PD-1-Expressing SARS-CoV-2-Specific CD8+ T Cells Are Not Exhausted, but Functional in Patients with COVID-19

Abstract

Memory T cell responses have been demonstrated in COVID-19 convalescents, but ex vivo phenotypes of SARS-CoV-2-specific T cells have been unclear. We detected SARS-CoV-2-specific CD8⁺ T cells by MHC class I multimer staining and examined their phenotypes and functions in acute and convalescent COVID-19. Multimer⁺ cells exhibited early differentiated effector-memory phenotypes in the early convalescent phase. The frequency of stem-like memory cells was increased among multimer⁺ cells in the late convalescent phase. Cytokine secretion assays combined with MHC class I multimer staining revealed that the proportion of interferon-γ (IFN-γ)-producing cells was significantly lower among SARS-CoV-2-specific CD8⁺ T cells than those specific to influenza A virus. Importantly, the proportion of IFN-γ-producing cells was higher in PD-1⁺ cells than PD-1^- cells among multimer⁺ cells, indicating that PD-1-expressing, SARS-CoV-2-specific CD8⁺ T cells are not exhausted, but functional. Our current findings provide information for understanding of SARS-CoV-2-specific CD8⁺ T cells elicited by infection or vaccination.

Keywords: CD8(+) T cell; COVID-19; IFN-γ; MHC class I multimer; Memory T cell; PD-1; SARS-CoV-2.

Graphical abstract

Figure 1

Ex Vivo Detection of SARS-CoV-2-Specific CD8⁺ T Cells Using MHC Class I Multimers (A) Study scheme for the detection of SARS-CoV-2-specific MHC class I multimer⁺CD8⁺ T cells in PBMCs from individuals with SARS-CoV-2 infection. (B) SARS-CoV-2-specific MHC class I multimers used in this study. (C) Summary data for the detection rate of SARS-CoV-2-specific multimer⁺CD8⁺ T cells. (D) Representative flow cytometry plots (S₂₆₉, n = 37; S_1220, n = 2) showing the ex vivo detection of SARS-CoV-2 S₂₆₉ and S₁₂₂₀ multimer⁺CD8⁺ T cells in the gate of CD3⁺ T cells. (E) Alignment of the amino acid sequences of SARS-CoV-2 S₂₆₉ and S₁₂₂₀ peptides with other human coronaviruses. See also Figure S1.

Figure 2

Phenotypes of SARS-CoV-2-Specific Multimer⁺CD8⁺ T Cells in the Convalescent Phase of COVID-19 (A–H) PBMCs from convalescent patients were analyzed by flow cytometry. (A) Representative flow cytometry plots (n = 32) showing the percentages of indicated subsets among SARS-CoV-2 S₂₆₉ multimer⁺CD8⁺ T cells. (B and C) The frequency of SARS-CoV-2 S₂₆₉ multimer⁺CD8⁺ T cells among total CD8⁺ T cells (B) and the percentages of the indicated subsets among SARS-CoV-2 S₂₆₉ multimer⁺CD8⁺ T cells (C) in the early convalescent samples were compared between the non-severe (n = 11) and severe (n = 8) groups. (D and E) The frequency of SARS-CoV-2 S₂₆₉ multimer⁺CD8⁺ T cells among total CD8⁺ T cells (D) and the percentages of the indicated subsets among SARS-CoV-2 S₂₆₉ multimer⁺CD8⁺ T cells (E) were compared between the early (n = 19) and late (n = 13) convalescent groups. (F) Correlation between the expression of the indicated markers in SARS-CoV-2 S₂₆₉ multimer⁺CD8⁺ T cells and days post-symptom onset (n = 30). (G and H) The phenotypes of SARS-CoV-2 S₂₆₉ multimer⁺CD8⁺ T cells (early convalescent phase, n = 19; late convalescent phase, n = 13) were compared with those of IAV MP₅₈ multimer⁺CD8⁺ T cells (n = 11; 3 HDs and 8 convalescent individuals). Representative flow cytometry plots (early convalescent phase, n = 19; late convalescent phase, n = 13; IAV, n = 11) (G) and summary data (H) of the percentages of all indicated subsets among CD8⁺ T cells specific for each virus. Data are presented as median and interquartile range (IQR). Statistical analysis was performed using the Mann–Whitney U test (B–E and H) or Spearman correlation test (F). ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001. See also Figure S2.

