Regulatory CD8+ T cells are Preferentially Recruited by Pro-Regenerative ECM Biomaterials After Muscle Trauma

Introduction: T cells that react to self-antigens are deleted within the thymus during development. However, a fraction of T cells escape thymic deletion and recognize peripheral tissue antigens leading to autoimmunity. It is known that a subset of patients who receive synthetic biomaterial implants develop autoimmune disease. However, mechanisms that prevent peripheral autoimmunity in the vast majority of patients are not well understood. Here, we sought to uncover mechanisms of antigen presentation and role of the regulatory T cells in maintaining self-tolerance after trauma.

Materials and

Methods: We used a model of volumetric muscle loss injury and reconstruction (wherein wild-type C57BL/6 mice and Aire -/- mice received either an untreated control injury, pro-inflammatory polyethylene (PE) or pro-regenerative decellularized extracellular matrix (ECM) treatment), and collected muscle tissue 1 week post-injury and performed flow cytometry on single cell suspensions of all muscle cells, and single cell RNA sequencing on sorted CD45+ CD11c+ MHC-II+ dendritic cells (DCs) and single cell- T cell receptor sequencing on CD45+ CD11b- CD11c- lymphocytes. Spatial transcriptomics was performed on 5μm cross-sections of muscle with all treatment groups as well as uninjured muscle. Soluble proteins were extracted in RIPA buffer in the presence of protease inhibitors. Proteins were diluted to 1 μg/mL and loaded on bone marrow derived, FLT3-L induced dendritic cells which were co-cultured for 72 hours with sorted CD8+ Ly49+, CD8+Ly49- and CD4+ populations separately.

Results, Conclusions, and Discussions: Single cell RNA sequencing of DCs revealed 10 subsets (Fig. 1A) that were recruited to the muscle in a material dependent manner. Tolerogenic DCs including cDC1s expressing XCR1 and CD103 as well as DCs enriched in scavenger receptors along with plasmacytoid DCs were enriched by ECM treatment. In contrast polyethylene treatment enriched DCs that had poor efferocytosis capacity. From muscle lymphocytes, we observed that a non-canonical, receptor diverse CD8 T cell subset expressing Ikzf2, Xcl1 and Ccl5 (CD8 Tregs) was enriched by ECM and outnumbered conventional Foxp3+ CD4+ Tregs (Fig.1B). Mice which received PE displayed attenuated recruitment of CD8 Tregs in the muscle which led to establishment of autoreactive B cells and T cells. We Also found the transcriptional signature derived from our dataset in publicly available datasets of mouse skin wounds and inflammation induced myometrium damage suggesting that CD8 Tregs are part of a tissue conserved damage response. Further, transcriptional markers of CD8 Tregs were spatially coincident with cDC1 signature (Fig.1C). Additionally, CD8 Tregs are largely depleted but not totally abrogated in the blood, spleen and inguinal lymph node of Aire -/- mice which underwent VML suggesting thymic origin (Fig.1D). Lastly, these cells exhibited a more robust proliferation reaction to soluble ECM loaded FLT3-L induced cDC1s in-vitro (Fig.1E) suggesting that cDC1s cross present ECM peptides to CD8 Tregs. Taken together, our analyses suggest a tolerogenic role for CD8 Tregs in the skeletal muscle after trauma.

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