Robust T Cell Immunity in Convalescent Individuals with Asymptomatic or Mild COVID-19

SARS-CoV-2-specific memory T cells will likely prove critical for long-term immune protection against COVID-19. Here, we systematically mapped the functional and phenotypic landscape of SARS-CoV-2-specific T cell responses in unexposed individuals, exposed family members, and individuals with acute or convalescent COVID-19. Acute-phase SARS-CoV-2-specific T cells displayed a highly activated cytotoxic phenotype that correlated with various clinical markers of disease severity, whereas convalescent-phase SARS-CoV-2-specific T cells were polyfunctional and displayed a stem-like memory phenotype. Importantly, SARS-CoV-2-specific T cells were detectable in antibody-seronegative exposed family members and convalescent individuals with a history of asymptomatic and mild COVID-19. Our collective dataset shows that SARS-CoV-2 elicits broadly directed and functionally replete memory T cell responses, suggesting that natural exposure or infection may prevent recurrent episodes of severe COVID-19.


In Brief
Sekine et al. provide a functional and phenotypic map of SARS-CoV-2-specific T cells across the full spectrum of exposure, infection, and COVID-19 severity. They observe that SARS-CoV-2 elicits broadly directed and functionally replete memory T cells that may protect against recurrent episodes of severe COVID-19.

INTRODUCTION
The world changed in December 2019 with the emergence of a new zoonotic pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes a variety of clinical syndromes collectively termed coronavirus disease 2019 . At present, there is no vaccine against SARS-CoV-2, and the excessive inflammation associated with severe COVID-19 can lead to respiratory failure, septic shock, and ultimately, death (Guan et al., 2020;Wolfel et al., 2020;Wu and McGoogan, 2020). The overall mortality rate is 0.5%-3.5% (Guan et al., 2020;Wolfel et al., 2020;Wu and McGoogan, 2020). However, most people seem to be affected less severely and remain asymptomatic or develop only mild symptoms during COVID-19 (He et al., 2020b;Wei et al., 2020;Yang et al., 2020). It will therefore be critical for public health reasons to determine whether people with milder forms of COVID-19 develop robust immunity against SARS-CoV-2.
Global efforts are currently underway to map the determinants of immune protection against SARS-CoV-2. Recent data have shown that SARS-CoV-2 infection generates near-complete protection against rechallenge in rhesus macaques (A) Dot plots summarizing the absolute counts and relative frequencies of CD3 + (left), CD4 + (center), and CD8 + T cells (right) in healthy blood donors from 2020 (2020 BD ) and patients with acute moderate (AM ) or acute severe COVID-19 (AS). Each dot represents one donor. Data are shown as median ± IQR. *p < 0.05, ***p < 0.001. Kruskal-Wallis rank-sum test with Dunn's post hoc test for multiple comparisons. Article (Chandrashekar et al., 2020), and similarly, there is limited evidence of reinfection in humans with previously documented COVID-19 (Kirkcaldy et al., 2020). Further work is therefore required to define the mechanisms that underlie these observations and evaluate the durability of protective immune responses elicited by primary infection with SARS-CoV-2. Most correlative studies of immune protection against SARS-CoV-2 have focused on induction of neutralizing antibodies (Hotez et al., 2020;Robbiani et al., 2020;Seydoux et al., 2020;Wang et al., 2020). However, antibody responses are not detectable in all patients, especially those with less severe forms of COVID-19 (Long et al., 2020;Mallapaty, 2020;Woloshin et al., 2020). Previous work has also shown that memory B cell responses tend to be short lived after infection with SARS-CoV-1 (Channappanavar et al., 2014;Tang et al., 2011). In contrast, memory T cell responses can persist for many years (Le Bert et al., 2020;Tang et al., 2011;Yang et al., 2006) and, in mice, protect against lethal challenge with SARS-CoV-1 (Channappanavar et al., 2014).
