Phenotypical and functional alteration of unconventional T cells in severe COVID-19 patients

Abstract

COVID-19 includes lung infection ranging from mild pneumonia to life-threatening acute respiratory distress syndrome (ARDS). Dysregulated host immune response in the lung is a key feature in ARDS pathophysiology. However, cellular actors involved in COVID-19-driven ARDS are poorly understood. Here, in blood and airways of severe COVID-19 patients, we serially analyzed unconventional T cells, a heterogeneous class of T lymphocytes (MAIT, γδT, and iNKT cells) with potent antimicrobial and regulatory functions. Circulating unconventional T cells of COVID-19 patients presented with a profound and persistent phenotypic alteration. In the airways, highly activated unconventional T cells were detected, suggesting a potential contribution in the regulation of local inflammation. Finally, expression of the CD69 activation marker on blood iNKT and MAIT cells of COVID-19 patients on admission was predictive of clinical course and disease severity. Thus, COVID-19 patients present with an altered unconventional T cell biology, and further investigations will be required to precisely assess their functions during SARS-CoV-2-driven ARDS.

Graphical abstract

Figure 1.

Inflammatory status of study patients. (A) Lymphocyte count in whole blood from severe COVID-19 (n = 30) and non–COVID-19 patients (n = 16; the lymphocyte count of one patient was not determined) on day 1 after admission. Individuals and means ± SEM are depicted. Dashed line represents the threshold for lymphopenia. Mann–Whitney U test. (B) SOFA score of COVID-19 (n = 30) and non–COVID-19 (n = 17) patients. Individuals and means ± SEM are shown. Mann–Whitney U test. (C) Spearman’s rank correlation of SOFA and lymphocyte counts in COVID-19 patients. (D) Levels of IL-1β, IL-6, IL-1RA, and IFN-α2 in the plasma of healthy donors (n = 20) and COVID-19 (n = 30) and non–COVID-19 (n = 16) patients. Individuals and means ± SEM are shown. Kruskal–Wallis test with Dunn's multiple comparisons test. (E) Levels of IL-1β, IL-6, IL-1RA, and IFN-α2 in the plasma and ETA supernatants of matched COVID-19 patients (n = 20). Paired individual values are shown. Wilcoxon matched-pairs signed rank test. (F) Comparison of airway levels of IL-1β, IL-6, IL-1RA, and IFN-α2 in ETA supernatants of COVID-19 (n = 20) and non–COVID-19 (n = 7) patients. Mann–Whitney U test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure S1.

Frequency, confounding effects, and TCR levels of T cell subsets in COVID-19 patients. (A) Frequency of circulating conventional T cells (within the lymphocyte gate) in healthy controls (n = 20) and COVID-19 (n = 30) and non–COVID-19 (n = 17) patients. Individuals and means ± SEM are shown. Kruskal–Wallis test with Dunn's multiple comparisons test. (B) Spearman’s rank correlation of age of COVID-19 patients and the frequency of blood MAIT (left panel) or iNKT (right panel) cells (n = 30). (C) Comparative analyses of MAIT and iNKT frequencies regarding the obese status (BMI > 30) of patients. Individuals and means ± SEM are shown. Mann–Whitney U test. (D) Mean intensity fluorescence (MFI) of TCR expression on MAIT and iNKT cells from controls (n = 20) or COVID-19 patients (n = 30) based on TCR Vα7.2 and PBS57-CD1d tetramer staining, respectively. Individuals and means ± SEM are shown. Mann–Whitney U test. (E and F) Comparative analysis of T cells in blood and ETA of COVID-19 (n = 20; E) or non–COVID-19 (n = 7; F) patients. Individuals and means ± SEM are shown. Wilcoxon matched-pairs signed rank test. ns, not significant; *, P < 0.05; **, P < 0.01.

Figure 2.

Relative proportion of uT cells in PBMCs and ETA of COVID-19 patients. (A) Flow cytometry analyses of uT cells in the blood of healthy donors (n = 20) and COVID-19 (n = 30) and non–COVID-19 (n = 17) patients. Representative dot plots of MAIT, iNKT, and γδT cells of the three groups are shown in the left panel as percentage of CD3⁺ live cells. Individuals and means ± SEM are shown in the right panel. Of note, iNKT cells could not be detected in four COVID-19 patients and one non–COVID-19 patient. Kruskal–Wallis test followed by a Dunn's multiple comparisons test. (B) Proportion of γδT subsets in the blood of healthy donors (n = 20) and COVID-19 (n = 30) patients. Representative dot plots of γδT subsets are shown in the left panel. Individuals as percentage of CD3⁺ live cells and means ± SEM are shown in the right panel. Mann–Whitney U test. (C) Comparative analysis of MAIT and γδT cell subsets in blood and ETA of COVID-19 patients with detected uT cells in ETA. Representative dot plots are shown in the left panel. Individuals and means ± SEM are shown in the right panel. Wilcoxon matched-pairs signed rank test. (D) Levels of CXCL10 and CXCL12 in ETA supernatants of COVID-19 patients (n = 20), according to the presence (n = 12) or not (n = 8) of uT cells, and non–COVID-19 (n = 7) patients. Individuals and means ± SEM are shown. Mann–Whitney U test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, not significant.

