key: cord-0751642-w693a2rm
authors: Flament, H.; Rouland, M.; Beaudoin, L.; Toubal, A.; Bertrand, L.; Lebourgeois, S.; Gouda, Z.; Rousseau, C.; Soulard, P.; Hurtado-Nedelec, M.; Luce, S.; Bailly, K.; Andrieu, M.; Boitard, C.; Vallet-Pichard, A.; Gautier, J.-F.; Ajzenberg, N.; Terrier, B.; Pene, F.; Ghosn, J.; Yazdanpanah, Y.; Visseaux, B.; Descamps, D.; Timsit, J.-F.; Monteiro, R. C.; Lehuen, A.
title: Outcome of SARS-CoV-2 infection linked to MAIT cell activation and cytotoxicity: evidence for an IL-18 dependent mechanism
date: 2020-09-02
journal: nan
DOI: 10.1101/2020.08.31.20185082
sha: 3ab8cd9d09359b705ab8475fbd06a501dcfd8d7c
doc_id: 751642
cord_uid: w693a2rm

Immune system dysfunction is paramount in Coronavirus disease 2019 (COVID-19) severity and fatality rate. Mucosal-Associated Invariant T (MAIT) cells are innate-like T cells involved in mucosal immunity and protection against viral infections. Here, we studied the immune cell landscape, with emphasis on MAIT cells, in a cohort of 182 patients including patients at various stages of disease activity. A profound decrease of MAIT cell counts in blood of critically ill patients was observed. These cells showed a strongly activated and cytotoxic phenotype that positively correlated with circulating pro-inflammatory cytokines, notably IL-18. MAIT cell alterations markedly correlated with disease severity and patient mortality. SARS-CoV-2-infected macrophages activated MAIT cells in a cytokine-dependent manner involving an IFN-dependent early phase and an IL-18-induced later phase. Therefore, altered MAIT cell phenotypes represent valuable biomarkers of disease severity and their therapeutic manipulation might prevent the inflammatory phase involved in COVID-19 aggravation.

perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. . https://doi.org/10.1101/2020.08.31.20185082 doi: medRxiv preprint Introduction: 65 The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the etiologic agent 83 and chronic viral infections, MAIT cell blood frequency is reduced while expression of HLA-DR, 84 PD-1, CD38 and CD69 is upregulated 18-20, [22] [23] [24] . Upon acute viral infection, they produce high 85 levels of granzyme B (GzB) 20 . 86 In COVID-19 patients, alteration of peripheral lymphocyte and myeloid subsets is associated 87 with clinical characteristics and treatment efficiency [25] [26] [27] . However, status of MAIT cells, 88 remain unknown in COVID-19 patients. Here, we analyzed blood MAIT cells of COVID-19 89 patients with different disease severity status from Infectious Disease Unit (IDU) (n=51) and 90 Intensive Care Unit (ICU) (n=51). These patients were compared with uninfected controls Adaptive and innate T cell frequency in the blood of COVID-19 patients. 98 We first began our study of immune cells in SARS-CoV-2 infection by analyzing the frequency 99 and phenotype of lymphocytes in blood samples from COVID-19 patients as well as (age-100 matched BMI-matched) non-infected donors (Fig. 1a) . Fifty-one COVID-19 patients had been 101 admitted in IDU (moderate cases) and 51 patients in ICU (severe cases) (with a death rate of 102 41% in ICU). As controls, we included 80 healthy non-infected donors as well as donors with 103 various pathologies (diabetes, obesity) to match those affecting hospitalized COVID-19 104 patients (Fig. 1a) . All characteristics and health data of recruited patients and controls are 105 listed in Table 1 . Whole blood was collected and stained for flow cytometry to analyze the 106 frequencies of both innate and adaptive T lymphocytes following the gating strategy explained 107 in Supplementary Figure 1 . A majority of patients with COVID-19 presented a significant 108 reduction of T cells in IDU, more pronounced in ICU patients (Fig. 1b) , confirming earlier reports 28, 29 . Among CD3 + T cells, there was a slight increase of blood αβ T cells frequency in 110 ICU patients, mirrored by a decrease of γδT cells (Fig. 1c) . Both CD4 + and CD8 + T cell frequency 111 among CD3 + T cells were not significantly impacted by SARS-CoV-2 infection whereas T 112 regulatory cell frequency was significantly reduced in both IDU and ICU patients compared to 113 uninfected controls (Fig. 1d) . We observed a collapse of Vα7.2 + CD161 + MAIT cell frequency 114 among CD3 + cells in COVID-19 patients, down to a tenth of those observed in uninfected 115 controls (Fig. 1e) . We confirmed that MAIT cell markers CD161 and Vα7.2 allowed similar MAIT 116 cell identification as with MR1 tetramers loaded with the active ligand 5-(2-117 oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) in all three groups of patients 118 ( Supplementary Fig. 2) . Among MAIT cells, the CD8 + subset was further reduced in COVID-19 119 patients, but to a lesser extent in ICU compared to IDU (Fig. 1f) . We therefore observed a 120 dramatic decrease of blood MAIT cells in COVID-19 patients, with an alteration of MAIT cell 121 subsets repartition. 122 123 MAIT cells are activated and cytotoxic in COVID-19 patients. 124 As MAIT cell frequency is extremely reduced in blood of COVID-19 patients, we analyzed both 125 their surface markers expression as well as their cytokine production. An activated phenotype 126 with a dramatic increase of CD69 expression was observed in COVID-19 patients, with a 127 median of 41% CD69 + MAIT cells in IDU and 67% in ICU patients, with some reaching 100% 128 (Fig. 2a) . Blood MAIT cells also displayed a significant increase of the NK cell-associated 129 activation CD56 + marker compared to controls, that was highest in ICU patients (Fig. 2a) .

