key: cord-0995708-7zr0bol6
authors: Sami, Ramin; Fathi, Farshid; Eskandari, Nahid; Ahmadi, Meysam; ArefNezhad, Reza; Motedayyen, Hossein
title: Characterizing the immune responses of those who survived or succumbed to COVID-19: Can immunological signatures predict outcome?
date: 2021-01-15
journal: Cytokine
DOI: 10.1016/j.cyto.2021.155439
sha: 17b0173c210cc91fc43aacc58624a1831b2f3896
doc_id: 995708
cord_uid: 7zr0bol6

Background Immunodeficiency has pivotal role in the pathogenesis of coronavirus disease 2019 (COVID-19). Several studies have indicated defects in the immune system of COVID-19 patients at different disease stages. Therefore, this study investigated whether alters in immune responses of COVID-19 patients may be considered as predicting factors for disease outcome. Methods The percentages of innate and adoptive immune cells in the recovered and dead patients with COVID-19, and healthy subjects were determined by flow cytometry. The levels of pro- and anti-inflammatory cytokines and other immune factors were also measured by enzyme-linked immunosorbent assay. Results At the first day of hospitalization, the frequencies of CD56dim CD16+ NK cells and CD56bright CD16dim/- NK cells in patients who died during treatment were significantly increased compared to recovered and healthy individuals (P < 0.0001). The recovered and dead patients had a significant increase in monocyte number in comparison with healthy subjects (P < 0.05). No significant change was observed in Th1 cell numbers between the recovered and dead patients while Th2, Th17 cell, and Treg percentages in death cases were significantly lower than healthy control and those recovered, unlike exhausted CD4+ and CD8+ T cells and activated CD4+ T cells (P < 0.0001-0.05). The activated CD8+ T cell was significantly higher in the recovered patients than healthy individuals (P < 0.0001-0.05). IL-1α, IL-1β, IL-6, and TNF-α levels in patients were significantly increased (P < 0.0001- 0.01). However, there were no differences in TNF-α and IL-1β levels between dead and recovered patients. Unlike TGF-β1 level, IL-10 was significantly increased in recovered patients (P < 0.05). Lymphocyte numbers in recovered patients were significantly increased compared to dead patients, unlike ESR value (P <0.001-0.01). CRP value in recovered patients significantly differed from dead patients (P < 0.001). Conclusion Changes in frequencies of some immune cells and levels of some immune factors may be considered as predictors of mortality in COVID-19 patients.

In December 2019, Wuhan city, China, experienced the prevalence of novel viral pneumonia with unknown reasons. This disease was then recognized as a zoonotic disorder like Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS) coronaviruses and called COVID-19 which is caused by a severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) (1) (2) (3) (4) . Coronavirus is one of the enveloped RNA viruses with a diameter of 60-140 nm which are crone-shape with having spike-shape projections (5) . Coronavirus can be categorized into the kinds of alpha, beta, delta, and gamma. This virus has the ability to infect various species and leads to serious disorders such as septic shock and acute respiratory distress syndrome (ARDS) (5, 6) . COVID-19 can appear with fever, dry cough, fatigue, myalgia, sore throat, conjunctivitis, headache, smell and taste impairments (7, 8) . It also can spread in many ways, including mouth mucus membranes, feces, aerosols, and droplets (6) . Old individuals with critical conditions such as cerebrovascular disorder, cardiovascular disorder, hypertension, and diabetes are more vulnerable to COVID-19 compared to other populations (9) . Although, there is no particular antiviral treatment or certain vaccine for the disease, comprehending the pathogenesis of COVID-19 can be helpful in the management of this viral condition (10) . The pathogenesis of this viral agent is not fully explained yet, but there is evidence that a failure in the immune system is present in these patients such as inflammatory cytokine storm and lymphocytopenia, and probably can be lead to death cases because of SARS-COV2 (11) (12) (13) . Viral agents can activate immune reactions via the host immune system. Several respiratory viruses inhibit innate immune reactions which create a chance for effective viral replication and infection enhancement. As a result of an interaction between the cells and COVID-19, affected cells elevate the releasing a great number of cytokines and chemokines, like IFN-I (14, 15) . But the exact role of IFN-I in destructing or protecting immune system reactions in coronavirus infections is a challenge (16). It is reported that in the great numbers of severe cases, there were the increased productions of some immune factors such as TNFα, IL-2R, IL-6, and IL-10 (14) . Moreover, it is documented that the increased levels of C-reactive protein (CRP), IL-1α, IL-1β, IL-6, and TNF-α can participate in ARDS in these patients (11, 17) . Previous studies have revealed that peripheral level of the natural killer cell (NK cell) was significantly decreased in the severe stage of the disease compared to the mild disease stage, but it is unknown whether this reduction was due to cell death and/or cell accumulation in the infected tissues (18, 19) . In a study on patients in severe and nonsevere stages of COVID-19, it was revealed that severe COVID-19 patients had statistically significant reductions in the percentages of basophils (0.1 versus 0.2%), eosinophils (0.0 versus 0.2%), and monocytes (6.6 versus 8.4 %) in comparison with nonsevere stages (20) .

