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Antigen presentation by dendritic cells after immunization with DNA encoding a major histocompatibility complex class II-restricted viral epitope.

Casares S, Inaba K, Brumeanu TD, Steinman RM, Bona CA - J. Exp. Med. (1997)

Bottom Line: We used DNA that encodes an A/PR/8/34 influenza peptide for CD4 T cells and that elicits protective antiviral immunity.When DNA was injected into muscles or skin, and antigen-presenting cells were isolated from either the draining lymph nodes or the skin, dendritic, but not B, cells presented antigen to T cells and carried plasmid DNA.We suggest that the uptake of DNA and/or the protein expressed by dendritic cells triggers immune responses to DNA vaccines.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Mount Sinai School of Medicine, New York 10029, USA.

ABSTRACT
Intramuscular and intracutaneous immunization with naked DNA can vaccinate animals to the encoded proteins, but the underlying mechanisms of antigen presentation are unclear. We used DNA that encodes an A/PR/8/34 influenza peptide for CD4 T cells and that elicits protective antiviral immunity. DNA-transfected, cultured muscle cells released the influenza polypeptide, which then could be presented on the major histocompatibility complex class II molecules of dendritic cells. When DNA was injected into muscles or skin, and antigen-presenting cells were isolated from either the draining lymph nodes or the skin, dendritic, but not B, cells presented antigen to T cells and carried plasmid DNA. We suggest that the uptake of DNA and/or the protein expressed by dendritic cells triggers immune responses to DNA vaccines.

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Detection of VH– TB gene in dendritic cells from  mice immunized intramuscularly  with pVH–TB plasmid. DNA extracted from 2 × 105 dendritic or  B cells from brachial and axillary  lymph nodes was amplified by  PCR using VH–TB-F and VH– TB-R primers annealing in the  5′ and 3′ end regions of the  VH–TB gene, respectively (top)  or, as control of integrity of  DNA, primers annealing in the  flanking regions of the genomic  IgG2a-CH1 exon (bottom). Lane  1, DNA markers (λ/HindIII);  lane 2, nonfractionated cells  from mice immunized with pC;  lane 3, B cells from mice immunized with pC; lane 4, dendritic cells from  mice immunized with pC; lane 5, nonfractionated cells from mice immunized with pVH–TB; lane 6, B cells from mice immunized with pVH–TB;  lane 7, dendritic cells from mice immunized with pVH–TB; lane 8, purified pVH–TB plasmid (5 ng); lane 9, negative control. Specific bands are  shown by arrows.
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Figure 3: Detection of VH– TB gene in dendritic cells from mice immunized intramuscularly with pVH–TB plasmid. DNA extracted from 2 × 105 dendritic or B cells from brachial and axillary lymph nodes was amplified by PCR using VH–TB-F and VH– TB-R primers annealing in the 5′ and 3′ end regions of the VH–TB gene, respectively (top) or, as control of integrity of DNA, primers annealing in the flanking regions of the genomic IgG2a-CH1 exon (bottom). Lane 1, DNA markers (λ/HindIII); lane 2, nonfractionated cells from mice immunized with pC; lane 3, B cells from mice immunized with pC; lane 4, dendritic cells from mice immunized with pC; lane 5, nonfractionated cells from mice immunized with pVH–TB; lane 6, B cells from mice immunized with pVH–TB; lane 7, dendritic cells from mice immunized with pVH–TB; lane 8, purified pVH–TB plasmid (5 ng); lane 9, negative control. Specific bands are shown by arrows.

Mentions: The above results (Fig. 2) demonstrated that, after intramuscular immunization, dendritic cells expressed T cell epitopes that were encoded by the naked DNA. It is possible that the dendritic cells had either taken up the plasmid itself or were expressing epitopes acquired from proteins contained in transfected myoblasts (shown in Fig. 1). To assess the former possibility, we used PCR to look for DNA in the dendritic and B cells from mice given the pC plasmid control or the pVH–TB vaccine. We found that dendritic cells, which had been purified by sorting as shown in Fig. 2 a, selectively expressed pVH–TB sequences (Fig. 3, lane 7). B cells and preparations that were partially enriched in dendritic cells did not contain detectable vaccine sequences (Fig. 3, lanes 6 and 5, respectively). As a control for the integrity of the DNA in each cell preparation, we amplified the genomic IgG2a-CH1 exon (Fig. 3, bottom). Using primers annealing in plasmid sequences (T7 and SP6), we also detected transfected cells only in the sorted dendritic cell fraction of mice immunized with pVH–TB and pC plasmid.


