Assessment of CD39 expression in regulatory T-cell subsets by disease severity in adult and juvenile COVID-19 cases

Abstract

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.

Keywords: CD39; COVID-19; FoxP3; Tregs; infectious diseases.

Figure 1

Flow cytometry gating strategy for analysis of Treg subsets. (A) Lymphocytes were separated based on CD45 and SSC characteristics. (B) CD39⁺, CD25^highFoxP3⁺, CD25^highCD127^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^lowCD39⁺ cells were separated at the CD25^highFoxP3⁺ gate. (D) Helios and FoxP3 were used to separate thymic Tregs

Figure 2

Graphical representation of statistical significance (p) and cell percentages (median and IQR) of Treg subsets of adult 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^highFoxP3⁺ and CD25^highCD127^low Tregs were increased in COVID‐19 cases. (B) CD4⁺CD39⁺ cells were also significantly increased. (C) CD39⁺ cells in the CD25^highFoxP3⁺ gate increased with disease severity. (D) As a result of classification based on CD45RA and FoxP3, nTregs, eTregs, and non‐Tregs increased with disease severity. (E) Helios⁺FoxP3⁺ cells increased with the severity of the disease. AD‐HC, adult healthy control; AD‐M, mild pneumonia adult cases; AD‐NC, noncomplicated adult cases; AD‐SEV, severe pneumonia adult cases; IQR, interquartile range

Figure 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^highFoxP3⁺ and CD25^highCD127^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^highFoxP3⁺ 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