key: cord-0959481-7qos61t4
authors: Simsek, Abdurrahman; Kizmaz, Muhammed A.; Cagan, Eren; Dombaz, Fatma; Tezcan, Gulcin; Asan, Ali; Demir, H. Ibrahim; Bal, S. Haldun; Ermis, Digdem Y.; Dilektaslı, Aslı G.; Kazak, Esra; Akalin, E. Halis; Oral, H. Barbaros; Budak, Ferah
title: Assessment of CD39 expression in regulatory T‐cell subsets by disease severity in adult and juvenile COVID‐19 cases
date: 2022-01-26
journal: J Med Virol
DOI: 10.1002/jmv.27593
sha: 3a0e3c2200852e35af180a05ac4ee71681aa5901
doc_id: 959481
cord_uid: 7qos61t4

COVID‐19 is a disease characterized by acute respiratory failure and is a major health problem worldwide. Here, we aimed to investigate the role of CD39 expression in Treg cell subsets in COVID‐19 immunopathogenesis and its relationship to disease severity. One hundred and ninety COVID‐19 patients (juveniles, adults) and 43 volunteers as healthy controls were enrolled in our study. Flow cytometric analysis was performed using a 10‐color monoclonal antibody panel from peripheral blood samples. In adult patients, CD39(+) Tregs increased with disease severity. In contrast, CD39(+) Tregs were decreased in juvenile patients in an age‐dependent manner. Overall, our study reveals an interesting profile of CD39‐expressing Tregs in adult and juvenile cases of COVID‐19. Our results provide a better understanding of the possible role of Tregs in the mechanism of immune response in COVID‐19 cases.

of these studies suggest that the effector activity of CD8 + T (Cytotoxic T) cells decreases during viral infections. [4] [5] [6] CD25 and CD127 have been suggested as two reliable markers for Tregs. Although these markers are not unique to Tregs, they allow the identification of a large population of Tregs. CD4 + CD25 high C-D127 low cells are often considered as bona fide Tregs. 7, 8 In addition, the transcription factor forkhead box P3 (FoxP3) has been identified as a specific marker of Tregs. 9 FoxP3 has been shown to be involved in the essential immunosuppressive properties of Tregs in autoimmune diseases. 10 Taking into consideration the heterogeneous phenotypes of these cells, two phenotypes seem to be beneficial in identifying Tregs. The determination of CD4 + CD25 high and CD4 + CD25 high CD127 low cells with the expression of the transcription factor FoxP3 is prime in determining the Treg cell pool. 11 Another marker, Helios, a transcription factor belonging to the Ikaros family, has been proposed as a marker for thymic Tregs (tTregs). In addition, it has been reported that it can be used to distinguish between peripheral and thymus-derived Tregs. 12 Moreover, Zabransky et al. 13 reported that Helios + FoxP3 + Tregs have stronger immunosuppressive properties than Helios -FoxP3 + Tregs.

Tregs are divided into three subsets based on the suppressive functions of the cells. CD4 + CD45RA + FoxP3 low cells represent naïve or resting Tregs (nTreg), whereas CD4 + CD45RA -FoxP3 high cells are known as activated effector Tregs (eTreg). 14, 15 Both Treg cell subsets have been reported to have immunosuppressive effects in vitro.

Another subset of FoxP3 + T cells, CD4 + CD45RA − FoxP3 low cells, are functionally distinct from the Treg subsets often referred to as non-Tregs. These cell subsets are characterized by expressing proinflammatory cytokines and having no immunosuppressive effects. 16, 17 CD39 is an ectoenzyme that can hydrolyze adenosine triphosphate (ATP) and adenosine diphosphate to adenosine monophosphate (AMP). The hydrolyzed AMPs are then converted by CD73 into anti-inflammatory adenosine (ADO), which can bind to adenosine receptors on T cells and antigen-presenting cells. 18 ADO, which occurs particularly as a result of changes in ATP metabolism, plays an important role in the immunosuppressive and anti-inflammatory effects of Tregs. As CD39 can directly contribute to the suppressive capacity of Tregs, it is considered a functional Treg marker. 19 Some studies have reported that CD4 + CD25 + CD39 + Tregs have more immunosuppressive effects than CD4 + CD25 + CD39 − Tregs. 20 Tregs in COVID- 19 have been a notable area of research recently.

