Medicine, Faculty of
Non UBC
Biomedical Engineering, School of
Surgery, Department of
DSpace
Children's Hospital (Vancouver, B.C.). Research Institute
Kennedy, Allison E.
Cook, Laura
Breznik, Jessica A.
Cowbrough, Braeden
Wallace, Jessica G.
Huynh, Angela
Smith, James W.
Son, Kiho
Stacey, Hannah
Ang, Jann
McGeer, Allison
Coleman, Brenda L.
Larché, Maggie
Larché, Mark
Hambly, Nathan
Nair, Parameswaran
Ask, Kjetil
Miller, Matthew S.
Bramson, Jonathan
Levings, Megan K.
Nazy, Ishac
Svenningsen, Sarah
Mukherjee, Manali
Bowdish, Dawn M. E.
Survivors of severe SARS-CoV-2 infections frequently suffer from a range of post-infection sequelae. Whether survivors of mild or asymptomatic infections can expect any long-term health consequences is not yet known. Herein we investigated lasting changes to soluble inflammatory factors and cellular immune phenotype and function in individuals who had recovered from mild SARS-CoV-2 infections (n = 22), compared to those that had recovered from other mild respiratory infections (n = 11). Individuals who had experienced mild SARS-CoV-2 infections had elevated levels of C-reactive protein 1–3 months after symptom onset, and changes in phenotype and function of circulating T-cells that were not apparent in individuals 6–9 months post-symptom onset. Markers of monocyte activation, and expression of adherence and chemokine receptors indicative of altered migratory capacity, were also higher at 1–3 months post-infection in individuals who had mild SARS-CoV-2, but these were no longer elevated by 6–9 months post-infection. Perhaps most surprisingly, significantly more T-cells could be activated by polyclonal stimulation in individuals who had recently experienced a mild SARS-CoV-2, infection compared to individuals with other recent respiratory infections. These data are indicative of prolonged immune activation and systemic inflammation that persists for at least three months after mild or asymptomatic SARS-CoV-2 infections.
virusesArticleLasting Changes to Circulating Leukocytes in People with MildSARS-CoV-2 InfectionsAllison E. Kennedy 1,2,3,† , Laura Cook 4,5,† , Jessica A. Breznik 1,2,3 , Braeden Cowbrough 1,2,3,Jessica G. Wallace 1,2,3, Angela Huynh 3, James W. Smith 3, Kiho Son 3,6 , Hannah Stacey 1,2,7, Jann Ang 1,2,7,Allison McGeer 8,9, Brenda L. Coleman 8,9 , Maggie Larché 3, Mark Larché 3, Nathan Hambly 3,6,Parameswaran Nair 3,6, Kjetil Ask 3,6, Matthew S. Miller 1,2,7 , Jonathan Bramson 1,3, Megan K. Levings 4,10,11,Ishac Nazy 3,12, Sarah Svenningsen 3,6, Manali Mukherjee 3,6 and Dawn M. E. Bowdish 1,2,3,6,*Citation: Kennedy, A.E.; Cook, L.;Breznik, J.A.; Cowbrough, B.; Wallace,J.G.; Huynh, A.; Smith, J.W.; Son, K.;Stacey, H.; Ang, J.; et al. LastingChanges to Circulating Leukocytes inPeople with Mild SARS-CoV-2Infections. Viruses 2021, 13, 2239.https://doi.org/10.3390/v13112239Academic Editor: Andrew DavidsonReceived: 13 October 2021Accepted: 1 November 2021Published: 8 November 2021Publisher’s Note: MDPI stays neutralwith regard to jurisdictional claims inpublished maps and institutional affil-iations.Copyright: © 2021 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).1 McMaster Immunology Research Centre, McMaster University, Hamilton, ON L8S 4K1, Canada;allkennedy@nosm.ca (A.E.K.); breznikj@mcmaster.ca (J.A.B.); cowbroub@mcmaster.ca (B.C.);wallacjg@mcmaster.ca (J.G.W.); staceyhd@mcmaster.ca (H.S.); ang.jann.catherine@gmail.com (J.A.);mmiller@mcmaster.ca (M.S.M.); bramson@mcmaster.ca (J.B.)2 Michael G. DeGroote Institute for Infectious Disease Research, McMaster University,Hamilton, ON L8S 4L8, Canada3 Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University,Hamilton, ON L8S 4K1, Canada; huynha8@mcmaster.ca (A.H.); smithjw@mcmaster.ca (J.W.S.);sonk@mcmaster.ca (K.S.); mlarche@mcmaster.ca (M.L.); larche@mcmaster.ca (M.L.);hamblyn@mcmaster.ca (N.H.); parames@mcmaster.ca (P.N.); askkj@mcmaster.ca (K.A.);nazyi@mcmaster.ca (I.N.); svennins@mcmaster.ca (S.S.); mukherj@mcmaster.ca (M.M.)4 BC Children’s Hospital Research Institute, University of British Columbia, Vancouver, BC V5Z 4H4, Canada;lcook@bcchr.ca (L.C.); megan.levings@ubc.ca (M.K.L.)5 Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity,University of Melbourne, Melbourne, VIC 3000, Australia6 Firestone Institute of Respiratory Health, St. Joseph’s Healthcare, Hamilton, ON L8N 4A6, Canada7 Department of Biochemistry & Biomedical Sciences, McMaster University, Hamilton, ON L8S 4K1, Canada8 Department of Microbiology, Sinai Health, Toronto, ON M5G 1X5, Canada;allison.mcgeer@sinaihealth.ca (A.M.); Brenda.Coleman@sinaihealth.ca (B.L.C.)9 Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto,Toronto, ON M5S 1A1, Canada10 School of Biomedical Engineering, University of British Columbia, Vancouver, BC V6T 1Z3, Canada11 Department of Surgery, University of British Columbia, Vancouver, BC V5Z 1M9, Canada12 McMaster Centre for Transfusion Research, Faculty of Health Sciences, Hamilton, ON L8S 4L8, Canada* Correspondence: bowdish@mcmaster.ca† A.E.K. and L.C. should be considered joint first author.Abstract: Survivors of severe SARS-CoV-2 infections frequently suffer from a range of post-infectionsequelae. Whether survivors of mild or asymptomatic infections can expect any long-term healthconsequences is not yet known. Herein we investigated lasting changes to soluble inflammatoryfactors and cellular immune phenotype and function in individuals who had recovered from mildSARS-CoV-2 infections (n = 22), compared to those that had recovered from other mild respiratoryinfections (n = 11). Individuals who had experienced mild SARS-CoV-2 infections had elevated levelsof C-reactive protein 1–3 months after symptom onset, and changes in phenotype and function ofcirculating T-cells that were not apparent in individuals 6–9 months post-symptom onset. Markersof monocyte activation, and expression of adherence and chemokine receptors indicative of alteredmigratory capacity, were also higher at 1–3 months post-infection in individuals who had mild SARS-CoV-2, but these were no longer elevated by 6–9 months post-infection. Perhaps most surprisingly,significantly more T-cells could be activated by polyclonal stimulation in individuals who had recentlyexperienced a mild SARS-CoV-2, infection compared to individuals with other recent respiratoryinfections. These data are indicative of prolonged immune activation and systemic inflammation thatpersists for at least three months after mild or asymptomatic SARS-CoV-2 infections.Keywords: COVID-19; SARS-CoV-2; immunophenotype; inflammation; immune activationViruses 2021, 13, 2239. https://doi.org/10.3390/v13112239 https://www.mdpi.com/journal/virusesViruses 2021, 13, 2239 2 of 161. IntroductionInfection with SARS-CoV-2 has unusual effects on circulating immune cells. In general,respiratory infections are associated with rapid and sustained release of neutrophils andmonocytes from the bone marrow, with no