key: cord-0929203-sc0frc0g
authors: Mo, Yufei; To, Kelvin Kai-Wang; Zhou, Runhong; Liu, Li; Cao, Tianyu; Huang, Haode; Du, Zhenglong; Lim, Chun Yu Hubert; Yim, Lok-Yan; Luk, Tsz-Yat; Chan, Jacky Man-Chun; Chik, Thomas Shiu-Hong; Lau, Daphne Pui-Ling; Tsang, Owen Tak-Yin; Tam, Anthony Raymond; Hung, Ivan Fan-Ngai; Yuen, Kwok-Yung; Chen, Zhiwei
title: Mitochondrial Dysfunction Associates With Acute T Lymphocytopenia and Impaired Functionality in COVID-19 Patients
date: 2022-01-14
journal: Front Immunol
DOI: 10.3389/fimmu.2021.799896
sha: 65c7b9784ad0a1bf8fedfb8a1f4538baaede2365
doc_id: 929203
cord_uid: sc0frc0g

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection results in rapid T lymphocytopenia and functional impairment of T cells. The underlying mechanism, however, remains incompletely understood. In this study, we focused on characterizing the phenotype and kinetics of T-cell subsets with mitochondrial dysfunction (MD) by multicolor flow cytometry and investigating the association between MD and T-cell functionality. While 73.9% of study subjects displayed clinical lymphocytopenia upon hospital admission, a significant reduction of CD4 or CD8 T-cell frequency was found in all asymptomatic, symptomatic, and convalescent cases. CD4 and CD8 T cells with increased MD were found in both asymptomatic and symptomatic patients within the first week of symptom onset. Lower proportion of memory CD8 T cell with MD was found in severe patients than in mild ones at the stage of disease progression. Critically, the frequency of T cells with MD in symptomatic patients was preferentially associated with CD4 T-cell loss and CD8 T-cell hyperactivation, respectively. Patients bearing effector memory CD4 and CD8 T cells with the phenotype of high MD exhibited poorer T-cell responses upon either phorbol 12-myristate-13-acetate (PMA)/ionomycin or SARS-CoV-2 peptide stimulation than those with low MD. Our findings demonstrated an MD-associated mechanism underlying SARS-CoV-2-induced T lymphocytopenia and functional impairment during the acute phase of infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID -19) (1), has shocked the world with more than 240 million confirmed cases and more than 4.9 million deaths by October 20, 2021 , as reported by the World Health Organization (2) . SARS-CoV-2 became the most devastating human coronavirus, which was associated with its extremely high transmissibility (3, 4) , hidden asymptomatic spread (4, 5) , and emerged viral variants of concern escaping from neutralizing antibodies during natural infection or post vaccination (6) (7) (8) . Clinically, COVID-19 patients might develop acute respiratory distress syndrome or be admitted to the intensive care unit (ICU) at around 8-15 days post symptom onset (p.s.o.) (9, 10) . Most severe COVID-19 patients also suffered from prompt lymphocytopenia (11) . Low T-cell count on hospital admission may predict disease severity (12) and relate to the increased peripheral pro-inflammatory cytokines among COVID-19 patients (13, 14) . Particularly, the low CD8 T-cell count has been suggested to be a predictor for high mortality and severity of COVID-19 pneumonia (13, 15, 16) . Furthermore, T-cell lymphocytopenia is associated with higher saliva viral load (17) . These findings demonstrated that T lymphocytopenia might have detrimental effects on acute COVID-19 patients. Considering that T-cell immunity is important for host immune defense by eliminating virusinfected cells and assisting antibody responses (18, 19) , we sought to investigate possible causes of T lymphocytopenia during the natural course of acute SARS-CoV-2 infection. We focused on the characterization of T-cell subsets with mitochondrial dysfunction (MD) by flow cytometry and the investigation of the association between MD and Tcell functionality.

