Author response: Prolonged T-cell activation and long COVID symptoms independently associate with severe COVID-19 at 3 months Article Swipe

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Marianna Santopaolo , Michaela Gregorová , Fergus Hamilton , David Arnold , Anna E. Long , Aurora Lacey , Elizabeth Oliver , Alice Halliday , Holly E. Baum , Kristy L. Hamilton , Rachel Milligan , Olivia Pearce , Lea Knežević , Begonia Morales Aza , Alice Milne , Emily Milodowski , Eben Jones , Rajeka Lazarus , Anu Goenka , Adam Finn , Nick Maskell , Andrew D. Davidson , Kathleen M. Gillespie , Linda Wooldridge , Laura Rivino · YOU? · · 2023 · Open Access · · DOI: https://doi.org/10.7554/elife.85009.sa2

Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Coronavirus disease-19 (COVID-19) causes immune perturbations which may persist long term, and patients frequently report ongoing symptoms for months after recovery. We assessed immune activation at 3–12 months post hospital admission in 187 samples from 63 patients with mild, moderate, or severe disease and investigated whether it associates with long COVID. At 3 months, patients with severe disease displayed persistent activation of CD4+ and CD8+ T-cells, based on expression of HLA-DR, CD38, Ki67, and granzyme B, and elevated plasma levels of interleukin-4 (IL-4), IL-7, IL-17, and tumor necrosis factor-alpha (TNF-α) compared to mild and/or moderate patients. Plasma from severe patients at 3 months caused T-cells from healthy donors to upregulate IL-15Rα, suggesting that plasma factors in severe patients may increase T-cell responsiveness to IL-15-driven bystander activation. Patients with severe disease reported a higher number of long COVID symptoms which did not however correlate with cellular immune activation/pro-inflammatory cytokines after adjusting for age, sex, and disease severity. Our data suggests that long COVID and persistent immune activation may correlate independently with severe disease. Editor's evaluation This is an important paper that presents convincing evidence that certain cellular and molecular immune fingerprints at day 30 post-infection are associated with severe prior infection and may be risk factors for prolonged symptoms. The work will be of broad interest to clinicians, immunologists, and virologists. https://doi.org/10.7554/eLife.85009.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease-19 (COVID-19), can be asymptomatic or lead to a broad spectrum of disease manifestations from mild to severe disease and death. There is evidence showing that acute COVID-19 causes a profound activation of innate and adaptive immune cells, and distinct immunological signatures are shown to correlate with disease outcomes (Mathew et al., 2020). Viral control and mild disease associate with the rapid generation of T-cells and antibodies targeting SARS-CoV-2 (Lucas et al., 2021; Tan et al., 2021), while severe disease is characterised by immune cell hyperactivation and a ‘cytokine storm’ (Chen et al., 2020; Kuri-Cervantes et al., 2020; Ruan et al., 2020). Commonly observed biomarkers of severe COVID-19 include lymphopenia, increased neutrophil to T-cell ratio, and elevated levels of pro-inflammatory cytokines/mediators such as interleukin-6 (IL-6), IL-10, IL-17, monocyte chemoattractant protein-1 (MCP-1), interferon gamma-induced protein 10 (IP-10), CRP, IL-1Rα, and IL-1β. In a smaller proportion of individuals, severe COVID-19 may be driven by pre-existing anti-interferon gamma (anti-IFN-γ) autoantibodies (Bastard et al., 2020; Liu et al., 2020; Lucas et al., 2020; Zhao et al., 2020). Other distinctive features of severe disease include a dysregulation in the myeloid compartment consisting of increased circulating immature/dysfunctional neutrophils and immature Ki67+ COX-2low monocytes (Mann et al., 2020; Schulte-Schrepping et al., 2020; Wu et al., 2020; Mardi et al., 2021), while T-cell activation and proliferation appears to be heterogenous in severe patients (Mathew et al., 2020; Kuri-Cervantes et al., 2020). The kinetics of immune recovery following the changes occurring during acute COVID-19 are complex and not