Mapping and role of T cell response in SARS-CoV-2-infected mice

Abstract

Virus-specific T cells play essential roles in protection against multiple virus infections, including SARS-CoV and MERS-CoV. While SARS-CoV-2-specific T cells have been identified in COVID-19 patients, their role in the protection of SARS-CoV-2-infected mice is not established. Here, using mice sensitized for infection with SARS-CoV-2 by transduction with an adenovirus expressing the human receptor (Ad5-hACE2), we identified SARS-CoV-2-specific T cell epitopes recognized by CD4+ and CD8+ T cells in BALB/c and C57BL/6 mice. Virus-specific T cells were polyfunctional and were able to lyse target cells in vivo. Further, type I interferon pathway was proved to be critical for generating optimal antiviral T cell responses after SARS-CoV-2 infection. T cell vaccination alone partially protected SARS-CoV-2-infected mice from severe disease. In addition, the results demonstrated cross-reactive T cell responses between SARS-CoV and SARS-CoV-2, but not MERS-CoV, in mice. Understanding the role of the T cell response will guide immunopathogenesis studies of COVID-19 and vaccine design and validation.

Figure S1.

Mapping SARS-CoV-2 T cell epitopes in BALB/c mice. (A) Lymphocytes from vaccinated lungs were stimulated with 5 µM 20-mer (20 amino acids) peptides. Antigen-specific CD4⁺ T cell responses were determined by intracellular IFN-γ staining. (B) Lymphocytes from vaccinated lungs were stimulated with 5 µM 20-mer (20 amino acids) peptides. Antigen-specific CD8⁺ T cell responses were determined. (C) Lymphocytes from vaccinated lungs were stimulated with 5 µM 20-mer (20 amino acids) and corresponding truncated 13–15-mer peptides for 5–6 h in the presence of brefeldin A. Antigen-specific CD4⁺ T cell responses were determined. (D) Lymphocytes from vaccinated lungs were stimulated with 5 µM 20-mer (20 amino acids) and corresponding truncated 8–9-mer peptides for 5–6 h in the presence of brefeldin A. Antigen-specific CD8⁺ T cell responses were determined. Candidate truncated epitopes are labeled with # (n = 3; data are representative of at least two independent experiments). All results are expressed as mean ± SEM. Ag, antigen; pep, peptide.

Figure S2.

Mapping SARS-CoV-2 T cell epitopes in C57BL/6 mice. (A) Lymphocytes from vaccinated lungs were stimulated with 5 µM 20-mer (20 amino acids) peptides. Antigen-specific CD4⁺ T cell responses were determined by intracellular IFN-γ staining. (B) Lymphocytes from vaccinated lungs were stimulated with 5 µM 20-mer (20 amino acids) peptides. Antigen-specific CD8⁺ T cell responses were determined. (C) Lymphocytes from vaccinated lungs were stimulated with 5 µM 20-mer (20 amino acids) and corresponding truncated 13–15-mer peptides. Antigen-specific CD4⁺ T cell responses were determined. (D) Lymphocytes from vaccinated lungs were stimulated with 5 µM 20-mer (20 amino acids) and corresponding truncated 8–9-mer peptides. Antigen-specific CD8⁺ T cell responses were determined. Candidate truncated epitopes are labeled with # (n = 3; data are representative of at least two independent experiments). All results are expressed as mean ± SEM. Ag, antigen; pep, peptide.

Figure 1.

Identification of CD4⁺ and CD8⁺ T cell epitopes in SARS-CoV-2–infected WT BALB/c and C57BL/6 mice. (A and B) Confirmation of CD4⁺ T cell epitopes (A) and CD8⁺ T cell epitopes (B) in infected BALB/c mice. Flow plots and summary columns are shown (n = 3; data verified in two independent experiments). (C and D) Confirmation of CD4⁺ T cell epitopes (C) and CD8⁺ T cell epitopes (D) in infected C57BL/6 mice. Flow plots and summary columns are shown (n = 3; data verified in two independent experiments). All results are expressed as mean ± SEM. Ag, antigen; pep, peptide.

Figure 2.

