key: cord-1000191-zgujsi9c
authors: Kalfaoglu, Bahire; Almeida-Santos, José; Tye, Chanidapa Adele; Satou, Yorifumi; Ono, Masahiro
title: T-cell dysregulation in COVID-19
date: 2020-11-07
journal: Biochem Biophys Res Commun
DOI: 10.1016/j.bbrc.2020.10.079
sha: a47b656aa5a1563b9315ef836a442786edf60dfa
doc_id: 1000191
cord_uid: zgujsi9c

T-cells play key roles in immunity to COVID-19 as well as the development of severe disease. T-cell immunity to COVID-19 is mediated through differentiated CD4(+) T-cells and cytotoxic CD8(+) T-cells, although their differentiation is often atypical and ambiguous in COVID-19 and single cell dynamics of key genes need to be characterized. Notably, T-cells are dysregulated in severe COVID-19 patients, although their molecular features are still yet to be fully revealed. Importantly, it is not clear which T-cell activities are beneficial and protective and which ones can contribute to the development of severe COVID-19. In this article, we examine the latest evidence and discuss the key features of T-cell responses in COVID-19, showing how T-cells are dysregulated in severe COVID-19 patients. Particularly, we highlight the impairment of FOXP3 induction in CD4(+) T-cells and how the impaired FOXP3 expression can lead to the differentiation of abnormally activated (hyperactivated) T-cells and the dysregulated T-cell responses in severe patients. Furthermore, we characterise the feature of hyperactivated T-cells, showing their potential contribution to T-cell dysregulation and immune-mediated tissue destruction (immunopathology) in COVID-19.

T-cell numbers are regulated by proliferation and apoptosis during homeostasis [13] , and accordingly, T-cell reduction in COVID-19 can be due to either or both of increased apoptosis and reduced proliferation rates. While Fas expression is increased in T-cells from COVID-19 [14] , T-cell data in Zhu et al showed that Fas, FasL, and Caspase-3 [15] , which play key roles of T-cell apoptosis, were not significantly increased in COVID-19 patients [16] .

Interleukin (IL)-7 is a key cytokine for T-cell homeostasis, sustaining the naïve T-cell pool [17] .

However, serum IL-7 levels are increased in severe COVID-19 patients [18] , indicating that the IL-7-mediated compensatory mechanism is operating normally. IL-15 is important for maintaining the size of the CD8 + T-cell and memory T-cell pool [17] and could play a role in T-cell homeostasis in COVID-19, although data for IL-15 in COVID-19 is limited. Interestingly, T-cell numbers are negatively correlated with the serum concentration of cytokines including IL-6 and IL-10 in COVID-19 patients [7] . IL-6 is primarily produced by macrophages,

inflammatory conditions [19] . IL-10 is produced by a wide range of cells including dendritic

T-cells (Treg). IL-10 can suppress the proliferation of CD4 + and CD8 + T-cells in some contexts [20] while enhancing T-cell proliferation in the presence of other γ-chain cytokines i.e. IL-2, IL-4, IL-7, and IL-15 [21] . Given the increased cytokine production in severe COVID-19 patients, it is unlikely that the elevated IL-10 levels is the cause of T-cell reduction. These collectively suggest that T-cell reduction is a consequence of the activation of various innate and adaptive immune cells, which inevitably leads to the activation of T-cells as well.

SARS-CoV-2-infected epithelial cells can detect viral RNA by cytosolic sensors including RIG-1 and MDA5 and produce type-I IFNs, which induces IFN-mediated anti-viral responses [22] .

