key: cord-0845305-2josvfb5
authors: Govender, Melissa; Hopkins, Francis R.; Göransson, Robin; Svanberg, Cecilia; Shankar, Esaki M.; Hjorth, Maria; Augustinsson, Åsa Nilsdotter; Sjöwall, Johanna; Nyström, Sofia; Larsson, Marie
title: Long-term T cell perturbations and waning antibody levels in individuals needing hospitalization for COVID-19
date: 2022-03-17
journal: bioRxiv
DOI: 10.1101/2022.03.17.484640
sha: fcb5c0eb4ac0b1316c9e68d8e4d4025897326afd
doc_id: 845305
cord_uid: 2josvfb5

COVID-19 is being extensively studied, and much remains unknown regarding the long-term consequences of the disease on immune cells. The different arms of the immune system are interlinked, with humoral responses and the production of high-affinity antibodies being largely dependent on T cell immunity. Here, we longitudinally explored the effect COVID-19 has on T cell populations and the virus-specific T cells, as well as neutralizing antibody responses, for 6-7 months following hospitalization. The CD8+ TEMRA and exhausted CD57+CD8+ T cells were markedly affected with elevated levels that lasted long into convalescence. Further, markers associated with T-cell activation were upregulated at the inclusion, and in the case of CD69+CD4+ T cells this lasted all through the study duration. The levels of T cells expressing negative immune checkpoint molecules were increased in COVID-19 patients and sustained for a prolonged duration following recovery. Within 2-3 weeks after symptom onset, all COVID-19 patients developed anti-nucleocapsid IgG and spike-neutralizing IgG as well as SARS-CoV-2-specific T cell responses. In addition, we found alterations in follicular T helper (TFH) cell populations, such as enhanced TFH-TH2 following recovery from COVID-19. Our study revealed significant and long-term alterations in T cell populations and key events associated with COVID-19 pathogenesis.

More than two years since initial reports of the disease, the COVID-19 pandemic has claimed approximately six million lives world-wide leaving survivors with long-term health consequences [1] . The etiologic agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) gains entry into host cells after binding to angiotensin-converting enzyme 2 (ACE2) and transmembrane protease, serine 2 (TMPRSS2) via the receptor binding domain (RBD) of the viral spike (S) protein [2, 3] . While a vast majority of infected individuals remain asymptomatic or pauci-symptomatic, severe and life-threatening disease occurs in 14% and 5-6% of cases, respectively [4] . Approximately 20% of all infected individuals, especially the elderly and those with co-morbidities require hospitalization. In fatal cases of COVID-19, death often occurs due to organizing pneumonia (OP), acute respiratory distress syndrome (ARDS) or sepsis-like cytokine storm-associated multi-organ failure [5] [6] [7] . The hyperinflammation in COVID-19 is caused by the activated inflammatory cells recruited in response to infection and not by the virus per se [8] [9] [10] . Furthermore, the variations in initial host responses appear to determine the varied disease spectrum observed in COVID-19 [11, 12] .

Clearance of viral infection involves both cellular and humoral immune responses [13, 14] .

Virus-specific CD4 + and CD8 + T cells are present in almost all individuals who have had a prior episode of COVID-19 [15, 16] , with lower numbers of these cells linked to disease severity [17] [18] [19] . Furthermore, the rapid appearance of functional SARS-CoV-2-specific T cells is linked to mild disease presentation [14] . The humoral immune response also plays a critical role in viral clearance in the host. The SARS-CoV-2-specific antibody responses vary in levels, as well as quality between asymptomatic individuals and those presenting with severe disease. While some studies have found higher levels and longer duration in more severe cases, others have reported no observable difference [20] [21] [22] [23] [24] [25] [26] . Furthermore, individuals who have recovered from severe compared to non-severe COVID-19

reportedly have higher nAb titers and CXCR3 + TFH cell frequencies, and faster recovery of lymphocyte levels, a month after leaving the hospital [36] .