Kinetics of virus-specific T cell responses in BALF and lung of SARS-CoV-2–infected BALB/c and C57BL/6 mice. (A–D) Lymphocytes from airway and lung of transduced/infected WT BALB/c mice were harvested at indicated time points after infection and stimulated with 5 µM N351 (A and B) and 1 µM S535 (C and D) for 6 h in the presence of brefeldin A. The frequencies (left) and cell numbers of antigen-specific T cells (right) in BALF (A and C) and lung (B and D) are shown (n = 3 or 4 mice; data are representative of three independent experiments). (E–H) Lymphocytes from airway and lung of transduced/infected C57BL/6 mice were harvested at indicated time points and stimulated with 5 µM ORF3a 266 (E and F) and 1 µM S538 (G and H) for 6 h in the presence of brefeldin A. The frequencies (left) and cell numbers (right) of antigen-specific T cells are shown (n = 3 or 4 mice; data are representative of three independent experiments). All results are expressed as mean ± SEM. Ag, antigen; pep, peptide; p.i., post-infection.

Figure S3.

Kinetics of virus-specific T cell responses in DLNs and spleens of SARS-CoV-2–infected BALB/c and C57BL/6 mice. (A–D) Lymphocytes from DLN and spleen of transduced/infected WT BALB/c mice were harvested at indicated time points after infection and stimulated with 5 µM N351 (A and B) and 1 µM S535 (C and D) for 6 h in the presence of brefeldin A. The frequencies (left) and cell numbers of antigen-specific T cells (right) in DLN (A and C) and spleen (B and D) are shown (n = 3 mice; data are representative of one experiment). (E–H) Lymphocytes from DLN and spleen of transduced/infected C57BL/6 mice were harvested at indicated time points and stimulated with 5 µM ORF3a 266 (E and F) and 1 µM S538 (G and H) for 6 h in the presence of brefeldin A. The frequencies (left) and cell numbers (right) of antigen-specific T cells are shown (n = 3; data are representative of one experiment). All results are expressed as mean ± SEM. Ag, antigen; pep, peptide; p.i., post-infection.

Figure S4.

SARS-CoV-2–specific CD8⁺ T cells were polyfunctional in infected C57BL/6 mice. (A) Cells from BALF were stained with antibodies against the indicated markers. Histograms shown here were gated on SARS-CoV-2-S538–specific CD8⁺ T cells. (B) Cytokine expression of airway derived SARS-CoV-2-S538–specific CD8⁺ T cells are shown (n = 3 or 4 mice; data are representative of one experiment). (C) Functional avidity curves (left) of airway- and lung-derived SARS-CoV-2-S538–specific CD8⁺ T cells and the amount of peptide required for half-maximum response (EC₅₀) are shown (right; n = 3; data are representative of two independent experiments; Student’st tests; P value of C is 0.011). (D) Representative flow histograms (left) and killing rates (right) of in vivo cytotoxicity of SARS-CoV-2-S538–specific CD8⁺ T cells in SARS-CoV-2–infected mice and mock-infected mice are shown (n = 5; data are representative of one experiment; Student’st tests; P value of C is <0.0001). ****, P < 0.0001. All results are expressed as mean ± SEM. Max, maximum; pep, peptide.

Figure 3.

SARS-CoV-2–specific CD4⁺ T cells and CD8⁺ T cells were polyfunctional. (A and E) Phenotyping BALF-derived SARS-CoV-2-N351–specific CD4⁺ T cells (A) and SARS-CoV-2-S535–specific CD8⁺ T cells (E). (B and F) Cytokine expression of airway-derived N351-specific CD4⁺ T cells (B) and S535-specific CD8⁺ T cells (F) are shown (n = 3 mice; data are representative of two independent experiments). (C and G) Functional avidity curves (left) of airway- and lung-derived N351-specific CD4⁺ T cells (C) and S535-specific CD8⁺ T cells (G) and the amount of peptide required for half-maximum response (EC₅₀) are shown (right; n = 3 mice; data are representative of two independent experiments; Student’st tests; P value of C is 0.0004; P value of G is 0.0051). (D and H) Representative flow histograms (left) and killing rates (right) of in vivo cytotoxicity of N351-specific CD4⁺ T cells (D) and S535-specific CD8⁺ T cells (H) in SARS-CoV-2–infected mice and mock-infected mice are shown (n = 5 mice per group; data are representative of two independent experiments; Student’st tests; P value of D is 0.0062; P value of H is 0.0002). *, P < 0.05; ***, P < 0.0005. All results are expressed as mean ± SEM. Max, maximum; pep, peptide.

Figure 4.

