Long-lived effector/central memory T-cell responses to severe acute respiratory syndrome coronavirus (SARS-CoV) S antigen in recovered SARS patients

Abstract

The role of cell-mediated immunity in human SARS-CoV infection is still not well understood. In this study, we found that memory T-cell responses against the spike (S) protein were persistent for more than 1 year after SARS-CoV infection by detecting the production of IFN-gamma using ELISA and ELISpot assays. Flow cytometric analysis showed that both CD4(+) and CD8(+) T cells were involved in cellular responses against SARS-CoV infection. Interestingly, most of SARS-CoV S-specific memory CD4(+) T cells were central memory cells expressing CD45RO(+) CCR7(+) CD62L(-). However, the majority of memory CD8(+) T cells revealed effector memory phenotype expressing CD45RO(-) CCR7(-) CD62L(-). Thus, our study provides the evidence that SARS-CoV infection in humans can induce cellular immune response that is persistent for a long period of time. These data may have an important implication in the possibility of designing effective vaccine against SARS-CoV infection, specifically in defining T-cell populations that are implicated in protective immunity.

Figure 1

SARS-CoV S-peptide-specific IFN-γ production by PBMCs. Fresh PBMCs were isolated from fully recovered SARS patients and normal individuals and cultured in vitro with or without a pool of 169 SARS-CoV S peptides in the presence of anti-CD28 (1 μg/ml) and anti-CD49d mAbs (1 μg/ml). After incubation for 72 h, the level of IFN-γ in culture supernatants was assayed by ELISA (A). The frequency of IFN-γ-secreting cells was detected by ELISpot assay at the single cell level after stimulation for 20 h (B). Student's t test was used for statistical analysis. The significance was set at P < 0.01 (***). ns, not significant.

Figure 2

Expression of intracellular IFN-γ and IL-2 in PBMCs of recovered SARS patients after stimulation with SARS-CoV S peptides in vitro. Fresh PBMCs from recovered SARS patients were cultured for 5 h in vitro with a mixture of 169 SARS-CoV S peptides and stained with anti-CD4, CD8, IFN-γ and IL-2 mAbs. The expression of IFN-γ and IL-2 on CD4⁺ (A) and CD8⁺ (B) T cells was analyzed. The numbers in each quadrant were indicated as percentage. Data were representative of two of five recovered SARS individuals with similar results. The percentage (mean ± SE) of IFN-γ or IL-2-secreting cells from 5 independent experiments was shown within CD4 or CD8 T cells (C). Student's t test was used for statistical analysis. Statistical significance was set at P < 0.05 (*), ns, not significant.

Figure 3

The correlation of intensity of IFN-γ and IL-2 within SARS-CoV S-specific CD4 T cells. The subsets of SARS-CoV S-specific CD4⁺ T cells based on the expression of IFN-γ and IL-2 (A), the expression of IL-2 in both IFN-γ^high or IFN-γ^low subpopulations (B) and frequency as well as intensity of IFN-γ-producing cells in IL-2^high and IL-2^low subsets (C) (A, top panels, unstimulated cells; bottom panels, S-peptide-stimulated cells; B–C, left panels top quadrant, unstimulated cells; left panels bottom quadrant, S-peptide-stimulated cells). Percentage of indicated cells and the mean fluorescent intensity (MFI) were shown. Data were representative of two of five recovered SARS individuals.

Figure 4

Phenotypic analyses of memory CD4 T cells specific for SARS-CoV S peptides. Total CD4⁺ (A) or CD4⁺ IFN-γ⁺ cells (B) were analyzed for the expression of CD45RO and CCR7 (C) or CD45RO and CD62L (D). The numbers in each quadrant were indicated as percentage. Data were representative for 3 separate experiments with similar results.

Figure 5

Characteristics of SARS-CoV S peptides-specific memory CD8 T cells. Total CD8⁺ (A) or CD8⁺ IFN-γ⁺ (B) were analyzed for the expression of CD45RO and CCR7 expression (C) or CD45RO and CD62L (D). Data were representative of 1 of 3 independent experiments with similar results.