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AB0011 (2023)
CYTOMETRIC ANALYSIS OF ACTIVATED ENTHESEAL TISSUE RESIDENT T-CELLS REVEALS IL-17F AS THE DOMINANT IL-17 ISOFORM EXPRESSED BY INNATE AND ADAPTIVE LYMPHOCYTES
N. Mcdermott1, T. Macleod2, A. S. Rao3, V. Borse3, P. Loughenbury3, R. Dunsmuir3, A. Khan3, A. Maroof4, D. Mcgonagle2,5
1University of Leeds, Leeds Institute of Rheumatic and Musculoskeletal Medicine, Leeds, United Kingdom
2University of Leeds, Leeds Institute of Rheumatic and Musculoskeletal, Leeds, United Kingdom
3Leeds Teaching Hospitals NHS Trust, Department of Spine Surgery, Leeds, United Kingdom
4Exscientia, Immunology Research and Translational Immunology, Oxford, United Kingdom
5Leeds Teaching Hospital, National Institute for Health Research, Leeds, United Kingdom

 

Background The IL-17/IL-23 pathway has been implicated in many inflammatory diseases such as ankylosing spondylitis, psoriatic arthritis, inflammatory bowel disease and psoriasis, and has led to the development of several IL-17 inhibitors. The enthesis is connective tissue where the tendon or ligament attaches to the bone. Enthesitis is the cardinal pathological process in spondyloarthritis (SpA) related diseases and T-cell derived cytokines including IL-17A, IL-17F and TNFα and are thought to be central to SpA immunopathology [1-3]. Clinical advances from the inhibition of both IL-17A and IL-17F has shown great success in treating psoriasis. However, our understanding of IL-17F biology at the human enthesis is rudimentary.

Objectives Investigate the expression of IL-17A and IL-17F at the enthesis and identify innate and adaptive subsets capable of IL-23-independent production of IL-17.

Methods Peripheral blood and spinal enthesis samples collected from patients undergoing spinal decompression or surgery for scoliosis correction. Immune cell populations were isolated, for peripheral blood, the lymphocyte population was isolated using red cell lysis and mechanical isolation was used for the enthesis samples. T-cells were stimulated with anti-CD3 (100ng/ml) and soluble CD28 (100ng/ml) for 72hrs in RPMI media. 3-4hrs before the end of the stimulation GolgiPlug was added and the cells harvested for analysis of intracellular IL-17A & IL-17F via flow cytometry or were fixed and frozen for further CyTOF analysis. The same setup was used for IL-23 stimulations with IL-23 (50ng/ml) with or without anti-IL-23p19 (20ng/ml) added to the cells and downstream inflammatory cytokine production was assessed using a multiplex bead immunoassay.

Results IL-17F was preferentially expressed over IL-17A by CD4+ T-cells in peripheral blood and enthesis after 72hrs of inflammatory activation. Only a small percentage of cells were found to express IL-17A or co-express IL-17A and IL-17F. IL-17 expression by CD8+ T-cells was lower than their CD4+ counterpart, with IL-17F again being the predominant isoform expressed by this cell type. Analysis of IL-17 expression by cell types within the peri-entheseal bone revealed a similar trend to PBMC, albeit, lower expression of IL-17 was observed. CyTOF analysis revealed IL-17A and IL-17F expression from rare γδ T-cells in the enthesis. The additional IL-23 stimulation enhanced IL-17F production from CD4 T-cells; yet IL-23 alone could not induce IL-17F production. Addition of an IL-23p19 neutralising antibody reduced IL-17F levels equivalent to those seen with TCR activation showing that IL-17 production was not solely dependent on the presence of IL-23.

Conclusion This is the first study looking at IL-17A and IL17F induction from normal entheses where biological differences between these IL-17A and IL-17F family members has emerged.

References

  1. Tsukazaki H, Kaito T. The Role of the IL-23/IL-17 Pathway in the Pathogenesis of Spondyloarthritis. Int J Mol Sci 2020;21(17), doi:10.3390/ijms21176401
  2. Mei Y, Pan F, Gao J, et al. Increased serum IL-17 and IL-23 in the patient with ankylosing spondylitis. Clin Rheumatol 2011;30(2):269-73, doi:10.1007/s10067-010-1647-4
  3. Ceribelli A, Motta F, Vecellio M, et al. Clinical Trials Supporting the Role of the IL-17/IL-23 Axis in Axial Spondyloarthritis. Front Immunol 2021;12(622770, doi:10.3389/fimmu.2021.622770

Image/graph:

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Figure 1. Summary of the percentage of IL17F, IL17A and IL17A/F co-expression via flow cytometry. N=3. IL-17 expression by CD4 & CD8 T-cells in peripheral blood & peri-entheseal bone was measured following 72hrs stimulation with anti-CD3 & CD28 (100ng/ml). Error bars representative of the mean with range.

Acknowledgements This work is funded in collaboration with UCB who provided support and access for CyTOF work.

Disclosure of Interests Nicole McDermott Grant/research support from: UCB, Tom Macleod Grant/research support from: UCB, Abhay S Rao: None declared, Vishal Borse: None declared, Peter Loughenbury: None declared, Robert Dunsmuir: None declared, Almas Khan: None declared, Ash Maroof Employee of: UCB, Dennis McGonagle Grant/research support from: UCB.

Keywords: Spondyloarthritis, Adaptive immunity, Enthesitis

DOI: 10.1136/annrheumdis-2023-eular.4522


Citation: , volume 82, supplement 1, year 2023, page 1184
Session: Adaptive immunity (T cells and B cells) in rheumatic diseases (Publication only)