SARS-CoV-2-specific T cells have been identified in humans Ni et al., 2020). It has nonetheless remained unclear to what extent various features of the T cell immune response associate with serostatus and the clinical course of COVID-19. To address this knowledge gap, we characterized SARS-CoV-2-specific CD4 + and CD8 + T cells in outcomedefined cohorts of donors (total n = 206) from Sweden, which has experienced a more open spread of COVID-19 than many other countries in Europe (Habib, 2020).
To extend these findings, we concatenated all memory CD4 + T cells and all memory CD8 + T cells from healthy blood donors, convalescent individuals, and patients with acute moderate or severe COVID-19. Phenotypically related cells were identified using the clustering algorithm PhenoGraph, and marker expression patterns were visualized using the dimensionality reduction algorithm Uniform Manifold Approximation and Projection (UMAP). Distinct topographical clusters were apparent in each group ( Figures 1D, S2A, and S2B). In particular, memory CD8 + T cells from patients with acute moderate or severe COVID-19 expressed a distinct cluster of markers associated with activation and the cell cycle, including CD38, HLA-DR, Ki-67, and PD-1 ( Figures 1D and S2A). A similar pattern was observed among memory CD4 + T cells from patients with acute moderate or severe COVID-19 ( Figure S2B). These findings were confirmed via manual gating of the flow cytometry data (Figure 1E). Correlative analyses further demonstrated that the activated/cycling phenotype was strongly associated with SARS-CoV-2-specific IgG levels and various clinical parameters, including age, hemoglobin concentration, platelet count, and plasma levels of alanine aminotransferase, albumin, D-dimer, fibrinogen, and myoglobin ( Figures S2C and S2D), but less strongly associated with plasma levels of various inflammatory markers, including interleukin (IL)-1b, IL-10, and tumor necrosis factor (TNF) ( Table S1).

Phenotypic Characteristics of SARS-CoV-2-Specific T Cells in Acute and Convalescent COVID-19
Unphysiologically high expression frequencies of CD38, potentially driven by a highly inflammatory environment, were defined segregation of memory CD4 + and CD8 + T cells. Bottom: dot plots showing the group-defined distribution of markers in PC2. Each dot represents one donor. MC, individuals in the convalescent phase after mild COVID-19. **p < 0.01, ***p < 0.001. Kruskal-Wallis rank-sum test with Dunn's post hoc test for multiple comparisons. (C) Dot plots summarizing the expression frequencies of activation/cycling markers among memory CD4 + (top) and CD8 + T cells (bottom) by group. Each dot represents one donor. Data are shown as median ± IQR. *p < 0.05, **p < 0.01, ***p < 0.001. Kruskal-Wallis rank-sum test with Dunn's post hoc test for multiple comparisons.  observed consistently among memory CD8 + T cells from patients with acute moderate or severe . In line with these data, we found that CD8 + T cells specific for cytomegalovirus (CMV) or Epstein-Barr virus (EBV) more commonly expressed CD38, but not HLA-DR, Ki-67, or PD-1, in patients with acute moderate or severe COVID-19 compared with convalescent individuals and healthy blood donors, indicating limited bystander activation and proliferation during the early phase of infection with SARS-CoV-2 (Figures 2A, 2B, and S3C). Of note, actively proliferating CD8 + T cells, defined by expression of Ki-67, exhibited a predominant CCR7 À CD27 + CD28 + CD45RA À CD127 À phenotype in patients with acute moderate or severe COVID-19 ( Figure S3D), as reported previously in the context of vaccination and other viral infections Miller et al., 2008). On the basis of these findings, we used overlapping peptides spanning the immunogenic domains of the SARS-CoV-2 spike, membrane, and nucleocapsid proteins to stimulate peripheral blood mononuclear cells (PBMCs) from patients with acute moderate or severe COVID-19. A vast majority of responding CD4 + and CD8 + T cells displayed an activated/cycling (CD38 + HLA-DR + Ki67 + PD-1 + ) phenotype ( Figure 2C). These results were confirmed using an activation-induced marker (AIM) assay to measure upregulation of CD69 and 4-1BB (CD137), suggesting that most CD38 + PD-1 + CD8 + T cells were specific for SARS-CoV-2 ( Figure 2D).