Notably, In comparison to other respiratory viruses, SARS-CoV-2 induces lower levels of type-I IFNs (IFN-α, IFN-β) and type-III IFN (IFN-λ) and higher chemokine expression in primary human bronchial epithelial cells in comparison to other common respiratory viruses [23] . Still, IL-1β and type I and type III interferons (IFNs) are modestly induced in active COVID-19, while persisting and even increased during the recovery phase [24] . J o u r n a l P r e -p r o o f Type-I IFNs not only induce cell-intrinsic anti-viral states in infected and neighbour cells, but also promote innate immune responses. In viral infections, dendritic cells (DCs), particularly plasmacytoid dendritic cells (pDCs), play key roles in inducing innate immune responses following IFN responses, as pDCs can rapidly produce large amounts of type-I IFNs upon viral infection [25] . Importantly, pDCs can produce type-I IFNs not only when they are infected but also when adjacent cells are infected and produce type-I IFNs. However, Laing et al showed that pDCs were markedly reduced in PBMCs from severe COVID-19 patients in comparison to moderate patients and healthy controls [26] .

In addition, type-I IFN signalling promotes the maturation of DCs, increasing the expression of MHC and CD80/86 [27] , the latter of which enhances the activation of T-cells as discussed below. In severe COVID-19 patients, type-I IFN response is impaired in peripheral mononuclear blood cells (PBMCs) and BAL fluid, showing reduced type-I IFN production [28, 29] . Similarly, in SARS-CoV-1, delayed type-I IFN responses can promote the development of a severe disease through the accumulation of inflammatory monocytesmacrophages, which leads to impaired T-cell responses [30] . In fact, circulating monocytes from severe patients express more IL-1β and TNF-α [31] . In addition, macrophages in bronchoalveolar lavage (BAL) fluids from severe COVID-19 patients express higher levels of IL-1β, IL-6 and TNF-α than moderate patients [32] . Intriguingly, the chemokine CCL3 (MIP1A) is increased in both serum and BAL fluid-derived T-cells from severe COVID-19 patients [18, 33] . Thus, the combination of the impaired production of type-I IFNs and the increased production of proinflammatory cytokines may contribute to the development of the severe disease [22] .

Collectively, the impaired IFN responses in COVID-19 can result in the increased proinflammatory cytokine responses, changing the dynamics of antigen presentation and cytokine production in innate immune cells, which may contribute to the dysregulation of Tcell responses.

Antigen presenting cells such as mature DCs present antigens to T-cells on their MHCs, and highly express CD80/CD86, which interacts with CD28 on T-cells [34] . Then, antigen-specific J o u r n a l P r e -p r o o f T-cells receive TCR signals and costimulatory signals, resulting in their activation. Activated T-cells produce IL-2 while expressing the IL-2 receptor, which is composed of α (CD25), β (CD122), and γ chains [35] . CD25 is particularly important as an inducible protein to produce the high-affinity IL-2 receptor. Thus, CD25-expressing activated T-cells receive IL-2 signalling, which further promotes their proliferation and differentiation.

Interestingly, soluble CD25 is increased in COVID-19 patients [24] , and a study showed the increase of IL-2 as well [18] . Since both IL-2 and IL-2 receptors are expressed predominantly by T-cells, it is possible that the positive feedback loop for IL-2 signalling in T-cells is established in some patients. However, it is not known whether this is protective or contributes to the pathology of COVID-19. In normal situations, IL-2 signalling can induce FOXP3, which stops the IL-2 response as a negative feedback mechanism [36] , but this FOXP3 induction is impaired in severe COVID-19 as discussed below. Importantly, therapeutic administration of recombinant IL-2 in human patients can induce vascular leak syndrome, which is characterized by multiple organ failure due to increased vascular permeability [37] . haplotype [40] to understand the dynamics of virus-specific T-cells and self-reactive T-cells.

J o u r n a l P r e -p r o o f Th1 cells, which produce IFN-γ and TNF-α [41] , are important for viral infections, although the role of Th1 response in COVID-19 is not clear. SARS-CoV-2 spike (S) protein-specific T cells are induced during the acute phase of COVID-19 and these cells produce IFN-γ [42] .

