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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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Presentation of the HA110-120 peptide to the specific TcH.  (Right) 2PK3 B lymphoma cells (2 × 105) or bone marrow–derived dendritic cells (5 × 104) were cultured with G7/pVH–TB , G7/pC myoblasts  (2 × 105 cells), or HA110-120 peptide (30 μg/ml). After 48 h, 2 × 105  14.3.1 TcH cells were added for 12 h. (Left) 2PK3 B lymphoma cells (105)  or bone marrow–derived dendritic cells (105) were cultured for 12 h with  2 × 105 14.3.1 TcH cells in the presence of HA110-120 synthetic peptide  (30 μg/ml), or with the saturated ammonium sulfate fraction of the cell culture supernatants from either G7 myoblasts or G7/pVH–TB myoblasts. The  percentage of TcH activated cells was determined by FACS® analysis.
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Figure 1: Presentation of the HA110-120 peptide to the specific TcH. (Right) 2PK3 B lymphoma cells (2 × 105) or bone marrow–derived dendritic cells (5 × 104) were cultured with G7/pVH–TB , G7/pC myoblasts (2 × 105 cells), or HA110-120 peptide (30 μg/ml). After 48 h, 2 × 105 14.3.1 TcH cells were added for 12 h. (Left) 2PK3 B lymphoma cells (105) or bone marrow–derived dendritic cells (105) were cultured for 12 h with 2 × 105 14.3.1 TcH cells in the presence of HA110-120 synthetic peptide (30 μg/ml), or with the saturated ammonium sulfate fraction of the cell culture supernatants from either G7 myoblasts or G7/pVH–TB myoblasts. The percentage of TcH activated cells was determined by FACS® analysis.

Mentions: We then investigated the ability of various APCs to activate the 14.3.1 TcH when cocultured with G7/pVH–TB and G7/pC myoblasts. 14.3.1 TcH recognizes the HA110-120 in association with IEd molecules (15). Data presented in Fig. 1 (left) show that a substantial activation was obtained when the TcHs were cocultured with G7/pVH–TB myoblasts and either 2PK3 B lymphoma cells or bone marrow– derived dendritic cells. Similar results were obtained when purified CD4+ transgenic T cells expressing the HA110-120–specific TCR were used in the proliferation assay (data not shown). The ammonium sulfate–precipitated fraction of the cell culture supernatants from G7/pVH–TB cells, but not from G7/pC cells, also activated the specific TcH in the presence of either 2PK3 cells or bone marrow–derived dendritic cells (Fig. 1, right). The stronger TcH activation obtained with the ammonium sulfate fraction is due to the higher concentration of VH–TB polypeptide from cell culture supernatant. The observed activation of TcH by ammonium sulfate fraction of myoblast culture supernatant ruled out the extracellular processing of the polypeptide.


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)

Presentation of the HA110-120 peptide to the specific TcH.  (Right) 2PK3 B lymphoma cells (2 × 105) or bone marrow–derived dendritic cells (5 × 104) were cultured with G7/pVH–TB , G7/pC myoblasts  (2 × 105 cells), or HA110-120 peptide (30 μg/ml). After 48 h, 2 × 105  14.3.1 TcH cells were added for 12 h. (Left) 2PK3 B lymphoma cells (105)  or bone marrow–derived dendritic cells (105) were cultured for 12 h with  2 × 105 14.3.1 TcH cells in the presence of HA110-120 synthetic peptide  (30 μg/ml), or with the saturated ammonium sulfate fraction of the cell culture supernatants from either G7 myoblasts or G7/pVH–TB myoblasts. The  percentage of TcH activated cells was determined by FACS® analysis.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199124&req=5

Figure 1: Presentation of the HA110-120 peptide to the specific TcH. (Right) 2PK3 B lymphoma cells (2 × 105) or bone marrow–derived dendritic cells (5 × 104) were cultured with G7/pVH–TB , G7/pC myoblasts (2 × 105 cells), or HA110-120 peptide (30 μg/ml). After 48 h, 2 × 105 14.3.1 TcH cells were added for 12 h. (Left) 2PK3 B lymphoma cells (105) or bone marrow–derived dendritic cells (105) were cultured for 12 h with 2 × 105 14.3.1 TcH cells in the presence of HA110-120 synthetic peptide (30 μg/ml), or with the saturated ammonium sulfate fraction of the cell culture supernatants from either G7 myoblasts or G7/pVH–TB myoblasts. The percentage of TcH activated cells was determined by FACS® analysis.
Mentions: We then investigated the ability of various APCs to activate the 14.3.1 TcH when cocultured with G7/pVH–TB and G7/pC myoblasts. 14.3.1 TcH recognizes the HA110-120 in association with IEd molecules (15). Data presented in Fig. 1 (left) show that a substantial activation was obtained when the TcHs were cocultured with G7/pVH–TB myoblasts and either 2PK3 B lymphoma cells or bone marrow– derived dendritic cells. Similar results were obtained when purified CD4+ transgenic T cells expressing the HA110-120–specific TCR were used in the proliferation assay (data not shown). The ammonium sulfate–precipitated fraction of the cell culture supernatants from G7/pVH–TB cells, but not from G7/pC cells, also activated the specific TcH in the presence of either 2PK3 cells or bone marrow–derived dendritic cells (Fig. 1, right). The stronger TcH activation obtained with the ammonium sulfate fraction is due to the higher concentration of VH–TB polypeptide from cell culture supernatant. The observed activation of TcH by ammonium sulfate fraction of myoblast culture supernatant ruled out the extracellular processing of the polypeptide.

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