In further experiments, we used HLA class I tetramers as probes to detect CD8 + T cells specific for the predicted optimal epitopes from SARS-CoV-2 ( Figure S3E; Table S2). A vast majority of tetramer + CD8 + T cells in the acute phase of infection, but not during convalescence, displayed an activated/cycling phenotype ( Figure 2E). In general, early SARS-CoV-2-specific CD8 + T cell populations were characterized by expression of immune activation molecules (CD38, HLA-DR, and Ki-67), inhibitory receptors (PD-1 and TIM-3), and cytotoxic molecules (granzyme B and perforin), whereas convalescent-phase SARS-CoV-2-specific CD8 + T cell populations were skewed toward an early differentiated memory (CCR7 + CD127 + CD45RA À/+ TCF1 + ) phenotype ( Figure 2F). Importantly, the expression frequencies of CCR7 and CD45RA among SARS-CoV-2-specific CD8 + T cells were positively correlated with the number of symptom-free days after infection (CCR7: r = 0.79, p = 0.001; CD45RA: r = 0.70, p = 0.008), whereas the expression frequency of granzyme B among SARS-CoV-2-specific CD8 + T cells was inversely correlated with the number of symptom-free days after infection (r = 0.70, p = 0.007) ( Figure 2G). Time from exposure was therefore associated with emergence of stem-like memory SARS-CoV-2specific CD8 + T cells.

Functional Characteristics of SARS-CoV-2-Specific T Cells in Convalescent COVID-19
On the basis of these observations, we quantified functional SARS-CoV-2-specific memory T cell responses across five distinct cohorts, including healthy individuals who donated blood before or during the pandemic, family members who shared a household with convalescent individuals and were exposed at the time of symptomatic disease, and individuals in the convalescent phase after mild or severe COVID-19. We detected potentially cross-reactive T cell responses directed against the spike and/or membrane proteins in 28% of healthy individuals who donated blood before the pandemic, consistent with previous reports Le Bert et al., 2020), but nucleocapsid reactivity was notably absent in this cohort ( Figures 3A, S4A, and S4B). The highest response frequencies against any of these three proteins were observed in convalescent individuals who experienced severe COVID-19 (100%). Progressively lower response frequencies were observed in convalescent individuals with a history of mild COVID-19 (87%), exposed family members (67%), and healthy individuals who donated blood during the pandemic (46%) ( Figure 3A).
To assess the functional capabilities of SARS-CoV-2-specific memory CD4 + and CD8 + T cells in convalescent individuals, we stimulated PBMCs with the overlapping spike, membrane, and nucleocapsid peptide sets and measured a surrogate marker of degranulation (CD107a) along with production of interferon (IFN)-g, IL-2, and TNF ( Figures 3B and 3C). SARS-CoV-2-specific CD4 + T cells predominantly expressed IFN-g, IL-2, and TNF ( Figure 3B), whereas SARS-CoV-2-specific CD8 + T cells predominantly expressed IFN-g and mobilized CD107a (Figure 3C). We then used the AIM assay to determine the functional polarization of SARS-CoV-2-specific CD4 + T cells. Interestingly, spike-specific CD4 + T cells were skewed toward a circulating T follicular helper (cTfh) profile, suggesting a key role in the generation of potent antibody responses, whereas membrane-specific and nucleocapsid-specific CD4 + T cells were skewed toward a Th1 or a Th1/Th17 profile ( Figures 3D, S5A, and S5B).
Serological evaluations revealed a strong positive correlation between IgG responses directed against the spike protein of SARS-CoV-2 and IgG responses directed against the nucleocapsid protein of SARS-CoV-2 (r = 0.82, p < 0.001) ( Figure S5C). Moreover, SARS-CoV-2-specific CD4 + and CD8 + T cell responses were present in seronegative individuals, albeit at lower frequencies compared with seropositive individuals (41% versus  (right) showing the expression of activation/cycling markers among CD107a + and/or IFN-g + SARS-CoV-2-specific CD4 + and CD8 + T cells (n = 6 donors). Numbers indicate percentages in the drawn gates. Data are shown as median ± IQR. NC, negative control. *p < 0.05, **p < 0.01. Paired t test or Wilcoxon signed-rank test.