When a virus arrives at the cell, its antigens can be subjected to the antigen presentation cells (APC). The presentation of antigen induces adaptive immune system through involving T and B cells (21) . In infectious conditions, virus-specific CD8+ and CD4+ T cells are activated. In addition, T regulatory cells (Tregs) are able to decrease immune reactions and thereby reduce tissue injuries (22) (23) (24) (25) (26) . In this line, the reduced numbers of CD4+ and CD8+ T cells, and Tregs contribute to the exacerbation of tissue injuries (20) . The CD4+ T helper (Th) cells have a remark role in the function of coronaviruses through the interaction with Tregs (27) . Previous studies on MERS-CoV patients, a disease with similar pathogenesis to COVID-19, have shown that the number of Th1 was associated with the good prognosis of the disease, but Th2 cytokines were related to the high rate of death (17, 28) . Furthermore, Th17, as another subset of CD4+ T helper cells, can participate in the cytokine storm in ARS-CoV-2 and probably enhance pulmonary edema (29) . Despite known roles of Th cells, the functional roles and numbers of other T cells in COVID-19 are not understood so far (30) . There is not enough information about the immune reactions in COVID-19 patients (20) . Our previous study indicated that changes in the immune system of patients with COVID-19 during a recovery period have critical role in determining disease severity (31) . This study was therefore focused on clarifying whether changes in immune cell numbers and immune factor values may be considered as a predicting factor to determine the outcome of COVID-19. Hence, the frequencies of innate immune cells, for instance, CD56 bright CD16 dim/-NK cells and CD56 dim CD16 + NK cells, and monocytes, and adaptive immune cells, such as B cells, Tregs, Th1 cells, Th2 cells, Th17 cells, activated CD4+ and CD8+ T cells, exhausted CD4+ and CD4+ T cells at the first day of hospitalization in recovered patients and death cases due to COVID-19 up to ten days were compared to healthy subjects. Moreover, the plasma levels of proand anti-inflammatory cytokines including IL-1α, IL-1β, IL-6, IL-10, TNF-α, and TGF-β1 and other immune factors were measured.

The study was performed in Khorshid hospital, Isfahan, Iran from March 18 to May 24, 2020.

The diagnosis of COVID-19 was approved by the specialist according to clinical and laboratory criteria including: 1) clinical evaluation; 2) chest CT scan imaging; 3) real time-polymerase chain reaction (RT-PCR) assay. Patients were negative for health problems, autoimmunity, infectious diseases, and malignancy. Pulmonary involvement was observed in the results of chest CT scan imaging of all patients as previously described (32) . Nasopharyngeal swab samples were collected from all participants and COVID-19 were confirmed at the time of admission by RT-PCR assay.

