Differential antigen presentation regulates the changing patterns of CD8+ T cell immunodominance in primary and secondary influenza virus infections

Abstract

The specificity of CD8+ T cell responses can vary dramatically between primary and secondary infections. For example, NP366-374/Db- and PA224-233/Db-specific CD8+ T cells respond in approximately equal numbers to a primary influenza virus infection in C57BL/6 mice, whereas NP366-374/Db-specific CD8+ T cells dominate the secondary response. To investigate the mechanisms underlying this changing pattern of immunodominance, we analyzed the role of antigen presentation in regulating the specificity of the T cell response. The data show that both dendritic and nondendritic cells are able to present the NP366-374/Db epitope, whereas only dendritic cells effectively present the PA224-233/Db epitope after influenza virus infection, both in vitro and in vivo. This difference in epitope expression favored the activation and expansion of NP366-374/Db-specific CD8+ memory T cells during secondary infection. The data also show that the immune response to influenza virus infection may involve T cells specific for epitopes, such as PA224-233/Db, that are poorly expressed at the site of infection. In this regard, vaccination with the PA224-233 peptide actually had a detrimental effect on the clearance of a subsequent influenza virus infection. Thus, differential antigen presentation impacts both the specificity of the T cell response and the efficacy of peptide-based vaccination strategies.

Figure 1.

Kinetics of the NP_366–374/D^b and PA_224–233/D^b-specific responses after primary and secondary infection with influenza virus. C57BL/6 mice (three mice per time point) were infected intranasally with 300 EID₅₀ x31 on day 0 and given a secondary intranasal infection with 3,000 EID₅₀ PR8 on day 42 (indicated by arrow). Tissues were removed at the indicated time points and stained with anti-CD8 and NP_366–374/D^b- or PA_224–233/D^b-specific tetramers. Shown are the number of NP- (closed) and PA- (open) specific T cells. The data shown are one of two complete kinetic experiments with similar results.

Figure 2.

Specificity and sensitivity of Lac Z inducible hybridomas. Clones 53-A8 and 6291-B8 were screened using L-D^b, AM11, or JAWS II cells incubated overnight with either the NP_366–374, PA_224–233, PB1_703–711, or HA_192–207 peptides at the indicated concentrations. The graphs show the number of positive Lac Z hybridomas per well (no more than 2,000 spots were counted per well).

Figure 3.

Presentation of the NP_366–374/D^b and PA_224–233/D^b epitopes after in vitro infection with influenza virus. The EL4, AM11, and JAWS II cell lines were infected with influenza x31 (top panels) or PR8 (bottom panels) for four hours. The cells were infected with three doses of influenza virus or left uninfected: multiplicity of infection (MOI) of 0 (open circle), 2 (closed circle), 10 (open triangle), or 50 (closed triangle). After infection, the cells were irradiated and then used in a standard antigen presentation assay with the 53-A8 (NP_366–374/D^b) or 6291-B8 (PA_224–233/D^b) hybridomas.

Figure 4.

Kinetics of antigen presentation after in vitro infection of the JAWS II cell line. The JAWS II cell line was infected with influenza (x31) for the times shown with a MOI of 0 (open circle), 2 (closed circle), 10 (open triangle), or 50 (closed triangle). After infection, the cells were irradiated and then used in a standard antigen presentation assay with the 53-A8 (NP_366–374/D^b) or 6291-B8 (PA_224–233/D^b) hybridomas.

Figure 5.

Kinetics of antigen presentation after in vitro infection of the AM11 cell line. The AM11 cell line was infected with influenza (x31) for the times shown with a MOI of 0 (open circle), 2 (closed circle), 10 (open triangle), or 50 (closed triangle). After infection, the cells were irradiated and then used in a standard antigen presentation assay with the 53-A8 (NP_366–374/D^b) or 6291-B8 (PA_224–233/D^b) hybridomas.

Figure 6.

Presentation of the NP_366–374/D^b and PA_224–233/D^b epitopes after in vitro infection with the x31 strain of influenza virus. Bone marrow–derived dendritic cells, peritoneal exudate cells (PECs), B cells, and lung epithelial cells were collected from uninfected mice. The cells were then infected in vitro with influenza virus (x31, top panels; PR8, bottom panels) at an MOI of 0 (open circle), 2 (closed circle), 10 (open triangle), or 50 (closed triangle) for 4 h, irradiated, and then used in a standard antigen presentation assay with the 53-A8 (NP_366–374/D^b) or 6291-B8 (PA_224–233/D^b) hybridomas.