receptor for the CCL20 chemokine (a tissue-migrating marker) and survival CD127 marker was 133 decreased, suggesting that the low circulating MAIT cell frequency might reflect their 134 migration into inflamed infected tissues and/or activation-induced cell death (Supplementary 135 Fig. 3a) . MAIT cell activation in some patients might be associated with secondary infections 136 and presence of bacteria in the blood (Supplementary Fig. 3b) . 137 In contrast to MAIT cells, CD69 + expression on CD8 + T cell remained moderate (median of 10%) 138 in both IDU and ICU although increased compared to uninfected controls (Supplementary Fig.   139 3c). Frequency of CD56 + CD8 + T cells was similar in all three groups of patients. Consequently, 140 the frequency of effector CD56 + CD69 + CD8 + T cells was modestly increased in COVID-19 141 patients compared to uninfected controls (Supplementary Fig. 3c ). 142 We next investigated with unsupervised methods the phenotype of MAIT cells, on 9 flow-143 cytometry parameters. We first compared MAIT cells of COVID-19 patients to uninfected 144 controls by Multidimensional scaling (MDS) plots, which revealed a severity progression from 145 non-infected controls to IDU and ICU patients (Fig. 2b) . We next performed graphical 146 dimensional reduction by both t-distributed Stochastic Neighbor Embedding (t-SNE) and 147 Uniform Manifold Approximation and Projection (UMAP) that also returned a progressive 148 distribution of MAIT cells from controls to IDU and ICU patients ( Fig. 2c Fig. 4a,b) . Of note, both CD69 and CD56 markers were critical molecules in the differential 150 distribution ( Fig. 2d and Supplementary Fig. 4c ). 151 MAIT cell function was then assessed after PMA/ionomycin stimulation by analyzing IFNγ and 152 GzB production in all three groups of patients and IL-2, IL-4, IL-10, IL-17 and TNF in IDU and 153 ICU patients (Fig. 2e,f and Supplementary Fig. 5a) . Production of IFNγ was decreased in 154 COVID-19 IDU patients compared to non-infected controls. However, IFNγ and IL-2 increased 155 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. . https://doi.org/10.1101/2020.08.31.20185082 doi: medRxiv preprint in ICU patients compared to IDU patients ( Fig. 2e and Supplementary Fig. 5a ). In contrast, GzB 156 production progressively increased in stimulated MAIT cells in IDU and ICU patients as 157 compared to controls. Elevated GzB production was also detected in unstimulated MAIT cells 158 from infected patients (Fig. 2f,g and Supplementary Fig. 5b) . Such enhanced cytokine and GzB 159 production in ICU patients were not observed in conventional αβT cells, γδT cells and CD3 -160 cells (including NK cells) (Supplementary Fig. 5a,b) . Altogether, these results show that blood 161 MAIT cells from COVID-19 patients display an activated/effector phenotype and cytotoxic 162 function associated with disease severity.