Viral infections lead to the activation of T cells with upregulated expression of markers that define early, i.e., CD69 and late, i.e., CD38/HLA-DR, activation [37, 38] . Activated CD4 + and CD8 + T cells exist during active/ongoing COVID-19 and in certain individuals, even after recovery, activated CD8 + T cells persist for over 100 days [39] . A case study of mild reported activated CD38 + HLA-DR + CD8 + T cells at day 7, which surged to a peak at day 9 but remained high even at day 20. Activated CD4 + T cells were also present, but at lower levels [16] . Many studies support the activation of T cells and B cell responses during the onset of COVID-19 disease, but the infection also leads to transient immunosuppression, especially during severe disease [40] . The expression of negative immune checkpoint molecules on CD8 + and CD4 + T cells examined in 14 individuals with severe COVID-19 revealed an increased abundance of PD-1 and TIM-3 levels in most severe COVID-19 cases [17] . In addition, exhausted T cells expressing CD57 and PD-1, with impaired proliferation, were found in individuals with COVID-19 [41] .

It is clear that COVID-19 induces immune dysfunction and alterations within T cell populations, and in light of this, we have longitudinally investigated the T cell profile of COVID-19 patients over a 6-7 month period, to monitor the changes in dynamics and functional quality of T cell responses. To further understand the immune responses elicited during SARS-CoV-2 infection, we also explored its long-term consequences on B cell and antibody responses in the cohort.

We found an increase in TFH cells and alterations in TH1/TH2/TH17 TFH subsets that persisted following recovery from COVID-19, and a consistent rise in the CD8 + terminal effector memory RA + (TEMRA) subset throughout the 6-7 months of the study. Further, markers associated with T-cell activation were upregulated and different forms of T cell impairment were also evident that lasted for a prolonged time following recovery from COVID-19.

The present study was approved by the Swedish Ethical Review Authority (Ethics No. 2020-02580) and recruited hospitalized patients with COVID-19 (N=46; age range 32 -91 years) and healthy staff (N=31; age range 23 -62 years) at the Infectious Diseases Clinic at the Vrinnevi Hospital, Norrköping, Sweden. The participants were ≥18-years-old male and female subjects who provided written informed consent before enrollment. The study was carried out in compliance with good clinical practices, including the International Conference on Harmonization Guidelines and the Declaration of Helsinki. COVID-19 patients were enrolled into the study during hospitalization and first blood samples were drawn, which was followed by three additional samples collected at 2 weeks, 6 weeks, and 6-8 months, respectively after inclusion (Figure 1) . The clinical and demographic characteristics of the hospitalized COVID-19 cohort are summarized in Table 1 .

On enrollment, the COVID-19 patients were provided with an approved questionnaire to secure details regarding COVID-19-associated symptoms prior to hospitalization, and smoking habits. Relevant medical data for the study were collected from digital medical records and included date of COVID-19 diagnosis, length of hospital stay, highest level of care received, maximum need of oxygen supplementation (<5L/min or HFNOT/CPAP/mechanical ventilation), details of COVID-19-related medication (anti-coagulants, remdesivir and/or dexamethasone), current medication, underlying conditions (if any) such as cardiovascular disease (CVD), chronic pulmonary disease, chronic renal failure, diabetes mellitus, and immunosuppression. COVID-19 severity was determined in the hospitalized individuals as per the criteria defined by the National Institutes of Health [42] , i.e., approximated with respect to the maximum oxygen needed, and the highest level of care provided. Accordingly, the cases were classified as having mild (pandemic department, no oxygen supplementation), moderate (pandemic department, oxygen supplementation <5L/min), severe (pandemic department or intermediate care unit, oxygen need ≥5L/min supplemented by HFNO or CPAP) and critical (intensive care unit, with or without mechanical ventilation) COVID-19 illness.

Sera were prepared from venous whole blood collected in Vacuette® tubes (Freiner Bio-one GnbH, Kremsmunster, Austria) by centrifugation at 1000 g for 10 minutes at room temperature and stored at -80°C until further use. Peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood by Ficoll-Paque (GE Healthcare, ThermoFisher) density gradient centrifugation. PBMCs were aliquoted in freezing medium (8% DMSO in FBS) and cryopreserved at -150°C for later use in the experiments.

The levels of CD3 + CD4 + T cells and CD3 + CD8 + T cells/µl were measured using BD Trucount™ The Avidin-Peroxidase Complex was prepared and left to stand for 30 min at room temperature before use. The wells were washed 4 times with wash buffer, after which 50μl stable DAB (Invitrogen, Fisher Scientific, Sweden) was added to each well for 3-5 min. Finally, the plates were washed with tap water three times and left to air dry. Spots were counted manually using an inverted phase-contrast microscope (Nikon SMZ1500, Light source Zeiss KL1500 LCD).

