Figure 2.

Increased Magnitude of Cytokine Producing S-Protein-Reactive T Cells in ARDS Patients in the Initial Visit and at Follow-up

The presence and functional status of SARS-CoV-2-reactive T cells was evaluated using PBMCs, isolated from the peripheral blood of 27 patients (17 COVID-19 control, in white, and 10 ARDS, in gray). Defrosted PBMCs rested for 24 h before treatment with overlapping peptide pools covering immune-dominant regions of the SARS-CoV-2 S-protein. The cells were stimulated for a total of 16 h and in the presence of brefeldin A for the last 14 h. The complete gating strategy is presented in Figure S6. No measurements of IgG antibodies were available for ARDS patients. (A) CD4⁺CD154⁺ frequency (first row) for ARDS and COVID-19 control patients at the initial visit (left boxplots) and follow-up (right boxplots), and frequencies of CD4⁺CD154⁺ cells expressing granzyme B (GrB), INF-γ, IL-2, and TNF-α (rows two to four). (B) CD8⁺CD137⁺ frequency (first row) for ARDS and COVID-19 control patients at the initial visit (left boxplots) and follow-up (right boxplots), and frequencies of CD8⁺CD137⁺ cells expressing GrB, INF-γ, IL-2, and TNF-α (rows two to four). Boxplots depict the median and the first and third quartiles. The whiskers correspond to 1.5 times the interquartile range. (C) Left side: comparison of the relative titers of SARS-CoV-2 S-protein-specific IgG antibodies, measured by ELISA and evaluated as the ratio to an internal control for samples with SARS-CoV-2-specific CD4⁺ T cells; the comparison of the relative titers classified depending on whether they had detectable virus-specific CD4⁺ T cells and the correlation of the relative titers with the frequency of virus-specific CD4⁺ T cells are shown. Right side: comparison of the relative titers of SARS-CoV-2 S-protein-specific IgG antibodies with the 50% neutralization dose. For the analysis of the antibody neutralization dose, the data were log transformed, assigning a value of zero for those with a value below the detection limit.