However, another report showed that CD4 + T-cells had impaired IFN-γ and TNF-α production in severe COVID-19 patients [43] , although monocytes in severe COVID-19 patients produce higher levels of IL-12 [44] , which potently induces Th1 differentiation [41] .

Lucas et al showed that severe COVID-19 patients had higher serum levels of IFN-γ and IL-17A, the latter of which is produced by Th17 cells [8] . Th17 plays a key role in autoimmunity and infections including COVID-19 [45] . Notably, IL-6 signalling induces Th17 differentiation through activating STAT3 and inducing Th17 transcription factors including ROR-γt and AHR [46] . Intriguingly, T-cells from COVID-19 patients express higher levels of the IL-6 receptor than those from Influenza patients and healthy controls [16] . In addition, some T-cells from COVID-19 patients are identified as non-classical Th1 cells that highly express the Th17 markers CD161 and IL-1 receptor type I (IL-1RI) [24] . IL4R and MAF, which are required for Th2 differentiation, were more highly induced in BALderived CD4 + T-cells from severe COVID-19 patients [33] . Collectively, it is likely that T-cells show mixed Th1, Th2, and Th17 responses in COVID-19, and it is yet to be revealed which Th response is the most protective.

CD4 + T cell responses to S-protein correlated with the concentration of the anti-SARS-CoV-2

IgG and IgA titres in convalescent patients [1] , showing the importance of the T-cell help for B-cell maturation in recovery from COVID-19. This is most likely mediated by a subtype of CD4 + T-cells, T-follicular helper cells (Tfh), which provide the T-cell help that is required for B-cell differentiation and the affinity maturation of antibodies [47] . Tfh cells differentiate 48] including severe patients [49] . Interestingly, cTfh upregulate CCR6, a marker of Th17 [50] , upon stimulation with the S-protein of SARS-CoV-2, thus showing Th17-like cTfh features [51] . On the other hand, Kaneko et al showed that germinal centres were not formed in lymph nodes and that BCL-6-expressing Tfh were reduced in COVID-19 [52] . In addition, Kalfaoglu et al showed that BCL6 and Th17 genes (including RORC, IL17A, and CCR6) were repressed in BAL fluid-derived CD4 + T-cells from severe COVID-19 [33] . Collectively, these suggest that uncharacterised T-cell differentiation, which partially resemble Th17 and Tfh, may be important to control the infection. 

FOXP3 is widely recognised as the lineage-specific transcription factor of Treg, FOXP3expressing T-cells can suppress T-cell responses [58, 59] . In humans, ~5% of CD4 + T-cells highly express FOXP3 [60, 61] . Notably those FOXP3 high Treg highly express CD25 and downregulate IL-7 receptor α (CD127) [61] . The majority of such naturally-occurring FOXP3 + Treg recognise self-antigens or microbiome antigens [62] . In fact, FOXP3 + Treg regularly J o u r n a l P r e -p r o o f receive TCR signals in vivo [63] and show higher Ki67 expression, indicating their activated and proliferating status [61] . The expression of both CD25 and FOXP3 is dynamically regulated in vivo [60, 63] and is controlled by IL-2 and TCR signals [64] .

When TCR and TGF-β signals are available, IL-2 signalling in activated T-cells promotes not only their survival and proliferation but also the expression of the FOXP3 gene [65] . FOXP3

represses IL2 transcription, inducing hyporesponsiveness (anergy) to TCR signals [66] .

Importantly, natural FOXP3 + Treg also further upregulate FOXP3 transcription through its autoregulatory transcriptional loop during inflammation, increasing the expression of Tregassociated molecules including CD25 and CTLA-4, which is required for their suppressive function [67] . Thus, FOXP3 works as a negative feedback mechanism for suppressing T-cell activation whether in Treg and in non-Treg (Figure 1 ).

than unexposed individuals [68] , and the frequency of Treg is reduced in severe patients in comparison to moderate patients [26, 69] . This suggests that, paradoxically, FOXP3 induction is favourable to the outcome, suggesting a role of FOXP3 in anti-viral T-cell immunity.