Qualitative measurement of anti-SARS-CoV-2 spike IgG, including anti-spike neutralizing IgG antibodies directed against the receptor binding domain (RBD) of S1 subunit was performed 

Data and statistical analyses were carried out using GraphPad PRISM v9.0 (GraphPad Software, CA, USA). Differences among the study groups were analyzed with either unpaired, parametric T test with Welch's correction, or Brown-Forsythe and Welch ANOVA tests, with no correction for multiple comparisons. All differences with P values of <0.05 were considered statistically significant.

During a 15 month period (July 2020 to October 2021), blood samples were collected from hospitalized COVID-19 patients and healthy participants. From this cohort, we analyzed data from 46 COVID-19 patients and 31 healthy controls. Furthermore, a subset of donors was followed longitudinally up to 6-7 months. Our analysis focused on the effect of COVID-19 on various T cell subsets and SARS-CoV-2-specific T cell and B cell immune responses (Figure 1 ).

Our hospitalized COVID-19 cohort represents the infection in wider society in terms of biological sex distribution, encompassing two-thirds of males and one third of females [44] , and a median age of 56.6 (32 to 91) years ( Table 1) . The disease scores for the patients were moderate or severe as described by the National Institutes of Health [42] . Our control cohort included a majority of females with a median age of 45 years and an age range of 26-62 years.

The level of C-reactive protein (CRP) was significantly elevated (88.4 mg/L) in the hospitalized cohort at inclusion with an 8-fold increase as compared to the healthy controls. In addition, the level of lactate dehydrogenase (LDH) was much higher in most patients than in the healthy controls with median of 6.9 µKat/L (Table 1 and Figure 2A ). Both these findings are consistent with observations made by others [45, 46] . No association was found between disease severity and biological sex, age, or clinical characteristics (data not shown).

The number of lymphocytes/L was significantly decreased, and leukocytes/L was significantly increased at inclusion compared to the healthy controls, whereas monocyte and platelet counts were not significantly affected, although there was a minor fraction of COVID-19 patients with increased levels of platelets ( Figure 2B ). To further characterize the attrition in lymphocytes, we analyzed CD4 + T cells, CD8 + T cells, B cells, NK cells, and NKT cells in peripheral blood. There was a significant decrease in T cells, CD4 + T cells, CD8 + T cells, and NKT cells/µL at inclusion among COVID-19 patients relative to healthy controls. Furthermore, the lymphocyte populations experienced recovery with a significant increase in cell numbers at week 2, which remained stable until the 6 week time point (Figure 2B) . Regarding the CD8 + T cells, the increase was significantly higher at week 6 compared to healthy controls. We found no significant alteration in the absolute number of NK cells or B cells ( Figure 2B ). Together, these data are reflective of the alterations in the levels of various immune cell types during COVID-19, in line with observations made by others [25, [47] [48] [49] [50] .

To assess the longitudinal effect of COVID-19 on T cell populations, we sampled PBMCs from 21 patients with COVID-19 and 16 healthy controls over a period of 6-7months. PBMCs were analyzed by flow cytometry to investigate immunophenotype across the various T cell compartments. The percentages of CD3 + T cells were significantly decreased in patients at inclusion compared to the later time points and to healthy controls (Supplementary Figure   3A ). At week 2, the fraction of CD3 + T cells was still decreased in comparison with the healthy controls. While comparable percentages of CD4 + T cells were observed at hospitalization and week 2, there was a significant decrease in the levels after 6 weeks and 6-7 months, relative to the healthy controls (Supplementary Figure 3B) . Conversely, CD8 + T cells were significantly increased at 6 weeks and 6-7 months, in comparison to healthy individuals, while similar levels of these cells were seen at the first two time points (hospitalization and 2 weeks) (Supplementary Figure 3C) .

The environment locally and systemically created by an infection can give rise to alterations in the general memory T cell populations [51] . Here, we examined the effect COVID-19 had on naïve and memory CD4 + and CD8 + T cells (Figure 3 ). Distinct differences in the CD4 + and CD8 + T populations between a COVID-19 patient and a healthy control are shown in the representative pseudo-color dot plots ( Figure 3A) . A slight, but not significant, decrease was seen in the naïve fraction of the CD4 + TH cells over time, with no alterations in the CD4+ TEMRA (CCR7 -CD45RA + ) or T cell effector memory (TEM; CCR7 -CD45RA -) fractions ( Figure 3B ).