Several studies have provided insights into the role of CD4 + Tregs (CD25 + CD127 low , CD25 + FoxP3 high/+ , CD45RA − FoxP3 high ) in COVID-19.

In studies of peripheral blood, PBMCs, and lung samples, some authors reported that Tregs increased, while others thought that these cells decreased or remained unchanged. 21 In addition to phenotypic analyses, studies were supported by gene expression and transcriptome analyses. 22 For example, soluble CD25 in peripheral blood was reported to be elevated in COVID-19 patients. 23 In addition, FoxP3 expression has been proposed as a biomarker of disease progression. 24 However, some other studies have reported conflicting results in both cases. 25 In conclusion, the question of whether Tregs support or hinder COVID-19 immunopathogenesis remains to be investigated. Figure S1 . The adult control group consisted of healthy volunteers who applied to the blood donation center as donor candidates and were not diagnosed with COVID-19, had no known or recognized disease, and were aged between 18 and 84 years. The juvenile control group included healthy children under 18 years of age who came for routine checkups (growth and development monitoring) for reasons other than infection. Laboratory findings in the adult and juvenile patient groups of COVID-19 are shown in Table 2 . The hematologic abnormalities and comorbidity analysis of the patients are shown in Figure S2 . In addition, only unvaccinated individuals were included in the study.

The diagnoses of all patients included in the study were made by physicians specializing in infectious diseases based on the evaluation of laboratory and radiographic findings and the reverse-transcriptase polymerase chain reaction (RT-PCR) (Bio-Speedy Direct RT-qPCR SARS-CoV-2 nucleic acid detection kit; Bioeksen). Adult patient groups were classified according to pneumonia status. These groups were composed of cases of noncomplicated, mild, and severe pneumonia. In noncomplicated cases, patients with some nonspecific symptoms but without respiratory complications were selected.

Cases with mild pneumonia consisted of patients who had nonsevere pneumonia caused by SARS-CoV-2 and did not require oxygen support. Cases of severe pneumonia were included in the study as recommended by the WHO (rate > 30 breaths/min; severe respiratory distress; or SpO 2 ≤ 93% on room air). On the contrary, 

The normality of the obtained data was determined by 

Adult patients were evaluated in three groups according to their COVID-19 pulmonary complication status (noncomplicated, mild, and severe pneumonia). The gating strategy used in the flow cytometry method is shown in Figure 1 .

Tregs were identified in this study as CD25 high FoxP3 + and CD25 high CD127 low/− cells. In adult cases of COVID-19, these cell levels were statistically significantly elevated, especially in cases with severe pneumonia. CD25 high FoxP3 + cells were significantly higher in severe and mild pneumonia than in noncomplicated cases and healthy controls. CD25 high CD127 low/− cells also followed a similar profile and were significantly increased in severe and mild pneumonia ( Figure 2A ).

In addition, interesting results were found in the studies on CD39 expression. It was found that CD39 expression in CD3 + CD4 + cells was significantly higher in patients with COVID-19 than in the healthy control group ( Figure 2B ). CD39 expression in CD25 + FoxP3 + cells also followed a similar profile and was significantly higher in the patient groups than in the healthy group. Moreover, a CD39 expression signature was detected in these cell groups that increased with disease severity.

In CD25 high FoxP3 + CD127 low/− cells, CD39 expression was significantly higher in severe pneumonia than in the noncomplicated and healthy control groups ( Figure 2C ).