fully understood. Longitudinal studies show that immune abnormalities and inflammation may persist after severe COVID-19, with highly activated myeloid cells, pro-inflammatory cytokines, and persistently activated T-cells detected 8–12 months after COVID-19 (Bergamaschi et al., 2021; Phetsouphanh et al., 2022; Taeschler et al., 2022). The potential impact of prolonged immune activation post recovery and whether it may underlie the symptoms of post-acute sequelae of SARS-CoV-2 infection or long COVID - a syndrome characterised by long-lasting symptoms affecting multiple organs - remains unclear. Long COVID is observed in 8–21% of individuals following mild to severe COVID-19, although a higher prevalence of symptoms is reported in patients requiring ICU admission and/or mechanical ventilation (Alwan, 2021; Nalbandian et al., 2021; Ballering et al., 2022). Several studies have reported a correlation between long COVID symptoms and a variety of immune profiles including a more rapid decline of SARS-CoV-2 memory CD8+ T-cells and lower levels of cytotoxic SARS-CoV-2 N-specific CD8+ T-cells (Peluso et al., 2021), increased levels of cytotoxic CD8+ T-cells, and elevated plasma levels of type I cytokines, IL-1β and IL-6 (Shuwa et al., 2021; Schultheiß et al., 2022). Another longitudinal study conducted in 69 patients found long COVID to associate with an altered immune blood cell transcriptome which included significant changes in genes involved in cell cycle, but not with the phenotypic profiles of these cells (Ryan et al., 2022). A large UK cohort study (PHOSP-COVID) of hospitalised COVID-19 patients identified female sex, obesity, and invasive mechanical ventilation as factors associated with a lower likelihood of full recovery, with an overall recovery rate of 28.9% at both 5 and 12 months across all disease severities (Evans et al., 2022). Similarly, other cohort studies have identified associations between poor recovery from COVID-19 and demographic factors such as sex and age, but also highlight a possible role of immune dysregulation that occurs in older age, obesity and asthma, which warrants further investigation (Sneller et al., 2022; Halpin et al., 2021; Thompson et al., 2022). Other hypotheses proposed to explain the mechanisms underlying long COVID include unresolved lung tissue damage, the persistence of SARS-CoV-2 infection, the reactivation of latent viruses such as human cytomegalovirus (CMV) and Epstein-Barr virus and autoimmunity (Proal and VanElzakker, 2021; Gatto et al., 2022; Plüß et al., 2021; Zollner et al., 2022). Due to the limited effectiveness of COVID-19 vaccines in protecting from re-infection, SARS-CoV-2 continues to circulate within our populations and millions of people worldwide are at risk of experiencing long-term health complications from COVID-19. Studies suggest that COVID-19 vaccination may reduce the risk of developing long COVID (Al-Aly et al., 2022; Notarte et al., 2022). Nevertheless the burden of long COVID threatens to increase and further compromise our post-pandemic economic recovery. A better understanding of the mechanisms underlying long COVID and the design/repurposing of drugs to treat or prevent this syndrome is urgently needed. In this study we investigated whether patients who had COVID-19, 3 months post hospitalisation display persistent immune activation and ongoing inflammation and if there are potential links between the observed immune profiles, COVID-19 disease severity and long COVID symptoms. We also investigated whether persistent immune activation and disease severity have an impact on the capacity of patients to generate and maintain a memory T-cell response and antibodies to SARS-CoV-2. Our immunological analysis of 187 samples from 63 hospitalised patients recovering from mild, moderate, or severe disease showed increased levels of activated CD4+ and CD8+ T-cells and increased plasma levels of T-cell-related cytokines (IL-4, IL-7, IL-17, and tumor necrosis factor-alpha [TNF-α]) at 3 months in severe compared to moderate/mild patients. T-cell activation and cytokine levels decreased at 12 months and were comparable in