Gamma Delta T Cells Modulate the Foreign Body Response to Synthetic Biomaterial Implants

Introduction: Synthetic polymer materials are used widely in medical applications, but often suffer from fibrosis induced by the foreign body response (FBR) that can ultimately compromise clinical efficacy. The FBR is a chronic inflammatory host defense reaction mediated by diverse immune and stromal cell populations, which typically terminates in fibrotic encapsulation of the foreign material. A recent study in our group highlights the synergistic role of interleukin (IL)17-secreting cells (i.e., CD4 Th17, gamma delta T cells, and ILC3) and senescent stromal cells in potentiating FBR outcomes in human fibrotic capsules surrounding synthetic breast implants [1]. Using a pre-clinical murine volumetric muscle loss injury model with synthetic biomaterial implants (primarily polycaprolactone, PCL), our group has observed chronic inflammation and fibrosis linked to IL17, senescent cell accumulation, and prolonged granulocyte infiltration [1,2].

Gamma delta (gd) T cells contribute to numerous fibrotic diseases through IL17 production and myriad of other effector functions ranging from innate- to adaptive-like behavior [3]; however, their functional role in the FBR remains largely unknown. Furthermore, host factors such as obesity and associated comorbidities can exacerbate implant-associated fibrosis [4]. Interestingly, gd T cells have a unique ability to sense a broad antigen pool, including lipids, and utilize oxidative metabolism [5]. Thus, this work aims to better understand how gd T cells contribute to the FBR and how their response may be impacted by high-fat diet-induced obesity.

Materials and

Methods: PCL particulate ( < 600um, MW 50,000) was implanted into bilateral murine quadricep femoris muscle defects (3x3x3 mm^3). Muscles with associated PCL implants were harvested at various timepoints (1, 6, and 12-weeks post-implantation), dissociated (mechanical and enzymatic), and processed with density gradient for lymphocyte enrichment. T cells (LiveCD45+CD3+SSClo) were isolated via fluorescence-activated cell sorting (FACS) followed by paired single-cell RNA and TCR sequencing (10x Genomics, Chromium 5’v1.1). Sequencing data was aligned to mouse genome using Cell Ranger (10x Genomics) and gene expression analyzed in R with Seurat. gd T cells were identified by gamma-delta TCR and/or delta chain gene expression (without corresponding alpha-beta TCR or chain genes) followed by computational sub-clustering. High-parameter spectral flow cytometry was utilized to validate phenotype and kinetics of the identified gd T cell subsets including Vg chains, cluster-specific surface markers, and cytokine production following ex vivo stimulation. To assess the functional contribution of gd T cells, bulk RNA sequencing was conducted on FACS-isolated endothelial (CD45-CD29+CD31+) and stromal (CD45-CD29+CD31-) cells from wildtype and TCRdelta-/- mice 6-weeks after PCL implant. Sequencing data was aligned with STAR and differential gene expression analyzed with DESeq2. Formalin-fixed, paraffin-embedded tissue samples were stained using Masson’s Trichrome (collagen deposition), anti-ColXI antibody, and anti-CD31 antibody (endothelial cells). Communication between gd T cells and fibroblasts was modeled and perturbed using an in vitro Transwell co-culture platform. High fat diet-induced obesity studies fed mice 60 kcal% fat chow (control 10%).

Results, Conclusions, and Discussions: Single-cell transcriptomics identified several gd T cell clusters within the murine PCL implant encapsulating fibrotic tissue including multiple IL17-expressing subsets (tissue-resident Vg6, Vg4, and interferon-stimulated Vg6) and an interferon-gamma (IFNg)-expressing subset (Fig A). The gd17 subsets differentially upregulated Il17a, Rorc, Il7r (encodes CD127), while the gdIFNg uniquely expressed Ifng, Tbx21, and Klrb1c (encodes NK1.1) (Fig B). Flow cytometric analysis showed divergent kinetics of gd T cell subtypes with gdIFNg (CD44+NK1.1+, IFNg+) peaking acutely (3-days and 1-week) and gd17 (CD44hiCD127+, IL17a+) persisting at chronic (6- and 12-week) implant timepoints (Fig C). High fat diet-induced obesity promoted ectopic fat deposition within the implant-associated fibrotic tissue (Fig D), upregulation of inflammatory genes (e.g., Il1b, Tnf, and Nos2), and expansion of gd T cells (Fig E). Additionally, gd T cell production of IL17a following ex vivo stimulation significantly increased with high fat diet.

Bulk RNA sequencing analysis revealed differential expression of collagen- and extracellular matrix (ECM)-related genes in stromal cells isolated from TCRdelta-/- mice (Fig F). Immunofluorescence staining confirmed a decrease in collagen XI within the implant-associated fibrotic tissue of TCRdelta-/- mice (Fig G) and in vitro co-cultures demonstrated that gdIFNg versus gd17 can differentially influence fibroblast expression of Il6 and collagen (e.g., Col1a1, Col2a1, and Col3a1) genes. Additionally, TCRdelta-/- mice exhibited higher endothelial cell counts, enlarged vessels within the implant-associated fibrotic tissue (Fig H), and increased endothelial cell expression of prostaglandins and inflammatory genes.

In conclusion, gd T cells associated with fibrotic implant capsules primarily displayed a gd17 phenotype and obesity may further impact implant-associated fibrosis by driving gd17 expansion. Genetic elimination of gd T cells influenced the vascularity and ECM composition of the encapsulating fibrotic tissue suggesting that gd T cell-stromal interactions may modulate FBR intensity. Ultimately, further investigation of gd T cell phenotypes and their functional consequences in the FBR may help inform implant material design and develop gd T cell targeting therapies to mitigate chronic fibrosis.

Acknowledgements (Optional):

References: [1] Chung. Sci Trans Med. 2020;12, [2] Sadtler. Biomaterials. 2019;192, [3] Zhang. Front Imm. 2020;11, [4] Orellano. Obesity. 2018;26, [5] Lopes. Nat Imm. 2021;22.

The authors would like to acknowledge funding from Morton Goldberg Chair (JHE), Bloomberg Kimmel Institute for Cancer Immunotherapy and NIH Pioneer (DP1AR076959) (JHE). AR is funded by NSF GRFP DGE-1746891. JCM is funded by K99AG081564.