immediate change in production or functionof lymphocytes [1]. In contrast, SARS-CoV-2 infections are consistently associated withlymphopenia, which is a reliable predictor of mortality [2]. The loss of T-cells may bedue to apoptosis in the secondary lymphoid organs, or possibly inappropriate recruit-ment to the lungs and other organs [3,4]. Severe cases of SARS-CoV-2 infection are alsoassociated with increased granularity and changes in nuclear morphology in neutrophilsand monocytes, which are generally due to premature egress from the bone marrow andstunted maturation [5]. Whether myelopoiesis and egress are affected in mild COVID isnot yet known.Hospitalization with any respiratory infection is associated with long-term healthconsequences [6]; however, the consequences of COVID-19 appear to be particularly broadand include “long COVID” (a constellation of symptoms that can include severe fatigueoften exacerbated by exercise, pain, and respiratory complications) [7,8], and a higher thanexpected risk of re-hospitalization for a variety of causes [8–10]. Although it has beendocumented that there are differences in acute immune responses in asymptomatic, mild,moderate, and severe cases of SARS-CoV-2 infection [11,12], it is not clear whether thereis any risk of long-term immune dysregulation or health consequences in survivors ofasymptomatic or mild cases.In this study we investigated whether there are any changes in phenotype and fre-quency of circulating leukocytes in individuals who had experienced mild COVID-19 (i.e.,did not require medical attention), and compared them to those who had other mild respira-tory infections. We found evidence of transient changes in circulating NK cells, T-cells, andmonocytes 1–3 months post mild COVID-19, but not after other mild respiratory infections.These changes generally resolved 6–9 months post-COVID-19 infection, indicating thatthere is an early period of immune dysregulation greater than that of other respiratoryinfections, which occurs even after mild SARS-CoV-2 infection.2. Materials and Methods2.1. Participant Recruitment and Blood CollectionResearch participants with symptoms consistent with COVID-19 (as summarized inTable 1) were recruited from the greater Hamilton area (Ontario, Canada) between February2020 and January 2021. All protocols were approved by the Hamilton Research Ethics Board(#10757 and #11471), and all participants provided informed consent. Venous blood wasdrawn in anti-coagulant-free vacutainers for the isolation of serum, and in heparin-coatedvacutainers for experiments that required viable leukocytes. Serum and leukocytes wereisolated as per standard protocols [13]. Immunophenotyping was performed on fresh bloodwithin four hours of collection. None of the participants had been vaccinated against SARS-CoV-2 at the time of sample collection. Three participants who had a positive polymerasechain reaction (PCR) test for SARS-CoV-2 were either asymptomatic or had mild symptoms(i.e., headache or tiredness that lasted for a day). The remaining participants had symptomsthat they reported as consistent with a typical respiratory infection (e.g., cough, fever,chills), but these were generally resolved within 2–4 weeks. The duration of symptomsand demographics of the 38 participants in this study (i.e., sex, age, body mass index(BMI), comorbid conditions, medications) are summarized in Table 1. Participants werecharacterized as having had COVID-19, ‘other respiratory infection’, or ‘indeterminate’, asdetailed in Supplementary Table S1.Viruses 2021, 13, 2239 3 of 16Table 1. Participant demographics 1.Other RespiratoryInfections (NotCOVID-19) n = 11COVID-19Infectionsn = 22Indeterminaten = 5 p Value1Age (mean ± STDEV) 55 ± 16 55 ± 15 57 ± 11 0.9249Sex (% female) 8 (73%) 11 (50%) 3 (60%) 0.2783BMI (kg/m2) 24.0 ± 2.9 25.0 ± 3.6 24.5 ± 2.3 0.4172Health Conditions (frequency)Asthma 1 (9%) 1 (5%) 1 (20%) -COPD (includingemphysema and chronicbronchitis)1 (9%) 0 (0%) 0 (0%) -Other lung disease 0 (0%) 1 (5%) 0 (0%) -Diabetes 0 (0%) 3 (14%) 0 (0%) -Hypertension 1 (9%) 1 (5%) 1 (20%) -Heart disease 0 (0%) 2 (9%) 0 (0%) -Cancer 2 (18%) 2 (9%) 0 (0%) -Autoimmune condition 1 (9%) 1 (5%) 1 (20%) -MedicationsNumber of medications(mean ± STDEV) 1 ± 2 2 ± 2 1 ± 1 0.2049Symptoms (frequency)Cough 6 (55%) 13 (59%) 2 (40%) -Shortness of breath 4 (36%) 9 (41%) 1 (20%) -Chest pain 3 (27%) 9 (41%) 2 (40%) -Fever 3 (27%) 12 (55%) 1 (20%) -Feeling generally unwell 9 (82%) 21 (95%) 2 (40%) -Abnormally tired 7 (64%) 20 (91%) 2 (40%) -New confusion 4 (36%) 1 (5%) 1 (20%) -New generalized muscleaches and pains 5 (45%) 16 (73%) 2 (40%) -New joint pain 3 (27%) 5 (23%) 2 (40%) -Earache/infection 0 (0%) 1 (5%) 0 (0%) -Headache 6 (55%) 14 (64%) 1 (20%) -Runny/stuffy nose 7 (64%) 7 (32%) 2 (40%) -Sinus pain 2 (18%) 4 (18%) 1 (20%) -Sore/scratchy throat 7 (64%) 10 (45%) 2 (40%) -Loss of appetite 4 (36%) 8 (36%) 2 (40%) -Loss of taste/smell 2 (18%) 11 (50%) 1 (20%) -Duration of SymptomsNo symptoms—1 week 4 (36%) 3 (14%) 1 (20%) -2–4 weeks 3 (27%) 11 (50%) 4 (80%) -4 or more weeks 4 (36%) 8 (36%) 0 -Viruses 2021, 13, 2239 4 of 16Table 1. Cont.Other RespiratoryInfections (NotCOVID-19) n = 11COVID-19Infectionsn = 22Indeterminaten = 5 p Value1DiagnosisPCR test for SARS-CoV-2performed 0 (0%) 15 (68%) 0 (0%) -Told by a health careprofessional that they hadSARS-CoV-2/COVID-19without a PCR test1 (8%) 5 (25%) 0 (0%) -1 Statistical comparisons were made between ‘other respiratory infections’ and ‘COVID-19 infections’ groupsonly, since the ‘indeterminate’ group was not used in subsequent analyses. Student’s unpaired parametric t-testwas used to compare age and BMI, which were normally distributed. Sex distribution was measured by Fisher’sexact test to analyze a 2 × 2 contingency table. Differences in the number of medications, which was not normallydistributed, was measured using an unpaired non-parametric test not assuming Gaussian distribution.Most participants did not consult a medical professional about their condition, consis-tent with the public health advice in the early months of the pandemic that individualswho did not need medical attention should stay home, and since none were hospitalized,we defined these as ‘mild’ infections. Participants that had recovered from COVID-19 gaveblood at either 1–3 months post-symptom onset or 6–9 months post-symptom onset, butno participant gave blood at both time points. Therefore, there were no repeat measuresfrom the same participant.2.2. Measurements of Anti-SARS-CoV-2 AntibodiesAnti-SARS-CoV-2 full-length spike (S) protein and receptor binding domain (RBD) IgGand IgA seropositivity were identified via validated serology enzyme-linked immunosor-bent assay (ELISA), as described by Huynh et al., 2021 [14]. Briefly, NUNC™ Maxisorp384 well plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with S (5 µg/mL)or RBD (2 µg/mL) antigens in 50 mM carbonate buffer (pH 9.6) overnight at 