mild, moderate, and severe patients. The SARS-CoV-2-specific memory T-cell response to spike, membrane and nucleocapsid was robust and qualitatively similar across the disease severities, however the magnitude of the CD4+ and CD8+ T-cell and antibody response was higher in patients with moderate compared to mild disease, as were the plasma levels of IFN-γ, a key cytokine for anti-viral immunity. Long COVID symptoms at 3 months were reported in 80% of patients across all disease severities but were more frequent in patients with severe disease. Poisson regression analysis showed a significant association between ongoing symptoms and the frequency of a subset of moderately activated CD4+ T and non-cytotoxic HLA-DR+ CD8+ T-cells in unadjusted analyses, but these associations did not meet the criteria for statistical significance after adjusting for age, sex, and severity grades. Our results demonstrate the presence of immune perturbations in peripheral blood CD4+ and CD8+ T-cell populations in severe COVID-19 patients 3 months after hospitalisation with no direct association between long COVID symptoms and immune activation/pro-inflammatory cytokines, for the markers that were measured. Results DISCOVER patients To investigate immune profiles in convalescent COVID-19 patients, we obtained peripheral blood mononuclear cells (PBMCs) and plasma from 63 patients enrolled in the DISCOVER study at 3 months post admission for COVID-19, and where possible matched PBMCs at 12 months and plasma samples at 8 and 12 months. The demographics and clinical characteristics of the 63 patients included in this study are shown in Table 1. Patients were stratified into mild, moderate, and severe based on the type of respiratory support they required during the acute illness, as follows: mild patients did not require supplementary oxygen or intensive care; moderate patients required supplementary oxygen during admission, and severe patients required invasive mechanical ventilation, non-invasive ventilation, and/or admission to the intensive treatment unit. Of the 63 patients included in this analysis 17, 32, and 14 recovered from mild, moderate, and severe disease, respectively. Overall, a higher proportion of patients were male (40/63) and the median age (± SD) of patients was 53±14.5, 58±12.6, and 61.5±10 years for mild, moderate, and severe patients, respectively. Patient ethnicity was predominantly Caucasian with an Asian/Black minority. Body mass index (BMI) was largely within the unhealthy range across all disease severities (overweight, obese, and extremely obese) with 7.6%, 15.6%, and 14.3% of patients with respectively mild, moderate, and severe disease displaying healthy BMI. The comorbidities observed in these 63 patients were consistent with those known to be associated with higher risks for COVID-19 hospitalisation and included heart disease, diabetes (predominantly type-2), hypertension, and chronic lung disease. Overall, the percentage of patients with comorbidities tended to increase progressively with disease severity (mild: 52.9%; moderate: 56.2%, and severe: 85.7%). The duration of hospital admission ranged from 3.3±1.99 to 7.8±5.16 and 12±6.67 days (± SD) in mild, moderate, and severe patients, respectively. None of the patients had received COVID-19 vaccination at the time of blood collection. Table 1 Details of the patients included in the immunological analysis of this study. Disease severity (n)MildModerateSevere(n=17)(n=32)(n=14)Age (median, SD)53±14.558±12.661.5±10Sex, % (n)Female35.3 (6)31.2 (10)50 (7)Male64.7 (11)68.5 (22)50 (7)Ethnicity, % (n)Caucasian83.3% (14)78.1% (25)87.7% (12)Asian11.7% (2)12.5% (4)14.3% (2)Black5.9% (1)6.3% (2)0 (0)Missing data0 (0)3.1% (1)0 (0)BMI, kg/m2, % (average, SD)Healthy7.6% (22.3±0.57)12.5% (23.75±0.5)14.3% (23±0)Overweight35.3% (26.6±1.5)28.1% (27±1.39)28.6% (28.75±0.5)Obese35.3% (32.8±1.47)46.9% (32.7±2.46)28.6% (35±2.1)Extremely obese11.8% (61±28.2)9.38% (46.3±6.02)28.6% (46±7.34)Missing data03.1%0Comorbidity % (n)None47.1% (8)43.75% (14)14.3% (2)Heart disease23.5% (4)15.6% (5)14.3% (2)T1DM0 (0)3.1% (1)7.14% (1)T2DM5.88% (1)12.5% (4)14.3% (2)Hypertension17.65 (3)18.75% (6)50% (7)Chronic lung disease5.88% (1)21.88% (7)50% (7)Kidney disease5.88 (1)9.37% (3)14.3% (2)Mental health0 (0)6.25% (2)14.3% (2)Cancer5.88% (1)3.1% (1)7.14 (1)Asthma5.88% (1)0 (0)14.3% (2)Total obesity47.1% (8)53.1% (17)57.1% (8)Other47.1% (8)28.1% (9)50% (7)Hospital stay (average days, SD)3.3±1.997.8±5.1612±6.67Ongoing symptomsat 3 months(n, %)None3 (17.65%)8 (25%)2 (14.3%)Dyspnoea7 (41.2%)15 (47%)9 (64.3%)Excessive fatigue5 (29.4%)12 (37.5%)9 (64.3%)Muscle weakness3 (17.65)7 (22%)5 (35.7%)Sleeping difficulties3 (17.65)6 (18.75%)8 (57%)Psychiatric2 (11.8%)8 (25%)6 (42.9%)Anosmia2 (11.8%)4 (12.5%)3 (21.4%)Chest pain2 (11.8%)6 (18.75%)2 (14.3%)Cough2 (11.8%)4 (12.5%)1 (7.14%)Other5 (29.4%)4 (12.5%)3 (21.4%) Increased CD4+ and CD8+ T-cell activation in severe patients at 3 months To investigate the presence of ongoing immune activation following recovery from COVID-19, we assessed the phenotype and activation/proliferation profiles of conventional CD4+ and CD8+ T-cells, TCR-γδ T, NK, B-cells, and monocytes by flow cytometry in PBMC samples from 63 patients at 3 months post admission. T and NK cells were assessed for expression of markers of activation, differentiation, proliferation, and cytotoxicity (HLA-DR, CD38, CD69, CCR7, CD45RA, CXCR3, Ki67, granzyme B, CD56) after gating on single live cells, CD3+ CD4+ or CD3+ CD8+ T-cells and CD3- CD56bright CD16+/- or CD56dim CD16+ NK cell populations, respectively (Figure 1, gating strategies in Figure 1—figure supplement 1; list of antibodies in Key resources table). Data were analysed by FlowJo using a manual gating strategy as well as the dimensionality reduction algorithm uniform manifold approximation and projection (UMAP) for dimension reduction and cluster analysis using FlowSOM. Figure 1 with 2 supplements see all Download asset Open asset CD4+ T-cell profiles in convalescent coronavirus disease-19 (COVID-19) patients at 3 months post admission. (A–D) Percentage of CD4+ T-cells within the CD3+ gate (A), absolute number of CD4+ T-cells (cells/mm3) (B), and percentages of CD4+ T-cells expressing CXCR3 (C) and co-expressing Ki67/CD38 (D) are shown in mild, moderate, and severe patients. (E) Percentages of naïve (CCR7+ CD45RA+), T central memory (TCM, CCR7+ CD45RA-), T effector memory (TEM, CCR7- CD45RA-), and T effector memory RA re-expressing (TEMRA, CCR7- CD45RA+) CD4+ T-cells are shown for patients with mild, moderate, and severe disease. (F) Flow cytometry plot showing a representative staining from a mild, moderate, and severe patient of HLA-DR and CD38 expression in CD4+ TEM cells (overlaid and shown respectively in blue, black, and red). (G) Percentages of activated HLA-DR+CD38+ CD4+ T-cells within naïve, TCM, TEM, and TEMRA cells. (H) Flow cytometry plot with a representative staining from a mild, moderate, and severe patient of HLA-DR and Ki67 expression in CD4+ TEM cells. (I) Percentages of proliferating HLA-DR+ Ki67+ CD4+ T-cells within naïve, TCM, TEM, and TEMRA cells. (J) Flow cytometry plot with a representative staining from a mild, moderate, and severe patient of HLA-DR and granzyme B (GrzmB) expression in CD4+ TEM cells. (K) Percentages of proliferating HLA-DR+ GrzmB+ CD4+ T-cells within naïve, TCM, TEM, and TEMRA cells. (L) Unsupervised uniform manifold approximation and projection (UMAP) analysis showing the FlowSOM clusters in mild (N=17), moderate (N=25), and severe (N=14) patients. Plots are gated on CD4+ T-cells. (M) Heatmap with the expression of each analysed marker within the FlowSOM populations shown as mean fluorescence intensity (MFI). (N) Summary of the percentage of CD4+ T-cells within the indicated FlowSOM populations in mild, moderate, and severe patients. Data in graphs are visualised as mean ± SEM. Statistics are calculated by one-way ANOVA (Kruskal-Wallis test) with Dunn’s correction for multiple testing. Overall, at 3 months post admission patients’ blood lymphocyte and neutrophil counts, CRP, and albumin levels - which were mostly perturbated during the acute illness - had normalised to levels that remained similar at 8 months, suggesting a resolution of the peak