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. 2022 Mar 11;13:836646. doi: 10.3389/fimmu.2022.836646

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Copyright © 2022 Dohnke, Moehser, Surnov, Kurth, Jessberger, Kretschmer and Garbe

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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Treg-cell-mediated suppression of osteoclastogenesis in vitro. Bone marrow-derived macrophages (BMMs) were cultured in the absence or presence of FACS-purified Swap-70^+/- or Swap-70^-/- Treg cells. (A) Representative images of tartrate-resistant acid-phosphatase (TRAP) stained osteoclast differentiation cultures without (left) or with Treg cells (middle: Swap-70^+/- , right: Swap-70^-/- Treg cells) at day 6. (B). Graph illustrates the impact of Treg cells on osteoclast formation assessed by the analysis of osteoclast areas (the number of TRAP-positive multinucleated cells) from co-cultures with Swap-70^+/+ (blue) or Swap-70^-/- (red) Treg cells normalized to control cultures without Treg cells (black) (n= 3, mean values with SD shown). (C) Bone resorption in vitro assay: Pre-osteoclast-Treg cell co-culture was seeded on bone slices at different ratios, and carboxy-terminal collagen crosslinks (CTX) in the day 5 supernatant from one sample of each titration step were measured by ELISA. (D) The bone slices of the co-culture shown in (C) were harvested at day 7, dried, coated and analyzed by SEM. (E–G) Treg-cell-mediated downregulation of CD80/86 on pre-osteoclasts. BMMs were co-cultured with Swap-70^+/- or Swap-70^-/- Treg cells, harvested and analyzed by flow cytometry at the indicated time points. (E) Representative histogram overlays of CD80 (top), CD86 (middle) and MCSF receptor (cfms, bottom) expression on electronically gated CD4^-Foxp3^GFP-CD11b⁺ cells from osteoclast differentiation cultures cultured without (grey) or with Treg cells (Swap-70^+/- blue; Swap-70^-/- red) at indicated time points. Numbers in histograms indicate representative MFIs of gated cells. (F) Graphs show expression levels of CD80, CD86, cfms and CD11b (MFI) for indicated culture conditions and timepoints (one or two data points per condition from continuous co-culture). (G) Representative imaging flow cytometry of osteoclast-Treg cells co-culture derived cell suspensions that were analyzed for CD11b, Foxp3^GFP, CTLA-4 and CD80 expression, showing single cell (top left: Foxp3⁺ Treg cell, bottom left: pre-osteoclast) and cluster images (top right: pre-osteoclast - Swap-70^+/+ Treg cell interaction, bottom right: pre-osteoclast - Swap-70^-/- Treg cell interaction).