4 ◦C, andblocked with 3% skim milk in PBS-0.05% Tween-20. After washing with phosphate-bufferedsaline (PBS), plates were coated with diluted serum (1:100) for 1 h at room temperature,washed, and incubated with 25 µL alkaline phosphatase-conjugated antibodies goat anti-human IgG (1:2000) or goat anti-human IgA (1:500) (Jackson ImmunoResearch, West Grove,PA, USA). Plates were washed, and antibody levels were quantified by adding 50 µL ofsubstrate buffer (0.27 µM p-nitrophenyl phosphate/diethanolamine buffer, 1 M, pH 9.6)and reading optical density (OD) at 405 nm detection every 1 min, with 490 nm referenceon a BioTek 800TS microplate reader (BioTek Instruments Inc., Winooski, WT, USA).2.3. Assessment of T Cell Activation Induced Markers (AIM) for SARS-CoV-2 PeptidesTo detect antigen-specific T-cell recall responses, 100 µL of heparinized venous bloodwas cultured with an equal amount of Iscove’s Modified Dulbecco’s Medium, GlutaMAX™Supplement (Invitrogen Life Technologies, Carlsbad, CA, USA) and antigens for 44 h in96-well flat bottom plates at 37 ◦C. Wells were stimulated using SARS-CoV-2 peptide pools(PepTivator® products from Miltenyi Biotec, Bergisch Gladbach, Germany) containingoverlapping peptides covering the complete sequence of the membrane glycoprotein (M;#130-126-702), the nucleocapsid phosphoprotein (N; # 130-126-699), or the immunodomi-nant sequence domains of the spike glycoprotein (S; #130-126-701); each used 1 µg/mL.Unstimulated wells served as negative controls, and polyclonal stimulation with Cy-toStim™ (0.5 µL/well, Miltenyi Biotec) was included as positive control. The fluorescentlyconjugated monoclonal antibody panel used for analysis is within Supplementary Table S2.Samples were run on a CytoFLEX LX (4 laser, Beckman Coulter, Brea, CA, USA). In thisactivation-induced marker assay, the antigen-specific T cells (AIM-positive) are defined byco-expression of CD25 and CD134 (OX40) for CD4+ T cells [15–17], and by co-expression ofViruses 2021, 13, 2239 5 of 16CD69 and CD137 (4-1BB) for CD8+ T-cells [18]. A positive assay result was defined as >3standard deviations above an unstimulated sample (negative control well), consisting of atleast 20 events.2.4. Quantification of Peripheral ImmunophenotypeCirculating neutrophils, monocytes, T, B, and NK cells were quantitated by multi-color flow cytometry, as previously described [13,19]. Direct application of monoclonalantibodies (specificities outlined in Supplementary Table S2) to 100 µL of whole blood wasperformed for 30 min at room temperature. Following staining, samples were incubatedwith either 1 × Fix/Lyse Buffer (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA)for 10 min, or following standard protocols for the FOXP3 Transcription Factor StainingKit (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA), washed with PBS, andresuspended in FACS Wash (5 mM EDTA, 0.5% BSA in PBS) for analysis with a CytoFLEXLX (4 laser, Beckman Coulter). Absolute counts for circulating immune populations weredetermined using CountBright™ absolute counting beads (Invitrogen Life Technologies,Carlsbad, CA, USA). Gating strategies and representative FACS dot plots to determinecirculating immune populations are shown in Supplementary Figure S1.2.5. Measurements of Cytokines and C-Reactive ProteinSerum cytokines interleukin-6 (IL-6), tumor necrosis factor (TNF), and C-reactiveprotein (CRP) were measured using the Ella™ Automated Immunoassay System (Bio-Techne, Minneapolis, MN, USA). Serum was diluted 1:2, as per the manufacturer’s protocol.The lower limits of quantification for CRP, IL-6, and TNF are 32 pg/mL, 0.28 pg/mL, and0.3 pg/mL, respectively.2.6. Statistical AnalysisData and statistical analyses were done in FlowJo™ (Version 10.7.1 Ashland, OR, USA:Becton, Dickinson and Company; 2021), GraphPad Prism version 9 (San Diego, CA, USA),and R open-source software. Multiple group comparisons were tested using Welch’s One-Way ANOVA and the Games–Howell post-hoc test. As data were non-normally distributed,correlations were tested using the Spearman correlation. Outliers were removed usingGrubbs’ test (α = 0.05).3. Results3.1. Demographics and Symptoms of Individuals Who Had SARS-CoV-2 and Other RespiratoryInfectionsDuring the early phase of the COVID-19 pandemic (February–June 2020), diagnosticPCR testing in Ontario was generally limited to individuals who were hospitalized, hada confirmed exposure with an infected person, or had travelled to an area with activeinfections. At the time, public health advice was that people who did not need medicalattention should self-isolate at home without being tested. Serologic studies have beenused to estimate an infection rate of 0.5–1.5% during the March-June ‘first wave’ [20].Compounding the lack of testing were the high rates of influenza and unusually highrates of respiratory syncytial virus (RSV) infections [21]. Consequently, some peoplewith respiratory infections in this early period were told by a health care professionalthat they had COVID-19 without a nasopharyngeal swab PCR test, and not all of ourstudy participants with symptoms of mild respiratory illness had COVID-19 (Table 1).Participants were classified as having had COVID-19 if they received a positive diagnosticPCR test and/or had anti-SARS-CoV-2 antibodies (Supplementary Table S1). There were nostatistically significant differences in age, sex, BMI, co-morbidities, medications, or durationof symptoms between individuals that had mild COVID-19 (n = 22) and individuals thathad other mild respiratory infections (n = 11, Table 1).Viruses 2021, 13, 2239 6 of 163.2. Survivors of Mild SARS-CoV-2 Infections Have Reduced T Cell Responses to SARS-CoV-2Peptides 6–9 Months Post Symptom OnsetLymphopenia is commonly reported in severe SARS-CoV-2 infections; however, itis not clear whether mild infections impact T-cell numbers or function. We used a flowcytometric activation-induced marker assay to measure T-cell responses to the SARS-CoV-2structural membrane protein (M), nucleocapsid protein (N), and immunodominant regionsof the spike (S) protein in all participants (Figure 1a). After 44 h incubation with antigen, thisassay identifies antigen-specific CD4+ T-cells by induced co-expression of CD25 and CD134(OX40), and antigen-specific CD8+ T-cells by co-expression of CD69 and CD137 (4-1BB)(Figure 1a) [15,16]. We identified five individuals who had mild respiratory symptoms, didnot have a positive diagnostic PCR test, and were seronegative for SARS-CoV-2 antibodies,but had detectable levels of SARS-CoV-2 reactive T-cells to some, but not all, of the SARS-CoV-2 M, N, and S peptides (Supplementary Table S1). Consequently, we classified thesefive individuals as “indeterminate”, and did not include them in subsequent analyses (theirdemographic data is summarized in Table 1).Viruses 2021, 13, x FOR PEER REVIEW 6 of 16 Consequently, we classified these five individuals as “indeterminate”, and did