(legend continued on next page) ll OPEN ACCESS 99%, respectively) ( Figure 4F). These discordant responses were nonetheless pronounced in some convalescent individuals with a history of mild COVID-19 (3 of 31), exposed family members (9 of 28), and healthy individuals who donated blood during the pandemic (5 of 31) ( Figures 4F and S5D), often targeting the internal (nucleocapsid) and surface antigens (spike and/or membrane) of SARS-CoV-2 ( Figure 4G). Higher frequencies of SARS-CoV-2-specific T cells were also found in exposed seronegative family members compared with unexposed donors ( Figure S5E). Potent memory T cell responses were therefore elicited in the absence or presence of circulating antibodies, consistent with a non-redundant role as key determinants of immune protection against COVID-19 (Chandrashekar et al., 2020).

DISCUSSION
We are currently facing the biggest global health emergency in decades, namely the devastating outbreak of COVID-19. In the absence of a protective vaccine, it will be critical to determine whether exposed and/or infected people, especially those with asymptomatic or very mild forms of the disease who likely act inadvertently as major transmitters, develop robust adaptive immunity against SARS-CoV-2 (Long et al., 2020).
In this study, we used a systematic approach to map cellular and humoral immune responses against SARS-CoV-2 in patients with acute moderate or severe COVID-19, individuals in the convalescent phase after mild or severe COVID-19, exposed family members, and healthy individuals who donated blood before (2019) or during the pandemic (2020). Individuals in the convalescent phase after mild COVID-19 were traced after returning to Sweden from endemic areas (mostly northern Italy). These donors exhibited robust memory T cell responses months after infection, even in the absence of detectable circulating antibodies specific for SARS-CoV-2, that may contribute to protection against severe COVID-19.
We found that T cell activation, characterized by expression of CD38, was a hallmark of acute COVID-19. Similar findings have been reported previously in the absence of specificity data (Huang et al., 2020;Thevarajan et al., 2020;Wilk et al., 2020). Many of these T cells also expressed HLA-DR, Ki-67, and PD-1, indicating a combined activation/cycling phenotype, which correlated with early SARS-CoV-2-specific IgG levels and, to a lesser extent, plasma levels of various inflammatory markers. Our data also showed that many activated/cycling T cells in the acute phase were functionally replete and specific for SARS-CoV-2. Equivalent functional profiles have been observed early after immunization with successful vaccines (Blom et al., 2013;Miller et al., 2008;Precopio et al., 2007).
Accordingly, the expression of multiple inhibitory receptors, including PD-1, likely indicates early activation rather than exhaustion (Zheng et al., 2020a(Zheng et al., , 2020b. Virus-specific memory T cells have been shown to persist for many years after infection with SARS-CoV-1 (Le Bert et al., 2020;Tang et al., 2011;Yang et al., 2006). In line with these observations, we found that SARS-CoV-2-specific T cells acquired an early differentiated memory (CCR7 + CD127 + CD45RA À/+ TCF1 + ) phenotype in the convalescent phase, as reported previously in the context of other viral infections and successful vaccines (Blom et al., 2013;Demkowicz et al., 1996;Fuertes Marraco et al., 2015;Precopio et al., 2007). This phenotype has been associated with stem-like properties (Betts et al., 2006;Blom et al., 2013;Demkowicz et al., 1996;Fuertes Marraco et al., 2015;Precopio et al., 2007). Accordingly, we found that SARS-CoV-2-specific T cells generated anamnestic responses to cognate antigens in the convalescent phase, characterized by extensive proliferation and polyfunctionality. Of particular note, we detected similar memory T cell responses directed against the internal (nucleocapsid) and surface proteins (membrane and/or spike) in some individuals lacking detectable circulating antibodies specific for SARS-CoV-2. Indeed, almost twice as many healthy individuals who donated blood during the pandemic had memory T cell responses versus antibody responses, implying that seroprevalence as an indicator may underestimate the extent of adaptive immune responses against SARS-CoV-2.
