"Regulatory T cells (Tregs) are immuno-suppressive cells which have been recently rediscovered as pro-regenerative cells. When accumulating in injured tissue, Tregs express numerous pro-healing factors such as anti-inflammatory molecules and cytokines. Interestingly, we found that a growth factor (GF) is expressed by Tregs across multiple injured tissues in the mouse. Therefore, we hypothesized that delivering a recombinant form of the GF into damaged tissues would promote healing.
In order to optimize the activity of the GF following delivery in tissues, we first aimed at improving its signalling, utilizing rational protein engineering. As a strategy, we modified the receptor-binding site of the GF taking inspiration from the receptor-binding site of a high-affinity ligand of the same receptor, in order to generate a new high affinity variant (GFhi). The improved affinity of GFhi for the receptor was confirmed with various binding assays. A cell line was used to investigate the nature of the signalling through experiments of receptor internalization/degradation and phosphorylation. The regenerative capacity of GFhi compared to the wild-type GF was tested in mouse models of bone regeneration (calvarial defect), muscle regeneration (volumetric muscle loss of quadriceps) and skin repair (full-thickness wound). The GFs were delivered via a fibrin hydrogel in bone and muscle and via intradermal injections in the skin. To understand the mechanisms behind GFhi regenerative capacity, we tested its ability to affect proliferation of stem/progenitor cells which are important for the healing process such as keratinocytes, myoblasts, mesenchymal stem cells, and endothelial cells. We also investigated if GFhi could modulate macrophage activity, since they are key cells involved in tissue repair and regeneration. On this regard, we assessed GFhi regenerative potential in mice lacking its receptor on myeloid cells.
We show that GFhi has a higher binding affinity for its receptor compared to the wild-type GF. GFhi also shows a higher ability to induce receptor internalization/degradation and phosphorylation compared to the wild-type GF. In vivo experiments show that local delivery of GFhi promotes a more complete healing compared to the delivery of the wild-type GF in bone, muscle and skin models. Mechanistically, GFhi showed to have some proliferation effect on stem/progenitor cell. More interestingly, preliminary data where GFhi was delivered in mice lacking the GF receptor on myeloid cells indicates that the engineered GF likely promotes regeneration via modulating macrophage activity. Overall, this study shows that delivering a factor highly expressed by Tregs is able to induce tissue regeneration. Moreover, engineering the activity of key Treg-derived factors is a promising strategy for regenerative medicine applications."