CD8+ T cell response in HLA-A*0201 transgenic mice is elicited by epitopes from SARS-CoV S protein

Abstract

Cytotoxic CD8(+) T lymphocytes (CTLs) play an important role in antiviral immunity. Several human HLA-A*0201 restricted CTL epitopes of severe acute respiratory syndrome (SARS) spike (S) protein have been identified in HLA-A*0201 transgenic (Tg) mice, but the mechanisms and properties of immune responses are still not well understood. In this study, HLA-A*0201 Tg mice were primed intramuscularly with SARS S DNA and boosted subcutaneously with HLA-A*0201 restricted peptides. The lymphocytes from draining lymph nodes, spleens and lungs were stimulated with the cognate peptides. Three different methods (ELISA, ELISPOT and FACS) were used to evaluate the immune responses during short and long periods of time after immunization. Results showed that peptide-specific CD8(+) T cells secreted IFN-γ, TNF-α and IL-2 and expressed CD107a/b on cell surface. IFN-γ(+)CD8(+) T cells and CD107a/b(+)CD8(+) T cells distributed throughout the lymphoid and non-lymphoid tissues, but the frequency of peptide-specific CD8(+) T cells was higher in lungs than in spleens and lymph nodes. The phenotype of the CD8(+) T cells was characterized based on the expression of IFN-γ. Most of the HLA-A*0201 restricted peptide-specific CD8(+) T cells represented a memory subset with CD45RB(high) and CD62L(low). Taken together, these data demonstrate that immunization with SARS S DNA and HLA-A*0201 restricted peptides can elicit antigen-specific CD8(+) T cell immune responses which may have a significant implication in the long-term protection. We provide novel information in cellular immune responses of SARS S antigen-specific CD8(+) T cells, which are important in the development of vaccine against SARS-CoV infection.

Fig. 1

Cellular immune responses were elicited by HLA-A*0201 restricted peptides. Tg mice (3–12 mice/group) were vaccinated with different agents in a similar manner as described. Fresh isolated lymphocytes from spleens, lymph nodes and lungs were cultured with 2 μg/ml cognate peptides. (A) The production of IFN-γ in supernatants was detected by ELISA. (B) The production of TNF-α and IL-2 were detected respectively in different tissues from vaccinated mice with SARS S DNA and HLA-A*0201 peptides. Cultures were set up in triplicate. Each symbol represents one data point from one mouse. *P < 0.05; **P < 0.01; ***P < 0.001; N.S. P > 0.05.

Fig. 2

Frequency of HLA-A*0201 restricted peptide-specific IFN-γ producing cells. Mice were vaccinated with SARS S DNA and HLA-A*0201 restricted peptides as described in Section 2. Cells from spleens were cultured at a density of 4 × 10⁵ cells/well in 96 well pre-coated plates. Assay was performed following instruction. (A) Representation of ELISPOT results. (B) The frequency of IFN-γ producing cell (n = 4). Cultures were set up in triplicate. Each symbol represents one data point from one mouse. *P < 0.05. Horizontal bars represent mean values.

Fig. 3

CD8⁺ T cells but not CD4⁺ T cells express IFN-γ in HLA-A*0201 restricted peptides stimulated splenocytes. Cells were cultured with or without peptides for 6 h, and then were harvested and stained for FACS analysis. (A) The two subpopulations of T cells. (B) One representative result from three independent experiments showing the expressing of IFN-γ. The numbers in the left corner represent the mean percentage of positive cells in gated T cells.

Fig. 4

The correlation of cytokines produced by HLA-A*0201 restricted peptide-specific CD8⁺ T cells. Tg mice were vaccinated with SARS S DNA and HLA-A*0201 restricted peptides. 10 d after the vaccination, splenocytes were harvested and stimulated with cognate peptides. Then intracellular cytokine staining with flow cytometry was performed. The cells were initially gated at CD8⁺ T cells. (A) The expression of IL-2, TNF-α and IFN-γ in HLA-A*0201 restricted peptide-specific CD8⁺ T cells. The number of positive cells was analyzed by flow cytometry and shown in the corner of each panel. The results of one representative assay from four similar independent experiments are shown. (B) Summaries of the independent experiments. The data represents the means of four independent experiments.

Fig. 5

Characteristics of HLA-A*0201 restricted peptide-specific CD8⁺ T cells. Tg mice were primed with SARS S DNA and boosted with HLA-A*0201 restricted peptides. 10 d after boost vaccination, splenocytes were harvested and stimulated with cognate peptides ex vivo for 6 h. The cells were stained with cell surface anti-CD45RB and anti-CD62L Abs, and then the intracellular cytokine IFN-γ was marked. Based on the expression of IFN-γ, characteristics of HLA-A*0201 restricted peptide-specific CD8⁺ T cells were analyzed by flow cytometry. (A) The expression of CD45RB and CD62L by IFN-γ⁺CD8⁺ T cells. Representative results are shown from four independent experiments. (B) The data were quantified and presented in a ring chart. Each slice of the ring represents the fraction of the mean value of a given quadrant.

Fig. 6

The expression of cytolytic effector molecules by HLA-A*0201 restricted peptide-specific CD8⁺ T cells. (A) Expression of TNF-α, IFN-γ and CD107a/b by CD8⁺ T cells. Splenocytes were obtained from Tg mice after SARS S DNA and HLA-A*0201 peptides vaccination as described in Section 2. Then splenocytes were stimulated with cognate peptides in vitro for 6 h and stained for the expression of intracellular cytokines (TNF-α and IFN-γ) and cell surface CD107a/b. Representative results are shown from three independent experiments. (B) The percentage of positive cells in CD8⁺ T cells. The bars represent the mean ± SD.

Fig. 7

The distribution of peptide-specific CD8⁺ T cells in different tissues. Lymphocytes from lymph nodes, spleens and lungs were obtained from Tg mice 10 d after SARS S DNA and HLA-A*0201 restricted peptides vaccination. The cells from different tissues were stimulated with cognate peptides in vitro. (A) IFN-γ producing cells in lymphoid tissues and non-lymphoid tissues. Based on the expression of IFN-γ, the distributions of peptide-specific CD8⁺ T cells in different organs are shown. (B) CD107a/b⁺CD8⁺ T cells were determined in lymph nodes, spleens and lungs. (C) The percentage of IFN-γ producing cells in CD8⁺ T cells was analyzed by flow cytometry. (D) The percentage of CD107a/b⁺CD8⁺ T cells. One of representative flow cytometric analysis from three independent experiments is shown in the left. The bars represent the mean ± SD.

Fig. 8

Long-term persistence of effector/memory CD8⁺ T cells following SARS S DNA and HLA-A*0201 restricted peptides vaccination. Tg mice were vaccinated as described in Section 2. Cells were prepared from spleens 30 d after HLA-A*0201 restricted peptides boost vaccination and were incubated with cognate peptide-loaded DCs for 6 h. Then cells were stained with surface anti-CD8 Ab, anti-CD45RB Ab, anti-CD62L Ab, and intracellular cytokine Abs anti-IFN-γ and anti-TNF-α. CD8⁺ T cells were first gated. (A) The expression of IFN-γ and TNF-α by CD8⁺ T cells. (B) The phenotype of peptide-specific CD8⁺ T cells, based on IFN-γ production. The numbers at the corner represent the mean percentages of positive cells from three independent experiments.