Intriguingly, a significant percentage of CD4 + T-cells highly express CD25 while FOXP3 expression is repressed in the lung of severe COVID-19 patients [33] . Those CD25 + FOXP3 -Tcells highly express CTLA-4, GITR, and other activation/Treg markers, suggesting that their differentiation into Treg is prematurely blocked (Figure 1 ). In addition, Jeannet et al showed that severe patients in Intensive Care Unit (ICU) showed sustained reduction of all lymphocyte subsets including T-cells, while the expression of CD25, CTLA-4, and PD-1 in CD4 + T-cells was increased, although their study did not analyse FOXP3 expression [6] .

Although the mechanism of FOXP3 repression in severe COVID-19 is yet to be revealed, IL-6 produced by macrophages and monocytes may contribute to the repression. IL-6 signalling activates STAT3, which antagonises STAT5 activities and thereby represses IL-2-mediated FOXP3 transcription [70] . As discussed above, T-cells in severe COVID-19 show higher IL-6 receptor expression and thus are considered to be sensitive to IL-6 in the microenvironments. In addition, our scRNA-seq analysis showed that IL-2 was not detected in BAL fluid-derived CD4 + T-cells from severe COVID-19 patients [33] , and critically ill patients show reduced serum IL-2 [71] . These suggest that IL-6 overproduction and IL-2 deprivation contribute to FOXP3 repression in severe COVID-19.

J o u r n a l P r e -p r o o f FOXP3 inhibition generally induces inflammation, although this may not be beneficial in infections [36] . Rather, the inhibition of FOXP3 induction can lead to unfocused T-cell responses that induce immunopathology (i.e. immune-mediated tissue destruction) and

ineffective antiviral T-cell response. For example, in a mouse model of herpes simplex infection, the depletion of FOXP3 + Treg induces strong inflammation and prolong the infection [72] . Similarly, Treg depletion results in increased mortality in a murine model of coronavirus-induced acute encephalitis [73] .

In addition, depletion of FOXP3 + Treg can induce T-cell responses to self-antigens, which lead to the development of autoimmunity [59] . Interestingly, severe COVID-19 patients show B-cell repertoire features previously described in active systemic lupus erythematosus (SLE) patients, a systemic autoimmune disease [74] . Here it is worthwhile to note that active SLE patients have reduced FOXP3 high Treg in circulation [61] . These collectively suggest that the impairment of FOXP3 induction in severe COVID-19 induces autoimmune-like T-cell responses to self-antigens, which deplete immunological resources that could have been used by virus-specific T-cells.

Interestingly, CD25 + T-cells in severe COVID-19 patients specifically express the protease FURIN [33] . FURIN and the serine protease TMPRSS2 can cleave spike (S) protein of SARS-CoV-2 and enhance the viral entry into human cells [75] . FURIN is expressed by Treg and a knockout study showed that FURIN expression is required for Treg-mediated immune suppression [76] . In addition, FURIN expression is induced in T-cells by TCR signalling in vivo and in vitro [33] , and therefore, FOXP3 + Treg and highly activated CD25 + CD4 + T-cells could potentially enhance the activation of S protein in inflammatory tissues. Since a larger proportion of macrophages produce FURIN and the number of FOXP3 -CD25 + CD4 + T-cells is small in each individual, the contribution of FURIN expression in those T-cells towards enhancing viral entry, if any, could be mediated through either SARS-CoV-2 infection of Tcells themselves or the enhancement of viral entry into DCs with which antigen-specific Tcells may intimately interact [77] . Although T-cells do not express ACE2, it is possible that SARS-CoV-2 enters non-ACE2 expressing cells using alternative receptors [78] , and further investigation is needed.