Our study was approved by the institutional review board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster and Kowloon West Cluster Research Ethics Committee [UW 13-265 and KW/EX-20-038(144- 26) ]. We recruited 88 acute patients (APs), admitted to the Queen Mary Hospital and Princess Margaret Hospital from August 6, 2020, to December 23, 2020, into our study. In the meantime, 17 convalescent patients (CPs) and 31 healthy donors (HDs) were recruited by the Hong Kong Red Cross as controls. We primarily divided APs into three groups: asymptomatic (AS), mild APs (symptomatic without O 2 treatment), and severe APs (symptomatic with O 2 treatment). The clinical information of our subjects was summarized in Table 1 .

Fresh peripheral blood mononuclear cells (PBMCs) from HDs and patients were isolated using Lymphoprep ™ (Axis Shield, Dundee, Scotland) density gradient centrifugation in our BSL-3 laboratory. Freshly purified PBMCs were used for phenotyping by flow cytometry, T-cell proliferation assay, T-cell functionality assay, and/or antigen-specific assay. For all experiments, PBMCs were cultured in R10 Medium [RPMI 1640 medium (Gibco ™ , Thermo Fisher, MA, USA) supplemented with 10% fetal bovine 

Fresh PBMCs were pre-labeled with carboxyfluorescein 6 succinimidyl ester (CFSE; 5 µM; Thermo Fisher) at 37°C for 10 min. Cells were then stimulated with anti-CD3 (2 µg/ml; BioLegend, CA, USA) plus anti-CD28 (1 µg/ml; BioLegend) antibodies for 3 days before FACS analysis. Non-stimulated or non-labeled cells served as controls. Proliferation index was calculated by normalizing the total number of divisions to number of cells that went into division (referring to flowjo proliferation platform: https://docs.flowjo.com/flowjo/ experiment-based-platforms/proliferation/). 

As previously described (20, 21) , fresh PBMCs were stimulated by 1 µg/ml spike peptide pool or 5 µg/ml nucleocapsid protein (NP) peptide pool of SARS-CoV-2 (15-mer overlapping by 11, spanning the whole spike or NP; Genscript, NJ, USA) in the presence of 0.5 µg/ml anti-CD28 and anti-CD49d antibodies (BioLegend) overnight. PMA/ionomycin stimulation served as positive control, while media only served as negative control. BFA (7.5 µg/ml) was added at 6 h before harvesting cells for intracellular FACS analysis on IFNg expression.

Valinomycin-treated PBMCs from HDs (1 µM, 60 min) and thawed PBMCs from HDs or APs were stained with MitoTracker ™ Green (200 nM) and MitoTracker ™ Red CMXRos (200 nM) at 37°C for 15 min before blocking with 

All statistical analyses were performed using the SPSS or GraphPad Prism 7 software. Data represent mean or mean with SEM of at least three independent experiments unless indicated. To compare the frequency of MD + cells between patients with high and low levels of markers (including HLA-DR + CD38 + , PD-1 + , and TNFa +/-IFNg +/-IL-2 +/-) or proliferation index, patients with level higher than the median were regarded as the high group, while others as the low group. Significant differences were calculated using a one-way analysis of variance (ANOVA) or two-tailed unpaired Student's t test. p < 0.05 was considered statistically significant.

