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. 2022 Feb 14;13:856. doi: 10.1038/s41467-022-28338-0

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Red and blue histograms represent fluorescence of CM-XRos in unstained Tregs and stained ASCs, respectively. Representative histograms from 1 of 7 experiments are shown. Uptake of active mitochondria is visualized as an increase in CM-XRos fluorescence in unstained Tregs after coculture with labelled ASCs. The percentage of Tregs that internalized active mitochondria derived from ASCs is given for each histogram. Unstained Tregs cultured separately served as a negative control (Solo-MIX) and were mixed with stained ASCs immediately before the analysis with flow cytometry to show Treg autofluorescence and fluorescence of labelled ASCs on the same graph. Simultaneously, stained ASCs were cocultured with unstained Tregs for 72 h in direct (Direct) and indirect contact (Indirect). The effect of 18β-glycyrrhetinic acid (18β 200 μM) and latrunculin A (LA 10 μM) on mitochondria transfer was tested in direct cocultures. b Uptake of ASC-derived active (MIG⁺CM-XRos⁺, grey-shaded graphs) and inactive (MIG⁺CM-XRos⁻, white graphs) mitochondria by Tregs in various culture conditions is depicted. Data for seven independent experiments were calculated using a two-sided Mann–Whitney U test with correction. *p < 0.05 for uptake of active or inactive mitochondria by Tregs from direct cocultures with and without inhibitors vs Tregs from monocultures and indirect cocultures. **p < 0.05 for uptake of active vs inactive mitochondria by Tregs in the a given culture condition. c–f Representative confocal micrographs from 1 of 4 experiments show MIG and CM-H2XRos labelled ASCs and unstained Tregs that became MIG⁺CM-XRos⁺ after incorporation of mitochondria from labelled ASCs in direct cell-to-cell coculture. Exemplary Tregs are highlighted with the arrows. c Green fluorescence of MIG (total mitochondrial mass). d Red fluorescence of CM-XRos (active mitochondria). e Light micrograph. f Merged view from three channels. g Positive correlations between the uptake of active allogeneic mitochondria by Tregs and HLA eplet mismatch load calculated with Spearman’s rank correlation, R and p values are given. Box plots indicate median (symbol within the box), 25th, 75th percentile (box), minimum and maximum values (whiskers). Source data are provided as a Source Data file.