Collectively, FOXP3 -CD25 + CD4 + T-cells in severe COVID-19 patients are considered to be abnormally activated (hyperactivated), failing to differentiate into specific T-cell subsets.

Their activation and differentiation may also be prematurely stopped at an early activation stage and contribute to the infection and pathogenesis (Figure 1 ). 

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Please note that all Biochemical and Biophysical Research Communications authors are required to report the following potential conflicts of interest with each submission. If applicable to your manuscript, please provide the necessary declaration in the box above.

(1) All third-party financial support for the work in the submitted manuscript.

(2) All financial relationships with any entities that could be viewed as relevant to the general area of the submitted manuscript.

(3) All sources of revenue with relevance to the submitted work who made payments to you, or to your institution on your behalf, in the 36 months prior to submission. (4) Any other interactions with the sponsor of outside of the submitted work should also be reported. (5) Any relevant patents or copyrights (planned, pending, or issued). (6) Any other relationships or affiliations that may be perceived by readers to have influenced, or give the appearance of potentially influencing, what you wrote in the submitted work. As a general guideline, it is usually better to disclose a relationship than not.

Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals

Detection, phenotyping, and quantification of antigen-specific T cells using a peptide-MHC dodecamer

Comparative analysis of activation induced marker (AIM) assays for sensitive identification of antigen-specific CD4 T cells

Molecular Mechanism of Class Switch Recombination: Linkage with Somatic Hypermutation

Memory CD8 T-cell differentiation during viral infection

Severe COVID-19 is associated with deep and sustained multifaceted cellular immunosuppression

Reduction and Functional Exhaustion of T Cells in Patients With Coronavirus Disease 2019 (COVID-19)

Longitudinal analyses reveal immunological misfiring in severe COVID-19

Association between high serum total cortisol concentrations and mortality from COVID-19

Lymphopenia and neutrophilia in SARS are related to the prevailing serum cortisol

Mechanisms of corticosteroid action on lymphocyte subpopulations. III. Differential effects of dexamethasone administration on subpopulations of effector cells mediating cellular cytotoxicity in man

Mechanisms of corticosteroid action on lymphocyte subpopulations. I. Redistribution of circulating T and b lymphocytes to the bone marrow

Mathematical Modeling Reveals the Biological Program Regulating Lymphopenia-Induced Proliferation

Increased CD95 (Fas) and PD-1 expression in peripheral blood T lymphocytes in COVID-19 patients

Activation-induced cell death: The controversial role of Fas and Fas ligand in immune privilege and tumour counterattack

Single-Cell Sequencing of Peripheral Mononuclear Cells Reveals Distinct Immune Response Landscapes of COVID-19 and Influenza Patients

Homeostasis of naive and memory T cells

Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China, The Lancet

IL-6 Promotes T Cell Proliferation and Expansion under Inflammatory Conditions in Association with Low-Level RORγt Expression

Diversity in the contribution of interleukin-10 to T-cell-mediated immune regulation

Lanzavecchia Cytokine-driven Proliferation and Differentiation of Human Naive, Central Memory, and Effector Memory CD4+ T Cells

Interplay between SARS-CoV-2 and the type I interferon response

Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19

Next-Generation Sequencing of T and B Cell Receptor Repertoires from COVID-19 Patients Showed Signatures Associated with Severity of Disease

Plasmacytoid Dendritic Cells: Development, Regulation, and Function

A dynamic COVID-19 immune signature includes associations with poor prognosis

Type I IFN drives a distinctive dendritic cell maturation phenotype that allows continued class II MHC synthesis and antigen processing

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

Host-Viral Infection Maps Reveal Signatures of Severe COVID-19 Patients

Dysregulated Type I Interferon and Inflammatory Monocyte-Macrophage Responses Cause Lethal J o u r n a l P r e -p r o o f Pneumonia in SARS-CoV-Infected Mice