not include the in subsequent a alys s (their demographic data is summarized in Table 1). For analysis of the T-cell responses, we split our cohort into those without antib dy r T -cell responses to SARS-CoV-2 (other respiratory infection; n = 11), and those with ntibo y responses to SARS-CoV-2 (COVID-19; n = 22), the majority of whom also had detectable T-cell responses to SARS-CoV-2 M, N, and S antigens (90.9%; n = 20/22) (Figure 1; Supplementary Figure S1). Of the 22 participants who had COVID-19 infections, 14 pro-vided samples 1–3 months post-symptom onset, and 8 provided samples 6–9 months post-symptom onset. Consistent with other reports of lasting emory T-cell responses to SARS-CoV-2 infection [11], we observed that all COVID-19 participants that provided samples at 1–3 months or 6–9 months post-symptom onset had generated T-cell memory to at least one of the M, N, and S peptides pools (Figure 1b, Supplementary Table S3). Figure 1. CD4+ and CD8+ T-cell responses to the M, N, and S peptide pools after mild SARS-CoV-2 infection. (a) The number of SARS-CoV-2-specific T-cells is measured as a percent of CD4+ T-cells expressing both CD25 and OX40, or CD8+ T-cells expressing both CD69 and CD137, after activation with the S, M, or N peptide pools 1–3 months and 6–9 months after infection. The polyclonal acti-vator Cytostim is used as a positive control. (b) All COVID-19 seropositive participants had an in-crease in CD25+OX40+CD4+ T-cells in response to at least one of the M, N, or S antigens 1–3 months after mild COVID-19 infection, compared to seronegative individuals recovered from other mild respiratory infections. Each participant is indicated by a single data point: other respiratory infection n = 11; 1–3 months post COVID-19 infection n = 11; 6–9 months post COVID-19 infection n = 8. Multiple group comparisons were tested using Welch’s One-Way ANOVA and the Games–Howell post-hoc test; bars represent the mean ± standard deviation. * p < 0.05; ** p < 0.01; *** p < 0.001. We saw similar frequencies of CD4+ T-cell responses to all SARS-CoV-2 antigens, and these were highest in COVID-19 patients 1–3 months post symptom onset (means of ~3–4% of CD4+ T-cells), and were significantly higher than in individuals with other respira-tory infections (Figure 1b). Compared to COVID-19 patients 1–3 months post-symptom onset, responses were reduced in COVID-19 patients 6–9 months post-symptom onset, and were not significantly different to those in individuals recovered from other respira-tory infections. Similar results were found for CD8+ T-cell responses, being highest in COVID-19 patients 1–3 months post-symptom onset (means of ~0.4–0.5% of CD8+ T-cells), Fig re 1. 4+ an 8 -cell res onses to the , , an S e ti e ools after il S S- o -2infection. (a) The number of SARS-CoV-2-specific T-cells is measured as a percent of CD4+ T-cellsexpressing both CD25 and OX40, or CD8+ T-cells expressing both CD69 and CD137, after activationwith the S, M, or N peptide pools 1–3 months and 6–9 months after infection. The polyclonal activatorCytostim is used as a positive control. (b) All COVID-19 seropositive participants had an increase inCD25+OX40+CD4+ T-cells in response to at least one of the M, N, or S antigens 1–3 months after mildCOVID-19 infection, compared to seronegative individuals recovered from other mild respiratoryi fections. Each participant is indicated by a single data point: other respiratory infection n = 11;1–3 months post COVID-19 inf ction n = 11; 6–9 months post COVID-19 infection n = 8. Multiplegroup comparisons were tested using Welch’s One-Way ANOVA and the Games–Howell post-hoctest; bars represent the mean ± standard deviation. * p < 0.05; ** p < 0.01; *** p < 0.001.For analysis of the T-cell respons s, we split our cohort into those without antibodyor T -cell responses to SARS-CoV-2 (other respiratory infection; n = 11), and thos w thantibody responses to SARS- V-2 (COVID-19; n = 22), th majority of w om also had de-tectable T-cell r pons s to SARS-CoV-2 M, N, and S antigens (90.9%; n = 20/22) (Figure 1;Viruses 2021, 13, 2239 7 of 16Supplementary Figure S1). Of the 22 participants who had COVID-19 infections, 14 pro-vided samples 1–3 months post-symptom onset, and 8 provided samples 6–9 monthspost-symptom onset. Consistent with other reports of lasting memory T-cell responsesto SARS-CoV-2 infection [11], we observed that all COVID-19 participants that providedsamples at 1–3 months or 6–9 months post-symptom onset had generated T-cell memory toat least one of the M, N, and S peptides pools (Figure 1b, Supplementary Table S3).We saw similar frequencies of CD4+ T-cell responses to all SARS-CoV-2 antigens, andthese were highest in COVID-19 patients 1–3 months post symptom onset (means of ~3–4%of CD4+ T-cells), and were significantly higher than in individuals with other respiratoryinfections (Figure 1b). Compared to COVID-19 patients 1–3 months post-symptom onset,responses were reduced in COVID-19 patients 6–9 months post-symptom onset, and werenot significantly different to those in individuals recovered from other respiratory infections.Similar results were found for CD8+ T-cell responses, being highest in COVID-19 patients1–3 months post-symptom onset (means of ~0.4–0.5% of CD8+ T-cells), although therewere no significant differences between the groups. Together, these data indicate there arelong-term differences in T-cell functionality in COVID-19 survivors.3.3. Survivors of Mild SARS-CoV-2 Infections Have Evidence of Sustained Inflammation 1–3Months Post Symptom OnsetProlonged immune activation can occur after recovery from severe infections, and isthought to contribute to malaise and other symptoms in survivors [22]. One of the unusualfeatures of COVID-19 is that a significant number of patients with mild to moderate illnessreport symptoms weeks to months after infection, despite having cleared the virus [23].Although the participants in our study generally had symptoms resolve within 1 monthpost-infection (Table 1), we have nonetheless found evidence of prolonged inflammation.Seropositive individuals had higher levels of CRP, TNF, and IL-6 in circulation 1–3 monthspost-symptom onset; but in samples from individuals 6–9 months post-symptom onset,levels were equivalent to those individuals who were seronegative and were recoveredfrom other respiratory infections (Figure 2a–c).Viruses 2021, 13, x FOR PEER REVIEW 7 of 16 although there were no significant differences between the groups. Together, these data indicate there are long-term differences in T-cell functionality in COVID-19 survivors. 