The patients did not receive any immunosuppressive agents before entering the study. All patients had clinical signs and symptoms of COVID-19 at least 3-5 days prior to refer to Khorshid hospital to initiate disease treatments. The patients had age range from 30 to 92 years, while it was from 58 to 81 years in healthy individuals. Healthy subjects were recruited among those referred to a health screening center. The voluntaries had no signs and symptoms of respiratory infections, autoimmune and immunodeficiency syndromes according to laboratory and clinical evaluations. 

Heparinized blood samples (10 ml) were obtained from patient and control groups. The blood sampling from patients was performed at first day of hospitalization. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by Ficoll-Paque centrifugation according to the manufacturer's instructions (Lymphodex, Germany). The isolated cells were washed twice with phosphate buffered saline (PBS) at 300 ×g for 10 min. The viability of the cell was determined by trypan blue dye exclusion.

Lymphocyte numbers in peripheral blood of COVID-19 and healthy subjects were determined using an automated cell counter system UF-100® (Sysmex, Kobe, Japan) within two hours after collecting heparinized blood samples.

To assess the percentages of innate and adoptive immune cells of patient (at first day of hospitalization) and healthy groups, PBMCs were stained with different monoclonal antibodies or matched to isotype control IgG for 30 min at 4°C. Isotype-matched control antibodies served as negative controls. The staining of some intracellular molecules with different antibodies was carried out after the fixation and permebilization of the cells according to the manufacturer's guideline (eBiosciences, USA). The stained cells were washed several times with PBS and then centrifuged at 300 ×g for 10 min at room temperature. The percentages of the stained cells were assessed by a FACSCalibur system (Becton Dickinson, San Jose, CA). Firstly, lymphocyte population was isolated from debris or dead cells using forward and side scatter. The gating strategy was done using FlowJo software (v10.1, FlowJo, Ashland, OR, USA). Afterwards, the gated lymphocytes were analyzed to determine the frequencies of the CD3+ cells and CD4+ cells which were used to measure the percentages of CD4+ T-bet+ IFN-γ+ Th1, CD4+ IL-4+ GATA3+ Th2, CD4+ IL-17α+ RORγt+ Th17 cells, CD4+ CD127 low FoxP3+ Tregs, CD4+ CD25+ CD69+ activated T cells, CD3-CD19+ CD22 + B cells, CD3+ CD4+ PD-1+ exhausted T cells, CD3+ CD8+ PD-1+ exhausted T cells, CD3-CD56 dim CD16 + NK cells, and CD3-CD56 bright CD16 dim/-NK cells. In this study, the CD8+ activated T cells and monocytes were, respectively, considered as CD8+ CD25+ CD69+ cells and CD14+ CD16+ CD11b+ cells. The monoclonal and their isotype control antibodies used in the study are indicated in Table. 1.

To determine cytokine profiles of alive and death cases due to COVID-19 up to ten days of hospitalization, the plasma samples were isolated from whole blood of patients (at the first day of hospitalization) and healthy subjects. The levels of IL-1α, IL-1β, TNF-α, IL-6, TGF-β1, and IL- 

The results were analyzed by GraphPad Prism 6 (GraphPad software, San Diego, CA) and are represented as the mean ± standard error of the mean (SEM). Comparisons of the groups with normal distributions were performed using One-way analysis of variance (ANOVA) and unpaired t-tests, while those with non-normal distributions were compared by Kruskal-Wallis and Mann-Whitney tests. Pearson's and Spearman's tests were used to determine the correlation coefficients of the results with normal and non-normal distributions, respectively. p value < 0.05 was considered statistically significant.

