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. 2024 Feb 7;25(4):2031. doi: 10.3390/ijms25042031

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© 2024 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

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Treg phenotypical characterization and differentiation. (A) SARS-CoV-2 spike-specific Treg were phenotypically characterized 48 h after culture with peptides in PBMC of samples from subjects 13 and 16 and of non-spike Treg (specific for pool 1 and pool 2) in samples from subject 16. The markers were chosen to determine Treg ontogeny, possibly reverting from pro-inflammatory T cells (pTreg) or arising from naïve T cells primed in tissues that synthesize spike proteins. Surprisingly, FOXP3+ Treg expressed CTLA-4 in a very small percentage of cells, but not PD-1, and the results were negative for IL-7R and CD45RA, suggesting that they arise from naïve T cells, rather than reverting from pro-inflammatory T cells. (B) Treg from subject 14 were also studied to address the role of IL-2 in the expression of PD-1 and were studied under IL-2 feeding (12.5 and 50 U/mL) during T cell priming. Surprisingly, IL-2 does not influence the expression of PD-1 on Treg, which may be a unique feature in pregnancy. (C) The PBMC from subject 19 were kept for four days in culture in the absence of IL-2 after T cell priming to determine the possible conversion of pTreg from pro-inflammatory T cells over time. IL-7R + Treg expanded rapidly, with many reverting to double positive IL-7R+CD45RA+ as classical pTreg. In pTreg, CTLA-4 and PD-1 expression was also very low.

Treg phenotypical characterization and differentiation. (A) SARS-CoV-2 spike-specific Treg were phenotypically characterized 48 h after culture with peptides in PBMC of samples from subjects 13 and 16 and of non-spike Treg (specific for pool 1 and pool 2) in samples from subject 16. The markers were chosen to determine Treg ontogeny, possibly reverting from pro-inflammatory T cells (pTreg) or arising from naïve T cells primed in tissues that synthesize spike proteins. Surprisingly, FOXP3+ Treg expressed CTLA-4 in a very small percentage of cells, but not PD-1, and the results were negative for IL-7R and CD45RA, suggesting that they arise from naïve T cells, rather than reverting from pro-inflammatory T cells. (B) Treg from subject 14 were also studied to address the role of IL-2 in the expression of PD-1 and were studied under IL-2 feeding (12.5 and 50 U/mL) during T cell priming. Surprisingly, IL-2 does not influence the expression of PD-1 on Treg, which may be a unique feature in pregnancy. (C) The PBMC from subject 19 were kept for four days in culture in the absence of IL-2 after T cell priming to determine the possible conversion of pTreg from pro-inflammatory T cells over time. IL-7R + Treg expanded rapidly, with many reverting to double positive IL-7R+CD45RA+ as classical pTreg. In pTreg, CTLA-4 and PD-1 expression was also very low.