key: cord-0703575-kh8fk774
authors: Rha, Min-Seok; Kim, A Reum; Shin, Eui-Cheol
title: SARS-CoV-2-Specific T Cell Responses in Patients with COVID-19 and Unexposed Individuals
date: 2021-02-17
journal: Immune Netw
DOI: 10.4110/in.2021.21.e2
sha: 86fac8c4ff6022ea66065057219626667d216619
doc_id: 703575
cord_uid: kh8fk774

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes coronavirus disease 2019 (COVID-19), an ongoing pandemic disease. In the current review, we describe SARS-CoV-2-specific CD4(+) and CD8(+) T-cell responses in acute and convalescent COVID-19 patients. We also discuss the relationships between COVID-19 severity and SARS-CoV-2-specific T-cell responses and summarize recent reports regarding SARS-CoV-2-reactive T cells in SARS-CoV-2-unexposed individuals. These T cells may be cross-reactive cells primed by previous infection with human common-cold coronaviruses. Finally, we outline SARS-CoV-2-specific T-cell responses in the context of vaccination. A better understanding of SARS-CoV-2-specific T-cell responses is needed to develop effective vaccines and therapeutics.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes coronavirus disease 2019 (COVID- 19) , which was declared a pandemic disease by the World Health Organization. As of January 3, 2021, there have been more than 84 million confirmed cases and more than 1.8 million deaths attributed to COVID-19 worldwide. SARS-CoV-2 infection results in diverse clinical manifestations, from asymptomatic or mild disease to severe disease that is associated with hyper-inflammatory responses (1) (2) (3) . Although the case fatality rate of COVID-19 is lower than that of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), it is much higher than that of seasonal influenza (4, 5) .

Patients with COVID-19 tend to have lymphopenia that preferentially affects T-cell populations (6, 7) , and T-cell lymphopenia is associated with severe COVID-19 (7, 8) . Early studies reported activation and exhaustion phenotypes for T cells in patients with COVID-19 and relationships between the T cell phenotype and disease severity (6, 7, (9) (10) (11) (12) (13) (14) . However, those studies examined pan-CD4 + and/or CD8 + T-cell populations without information on Ag specificity.

Later, SARS-CoV-2-reactive T-cell responses in acute and/or convalescent COVID-19 patients were reported. SARS-CoV-2-reactive T cells were examined by functional assays, including IFN-γ ELISpot assays, intracellular cytokine staining (ICS), and activation-induced marker (AIM) assays performed following ex vivo stimulation with SARS-CoV-2 proteins or peptides. More recently, SARS-CoV-2-specific CD8 + T cells were detected, and their phenotypes were examined by an MHC-I multimer technique (15) (16) (17) (18) (19) (20) (21) . In addition, SARS-CoV-2-reactive T cells were enriched using an Ag-reactive T cell enrichment (ARTE) technique and subsequently analyzed by single-cell RNA sequencing (scRNA-seq) (22) .

In the present review, we summarize recent reports regarding SARS-CoV-2-specific T-cell responses in acute and convalescent COVID-19 patients and unexposed individuals. We also describe SARS-CoV-2-specific T-cell responses in the context of vaccination.

In COVID-19 convalescent individuals, SARS-CoV-2-specific T-cell responses are detected by IFN-γ ELISpot assays or ICS following ex vivo stimulation of PBMCs with recombinant proteins (23) or overlapping peptide pools (24) . When AIM assays were performed with peptides predicted for MHC-I or MHC-II binding, SARS-CoV-2-specific CD4 + and CD8 + T-cell responses were detected in 100% and up to 70% of COVID-19 convalescent individuals, respectively (25) . These responses targeted not only spike (S) protein, but also membrane (M), nucleocapsid (N), and other open reading frames (ORFs). In these studies, SARS-CoV-2-specific T-cell responses significantly correlated with SARS-CoV-2 S-specific Ab titers (15, 23, 25, 26) . However, memory T-cell responses can be elicited even in the absence of SARS-CoV-2-specific Abs (16) .

In COVID-19 convalescent individuals, SARS-CoV-2-specific CD4 + T cells predominantly produce IFN-γ, IL-2, and TNF, whereas SARS-CoV-2-specific CD8 + T cells predominantly produce IFN-γ and exert a cytotoxic function that can be evaluated by degranulation marker CD107a following ex vivo stimulation (16) . CD8 + T cells targeting different viral proteins exhibit different degrees of polyfunctionality. M-or N-specific CD8 + T cells had higher polyfunctionality, a greater ability to produce multiple cytokines when stimulated, than S-specific CD8 + T cells (15) . CD4 + T cells targeting different viral proteins exhibited different differentiation patterns in convalescents. S-specific CD4 + T cells had a profile of follicular helper T (T FH ) cells, whereas M-or N-specific CD4 + T cells had a profile indicative of Th1/ Th17 cells (16) . However, little or no Th2 cytokines were produced by S-, M-, or N-specific T cells from COVID-19 convalescents (25, 27) although Th2 cell responses play a role in some pulmonary diseases.