Immunophenotyping of COVID-19 and influenza highlights the role of type I interferons in development of severe COVID-19

Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19

T-Cell Hyperactivation and Paralysis in Severe COVID-19 Infection Revealed by Single-Cell Analysis

CD28 induces immunostimulatory signals in dendritic cells via CD80 and CD86

Structure and function of the interleukin 2 receptor: affinity conversion model

Controversies concerning thymus-derived regulatory T cells: fundamental issues and a new perspective

Constitutive expression of IL-2Rbeta chain and its effects on IL-2-induced vascular leak syndrome

Single-cell TCRseq: paired recovery of entire T-cell alpha and beta chain transcripts in T-cell receptors from single-cell RNAseq

T-cell receptor repertoires share a restricted set of public and abundant CDR3 sequences that are associated with self-related immunity

Molecular Mechanisms RegulatinG Th1 Immune Responses

Phenotype and kinetics of SARS-CoV-2-specific T cells in COVID-19 patients with acute respiratory distress syndrome

Elevated exhaustion levels and reduced functional diversity of T cells in peripheral blood may predict severe progression in COVID-19 patients

Single-cell RNA-seq and V(D)J profiling of immune cells in COVID-19 patients

Marked T cell activation, senescence, exhaustion and skewing towards TH17 in patients with COVID-19 pneumonia

Regulation of the Immune Response by the Aryl Hydrocarbon Receptor

T follicular helper cell differentiation, function, and roles in disease

Breadth of concomitant immune responses prior to patient recovery: a case report of non-severe COVID-19

Comprehensive mapping of immune perturbations associated with severe COVID-19

RORC2: The master of human Th17 cell programming

Humoral and circulating follicular helper T cell responses in recovered patients with COVID-19

Oxford Immunology Network Covid-19 Response, I.C. Investigators, Broad and strong memory CD4+ and CD8+ T cells induced by SARS-CoV-2 in UK convalescent individuals following COVID-19

Aberrant hyperactivation of cytotoxic T-cell as a potential determinant of COVID-19 severity

Impaired Cytotoxic CD8+ T Cell Response in Elderly COVID-19 Patients

Follicular CXCR5-expressing CD8+ T cells curtail chronic viral infection

T cell receptor signalling in the control of regulatory T cell differentiation and function

Regulatory T cells and immune tolerance

Regulatory T Cells in Melanoma Revisited by a Computational Clustering of FOXP3+ T Cell Subpopulations

Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor

Regulatory T cells and immune tolerance in the intestine

A timer for analyzing temporally dynamic changes in transcription during differentiation in vivo

Control of regulatory T-cell differentiation and function by T-cell receptor signalling and Foxp3 transcription factor complexes

Critical Role of TGF-β and IL-2 Receptor Signaling in Foxp3 Induction by an Inhibitor of DNA Methylation

Control of Regulatory T Cell Development by the Transcription Factor Foxp3

From stability to dynamics: understanding molecular mechanisms of regulatory T cells through Foxp3 transcriptional dynamics

The analysis of the long-term impact of SARS-CoV-2 on the cellular immune system in individuals recovering from COVID-19 reveals a profound NKT cell impairment

Clinical and immunological features of severe and moderate coronavirus disease

Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells

The inhibition of IL-2/IL-2R gives rise to CD8+ T cell and lymphocyte decrease through JAK1-STAT5 in critical patients with COVID-19 pneumonia

Coordination of early protective immunity to viral infection by regulatory T cells

Role of regulatory T cells in coronavirus-induced acute encephalitis

Extrafollicular B cell responses correlate with neutralizing antibodies and morbidity in COVID-19

A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells

T-cell-expressed proprotein convertase furin is essential for maintenance of peripheral immune tolerance

Characterization of CD4+ T-cell-dendritic cell interactions during secondary antigen exposure in tolerance and priming

A Single-Cell RNA Expression Map of Human Coronavirus Entry Factors