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. 2021 Nov 18;9:751590. doi: 10.3389/fcell.2021.751590

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Copyright © 2021 Ou, Hamo, Schulze, Roemhild, Kaiser, Gasparoni, Salhab, Zarrinrad, Amini, Schlickeiser, Streitz, Walter, Volk, Schmueck-Henneresse, Reinke and Polansky.

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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(A) Example manufacturing run for first-generation Treg products. Briefly, Tregs were enriched through CD8⁺ depletion followed by CD25⁺ enrichment using the CliniMACS^® system from whole blood. Tregs are cultured over the course of 23 days, with 8 total stimulations with anti-CD3/CD28 microbeads. Time points used for DNA methylation analysis are indicated with the gray arrows. Figure created with BioRender.com. (B) PCA analysis of samples taken from all available time points from both manufacturing runs. (C,D) Median methylation values of partially methylated domains (PMDs) (left) and cumulated expansion rate (right) during Treg product generation.