inflammatory events occurring during the acute illness (Figure 1—figure supplement 2). The frequencies and absolute numbers of CD4+ T-cells were similar in patients across disease severities (Figure 1A and B). However, we observed an increased frequency of CD4+ T-cells expressing the peripheral homing receptor CXCR3 in severe compared to moderate patients and an increased frequency of CD4+ T-cells co-expressing the proliferation/activation markers Ki67 and CD38 in severe compared to mild patients (Figure 1C and D). The overall differentiation status of CD4+ T-cells, defined by the coordinated expression of CCR7 and CD45RA, was similar in mild, moderate, and severe patients, with high proportions of naïve (CCR7+ CD45RA+) and T central memory cells (CCR7+ CD45RA-, TCM) followed by detectable levels of T effector memory cells (CCR7- CD45RA-, TEM) and low frequencies of T effector memory re-expressing RA cells (CCR7- CD45RA+, TEMRA) (Figure 1E). The most abundant naïve and TCM CD4+ T-cell subsets contained significantly higher levels of cells co-expressing markers of activation, proliferation, and cytotoxic potential (HLA-DR+ CD38+, HLA-DR+ Ki67+, CD38+ granzyme B+) in severe compared to moderate/mild patients (Figure 1F–K). An unsupervised analysis by UMAP and FlowSOM revealed the presence of 15 distinct clusters of cells characterised by a unique, coordinated expression of the analysed markers (Figure 1L), shown as mean fluorescence intensity in the heatmap (Figure 1M). These analyses revealed significantly higher frequencies in severe compared to moderate patients of clusters of cells expressing HLA-DR, CD38, and Ki67 (populations 8 and 13) as well as of cells that express CCR7 but lack expression of all other markers analysed, and which resemble undifferentiated, resting CD4+ T-cells (population 2) (Figure 1N). The latter population accounted for a large proportion of CD4+ T-cells (up to 60% of cells in severe patients). A population characterised by low levels of expression of all markers analysed is decreased in severe compared to moderate patients (population 1, Figure 1N). In summary, both manual gating and unsupervised analysis reveal the presence of higher frequencies of CD4+ T-cell populations expressing markers of activation, proliferation, and peripheral tissue homing in patients who recovered from severe versus mild and/or moderate disease. Similarly, the analyses of CD8+ T-cells showed comparable frequencies and absolute numbers of circulating CD8+ T-cells in patients across disease severities and increased levels of CD8+ T-cells expressing markers of activation (HLADR+CD38+) in severe compared to moderate patients and cytotoxicity (granzyme B+) in severe compared to mild patients (Figure 2A–D). The overall differentiation status of CD8+ T-cells was comparable in patients across disease severities with detectable frequencies of naïve, TCM, TEM, and TEMRA cells (Figure 2E). CD8+ T-cells expressing markers of activation and cytotoxicity (HLADR+ CD38+ and CD38+ granzyme B+) could be detected at higher frequencies within TCM, TEM, and TEMRA subsets in severe compared to moderate and/or mild patients (Figure 2F–I). Unsupervised analysis by UMAP and FlowSOM of CD8+ T-cells showed the presence of 15 clusters of cells expressing the analysed markers (Figure 2J and K). The frequencies of a cluster of CD8+ T-cells expressing high levels of granzyme B and CD45RA (cluster 3) were significantly increased in both severe and moderate compared to mild patients and represented up to 50% of all CD8+ T-cells in severe/moderate groups and up to 30% in mild group (Figure 2L). CD8+ T-cells expressing high levels of CCR7 and intermediate levels of granzyme B, CXCR3, and CD56 were increased significantly in both mild and severe compared to moderate patients (cluster 12). In summary, manual and unsupervised analysis revealed the presence of CD8+ T-cells expressing markers of activation and cytotoxicity which are increased in severe compared to moderate and/or mild patients. Analysis of the T-cell phenotypes in the same patients at 12 months showed that CD4+ and CD8+ T-cell activation and proliferation