A total of 71 subjects with COVID-19, who recovered (60 cases) or died (11 cases) up to ten days of hospitalization, and 50 healthy individuals were participated in the study (Table. 2). The mean age of subjects and number of lymphocytes were significantly higher in recovered patients than those who died during the disease recovery, unlike ESR value (P <0.001-0.01, Table. 2). The frequencies of background diseases and the value of CRP in recovered patients significantly differed from dead patients (P < 0.001, Table. 2). Table. 2 shows the demographic and other information of healthy individuals and COVID-19 patients.

At the first day of hospitalization, the frequencies of CD56 dim CD16 + NK cells and CD56 bright CD16 dim/-NK cells in death cases up to ten days of hospitalization were significantly increased compared to alive patients and healthy individuals ( Figure. 1A, C, and D, P <0.0001). Furthermore, the numbers of these cells were significantly higher in alive patients than control group ( Figure. 1A, C, and D, P <0.0001). Our results indicated that death cases had a significant increase in the number of monocyte compared to healthy subjects ( Figure. 1B and E, P < 0.05). The same trend was also observed in alive patients ( Figure. 1B and E, P < 0.05).

To compare the situations of adaptive immunity in the recovered patients and those who died up to ten days of hospitalization, adoptive immune cell frequencies of patients were assessed at the first day of hospitalization. In the early recovery stage, no significant change was observed in Th1 cell numbers between the recovered patients and death cases due to COVID-19 up to ten days ( Figure. 

Regarding the fact that cytokine storming plays an indispensable role in pathogenesis of COVID-19, cytokine profiles were studied in COVID-19 patients who recovered and dead during hospitalization. As shown in Fig. 3A -D, statistically significant increases in the levels of pro-inflammatory cytokines (IL-1α, IL-1β, IL-6, and TNF-α) in patients were observed compared to healthy individuals (P < 0.0001-0.01). However, these significant increases in TNF-α and IL-1β levels were not found in the cases who died up to ten days of hospitalization in comparison with those recovered. The level of IL-10 was significantly higher in the recovered patients than healthy group, unlike death cases ( Figure. E, P < 0.05). However, there is no significant change in TGF-β1 level among the recovered patients, death cases, and healthy subjects ( Figure. F ).

COVID-19 originated from SARS-COV2, is a prominent public health problem around the world (33) . The pathogenesis of this disease is not exactly declared yet, but according to the recent documents, a defect in the function and/or modulation of the immune system can be involved in the pathogenic mechanisms of COVID-19 (34, 35) . Hereby, the present study was accomplished for investigating some innate and adaptive immune cells and immune agents of COVID-19 patients in the course of ten days after hospitalization, which is approximately a period that the disease is worsen and may lead to death or recovery from COVID-19 (12, 36) . Having considered that the innate immune system is related to the early reactions versus viral infections, some agents of innate immunity were investigated. At the first day of hospitalization, we observed significant increases in the frequencies of CD56 dim CD16 + NK cells and CD56 bright CD16 dim/-NK cells in patients who died up to ten days than the recovered patients and healthy subjects. Unlike our findings, Wang et al. declared that the amount of NK cell was decreased in COVID-19 patients, and in the severe stage of the disease, these cells are lower than the mild stage (37) . Regarding the expressions of CD16 and CD56, NK cells are categorized into various populations, and two main types of them are CD56 dim CD16 + NK cells and CD56 bright CD16 dim/-NK cells (38) . CD56 dim CD16 + NK cells can express high levels of the maturation marker CD57 and killer inhibitory receptors (KIR) (39) . showed that the number of monocytes was in a normal range (41) . This difference in the results may be attributed to the disease stage when the patients were studied.

