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Human memory T cell responses to SARS-CoV E protein - PubMed

Human memory T cell responses to SARS-CoV E protein

Hui Peng et al. Microbes Infect. 2006 Aug.

Abstract

E protein is a membrane component of severe acute respiratory syndrome coronavirus (SARS-CoV). Disruption of E protein may reduce viral infectivity. Thus, the SARS-CoV E protein is considered a potential target for the development of antiviral drugs. However, the cellular immune responses to E protein remain unclear in humans. In this study, we found that peripheral blood mononuclear cells (PBMCs) from fully recovered SARS individuals rapidly produced IFN-gamma and IL-2 following stimulation with a pool of 9 peptides overlapping the entire E protein sequence. Analysis of the immune responses by flow cytometry showed that both CD4+ and CD8+T cells were involved in the SARS-CoV E-specific immune responses after stimulation with SARS-CoV E peptides. Moreover, the majority of IFN-gamma+CD4+T cells were central memory cells expressing CD45RO+CCR7+CD62L-; whereas IFN-gamma+CD8+ memory T cells were mostly effector memory cells expressing CD45RO-CCR7-CD62L-. The results of T-cell responses to 9 individual peptides indicated that the E protein contained at least two major T cell epitopes (E2 amino acid [aa] 9-26 and E5-6: aa 33-57) which were important in eliciting cellular immune response to SARS-CoV E protein in humans.

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Figures

Fig. 1
Fig. 1

IFN-γ production by PBMCs from SARS-recovered individuals in response to a pool of E peptides. (A) PBMCs from six SARS-recovered donors were cultured in 96-well plates with or without a pool of E peptides for 72 h. The culture supernatants were collected and assessed for the production of IFN-γ by ELISA. All assays were performed in triplicate. (B) PBMCs from eight SARS-recovered donors were stimulated with or without a pool of E peptides. IFN-γ-producing cells were determined by ELISPOT assay. The number of spots in the medium control wells ranged from 0 to 2. All assays were performed in triplicate. Normal subjects without any contact history with SARS patients were used as negative controls. Statistical analysis was performed using the Student's t-test, and a statistically significant difference was set at P < 0.05(**). Bars indicate mean values.

Fig. 2
Fig. 2

SARS-CoV E peptide-specific IFN-γ-producing T cells in PBMCs, intracellular staining. PBMCs from SARS-recovered donors were incubated with a pool of peptides for 6 h. Cell surface and intracellular cytokine staining for IFN-γ was performed. The cells were first gated on CD8+T and CD4+T cells and subsequently analyzed for IFN-γ expression. (A) Results shown are from one experiment performed on PBMCs from a SARS patient and are representative of 5 independent experiments from 5 SARS patients. (B) Results represent the expression of IFN-γ as percentage of total CD4+ or CD8+T cells in different patients. Bars indicate mean values.

Fig. 3
Fig. 3

IL-2 and IFN-γ expression in CD4+T cells in response to E peptides. PBMCs from SARS-recovered donors were stimulated with a pool of E peptides for 6 h. The expression of IL-2 and IFN-γ was assessed by flow cytometry. (A) Dot plots show intracellular staining for IL-2 in the CD4+T cells. (B) The frequency of cells expressing both IL-2 and IFN-γ in CD4+T cells from SARS-recovered donors. The data are representative of three independent experiments with similar results. (C) Results presented as a percentage of the total number of cells expressing cytokines.

Fig. 4
Fig. 4

Functional characterization of E protein-specific IFN-γ-producing memory T cells. PBMCs from SARS-recovered donors were stimulated with a pool of E peptides for 6 h. The expression of CCR7, CD62L and CD45RO in IFN-γ+CD4+ or IFN-γ+CD8+T cells was assessed by flow cytometry. CD4+CD8− or CD4−CD8+ cells were gated and analyzed. Results shown are from one experiment performed on PBMCs from a SARS patient.

Fig. 5
Fig. 5

Screening of T cell responses to individual peptide of SARS-CoV E protein. (A) PBMCs from 3 SARS-recovered donors were stimulated with an individual peptide of SARS-CoV E protein (1 μg/ml of each peptide). The frequency of IFN-γ producing cells was determined by ELISPOT assay. PBMCs incubated with media alone were used as controls. The experiments were carried out in triplicate.

Fig. 6
Fig. 6

Memory T cell responses specific to peptides E2, E5 and E6. PBMCs from SARS-recovered donors were incubated with the indicated peptides (E2, E5 or E6) for 6 h. Intracellular staining for IFN-γ was performed. CD4+ and CD8+T cells were gated and subsequently analyzed for IFN-γ expression by flow cytometry. Results shown are representative of 3 independent experiments from 3 SARS patients. Similar results were obtained in other experiments.

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