Transcriptional characterization of tissue-Tregs and whole tissues of skeletal muscles in muscle Treg TCR transgenic mice.

Foxp3+CD4+ regulatory T cells (Tregs) play important roles in controlling both homeostatic processes and immune responses at the tissue and organismal levels. For example, Tregs promote muscle regeneration in acute or chronic injury models by direct effects on local muscle progenitor cells as well as on infiltrating inflammatory cells. Muscle Tregs have a transcriptome, a T cell receptor (TCR) repertoire and effector capabilities distinct from those of classical, lymphoid-organ Tregs, but it has proven difficult to study the provenance and functions of these unique features due to the rarity of muscle Tregs and their fragility upon isolation. Here, we attempted to side-step these hindrances by generating, characterizing and employing a line of mice carrying rearranged transgenes encoding the TCRα and TCRβ chains from a Treg clone rapidly and specifically expanded within acutely injured hindlimb muscle of young mice. Tregs displaying the transgene-encoded TCR preferentially accumulated in injured hindlimb muscle in a TCR-dependent manner both in the straight transgenic model and in adoptive-transfer systems; non-Treg CD4+ T cells expressing the same TCR did not specifically localize in injured muscle. The definitive muscle-Treg transcriptome was not established until the transgenic Tregs inhabited muscle. When crossed onto the mdx model of Duchenne muscular dystrophy, the muscle-Treg TCR transgenes drove enhanced accumulation of Tregs in hindlimb muscles and improved muscle regeneration. These findings invoke the possibility of harnessing muscle Tregs or their TCRs for treatment of skeletal muscle pathologies. For RNA-seq library construction of Tregs, Vβ8+ Tregs or total Tregs were double-sorted by Moflo from muscle and spleen of Tg+ or Tg-, respectively, Foxp3-GFP mice 3 days after Ctx-injury. 2x10^3 cells were lysed with TCL buffer (Qiagen) containing 1% 2-mercaptoethanol (Sigma) and used for generating libraries. For whole-muscle RNA-seq library construction, muscles of Tg+ or Tg- mice were collected 8 days after Ctx-injury. Total RNAs were isolated using Trizol (Invitrogen) according to the manufacturer’s instructions. 5ng of RNA was used for generating each library, as above. Sequencing was performed on an Illumina NextSeq500 using the 2x25 base-pair read option.