The phenotypes of SARS-CoV-2-specific T cells was initially characterized by stimulationbased functional assays, such as ICS, and SARS-CoV-2-specific T cells exhibited mainly the effector memory (EM) phenotype (26) . However, the phenotypes of T cells can change during ex vivo stimulation. More importantly, virus-specific, non-functioning T cells cannot be detected by stimulation-based functional assays. These limitations can be overcome by using MHC multimer techniques, which require information on the immunodominant epitopes and their MHC restriction. Recently, data from MHC multimer staining became available in terms of the phenotypes of SARS-CoV-2-specific T cells, particularly CD8 + T cells. A list of SARS-CoV-2-specific MHC-I multimers with positive staining results is presented in Table 1 .

MHC-I multimer staining revealed that SARS-CoV-2-specific CD8 + T cells exhibit the EM (CCR7 − CD45RA − ; up to 50%) or central memory (CM) phenotypes (CCR7 + CD45RA − ; up to 20%) with early (CD27 + CD28 + ; up to 40%) or intermediate (CD27 + CD28 − ; up to 50%) differentiation in COVID-19 convalescent individuals (15) . In another study, SARS-CoV-2specific MHC-I multimer + CD8 + T cells exhibited an early differentiated memory phenotype (CCR7 + CD127 + CD45RA −/+ TCF1 + ) in convalescents (16) .

Our group also performed SARS-CoV-2-specific MHC-I staining in combination with the cytokine secretion assay (CSA) to examine the phenotypes and functions of SARS-CoV-2specific CD8 + T cells (19) . SARS-CoV-2-specific MHC-I multimer + CD8 + T cells exhibited early differentiated EM phenotypes in the early convalescent phase. Interestingly, the frequency of CCR7 + CD45RA + cells among SARS-CoV-2-specific MHC-I multimer + CD8 + T cells increased in the late convalescent phase, suggesting the development of SARS-CoV-2-specific, stem-like memory CD8 + T cells. In terms of their functions, SARS-CoV-2-specific MHC-I multimer + CD8 + T cells from convalescent patients exhibited a high proliferative capacity even though IFN-γ was produced by less than half of them. The relative frequency of IFN-γproducing cells was significantly lower among SARS-CoV-2-specific CD8 + T cells than among influenza A virus-specific CD8 + T cells. However, SARS-CoV-2-specific MHC-I multimer + CD8 + T cells produced IFN-γ regardless of their PD-1 expression.

In patients with COVID-19, T-cell exhaustion or dysfunction has been reported since early in the emergence of SARS-CoV-2. Several studies reported an exhausted phenotype of CD8 + T cells in patients with COVID-19, particularly in severe cases, with up-regulation of immune checkpoint inhibitory receptors, such as PD-1 (10-13). However, the expression of PD-1 in CD8 + T cells can be up-regulated not only by functional exhaustion, but also recent activation by the T-cell receptor. Recently, our group demonstrated that IFN-γ is produced by SARS-CoV-2-specific MHC-I + CD8 + T cells regardless of PD-1 expression in acute and convalescent COVID-19 patients (19) . This result indicates that SARS-CoV-2-specific PD-1 + CD8 + T cells are functional, not exhausted. Delayed induction of SARS-CoV-2-specific T-cell responses during acute infection has also been reported (26) . In this study, acute SARS-CoV-2 infection resulted in functional impairment of dendritic cells and delayed virus-specific T-cell responses comprising more of a CD4 + T-cell response than a CD8 + T-cell response while neutralizing Abs were rapidly developed.

In patients with acute moderate or severe COVID-19, the CD4 + and CD8 + T-cells responding upon ex vivo stimulation with SARS-CoV-2 overlapping peptides, as detected by IFN-γ or CD107a positivity, were CD38 + HLA-DR + Ki-67 + PD-1 + (16), indicating that they are fully activated and proliferating. MHC-I multimer staining confirmed this result. SARS-CoV-2specific MHC-I multimer + CD8 + T cells expressed activation markers (CD38, HLA-DR, and Ki-67), inhibitory receptors (PD-1 and TIM-3), and cytotoxic molecules (perforin and granzyme B) during acute infection (16) .

