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

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.

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/pV_H–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/pV_H–TB myoblasts. The percentage of TcH activated cells was determined by FACS^® analysis.

Figure 2

Activation of the HA110-120–specific TcH by various numbers of dendritic and B cells. Various numbers of brachial and axillary lymph node, dendritic cells, or B cells were purified by cell sorting (a) from BALB/c mice immunized in the biceps and scapular muscles with pC and cultured (b) with TcH (2 × 10⁵) for 24 h in the presence of HA110-120 peptide (25 μg/ ml). Dendritic or B cells isolated from mice immunized with pV_H–TB or pC were cultured for 24 h with TcH (2 × 10⁵) in the absence of exogenous antigen (c). The percentage of TcH-activated cells was determined by FACS^® analysis.

Figure 3

Detection of V_H– TB gene in dendritic cells from mice immunized intramuscularly with pV_H–TB plasmid. DNA extracted from 2 × 10⁵ dendritic or B cells from brachial and axillary lymph nodes was amplified by PCR using V_H–TB-F and V_H– TB-R primers annealing in the 5′ and 3′ end regions of the V_H–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 pV_H–TB; lane 6, B cells from mice immunized with pV_H–TB; lane 7, dendritic cells from mice immunized with pV_H–TB; lane 8, purified pV_H–TB plasmid (5 ng); lane 9, negative control. Specific bands are shown by arrows.

Figure 4

Activation of a HA110-120–specific TcH by enriched Langerhans cells transfected in vivo with pV_H– TB plasmid. Langerhans cells (2 × 10⁵) were obtained from mice immunized intracutaneously with pV_H–TB plasmid and cultured for 24 h with 14.3.1 TcH (2 × 10⁵). FACS^® analysis shows that Langerhans cells from animals immunized with pV_H–TB activated 26% of the HA110-120–specific TcH (a), but Langerhans cells from animals immunized with pC did not activate any of the TcH (b). (c), PCR analysis of pV_H– TB and pC plasmids using T7 and Sp6 primers in skin cells from mice immunized intracutaneously. Lane 1, molecular markers; lane 2, purified pV_H–TB plasmid; lane 3, purified pC plasmid; lane 4, skin cells from mice immunized with pC; lane 5, skin cells from mice immunized with pV_H–TB; lane 6, MHC class II–positive Langerhans cells from mice immunized with pV_H–TB; lane 7, MHC class II–negative skin cells from mice immunized with pV_H– TB; lane 8, negative control.

Figure 4

Activation of a HA110-120–specific TcH by enriched Langerhans cells transfected in vivo with pV_H– TB plasmid. Langerhans cells (2 × 10⁵) were obtained from mice immunized intracutaneously with pV_H–TB plasmid and cultured for 24 h with 14.3.1 TcH (2 × 10⁵). FACS^® analysis shows that Langerhans cells from animals immunized with pV_H–TB activated 26% of the HA110-120–specific TcH (a), but Langerhans cells from animals immunized with pC did not activate any of the TcH (b). (c), PCR analysis of pV_H– TB and pC plasmids using T7 and Sp6 primers in skin cells from mice immunized intracutaneously. Lane 1, molecular markers; lane 2, purified pV_H–TB plasmid; lane 3, purified pC plasmid; lane 4, skin cells from mice immunized with pC; lane 5, skin cells from mice immunized with pV_H–TB; lane 6, MHC class II–positive Langerhans cells from mice immunized with pV_H–TB; lane 7, MHC class II–negative skin cells from mice immunized with pV_H– TB; lane 8, negative control.