is shown, consisting of a single plane of focus through the center of a cell; f 72 h post-transduction, SLC expression in Hepal-6 cells were analyzed by fluorescence microscopy for GFP detection

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


The anti-tumor effect and increased tregs infiltration mediated by rAAV-SLC vector.

Li R, Hu H, Ma H, Chen L, Zhou B, Liu Y, Liang C - Mol. Biol. Rep. (2013)

rAAV-SLC was constructed and infected Hepal-6 cells at high efficiency. a The entire nucleotide sequence of SLC was cloned into the MCS of the pAAV-IRES-hrGFP vector; Positive clones were demonstrated by restriction digestion (b) and by PCR (c); (d) rAAV-SLC was packaged in AAV293 cells by using the three-plasmid co-transfection system. GFP expression was served as a useful expression marker; e distribution of Cy3-rAAV-SLC particles (red) in Hepal-6 cells 2 h post-transduction were observed to shift toward the nucleus and begin to accumulate at the nuclear envelope, detected by laser scanning confocal microscope. The position of the cell nucleus was assessed by DAPI (blue) staining. A representative image is shown, consisting of a single plane of focus through the center of a cell; f 72 h post-transduction, SLC expression in Hepal-6 cells were analyzed by fluorescence microscopy for GFP detection
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3824217&req=5

Fig1: rAAV-SLC was constructed and infected Hepal-6 cells at high efficiency. a The entire nucleotide sequence of SLC was cloned into the MCS of the pAAV-IRES-hrGFP vector; Positive clones were demonstrated by restriction digestion (b) and by PCR (c); (d) rAAV-SLC was packaged in AAV293 cells by using the three-plasmid co-transfection system. GFP expression was served as a useful expression marker; e distribution of Cy3-rAAV-SLC particles (red) in Hepal-6 cells 2 h post-transduction were observed to shift toward the nucleus and begin to accumulate at the nuclear envelope, detected by laser scanning confocal microscope. The position of the cell nucleus was assessed by DAPI (blue) staining. A representative image is shown, consisting of a single plane of focus through the center of a cell; f 72 h post-transduction, SLC expression in Hepal-6 cells were analyzed by fluorescence microscopy for GFP detection
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

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.

View Article: PubMed Central - PubMed

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus