key: cord-0984028-j008qh26
authors: Chu, C.-F.; Sabath, F.; Sun, S.; Chao, Y.-Y.; Zielinski, C. E.
title: In-depth phenotyping of human peripheral blood mononuclear cells in convalescent COVID-19 patients following a mild versus severe disease course
date: 2020-05-25
journal: nan
DOI: 10.1101/2020.05.25.20112763
sha: 8307793d5f7e91570c97776e8b13b6e35ca61b1c
doc_id: 984028
cord_uid: j008qh26

Background: Covid-19, the disease caused by infection with SARS-CoV-2, has developed to a pandemic causing more than 239, 000 deaths worldwide as of 6th May according to the World Health Organization (WHO). It presents with a highly variable disease course ranging from a large proportion of asymptomatic cases to severe respiratory failure in 17-29% of cases even in the absence of apparent comorbidities 1, 2. This implies a diverse host immune response to SARS-CoV-2. The immunological characteristics underlying these divergent disease courses, however, still remain elusive. While insights into abrogations of innate immunity begin to emerge, adaptive immune responses towards SARS-CoV-2 are poorly investigated, although they serve as immune signatures of protection and vaccine responses. We therefore set out to characterize immune signatures of convalescent COVID-19 patients stratified according to their disease severity. Methods: We performed high-dimensional flow cytometric profiling of peripheral blood mononuclear cells of convalescent COVID-19 patients who we stratified according to their disease severity by a physician-assisted questionnaire based assessment of COVID-19 symptoms. Results: Surprisingly, we did not observe any difference in the relative proportions of any major immune cell type in convalescent patients presenting with different severity of COVID-19 disease except for a reduction in monocytes. The frequency of Tnaive T cells was significantly reduced in CD4+ and CD8+ T cells, whereas other T cell differentiations states (TCM, TEM, TEMRA) remained relatively unaffected by COVID-19 severity as assessed approximately two weeks after infection. Conclusions In our COVID-19 patient cohort, which is characterized by absence of comorbidities and therapeutic interventions other than symptomatic antipyretics, the immunophenotype is similar irrespective of a highly variable disease severity. Convalescence is therefore associated with a rather uniform immune signature. Abrogations, which were previously identified in the innate and adaptive immune compartment of COVID-19 patients should be scrutinized for direct associations with a preconditioned immune system shaped and made vulnerable for SARS-CoV-2 by preexisting comorbidities.

The pandemic caused by SARS-CoV-2 is a global health emergency. The World Health Organization (WHO) declared the outbreak with this novel virus a pandemic. As of now (23 rd May 2020), around 5 million people are affected by this virus, with an estimated 3-4 % casefatality rate with large regional variations reaching up to 10% (WHO status report) 3 . Countries all over the world are currently at unprecedented speed installing regulatory measures to prevent human-to-human viral spread. SARS-CoV-2 causes the disease COVID-19, which is characterized by severe respiratory symptoms. Most patients infected with SARS-CoV-2 present with mild to moderate symptoms such as fever, fatigue, cough and anosmia. A small number of patients (17%), however, progress to severe disease courses characterized by acute respiratory distress syndrome that might ultimately lead to multiple organ failure and death 1 .

Comorbidities, such as cardiovascular disease, diabetes, hypertension, old age and immunosuppressive therapies have been shown to predispose to severe COVID-19 by epidemiological investigations in a large proportion of patients 1, 4 . But variable severity in the disease course has also been reported even in young and healthy patients [5] [6] [7] [8] [9] . Asymptomatic infected patients pose a great threat to our society since their activity radius increases viral . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 25, 2020. . https://doi.org/10.1101/2020.05. 25.20112763 doi: medRxiv preprint spread. It remains enigmatic what factors predispose to mild versus severe disease in the absence of apparent comorbidities.

The pathogenesis of COVID-19 is strongly associated with a profound impact of host immune responses to the virus. Severe respiratory distress syndrome is associated with a cytokine storm 10, 11 . Proinflammatory cytokines such as IL-2, TNF-a, GM-CSF and IL-6, IL-1β, IL-8, G-CSF and others have been detected in the plasma of patients with severe illness 2, 12 . Cytokine release is less pronounced in settings of mild disease courses indicating a correlation with clinical features rather than infection with the virus itself 13 . The innate immune compartment is rapidly activated resulting in cytokine release and tissue damage. This has initiated clinical trials with specific innate cytokine blocking drugs, such as tocilizumab (IL-6R

blocking antibody). They demonstrated improvement in respiratory and laboratory parameters 14, 15 . ACE2, the entry receptor of SARS-CoV-2, has been reported to be expressed in several innate immune cell types such as monocytes [16] [17] [18] . Despite the cross-talk of innate and adaptive immunity, little is known about T cell abrogations in COVID-19 until now. In particular, their differential impact on mild versus severe disease remains elusive.

