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. 2022 May 18;12:893044. doi: 10.3389/fcimb.2022.893044

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Copyright © 2022 Russo, Raiden, Algieri, De Carli, Davenport, Sarli, Bruera, Seery, Sananez, Simaz, Bayle, Nivela, Ferrero, Geffner and Arruvito

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Extracellular ATP-P2X7R axis in children with COVID-19. (A) Left panel: Levels of ATP in plasma from healthy children (controls, n = 14), and children with COVID-19 (non-severe, n = 27 and severe, n = 9) measured by luminometry. Right panel: PBMCs (5x10⁶/mL) from healthy children (controls, n = 12), and children with COVID-19 (non-severe, n = 25 and severe, n = 12) were stimulated with anti-CD2/CD3/CD28 coated beads (0.3 µg/mL) during 4 min. Levels of extracellular ATP were measured in the supernatant by luminometry. (B) Left panel: Confocal microscopy of P2X7R expression in purified CD4+ T cells (green: CD4, red: P2X7R). Nuclear counterstain with DAPI was used. Representative images from a child with non-severe COVID-19 are shown at x300. Right panel: Basal P2X7R expression in PBMCs from healthy children (controls, n = 10) and children with COVID-19 (non-severe, n = 17 and severe, n = 7) quantified by qRT-PCR. (C) Left panel: Representative dot plot showing the expression of FoxP3 and CD39 in CD4+ T cells of heathy children and children with COVID-19 (non-severe and severe). Middle and Right panels: Frequency of CD4+CD39+FoxP3- T cells (middle) and CD4+CD39+FoxP3+ T cells (right) in healthy children (n = 12), and children with COVID-19 (non-severe, n = 29 and severe, n = 11). Data are expressed as percentage of CD4+ T cells. (D) Purified CD4+ T cells (1×10⁶/mL) from children with COVID-19 (n = 15) were stimulated with anti-CD2/CD3/CD28 coated beads (0.035 µg/mL) and treated or not with BzATP (100 µM) during 3 days. Levels of cytokines were quantified in the culture supernatant by multiplex flow cytometric bead array. (E) Graphs showing correlations between the frequency of CD4+CD25+FoxP3+ Tregs (n = 40, left), CD4+ROR-γt+ T cells (n = 24, middle), and CD4+CD39+FoxP3- T cells (n = 40, right) and the levels of ATP in plasma. Mean ± SEM of n donors are shown in (A, B) (right), (C) (middle and right) and (D) P values were determined by Mann-Whitney U test, Kruskal-Wallis test and Wilcoxon test. Correlations were evaluated by using Spearman rank correlation coefficient test. *p<0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.