COVID-19 immune signatures reveal stable antiviral T cell function despite declining humoral responses

Abstract

Cellular and humoral immunity to SARS-CoV-2 is critical to control primary infection and correlates with severity of disease. The role of SARS-CoV-2-specific T cell immunity, its relationship to antibodies, and pre-existing immunity against endemic coronaviruses (huCoV), which has been hypothesized to be protective, were investigated in 82 healthy donors (HDs), 204 recovered (RCs), and 92 active COVID-19 patients (ACs). ACs had high amounts of anti-SARS-CoV-2 nucleocapsid and spike IgG but lymphopenia and overall reduced antiviral T cell responses due to the inflammatory milieu, expression of inhibitory molecules (PD-1, Tim-3) as well as effector caspase-3, -7, and -8 activity in T cells. SARS-CoV-2-specific T cell immunity conferred by polyfunctional, mainly interferon-γ-secreting CD4⁺ T cells remained stable throughout convalescence, whereas humoral responses declined. Immune responses toward huCoV in RCs with mild disease and strong cellular SARS-CoV-2 T cell reactivity imply a protective role of pre-existing immunity against huCoV.

Keywords: COVID-19; SARS-CoV-2; antibody; antiviral T cell immunity; caspases; cell death; chemokine receptors; convalescence; endemic human coronavirus; humoral immunity.

Graphical abstract

Figure 1

SARS-CoV-2 IgG ratios decline and COVID-19-associated humoral and cellular immune profile regresses throughout recovery Shown are the SARS-CoV-2 humoral and cellular immune profiles of healthy donors (HD, green), recovered COVID-19 patients (RC, red), and patients with active COVID-19 (AC, blue). (A) SARS-CoV-2 N and S1 IgG ratios in HDs, RCs, and ACs (first sample). (B) Seroconversion during COVID-19, expressed as anti-N and -S1 IgG ratios during weekly follow-up of ACs. Dashed lines indicate cutoff values for negative (< 0.8) and intermediate IgG ratios (0.8–1.1). (C) Left: Correlation between anti-N and -S1 IgG ratios in RCs and ACs (first sample). Right: Association between WHO score and SARS-CoV-2 antibodies. Dashed lines indicate cutoff values for negative and intermediate IgG ratios. (D) Left: Correlation between disease severity score (DSS) or time of convalescence and SARS-CoV-2 IgG ratios. Right: SARS-CoV-2 IgG ratios over time in individual RCs. Each symbol and color represent data from one RC; numbers behind the lines indicate p values for the corresponding individual. (E and F) Cellular profile was determined by flow cytometric analysis of whole blood. (A–C) n = 76–82 (HD), n = 199–204 (RC), and n = 92 (AC; first sample). (D) Left: n = 199; right: n = 16. (E–F) n = 63–79 (HD), n = 171 (RC), and n = 91–92 (AC). (A, E, and F) Kruskal-Wallis test and Dunn’s test for multiple comparisons or (C and D) linear regression analysis was used to calculate statistical significance: ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001. See also Figures S1–S3.

Figure 2

Recovery from mild COVID-19 is associated with a broad anti-SARS-CoV-2 T cell repertoire (A–C) IFN-γ ELISPOT data from (A) healthy donors (HD, green, n = 48–82), (B) recovered COVID-19 patients (RC, red, n = 110–204), and (C) patients with active COVID-19 (AC, blue, n = 86–92; first sample) are depicted as the number of spots per well (spw)/2.5x10⁵ PBMCs on the left side. For CMV_pp65 only values for seropositive individuals are depicted. The corresponding frequencies of non- (NR), low (LR), intermediate (IR), and high responders (HR) are shown on the right side. (D) Inter-cohort comparison of antiviral T cell frequencies determined by ELISPOT assay normalized to T cell frequencies within PBMCs, depicted as spw/1.0x10⁴ CD3⁺ T cells. Statistical significance was calculated by Kruskal-Wallis test and Dunn’s test for multiple comparisons. (A–C) Statistically significant differences to negative control (NC) are depicted. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001. See also Figure S4.

