Figure 4. Abundance of polyfunctional PD-L1⁺CXCR3⁺CD8⁺ T cells and NC-specific, cytokine-producing T cells define survival vs. fatal outcome after critical SARS-CoV-2 infection.

PBMCs from critical patients with COVID-19 were incubated with brefeldin A (16 h) and stained using multiparametric mass cytometry panel 2 (n = 21, R = 12, D = 9). (A) CD3⁺CD8⁺ T cell (20,000 events) were randomly taken among sample for unsupervised cluster using FlowSOM. Density plot t-SNE representing the expression of indicated markers. (B) Heatmap representation of mean signal intensity of each marker in CD3⁺CD8⁺T cell subsets. (C) Density plot t-SNE representing abundance of events using concatenated files of 12 R and 9 D patients. (D) Radar representing mean (min/max normalized) abundances of CD3⁺CD4⁺T cell subsets in 12 R (blue) and 9 D (red) patients. Multiple Mann-Whitney U test using Benjamini, Krieger, and Yekutieli FDR correction was performed, with significance set at adjusted P <.05. (E) SARS-CoV-2–specific T cell responses were measured in PBMCs from 46 ICU patients on day 15 ± 0.85 (mean ± SEM) after symptom onset. PBMCs were stimulated for 16 hours with SARS-CoV-2 overlapping 15-mer peptides (S1, S2, and NC). The frequency of specific CD8⁺ T cells (Boolean gating of IFN-γ, IL-2, and/or TNF-α) is represented with box-and-whisker plots (min to max) after background subtraction according to background control (left). Color-code (green) symbols represent individuals that were under immunosuppressive treatment when SARS-CoV-2–specific responses were studied. Individuals were considered responders when the frequency of cytokines produced was >0.005% of CD3⁺CD8⁺ cells. The frequency of nonresponders is represented among R (blue, S1 and S2: n = 31; NC n = 28) and D (red, S1 and S2 n = 15; NC n = 11) patients (right). χ² test was performed, with significance set at *P < 0.05. (F) Frequency of patients with detectable cells producing cytokines (0, 1, 2, 3 F) after stimulation in R (blue) and D patients (red). χ² test was performed, with significance set at *P < 0.05. R, recovered; D, deceased; NC, nucleocapsid; F, function.