Links between MAIT cell activation and other innate immune cell alterations 165 Following MAIT cell analyses, we further investigated other innate-like immune cell frequency, 166 phenotype and activation in the blood of SARS-CoV-2 infected patients. A significant NK cell, 167 ILC2 and ILC3 frequency reduction in IDU and ICU patients was observed, which was more 168 pronounced for NK cells and ILC2 in COVID-19 patients from ICU (Fig. 3a) . CD69 expression 169 was higher in all infected patients on NK cells, ILC3, and γδT cells whereas it was reduced on 170 ILC2 (Fig. 3b) . 171 We sought to identify correlations between MAIT cells and other innate cells in COVID-19 172 patients. A multiparametric matrix correlation plot showed strong positive correlations 173 between the frequencies of activated MAIT cells (both CD56 + and/or CD69 + MAIT cells) with 174 CD69 + ILC3, NK and γδT cells frequencies as well as CD69 + CD56 + γδT cells (Fig. 3c,d) . Several 175 negative correlations were also observed between activation of these populations and their 176 frequencies. CD69 expression on MAIT cells was negatively correlated with ILC3, ILC2 and NK 177 cell frequencies (Fig. 3c) . Moreover, cytokine production by MAIT cells was also negatively 178 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. . https://doi.org/10.1101/2020.08.31.20185082 doi: medRxiv preprint correlated with ILC3 frequencies and to a lesser extent with ILC2 and NK cell frequencies. 179 Taken together, these data suggest that inflammatory processes in SARS-CoV-2-infected   180   patients involve concomitant activation of MAIT cells with other innate-immune cells   181 associated with loss of these populations' frequencies in blood. COVID-19 patients compared to surviving IDU and/or ICU patients (Fig. 4a) . MAIT cells 188 displayed the highest activation level in all patients, particularly those with fatal outcomes. 189 We next examined immune cell function relative to disease outcome by measuring surviving patients with SARS-CoV-2 infection ( Fig. 4b and Supplementary Fig. 6a,b) . production by stimulated γδT cells in patients who succumbed was greater compared to 195 surviving COVID-19 patients ( Fig. 4b and Supplementary Fig. 6a) . Thus, MAIT cell function was 196 more associated to mortality than functions of other T cell populations. No modification of IL-197 2, IL-4, and IL-17 production in CD4 + , CD8 + , CD3 -, γδT, and MAIT cells was observed in fatal 198 cases ( Fig. 4b and Supplementary Fig. 6a) . Moreover, statistical regression analyses identified 199 four MAIT cell markers (IFNγ production, stimulated GzB production, unstimulated GzB 200 production, CD69) that defined predictive models for COVID-19 outcome, as tested on a 201 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. . https://doi.org/10.1101/2020.08.31.20185082 doi: medRxiv preprint receiver-operating-characteristic (ROC) curve (Fig. 4c) . In order to have a more global view of 202 individual patients, we generated an heatmap based on multiple immune cell and blood 203 parameters from single uninfected, infected and fatal COVID-19 patients. This analysis 204 highlights activation signatures of non-surviving patients compared to surviving COVID-19 205 patients and controls (Fig. 4d) .