3.3. Survivors of Mild SARS-CoV-2 Infections Have Evidence of Sustained Inflammation 1–3 o ths ost y pto nset r l e i e acti atio can occ r after recovery fro severe infections, and is t ght to c tribute to alaise and other sy ptoms in survivors [22]. ne of t e sual feat res of I -19 is t at i ificant ber of atients ith ild to erate i lness re rt s t t t ft i f . lt t e partici ts i our study genera ly had sy pto s resolve ithin 1 onth ost-i fecti ( able 1), e a e et eless f i l fl eropositive individuals had higher levels of CRP, TNF, and IL-6 in circulation 1–3 months post-symptom onset; but in samples from individuals 6–9 m n hs pos -symptom onset, levels were equivalent to those individuals ho were seronegative and w re r cov-ered from other respiratory infections (Figure 2a–c). Figure 2. Transient increases in soluble mediators of inflammation occur after mild SARS-CoV-2 infection. (a) CRP levels in serum were higher 1–3 months after mild COVID-19 infection, and there was a trend towards remaining elevated 6–9 months after mild COVID-19 infection, compared to levels observed in seronegative individuals after other respiratory infections. Serum TNF levels (b) and IL-6 levels (c) were higher 1–3 months after COVID-19 infection, but returned to levels seen in individuals who had other respiratory infections by 6–9 months. Each participant is indicated by a single data point: other respiratory infection n = 11; 1–3 months post-COVID n = 9–12; 6–9 months post-COVID n = 8. Multiple group comparisons were tested using Welch’s One-Way ANOVA and the Games–Howell post-hoc test; bars are presented as mean ± standard deviation. * p < 0.05; ** p < 0.01. We observed that there were differences in the degree of epitope independent poly-clonal activation of T-cells between the COVID-19 and non-COVID-19 groups. When whole blood was stimulated with Cytostim, which acts as a superantigen by crosslinking the T-cell receptor (TCR) (regardless of TCR variable β gene usage) and major histocom-patibility complex (MHC), there was a statistically significant difference in the percent of CD25+OX40+CD4+ T-cells and CD69+CD137+CD8+ T-cells in COVID-19 patients 1–3 months post-symptom onset (Figure 3a,b). Whether this superantigen-like crosslinking activation contributes to, or is a result of, systemic inflammation is not clear, but CRP levels corre-lated with the percent of Cytostim-activated CD4+ T-cells (Figure 3c). CD4+ T-cells in the COVID-19 group also had higher expression levels of the activation markers OX40 and CD69, as well as the chemokine receptors CCR6 and CCR4, than the other respiratory infection group, implying a stronger activation (Figure 3d; Supplementary Figure S2). Alt-hough the levels of OX40 and CD69 were reduced in samples collected 6–9 months post-symptom onset compared to samples collected 1–3 months post-symptom onset, levels of CCR4 and CCR6 remained similar. Collectively, these data imply that there may be subtle Figure 2. Transient increases in soluble ediators of infla ation occur after ild SARS-CoV-2infection. (a) CRP levels in serum were higher 1–3 months after mild COVID-19 infection, and therewas a trend towards remaining elevated 6–9 months after mild COVID-19 infection, compared tolevels observed in seronegative individuals after other respiratory infections. Serum TNF levels(b) and IL-6 levels (c) were higher 1–3 months after COVID-19 infection, but returned to levels seenin individuals who had other respiratory infections by 6–9 months. Each participant is indicated by asingle data point: other respiratory infection n = 11; 1–3 months post-COVID = 9–12; 6–9 monthspost-COVID n = 8. Multiple group comparisons were te ted using Welch’s One-Way ANOVA and theGames–Howell post-hoc test; bars are presented as mean ± standard deviation. * p < 0.05; ** p < 0.01.e observed that there were differences in the degree of epitope independent poly-clonal activation of T-cells between the COVID-19 and non-COVID-19 groups. henwhole blood was stimulated with Cytostim, which acts as a superantigen by crosslinkingViruses 2021, 13, 2239 8 of 16the T-cell receptor (TCR) (regardless of TCR variable β gene usage) and major histo-compatibility complex (MHC), there was a statistically significant difference in the per-cent of CD25+OX40+CD4+ T-cells and CD69+CD137+CD8+ T-cells in COVID-19 patients1–3 months post-symptom onset (Figure 3a,b). Whether this superantigen-like crosslinkingactivation contributes to, or is a result of, systemic inflammation is not clear, but CRP levelscorrelated with the percent of Cytostim-activated CD4+ T-cells (Figure 3c). CD4+ T-cells inthe COVID-19 group also had higher expression levels of the activation markers OX40 andCD69, as well as the chemokine receptors CCR6 and CCR4, than the other respiratory infec-tion group, implying a stronger activation (Figure 3d; Supplementary Figure S2). Althoughthe levels of OX40 and CD69 were reduced in samples collected 6–9 months post-symptomonset compared to samples collected 1–3 months post-symptom onset, levels of CCR4 andCCR6 remained similar. Collectively, these data imply that there may be subtle changesin inducible T-cell activation in convalescent SARS-CoV-2 patients that do not occur inresponse to other respiratory infections.Viruses 2021, 13, x FOR PEER REVIEW 8 of 16 changes in inducible T-cell activation in convalescent SARS-CoV-2 patients that do not occur in response to other respiratory infections. Figure 3. Evidence of prolonged T-cell activation after mild SARS-CoV-2 infection. The polyclonal activator Cytostim was used to measure T-cell responses. COVID-19 seropositive individuals had a higher proportion of CD4+ (a) and CD8+ (b) activated T-cells 1 to 3 months after infection, compared to seronegative individuals after other respiratory infections. (c) The number of CD4+ T-cells that responded to polyclonal stimulation correlated with CRP in COVID-19 seropositive individuals. (d–g) Activation markers OX40, CCR6, CCR4, and CD69 on CD4+ T-cells were higher 1–3 months after COVID-19 infection, compared to after other respiratory infections, after Cytostim exposure. Each participant is indicated by a single data point: other respiratory infection n = 7–11; 1–3 months post COVID-19 infection n = 11–12; 6–9 months post COVID-19 infection n = 5–8. Multiple group com-parisons in (a,b) and (d–g) were tested using Welch’s One-Way ANOVA and the Games–Howell post-hoc test; bars are presented as mean ± standard deviation. Data in C was assessed by Spear-man’s rank correlation. * p < 0.05; ** p < 0.01. 