Limits...
Role of TLR3 in the immunogenicity of replicon plasmid-based vaccines.

Diebold SS, Schulz O, Alexopoulou L, Leitner WW, Flavell RA, Reis e Sousa C - Gene Ther. (2008)

Bottom Line: Toll-like receptor 3 (TLR3) is a dsRNA innate immune receptor expressed by antigen-presenting dendritic cells (DCs).Here, we test the hypothesis that TLR3 is necessary for the immunogenicity of replicon plasmid-based DNA vaccines.We show that mouse CD8 alpha(+) DC phagocytose dying replicon plasmid-transfected cells in vitro and are activated in a TLR3-dependent manner by dsRNA present within those cells.

View Article: PubMed Central - PubMed

Affiliation: Immunobiology Laboratory, Cancer Research UK, London Research Institute, London, UK. sandra.diebold@kcl.ac.uk

ABSTRACT
Replicon plasmids encoding an alphavirus RNA replicase constitute an alternative to conventional DNA plasmids with promise for DNA vaccination in humans. Replicase activity amplifies the levels of transgene mRNA through a copying process involving double-stranded (ds) RNA intermediates, which contribute to vaccine immunogenicity by activating innate antiviral responses. Toll-like receptor 3 (TLR3) is a dsRNA innate immune receptor expressed by antigen-presenting dendritic cells (DCs). Here, we test the hypothesis that TLR3 is necessary for the immunogenicity of replicon plasmid-based DNA vaccines. We show that mouse CD8 alpha(+) DC phagocytose dying replicon plasmid-transfected cells in vitro and are activated in a TLR3-dependent manner by dsRNA present within those cells. However, we find that cytotoxic T-cell responses to a replicon plasmid intramuscular vaccine are not diminished in the absence of TLR3 in vivo. Our results underscore the potential role of TLR3 in mediating immune activation by dsRNA-bearing replicon plasmid-transfected cells and indicate that other innate sensing pathways can compensate for TLR3 absence in vivo.

Show MeSH

Related in: MedlinePlus

Uptake of transfected cells by splenic DCVERO cells were transfected with the replicon plasmid pSIN-GFP or the conventional plasmid pEGFP by electroporation and cultured for 6 hours. Cells were labeled with PKH26 and co-cultured with CD11c-enriched splenic DC for 4 hours. Uptake of material from PKH26-labeled VERO cells was determined by flow cytometry gating on CD11c+ CD8α+ splenic DC. (a) Representative data from one of two independent experiments. (b) Pooled data from the two experiments showing the frequency of PKH26+ CD8α+ splenic DC. No significant difference was observed between uptake of conventional or replicon plasmid transfected cells (p>0.05, two tailed student’s t test).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2655288&req=5

Figure 3: Uptake of transfected cells by splenic DCVERO cells were transfected with the replicon plasmid pSIN-GFP or the conventional plasmid pEGFP by electroporation and cultured for 6 hours. Cells were labeled with PKH26 and co-cultured with CD11c-enriched splenic DC for 4 hours. Uptake of material from PKH26-labeled VERO cells was determined by flow cytometry gating on CD11c+ CD8α+ splenic DC. (a) Representative data from one of two independent experiments. (b) Pooled data from the two experiments showing the frequency of PKH26+ CD8α+ splenic DC. No significant difference was observed between uptake of conventional or replicon plasmid transfected cells (p>0.05, two tailed student’s t test).

Mentions: We next compared the uptake of VERO cells transfected with replicon plasmid or conventional plasmid DNA by DC. Six hours after electroporation, VERO cells were labeled with the membrane dye PKH26 and were co-cultured with CD11c-enriched splenic cells. Uptake of PKH26-labelled cellular material by CD8α+ DC was determined by flow cytometry19. 72% and 64% of CD8α+ DC stained positive for the membrane dye PKH26 after incubation with VERO cells transfected with pSIN-GFP and pEGFP, respectively (Fig. 3 A). The dye transfer reflected true uptake of cells or cell debris by DC as it was blocked by latrunculin, which prevents actin-dependent phagocytic and macropinocytic processes (data not shown; see reference 19). In multiple experiments, no significant differences were seen in uptake of replicon plasmid-versus conventional plasmid-transfected VERO cells (Fig. 3B). Thus, CD8α+ DC have equal access to cellular material from replicon plasmid- or conventional plasmid-transfected VERO cells despite the increased frequency of apoptosis in the former population.