Increased pro-inflammatory cytokine levels correlate with blood MAIT cell alterations. 208 We next analyzed plasma levels of several cytokines by Cytometric Bead Array (CBA) and MSD 209 Quickplex in both surviving IDU and ICU patients as well as non-surviving patients (Fig. 5a,b) . 210 IL-6, IL-8, IL-10, IL-15, and IL-18 levels were significantly increased in the plasma of non-211 surviving patients compared to surviving patients, confirming a state of widespread, 212 pronounced inflammation in severe COVID-19 cases 28 (Fig. 5a,b) . IFNα2 levels were 213 significantly decreased in ICU compared to IDU, although no significant difference between 214 surviving ICU and deceased patients was detected (Fig. 5b) . IL-1β levels were similar between 215 all three groups (Fig. 5b) . Multiparametric matrix correlation plot showed strong positive 216 correlation of IL-6, IL-8, IL-10, IL-15, IL-18 levels with frequencies of CD69 + , and CD69 + CD56 + 217 MAIT cells in the blood of all COVID-19 patients (Supplementary Fig. 7a,b) . IFNα2 blood level 218 correlated positively with the production of IFNγ, TNF, and IL-2 by MAIT cells (Supplementary 219 Fig. 7a) . 220 Separated matrix correlation plot between surviving and non-surviving ICU patients showed 221 different relationships between blood cytokines and GzB production by MAIT cells. In surviving 222 patients, there was a strong negative correlation between blood IL-15 level and spontaneous 223 GzB production by MAIT cells whereas in non-surviving patients there was a strong positive 224 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. . https://doi.org/10.1101/2020.08.31.20185082 doi: medRxiv preprint correlation between blood IL-18 level and GzB MAIT production. The link between IL-18 and 225 MAIT cell activation is further supported by high IL-18Rα expression on all blood MAIT cells in 226 control and COVID-19 infected compared to other immune populations ( Supplementary Fig.   227   8a,b,c) . Interestingly, in non-surviving ICU patients, blood IL-18 level was negatively correlated 228 with IFNα2 blood levels (Fig. 5c) . Accordingly, IL-18 blood level was increased in long-term 229 infected ICU patients whereas IFNα2 blood level was decreased in long-term in these patients 230 (Fig. 5d) . Of note, CD69 + MAIT cell frequency increased as well with time and was highest one 231 month after symptoms onset, when death rate was highest among ICU patients. Our data 232 therefore reveal that pro-inflammatory cytokines and IL-10 levels are associated with MAIT 

