Regulatory T Cell Dynamics in Murine Model of Foreign Body Response

Introduction: The foreign body response (FBR) is an inflammatory process that occurs after implantation of a material in the body. Implanted medical devices are a rapidly growing industry, and a conservative estimate of device failure due to fibrosis is 10% [1]. The FBR has conventionally been described as an innate immune response. Recently, it has been revealed that T cells, part of the adaptive immune system, are involved in fibrosis in many different tissues and disease contexts [2]. Regulatory T cells (Tregs) play a role in tissue repair and regeneration and modulate immune responses [3]. As part of the adaptive immune response, T cells possess a T cell receptor (TCR) that recognizes specific antigens; despite increased interest in the role of T cells, little is known about the potential role of antigen specificity in the context of the FBR. Previous work suggests TCR repertoire may play a role in the T cell response to injury and material implant [4].

Paired single-cell RNA sequencing (scRNAseq) and TCR sequencing (scTCRseq) collected from a murine model of fibrosis over multiple time points gives insight into the dynamics of T cell phenotype and TCR repertoire over time. Through analysis of sequencing data, we identify the role that Tregs play in the development of fibrosis and infer methods through which they impact other cells. Increased understanding of the role of Treg phenotype and antigen specificity in fibrosis, along with predicted communication pathways, may provide potential targets for therapeutic interventions to alter the FBR.

Materials and

Methods: Volumetric muscle loss (VML): Mice given bilaterial defect to quadricep femoris, treated with saline (as injury control) or synthetic material polycaprolactone (PCL, < 600μm, MW 50k) to induce fibrosis.

T cell scRNAseq/scTCRseq: C57BL6 mice given VML; muscle harvested at 1- (saline, PCL), 6- and 12- week (PCL only) timepoints then enzymatically and mechanically dissociated. Using fluorescence activated cell sorting for live, CD45+ CD3+ low side scatter cells, T cells were isolated then sequenced using 10x Genomics Chromium 5’v1.1 for gene expression and TCR sequence.

Bulk sequencing: VML with PCL given to C57BL6 and Foxp3-DTR mice; DT administered 3 days before the quadricep harvested at 6 weeks for bulk mRNA sequencing. Data aligned with STAR and differential expression analyzed with DESeq2 [5]. Hallmark [6] gene sets used for ranked gene set enrichment analysis using fgsea [7].

FBR scRNAseq: VML with PCL administered to C57BL6 mice and harvested at 6 weeks, then dissociated into single cell suspension. Bead sorting for CD45 performed and CD45+/- populations mixed 50/50. Samples sequenced using 10x Genomics Chromium 3’ v3.1.

scRNAseq analysis: Data aligned to mouse genome using Cell Ranger (10x Genomics). Gene expression analyzed in R with Seurat [8]. TCRs analyzed with scRepertoire [9], transcription factor regulons inferred and scored using SCENIC [10], and cell-cell communication pathways predicted with dominoSignal [11].

Results, Conclusions, and Discussions: We conducted an analysis of T cell populations over a 12-week period in a murine model of the foreign body response (FBR). By sequencing more than 18,000 cells at 1-, 6-, and 12-weeks post-implantation, we identified CD8, CD4, and regulatory T cell (Treg) populations, along with unconventional T cells (Fig. 1A). Our compositional analysis revealed a significant increase in Tregs between 1 and 6 weeks, persisting through 12 weeks (Fig. 1B). Subclustering of Tregs identified distinct subpopulations with varied roles in the fibrotic environment, ranging from lymph node homing to effector to proliferating (Fig. 1C). Transcription factor analysis pinpointed the Treg 2 subset as recently activated through the T cell receptor (TCR), characterized by heightened Nfkb and Rel activation. Treg 1 and Treg 2 exhibited rapid expansion between 1 and 6 weeks that persisted through 12 weeks. (Fig. 1D).

TCR repertoire analysis unveiled Tregs as the population with the largest clones, and analysis across timepoints highlighted clonal expansion in Tregs consistent with the expanded cell numbers (Fig. 1E). Treg 2, activated through the TCR, contained the most expanded clones. Comparison of these expanded sequences with known antigen databases did not yield matches, suggesting that the TCRs may recognize novel antigens.

Bulk RNA sequencing of the fibrosis in a Treg depletion model identified numerous differentially expressed genes (Fig. 1F); while some genes are present in Tregs (Foxp3, Ctla4, Icos), other genes (Bcl2l11, Tgtp1, Cyp7b1) are involved in processes such as cell division, interferon response, and metabolism. Ranked gene set enrichment analysis highlights Tregs' impact on different functional pathways, including hypoxia, myogenesis, adipogenesis, and fatty acid metabolism (Fig. 1G). To elucidate Tregs' interactions within the FBR, we employed cell communication inference alongside scRNAseq data of all cell types in the environment. Further analysis will detect distinct communication pathways between different Treg populations, with unique ligand exchanges both within and across cell types. Identification of specific pathways of interest will facilitate the selection of perturbation targets. Our findings provide increased understanding of Treg dynamics and functionality in the FBR, offering potential avenues for therapeutic exploration.

Acknowledgements (Optional): The authors would like to acknowledge funding from Morton Goldberg Chair, Bloomberg Kimmel Institute for Cancer Immunotherapy, NIH Pioneer (DP1AR076959), and NIBIB R01 (R01EB028796) to JHE. JCM is funded by K99AG081564. AR was supported by NSF-GRFP award #DGE-1746891 at the time of study.

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