3.4. Survivors of Mild SARS-CoV-2 Infections Have Changes in Circulating Immunophenotype 1–3 Months Post-Symptom Onset The total numbers of circulating CD45+ cells, CD4+ T-cells and CD8+ T-cells were not different between individuals who had other respiratory infections and those who had COVID-19 at either 1–3 months or 6–9 months post-symptom onset (Figure 4a–c). Com-pared to participants who were recovered from other respiratory infections, there was an expansion of regulatory T-cells (Tregs, defined as CD45+CD3+CD4+CD25+CD127lowFOXP3+) and a decrease in natural killer (NK) cells (CD45+CD56+/NKp46+) in COVID-19 patients at 1–3 months post-symptom onset that returned by 6–9 months post-symptom onset to lev-els observed in individuals with non-COVID-19 respiratory infections (Figure 4d,g). We also measured the proportions of circulating CD4+ and CD8+ T-cells that were naïve cells (CD45RA+CCR7+), central memory cells (CD45RA−CCR7+), effector memory cells (CD45RA−CCR7−), terminally differentiated effector memory cells re-expressing CD45RA (TEMRA; CD45RA+CCR7−), and terminally differentiated cells (CD45RA+CCR7−CD57+CD28−). Compared to patients with non-COVID-19 respiratory Figure 3. Evidence of prolonged T-cell activation after ild SARS-CoV-2 infection. The polyclonalactivator Cytostim was used to measure T-cell responses. COVID-19 seropositive individuals had ahigher proportion of CD4+ (a) and CD8+ (b) activated T-cells 1 to 3 months after infection, comparedto seronegative individuals after other respiratory infections. (c) The number of CD4+ T-cells thatresponded to polyclonal stimulation correlated with CRP in COVID-19 seropositive individuals.(d–g) Activation markers OX40, CCR6, CCR4, and CD69 on CD4+ T-c lls were higher 1–3 mont safter COVID-19 infection, compared to after other res i s, after Cytostim exposure.Each participant is indicated by a single data point: other respiratory infection n = 7–11; 1–3 monthspost COVID-19 infection n = 11–12; 6–9 months post COVID-19 infection n = 5–8. Multiple groupcomparisons in (a,b) and (d–g) were tested using Welch’s One-Way ANOVA and the Games–Howellpost-hoc test; bars are presented as mean± standard deviation. Data in C was assessed by Spearman’srank correlation. * p < 0.05; ** p < 0.01.Viruses 2021, 13, 2239 9 of 163.4. Survivors of Mild SARS-CoV-2 Infections Have Changes in Circulating Immunophenotype1–3 Months Post-Symptom OnsetThe total numbers of circulating CD45+ cells, CD4+ T-cells and CD8+ T-cells were notdifferent between individuals who had other respiratory infections and those who hadCOVID-19 at either 1–3 months or 6–9 months post-symptom onset (Figure 4a–c). Com-pared to participants who were recovered from other respiratory infections, there was an ex-pansion of regulatory T-cells (Tregs, defined as CD45+CD3+CD4+CD25+CD127lowFOXP3+)and a decrease in natural killer (NK) cells (CD45+CD56+/NKp46+) in COVID-19 patientsat 1–3 months post-symptom onset that returned by 6–9 months post-symptom onset tolevels observed in individuals with non-COVID-19 respiratory infections (Figure 4d,g).We also measured the proportions of circulating CD4+ and CD8+ T-cells that were naïvecells (CD45RA+CCR7+), central memory cells (CD45RA−CCR7+), effector memory cells(CD45RA−CCR7−), terminally differentiated effector memory cells re-expressing CD45RA(TEMRA; CD45RA+CCR7−), and terminally differentiated cells (CD45RA+CCR7−CD57+CD28−).Compared to patients with non-COVID-19 respiratory infections, there was an expansionof central memory CD4+ T-cells and terminally differentiated CD8+ T-cells in COVID-19patients at 1–3 months post-symptom onset that returned to levels observed in patientswith other respiratory infections by 6–9 months post-symptom onset (Figure 4e,f). Therewere no significant differences between participants who had other respiratory infectionsand those who had COVID-19 for any other measured CD4+ and CD8+ T-cell subsets.Although six months after SARS-CoV-2 infection there was a statistical trend towardsa lower ratio of myeloid to lymphoid cells, this was due to a non-significant decreasein neutrophil numbers rather than changes in the number of monocytes (Figure 5a–c).However, as circulating monocytes are a sensitive marker of chronic inflammation, weassessed monocyte subsets, as well as their expression of migratory and activation mark-ers (Figure 5d, Supplementary Tables S4 and S5). Classical monocytes (CD14+CD16−)expressing CCR2 are the first to leave the bone marrow. They have a half-life in the cir-culation of less than 24 h, since they are either recruited to sites of acute inflammationin response to CCL2/monocyte chemoattractant protein-1 (MCP-1), or differentiate intoCX3CR1-expressing intermediate (CD14+CD16+) monocytes [24,25]. Classical monocytesincrease in the circulation during acute infection so, as expected, there was no difference inthe number of circulating monocytes after COVID-19 infection had resolved (Figure 5c). At1–3 months after infection, levels of CCR2 were lower on intermediate and non-classicalmonocyte populations (Figure 5d), implying that cells with the highest levels of CCR2emigrated from the circulation.In general, the chemokine receptor CX3CR1 is expressed more highly on intermediateand non-classical (CD14lowCD16+) monocytes, and is associated with recruitment to thetissues or vasculature and repair of damage in response to CX3CL1/fractalkine [26,27].A transient decrease in CX3CR1-expressing classical monocytes was found 1–3 monthspost COVID-19 infection, which likely indicates that monocytes with the highest levelsof CX3CR1 (and therefore the most responsive to CX3CL1) had emigrated from the cir-culation. Expression of the integrin CD11b was also transiently increased on monocytesat 1–3 months post-symptom onset, but not neutrophils, providing further evidence oftransient but systemic immune activation, and possible changes in the migratory capacityof monocytes after mild SARS-CoV-2 infection.Changes in monocyte activation markers could be due to the increase in basal inflam-mation, or could be proportionate to the SARS-CoV-2 specific immune response. We foundthat decreasing CX3CR1 expression was most strongly associated with the percentage ofS-antigen specific CD4+ T-cells (p = 0.0389), and that there was a strong association betweenM-antigen specific CD8+ T-cells and human leukocyte antigen-DR isotype (HLA-DR) ex-pression on non-classical/patrolling monocytes (p = 0.0198) (Figure 5e). There was norelationship between polyclonal T-cell activation (i.e., Cytostim) and myeloid activation,implying that these changes are not due to the general increase in T-cell activation observedin convalescent patients, but rather SARS-CoV-2 specific responses.Viruses 2021, 13, 2239 10 of 16Viruses 2021, 13, x FOR PEER REVIEW 9 of 16 infections, there was an expansion of central memory CD4+ T-cells and terminally differ-entiated CD8+ T-cells in COVID-19 patients at 1–3 months post-symptom onset that re-turned to levels observed in patients with other respiratory infections by 6–9 months post-symptom onset (Figure 4e,f). There were no significant differences between participants who had other respiratory infections and those who had COVID-19 for any other meas-ured CD4+ and CD8+ T-cell subsets. Figure 4. Transient changes in circulating lymphocytes occur 1–3 months after COVID-19 infection. Absolute numbers of circulating CD45+ T-cells (a), CD4+ T-cells (b), and CD8+ T-cells (c) were not different after 1–3 months or 6–9 months post-symptom presentation in seropositive individuals recovered from mild COVID-19 infection, compared to seronegative individuals recovered from