T cells participate in viral clearance through antigen specific cytotoxicity and B cell help 19, 20 . Their polyfunctional cytokine release contributes to an overall immune activation with downstream inflammatory actions on immune and stromal cells 21, 22 . An individual's T cell signature could either predispose or protect from infection or severe COVID-19 illness. The T cell post-activation fingerprint represents an immune signature of protection and is expected to correspond to future vaccination success. Dissecting human T cell responses to SARS-CoV-2 will therefore yield predictive markers for the disease course as well as information on the effect of the viral infection on adaptive immunity. Recently, it has been shown that lymphopenia in both the CD8 and CD4 T cell compartment correlates with severe disease 13, 23, 24 . T cells displayed increased activation as judged by HLA-DR upregulation 13, 25 . But also the upregulation of inhibitory receptors such as NKG2A, PD-1 and TIM-3 has been reported, . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted May 25, 2020. . https://doi.org/10.1101/2020.05.25.20112763 doi: medRxiv preprint implying T cell exhaustion in response to SARS-Cov2 infection in settings of severe illness [25] [26] [27] . Cytokine expression in T cells from COVID-19 patients showed variable results. The percentage of IFN-g producing CD4 + T cells was increased in severely affected patients, in line with the previously reported contribution of IFN-g to the cytokine storm in SARS patients 28 . Others reported IFN-g to be unaltered or even decreased as part of reduced functional T cell diversity 26, [29] [30] [31] . Other T cell related cytokines showed increased expression levels upon analysis in the serum 27, 32 . These results imply that productive T cell effector function might precede a state of T cell exhaustion.

In this study we set out to identify immune signatures of mild versus severe COVID- 19 . We therefore implemented a high-dimensional analytical approach to characterize the composition of peripheral blood mononuclear cells with a focus on T cell differentiation states in convalescent COVID-19 patients stratified according to the severity of their disease course. Importantly, our patient cohort excludes study participants with comorbidities, who might have skewed previous studies towards a more severe disease course and to immune signatures characteristic of the underlying comorbidity and baseline medication.

Subjects were recruited after announcement of the study via social media (Facebook site of Klinikum r.d. Isar). More than 7000 shares were recorded by the end of the patient recruitment. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 25, 2020. . https://doi.org/10.1101/2020.05.25.20112763 doi: medRxiv preprint

We established a clinical COVID-19 score including 15 symptoms this disease has been reported to present with. Quantitative scoring of each symptom (range 0-3) allowed to achieve a final COVID-19 severity score with a mean of 14,5 in our cohort (range: 0-45) (Figure 1c ).

More than 50% of all patients presented with unspecific symptoms such as headache, cough, myalgia, fever and a sore throat. But also more than 50% of all patients presented with chemosensory alterations such as ageusia and anosmia, which have been proposed to be more characteristic of SARS-CoV-2 infection and to even occur in otherwise asymptomatic infections (Figure 1d ) 33 . In our patient cohort the chemosensory loss of taste and smell was consistently associated with other symptoms and always occurred very early in COVID-19 (not shown). Increased age has previously been reported to represent an independent risk factor for severe COVID-19 disease course and death 6 . Accordingly, we observed slightly higher disease scores in correlation with higher age in men. Interestingly, this age-severity correlation was not significant for women (Figure 1e ). Although men have occasionally been reported to develop more severe COVID-19, severity between men and women was equally distributed in our cohort ( Figure 1f ).

In sum, our patient cohort covered a wide range of COVID-19 severity in the absence of comorbidities and immunosuppression, thus excluding critical predictive markers and immunological abrogations that have previously been correlated with a severe disease course.

There is substantial evidence that a dysregulated host immune response plays a critical role in disease severity in several viral infections including SARS-CoV-2. Comorbidities and associated therapies might predispose an individual's immune system to a more severe disease course and even death as suggested by multiple previous studies 34 . However, how SARS-CoV-2 infection translates into a mild versus severe disease course in healthy individuals has remained elusive. The host's response to infection is mirrored by its immune signatures. We . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 25, 2020. . https://doi.org/10.1101/2020.05.25.20112763 doi: medRxiv preprint therefore set out to characterize the cellular immune cell composition in our convalescent COVID-19 patient cohort and to correlate the immunophenotype with disease severity.

