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. 2010 Feb;84(3):1289-301.
doi: 10.1128/JVI.01281-09. Epub 2009 Nov 11.

Cellular immune responses to severe acute respiratory syndrome coronavirus (SARS-CoV) infection in senescent BALB/c mice: CD4+ T cells are important in control of SARS-CoV infection

Jun Chen  1 , Yuk Fai Lau, Elaine W Lamirande, Christopher D Paddock, Jeanine H Bartlett, Sherif R Zaki, Kanta Subbarao
Affiliations

Cellular immune responses to severe acute respiratory syndrome coronavirus (SARS-CoV) infection in senescent BALB/c mice: CD4+ T cells are important in control of SARS-CoV infection

Jun Chen et al. J Virol. 2010 Feb.

Abstract

We characterized the cellular immune response to severe acute respiratory syndrome coronavirus (SARS-CoV) infection in 12- to 14-month-old BALB/c mice, a model that mimics features of the human disease. Following intranasal administration, the virus replicated in the lungs, with peak titers on day 2 postinfection. Enhanced production of cytokines (tumor necrosis factor alpha [TNF-alpha] and interleukin-6 [IL-6]) and chemokines (CXCL10, CCL2, CCL3, and CCL5) correlated with migration of NK cells, macrophages, and plasmacytoid dendritic cells (pDC) into the lungs. By day 7, histopathologic evidence of pneumonitis was seen in the lungs when viral clearance occurred. At this time, a second wave of enhanced production of cytokines (TNF-alpha, IL-6, gamma interferon [IFN-gamma], IL-2, and IL-5), chemokines (CXCL9, CXCL10, CCL2, CCL3, and CCL5), and receptors (CXCR3, CCR2, and CCR5), was detected in the lungs, associated with an influx of T lymphocytes. Depletion of CD8(+) T cells at the time of infection did not affect viral replication or clearance. However, depletion of CD4(+) T cells resulted in an enhanced immune-mediated interstitial pneumonitis and delayed clearance of SARS-CoV from the lungs, which was associated with reduced neutralizing antibody and cytokine production and reduced pulmonary recruitment of lymphocytes. Innate defense mechanisms are able to control SARS-CoV infection in the absence of CD4(+) and CD8(+) T cells and antibodies. Our findings provide new insights into the pathogenesis of SARS, demonstrating the important role of CD4(+) but not CD8(+) T cells in primary SARS-CoV infection in this model.