There was, notwithstanding, a significant upregulation in the T cell central memory (TCM; CCR7 + CD45RA -) compartment of CD4 + T cells at 6-7 months in comparison with hospitalization.

We found that CD8 + T cells displayed more sustained alterations in their naïve and TEMRA compartments compared with healthy controls. However, the frequencies of TCM and TEM were not affected ( Figure 3B) . A gradual decrease over time occurred in the naïve CD8 + T cells of COVID-19 patients, with the 2 weeks, 6 weeks, and 6-7 month time points presenting significantly decreased fractions of these cells compared to healthy controls ( Figure 3B ). In contrast, we found a sustained increased percentage of CD8 + TEMRA cells throughout the investigation, in comparison to healthy controls.

Next, we characterized the regulatory T cell (Treg; CD25 + CD127 -) fraction of CD4 + T cells ( Figure 3C and 3D) and found comparable levels of CD4 + Tregs in patient and control samples ( Figure 3D) . The TFH subset amongst Tregs was significantly increased in COVID-19 patients at hospitalization but decreased to a comparable level relative to healthy controls by the 2 week time point (Figure 3D) . Overall, we found alterations in the memory T cell pool and this was most evident in the CD8 + TEMRA subset, where the changes were sustained until the end of the study.

Phenotype Follicular helper T cells interact with B cells and facilitate the humoral arm of immunity by enhancing B cell responses, ultimately resulting in the generation of long-lasting, high-affinity antibodies, especially in the context of viral infections [52] [53] [54] . We evaluated TFH responses ( Figure 3E and 3F) and found that TCM and TEM levels among the TFH remained unchanged ( Figure 3F ). In contrast, the TFH subsets had significant alterations in the TH1 (CXCR3 + CCR6 -), TH2 (CXCR3 -CCR6 -) and TH17 (CXCR3 -CCR6 + ) proportions ( Figure 3F ). There was a significant drop in CD4 + TFH-TH1 cells at hospitalization among COVID-19 patients as compared to healthy controls, which consistently increased throughout the infection, reaching a level at 6-7 months that was comparable to healthy controls ( Figure 3F) . Conversely, TFH-TH2 cells were significantly upregulated at hospitalization in comparison to healthy controls. The fractions of these cells were consistently elevated during the infection. Although their levels eventually decreased, they remained higher compared to healthy controls after 6-7 months ( Figure 3F) . The level of TFH-TH17 cells was not significantly different from the control group at any time point, despite appearing lower after 6-7 months. Nonetheless, there was a gradual decrease of TFH-TH17 over time, which reached significance by the final time point ( Figure   3F ). There was a clear change in the TFH population, with a sustained increase in the TH2 subset in particular.

Classical markers of early and late activation of T cells are represented by CD69 and CD38/HLA-DR, respectively [37, 38] . Persistent expression of these markers is suggestive of a hyperimmune activation state and affirms an ongoing phase of systemic inflammation and tissue injury occurring in severe COVID-19 [55] . Hence, to examine the activation status of CD4 + and CD8 + T cells, we assessed the cells for CD38 + HLA-DR + and CD69 + expression (Figure 4) . We found clear differences in the activation status of the T cells between the COVID-19 patient and healthy control as illustrated in the representative scatter plots (Figure 4A) . The fraction of CD38 + HLA-DR + activated CD4 + T cells in the COVID-19 patients significantly decreased over the study duration and appeared to be lower than healthy controls at 6-7 months although this was not significant (Figure 4B) . The levels of CD69 + activated CD4 + T cells were significantly higher at hospitalization than in healthy controls. This elevation was sustained until the 6-7 month time point despite having dropped significantly from the initial levels ( Figure 4B ).

Activated CD8 + T cells in COVID-19 patients were also abundantly increased at hospitalization when compared to healthy controls. We observed a decrease over the 6-7 months period to the same level as healthy controls, although the CD38 + HLA-DR + phenotype was maintained up to 6 weeks where the CD69 + phenotype declined earlier ( Figure 4C) . The data collectively illustrates a significantly increased activation of both CD4 + and CD8 + T cells in COVID-19 patients at the initial sampling period that is sustained for some but not all markers in each subset.

