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Normal human enthesis harbours conventional CD4+ and CD8+ T cells with regulatory features and inducible IL-17A and TNF expression
  1. Abdulla Watad1,2,3,4,
  2. Hannah Rowe1,
  3. Tobias Russell1,
  4. Qiao Zhou1,5,
  5. Lisa K Anderson6,
  6. Almas Khan7,
  7. Robert Dunsmuir7,
  8. Peter Loughenbury7,
  9. Vishal Borse7,
  10. Abhay Rao7,
  11. Peter A Millner7,
  12. Nicola Luigi Bragazzi8,9,
  13. Howard Amital2,3,4,
  14. Richard Cuhtbert1,
  15. Miriam Wittmann1,10,
  16. Kassem Sharif1,2,4,
  17. Tony Kenna6,
  18. Matthew A Brown6,11,
  19. Darren Newton12,
  20. Charlie Bridgewood1,
  21. Dennis G McGonagle1
  1. 1 University of Leeds Leeds Institute of Rheumatic and Musculoskeletal Medicine, Leeds, UK
  2. 2 Internal Medicine "B", Sheba Medical Center, Tel Hashomer, Israel
  3. 3 The Zabludowicz Center for Autoimmune Diseases, Rheumatology Unit, Sheba Medical Center, Tel-Hashomer, Israel
  4. 4 Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
  5. 5 Department of Rheumatology & Immunology, Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital, Chengdu, Sichuan, China
  6. 6 Queensland University of Technology, Institute for Health and Biomedical Innovation, Translational Research Institute, Princess Alexandra Hospital, Brisbane, Queensland, Australia
  7. 7 Leeds Teaching Hospitals NHS Trust, Leeds, UK
  8. 8 Universita degli Studi di Genova Dipartimento di Medicina Interna e Specialita Mediche, Genova, Liguria, Italy
  9. 9 Department of Mathematics and Statistics, Laboratory for Industrial and Applied Mathematics (LIAM), York University, Toronto, Ontario, Canada
  10. 10 Leeds Teaching Hospitals NHS Trust, Leeds, NIHR Leeds Biomedical Research Centre, UK
  11. 11 Guy’s and St Thomas’ NHS Foundation Trust, King’s College London NIHR Biomedical Research Centre, London, United Kingdom
  12. 12 Division of Haematology and Immunology, University of Leeds, Leeds, West Yorkshire, UK
  1. Correspondence to Dr Dennis G McGonagle, Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, United Kingdom; d.g.mcgonagle{at}leeds.ac.uk

Abstract

Background The human enthesis conventional T cells are poorly characterised.

Objectives To study the biology of the conventional T cells in human enthesis.

Methods CD4+ and CD8+ T cells were investigated in 25 enthesis samples using immunofluorescence, cytometrically, bulk RNAseq and quantitative real-time PCR following anti-CD3/CD28 bead stimulation to determine interleukin (IL)-17A and tumour necrosis factor (TNF) levels. T-cell receptor (TCR) repertoires were characterised and a search for putative T-cell reactivity was carried out using TCR3 database. The impact of pharmacological antagonism with retinoic acid receptor-related orphan nuclear receptor gamma t inhibitor (RORγti), methotrexate and phosphodiesterase type 4 inhibitor (PDE4i) was investigated.

Results Immunofluorescence and cytometry suggested entheseal resident CD4+ and CD8+ T cells with a resident memory phenotype (CD69+/CD45RA-) and tissue residency gene transcripts (higher NR4A1/AhR and lower KLF2/T-bet transcripts). Both CD4+ and CD8+ T cells showed increased expression of immunomodulatory genes including IL-10 and TGF-β compared with peripheral blood T cells with entheseal CD8+ T cells having higher CD103, CD49a and lower SIPR1 transcript that matched CD4+ T cells. Following stimulation, CD4+ T cells produced more TNF than CD8+ T cells and IL-17A was produced exclusively by CD4+ T cells. RNAseq suggested both Cytomegalovirus and influenza A virus entheseal resident T-cell clonotype reactivity. TNF and IL-17A production from CD4+ T cells was effectively inhibited by PDE4i, while RORγti only reduced IL-17A secretion.

Conclusions Healthy human entheseal CD4+ and CD8+ T cells exhibit regulatory characteristics and are predicted to exhibit antiviral reactivity with CD8+ T cells expressing higher levels of transcripts suggestive of tissue residency. Inducible IL-17A and TNF production can be robustly inhibited in vitro.

  • ankylosing spondylitis
  • spondyloarthritis
  • T cells
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Footnotes

  • Handling editor Josef S Smolen

  • Twitter @tonykenna3

  • AW and HR contributed equally.

  • CB and DGM contributed equally.

  • Correction notice This article has been corrected since it published Online First. The ORCD ID for Abdulla Watad has been corrected.

  • Contributors AW, CB, RC, DN and DGM have contributed to the study planning and design. AW, HR, RC, CB, LA, MW, TK, DN, MB and DGM contributed to the acquisition, analysis or interpretation of data and critical revision of the manuscript. TR, QZ, AK, RD, PL, VB, AR, PM, NB, HA, MW, KS, TK, MB, DN and DGM contributed to the acquisition of data and drafting of the manuscript. All the authors have read and approved the final version of the manuscript.

  • Funding DGM is funded by the Leeds NIHR Biomedical Research Centre. Funding: Novartis UK -investigator-initiated non-clinical research funding support (HR, TR, CB). Research funded by a Pfizer investigator-initiated research grant (RC). AW QZ, and KS were funded by the Celgene supported PARTNER fellowship programme.

  • Competing interests None declared.

  • Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.

  • Patient consent for publication Not required.

  • Ethics approval The current study has been approved by the University of Leeds ethical approval committee.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data availability statement All data relevant to the study are included in the article or uploaded as supplementary information. All the relevant data is included in the article or supplementary file.

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