Mucosal-Associated Invariant T (MAIT) Cells Are Highly Activated and Functionally Impaired in COVID-19 Patients

Abstract

Coronavirus disease 2019 (COVID-19), caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprises mild courses of disease as well as progression to severe disease, characterised by lung and other organ failure. The immune system is considered to play a crucial role for the pathogenesis of COVID-19, although especially the contribution of innate-like T cells remains poorly understood. Here, we analysed the phenotype and function of mucosal-associated invariant T (MAIT) cells, innate-like T cells with potent antimicrobial effector function, in patients with mild and severe COVID-19 by multicolour flow cytometry. Our data indicate that MAIT cells are highly activated in patients with COVID-19, irrespective of the course of disease, and express high levels of proinflammatory cytokines such as IL-17A and TNFα ex vivo. Of note, expression of the activation marker HLA-DR positively correlated with SAPS II score, a measure of disease severity. Upon MAIT cell-specific in vitro stimulation, MAIT cells however failed to upregulate expression of the cytokines IL-17A and TNFα, as well as cytolytic proteins, that is, granzyme B and perforin. Thus, our data point towards an altered cytokine expression profile alongside an impaired antibacterial and antiviral function of MAIT cells in COVID-19 and thereby contribute to the understanding of COVID-19 immunopathogenesis.

Keywords: COVID-19; SARS-CoV-2; mucosal-associated invariant T (MAIT) cells.

Figure 1

Alterations in adaptive immune cell frequency in COVID-19 patients. (A) Frequency of conventional T cells (HC n = 15; COVID mild n = 17; COVID severe n = 9), B cells (HC n = 15; COVID mild n = 10; COVID severe n = 9) and Treg cells (HC n = 10; COVID mild n = 9; COVID severe n = 8) in peripheral blood of COVID-19 patients and healthy controls (HC); (B) Flow cytometry gating strategy for identification of T cell subsets; Frequency of CD4⁺ (C) and CD8⁺ (D) T cell subsets in peripheral blood of COVID-19 patients and healthy controls (HC n = 10; COVID mild n = 9; COVID severe n = 8). Data are presented as mean ± SEM and were pooled from three independent experiments; each symbol represents one patient; ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. HC or as indicated, data were assessed using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test, ns = not significant; T_cm= central memory T cells, T_em = effector memory T cells and T_te = terminally differentiated T effector cells.

Figure 2

MAIT cells are significantly and continuously reduced in peripheral blood of COVID-19 patients, regardless of disease severity. (A) Frequency of natural killer (NK) cells ( HC n = 12; COVID mild n = 9; COVID severe n = 10), NKT-like cells (HC n = 13, COVID mild n = 12; COVID severe n = 10) and (B) γδ2 T cells (HC n = 12; COVID mild n = 12; COVID severe n = 10) in peripheral blood of COVID-19 patients and healthy controls (HC); (C,D) mucosal-associated invariant T (MAIT) cell frequency (C: HC n = 26 or 12; COVID n = 38 or 20; D: HC n = 26; COVID mild total n = 22, acute n = 13, convalescent n = 9; COVID severe total n = 17, acute n = 6, convalescent n = 11), (E) T cell receptor (TCR) co-receptor expression (HC n = 16; COVID n = 26) and (F) IL-12 receptor (IL-12R): HC n = 12; COVID n = 20) and IL-18 receptor (IL-18R): HC n = 12; COVID n = 22) expression in COVID-19 patients and HC. Data are presented as mean ± SEM and were pooled from 3 (A,B) or >10 (C,D) independent experiments; each symbol represents one patient; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs. HC or as indicated; data were assessed using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (A,B,D), unpaired t-test (C,F) or two-way ANOVA with Sidak’s multiple comparisons test (E). ns = not significant; DN = CD4/CD8 double-negative.

Figure 3

MAIT cells of COVID-19 patients express high levels of activation markers and cytokines ex vivo. (A–D) Ex vivo expression of surface activation markers in MAIT cells from peripheral blood of COVID-19 patients and healthy controls (HC); (E) Spearman correlation between expression of HLA-DR in MAIT cells in peripheral blood of COVID-19 patients and SAPS II score at day of sampling; (F) Ex vivo intracellular cytokine expression in MAIT cells from peripheral blood of COVID-19 patients and HC, representative data from one patient and summary data; (A,E,F) each symbol represents one patient. (A) CD38: HC n = 18 and COVID n = 30; CD69: HC n = 21 and COVID n = 38; HLA-DR: HC n = 21 and COVID = 35; CTLA-4: HC n = 20 and COVID n = 36; PD-1: HC n = 23 and COVID n = 34. (B) HC n = 13, COVID mild n = 22 and COVID severe n = 18; (C) HC n = 26, COVID mild n = 17 and COVID severe n = 9; (D) HC n = 19, COVID acute n = 13 and COVID convalescent n = 9; (E) n = 8; (F) IFNγ: HC n = 19 and COVID n = 33; IL-17A: HC n = 23 and COVID n = 33; TNFα: HC n = 23 and COVID n = 34; GrzB: HC n = 23 and COVID n = 34. Data are presented as mean ± SEM and were pooled from six (A–D) or four (E) independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs. HC or as indicated. Data were assessed using unpaired t-test (A,F), two-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (B,D) or one-way ANOVA with Tukey’s multiple comparisons test (C). ns = not significant.

Figure 4

MAIT cells from peripheral blood of COVID-19 patients show an altered cytokine expression profile. Intracellular cytokine expression in MAIT cells in a PBMC pool from peripheral blood of COVID-19 patients and healthy controls (HC) following co-culture with E. coli at 10 bacteria per cell (A,C,D) or stimulation with 50 ng/mL IL-12/IL-18 (B,C,D) for 24 h. Data are presented as mean ± SEM and were pooled from three independent experiments; each symbol represents one patient. (A) IFNγ: HC n = 16 and COVID n = 22; IL-17A/TNFα: HC n = 20 and COVID n = 24; (B) IFNγ: HC n = 16 and COVID n = 21; IL-17A/TNFα: HC n = 26 and COVID n = 20; (C,D) HC n = 16 and COVID n = 21; * p < 0.05 and ** p < 0.01 vs. unstimulated control. Data were assessed using paired t-test (A,B) or one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test (C,D). ns = not significant.