Figure 3

Antiviral T cell repertoire remains stable during recovery from mild COVID-19 (A) Antiviral T cell frequencies in relation to disease severity in recovered COVID-19 patients (RCs; red, n = 136–204). (B) SARS-CoV-2_M-, N-, S-, S1-, and S2-specific T cell frequencies during convalescence in RCs (n = 110–178). Statistical significance was calculated by linear regression analysis. (C) Frequencies of T cells specific for SARS-CoV-2 (upper panel), OC43, 229E, respiratory syncytial virus (RSV) and influenza A virus (IAV) (lower panel) in follow-up samples from RCs (n = 4–15). See also Figure S4.

Figure 4

SARS-CoV-2 T cell frequencies partially correlate with SARS-CoV-2 IgG ratios Recovered (RC, red, n = 131–199) and active COVID-19 patients (AC, blue, n = 90; first sample) were classified as non- (NR), low (LR), intermediate (IR), and high responders (HR) for the indicated antigens based on ELISPOT results. Dashed lines indicate cutoff values for negative and intermediate IgG ratios. (A and B) Association between SARS-CoV-2-specific T cells and (A) anti-N or (B) anti-S1 IgG ratios. Statistical significance was calculated by Kruskal-Wallis test and Dunn’s test for multiple comparisons. ^∗p < 0.05.

Figure 5

COVID-19 patients with pre-existing T cell immunity against endemic coronaviruses have higher SARS-CoV-2_S-specific T cell frequencies (A) Correlation between OC43_S- and SARS-CoV-2_S-specific T cells in healthy donors (HD, green, n = 69), recovered (RC, red, n = 136) and active COVID-19 patients (AC, blue, n = 92; first sample). (B) Correlation between 229E_S- and SARS-CoV-2_S-specific T cells in HDs (green, n = 69), RCs (red, n = 136) and ACs (blue, n = 92; first sample). (A and B) Star-shaped symbols indicate RCs tested for huCoV-specific antibodies. Statistical significance was calculated by linear regression analysis. (C) Exemplary western blots for SARS-CoV-2 and huCoV and summarized results from selected HDs (green), RCs (red) and ACs (blue, first sample). Each column represents data from one subject; the legend indicates signal strength.

Figure 6

Overall impairment of T cell function contributes to reduced antiviral T cell immunity during COVID-19 Chemokine and immune checkpoint receptor expression on T cells as well as caspase-3 and/or -7 and caspase-8 activity in T cells were determined by flow cytometry, and plasma of healthy donors (HD, green, n = 24–43), recovered COVID-19 patients (RC, red, n = 47–66) and active COVID-19 patients (AC, blue, n = 65–92; first sample) was analyzed by LEGENDPlex assay. Statistical significance was calculated by Kruskal-Wallis test and Dunn’s test for multiple comparisons: ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001. See also Figure S5.

Figure 7

The signature of SARS-CoV-2-specific T cell recall responses is diverse and CD4-mediated (A–C) Cell culture supernatants of PBMCs of recovered COVID-19 patients (RCs) stimulated with the specified peptide pools for 20 h were analyzed by (A and B) LEGENDPlex assay (n = 8; CMV_pp65: seropositive subjects only [n = 6]) or (C) ELISA (n = 7; CMV_pp65: seropositive subjects only [n = 5]). Error bars indicate standard deviation. (B) Data are shown as fold change to unstimulated controls. For key to color code, see legend (n = 8). (C) Statistical significance was calculated by Friedman’s test and Dunn’s test for multiple comparisons; CMV_pp65 was excluded from the analysis. ^∗p < 0.05. (D) Frequencies of IFN-γ and granzyme B (Gzmb)-producing cells in response to the specified peptide pools were determined by FluoroSpot. Exemplary and summarized results for n = 5 cases are shown. (E and F) PBMCs from RC were stimulated with the specified peptide pools for 5 h, followed by flow cytometric analysis. Graphs show frequencies of IFN-γ⁺ TNF-α^- (yellow), IFN-γ⁺ TNF-α⁺ (green), and IFN-γ^- TNF-α⁺ (blue) cells within CD4⁺ (E) and CD8⁺ (F) T cell subsets (n = 8; CMV_pp65: seropositive subjects only [n = 6]). See also Figure S6.