We first quantified the relative proportions of major immune cell types by multi-color flow cytometry of fresh PBMC and correlated them to the respective COVID-19 disease scores of each individual patient (Figure 2) . Surprisingly, we did not observe any change in the frequency of CD3 + T cells within the lymphocyte population over a wide range of disease severities. This also applied to the frequency of CD4 + and CD8 + ab T cells subsets, Treg cells as well as gd-T cells within the CD3 + T cell population. Relative CD19 + B cell and CD56 + NK cell frequencies within the lymphocyte population were also unaffected by disease severity.

The proportion of myeloid cells within the PBMC did, however, decline in correlation with increased COVID-19 (r=0.29, p=0.03). This was also the case for the fraction of CD14 + monocytes within the myeloid cell population (r=0.28, p=0.03). We then excluded patients aged 60 and older to test if absence of this independent risk factor, as we showed in the case of men, would explain the negative correlation with myeloid cells or even unravel any new correlation with immune cell frequencies. In fact, the same results could be recapitulated with a patient cohort spanning the age of 19 to 59 (mean 39 years) (data not shown). In sum, this demonstrates absence of any major perturbations in the relative frequencies of the main immune cell types, in particular adaptive immune cells, over a wide range of COVID-19 severity in patients without relevant comorbidities. Only myeloid cells, and in particular CD14 + monocytes, correlated negatively with COVID-19 disease severity.

Memory T cells represent the repository of an individual´s antigen experience 21 . A gradual loss of antigen inexperienced naïve T cells over a lifetime in accordance with a history of antigenic challenges is associated with increased frequencies of memory and effector T cells 35 . The memory T cell pool contains distinct populations of central and effector memory T cells . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 25, 2020. . https://doi.org/10.1101/2020.05.25.20112763 doi: medRxiv preprint that differ in their homing capacities, functions and history of antigen encounters 20, 36 . Evidence of T cell activation in both CD4 + and CD8 + T cells has recently been reported in COVID-19 patients 13, 37 . We therefore decided to investigate the distribution of distinct T cell differentiation states in the CD4 + and CD8 + T cell compartment in patients following SARS-CoV-2 infection. Naïve (TNaive), central memory (TCM), effector memory (TEM) and terminally differentiated effector memory cells expressing CD45RA (TEMRA) were identified based on the differential expression of CD45RA and CCR7 within CD4 + and CD8 + CD3 + T cells (CD19 -, CD56 -, gd TCR -) as described before 36 . Interestingly, we observed that reduced frequencies of TNaive cells correlated with more severe COVID-19 disease scores in convalescent patients in both the CD4 + and CD8 + T cell compartments (Figure 3a,b) . In CD8 + but not CD4 + T cells, this was associated with a concomitant increase in TEM cells. TCM and TEMRA cell frequencies did not differ upon correlation with disease scores. Taken together, the distribution of distinct T cell differentiation states in early convalescent COVID-19 patients was overall similar across disease severity scores, with significant changes mainly observable within TNaive cells.

The SARS-CoV-2 pandemic has stirred research efforts with tremendous speed and dedication all over the world. Several studies, which investigated innate and adaptive immunity, have also been initiated 38, 39 . Comparisons are complicated by variable patient cohorts, which differ in age, sex, time after infection, disease course, comorbidities and medical treatment. We have therefore set out to provide a patient cohort that excludes previously identified risk factors for a severe COVID-19 disease course. Importantly, patients were all investigated at similar timepoints defined by the end of quarantine, which was legally imposed by the German government for two weeks after RT-PCR confirmed diagnosis of SARS-CoV-2 infection.

Despite a wide range of disease severity, our patient cohort presented with overall very similar convalescent immunophenotypes that did not correlate with disease scores. The . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 25, 2020. . https://doi.org/10.1101/2020.05.25.20112763 doi: medRxiv preprint immunophenotypes were reproducible in our COVID-19 cohort after exclusion of patients older than 60 years of age (mean age 39 years) and absence of relevant comorbidities, medication and immunosuppression. Although we did not perform a direct comparison to healthy PBMC samples, the immunophenotypes, which we obtained, were within the physiological ranges reported before, thus excluding any major immune perturbations following SARS-CoV-2 infection. Together, this indicates stability of the immune composition in early convalescent patients over a wide range of disease severities.

Significant alterations were, however, reported within the myeloid cell comportment, which demonstrated reduced cells and, in particular, reduced frequencies of CD14 + monocytes in correlation with increased disease severity. This could be due to activation triggered loss of these short lived innate immune cells and would be consistent with the cytokine storm that is mainly composed of innate cytokines such as IL-6 39, 40 . Alternatively, monocytes could still be sequestered in peripheral organs, such as the lung, at this early timepoint after clinical recovery in severely affected individuals.