In the investigation of the numbers of adaptive immune cells, we observed that Th2, Th17 cells, and Tregs in dead patients were significantly lower than healthy and recovered patients. However, there was no remark change in Th1 cell frequency between recovered and dead patients at the first day of recovery. In this respect, a study by Qin et al. manifested that the percentages of suppressor and helper T cells in the patients were lower than healthy individuals. Moreover, in their study on the severe stage of the disease, naive Th cell frequency were increased, but memory Th cells and Tregs were decreased (42) . In our study, the number of Tregs in the early stage of recovery was significantly higher in dead and recovered patients than the control group. Similar to this, the results of Tan et al. implicated that Treg number was elevated in the mild stage of COVID-19 in comparison with healthy subjects (43) . We also indicated that the numbers of the exhausted CD4+ and CD8+ T cells and activated CD4+ T cells at the first day of recovery were significantly higher than recovered and control groups, which is consistent with previous studies showing higher percentages of senescent/exhausted cells (PD1+CD57+) in COVID-19 patients (44) . Furthermore, the percentage of activated CD8+ T cells was dramatically higher in the recovered group than dead and healthy individuals, and totally, the percentage of this cell in patients (recovered and dead patients) were higher than the healthy group. In contrast with our findings, Wang et al. by analyzing subsets of peripheral lymphocyte from 60 patients with COVID-19 before and after treatment indicated that total lymphocytes, CD4+ T cells, CD8+ T cells were reduced in patients, and these cells were lower in cases with the severe disease stage than those with the mild disease stage (37) . This discrepancy may be pertinent to different therapeutic methods employed in diverse stages of the disease.

Regarding the fact that one of the mechanisms involved in the pathogenesis of COVID-19 may be related to cytokine storm, as extreme and uncontrolled secretion of pro-inflammatory cytokines such as TNF-α, IFN-γ, IL-1 and IL-6 which can lead to death (11, 45, 46) , we measured some of pro-and anti-inflammatory cytokines such as IL-1α, IL-1β, IL-6, IL-10, TNF-α, and TGF-β1.

According to our observations, the plasma levels of IL-1α, IL-1β, IL-6, and TNF-α in patients at the first day of recovery were dramatically higher than the healthy group. In line with the role of inflammation in COVID-19 pathogenesis, it is reported that the increased level of CRP, as a marker of inflammation, was significantly correlated to mortality (47) . Other data demonstrated that the level of IL-10 was significantly higher in recovered patients than healthy subjects. However, no 

The authors thank all subjects who participated in the study.

This study was financially supported by Isfahan University of Medical Sciences (grant number: 199422).

The authors declare no conflict of interests. 

 The frequencies of CD56 dim CD16 + NK cells and CD56 bright CD16 dim/-NK cells were significantly higher in death cases due to COVID-19 than recovered patients and healthy individuals.  No significant change was observed in Th1 cell numbers between the recovered and dead patients  Th2, Th17 cell, and Treg percentages in death cases were significantly lower than recovered patients and healthy controls.  The activated CD8+ T cell was significantly higher in recovered patients than healthy individuals, unlike exhausted CD4+ T cells, exhausted CD8+ T cells, and activated CD4+ T cells.  Unlike TGF-β1 level, IL-1α, IL-1β, IL-6, and TNF-α levels in patients were significantly increased.

The reproductive number of COVID-19 is higher compared to SARS coronavirus

Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. The lancet. 2020. 3. Remuzzi A, Remuzzi G. COVID-19 and Italy: what next? The lancet

Association of cardiac injury with mortality in hospitalized patients with COVID-19 in Wuhan, China

Clinical and Paraclinical Characteristics of COVID-19 patients: A systematic review and meta-analysis. medRxiv

Clinical features and treatment of COVID-19 patients in northeast Chongqing

Clinical characteristics of COVID-19 patients with digestive symptoms in Hubei, China: a descriptive, cross-sectional, multicenter study. The American journal of gastroenterology

The optimal diagnostic methods for COVID-19

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

Immune cell profiling of COVID-19 patients in the recovery stage by single-cell sequencing

Molecular immune pathogenesis and diagnosis of COVID-19

The use of anti-inflammatory drugs in the treatment of people with severe coronavirus disease 2019 (COVID-19): The experience of clinical immunologists from China