Figure 3

Proliferation and Effector Function of SARS-CoV-2-Specific CD8⁺ T Cells from Convalescent Patients (A) CTV-labeled PBMCs from convalescent COVID-19 patients (n = 6) were stimulated with S₂₆₉ peptide (10 μg/mL) for 7 days and analyzed by flow cytometry. Flow cytometry plots show the frequency of CTV^low cells among SARS-CoV-2 S₂₆₉ multimer⁺CD8⁺ T cells. (B–E) PBMCs were stimulated with SARS-CoV-2 S₂₆₉ peptide (n = 10; 3 early convalescent samples with non-severe disease, 4 early convalescent samples with severe disease, and 3 late convalescent samples), CMV pp65₄₉₅ peptide (n = 7; 4 HDs and 3 convalescent individuals), or IAV MP₅₈ peptide (n = 7; 2 HDs and 5 convalescent individuals). The concentration of each peptide was 1 μg/mL. After 5.5 h of stimulation, CSAs were performed in combination with MHC class I multimer staining to analyze the frequency of IFN-γ-producing cells among MHC class I multimer⁺ cells. Representative flow cytometry plots (early convalescent samples with non-severe disease, n = 3; early convalescent samples with severe disease, n = 4; late convalescent samples, n = 3; IAV, n = 7) (B) and summary data (C) are presented. Horizontal lines represent the median. (D) Representative flow cytometry plots (n = 10) and (E) summary data showing the frequency of IFN-γ-producing cells among PD-1⁺ and PD-1⁻ subpopulations of SARS-CoV-2 S₂₆₉ multimer⁺CD8⁺ T cells from convalescent COVID-19 patients. (F and G) Sorted PD-1⁺CD8⁺ and sorted PD-1⁻CD8⁺ T cells from PBMCs of COVID-19 patients (n = 3) were stimulated with SARS-CoV-2 S₂₆₉ peptide (1 μg/mL) for 5.5 h and CSAs performed in combination with MHC class I multimer staining. Representative flow cytometry plots (n = 3) (F) and summary data (G) showing the frequency of IFN-γ-producing cells in the SARS-CoV-2 S₂₆₉ multimer⁺CD8⁺ T cell population among sorted PD-1⁺ and PD-1⁻CD8⁺ T cells. Statistical analysis was performed using the Mann–Whitney U test (C) or the Wilcoxon signed-rank test (E). ^∗∗p < 0.01.

Figure 4

Phenotypes, Kinetics, and Effector Function of SARS-CoV-2-Specific Multimer⁺CD8⁺ T Cells in the Acute Phase of COVID-19. (A) PBMCs from patients in the acute phase of COVID-19 (n = 10) were analyzed by flow cytometry. Left, Representative flow cytometry plots (n = 10). Right, summary data for the expression of indicated markers in SARS-CoV-2 S₂₆₉ multimer⁺CD8⁺ T cells. Data are presented as median and IQR. (B) The frequency and phenotypes of SARS-CoV-2-specific CD8⁺ T cells were examined during the course of SARS-CoV-2 infection in seven patients (P5, P15, P18, P74, and P76, severe disease; P12, non-severe disease; and P11, non-severe, asymptomatic). Viral titers (Ct values) were determined by real-time RT-PCR of RdRP (P5, P15, P18, P74, P76, and P12) or N (P11) genes in specimens from nasopharyngeal swabs serially obtained during the course of COVID-19. The viral titer and frequency of SARS-CoV-2 S₂₆₉ multimer⁺ cells among total CD8⁺ T cells were plotted against days post-symptom onset (P5, P15, P18, P74, P76, and P12) or days post-admission (P11). The percentages of Ki-67⁺ (orange), perforin⁺granzyme B⁺ (blue), CD38⁺HLA-DR⁺ (pink), and PD-1⁺ (dark red) cells among multimer⁺ cells are presented. (C and D) PBMCs from P5 (14 days post-symptom onset), P12 (16 days post-symptom onset), P15 (15 days post-symptom onset), P74 (19 days post-symptom onset), and P76 (17 days post-symptom onset) were stimulated with S₂₆₉ peptide (1 μg/mL) for 5.5 h and CSAs performed in combination with MHC class I multimer staining to analyze the phenotypes of IFN-γ-producing cells among MHC class I multimer⁺ cells. Representative flow cytometry plots (n = 5) (C) and summary data (D) show IFN-γ production according to the expression of PD-1, CD38, and HLA-DR in the gate of multimer⁺ cells.