Furthermore, nTregs, eTregs, and non-Tregs were assessed by the expression of CD45RA and FoxP3. It was found that nTregs were significantly higher in patients with severe and mild pneumonia compared with healthy controls. On the contrary, eTregs were significantly increased in patients with severe and mild pneumonia, but there was no difference between the noncomplicated and healthy control groups. Moreover, non-Tregs differed significantly from each other in each disease group in direct proportion to the severity of the disease. In severe pneumonia cases, eTregs were statistically significantly higher than even in mild pneumonia cases. For mild pneumonia cases, eTregs were also significantly higher than for noncomplicated cases. In severe and mild pneumonia cases, these cells were also significantly elevated compared to healthy controls ( Figure 2D ).

Changes in Helios + FoxP3 + cells were also observed in a similar profile to non-Tregs ( Figure 2E ). In direct proportion to the severity of disease, from the healthy control group to the cases with severe pneumonia, each group was found to be significantly higher than the disease severity group below. There was no statistically significant difference in Treg subsets in patients followed up after recovery compared to initial flow cytometry data ( Figure S3 ).

Moreover, interesting results were obtained in correlation analysis of Treg profile and hematological parameters in adults. In severe pneumonia cases, lactate dehydrogenase (LDH) and ferritin levels were negatively correlated with CD3 + CD4 + cells. Interestingly, LDH and ferritin levels were significantly positively correlated with Tregs.

Conversely, platelet count was positively correlated with CD3 + CD4 + cells while negatively correlated with Treg cells. In addition, the group of Helios + FoxP3 + cells was positively correlated with CRP (- Figure S5A ). In mild pneumonia cases, a negative correlation was found between non-Tregs and lymphocytes ( Figure S5B ) Similarly, Tregs (CD25 high FoxP3 + cells) and lymphocytes were negatively correlated in noncomplicated cases ( Figure S5C ).

F I G U R E 1 Flow cytometry gating strategy for analysis of Treg subsets. (A) Lymphocytes were separated based on CD45 and SSC characteristics. (B) CD39 + , CD25 high FoxP3 + , CD25 high CD127 low , 45RA + , and FoxP3 + cells were separated at the CD3 + CD4 + lymphocyte gate. Naive Tregs (nTreg), effector Tregs (eTreg), and non-Tregs were separated using CD45RA and FoxP3 gated on CD3 + CD4 + cells. (C) CD39 + and CD127 low CD39 + cells were separated at the CD25 high FoxP3 + gate. (D) Helios and FoxP3 were used to separate thymic Tregs 

Serious illness and associated deaths are extremely rare in pediatric COVID-19 patients. In almost all pediatric patients included in our study, the disease was asymptomatic or with mild symptoms. In this regard, we found an age-based distinction more useful in pediatric COVID-19 cases. We examined Treg and CD39 expression in juvenile patients in two different age groups (0-12 vs. 13-18).

In examining our juvenile patients, we used the same flow cytometric panels as in the adult patients and applied the same gating strategy. The first striking point we observed in the juvenile patients was that CD25 high FoxP3 + cells did not change in all age groups, whereas there was a tendency for CD25 high CD127 low/− cells to decrease, especially in the 0-12 age group ( Figure 3A ).

This result in Tregs aroused curiosity about how CD39 expression evolves. We observed that patients aged 0-12 years had lower CD39 expression in CD4 + cells than patients aged 13-18 years ( Figure 3B ). However, no statistical significance was found between juvenile patients and healthy controls. A similar profile was observed for CD39 expression in CD25 high FoxP3 + cells. Interestingly, COVID-19 cases in the 0-12 age group tended to decrease, while cases in the 13-18 age group tended to increase compared with the age-matched healthy controls. The CD39 expression profile of CD25 high CD127 low/− cells also showed the same signature ( Figure 3C ). According to these results, it can be assumed that patients aged 0-12 years with COVID-19 express less CD39 in Tregs. When comparing all juvenile COVID-19 and healthy groups participating in the study, CD39 + Tregs tended to decrease overall.