T cells specific for SARS-CoV2 have recently been detected within TCM, TEM and TEMRA cells, but their characteristics and role in COVID-19 pathogenesis remained undefined 41 .

severity. This could be due to antigen specific stimulation with SARS-CoV2 antigens and recruitment into the memory T cell pool. This would be in line with the concomitant increase in CD8 + TEM cells. Antigen specific T cell assays and T cell receptor clonality analyses will further educate about the dynamics of SARS-CoV-2 immunological memory formation, which is relevant for the design of future vaccines. Longitudinal investigations within the same patient cohort will be critical to assess the stability of the immune response to SARS-CoV2 infection in correlation with disease severity.

Taken together, this study has highlighted a wide distribution of disease severities in a well characterized and standardized COVID-19 patient cohort despite absence of any relevant . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 25, 2020. . https://doi.org/10.1101/2020.05.25.20112763 doi: medRxiv preprint previously reported risk factors, in particular old age, comorbidities and therapeutic immunosuppression. Despite the wide spread of disease severity, the immune composition in convalescent patients was fairly stable across disease severity scores except for reduced myeloid cells and naïve T cell frequencies.

Patients were randomly selected based on positive SARS-CoV-2-RNA test results by RT-PCR from throat swab samples as well as absence of co-morbidities or pregnancy as assessed by a physician (lung diseases, cardiovascular diseases, cancer, autoimmune diseases, therapeutic . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

Error bars indicate the standard error of the mean (SEM); p values of 0.05 or less were considered significant. Analyses were performed using GraphPad Prism 8. 

Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study

Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China

Understanding the asymmetric spread and case fatality rate (CFR) for COVID-19 among countries

Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan, China

Clinical features of COVID-19 in elderly patients: A comparison with young and middle-aged patients

Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

Detection of Covid-19 in Children in Early

Epidemiological Characteristics of 2143 Pediatric Patients With 2019 Coronavirus Disease in China

Epidemiology and clinical features of coronavirus disease 2019 in children

Acute Respiratory Distress Syndrome

COVID-19: consider cytokine storm syndromes and immunosuppression

Breadth of concomitant immune responses prior to patient recovery: a case report of non-severe COVID-19

Pilot prospective open, single-arm multicentre study on off-label use of tocilizumab in patients with severe COVID-19

Effective treatment of severe COVID-19 patients with tocilizumab

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Human monocyte subsets exhibit divergent angiotensin I-converting activity

The novel coronavirus 2019 (2019-nCoV) uses the SARS-coronavirus receptor ACE2 and the cellular protease TMPRSS2 for entry into target cells

COVID-19: risk for cytokine targeting in chronic inflammatory diseases?

Dissecting the human immunologic memory for pathogens

Human T cell immune surveillance: Phenotypic, functional and migratory heterogeneity for tailored immune responses

Functional signatures of protective antiviral T-cell immunity in human virus infections

Longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of SARS-CoV-2 infected patients

Dysregulation of immune response in patients with COVID-19 in

The laboratory tests and host immunity of COVID-19 patients with different severity of illness

Functional exhaustion of antiviral lymphocytes in COVID-19 patients

Reduction and Functional Exhaustion of T Cells in Patients with Coronavirus Disease

An interferon-gamma-related cytokine storm in SARS patients

High IL-6/IFN-gamma ratio could be associated with severe disease in COVID-19 patients

The clinical course and its correlated immune status in COVID-19 pneumonia

The Prevalence of Olfactory and Gustatory Dysfunction in COVID-19 Patients: A Systematic Review and Meta-analysis

Risk factors of critical & mortal COVID-19 cases: A systematic literature review and meta-analysis

Central memory and effector memory T cell subsets: function, generation, and maintenance

Two subsets of memory T lymphocytes with distinct homing potentials and effector functions

Reduction and Functional Exhaustion of T Cells in Patients With Coronavirus Disease 2019 (COVID-19)

Detection of SARS-CoV-2-Specific Humoral and Cellular Immunity in COVID-19 Convalescent Individuals

Advancing scientific knowledge in times of pandemics

Detectable serum SARS-CoV-2 viral load (RNAaemia) is closely associated with drastically elevated interleukin 6 (IL-6) level in critically ill COVID-19 patients

Phenotype of SARS-CoV-2-specific T-cells in COVID-19 patients with acute respiratory distress syndrome

We thank Andrea Werner and Gustavo Almeida for coordination of the patient recruitment and technical assistance and all members of the Zielinski lab for discussions.