Antigen presentation by dendritic cells after immunization with DNA encoding a major histocompatibility complex class II-restricted viral epitope.

Casares S, Inaba K, Brumeanu TD, Steinman RM, Bona CA - J. Exp. Med. (1997)

Detection of VH– TB gene in dendritic cells from  mice immunized intramuscularly  with pVH–TB plasmid. DNA extracted from 2 × 105 dendritic or  B cells from brachial and axillary  lymph nodes was amplified by  PCR using VH–TB-F and VH– TB-R primers annealing in the  5′ and 3′ end regions of the  VH–TB gene, respectively (top)  or, as control of integrity of  DNA, primers annealing in the  flanking regions of the genomic  IgG2a-CH1 exon (bottom). Lane  1, DNA markers (λ/HindIII);  lane 2, nonfractionated cells  from mice immunized with pC;  lane 3, B cells from mice immunized with pC; lane 4, dendritic cells from  mice immunized with pC; lane 5, nonfractionated cells from mice immunized with pVH–TB; lane 6, B cells from mice immunized with pVH–TB;  lane 7, dendritic cells from mice immunized with pVH–TB; lane 8, purified pVH–TB plasmid (5 ng); lane 9, negative control. Specific bands are  shown by arrows.
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Figure 3: Detection of VH– TB gene in dendritic cells from mice immunized intramuscularly with pVH–TB plasmid. DNA extracted from 2 × 105 dendritic or B cells from brachial and axillary lymph nodes was amplified by PCR using VH–TB-F and VH– TB-R primers annealing in the 5′ and 3′ end regions of the VH–TB gene, respectively (top) or, as control of integrity of DNA, primers annealing in the flanking regions of the genomic IgG2a-CH1 exon (bottom). Lane 1, DNA markers (λ/HindIII); lane 2, nonfractionated cells from mice immunized with pC; lane 3, B cells from mice immunized with pC; lane 4, dendritic cells from mice immunized with pC; lane 5, nonfractionated cells from mice immunized with pVH–TB; lane 6, B cells from mice immunized with pVH–TB; lane 7, dendritic cells from mice immunized with pVH–TB; lane 8, purified pVH–TB plasmid (5 ng); lane 9, negative control. Specific bands are shown by arrows.
Mentions: The above results (Fig. 2) demonstrated that, after intramuscular immunization, dendritic cells expressed T cell epitopes that were encoded by the naked DNA. It is possible that the dendritic cells had either taken up the plasmid itself or were expressing epitopes acquired from proteins contained in transfected myoblasts (shown in Fig. 1). To assess the former possibility, we used PCR to look for DNA in the dendritic and B cells from mice given the pC plasmid control or the pVH–TB vaccine. We found that dendritic cells, which had been purified by sorting as shown in Fig. 2 a, selectively expressed pVH–TB sequences (Fig. 3, lane 7). B cells and preparations that were partially enriched in dendritic cells did not contain detectable vaccine sequences (Fig. 3, lanes 6 and 5, respectively). As a control for the integrity of the DNA in each cell preparation, we amplified the genomic IgG2a-CH1 exon (Fig. 3, bottom). Using primers annealing in plasmid sequences (T7 and SP6), we also detected transfected cells only in the sorted dendritic cell fraction of mice immunized with pVH–TB and pC plasmid.

Bottom Line: We used DNA that encodes an A/PR/8/34 influenza peptide for CD4 T cells and that elicits protective antiviral immunity.When DNA was injected into muscles or skin, and antigen-presenting cells were isolated from either the draining lymph nodes or the skin, dendritic, but not B, cells presented antigen to T cells and carried plasmid DNA.We suggest that the uptake of DNA and/or the protein expressed by dendritic cells triggers immune responses to DNA vaccines.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Mount Sinai School of Medicine, New York 10029, USA.

ABSTRACT
Intramuscular and intracutaneous immunization with naked DNA can vaccinate animals to the encoded proteins, but the underlying mechanisms of antigen presentation are unclear. We used DNA that encodes an A/PR/8/34 influenza peptide for CD4 T cells and that elicits protective antiviral immunity. DNA-transfected, cultured muscle cells released the influenza polypeptide, which then could be presented on the major histocompatibility complex class II molecules of dendritic cells. When DNA was injected into muscles or skin, and antigen-presenting cells were isolated from either the draining lymph nodes or the skin, dendritic, but not B, cells presented antigen to T cells and carried plasmid DNA. We suggest that the uptake of DNA and/or the protein expressed by dendritic cells triggers immune responses to DNA vaccines.

Show MeSH
Related in: MedlinePlus