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• The demographic, laboratory, and clinical characteristics of COVID-19 andhealthy subjects.
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• The frequencies of different immune cells and levels of pro-and anti-inflammatory cytokines in whole blood of participants were determinedby flow cytometry and enzyme-linked immunosorbent assay, respectively.
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• Moreover, Qinet al. indicated that suppressor and helper T cell percentages were lower inpatients than normal group.

The CD3+ cell population was also determined using the gating oflymphocyte population and was then used to measure the percentages of B cells(CD3− CD19+ CD22+ cells), exhausted CD4+ T cells (CD3+ CD4+ PD-1+ cells),exhausted CD8+ T cells (CD3+ CD8+ PD-1+ cells), CD56lowCD16+ NK cells (CD3− CD56lowCD16+ cells), and CD56high CD16+/− NK cells(CD3−CD56high CD16+/− cells). Afterwards, the lymphocyte population was gatedto assess the frequencies of the CD4+ cells which were used to determine thepercentages of Th1 cells (CD4+ T-bet+ IFN-γ+ cells), Th2 cells (CD4+ IL-4+GATA3+ cells), Th17 cells (CD4+ IL-17α+ RORγt+ cells), Tregs (CD4+CD127low FoxP3+ cells), and activated CD4+ T cells (CD4+ CD25+CD69+ cells). At the first day (the early recovery stage) and 10 days of initiation oftherapeutic methods (the late recovery stage), heparinized blood samples (5 ml)were obtained from patients. All patients had pulmonary involvement and were not on treatment withdrugs influencing the immune system and antibodies production (i.e. steroids,sulfasalazine, phenytoin, and antimalarial drugs) prior to study initiation.
CD56highCD16+/− NK cells aredescribed by NKG2A, low level of perforin, and are primarily characterized bycytokine production.16,30,31 Therefore, it is likely that the changes in the frequencies oftwo subsets of NK cells during the disease recovery are protective mechanisms toeliminate the SARS-COV2 and thereby reducing inflammation occurred in the earlystages of disease. Our data revealed that the percentages of Th1, Th2, andTh17 cells were significantly lower in patients than healthy control (Figure 3(a)–(c) and (j)–(l), P Figure 3(d)–(h) and (m)–(q), P Figure 3(f), (h), (o), and (q), P Figure3(d), (e),(g), (m), (n), and (p)). The number of CD56low CD16+ NK cells in patients wassignificantly increased compared to healthy subjects. To determine the percentages of activated T cells, exhausted T cells, Th1 cells,Th2 cells, Th17 cells, Tregs, B cells, NK cells, and monocytes in peripheralblood of COVID-19 patients (the first day and 10 days of initiation oftherapeutic approaches) and healthy subjects, PBMCs were stained with differentmonoclonal antibodies or matched to isotype control IgG for 30 min at4○C. These changes may play afundamental role in reducing disease severity through regulating cytokineproductions involved in the inflammation and functions of various immune cells.Nevertheless, larger and more multicenter studies are needed to validate theseconclusions. A limitation of the study was the lack of determinationof immune system differences between alive and dead patients with COVID-19 during arecovery period.

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• The levels of erythrocyte sediment rate (ESR) and C-reactive protein (CRP) ofCOVID-19 patients were measured using the erythrocyte sedimentation rate (ESR)analyzer (Parsian Teb, Iran) and Mindray BS-800 automated biochemistry analyzer(Shenzhen Mindray Bio-Medical Electronics, China), respectively.
• This is an analytical observational (case-control) study performed on 57 patientswith COVID-19, who were referred to a COVID-19 center, Isfahan, Iran from March2020 to April 2020, and 40 healthy individuals without any the signs andsymptoms of acute respiratory infections and other health problems affected theimmune system.

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In this regard, the FlowJosoftware (v10.1, FlowJo, Ashland, OR, USA) was used to gate lymphocytepopulation using forward and side scatter to exclude debris or dead cells fromthe analysis of different cells. The cell markers used to determine thefrequencies of the stained cells are indicated in Table 1. The percentages of the stained cells were measured by a FACSCalibursystem (Becton Dickinson, San Jose, CA).

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Assessments of innate immune cells in patients with COVID-19 during a

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The RRF funding amounts shown for measures are based on the initial cost estimates provided by the Member States included in the recovery and resilience plans and may as such differ from finalized Commission financial reports, which follow eventual project implementation. The map exclusively serves information purposes and is not an exhaustive database of projects supported by the Recovery and Resilience Facility. Improving access to and the quality of general, vocational, and higher education; focusing on digital education, early childhood education and care; supporting youth employment. The funding amounts shown reflect the initial cost estimates included in the national recovery and resilience plans. It is not an exhaustive database of projects supported by the Facility and will be regularly updated as the implementation progresses.
The resultsare representative of 57 independent experiments for COVID-19 patientsat the first day of treatment, 51 independent experiments for COVID-19patients in 10 days of treatment, and 40 independent experiments forhealthy individuals. The demographic, laboratory, and clinical characteristics of COVID-19 andhealthy subjects. Table2 depicts the demographic and other characteristics of COVID-19 andhealthy subjects. Of the 57 patients, 51 (89.48%) weredischarged from hospital and 6 (10.52%) died during the study. Antibodies used for determing the changes of the immune system ofCOVID-19 patients by flow cytometry.

