An SV40 VP1-derived epitope recognized by CD8+ T cells is naturally processed and presented by HLA-A*0201 and cross-reactive with human polyomavirus determinants

Abstract

The CD8+ T cell responses directed toward the VP1 antigens of human polyomaviruses JC and BK recently were shown to be cross-reactive. Two HLA-A0201-restricted determinants from each virus have been defined and include JCp100-108 (ILMWEAVTL) and BKp108-116 (LLMWEAVTV) as well as JCp36-44 (SITEVECFL) and BKp44-52 (AITEVECFL). We asked whether VP1 from the related SV40 contains similar HLA-A0201-restricted determinants. In this study, we demonstrate that CD8+ T cells specific for SV40 VP1 p110-118 (ILMWEAVTV), but not p46-54 (SFTEVECFL), can be induced in HLA-A0201-transgenic mice and that these CD8+ T cells cross-react with the corresponding determinants from JC and BK virus. The SV40 p110 determinant was found to be processed and presented in SV40-infected cells. These results indicate that the JCp36/BKp44 determinants are distinctive for the human polyomaviruses while the JCp100/BKp108/SVp110 determinants are shared by all three viruses, providing a target for CD8+ T cell cross-reactivity.

Figure 1

Sequence and peptide/MHC stability of HLA-A*0201-restricted polyomavirus VP1 determinants. A: Comparison of human polyomavirus VP1 determinants and the corresponding sequences from SV40 VP1. Residues with underlines vary from the JC virus sequence. B: VP1 peptide-induced stabilization of HLA-A*0201 surface expression. Peptides were incubated with T2 cells at the indicated concentrations overnight at 37°C and then stained for expression of HLA-A*0201. The control H-2K^b-binding peptide gB498–505 from Herpes simplex virus was used as a negative control.

Figure 2

The SVp110 determinant is immunogenic in HHDII mice. Groups of 3 HHDII mice were immunized subcutaneously with 100 µg of peptides SVp110 or SVp46 emulsified in IFA. Mice received a booster immunization after two weeks and spleens were harvested after a further 7 days. Spleen cells were restimulated in vitro with syngeneic DCs pulsed with the immunizing peptide and cultured for 6 days. Responder cells were tested for their ability to produce IFN-γ following stimulation with the indicated peptides and stained by ICS. The percentage of CD8+ T cells that produced IFN-γ is indicated in each dot plot. This experiment was performed three times using three mice per group and achieved similar results.

Figure 3

CD8+ T cells responsive to SVp110 are detected directly ex vivo and are highly cross-reactive with determinants from the human polyomaviruses. A: Groups of three HHDII mice were immunized with the indicated VP1 peptides as described in figure 2 and received a booster immunization after one week. After a further 7 days, spleen cells were harvested and subjected to ICS for IFN-γ following a 6 hour stimulation with the same peptide used for immunization or the Gag p17 peptide derived from HIV. The percentage of CD8+ T cells that produced IFN-γ is indicated in each dot plot. B. Splenocytes from A were restimulated in vitro for 6 days with syngeneic DCs pulsed with the immunizing peptide and then tested by ICS for IFN-γ production following stimulation for 6 hours with titrated amounts of each of the indicated peptides. Data are plotted as the percentage of CD8+ T cells that produced IFN-γ at a given peptide concentration. This experiment was performed twice using three mice per group and gave similar results to those shown.

Figure 4

Endogenously expressed VP1 determinants induce cross-reactive CD8+ T cell populations. HHDII mice were immunized with rVVs expressing ES-JCp100, ES-BKp108, ES-SVp110 or ES-SVT281. Ten days following immunization, spleens were harvested and evaluated by ICS for IFN-γ production following stimulation with the indicated peptides. The percentage of CD8+T cells responsive to each peptide is indicated in the dot plots. Data are representative of two independent experiments with three mice per group.

Figure 5

The SV40 epitope recognized by SVp110-induced CD8+ T cells is naturally processed and presented from SV40 infected cells. HHDII CD8+ T cell lines induced by peptide immunization with SVp110 (A) or SVT281 (B) and maintained by in vitro restimulation were tested for their ability to lyse SV40-infected (48 hrs) TC7 monkey cells co-infected with either rVV-A2.1 or rVV-Kd in a 4-hour ⁵¹Cr-release assay. The effector:target ratio (E:T) is indicated.