Figure 3.

Functional analysis of uT cells during severe COVID-19. (A) Flow cytometry analyses of CD69 and PD-1 expression on MAIT, γδT, and iNKT cells in the blood of healthy donors (n = 20) and COVID-19 (n = 30) and non–COVID-19 (n = 17) patients. Individuals and means ± SEM are shown. Kruskal–Wallis test followed by a Dunn's multiple comparisons test. (B) Levels of IL-18 in the plasma of healthy donors (n = 20) and COVID-19 (n = 30) and non–COVID-19 (n = 17) patients. Individuals and means ± SEM are shown. Kruskal–Wallis test followed by a Dunn's multiple comparisons test. (C) Spearman’s rank correlation of CD69 expression on blood uT cells and levels of IL-18 in COVID-19 patients. (D) Relative proportions of CD69⁺ and PD-1⁺ MAIT and γδT cells in the blood and ETA of matched patients. Paired individual values are shown. Wilcoxon matched-pairs signed-rank test. (E) Intracellular staining for IFN-γ and IL-17A of PMA/ionomycin-activated PBMCs. Individuals and means ± SEM are shown. Mann–Whitney U test. (F) Levels of IFN-γ and IL-17A in the plasma and ETA supernatants of matched patients. Paired individual values are shown. Wilcoxon matched-pairs signed rank test. (G) Levels of IFN-γ and IL-17A in the ETA supernatants of COVID-19 patients, according to the presence (n = 12) or not (n = 8) of uT cells, and non–COVID-19 patients. Kruskal–Wallis test followed by a Dunn's multiple comparisons test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, not significant.

Figure S2.

Phenotype and cytokine production of blood uT cells in COVID-19 patients. (A) Representative histogram plots of CD69 and PD-1 expression on uT cell subsets in healthy controls and COVID-19 patients. (B) Flow cytometry analyses of CD69 (upper panel) and PD-1 (lower panel) expression on γδT subsets in the blood of healthy donors (n = 20) and COVID-19 patients (n = 30). Individuals and means ± SEM are shown. Mann–Whitney U test. (C) Spearman’s rank correlation of CD69 expression on blood uT cells and levels of IL-1β or IFN-α2 in COVID-19 patients. (D) Mean intensity fluorescence (MFI) of CD3 expression on MAIT and iNKT cells from healthy controls (n = 20) and COVID-19 patients (n = 30). Individuals and means ± SEM are shown. Mann–Whitney U test. (E) Mean intensity fluorescence of CD161 expression on MAIT cells from healthy controls (n = 20) and COVID-19 patients (n = 30). Individuals and means ± SEM are shown. Mann–Whitney U test. (F) Representative dot plots of intracellular staining for IFN-γ (left panel) and IL-17A (right panel) of blood iT cells from healthy donors and COVID-19 patients following PMA/ionomycin activation. *, P < 0.05; ***, P < 0.001. ns, not significant.

Figure S3.

Analysis of uT cell biological parameters regarding the clinical evolution of COVID-19 patients. (A) Comparative analyses of the frequencies of blood MAIT, iNKT, and γδT subsets in COVID-19 patients who were discharged (n = 14) or not discharged (n = 15) from ICU at day 14. Individuals and means ± SEM are shown. Mann–Whitney U test. (B) Comparative analyses of the CD69 expression of blood γδT subsets in COVID-19 patients who were discharged (n = 14) or not discharged (n = 15) from ICU at day 14. Individuals and means ± SEM are shown. Mann–Whitney U test. (C) Comparative analyses of PD-1 expression on blood MAIT, iNKT, and γδT subsets in COVID-19 patients who were discharged (n = 14) or not discharged (n = 15) from ICU at day 14. Individuals and means ± SEM are shown. Mann–Whitney U test. (D) Comparative analysis of IFN-γ and IL-17A production by uT cells in COVID-19 patients who were discharged (n = 14) or not discharged (n = 15) from ICU at day 14. Individuals and means ± SEM are shown. Mann–Whitney U test. (E) Kinetics plots showing individual values of uT cell frequency and phenotype for each rapidly discharged (before day 15) patients (n = 14) at days 1 and 7 after admission. Wilcoxon matched-pairs signed rank test. (F) Spearman’s rank correlation of CD69 expression on blood γδT subsets on admission and hypoxemia levels at day 7 in COVID-19 patients (n = 20). *, P < 0.05; **, P < 0.01. ns, not significant.

Figure 4.

Predictive role and kinetics analysis of uT cell parameters in the clinical course of COVID-19. (A) Comparative analyses of CD69 expression on blood MAIT, γδT, and iNKT cells in COVID-19 patients who were discharged (n = 14) or not discharged (n = 15) from ICU at day 14. Individuals and means ± SEM are shown. Mann–Whitney U test. (B) Kinetics plots showing individual values of uT cell frequency and phenotype at days 1, 7, and 14 after admission for each patient still in critical condition at day 14 (n = 13). One-way ANOVA (Friedman test) with Dunn’s multiple comparison. (C) Spearman’s rank correlation of CD69 expression on blood uT cells on admission and hypoxemia levels at day 7 in COVID-19 patients (n = 20). *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, not significant.