As previously reported in our cohort, COVID-19 severity was associated with extended 239 pulmonary damage as evaluated on chest computed tomography (CT) (score from 1:mild to 240 5:critical) (Fig. 6a) . PaO2/FiO2 ratio was significantly reduced and C-reactive protein (CRP) 241 levels were significantly increased in ICU patients. Both parameters were even more impacted 242 in fatal cases (Fig. 6b,c) . 243 A correlation matrix of MAIT cells activation, function and clinical parameters was established 244 for all COVID-19 patients (Fig. 6d) . It included age, body mass index (BMI), CRP, Simplified 245 Acute Physiology Score II (SAPSII) (an estimator of patient mortality risk at ICU admission), 246 PaO2/FiO2 ratio, polymorphonuclear neutrophil (PMN), disease duration and pulmonary 247 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. . https://doi.org/10.1101/2020.08.31.20185082 doi: medRxiv preprint lesions. Analysis of these clinical indicators in light of MAIT cells revealed that on vital 248 parameters, PaO2/FiO2 strongly negatively correlated with CD69 + expression, IFNγ and TNF 249 production by MAIT cells, whereas SAPSII score positively correlated with CD69 + MAIT cell 250 frequency (Fig. 6d,e) . Concerning inflammatory markers, CRP level correlated with MAIT cell 251 activation phenotype and cytokine secretion (IL-2, IFNγ and TNF) while PMN frequency 252 positively correlated with CD69 + MAIT cell frequency (Fig. 6d,e) . perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. were always activated, whereas it was not the case for other innate immune cells (Fig. 7e) . 281 Since MAIT cells exert their antiviral activity in a cytokine dependent manner 18,20 , we next perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. . https://doi.org/10.1101/2020.08.31.20185082 doi: medRxiv preprint Discussion: 294 Our study reveals major MAIT cells alteration in numerous COVID-19 cases. MAIT cell 295 frequency is strongly reduced in greater proportion than all other major T cell subsets and 296 they are highly activated with secretion of critical pro-inflammatory cytokines, such as IFNγ. 297 MAIT cells possess a strong cytotoxic capability with an increased expression of GzB. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. . https://doi.org/10.1101/2020.08.31.20185082 doi: medRxiv preprint as previously reported 28 . Presence of elevated levels of IL-1β, IL-6, IL-8, and IL-10 are indicative 339 of cytokine storms that are major potential complications in severe COVID-19 patients. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. . https://doi.org/10.1101/2020.08.31.20185082 doi: medRxiv preprint association between the activation of MAIT cells and the onset of severe disease 20 . It is 363 interesting to note that both the DV and the SARS-CoV-2 are able to infect macrophages, 364 which then can activate MAIT cells 47 . Activation of MAIT cells by infected macrophages 365 through IL-18 may switch MAIT cells toward a detrimental role in these infections. 366 In conclusion, human MAIT cells are activated, displaying a cytotoxic profile in the blood of 367 SARS-CoV-2 infected patients, which is associated with other innate immune cell activation, 368 and a pro-inflammatory environment. Together, these data extend the knowledge of the perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. CoV-2 stock titer was 2.10 7 PFU.mL -1 . Supernatant was aliquoted for storage at −80°C. 461 For viral titration, SARS-CoV-2 was titrated by lysis plaque assay as previously described 49 . 462 Vero E6 cells were plated onto 12-well plate at a density of 5.10 4 cells per well in DMEM with 463 10% FBS. 24h later, cells were infected by 10 to 10 serial viral dilutions. After virus adsorption 464 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. . https://doi.org/10.1101/2020.08.31.20185082 doi: medRxiv preprint for 1h at 37°C with plate rocking every 15 min, the viral inoculum was removed and Vero cells 465 were washed with PBS free medium. After, 500μL of an agarose medium mix was added. After 466 three days of incubation at 37°C with 5% CO2, supernatant was removed and cells were fixed 467 with 1 mL of 6% formalin solution for 30 minutes. The formalin solution was removed and cells 468 were colored with a 10% crystal violet solution for 15 minutes. All wells were then washed 469 with distilled water and dried on bench-coat paper before analysis. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. table   525 values were computed with the atable R library. All R code written and used from our study is 526 available at github.com/MatthieuRouland/MAIT-COVID19. 527 Statistical analyses were performed with GraphPad Prism software version 8.0 and R software 528 version 4.0. All datasets were tested for normal distribution using Shapiro-Wilk normality test. 529 Since all normality tests returned negative, all datasets were compared using nonparametric 530 two-tailed Mann-Whitney. Correlation calculation between two parameters has been 531 performed using the Spearman's correlation test corrected for multiple inferences using 532 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. . https://doi.org/10.1101/2020.08.31.20185082 doi: medRxiv preprint Holm's method. Logistic regression and ROC (Receiver Operating Curve) were produced with 533 XLSTATS 2020.4 and confirmed with a randomly-split cohort on R with the RORC package. 534 Prognostic validity of the model was evaluated by analysis of the ROC curve and was measured 535 using the area under the curve (AUC). Differences were considered significant at P < 0.05 536 (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). 537 538 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. . https://doi.org/10.1101/2020.08.31.20185082 doi: medRxiv preprint Acknowledgments: 539 We thank all the patients and their physicians, nurses and technician staff who helped with 540 the study. We thank the Department of Biological Hematology and the Department of perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. Data availability statement: Data generated during this study will be available before 571 publication in a repository. Access code will be given upon request. 572 573 Code availability statement: Code used during this study will be available before publication 574 in a repository. Access code will be given upon request. 575 576 577 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. . https://doi.org/10.1101/2020.08.31.20185082 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. . https://doi.org/10.1101/2020.08.31.20185082 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. . https://doi.org/10.1101/2020.08.31.20185082 doi: medRxiv preprint perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. . https://doi.org/10.1101/2020.08.31.20185082 doi: medRxiv preprint perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. . https://doi.org/10.1101/2020.08.31.20185082 doi: medRxiv preprint perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 2, 2020. 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preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

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preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

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preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

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preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

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Cryo-EM structure of the 2019-nCoV spike in the prefusion 579 conformation

The trinity of COVID-19: 581 immunity, inflammation and intervention

Epidemiological and clinical characteristics of 99 cases of 2019 novel 584 coronavirus pneumonia in Wuhan, China: a descriptive study

Clinical features of patients infected with

Acute Respiratory Distress Syndrome

Acute respiratory distress syndrome

The species Severe acute respiratory syndrome-related 593 coronavirus : classifying 2019-nCoV and naming it SARS-CoV-2

Tissue-specific functions of invariant natural killer T cells

Human γδ T-Cell Control of Mucosal Immunity and 598 Inflammation

Mucosal-associated invariant T cells and 600 disease

Natural Killer T Cells and Mucosal-Associated Invariant T Cells in 602 Lung Infections

MAIT cells are imprinted by the microbiota in early life and 604 promote tissue repair

TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific 606

Tissue Repair and Effector Functions

Innate lymphoid cells in lung infection and 608 immunity

Innate Lymphoid Cells: 10 Years On

Selection of evolutionarily conserved mucosal-associated invariant T 611 cells by MR1

T-cell activation by transitory neo-antigens derived from distinct 613 microbial pathways