To investigate the magnitude of T cell impairment during SARS-CoV-2 infection and recovery, we measured the frequency of co-inhibitory markers that are normally upregulated during exhaustion and suppression on CD4 + and CD8 + T cell subsets. CD57 is a marker of terminally differentiated non-proliferative T cells (senescence and exhaustion) and the frequency of CD57 + CD4 + and CD57 + CD8 + T cells increases with age as well as with cancer and chronic infections [56] . Both CD4 + and CD8 + T cell populations were examined for expression of CD57 as shown by a representative pseudo color dot plot (Figure 5A) . We found no significant difference in the proportion of CD57 + CD4 + T cells between COVID-19 patients and healthy controls ( Figure 5B) . Interestingly, we observed a significant surge in the percentage of CD57 + CD8 + T cells among COVID-19 patients at inclusion compared to healthy controls, which increased further by the 6 week time point and was sustained up until 6-7 months (Figure 5B) .

Taken together, our data clearly shows a long-lasting effect on the CD8 + T cell population with increased level of exhausted CD57 + CD8 + T cells.

Next, we examined the expression of three co-inhibitory markers i.e., LAG-3, which inhibits T cell activation and promotes a suppressive phenotype [57] ; TIM-3, a T cell exhaustion marker [58] and PD-1, which down-regulates functional T cell activation [59] . These markers of T cell impairment are all expressed during chronic infections. We observed a significant increased level of LAG-3 + CD4 + T cells among COVID-19 patients at hospitalization and again at the 6-7 month time point when compared to healthy controls ( Figure 5C) . Similarly, the proportion of TIM-3 + CD4 + T cells was elevated at hospitalization, and again at the 6 week time point. At the 6-7 month time point, the level appeared to rise but was not significant (Figure 5C ). The percentage of PD-1 + CD4 + T cells was significantly raised compared to healthy controls at all time points except for a significant dip at 6 weeks ( Figure 5C) .

A significant elevation of LAG-3 + CD8 + T cells was observed at hospitalization and again after 2 weeks, and 6-7 months (Figure 5D) . The fraction of TIM-3 + CD8 + T cells was significantly raised at hospitalization, 6 weeks, and 6-7 months (Figure 5D) . The levels of PD-1 + CD8 + cells were significantly raised at hospitalization and reverted to normal levels over the following 6-7 months (Figure 5D) . Overall, there was a general trend within all three markers of being elevated among COVID-19 patients at hospitalization when compared to healthy controls across both CD4 + and the CD8 + T cell subsets. Of note, there was a pattern within most of the data, of levels decreasing after the first time point, but increasing again by the 6-7 month time point.

ELISPOT assay measures the antigen-specific responses of T cells [60] [61] [62] , and has been used to determine the functional responses of SARS-CoV-2-specific T cells [63] [64] [65] . Here, we evaluated the level of SARS-CoV-2-specific IFN-γ producing bulk T cells in the COVID-19 patients, following stimulation with overlapping peptides covering the spike (containing the immunodominant RBD), spike+ (containing a portion of the spike region) and the nucleocapsid (covering full sequence) proteins (Figure 6) . We found SARS-CoV-2 specific T cell responses in all COVID-19 patients (Figure 6 and Supplementary Figure 4) . The SARS-CoV-2 specific response from inclusion to the 6-7 month time point in a representative donor is presented in Figure 6A . We observed a gradual and significant increase in the number of spike ( Figure   6B ), spike+ (Figure 6C) , and nucleocapsid ( Figure 6D ) SARS-CoV-2-specific T cells, which was sustained for 6-7 months. The antigen specific T cell responses in individual donors are illustrated in Supplementary Figure 4 . Altogether, we found the frequency of bulk SARS-CoV-2-specific T cells to be increased over the duration of the investigation.

The levels of anti-SARS-CoV-2 nucleocapsid and spike-neutralizing Abs in the COVID-19 hospitalized patients were assessed at inclusion, 2 weeks, 6 weeks, and 6-8 months. The levels of antibodies increased significantly from inclusion to the 2 week time point and were also significantly higher at 6 weeks, before declining back to inclusion levels by 6-8 months ( Figure  7A , left). The spike-nAbs were of the same profile as the anti-nucleocapsid Abs, which increased significantly from inclusion compared to the 2 and 6 week time points and eventually declined at the 6-8 month time point (Figure 7A, right) . We ensured that the SARS-CoV-2 vaccinated participants were not tested at the final time point.