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Having considered that innate immunity provides the early line of defense againstviral infections, some innate immune cells were studied in the course of 10 daysafter initiation of treatment, which is almost a period that the disease isdeteriorated and may result in death or recovery from COVID-19.27,28 Our datashowed that CD56lowCD16+ NK cell number was significantlylower in the early stage of recovery than the late stage of recovery; however itsfrequency was noticeably increased compared to healthy subjects. Moreover, the authors have shown that CD4+and CD8+ T cell numbers were notably decreased.17 In agreement with previous study, our data revealed that the frequencies ofTh cells (Th1, Th2, and Th17 cells) in patients were significantly lower in theearly recovery stage than the late recovery stage and healthy individuals. B cells showed an increasedpercentage in patients compared to healthy subjects, while this increase wassignificantly reduced in the late stage of recovery (Figure 3(i) and (r),P Open in a new tabThe percentages of adaptive immune cells in COVID-19 and healthyindividuals. Its frequency wassignificantly higher in the late recovery stage than early recovery stage (Figure 2(a) and (c),P highCD16+/− NK cells in the early stage of recovery was significantlyincreased in comparison with the late stage of recovery and healthy individuals(Figure 2(a) and(d),P Figure2(b) and (e),P Open in a new tabThe frequencies of innate immune cells in COVID-19 and control subjects.The percentages of CD56low CD16+ NK cells,CD56high CD16+/− NK cells, and monocytes werestudied by flow cytometry (a and b) and then analyzed (c–e).

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In disagreement with other reports showing increasedfrequency of B cells in the late stage of recovery,17 we observed that the percentage of this cell was decreased followingrecovery. Moreinterestingly, the percentages of exhausted CD4+ T cells and exhausted CD8+ T cellswere higher in the early stage of recovery than the late stage of recovery. Thisobservation was in contrast with previous study showing severe cases of COVID-19tend to have lower percentages of monocytes.24 This discrepancy may be attributed to disease stage which patients wereevaluated.
The stained cells were washedtwice with PBS and centrifuged at 300 × g for 10 min at roomtemperature. Fixation and permebilization of the cells were performed for stainingsome intracellular molecules with different antibodies according to themanufacturer’s guideline (eBiosciences, USA). The isolated cells were washed twice with phosphate buffered saline(PBS) at 300 × g for 10 min. Peripheral bloodmononuclear cells (PBMCs) were isolated from whole blood by Ficoll-Paquecentrifugation according to the manufacturer’s instructions (Lymphodex,Germany).
Moreover, Qinet al. indicated that suppressor and helper T cell percentages were lower inpatients than normal group. In revery play login the next step, the adaptive immune system of COVID-19 subjects was studied after 1and 10 days of initiation of therapeutic methods. In an attempt to discover the frequency of other cells of innateimmunity, the number of monocytes was also assessed. As shown inFigure 4(a)–(d),statistically significant reduction in the levels of pro-inflammatory cytokines(IL-1α, IL-1β, IL-6, and TNF-α) in patients were observed during a recovery,with the exception of IL-1β level (P Figure 4(e),P Figure 4(f)). Having considered that severe COVID-19 is largely related to a cytokine storm,cytokine profiles of COVID patients were assessed during a recovery.

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Other results of the currentstudy revealed that the percentage of another subset of NK cells(CD56highCD16+/− NK cells) was significantly increased atthe first day of recovery. TheCD56lowCD16+ NK cells have high expression levels ofkiller inhibitory receptors, the maturation marker (CD57), and natural andantibody-dependent cellular cytotoxicity which is mediated by releasing high levelsof perforin and enhanced killing.16,30,31 These findings suggest thatthe reduced number of CD56lowCD16+ NK cells may contribute todisease susceptibility in the early stages of disease. COVID-19, as a pandemic disease, is responsible for considerable mortality and morbidity.25 Immune system functions have fundamental role in the pathogenesis and outcomeof disease.26 Therefore, the current study focused on determining how immune system changesduring a recovery were correlated to disease severity. Thepercentages of Th1, Th2, Th17, Tregs, exhausted CD4+ T cells, exhaustedCD8+ T cells, activated CD4+ T cells, activated CD8+ T cells, and Bcells were assessed using flow cytometry (a–i) and then analyzed (j–r).The depicted results are representative of 57 independent experimentsfor COVID-19 patients at the first day of treatment, 51 independentexperiments for COVID-19 patients in 10 days of treatment, and 40independent experiments for healthy groups. The frequencies of innate immune cells in COVID-19 and control subjects.The percentages of CD56low CD16+ NK cells,CD56high CD16+/− NK cells, and monocytes werestudied by flow cytometry (a and b) and then analyzed (c–e).