other respiratory infections; however, NK cell numbers (d) were lower in the 1–3 months post-COVID-19 infection group. At 1–3 months post recovery from COVID-19 there was an increase in Figure 4. Transient changes in circulating lymphocytes occur 1–3 months after COVID-19 infection.Absolute numbers of circulating CD45+ T-cells (a), CD4+ T-cells (b), and CD8+ T-cells (c) were notdiff nt after 1–3 months or 6–9 months post-symptom prese tat on in s ropositive individualsrecovered from mild COVID-19 infection, compared to seronegative individuals recovered fromother respiratory infections; however, NK cell numbers (d) were lower in the 1–3 months post-COVID-19 infection group. At 1–3 months post recovery from COVID-19 there was an increase inCD45RA−CCR7+ central memory CD4+ T-cells (e), and an increase in CD45RA+CCR7−CD57+CD28−terminally differentiated CD8+ T-cells (f), compared to individuals recovered from other respiratoryinfections, but these differences were not apparent in individuals who had recovered from COVID-196–9 months prior. (g) Levels of circulating regulatory T-cells (measured as a % of CD4+ T-cells) werehigher in individuals 1–3 months post COVID-19 infection. Each participant is indicated by a singledata point: other respiratory infection n = 11; 1–3 months post COVID-19 infection n = 11–13; 6–9months post COVID-19 infection n = 8. ns—not statistically significant. Multiple group comparisonswere tested using Welch’s One-Way ANOVA and the Games–Howell post-hoc test; bars are presentedas mean ± standard deviation. * p < 0.05.Viruses 2021, 13, 2239 11 of 16Viruses 2021, 13, x FOR PEER REVIEW 11 of 16 Figure 5. Evidence of sustained cellular inflammation after mild SARS-CoV-2 infection. (a) Therewas a trend towards a decreasing ratio of myeloid to lymphoid cells after SARS-CoV-2 infection,Viruses 2021, 13, 2239 12 of 16compared to individuals recovered from other infections, which was driven by a decrease in circu-lating neutrophils (b). Although total monocyte numbers did not change after infection (c), surfaceexpression of the migratory markers CX3CR1 and CCR2 decreased transiently (d). Concurrentincreases in surface expression of the migration and activation marker CD11b implies that the mono-cytes were activated. (e) Correlation analysis (Spearman’s correlation) of SARS-CoV-2 specific CD4+and CD8+ T-cell responses with measures of monocyte activation and migratory potential. Multiplegroup comparisons in (a–d) were tested using Welch’s One-Way ANOVA and the Games–Howellpost-hoc test; in (a–c) bars are presented as mean ± standard deviation, and each dot indicates aparticipant. The spread of expression of monocyte surface markers in d was visualized by concate-nating uncompensated CD45+CD19−CD3−CD56−CD11b+HLADR+CD14+ events in FlowJo for eachinfection group prior to overlaying geometric mean fluorescence intensity expression data from allparticipants onto the same histogram plot. Other respiratory infection (grey) n = 11, 1–3 monthsafter COVID-19 infection (red) n = 14, 6–9 months from COVID-19 infection (pink) n = 8. ns—notstatistically significant. Data in (e) were assessed with the rcorr function in the Hmisc package in R,and only statistically significant associations are shown. * p < 0.05.4. DiscussionInfections severe enough to require hospitalization can have long-term health conse-quences, but one of the unusual features of COVID-19 is that a significant proportion of mildinfections have lasting cardiovascular, respiratory, and neurologic consequences [23,28].The range of long-term health consequences and the multi-organ immune pathologyobserved post-SARS-CoV-2 infection implies that there may be a greater degree of im-mune dysregulation than that commonly observed after infection with other respiratorypathogens. Importantly, the participants in this study did not have “long-COVID” in thatthey reported that their symptoms were mild, and had mostly resolved within 2–4 weeks(Table 1); however, participants with even mild COVID-19 symptoms experienced elevatedinflammation and immune activation that endured at least 1–3 months after their infectionhad resolved. The fact that these participants had increased measures of systemic inflam-mation (e.g., CRP), and lasting phenotypic and functional changes to both monocytesand T-cells, implies that inflammatory responses to mild COVID-19 infections are moreprotracted than expected.The proportions of memory CD4+ and CD8+ T-cells specific for SARS-CoV-2 S, M,and N proteins we observed were consistent with those reported for convalescent patientswho had had more severe infections, implying that disease severity is not proportionate toSARS-CoV-2 memory T-cell responses [29,30]. We also detected SARS-CoV-2 reactive T-cells for n = 5 individuals who had mild respiratory symptoms that did not have a positivediagnostic PCR test, and were seronegative for anti-SARS-CoV-2 antibodies. Although it ispossible these individuals had been infected with SARS-CoV-2 and had antibodies at somepoint, these data are also consistent with reports of pre-existing cross-reactive T-cells toSARS-CoV-2 antigens in as many as 30% of individuals [29]. Previous studies have reporteddifferences in the expression of activation markers on SARS-CoV-2 specific memory CD4+T-cells between exposed but SARS-CoV-2 seronegative individuals, compared to those whohad either mild or severe infections [11]; however, to our knowledge, this is the first reportof elevated activation of T-cells by MHC/TCR crosslinking (i.e., Cytostim) in convalescentCOVID-19 patients. Cytostim-treated CD4+ T-cells in participants with mild COVID-19 1–3 months post-symptom onset, compared to those in participants recovered fromother respiratory infections, had higher expression of the activation markers OX40, CCR4,CD69, and CCR6, and OX40 and CD69 expression remained elevated after 6–9 monthspost-symptom onset. There were some differences between participant groups in theexpression of activation markers on circulating unstimulated cells, demonstrating thatthese differences are not restricted to polyclonally activated cells. There are several possibleexplanations for this result. CD4+ T-cells from 1–3 months post-symptom onset of COVID-19 may be primed to respond quicker and more strongly to activation signals. Alternatively,since the number of activated CD4+ T-cells in this participant group correlated stronglyViruses 2021, 13, 2239 13 of 16with rising CRP levels, it is also possible that there may be circulating cytokines (e.g., IL-6)or other factors that lower the threshold for T-cell activation.Our observations of transient increases in central memory CD4+ T-cells and termi-nally differentiated CD8+ T-cells are consistent with T-cell responses to acute viral infec-tion [31,32], and with previous observations that survivors of mild SARS-CoV-2 infectionshave lasting memory responses [11]; however, a pronounced increase in regulatory