The frequency of activated B cells, defined by CD38 expression [66, 67] , was significantly higher at inclusion compared to the 2 and 6 week time points (Figure 7B) . The levels of memory B cells at inclusion were reduced, but not significantly so, when compared to healthy controls. Similarly, unswitched memory B cell levels were significantly decreased at hospitalization. The percentages of both the populations increased at the 2 week time point to levels comparable to healthy controls ( Figure 7B ). There were no significant alterations observed in the levels of switched memory B cells (Figure 7B) . Given there was no effect on the absolute numbers of B cells per µl (Figure 1C) , these data indicate an alteration in the proportion of the different sub-populations within the B cell compartment. Collectively, the data illustrate a decrease in the unswitched B cell population and subsequent waning of SARS-CoV-2-specific antibodies over time.

We have profiled T cell populations during COVID-19 in a longitudinal study cohort, where we followed them from inclusion up to 6-8 months post-recovery, using spectral flow cytometry and serological and immunological assays. Additionally, we have evaluated the SARS-CoV-2 specific T cell responses, B cell responses, and SARS-CoV-2 antibodies generated in the cohort.

We found wide-ranging alterations to the T cell compartment including a rise in CD8 + TEMRA and TFH TH2 cells, and different forms of functional impairments that lasted long into convalescence.

Our initial measurement of clinical parameters was consistent with others showing elevated CRP and LDH, common acute-inflammatory biomarkers [49, 68, 69] . These have been shown to have a positive correlation with the development of pulmonary lesions during early COVID-19, and consequently with severe COVID-19 and related respiratory complications [70, 71] . We recently found an association of soluble urokinase Plasminogen Activator Receptor with COVID-19 severity, and length of hospitalization [72] . Additionally, lymphopenia was observed in many of the COVID-19 patients in our cohort at inclusion (i.e., at hospitalization), and is a common clinical observation [73] [74] [75] [76] that may likely be attributed to the cells relocating or consequently dying at this stage of the disease. Indeed, a highly inflammatory form of cell death, i.e., pyroptosis, induced in infected and uninfected cells, appears to be a major contributing factor for the onset of strong inflammatory responses seen globally in many individuals with COVID-19 [8, 49, 69, 77, 78] . Furthermore, we and others have shown that the T cell compartment is highly affected, and to a higher degree than immune cells such as B or NK cells [79] [80] [81] [82] , which implies that T cell subsets play a paramount role in COVID-19 pathogenesis.

Memory T cells, both general and antigen-specific, in the context of SARS-CoV-2 infection have been widely studied, notwithstanding often for shorter time-periods [22, 75, 83, 84] with fewer long-term studies, i.e. 8-9 months [85] . Evidence from the SARS-CoV outbreak in 2005 suggests that anti-SARS-CoV antibodies fell below detection limits within two years [86] , and SARS-CoV-specific memory T cells were detectable 11 years after the SARS outbreak [87] .

Memory T cells are an important and diverse subset of antigen experienced T cells that are sustained long-term, and when needed are converted into effector cells during re-infection/exposure [88, 89] . Depending on their cellular programming and phenotype they are classified into different central and effector memory subtypes. The effector subsets contain the CD45RA + CCR7 -TEMRA, which are essentially TEM that re-express CD45RA after antigen stimulation [90] . Not much is known about the functionality of this population, but CD4 + TEMRA are implicated in protective immunity [90] . Furthermore, high levels of virus specific CD8 + TEMRA are maintained after dengue vaccination [91] . We found an elevation of CD8 + TEMRA that lasted throughout the study, i.e., 6-7 months, which contrasted with the CD4 + TEMRA that remained unaltered by COVID-19. Previous studies have shown the CD8 + TEMRA population to be increased at hospitalization [92, 93] and sustained for 6 weeks [92] .

Nonetheless, as far as we are aware, no studies have followed up beyond the 6 week time point. Currently, the exact role of CD8 + TEMRA in COVID-19 remains largely ambiguous, but Cohen et al [85] found an increase in SARS-CoV-2 specific CD8 + TEMRA over time. In our case, we have explored the whole expanded CD8 + TEMRA population and cannot confirm if there was a larger fraction of antigen-exposed, i.e., SARS-CoV-2-specific T cells, among the population.