decreased significantly from 3 to 12 months and activated/proliferating T-cells were largely absent at 12 months (Figure 2—figure supplement Analysis by manual gating showed a significant of activated/proliferating CD4+ and CD8+ T-cells CD38+, HLA-DR+ and of CD4+ and CD8+ T-cells expressing the peripheral receptor from 3 to 12 months (Figure 2—figure supplement An unsupervised analysis by UMAP revealed similar of T-cell clusters in patients with mild, moderate, and severe disease at 12 months, while were observed between patients from the groups at 3 months (Figure 2—figure supplement Our data suggests that the ongoing T-cell activation observed at 3 months in patients with severe disease had recovered by 12 months post admission to levels similar to those observed in patients with mild and moderate disease. Figure 2 with 2 supplements see all Download asset Open asset CD8+ T-cell profiles in convalescent coronavirus disease-19 (COVID-19) patients at 3 months post admission. (A–D) Percentage of CD8+ T-cells within the CD3+ gate (A), absolute number of CD8+ T-cells (cells/mm3) (B), and percentages of CD8+ T-cells co-expressing the activation markers (C) or granzyme B shown as mean fluorescence intensity are shown in mild, moderate, and severe patients. (E) Percentages of naïve (CCR7+ CD45RA+), T central memory (TCM, CCR7+ CD45RA-), T effector memory (TEM, CCR7- CD45RA-), and T effector memory RA re-expressing (TEMRA, CCR7- CD45RA+) CD8+ T-cells in patients with mild, moderate, and severe disease. (F) Flow cytometry plot with a representative staining from a mild, moderate, and severe patient (overlaid and shown respectively in blue, black, and of HLA-DR and CD38 expression in CD8+ TEM cells. (G) Percentages of activated HLA-DR+ CD38+ CD8+ T-cells within naïve, TCM, TEM, and TEMRA cells. (H) Flow cytometry plot with a representative staining from a mild, moderate, and severe patient of HLA-DR and granzyme B (GrzmB) expression in CD8+ TEM cells. (I) Percentages of proliferating HLA-DR+ GrzmB+ CD8+ T-cells within naïve, TCM, TEM, and TEMRA cells. (J) Unsupervised uniform manifold approximation and projection (UMAP) analysis showing the FlowSOM clusters in mild (N=17), moderate (N=25), and severe (N=14) patients. Plots are gated on CD8+ T-cells. (K) Heatmap with levels for each analysed marker within the FlowSOM (L) Summary of percentage of CD8+ T-cells within the indicated FlowSOM populations in mild, moderate, and severe patients. Data in the graphs are shown as mean ± SEM. Statistics were calculated by one-way ANOVA (Kruskal-Wallis test) with Dunn’s correction for multiple testing. Analysis of expression of the same markers of activation/proliferation in NK CD56dim and CD56bright cells did not show evidence of ongoing NK cell activation and NK cells were similar in patients who recovered from mild, moderate, and severe disease (Figure 2—figure supplement Similarly, analysis of the frequencies and expression of activation markers of intermediate and monocytes showed similar frequencies of these cells across the disease severities and lack of ongoing activation (Figure 2—figure supplement frequencies of TCR-γδ T-cells were detected in patients with mild, moderate, and severe disease and these cells expression of markers of activation/proliferation (Figure 2—figure supplement In B cells from severe patients showed an increase in the expression of the activation marker compared to patients with mild disease, although expression of other markers of activation/proliferation did not (HLA-DR, CD38, and (Figure 2—figure supplement In summary, both manual and unsupervised analysis showed increased frequencies of activated/proliferating CD4+ and CD8+ T-cells in severe compared to mild and/or moderate patients at 3 months post admission, suggesting the presence of ongoing immune activation in these patients. We did not significant activation of other immune cells analysed with the of B cells from severe patients which increased levels of compared to mild patients. pro-inflammatory in severe patients at 3 months To into the factors that may be CD4+ and CD8+ T-cell activation and proliferation