By assessing clinical test results, we found that 73.9% of the 88 recruited study subjects displayed lymphocytopenia upon hospital admission. Consistent with previous findings, men were more likely to become severely ill than women (Table 1 ) (22, 23) . A higher neutrophil count was found in severe APs than non-severe ones ( Table 1 ) (24) . There were no differences between recruited mild and severe APs in terms of other clinical presentations ( Table 1) . We then profiled T-cell frequency in APs [n = 88; 14 asymptomatic (AS), 48 mild APs, and 26 severe APs], as compared to the control groups including HDs (n = 31) and CPs (n = 17), by FACS analysis ( Figure 1A ). Consistent with results as previously described by us and others (13, 21) , T-cell frequency in APs declined dramatically as compared with HDs ( Figure 1B ). More CD8 T-cell reductions were found among severe APs ( Figure 1B ) than mild ones (15) . In the meantime, we used MitoTracker ™ Green (Mito Green, representing for mitochondrial density) and MitoTracker ™ Red CMXRos (Mito Red, representing for mitochondrial membrane potential) to assess mitochondrial function by flow cytometry. The MD + proportion ( Figure 1A ) was defined by cells of Mito Green high Mito Red Low (25) (26) (27) , which was validated by confocal study and Seahorse XF Cell Mito Stress Test using the MD-inducing drug, valinomycin, on HD PBMCs (Supplementary Figures S1A-E). As compared with HDs, T cells from both AS and symptomatic AP groups showed high frequencies of MD + cells ( Figure 1C) . Moreover, the frequencies of MD + CD8 T cells were relatively higher than those of MD + CD4 T cells in both mild and severe APs ( Figure 1C) . The elevated MD + T cell proportion was consolidated by correlation analysis with the Mito Green high TMRM Low population [TMRM, another indicator for mitochondrial membrane potential (28)]. Both Mito Green high Mito Red Low and Mito Green high TMRM Low correlated positively with the Annexin V + apoptotic proportion in CD4 or CD8 T cells from APs (Supplementary Figures S2A, B) . Besides, the phenotype of MD + T cells was supported by moderately less fission under confocal analysis (29) and by relatively lower ATP production and maximal respiration by Seahorse XF Cell Mito Stress Test (Supplementary Figures S3A-F) .

Subsequently, we analyzed the kinetics of MD + T cells among symptomatic patients. In the first 5 days p.s.o., frequencies of Tcell subsets decreased quickly, while proportions of MD + T-cell subsets increased (Figures 1D, E) . MD + T-cell proportion, therefore, was negatively correlated with T-cell frequency, especially with a steeper slope of curve in CD4 than CD8 Tcell subsets ( Figure 1F ). Significant reduction of CD4 T-cell frequency was found in severe APs during the period of 6-10 days p.s.o., albeit that a stronger negative correlation between MD + proportion and CD4 T-cell frequency occurred in mild APs than severe APs ( Figures 1D, G) . During 11-15 days p.s.o., significantly lower MD + proportion in CD8 T cells was found in severe than mild APs, although no significant correlation was found between MD + proportion and CD8 T-cell frequency ( Figures 1E, H) . These results demonstrated that acute SARS-CoV-2 infection resulted in significant CD4 T-cell loss and subsequent CD8 T-cell loss especially among severe APs, and that MD correlated more negatively with CD4 than CD8 T-cell loss regardless of disease severity.

Next, we further analyzed MD phenotype in various T-cell subsets. On average, we found that both central (23.4%) and effector (44.9%) memory CD4 T cells (CD4 T CM and CD4 T EM ) were among total MD + CD4 T cells (Figures 2A, B) . Similarly, both effector memory (34.1%) and effector (44.3%) CD8 T cells were among total MD + CD8 T cells (Figures 2A, B) . To further investigate the role of MD in regulating T-cell activation and function, we examined biomarkers of T-cell hyperactivation (HLA-DR + CD38 + ) and activation/exhaustion (PD-1 + ) in memory subsets along with monitoring MD in APs (8 mild and 9 severe APs during 11-15 days p.s.o.) as compared to HD controls ( Figure 4A ). Like previous findings (21), CD4 T CM , CD4 T EM , CD8 T CM , and CD8 T EM cells from APs showed moderately increased percentage of HLA-DR + CD38 + cells and significantly increased percentage of PD-1 + cells as compared to HD ( Figure 4B ). Mild APs were likely to have higher frequencies of PD-1 + CD4 T CM , CD4 T EM , and CD8 T CM cells than severe ones ( Figure 4B) . Interestingly, when comparing MD + vs. HLA-DR + CD38 + cells, APs with highly hyperactivated CD4 T EM , CD8 T CM , and CD8 T EM cells also exhibited significantly higher MD + ( Figure 4C ). For PD-1 expression, however, PD-1 + memory T cells showed little association with MD + proportion except for T EM cells ( Figure 4D) . These results indicated that APs with hyperactivated memory T cells, especially memory CD8 T cells, displayed high MD phenotype at the time of 11-15 days p.s.o.. These findings implicated that mitochondria were likely involved in regulating T-cell hyperactivation and immune responses at the later stage of SARS-CoV-2 infection. 