Role of TLR3 in the immunogenicity of replicon plasmid-based vaccines.

Diebold SS, Schulz O, Alexopoulou L, Leitner WW, Flavell RA, Reis e Sousa C - Gene Ther. (2008)

Uptake of transfected cells by splenic DCVERO cells were transfected with the replicon plasmid pSIN-GFP or the conventional plasmid pEGFP by electroporation and cultured for 6 hours. Cells were labeled with PKH26 and co-cultured with CD11c-enriched splenic DC for 4 hours. Uptake of material from PKH26-labeled VERO cells was determined by flow cytometry gating on CD11c+ CD8α+ splenic DC. (a) Representative data from one of two independent experiments. (b) Pooled data from the two experiments showing the frequency of PKH26+ CD8α+ splenic DC. No significant difference was observed between uptake of conventional or replicon plasmid transfected cells (p>0.05, two tailed student’s t test).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2655288&req=5

Figure 3: Uptake of transfected cells by splenic DCVERO cells were transfected with the replicon plasmid pSIN-GFP or the conventional plasmid pEGFP by electroporation and cultured for 6 hours. Cells were labeled with PKH26 and co-cultured with CD11c-enriched splenic DC for 4 hours. Uptake of material from PKH26-labeled VERO cells was determined by flow cytometry gating on CD11c+ CD8α+ splenic DC. (a) Representative data from one of two independent experiments. (b) Pooled data from the two experiments showing the frequency of PKH26+ CD8α+ splenic DC. No significant difference was observed between uptake of conventional or replicon plasmid transfected cells (p>0.05, two tailed student’s t test).
Mentions: We next compared the uptake of VERO cells transfected with replicon plasmid or conventional plasmid DNA by DC. Six hours after electroporation, VERO cells were labeled with the membrane dye PKH26 and were co-cultured with CD11c-enriched splenic cells. Uptake of PKH26-labelled cellular material by CD8α+ DC was determined by flow cytometry19. 72% and 64% of CD8α+ DC stained positive for the membrane dye PKH26 after incubation with VERO cells transfected with pSIN-GFP and pEGFP, respectively (Fig. 3 A). The dye transfer reflected true uptake of cells or cell debris by DC as it was blocked by latrunculin, which prevents actin-dependent phagocytic and macropinocytic processes (data not shown; see reference 19). In multiple experiments, no significant differences were seen in uptake of replicon plasmid-versus conventional plasmid-transfected VERO cells (Fig. 3B). Thus, CD8α+ DC have equal access to cellular material from replicon plasmid- or conventional plasmid-transfected VERO cells despite the increased frequency of apoptosis in the former population.

Bottom Line: Toll-like receptor 3 (TLR3) is a dsRNA innate immune receptor expressed by antigen-presenting dendritic cells (DCs).Here, we test the hypothesis that TLR3 is necessary for the immunogenicity of replicon plasmid-based DNA vaccines.We show that mouse CD8 alpha(+) DC phagocytose dying replicon plasmid-transfected cells in vitro and are activated in a TLR3-dependent manner by dsRNA present within those cells.

View Article: PubMed Central - PubMed

Affiliation: Immunobiology Laboratory, Cancer Research UK, London Research Institute, London, UK. sandra.diebold@kcl.ac.uk

ABSTRACT
Replicon plasmids encoding an alphavirus RNA replicase constitute an alternative to conventional DNA plasmids with promise for DNA vaccination in humans. Replicase activity amplifies the levels of transgene mRNA through a copying process involving double-stranded (ds) RNA intermediates, which contribute to vaccine immunogenicity by activating innate antiviral responses. Toll-like receptor 3 (TLR3) is a dsRNA innate immune receptor expressed by antigen-presenting dendritic cells (DCs). Here, we test the hypothesis that TLR3 is necessary for the immunogenicity of replicon plasmid-based DNA vaccines. We show that mouse CD8 alpha(+) DC phagocytose dying replicon plasmid-transfected cells in vitro and are activated in a TLR3-dependent manner by dsRNA present within those cells. However, we find that cytotoxic T-cell responses to a replicon plasmid intramuscular vaccine are not diminished in the absence of TLR3 in vivo. Our results underscore the potential role of TLR3 in mediating immune activation by dsRNA-bearing replicon plasmid-transfected cells and indicate that other innate sensing pathways can compensate for TLR3 absence in vivo.

Show MeSH
Related in: MedlinePlus