and the of persistent inflammation in these patients, we investigated the presence of circulating pro-inflammatory in the plasma of COVID-19 patients 3 months after admission. The plasma levels of the following were using a IFN-γ, IL-10, IL-7, inflammatory and Our results show that at 3 months the levels of IL-7, IL-17, and were significantly increased in the plasma of patients with severe compared to mild and/or moderate disease, while was increased in moderate compared to mild patients. was largely in the plasma of severe patients (Figure A for increased levels of was observed in the plasma of severe compared to mild and moderate patients but the was not significantly The plasma levels of other did not between patients with disease severities at 3 months (Figure supplement We investigated the kinetics of expression of these cytokines in patients at and 12 months post admission and observed a significant of plasma levels of and from 3 to 8 and/or 12 months, suggesting that at 3 months the levels of these cytokines may be by events occurring during the acute of levels were similar at 3 and 8 months and decreased (Figure and levels were higher at 3 months and progressively although were not In levels were higher at 8 and 12 months compared to 3 months. The plasma levels of the other cytokines analysed including IL-7, IFN-γ, and were comparable at 3–12 months post admission. Figure 3 with 1 supplement see all Download asset Open asset Plasma pro-inflammatory at and 12 months. Plasma at 3 months post admission which significantly between patients with mild, moderate, and severe disease are shown moderate: severe: in and in matched samples in patients at 3 and 12 months post admission samples for each time are Data from that significantly between time in B are shown in for each CD3+ T-cells from healthy donors for 3 for 12 were with plasma from healthy mild, and severe patients at 3 months post is expression in T-cells from a representative at 3 months (D) and the expression of by T-cells from each peripheral blood mononuclear cell after with plasma from mild and severe patients, where each data a single patient Statistics were calculated by one-way ANOVA (Kruskal-Wallis test) with Dunn’s multiple and by one-way analysis with the multiple Data are visualised as mean ± SEM. on our results in Figures showing increased levels of circulating activated/proliferating CD4+ and CD8+ T-cells in severe patients and on the elevated levels of cytokines involved in T-cell proliferation at 3 months, we whether factors in the plasma of severe patients T-cells more to activation, which occurs during infection and is to be driven by To we T-cells from healthy donors for days with plasma from healthy donors or patients with mild or severe COVID-19 at 3 and 12 months We observed a significant of the the that to in T-cells in the presence of plasma from severe patients at 3 months compared to healthy donors (Figure and In from severe patients at 12 months to upregulate These suggest that 3 months after severe COVID-19, peripheral blood CD4+ and CD8+ T-cells are in to plasma factors that more to by

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Author response: Prolonged T-cell activation and long COVID symptoms independently associate with severe COVID-19 at 3 months

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Marianna Santopaolo, Michaela Gregorová, Fergus Hamilton, David Arnold, Anna E. Long, Aurora Lacey, Elizabeth Oliver, Alice Halliday, Holly E. Baum, Kristy L. Hamilton, Rachel Milligan, Olivia Pearce, Lea Knežević, Begonia Morales Aza, Alice Milne, Emily Milodowski, Eben Jones, Rajeka Lazarus, Anu Goenka, Adam Finn, Nick Maskell, Andrew D. Davidson, Kathleen M. Gillespie, Linda Wooldridge, Laura Rivino

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Immune system, CD38, Disease, Medicine, CD8, Coronavirus disease 2019 (COVID-19), Cytokine storm, Granzyme B, Immune dysregulation, T cell, Immunology, Tumor necrosis factor alpha, Internal medicine, Biology, Infectious disease (medical specialty), Stem cell, Genetics, CD34

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