Our study was cross-sectional in nature and limited in terms of clinical follow-up and overall donor numbers in each outcomedefined group. It therefore remains to be determined whether robust memory T cell responses in the absence of detectable circulating antibodies can protect against severe forms of COVID-19. This scenario has nonetheless been inferred from previous studies of MERS and SARS-CoV-1 (Channappanavar et al., 2014;Li et al., 2008;Zhao et al., 2016Zhao et al., , 2017, both of which have been shown to induce potent memory T cell responses that persist for years, in contrast to the corresponding antibody responses (Alshukairi et al., 2016;Shin et al., 2019;Tang et al., 2011). Antibody responses have also been shown to wane after infection with SARS-CoV-2 (Ibarrondo et al., 2020;Long et al., 2020), suggesting that transient humoral immunity is a general feature of coronavirus infections (Callow et al., 1990). However, the fact that memory B cells (Juno et al., 2020) and memory T cells are generated in response to SARS-CoV-2 suggests that natural infection may elicit protection from severe COVID-19. In line with these observations, none of the convalescent individuals in this study, including those with previous mild disease, experienced further episodes of COVID-19.  . Numbers indicate percentages in the drawn gates. Right: bar graphs and pie charts summarizing the distribution of individual functions among SARS-CoV-2-specific CD4 + (B) and CD8 + T cells (C) from convalescent individuals in groups MC (n = 12) or SC (n = 14). Data are shown as median ± IQR. Key as in (A). *p < 0.05. Unpaired t test or Mann-Whitney U test. (D) Left: bar graphs summarizing the functional polarization of SARS-CoV-2-specific CD4 + T cells from convalescent individuals in groups MC (n = 5) and SC (n = 6). Subsets were defined as CXCR5 + (cTfh), CCR4 À CCR6 À CXCR3 + CXCR5 À (Th1), CCR4 + CCR6 À CXCR3 À CXCR5 À (Th2), CCR4 À CCR6 + CXCR3 À CXCR5 À (Th17), CCR4 À CCR6 + CXCR3 + CXCR5 À (Th1/17), and CCR4 À CCR6 À CXCR3 À CXCR5 À (non-Th1/2/17). Data are shown as median ± IQR. *p < 0.05. Unpaired t test or Mann-Whitney U test. Right: line graph comparing cTfh versus Th1 polarization by specificity in convalescent individuals from groups MC and SC. Each dot represents one donor. Key as in (A). *p < 0.05, **p < 0.01. Paired t test. Of note, we detected cross-reactive T cell responses directed against the spike and/or membrane proteins of SARS-CoV-2 in 28% of unexposed healthy blood donors, consistent with a high degree of pre-existing immunity in the general population (Braun et al., 2020;Grifoni et al., 2020;Le Bert et al., 2020). Moreover, these particular data were derived from cryopreserved samples, so this figure might be considered as a lower bound estimate of overall prevalence (Owen et al., 2007). In this context, our findings most likely reflect widespread exposure to seasonal coronaviruses, which could shape the subsequent immune response to SARS-CoV-2. As such, it remains likely that a fraction of the anamnestic SARS-CoV-2-specific T cell response was initially induced by seasonal coronaviruses in seronegative individuals . It is also tempting to speculate that such responses may provide at least partial protection against SARS-CoV-2, given that pre-existing T cell immunity has been associated with beneficial outcomes after challenge with the pandemic influenza virus strain H1N1 (Sridhar et al., 2013;Wilkinson et al., 2012).
Collectively, our data provide a functional and phenotypic map of SARS-CoV-2-specific T cell immunity across the full spectrum of exposure, infection, and disease. The observation that many individuals with asymptomatic or mild COVID-19 had highly durable and functionally replete memory T cell responses, not uncommonly in the absence of detectable humoral responses, further suggests that natural exposure or infection could prevent recurrent episodes of severe COVID-19.