The epidemiology and pathogenesis of coronavirus disease (COVID-19) outbreak

Down-regulated gene expression spectrum and immune responses changed during the disease progression in COVID-19 patients

2020:104791. 16. Cao X. COVID-19: immunopathology and its implications for therapy

From sepsis to acute respiratory distress syndrome (ARDS): emerging preventive strategies based on molecular and genetic researches

The innate immune system: fighting on the front lines or fanning the flames of COVID-19? Cell Host & Microbe

Functional exhaustion of antiviral lymphocytes in COVID-19 patients

Dysregulation of immune response in patients with COVID-19 in Wuhan, China. Clinical Infectious Diseases

Profile of specific antibodies to the SARS-associated coronavirus

Regulatory T-cells and their impacts on cytokine profile of end-stage renal disease patients suffering from systemic lupus erythematosus

Comparison of the percentage of regulatory T cells and their p-STAT5 expression in allergic and non-allergic common variable immunodeficiency patients

The effects of silymarin and cyclosporine A on the proliferation and cytokine production of regulatory T cells

Regulatory T cells in arterivirus and coronavirus infections: do they protect against disease or enhance it?

Increased expression of lymphocyte activation gene-3 by regulatory t cells in multiple sclerosis patients with fingolimod treatment Multiple sikleroz hastalar ฤ ฑ nda

lenfosit aktivasyon gen-3 ekspresyonunda art ฤ ฑ ล

Immune-epidemiological parameters of the novel coronavirus-a perspective

MERS-CoV infection is associated with downregulation of genes encoding Th1 and Th2 cytokines/chemokines and elevated inflammatory innate immune response in the lower respiratory tract

TH17 responses in cytokine storm of COVID-19: An emerging target of JAK2 inhibitor Fedratinib

Reduction and functional exhaustion of T cells in patients with coronavirus disease 2019 (COVID-19)

Immune system changes during COVID-19 recovery play key role in determining disease severity

Radiological Society of North America Expert Consensus Statement on Reporting Chest CT Findings Related to COVID-19. Endorsed by the Society of Thoracic Radiology, the American College of Radiology, and RSNA. Radiology: Cardiothoracic Imaging

The epidemiology and pathogenesis of coronavirus disease (COVID-19) outbreak

Molecular immune pathogenesis and diagnosis of COVID-19

Immune cell profiling of COVID-19 patients in the recovery stage by single-cell sequencing

Real estimates of mortality following COVID-19 infection. The Lancet infectious diseases

Characteristics of peripheral lymphocyte subset alteration in COVID-19 pneumonia. The Journal of infectious diseases

CD56bright natural killer (NK) cells: an important NK cell subset

Immunoregulatory Effects of Human Amnion Epithelial Cells on Natural Killer and T Cells in Women with Recurrent Spontaneous Abortion (RSA)

Role of distinct natural killer cell subsets in anticancer response

Clinical characteristics of 82 death cases with COVID-19

Dysregulation of immune response in patients with COVID-19 in Wuhan, China

Immunopathological characteristics of coronavirus disease

Marked T cell activation, senescence, exhaustion and skewing towards TH17 in patients with COVID-19 pneumonia

Cytokine storm and immunomodulatory therapy in COVID-19: role of chloroquine and anti-IL-6 monoclonal antibodies. International journal of antimicrobial agents

Single-cell RNA-seq and V (D) J profiling of immune cells in COVID-19 patients. medRxiv

The role of biomarkers in diagnosis of COVID-19-A systematic review

Farshid Fathi and Meysam Ahmadi carried out some of the experiments and participated in statistical analysis of the data

Nahid Eskandari participated in the design of some experiments and obtained funding for the work

Reza ArefNezhad performed some of the experiments and drafted the manuscript

Hossein Motedayyen participated in the study design and revised the manuscript. The authors read and approved the final version of manuscript

× The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.×The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:The authors declare that there is no conflict of interests