Since memory T-cell hyperactivation was likely to be associated with high MD in APs at the time of 11-15 days p.s.o., we investigated the relationship between MD and T-cell functionality at this stage. After 3-day non-antigen T-cell receptor (TCR) activation, PBMCs from APs (8 mild and 5 severe) and HDs were harvested for proliferation analysis. Significantly lower proliferation index was found in both CD4 and CD8 T cells of mild rather than severe APs (Supplementary Figures S4A, B) . Moreover, APs with lower proliferation index in CD8 T cells showed significantly higher MD, which was not shown in CD4 T cells (Supplementary Figures S4C, D) . Subsequently, we analyzed T-cell functionality upon 6-h non-antigen PMA/ionomycin activation on PBMCs from these APs by evaluating intracellular cytokine expression levels of TNFa, IFNg, and IL-2 (21, 30) (Supplementary Figure 5) . Both CD4 T EM and CD8 T EM cells from mild APs exhibit a significantly lower percentage of single functional TNFa + IFNg -IL2cells compared to HDs ( Figure 5A) . Similarly, lower percentage of dual functional TNFa + IFNg + IL2cells was detected in both CD8 T CM and CD8 T EM cells from mild APs, which was not found in severe APs ( Figure 5A ). Among aforementioned functionally defective subsets of memory T cells, dramatically higher MD + proportion was observed in those subsets with lower percentage of single functional TNFa + IFNg -IL2cells ( Figures 5B, C) or dual functional TNFa + IFNg + IL2cells ( Figures 5D, E) . Basically, MD in T EM cells of APs was associated with reduced T-cell functionality upon PMA/ionomycin activation.

Considering that acute SARS-CoV-2 infection preferentially induced antigen-specific CD4 T-cell response (21, 31) , we investigated such responses in memory T-cell subsets upon Figure S6B ) (32) . We also compared the MD + proportion in these subsets specific to spike or NP among these 25 APs. Surprisingly, poor responders to antigen-specific responses of CD4 T EM exhibited significantly higher MD + proportion, which was not shown in CD4 T CM , CD8 T CM , and CD8 T EM cells ( Figure 6C ). Figure S7) . Therefore, MD in CD4 T EM cells in APs was preferentially related to poor SARS-CoV-2specific responses.

In this study, we demonstrated that SARS-CoV-2 infection resulted in rapidly increased frequencies of MD + T cells by testing freshly isolated PBMCs from COVID-19 patients.