Human mucosal-associated invariant T cells contribute to antiviral influenza 615 immunity via IL-18-dependent activation

MAIT cells and viruses

MAIT cells are activated during human viral infections

CD161++CD8+ T cells, including the MAIT cell subset

Peripheral loss of CD8(+) CD161(++) TCRVα7·2(+) mucosal-associated 625

invariant T cells in chronic hepatitis C virus-infected patients

Activation, exhaustion, and persistent decline of the antimicrobial 628

MR1-restricted MAIT-cell population in chronic HIV-1 infection

Arming of MAIT Cell Cytolytic Antimicrobial Activity Is Induced by IL-7 631 and Defective in HIV-1 Infection

Characteristics of Peripheral Lymphocyte Subset Alteration in COVID-19

Elevated calprotectin and abnormal myeloid cell subsets discriminate 635 severe from mild COVID-19

Severe COVID-19 is marked by a dysregulated myeloid cell 637 compartment

Impaired type I interferon activity and inflammatory responses in severe 639 COVID-19 patients

Single-cell landscape of immunological responses in patients with 641 COVID-19

Functional alteration of innate T cells in critically ill Covid-19 patients

COVID-19 severity correlates with airway epithelium-immune cell 645 interactions identified by single-cell analysis

Human MAIT cells are xenobiotic-resistant, tissue-targeted, CD161hi 647 43. Ishimori, A. et al. Circulating activated innate lymphoid cells and mucosal-associated 671 invariant T cells are associated with airflow limitation in patients with asthma

Activation and evasion of type I interferon responses by SARS-CoV-2

Multiple layers of heterogeneity and subset 676 diversity in human MAIT cell responses to distinct microorganisms and to innate 677 cytokines

TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific 679

Tissue Repair and Effector Functions

Primary human splenic macrophages, but not T or B cells, are the 681 principal target cells for dengue virus infection in vitro

Host-pathogen interactions during apoptosis

A SARS-CoV-2 protein interaction map reveals targets for drug 685 repurposing

Vaccinia virus encodes a soluble type I interferon 687 receptor of novel structure and broad species specificity

Quantification of human immunodeficiency virus type 1 proviral load by 689 a TaqMan real-time PCR assay

CyTOF workflow: differential discovery in high-throughput high-691 dimensional cytometry datasets

IL-8, and IL-10 in the blood of surviving COVID-19 cases

Infectious Disease Unit (IDU) (n=19-22) or hospitalized in Intensive Care Unit (ICU) (n=19-28) 765 and fatal cases (n=25). (b) MSD Quickplex cytokine quantification of IFNα2, IL-1β, IL-15, and 766 IL-18, in the blood of surviving COVID-19 cases hospitalized in IDU

Values under the limit of detection are not statistically 768 computed and are not displayed. (c) Multiparametric matrix correlation plot of IL-6, IL-8, IL-769 15, IL-18, IFNα cytokines blood level; frequencies of CD69 + , CD56 + , CD69 + CD56 + , and GzB + 770 MAIT cells; frequencies of IFNγ + , IL-2 + , TNF + , and GzB + stimulated MAIT cells

Variables 773 are ordered by alphabetical order. (d) CD69 + MAIT cells, IL-18, and IFNα blood levels according 774 to symptoms duration (days) in COVID-19 patients, starting at the first clinical sign

001 (two-sided Mann-Whitney nonparametric test and Spearman 777 nonparametric correlation test corrected for multiple inferences using Holm's method)

MAIT cells; frequencies 787 of IFNγ + , IL-2 + , TNF + , and GzB + stimulated MAIT cells

CRP (n=94), and PMN (n=101) and the frequency of CD69 + MAIT cells in blood 791 (presented as a % value of total MAIT cells) from all COVID-19 patients. (f-g) Principal 792 component analysis (PCA) of 50 variables (listed in Supplementary Fig. 9) including clinical data 793 and frequencies of immune cell phenotype in IDU (n=46), ICU (n=30), and fatal cases from IDU 794 (n=6) and ICU (n=21) infected patients. Each point represents a single patient. Mean value of 795 each group is symbolized by a bigger symbol (f)

Small horizontal lines indicate the median, each symbol represents one 799 patient. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001 (two-sided Mann-Whitney 800 nonparametric test and Spearman nonparametric correlation test corrected for multiple 801 inferences using Holm's method)