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Figures

FIG. 1.
FIG. 1.
Kinetics of inflammatory mediators in the lungs following SARS-CoV infection. Protein levels of cytokines (A) and chemokines (B) and fold increase in gene transcripts of chemokines and the receptors (C) were measured in the lungs at the indicated time points following i.n. inoculation of 105 TCID50 SARS-CoV. Data are shown as mean ± standard error of the mean (SEM) for four mice at each time point, and the units are reported next to the name of the protein. *, P < 0.05 for the comparison between mock- and SARS-infected groups by Student's t test.
FIG. 2.
FIG. 2.
Presence of inflammatory cells in the lungs of mice following SARS-CoV infection. (A) FACS analysis of inflammatory cells (CD11c+ PDCA1+ for pDC, CD3− DX5+ for NK, CD3+ DX5+ for NKT, CD11b+ F4/80+ for macrophages, and CD11c− Gr1+ for neutrophils) in mock-infected and SARS-CoV-infected lungs. Cells for analysis are gated on a CD45+ population. (B) Total number of inflammatory cells isolated from the lungs on the indicated day p.i. Data are the means ± SEMs for three to five mice analyzed at each time point. Results from one representative experiment out of three are shown. *, P < 0.05; **, P < 0.01 (for the comparison between mock-and SARS-CoV-infected groups) by Student's t test. (C) FACS analysis of IFN-γ-producing CD8+ and CD4+ T cells isolated from the lungs of SARS-CoV-infected mice on day 7 p.i., with or without ex vivo stimulation with PMA/ionomycin or SARS-CoV S or N peptides. Cells for analysis are gated on a CD3+ population. (D) Total number of CD8+ and CD4+ T cells producing IFN-γ in the lungs of mock- or SARS-CoV-infected mice, with or without ex vivo stimulation with PMA/ionomycin or SARS-CoV S or N peptides. Data are from pools of cells from four mice analyzed at day 7 p.i. Results from a representative experiment out of two is shown.
FIG. 3.
FIG. 3.
Delayed viral clearance in the lungs of SARS-CoV-infected mice depleted of CD4+ T cells. (A) Depletion of T-cell subsets in senescent mice infected with SARS-CoV. Mice were treated with MAbs i.p. at 3 days before and 3 and 7 days after SARS-CoV infection. (B) FACS analysis of CD4 and CD8 expression on lymphocytes isolated from lungs and spleen on day 9 p.i. The recorded numbers denote the percentages of CD4+ or CD8+ cells in the total lymphocyte population. (C) Virus titers in the lungs were determined on the indicated day p.i. The limit of detection was 101.5 TCID50/g of tissue (dashed line). Data are means ± SEMs for four mice at each time point. (D) IHC staining of SARS-CoV antigen in the lungs of mice depleted of T-cell subsets. By day 9 p.i., SARS-CoV antigens (red) are extremely sparse or entirely absent from the lungs of infected mice (a) and are rare in the lungs of mice depleted of CD8+ T cells (c); however, abundant viral antigens remain in the lungs of mice depleted of CD4+ T cells (b) or both CD4+ and CD8+ T cells (d) at day 9 (immunoalkaline phosphatase stain with naphthol-fast red and hematoxylin counterstain).
FIG. 4.
FIG. 4.
Reduced Ab production in mice depleted of CD4+ T cells. Mice were treated with isotype control and MAbs against T-cell subsets as described in Materials and Methods. (A) Neutralizing Ab titers in the serum were determined on the indicated day p.i. Geometric mean neutralizing Ab titers from four mice per group are expressed as log2. (B) Virus-specific IgG titers in the serum were determined by ELISA on the indicated day p.i. Data are means ± SEMs for four mice at each time point. (C) HIS or NMS (500 μl per mouse) was given i.p. to mice depleted of CD4+ or CD4+ and CD8+ T cells on day 7 p.i., and virus titers in the lungs were determined on day 9 p.i. The limit of detection was 101.5 TCID50/g of tissue (dashed line). Data are means ± SEMs for four mice at each time point.
FIG. 5.
FIG. 5.
Reduced T-cell responses in the lungs of SARS-CoV-infected mice depleted of CD4+ T cells. Mice were treated with isotype control and MAbs against T-cell subsets as described in Materials and Methods. (A) Cytokines and chemokines were determined in lung homogenates by Cytokine Beads Array and Bioplex on day 7 p.i. Data are shown as means ± SEMs for four mice. (B) Number of cells isolated from the lungs of mice treated with MAbs for FACS analysis by day 9 p.i. Data are presented as means ± SEMs for four mice from a typical experiment. (C) Intracellular staining for IFN-γ on CD4+ and CD8+ T cells isolated from the lungs by day 9 p.i., with ex vivo stimulation with SARS-CoV antigen. Data are presented as means ± SEMs for four mice. (D) FACS analysis of CD107 expression on IFN-γ-negative (subset A) and IFN-γ-positive (subset B) T cells isolated from the lungs on day 9 p.i., with ex vivo stimulation with viral antigen. The recorded numbers denote the percentages of IFN-γ-positive CD4+ or CD8+ T cells expressing CD107.
FIG. 6.
FIG. 6.
Histopathologic changes in the lungs of SARS-CoV-infected mice at day 9 p.i. (A) Mice were treated with isotype control Ab (a and b), anti-CD8 (c and d), anti-CD4 (e and f), and anti-CD4 and CD8 Ab (g and h) as described in Materials and Methods. Compared to isotype control-treated mice (a), perivascular lymphocytic infiltrates are diminished in mice by treatment with anti-CD8 (c), anti-CD4 (e), and anti-CD4 and CD8 Ab (g) MAbs. Multifocal interstitial infiltrates are identified in the lungs of all infected mice (b, d, f, and h) but are most prominent in the mice depleted of CD4+ T cells (f). (B) Pathology scores of pulmonary inflammation in the lungs were assessed on day 9 p.i. Data represent means ± SEMs for four mice at the indicated time point.
FIG. 7.
FIG. 7.
Time course of host responses to primary SARS-CoV infection in senescent mice. A biphasic expression of inflammatory mediators associated with cellular infiltration into the lungs of infected mice, coincident with peaks in viral replication and clearance, respectively, is seen. Clinical illness such as weight loss was observed coincident with pulmonary viral replication, while lung pathology (pneumonitis) was associated with T-cell infiltration when virus was cleared from the lung.
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