Our group also reported phenotypes of SARS-CoV-2-specific MHC-I multimer + CD8 + T cells in acute COVID-19 patients (19) . SARS-CoV-2-specific CD8 + T cells from patients in the acute phase exhibited an activated phenotype with high expression of CD38, HLA-DR, PD-1, perforin, and granzyme B. The proliferation marker Ki-67 was highly expressed in MHC-I multimer + cells in the acute phase, whereas CD127 was rarely expressed. In a kinetic analysis during the acute phase, the relative frequency of MHC-I multimer + cells among total CD8 + T cells tended to follow the kinetics of viral titers in nasopharyngeal swabs. The relative frequency of Ki-67 + proliferating cells and CD38 + HLA-DR + activated cells among MHC-I multimer + cells paralleled changes in the frequency of MHC-I multimer + cells. However, the relative frequency of perforin + granzyme B + cells and PD-1 + cells among MHC-I multimer + cells was sustained during the course of COVID-19.

A possibility of bystander activation of T cells, which has been shown in other viral infections (28, 29) , was investigated in acute SARS-CoV-2 infection. Early studies reported hyperactivated T-cell phenotypes without information on Ag specificity (30, 31) . However, a study using MHC-I multimers in patients with COVID-19 demonstrated that the expression of HLA-DR and Ki-67 were not up-regulated in CD8 + T cells specific to SARS-CoV-2-unrelated viruses, including cytomegalovirus (CMV) and Epstein-Barr virus (EBV), though the expression of CD38 was markedly increased (16) , indicating that bystander CD8 + T cells are activated to a limited degree during acute SARS-CoV-2 infection.

Regarding a relationship between SARS-CoV-2-specific T-cell responses and disease severity, there have been contradictory results. Among COVID-19 convalescent individuals, the magnitude of overall T-cell responses was significantly greater in individuals with severe disease compared to individuals with mild disease, as evaluated by IFN-γ ELISpot assays that measure both CD4 + and CD8 + T cell responses (15) . However, the frequency of cytokineproducing cells among total CD8 + T cells following stimulation with SARS-CoV-2 peptide pools is lower in severe cases compared with mild cases in the ICS results from the same study (15) . More detailed analyses have been performed with several groups of individuals, including healthy individuals who donated blood before or during the COVID-19 pandemic, exposed family members of symptomatic patients, and convalescent individuals who experienced mild or severe COVID-19 (16) . SARS-CoV-2-specific T-cell responses against S, M, 4/11 https://doi.org/10.4110/in.2021.21.e2

and N were highest in convalescent individuals with severe disease, with a stepwise decrease in convalescent individuals with mild disease, exposed family members, blood donors during the pandemic, and blood donors before the pandemic. Similar results were observed when SARS-CoV-2-specific CD4 + T-cell proliferation was evaluated by CTV dilution assays (16) .

In our recent study, we examined the relative frequency of CD127 + KLRG1 − cells, which are known as memory-precursor effector cells during viral infection and are able to differentiate into multiple memory cell lineages (32, 33) , among SARS-CoV-2-specific MHC-I multimer + CD8 + T cells from early COVID-19 convalescent individuals (19) and found that it was significantly lower in individuals who recently recovered from severe disease than in those who recovered from mild disease. These data suggest that the generation of long-term memory CD8 + T cells after recovery from COVID-19 may be determined by peak disease severity in the acute phase.

A study has comprehensively evaluated all three arms of the adaptive immunity in acute and convalescent COVID-19 patients (27) . In this study, SARS-CoV-2-specific CD4 + and CD8 + T-cell responses were associated with mild disease, indicating the roles of CD4 + and CD8 + T cells in protective immunity against SARS-CoV-2 infection. Moreover, the coordination in SARS-CoV-2-specific adaptive immune responses, including Ab and CD4 + and CD8 + T-cell responses, was associated with mild disease. Interestingly, as a single parameter, the relative frequency of SARS-CoV-2-specific IFN-γ-producing cells among total CD8 + T cells showed the strongest correlation with peak disease severity in acute COVID-19 patients.