IFN-I signaling was critical for the generation of robust T cell responses against SARS-CoV-2 infection. (A and B) Frequencies (left) and cell numbers (right) of airway-derived N351-specific CD4⁺ T cells (A) and S535-specific CD8⁺ T cells (B) at indicated time points are shown (n = 3 or 4 mice per time point; data are representative of three independent experiments). (C and D) Airway-derived N351-specific CD4⁺ T cell responses (C) and S535-specific CD8⁺ T cell responses (D) in WT and KO BALB/c mice are compared (n = 3 or 4 mice; data are representative of two independent experiments; Student’st tests; P values of C are 0.0006 and 0.0013; P values of D are 0.0005 and 0.0029). (E and F) Representative flow plots of N351-specific CD4⁺ T cells (E) and S535-specific CD8⁺ T cells (F) are shown (left). Bi-cytokine expression capability (right three panels) is statistically different between WT and KO mice (n = 3 or 4 mice; data are representative of two independent experiments; Student’st tests; P values of E are 0.0003, 0.0057, and 0.0004; P values of F are <0.0001, 0.0445, and <0.0001). (G and H) Functional avidity curves (left) of N351-specific CD4⁺ T cells (G) and S535-specific CD8⁺ T cells (H) in KO and WT mice and the amount of peptide required for half-maximum response (EC₅₀) are shown (right; n = 3 mice; data are representative of two independent experiments; Student’st tests; P value of G is 0.050; P value of H is 0.052). (I) Weight loss and viral titers in the lungs were measured at the indicated time points (n = 3–5 mice per group per time point for viral titer; data are representative of two independent experiments; Student’st tests; P values are 0.0291 and 0.0394). *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.0001. All results are expressed as mean ± SEM. Ag, antigen; FFU, focus-forming unit; Max, maximum; pep, peptide; p.i., post-infection.

Figure 5.

Epitope-specific CD4⁺ and CD8⁺ T cells partially protected SARS-CoV-2–infected mice from severe disease. (A) Strategy of VRP vaccination and SARS-CoV-2 challenge. (B and C) Effects of N351-specific CD4⁺ T cells (B) and S535-specific CD8⁺ T cells (C) in BALB/c mice. Cell numbers of airway-derived antigen-specific T cells are shown (n = 3 or 4 mice per group per time point; left). Viral titers in the lungs were measured at the indicated time points (n = 4 mice per group per time point; middle; data are representative of at least two independent experiments; Student’st tests; P values of B are 0.0051 and 0.0403; P values of C are 0.0026 and 0.0804). Sections of paraffin-embedded lungs from infected mice at 4 d.p.i. were stained with hematoxylin/eosin (n = 3 mice per group per time point; right; data are representative of at least two independent experiments). Scale bar, 100 mm. (D) Effects of S538-specific CD8⁺ T cells in C57BL/6 mice. Cell numbers at indicated time points are shown (n = 3 mice per group per time point; left). Viral titers in the lungs were measured at the indicated time points (n = 4 mice per group per time point; middle; data are representative of at least two independent experiments; Student’st tests; P values of D are 0.0372 and 0.0043). Sections of paraffin-embedded lungs from infected mice at 6 d after infection were stained with hematoxylin/eosin (n = 3 mice per group per time point; right; data are representative of at least two independent experiments). Scale bar, 100 mm. *, P < 0.05; **, P < 0.005. All results are expressed as mean ± SEM. Arrowheads, hemorrhage; asterisks, edema; Ag, antigen; D, day; FFU, focus-forming unit; p.i., post-infection.

Figure S5.

No neutralizing antibodies in BALB/c mice at the time of challenge. Neutralizing antibodies titers in the sera of vaccinated and infected BALB/c mice at time of challenge and 14 d.p.i. (n = 6; data are representative of one experiment). All results are expressed as mean ± SEM. LOD, limit of detection; p.i., post-infection.

Figure 6.

Cross-reactive T cell responses were found between SARS-CoV-2 and SARS-CoV in SARS-CoV-2–infected mice. (A) Characteristics of conserved T cell epitopes in SARS-CoV-2, SARS-CoV, and MERS-CoV. (B and C) BALB/c mice were transduced and infected with SARS-CoV-2. Lymphocytes derived from airway were prepared at 8 d.p.i. and stimulated with conversed epitopes. CD4⁺ (B) and CD8⁺ T cell responses (C) were detected by IFN-γ expression. Flow plots (left) and cross-reactivity rate (right) are shown (n = 3 or 4 mice per group; data are representative of two independent experiments). (D) Functional avidity curves (left) of S535-specific CD8⁺ T cells and S521–cross-reactive CD8⁺ T cells and the amount of peptide required for half-maximum response (EC₅₀) are shown (right; n = 3 mice; data are representative of two independent experiments; Student’st tests; P value of D is 0.0182). *, P < 0.05. All results are expressed as mean ± SEM. Max, maximum; pep, peptide.