Figure 7.

Ex vivo presentation of the NP_366–374/D^b and PA_224–233/D^b epitopes after infection with influenza virus. Mice were infected intranasally with x31 and cells from the lung airways (closed circle) and MLN (open triangle) were collected on day 6 after infection. (A) Twofold serial dilutions of cells were made in flat-bottom, 96-well plates starting at 10⁵ cells/well and a standard antigen presentation assay was performed using the 53-A8 (NP_366–374/D^b) or 6291-B8 (PA_224–233/D^b) Lac Z-inducible hybridomas. (B) Cells from the lung were stained with anti-B220-CyChrome, anti-CD11c-FITC, and anti-CD11b-PE. The macrophage (B220⁻CD11b⁺) and dendritic cell (B220⁻CD11c⁺) populations were collected using a FACSVantage™ flow cytometer with DiVa options. Twofold serial dilutions of cells were made in flat-bottom, 96-well plates, and a standard Lac Z APC assay was performed using the 53-A8 (NP_366–374/D^b) or 6291-B8 (PA_224–233/D^b) Lac Z–inducible hybridomas. Cells from uninfected control mice did not elicit a response following incubation with either the NP_366–374/D^b or PA_224–233/D^b hybridomas.

Figure 8.

Simultaneous detection of influenza virus-specific naive and memory cells. Cells were collected from the spleens of C57BL/6 mice infected 32 d previously with 300 EID₅₀ x31. The cells were made into a single-cell suspension, enriched for CD8⁺ cells and stained with anti-CD8-PE and anti-CD44-FITC. The memory cells (CD8⁺CD44⁺) were collected using a FACSVantage™ flow cytometer with DiVa options. 2.5 × 10⁵ purified memory cells were then injected into naive PepBoy/J mice. 1 d after transfer, the recipient mice were infected with 300 EID₅₀ x31. 10 d after infection (11 d after transfer), cells from the lung airways and spleen were collected and stained with anti-CD8-PerCP, anti-CD45.2-FITC and anti- NP_366–374/D^b-APC or anti- PA_224–233/D^b-APC. A and B show the percentage of NP_366–374/D^b- and PA_224–233/D^b-specific CD8 cells, respectively, within the donor (CD45.2⁺) or host (CD45.2⁻) lung airway populations. C shows the average (± standard deviation) percent NP_366–374/D^b- (solid bars) and PA_224–233/D^b- (striped bars) specific CD8 cells in the lung airways or spleen.

Figure 9.

Vaccination with the PA_224–233 peptide results in delayed viral clearance after influenza virus challenge. Mice were injected i.v. with 0.5 × 10⁶ dendritic cells pulsed with either the PA_224–233 peptide of influenza or a Sendai virus peptide. As a further control, one group of mice did not receive any dendritic cells (un-vaccinated). 2 wk after vaccination, the mice were infected with 300 EID₅₀ x31. 10 d after infection, cells from the lung airways were collected and stained with anti-CD8-PerCP, and anti-NP_366–374/D^b-APC or anti-PA_224–233/D^b-APC. Panel A shows the percentage of NP_366–374/D^b- and PA_224–233/D^b-specific cells of total CD8s after infection of the Sendai peptide or PA_224–233 peptide vaccinated mice. B shows the viral load in the lungs of individual mice on day 10 after infection.

Figure 10.

A model of antigen presentation and T cell responses after influenza virus infection. After infection with influenza virus, the NP_366–374/D^b epitope is processed and presented by both macrophages (MΦ) and dendritic cells (DC). The PA_224–233/D^b epitope, however, is only presented by the dendritic cells. During the primary response, the naive T cells respond only to dendritic cells resulting in an equivalent NP_366–374/D^b- and PA_224–233/D^b-specific T cell response. After secondary infection, the memory T cells are able to respond to both macrophages and dendritic cells resulting in a predominance of NP_366–374/D^b specific T cells.