Elucidating T cell phenotype and function in frozen shoulder

themes

2, 5

project overview

Frozen shoulder is a disabling condition affecting 10% of the working population. Disease causes significant pain and immobility of the shoulder joint, reducing life quality of affected patients. Frozen Shoulder is an inflammatory fibrotic disease localised to the shoulder joint capsule. Curiously the disease is self-limiting, as symptoms almost always resolve, albeit over 2-3 years. Frozen shoulder is therefore a unique example of a chronic inflammatory fibrotic disease that resolves. The cellular basis underpinning how inflammatory fibrosis resolves in frozen shoulder is currently unknown. Understanding this cellular basis of resolution will 1)identify new treatments to accelerate resolution of frozen shoulder and 2)inform the biological cues to push persistent inflammatory fibrotic diseases like arthritis down a resolving pathway.

In the absence of animal models that accurately recapitulate human disease, we set up the ICECAP clinical study, enabling us to collect well-phenotyped shoulder capsule tissues from patients undergoing surgery for frozen shoulder. We also collect comparator capsular tissues from patients undergoing shoulder stabilisation or arthroplasty procedures. Our pilot scRNAseq data identify that the human shoulder capsule is comprised of distinct tissue-resident stromal cell subsets. We have identified a unique subset of CD3+CD8+CD69+ T cells which appear to be resident in the capsule. These cells also highly express GRANZYME K, GRANULYSIN, IL7R, CXCR4 and KLRB1 (Figure 1). We confirmed expression of these proteins in sections of frozen shoulder patient tissues using ChipCytometry (Figure 2A&B). These T cells exhibit a profile akin to the SCT5 subset identified by Zhang et al. in synovial tissues from patients with rheumatoid arthritis¹. This preliminary data suggests that T cells in frozen shoulder may be enriched for cytotoxicity. However their precise phenotype(s), biological function(s) and how these cells might change in frozen shoulder remain unknown. Pereira et al. identified that Sestrins can induce the re-programming of non-proliferative senescent-like CD8+ T cells, enabling them to acquire broad-spectrum, innate-like killing activity². We therefore hypothesise that T cells in the shoulder capsule are implicated in killing senescent capsular fibroblasts, contributing to resolution processes during frozen shoulder.

The over-arching aim of this project is to elucidate the biological role of T cells in the resolution of frozen shoulder. The specific objectives to address this aim are to:

1. Expand the scRNAseq dataset to identify transcriptomic T cell signature(s) in capsular tissues collected from non-diseased comparator and frozen shoulder patient tissues.
2. Confirm T cell protein signatures in sections of capsular tissues from comparator and frozen shoulder patients
3. Use organoid cultures comprised of patient-derived cells to understand how T cells interact with capsular stromal cells to resolve inflammatory fibrosis in frozen shoulder
4. Bioinformatically compare the profiles of capsular T cells in resolving frozen shoulder with T cells in non-resolving fibrotic diseases

In addition to discovering new therapeutic strategies for frozen shoulder, this work will also provide novel insights into the cellular mechanisms of intractable soft tissue inflammatory and fibrotic diseases affecting the lung, liver, kidney and skin which ultimately contribute to 45% of all-cause mortality³, leading towards potential new treatment paradigms.

Figure 1. Profile of capsular T cells identified by scRNAseq. Violin plots showing differentially expressed genes in T cells residing within the shoulder joint capsule. Data are generated from tissues collected from 6 non-diseased comparator and 3 frozen shoulder donors.

Figure 2A. ChipCytometry immunostaining of T cell markers in cryosections of frozen shoulder patient tissues.

Figure 2A. Immunostaining of frozen shoulder patient tissues for T cell markers. Representative image shows GZMK (yellow), CD18 (green), CD161 (KLRB1, white), CD3 (violet), CD45 (red) and vascular endothelial marker CD31 (blue). Note the perivascular location of identified T cells. Scale bar = 50μm.

Figure 2B. Immunostaining of frozen shoulder patient tissues for T cell markers. Representative image shows CD18 (green) CD161 (KLRB1, red), CD2 (blue), CD5 (orange), CD3 (magenta), CD8 (white). Scale bar = 50μm.

keywords

Musculoskeletal, inflammation, fibrosis, T cells, frozen shoulder

training opportunities

This project represents an excellent training opportunity for a young scientist with an interest in biology and bioinformatics. Training will be provided in the following aspects:

1. Preparation of capsular patient tissues for NGS and immunostaining
2. Analysis of Next Generation Sequencing (NGS) data sets for mechanistic study of T cell gene function
3. Bioinformatic modelling of T cell focused ligand-receptor and protein-protein interactions
4. Multiplex imaging of stained capsular tissues

Dakin has significant experience in DPhil supervision, having successfully supervised 8 DPhil students over the past 6 years and has 2 current DPhil students (due to complete in 2022 and 2023). Buckley and Coles have extensive supervision experience, having successfully supervised 14 & 20 DPhil students respectively. The Dakin, Buckley & Coles labs possess the expertise and access to necessary patient tissue samples, resources and equipment required for wet-lab based experiments to complete this project.

key publications

1. Croft AP, Campos J, Jansen K, Turner JD, Marshall J, Attar M, Savary L, Wehmeyer C, Naylor AJ, Kemble S, Begum J, Dürholz K, Perlman H, Barone F, McGettrick HM, Fearon DT, Wei K, Raychaudhuri S, Korsunsky I, Brenner MB, Coles M, Sansom SN, Filer A, Buckley CD. Distinct fibroblast subsets drive inflammation and damage in arthritis. Nature. 2019 Jun;570(7760):246-251. doi: 10.1038/s41586-019-1263-7. Epub 2019 May 29. PMID: 31142839; PMCID: PMC6690841.
2. Dakin SG, Coles M, Sherlock JP, Powrie F, Carr AJ, Buckley CD (2018). Pathogenic stromal cells as therapeutic targets in joint inflammation. Nat Rev Rheumatol. Dec;14(12):714-726. doi: 10.1038/s41584-018-0112-7.
3. Dakin SG, Rangan A, Martinez F, Brealey S, Northgraves M, Kottam L, Cooper C, Buckley CD, Carr AJ. (2019) Tissue inflammation signatures point towards resolution in adhesive capsulitis. Rheumatology (Oxford). 2019 Jan 27. doi: 10.1093/rheumatology/kez007.
4. Dakin SG, Martinez FO, Yapp C, Wells G, Oppermann U, Dean BJF, Smith RDJ, Wheway K, Watkins B, Roche L, Carr AJ. (2015) Inflammation activation and resolution in human tendon disease. Sci. Transl. Med. 7 (311); 311ra173. doi: 10.1126/scitranslmed.aac4269.
5. Kendal AR, Layton T, Al-Mossawi H, Appleton L, Dakin SG, Brown R, Loizou C, Rogers M, Sharp R, Carr AJ. Multi-omic single cell analysis resolves novel stromal cell populations in healthy and diseased human tendon. Sci Rep. 2020 Sep3;10(1):13939.

Contact information of all supervisors

Email: stephanie.dakin@ndorms.ox.ac.uk

Email: christopher.buckley@kennedy.ox.ac.uk

Email: markcoles2@kennedy.ox.ac.uk