Interesting results also emerged from the analysis of nTregs, eTregs, and non-Tregs by examining the expression of CD45RA and FoxP3 together. nTregs were significantly lower in both the 0-12 years age group and the 13-18 years age group than in the healthy control groups in the corresponding age range ( Figure 3D ). Although no statistically significant results were obtained for eTregs and non-Tregs, a decreasing trend was observed for these cells in the 0-12 years age group. In addition, when Helios and FoxP3 expressions were examined, a slight decrease was observed in the patient groups for Helios + Tregs ( Figure 3E ).

After recovery, no major change was observed in juvenile patients who were followed up. However, in patients aged 13-18 years, non-Treg levels were found to be significantly lower after recovery ( Figure S4 ).

The course of COVID-19 infections varies from asymptomatic to acute respiratory failure syndrome and multiorgan dysfunction. 26 Tregs have been shown to have an important impact on the immune response in many acute and chronic viral infections, but their role in COVID-19 immunopathogenesis remains to be elucidated. 27 Here, 28 reported an increased proportion of CD25 high CD127 low cells in COVID-19 patients. In another study, Tan et al. 29 reported that these cell subsets increased in both mild and severe cases, with a relatively smaller increase in severe cases compared with mild cases.

Peña et al. 30 Ahmadi et al. 36 examined the expression of CD39 and CD73 in CD8 + and CD4 + cell subsets in 14 COVID-19 patients. Although they found a significant increase in CD73 expression in CD8 + cell subsets, they found no correlation between CD39 expression and these cell subsets.

In this study, we observed a significant increase in CD39expressing Tregs in cases of mild and severe pneumonia in COVID-19, which may be a sign of the development of a strong immunosuppressive response that seems to be particularly beneficial in | 2097 a disease whose severe cases are characterized by aggressive inflammation and cytokine storms. In contrast to this profile we observed in Tregs, why does aggressive inflammation in COVID-19 not tend to decrease with disease severity? First, circulating Tregs can be expected to proliferate more in severe cases and have a stronger immunosuppressive effect, but there may be abnormalities in their migration to tissues and organs (especially the lungs). Some molecules with immunomodulatory properties, such as sphingosine-1-phosphate (S1P), can mediate migration of Tregs to the lungs. 37 Besides, the S1P molecule was found to be inversely correlated with disease severity in COVID-19, which may indicate a discrepancy in the efficient migration of Treg cells. 38 In addition, some membrane receptors enable

Tregs to undergo chemotaxis. Some chemokine receptors, such as CCR4, CCR5, CCR7, and CCR8, are effective for migration to the lung. 39 It is possible that there is a dysfunction in these membrane receptors that may affect migration to the lung. However, He et al. 40 and Ronit et al. 41 On the contrary, CD39-expressing Tregs decreased in the juvenile patients in an age-dependent manner (0-12 vs. 13-18).

Pediatric-specific risk factors for COVID-19 have not been adequately explored. A physiologic increase in immunoregulatory cells and a well-regulated immune system appear to be beneficial in SARS-CoV-2 infections in children and infants. 45 However, our current understanding of the immunopathogenesis of COVID-19 is still insufficient to clarify how children are protected from the symptoms of SARS-CoV-2 infection.

We also analyzed nTregs, eTregs, and non-Tregs based on CD45RA and FoxP3 expression. In adults, we observed an increase in these cell groups in direct proportion to the severity of the disease.

Interestingly, the percentage of non-Treg cells was increased 2.5-fold in the severe pneumonia group compared with the healthy group.

A downward trend in this cell group was observed in patients who followed up after recovery ( Figure S3 ). However, in juvenile patients, nTregs were significantly reduced at the time of illness. After recovery, a slight decrease in nTregs continued and a significant decrease in non-Tregs was observed ( Figure S4 ). Although the results in the eTreg and nTreg profiles in adults were fairly consistent throughout the study, the significant results in non-Tregs are of particular interest. Notably, non-Tregs have no immunosuppressive properties and produce proinflammatory cytokines, such as IL-2, IFN-γ, and IL-17. 17 These results may support the theory of "proinflammatory Tregs" in COVID-19.

