Memory CD8+ T cells specific for a single immunodominant or subdominant determinant induced by peptide-dendritic cell immunization protect from an acute lethal viral disease

Abstract

The antigens recognized by individual CD8(+) T cells are small peptides bound to major histocompatibility complex (MHC) class I molecules. The CD8(+) T cell response to a virus is restricted to several peptides, and the magnitudes of the effector as well as memory phases of the response to the individual peptides are generally hierarchical. The peptide eliciting a stronger response is called immunodominant (ID), and those with smaller-magnitude responses are termed subdominant (SD). The relative importance of ID and SD determinants in protective immunity remains to be fully elucidated. We previously showed that multispecific memory CD8(+) T cells can protect susceptible mice from mousepox, an acute lethal viral disease. It remained unknown, however, whether CD8(+) T cells specific for single ID or SD peptides could be protective. Here, we demonstrate that immunization with dendritic cells pulsed with ID and some but not all SD peptides induces memory CD8(+) T cells that are fully capable of protecting susceptible mice from mousepox. Additionally, while natural killer (NK) cells are essential for the natural resistance of nonimmune C57BL/6 (B6) to mousepox, we show that memory CD8(+) T cells of single specificity also protect B6 mice depleted of NK cells. This suggests it is feasible to produce effective antiviral CD8(+) T cell vaccines using single CD8(+) T cell determinants and that NK cells are no longer essential when memory CD8(+) T cells are present.

Fig 1

Identification of H-2 K^b-restricted ECTV determinants. (A) B6 mice were infected with 3,000 PFU of ECTV in the footpad, and splenocytes were analyzed 7 dpi. Representative flow cytometry plots show the frequencies of CD8⁺ cells positive for H-2 K^b-TSYKFESV, -SIFRFLNI, -KSYNYMLL, -ITYRFYLI, and -STLNFNNL. The dot plots represents the frequency of CD8⁺ cells that were stained with the indicated K^b-peptide dimer in 3 independent experiments for all peptides except for ITYRFYLI, for which data are from 2 independent experiments. (B) Graphs showing summary data of flow cytometry plots from panel A. Open symbols represent naïve mice, and closed symbols represent ECTV-infected mice. Each data point corresponds to pooled splenocytes from 2 to 3 mice in one experiment. (C) Representative in vivo cytotoxicity data. Naïve and VACV-immune mice were inoculated i.v. with a 1:1 mixture of B6 splenocytes labeled with 0.8 μM CFSE (CFSE^low) or splenocytes labeled with 4 μM CFSE and pulsed with the TSYKFESV (CFSE^high). Mice were killed 4 h after target inoculation, and the proportions of CFSE^low and CFSE^high cells were determined by flow cytometry in spleens of individual mice. Histograms are gated on CFSE-positive cells of naïve and ECTV-infected B6 mice, as indicated. (D) Summary of in vivo cytotoxicity assays using the indicated ECTV/VACV peptide-pulsed targets. Numbers indicate percentages of specific killing of CFSE^high cells, calculated as detailed in Materials and Methods. Data correspond to means ± SEM for 3 or more independent experiments, where n was 15 for TSYKFESV, n was 12 for SIFRFLNI, n was 12 for KSYNYMLL, n was 10 for ITYRFYLI, n was 3 for STLNFNNL, and n was 11 for SIINFEKL. P values (*, P < 0.05; ***, P < 0.001) shown were determined for comparisons TSYKFESV and all other groups. The P value determined for SIINFEKL versus all other groups was <0.001 (data not shown).

Fig 2

Variable responses to immunization with DCs pulsed with ECTV/VACV peptides. (A) Representative flow cytometry plots of the frequencies of positive K^b-TSYKFESV, -SIFRFLNI, -KSYNYML, -ITYRFYLI, -STLNFNNL, and -SIINFEKL CD8⁺ PBMC obtained from B6.D2-D6 mice 1 month after peptide-DC booster immunization. (B) Column graphs showing summary data of flow cytometry plots from panel A. Data correspond to 3 or more independent experiments, with n ≥ 5 per group. (C) Representative in vivo cytotoxicity data of naïve and peptide-DC-immunized mice (>2 months after vaccination) that received SIINFEKL-pulsed CFSE^low cells (control) or ECTV peptide-pulsed CFSE^high cells i.v. Mice were killed 18 h after target inoculation, and the proportions of CFSE^low and CFSE^high cells were determined by flow cytometry in pooled LNs and spleens. Histograms are gated on CFSE-positive cells. Numbers indicate percentages of specific killing of CFSE^high cells, calculated as detailed in Materials and Methods. (D and E) Column graphs showing summary data from panel C in pooled LNs (D) and in spleens from individual mice (E). Data correspond to 5 vaccinated mice per group ± the SEM. Data are representative of two (SIFRFLNI and ITYRFLI) or three (TSYKFESV) experiments. All samples are compared to results for animals that received DC-SIINFEKL (*, P < 0.05; **, P < 0.01; ***, P < 0.001), and where statistical differences are indicated, those groups were also significantly different from uninfected animals (data not shown).