The presence of SARS-CoV-2-reactive T-cell responses in unexposed individuals has been reported by several studies (34) . SARS-CoV-2-reactive T-cell responses in unexposed individuals were first detected by AIM assays using peptides predicted for MHC-I or MHC-II binding (25) . In this study, SARS-CoV-2-reactive CD4 + and CD8 + T-cell responses were detected in up to 50% and 20% of unexposed individuals, respectively, suggesting that these responses may result from previous infection by cross-reactive common-cold coronaviruses (CCCoVs), including HCoV-OC43, HCoV-HKU1, HCoV-229E, and HCoV-NL63. In another study, a SARS-CoV-2 S-reactive CD4 + T-cell response was detected in 35% of unexposed individuals (35) . This response primarily targeted the C-terminal part of S, which has a similarity with the S protein of CCCoVs. The SARS-CoV-2-reactive CD4 + T-cell response has been mapped at epitope levels across the SARS-CoV-2 proteome (36) . In this study, several epitopes of the memory CD4 + T-cell response in unexposed individuals were cross-reactive to SARS-CoV-2 and CCCoVs with comparable affinity.

reactive T-cell responses were detected against N, NSP7, and NSP13 proteins in unexposed individuals. Intriguingly, NSP7-specific T-cell response among unexposed individuals showed reactivity to regions conserved among animal β-coronaviruses rather than human CCCoVs (24) , suggesting that exposure to unknown coronaviruses of animal origin other than the four CCCoVs may induce memory T-cell responses cross-reactive to SARS-CoV-2. 

Given the presence of a certain degree of homology between SARS-CoV-2 and CCCoVs, we need to consider a similarity and difference in the amino acid sequence of epitope peptides when we focus on T-cell response at a single epitope level. Table 2 shows a list of SARS-CoV-2-derived CD4 + T-cell epitopes that have been tested in unexposed individuals or examined for cross-reactivity with human CCCoVs (24, 36, 37) .

It is unclear whether pre-existing SARS-CoV-2-reactive T cells contribute to protective immunity against SARS-CoV-2 during acute infection or interfere with the development of a competent SARS-CoV-2-specific T-cell response by an 'original antigenic sin' phenomena (38) . Intriguingly, a recent study reported that COVID-19 patients with a recent history of CCCoV infection have significantly milder disease than those without a recent history of CCCoV infection (39) . This result suggests that pre-existing SARS-CoV-2-reactive memory T cells primed by recent CCCoV infection may prevent the development of severe disease when hosts encounter SARS-CoV-2 infection. Further studies are required to clarify a role of SARS-

Since December 2020, COVID-19 prophylactic vaccines have been approved and started to be administered to high-risk populations. Although various vaccine platforms were used, including mRNA, viral vectors, and recombinant proteins, vaccinations were shown to successfully elicit not only neutralizing Abs, but also SARS-CoV-2-specific CD4 + and CD8 + T-cell responses (40) (41) (42) (43) A recent study using a SARS-CoV-2 infection model of rhesus macaques examined the contribution of Ab and T-cell immunity to protection against SARS-CoV-2 infection (44) . They demonstrated the importance of Abs by adoptive transfer of purified IgG from convalescent animals to naïve animals, which protects recipients against SARS-CoV-2 challenge. They also showed the importance of CD8 + T cells in a depletion study. Depletion of CD8 + T cells in convalescent animals partially abolished the protective immunity against SARS-CoV-2 rechallenge. They suggested that T-cell responses are important, particularly when Ab responses are suboptimal. Although this study was performed using animals recovered from previous SARS-CoV-2 infection, the conclusion can be applied to the setting of vaccination. Further studies are required to determine the individual roles of Abs and CD4 + and CD8 + T cells in the protective immunity against SARS-CoV-2 infection following vaccination. This question is also important for determining immune correlates of protection against SARS-CoV-2 infection.

Ending the current COVID-19 pandemic relies on herd immunity that blocks the spread of SARS-CoV-2 within the population. Herd immunity can be achieved by eliciting protective immunity via vaccination or natural infection in a considerable proportion of the population and the maintenance of this protective immunity. In SARS-CoV-2 infection, the roles of virus- (Continued) A list of SARS-CoV-2-derived CD4 + T-cell epitopes that have been tested in unexposed individuals or examined for cross-reactivity with human CCCoVs specific CD4 + and CD8 + T cells in protective immunity are not as well clarified as the roles of neutralizing Abs. In addition, the longevity of SARS-CoV-2-specific CD4 + and CD8 + T cells needs to be investigated further. Whether pre-existing SARS-CoV-2-reactive T cells elicited by CCCoVs are beneficial or harmful to hosts remains to be elucidated. Immunological research of these questions will help us end the current COVID-19 pandemic and prepare for the next pandemic by developing effective prophylactics and therapeutics and establishing proper epidemiological strategies.

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This research was supported by the 2020 Joint Research Project of Institutes of Science and Technology.