Helios + Treg cells are known as thymus-derived Treg cells. Coexpression of Helios and FoxP3 enhances the immunosuppressive effect of Tregs. 46 CD45RA + Helios + FoxP3 + Tregs have been observed to increase in HIV-1 infection and have been reported to cause the release of PD-1 from monocytes. 47 In a study conducted in murines, an increase in Helios + FoxP3 + Tregs proliferating rapidly in the lungs and a correlation with TGF-β was found in pneumococcal pneumonia. 48 Moreover, in this study, Helios + FoxP3 + (gated on CD3 + CD4 + CD45RA + ) cells were found to be significantly higher in adult COVID-19 cases than in healthy controls. This raises the possibility that Helios + FoxP3 + Tregs expand rapidly in the peripheral circulation and lungs in COVID-19 and can be an early marker of disease progression.

Treg cell subsets, three important parameters emerged in severe pneumonia cases. It has been reported that LDH and ferritin levels F I G U R E 3 Graphical representation of statistical significance (p) and cell percentages (median and IQR) of Treg subsets of juvenile patient groups according to flow cytometry analysis data. In all analyses, p < 0.05 was considered to indicate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001). (A) CD25 high FoxP3 + and CD25 high CD127 low Tregs were not significantly altered in juvenile COVID-19 cases. (B) CD4 + CD39 + cells were significantly higher in the 13-18 age group than in the 0-12 age group. (C) CD39 + cells in the CD25 high FoxP3 + gate showed age-related changes. Moreover, the percentage in the age group 0-12 years was significantly lower than that in the healthy control group. (D) As a result of classification based on CD45RA and FoxP3, significant age-related changes were observed in nTregs. Moreover, nTregs were significantly lower in the patient groups than in the age-matched healthy controls. (E) Although no significant changes were observed in Helios + FoxP3 + cells, a decreasing trend was observed in juvenile patients. Moreover, CD45RA cells were significantly decreased in the patients of age group 13-18 years as compared to the patients of age group 0-12 years. J-COV 0-12, Juveniles 0-12 age; J-COV 13-18, Juveniles 13-18 age, J-HC 0-12, healthy juveniles 0-12 age; J-HC 13-18, healthy juveniles 13-18 age, J-COV Total, total juvenile patients; J-HC Total, total healthy controls increase with the severity of the disease in COVID-19 cases. 49, 50 In our study, Tregs were found to increase with disease severity and positively correlated with these parameters. Decreased platelet count at COVID-19 was associated with disease severity. 51 Consequently, a negative correlation between Tregs and platelet count was found in this study. In mild pneumonia and noncomplicated cases, there is a negative correlation between lymphocytes and Treg subsets (non-Tregs and CD25 high FoxP3 + cells, respectively) ( Figure S5 ). This may indicate an increase in Treg cell subsets despite lymphocytopenia.

Finally, we are well aware of some limitations of our study. First, the major limitation of this study is the small sample size of healthy controls. Second, the possible role of memory Tregs in COVID-19 was also a matter of interest to us. Patient samples were collected not only during the acute phase of COVID-19 but also during the con- Taken together, in our study, significant increases were discerned in Tregs and expression of CD39 in adult COVID-19 patients.

In juvenile patients, it was noticed that CD39 expression of Tregs tended to change in an age-dependent manner. We speculate that changes in the Treg profile of juveniles and adults at COVID-19 may 

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We would like to thank Deniz Gulkaya and Tugce Bozkurt for their technical support. This project was supported by a grant from the Foundation for Scientific Research Projects (BAP) of the Bursa Uludag University of Turkey (Project No. OUAP [T]-2020/6).

The authors declare that there are no conflict of interests.

This study protocol was approved by the Ethics Committee of Bursa Uludag University, Faculty of Medicine (Permit number: 2020-7/9) Bursa, Turkey, and all subjects provided written informed consent. 

Budak. All listed authors reviewed and revised the manuscript.

The data that support the findings of this study are available from the corresponding author upon reasonable request.