Fig 3

Memory CD8⁺ T cells elicited by immunization with peptide-pulsed DCs are present in the liver and spleen after ECTV infection. B6.D2-D6 mice were immunized with peptide-DC and 2 months later infected with 3,000 PFU of ECTV in the footpad. At 7 dpi, livers and spleens were analyzed in individual mice. (A) Representative flow cytometry plots showing frequencies of CD8⁺ cells in the livers that were K^b-TSYKFESV⁺ or K^b-SIFRFLNI⁺. (B) Column graphs correspond to summary data presented in the flow cytometry plots of panel A. Frequencies and absolute numbers of positive CD8⁺ cells are shown for mice immunized with K^b-TSYKFESV as indicated. Data correspond to five mice per group ± the SEM and are representative of two independent experiments. (C) Summary data as described for panel B, but showing the frequencies and absolute numbers of CD8⁺ cells that were K^b-SIFRFLNI⁺. (D) Representative flow cytometry plots showing frequencies of CD8⁺ cells that were K^b-TSYKFESV⁺ or K^b-SIFRFLNI⁺ in the spleen. (E) Column graphs corresponding to summary data for the frequencies of CD8⁺ cells that were K^b-TSYKFESV⁺. Data correspond to results for five mice per group ± SEM and are representative of two independent experiments. (F) Summary data as described for panel B, but for K^b-SIFRFLNI⁺ cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 4

Memory CD8⁺ T cells elicited by immunization with peptide-pulsed DCs become activated in the liver and spleen in response to ECTV infection. B6.D2-D6 mice were immunized with DCs pulsed with the indicated peptides and 2 months later infected with 3,000 PFU of ECTV in the footpad. (A and B) At 7 dpi, livers and spleens were analyzed in individual mice. Representative flow cytometry dot plots are shown for IFN-γ and GzB expression in liver mononuclear cells (A) and splenocytes (B). The top row shows control unimmunized, uninfected mice. All graphs are gated on CD8⁺ CD4⁻ cells. (C to F) Graphs show the summary data for the frequencies of CD8⁺ T cells expressing IFN-γ (C and D) or GzB (E and F) in the livers and spleens. Data correspond to five mice per group ± the SEM and are representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 5

Productive peptide-DC immunization results in protective immunity against lethal mousepox infection. B6.D2-D6 mice were immunized and boosted (1 week apart) with the indicated peptide-DC dimer and challenged more than 1 month later with 3,000 PFU of ECTV in the footpad. (A) Survival curve of B6.D2-D6 mice. The experiment is representative of three, with n of 5 per group. (B) Body weights over the course of infection. Data are expressed as the percent initial weight ± the SEM. (C and D) Virus titers in spleen and liver, respectively. Data correspond to 5 mice per group ± SEM and are representative of three independent experiments. (E) Liver histopathology (H&E stain) and immunohistochemistry (anti-EVM135 stain). Original magnifications are indicated. Data correspond to 5 mice per group ± the SEM and are representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 6

Memory CD8⁺ T cells induced by peptide-DC immunization protect mice against lethal mousepox infection in the absence of NK cells. B6 mice were immunized with peptide-pulsed DCs. (A) Representative data for the percentage of K^b-TSYKFESV-positive CD8⁺ in PBMCs from naïve B6 mice or B6 mice vaccinated with TSYKFESV-DC. (B) Column graphs correspond to summary data presented in the panel A flow cytometry plots for frequencies of CD8⁺ cells that were K^b-TSYKFESV⁺. Data correspond to 3 independent experiments (± SEM; n = 7 for naïve mice and n = 15 for TSYKFESV-vaccinated mice). (C) Survival of naïve and TSYKFESV-DC-immunized B6 mice that were depleted of NK cells with the PK136 monoclonal antibody i.p. and challenged with ECTV. Data correspond to 2 independent experiments ± SEM for naïve (n = 8) and TSYKFESV-immunized (n = 10) mice. ***, P < 0.001.