We found that all COVID-19 patients developed SARS-CoV-2 antigen specific T cells, that increased over time. Our findings are in accordance with other studies that have shown that SARS-CoV-2 infection results in increased expansion of antigen specific CD4 + and CD8 + T cell subsets [63, 94] . It is still unclear if the lower antigen specific responses seen at 2 and 6 weeks compared to 6-7 months are due to an overall immunosuppression [95] or a natural development of the immune response over time [65] . Furthermore, there might be a . In addition, the TFH regulatory T cells were elevated at inclusion in the study but reverted to normal levels after a few weeks.

Our study clearly shows that activation of CD4 + and CD8 + T cells is a key finding in COVID-19 patients during active disease, and this is in agreement with others [39, 55] . Concerning the activated CD8 + T cell population expressing CD38 and HLA-DR, the levels were elevated compared to healthy controls and were sustained for more than 6 weeks, whereas the CD4 + T cells decreased over time within the COVID-19 patients. High levels of HLADR + CD38 + CD8 + T cells that persisted 30-60 days were detected in individuals with severe COVID-19 [39] , which is in accordance with our findings. We found elevated levels of activated CD69 + CD4 + T cells that were sustained at the 6-7 month follow-up, while the levels of CD69 + CD8 + T cells dropped to healthy control levels already at the 2 and 6 week time points. CD69 expression on T cells is considered an early activation marker [38] , and is also implicated in regulating mucosal inflammation [96] . It has also been shown that sustainment of CD69 on circulating CD8 + T cells eventually could also accelerate their destruction in the liver [97] , and hence it remains to be seen if the activated T cells would still be recruited into the effector or memory pool.

The hyper-activation of T cells is a well-studied mechanism in chronic viral infections [98] and is often dependent on the levels of viral persistence in the circulation. The reason for the prolonged activation of T cells in COVID-19 could be owing to the persistence of viral antigens in the bone marrow, that indirectly affect the circulating lymphocytes [99] . In addition, this may also be attributed to an ongoing chronic inflammation resulting from fibrosis/on-going tissue repair in the lung of patients' recovering from COVID-19 [100, 101] . Indeed, individuals that had been infected with SARS-CoV experienced dysregulation in lung function that persisted for 2 years [102] , and hence in our cohort, 6-7 months is seemingly too short to witness complete recovery. Chronic activation of T cells leads to impairment in their functionality and elevated levels of negative immune checkpoint molecules, which compromise both the antigen specific and bystander responses [103] . The negative immune checkpoint molecules PD-1, LAG-3, or TIM-3 expressing CD4 + and CD8 + T cells were elevated at inclusion. Additionally, the levels of LAG-3 and PD-1 on CD4 + T cells, and LAG-3 and TIM-3 on CD8 + T cells were elevated at the 6-7 month time point. The expression of negative immune checkpoint molecules TIM-3 and PD-1 mediates immunosuppression in lung, and was found to be a hallmark of severe COVID-19, particularly in men [104] . Besides the induction of the negative immune checkpoint molecules, chronic stimulation can give rise to CD57 + exhausted T cells. In addition, the frequency of CD57 + T cells increases with age [56] . There was a high impact on the CD8 + T cell population with an increase in CD57 + CD8 + T cells that lasted throughout the study (6-7 months), although we did not find any effect on the CD4 + T cell population. Increased CD57 CD8 + T cells has previously only been shown early during hospitalization in COVID-19 patients [41] .

Today it is clear that all immunocompetent individuals develop SARS-CoV-2-specific antibodies, which is also evident in our investigation. Some studies provide clear evidence that these antibodies are detected only for a few months after infection [105, 106] , whereas others support a minimum of 6 months [20, 107]. In our cohort, we found that the SARS-CoV-2 nucleocapsid-specific and spike-specific neutralizing antibody levels lasted 6-8 months post infection, even though there was a drastic decline at some time point after 6 weeks. Our findings are supported by Björkander et al. [94] , who have reported that the antibody responses lasted up to 8 months among young adults.