T-cellsis generally associated with minimizing pathology during the acute, not the convalescent,phase of respiratory viral infections [31,32]. Severe SARS-CoV-2 infections are associatedwith the development of autoantibodies that contribute to severity [33]. Mild SARS-CoV-2infections are associated with autoimmune inflammatory syndromes (e.g., arthritis, vas-culitis) after the primary infection has resolved [34,35]. Whether this expansion of Tregsprotects from autoimmune sequelae is not clear, but this has been reported to occur afterother zoonotic infections [36]. Appropriate regulation of Tregs may be especially impor-tant in SARS-CoV-2 infections, since pathology of these infections are caused in part bydysregulation of transforming growth factor-β production, a major cytokine produced byTregs [37] that is required for their appropriate differentiation [38].Severe COVID-19 infections are associated with a dysregulation of myelopoiesis thatis so extreme that monocytes and neutrophils are unrecognizable by blood smear [39],and have dramatic changes in granularity, size, and surface marker expression [40]. Bothmild and severe COVID-19 are associated with changes in the number and migratorypotential of dendritic cells for at least 7 months post-infection [41]. We do not knowif the transient changes in expression of CCR2 and CX3CR1 on circulating monocytes1–3 months after infection are due to emigration of cells with the highest expression ofthose markers to inflamed or damaged tissues; however, CCL2 has been implicated inrecruitment of monocyte to the lungs during infection [42]. The observation that decreasingCX3CR1 is associated with the level of SARS-CoV-2-specific T-cells may imply that thereis a relationship between immune responsiveness and innate immune activation. Therewas no relationship between polyclonal T-cell activation (i.e., Cytostim) and myeloidactivation, implying that this relationship is not due to the general increase in T-cellactivation observed in convalescent patients, but rather SARS-CoV-2-specific responses.Furthermore, we observed prolonged elevation of cytokines, perhaps a lasting effect ofthe cytokine storm present in many COVID-19 patients, even in mild infections [43,44].These observations in combination with the elevated expression of monocyte CD11b,which increases during acute and chronic inflammation and alters monocyte migration andadherence to the vasculature [45], imply even though the symptoms of mild COVID-19infection may resolve in weeks, immune activation persists for at least 1–3 months.Our study has identified key differences between immune responses following SARS-CoV-2 infection and other respiratory infections, although a limitation was that we wereunable to determine the type(s) of non-COVID-19 respiratory infections. All participants inthe study were ill before SARS-CoV-2 vaccines were available, and were unvaccinated atthe time of sample collection. Although we identify some intriguing differences at distincttime points post-infection, this was not a longitudinal study, so different individuals werecompared in the 1–3-month and 6–9-month post-infection groups. It will be interestingto compare our data on immune responses generated by primary infection to futurestudies examining post-infection immune activation in vaccinated individuals with mildbreakthrough infections.Collectively, these data provide evidence that mild, and, in some individuals, evenasymptomatic SARS-CoV-2 infections can lead to sustained immune activation after resolu-tion of symptoms, which is not observed in response to other mild respiratory infections.Whether this immune activation is more pronounced in patients with long-COVID, or moreserious infections, remains to be seen.Viruses 2021, 13, 2239 14 of 16Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3390/v13112239/s1, Figure S1: Gating strategies for flow cytometry data analysis, Figure S2:Expression of activation markers on unstimulated T cells, Table S1: Criteria and identification ofindividuals with SARS-CoV-2 and other respiratory infections, Table S2: Fluorophore-conjugatedantibodies used for flow cytometry, Table S3: Summary of SARS-CoV-2 PepTivator AIM assay T-cellactivation observations, Table S4: All immune parameter data analysis, Table S5: Monocyte surfacereceptor expression as mean fluorescence intensity (MFI).Author Contributions: Conceptualization, D.M.E.B., L.C., M.K.L. and M.M.; methodology, A.E.K.,L.C., A.H., J.W.S., H.S. and J.A.; formal analysis, A.E.K., L.C. and J.A.B.; investigation, A.E.K.;resources, A.M., B.L.C., M.L. (Maggie Larché), M.L. (Mark Larché), N.H., P.N., K.A., M.S.M., J.B.,M.K.L., S.S. and M.M.; data curation, J.G.W., B.C., A.H., J.W.S., K.S., H.S. and J.A.; writing—originaldraft preparation, A.E.K.; writing—review and editing, L.C., J.A.B., M.S.M., J.B., M.K.L., S.S., M.M.and D.M.E.B.; supervision, D.M.E.B.; project administration, J.G.W.; funding acquisition, M.K.L., I.N.and D.M.E.B. All authors have read and agreed to the published version of the manuscript.Funding: This work was supported by a grant from the W. Garfield Weston Family Foundationto D.M.E.B., the Canadian Institutes of Health Research (FDN-154304) to M.K.L., and OntarioResearch Fund (ORF) COVID-19 Rapid Research Fund (#C-191-2426729-NAZY) to IN. D.M.E.B. isthe Canada Research Chair in Aging & Immunity. A.E.K. was supported by a Thode Fellowship. L.C.was supported by a BC Children’s Hospital Research Institute Bertram Hoffmeister PostdoctoralFellowship. H.S. is supported by the CSV United Supermarket Studentship in SARS-CoV-2 Researchand an Ontario Graduate Scholarship. M.M. is supported by the Canadian Institutes of HealthResearch/Canadian Allergy Asthma Immunology Foundation/Emerging Researcher Award. P.N. issupported by the Frederick E. Hargreave Teva Innovation Chair in Airway Diseases. M.K.L. wassupported by a BCCHR salary award and is the Canada Research Chair in Engineered ImmuneTolerance.Institutional Review Board Statement: The study was conducted according to the guidelines ofthe Declaration of Helsinki and approved by the Hamilton Integrated Research Ethics Board ofMCMASTER UNIVERSITY (#10757 and #11471).Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.Data Availability Statement: Data are available from the corresponding author upon reasonable request.Acknowledgments: The authors would like to thank Brian Dixon, Marc Aucoin, Mark Bruder, andAaron Frenette (University of Waterloo) for the recombinant antigens used in the serology assays,and Carmen Venegas for assistance with participant recruitment and data collection.Conflicts of Interest: The authors declare no conflict of interest. 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immunophenotype
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Lasting Changes to Circulating Leukocytes in People with Mild SARS-CoV-2 Infections
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