Notably, this increase took place among all infected individuals including asymptomatic subjects within the first week of symptom onset and the symptomatic subjects without any drug treatment. Moreover, this increase was not restricted to a particular T-cell population because both CD4 and CD8 T cells with various phenotypic subsets displayed elevated MD. Among symptomatic patients, however, the frequency of MD + T cells was preferentially correlated with CD4 T-cell loss and CD8 T-cell hyperactivation, respectively. While lower proportion of MD + memory CD8 T cells was found among severe patients than in mild ones at the stage of disease severity, both mild and severe patients bearing effector memory MD hi CD4 or MD hi CD8 T cells exhibited poorer T-cell responses upon stimulation of either SARS-CoV-2 peptide or PMA/ionomycin. Our results, therefore, demonstrated that MD was associated with T lymphocytopenia and impaired T-cell functionality during acute phase of COVID-19. SARS-CoV-2 manipulated host T-cell mitochondria rapidly, contributing to T lymphocytopenia. The rapid burst of viral loads during the first week of symptom onset might have direct effects on mitochondria. SARS-CoV-2 proteins nsp2 and nsp4 were likely involved in endoplasmic reticulum calcium homeostasis and mitochondrial biogenesis (33) . SARS-CoV-2 RNA-protein interactions mediate virus-mediated mitochondrial dysfunction during early infection (34) . Growing evidence indicated that the causes of this rapid manipulation might involve multiple mechanisms in immune cells. Recently, elevated mitochondrial mass was found to be correlated with mitochondrial apoptosis in T cells from COVID-19 patients (35) . A highly expressed voltage-dependent anion channel 1, a mitochondrial membrane protein for transporting calcium, was suggested as a cause of mitochondrial dysregulation and apoptosis of T cells in COVID-19 patients (36) . Moreover, COVID-19 patients displayed depolarized mitochondria and abnormal mitochondrial ultrastructure in monocytes, which was associated with the production of several inflammatory cytokines and chemokines (37) . Inflammatory cytokines such as high levels of TNFa and IL-6 might then drive mitochondria-mediated T-cell apoptosis and lymphocytopenia (14, 38) . Consistently with other studies (35, 36, 38) , we observed a strongly negative correlation between profound T-cell loss and rapidly increased MD + T-cell proportion. Besides, with a large number of patients, our results further extend the impact of SARS-CoV-2 infection on the kinetics of T-cell frequency and mitochondrial dysfunction not only to asymptomatic subjects but also to a much broader range of T-cell subsets. It is, therefore, plausible that mitochondria-driven T-cell apoptosis could be one of the mechanisms underlying clinical T lymphocytopenia. To investigate the relationship between MD + proportion and T-cell activation, the percentages of HLA-DR + CD38 + cells and MD + proportion in CD4 T CM , CD4 T EM , CD8 T CM , and CD8 T EM cells were compared between patients with a high percentage of HLA-DR + CD38 + cells (higher than median) and those with a low percentage of HLA-DR + CD38 + cells (not higher than median). (D) Similarly, the percentages of PD-1 + cells and MD + proportion in CD4 T CM , CD4 T EM , CD8 T CM , and CD8 T EM cells were compared between patients with high percentage of PD-1 + cells (higher than median) and those with low percentage of PD-1 + cells (not higher than median). HDs served as control. Data represent mean ± SEM. Statistics were calculated based on one-way ANOVA test. *p < 0.05, **p < 0.01, ***p < 0.001. NS, not significantly different.

immune responses are essential for eliminating SARS-CoV-2infected cells and assisting antibody responses (18, 19) . Upon antigen binding to TCR, T cells undergo drastic metabolic reprogramming with increased glucose utilization and glycolysis, which was controlled by mitochondria to modulate cell proliferation and differentiation (39) . When mitochondrial function is compromised during cellular stress, the bioenergy and responses of immune cells will be reduced. Indeed, a The percentage of certain cytokine expression groups and profile of MD + proportion in CD4 T EM , CD8 T CM , and CD8 T EM cells were compared between patients with highly cytokine-expressed cells (higher than median) and those with impaired cytokine-expressed cells (not higher than median). Data represent mean ± SEM. Statistics were calculated based on one-way ANOVA test. *p < 0.05, **p < 0.01, ***p < 0.001.