With the continued burden of the current COVID-19 pandemic on the population, there is still a need for more insight into the SARS-CoV-2-specific immune response elicited during infection. Despite having multiple approved and licensed vaccines, the emergence of variants with multiple mutations [108] , and the long list of long-term symptoms following a natural SARS-CoV-2 infection [109] [110] [111] , are still cause for great concern. Further, given the likely It is important to note that the current investigation has limitations. A critical factor to consider is the pre-selection of patients for this cohort, with most of the patients included already having moderate to severe COVID-19. It is also noteworthy to consider that health systems vary across geographical locations so patients in other counties might be more diverse due to different thresholds of illness required for hospital admission. Additionally, due to severe disease presentations, there were challenges with obtaining clinical samples from some patients and, in some cases, the cell numbers were low and/or of poor quality. During the study some COVID-19 patients were vaccinated against SARS-CoV-2 and therefore were excluded from our data set as this interfered with some results.

Despite the immensely challenging conditions during the pandemic, we do believe that the cohort presented in this investigation is well characterized and of high quality and value. 

Peripheral blood was collected from patients with COVID-19 that required hospitalization (N=46) and healthy controls (N=31) and levels of clinical markers and white blood cells were measured at inclusion. PBMCs collected from COVID-19 patients that required hospitalization (N=23) and healthy donors (N=16), were stained for flow cytometry to assess the activation status of CD4 + and CD8 + T cells. (A) Representative dot plots for CD38 + /HLA-DR + and CD69 + activated CD4 + and CD8 + T cells. (B) Proportions of the CD38 + /HLA-DR + and CD69 + activated CD4 + T cells. (C) Proportions of the CD38 + /HLA-DR + and CD69 activated CD8 + T cells. Data is represented as mean with 95% Cl, with significance of *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001, determined using Brown-Forsythe and Welch ANOVA tests. Inc = Inclusion in study at the hospital, 2W = 2 weeks, 6W = 6 weeks, 6M = 6-7 months, HC = healthy control.

PBMCs collected from COVID-19 patients that needed hospitalization (N=23) and healthy controls (N=16), were stained for flow cytometry to assess the expression of negative immune checkpoint factors and exhausted status of CD4 + and CD8 + T cells. (A) Representative flow cytometry plots show how CD57 expression levels were determined for CD4 + and CD8 + T cells. (B) Expression frequency of CD57 among CD4 + and CD8 + T cells. (C) Expression frequencies of LAG-3 + , TIM-3 + and PD-1 + among CD4 + and (D) CD8 + T cells. Data is represented as mean with 95% Cl, with significance of *p≤0.05, **p≤0.01, ***p≤0.001, determined using Brown-Forsythe and Welch ANOVA tests. Inc = Inclusion in study at the hospital, 2W = 2 weeks, 6W = 6 weeks, 6M = 6-7 months, HC = healthy control. PBMCs collected from COVID-19 patients that needed hospitalization (N=23) were left unstimulated (M) or incubated with SARS-CoV-2 specific peptides: Spike (S), spike+ (S+), and nucleocapsid (N); and assessed for their production of IFN-γ by ELISPOT. Representative images of the IFN-γ production in a COVID-19 hospitalized patient at inclusion, 2 weeks, 6 weeks, and 6-7 months, each dot represent one antigen-specific T cell (A). Antigen specific T cells responses as spot forming cells (SFC) per 10 6 PBMCs, specific for spike (B), spike+ (C), and nucleocapsid antigens (D). Data is represented as mean with 95% Cl, with significance of *p ≤ 0.05, **p ≤ 0.01, determined using Brown-Forsythe and Welch ANOVA tests. Inc = Inclusion in study at the hospital, 2W = 2 weeks, 6W = 6 weeks, 6M = 6-7 months, HC = healthy control. . Data is represented as mean with 95% Cl, with significance of *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, determined using Brown-Forsythe and Welch ANOVA tests. Inc = Inclusion in study at the hospital, 2W = 2 weeks, 6W = 6 weeks, HC = healthy control. 

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Protein (mg/L) median (range)

We sincerely thank all the donors who participated in the study. We are grateful to all those who contributed to the study, especially the health care personnel at the Clinic of Infectious Diseases and at the Intensive Care Unit at the Vrinnevi Hospital, Norrköping, Sweden, and the cohort study coordinators. We would also like to thank Annette Gustafsson for study coordination and collecting samples from the donors, and Mario Alberto Cano Fiestas for assisting with processing of samples. We are grateful for the assistance from the Core Facility 

The authors declare no competing interest/conflict.