compromised mitochondrial function and deficient energy supply were observed in PBMCs from COVID-19 patients, which contributed to enhanced inflammatory responses causing disease severity (40) . MD in T cells might also involve a series of metabolic dysregulation including reduced ATPlinked respiration, dampened glycolysis, and decreased mitochondrial membrane potential (35, 40) . During these processes, the formation of oxysterols would increase mitochondrial oxidative stress (41) , and in return, the exacerbation of mitochondria-derived ROS might lead to the rupture of redox homeostasis and the induction of apoptosis (42) . To this end, we consistently found high calcium uptake and excessive production of mitochondrial ROS in T cells of COVID-19 patients. Under MD with excessive ROS and low mitochondrial membrane potential, fission is a critical way to remove damaged mitochondria via mitophagy (43) . Whether or not SARS-CoV-2 infection dampens the activities of mitophagy and mitochondrial fission in host T cells as potential causes of MD remains to be further investigated. Furthermore, among patients with MD hi T cells, our finding of poorer CD4 T EM or memory CD8 T-cell responses to SARS-CoV-2 peptide or PMA stimulation highlighted the reduced antiviral T-cell immune responses. Our patients with MD hi CD4 T EM and memory CD8 T cells exhibited functional exhaustion with reduced production of pro-inflammatory cytokines such as TNFa and IFNg, which was also in line with other reports on chronic hepatitis B or long-term tumor antigen exposure (44, 45) . Interestingly, since CD4 and CD8 T cells might undergo different metabolic processes (46) , it might explain why the frequency of MD + T cells was correlated preferentially and respectively with CD4 T-cell loss and CD8 T-cell hyperactivation in this study. Further studies are required to reveal why more robust CD4 but not CD8 T-cell responses were found during the natural course of SARS-CoV-2 infection (21) . were also compared between poor responders (-) and good responders (+) specific to spike or NP peptide pool. Data represent mean ± SEM. Statistics were calculated based on unpaired Student's t-test. *p < 0.05, **p < 0.01, ***p < 0.001. NS, not significantly different.

In terms of the association between MD and disease severity, it is possible that MD-related poor responses of CD4 T EM and memory CD8 T cells might be linked with COVID-19 disease progression or severity. Profound T-cell loss during 6-10 days p.s.o. predicts ICU admission and disease severity (13, 15, 16) , but it also potentiates thymopoiesis and results in naive T-cell overproduction in a compensative manner (47) . Partially reversed T-cell count in severe patients during 11-15 days p.s.o. might be due to freshly compensated T cells. A recent single-cell transcriptomics study pointed out that SARS-CoV-2specific CD8 T cells in mild patients were found to be proexhausted but those in severe APs were pro-survival (48) , which might be a consequence of the compensation manner. Increased SARS-CoV-2-reactive T cells were also suggested as a cause of immunopathogenesis in patients (48, 49) , which might be alleviated in patients with MD hi T cells along with impaired Tcell functionality. Furthermore, most severe patients received treatment of steroid, when oxygen supply is required from the second week p.s.o (50) . Unexpectedly, we found a relatively lower proportion of MD + T cell but better T-cell functionality in CD4 T EM and memory CD8 T cells of severe patients than mild ones during 11-15 days p.s.o. Steroid did not seem to be a distinct immunosuppressor for interrupting thymopoiesis and inhibiting T-cell responses (51) , but early administration of steroid is probably good for CD8 T-cell restoration in CPs after day 14 p.s.o (52) . It is, therefore, possible that MD in T cell may serve as a suppressive factor in immunopathogenesis during severity development.

The raw data supporting the conclusions of this article will be made available by the authors without undue reservation.

The studies involving human participants were reviewed and approved by the institutional review board of University of Hong Kong/Hospital Authority Hong Kong West Cluster and Kowloon West Cluster Research Ethics Committee [UW 13-265 and KW/EX-20-038(144- 26) ]. The patients/participants provided their written informed consent to participate in this study.

ZC and K-YY supervised the collaborative teams and gained the research grants for this study. YM, RZ, and ZC designed the experiments, performed the experiments, collected the data, analyzed the data, interpreted the data, and wrote the article. KK-WT collected the clinical data, analyzed the clinical data, interpreted both clinical and experimental data, and prepared the article. LL, TC and CYHL participated in the experimental design, data analysis, and data interpretation. 

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We thank Ms. Polly Pang for technical assistance.

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2021.

Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Publisher's Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.Copyright © 2022 Mo, To, Zhou, Liu, Cao, Huang, Du, Lim, Yim, Luk, Chan, Chik, Lau, Tsang, Tam, Hung, Yuen and Chen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.