Normal human enthesis harbours conventional CD4+ and CD8+ T cells with regulatory features and inducible IL-17A and TNF expression

Abstract

BackgroundThe human enthesis conventional T cells are poorly characterised.ObjectivesTo study the biology of the conventional T cells in human enthesis.MethodsCD4+ 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.ResultsImmunofluorescence 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.ConclusionsHealthy 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.

Revisiting the gut–joint axis: links between gut inflammation and spondyloarthritis

“…Whether HLA-B27 could be mediating IEL activation in patients with SpA, and whether it is possible for these cells to home to the inflamed joints of these patients, is interesting to speculate. Relevant to this point, recent studies have revealed the presence of resident-like CD8 + T cells in axial spinal ligaments 134 , whereas other studies have demonstrated high frequencies of granzyme-producing CD8 + T cells with gut-related homing markers, in the peripheral joints of SpA patients 123,135 .…”

IL-17A and TNF Modulate Normal Human Spinal Entheseal Bone and Soft Tissue Mesenchymal Stem Cell Osteogenesis, Adipogenesis, and Stromal Function

“…Additionally, human iliac crest MSCs and MSCs from murine femurs display enhanced osteogenic differentiation after exposure to either IL-17A or TNF [15,16], supporting the rationale for IL-17A being potentially involved in aberrant bone reactions in AS. Normal murine and human spinal entheses also have tissue resident innate and adaptive immune cells capable of either IL-17A and TNF production, depending on the system and conditions, which points towards the proximity of immune cells and cytokines that could enhance or fine tune tissue repair responses for neighboring MSCs [17][18][19][20]. Indeed, stromal cells from AS patients and a healthy facet capsule reported enhanced osteogenic potential of the AS patient-derived cells, with IL-17A also driving increased osteogenesis from these same cells [12].…”

Correspondence to ‘Normal human enthesis harbours conventional CD4+ and CD8+ T cells with regulatory features and inducible IL-17A and TNF expression’

“…We read with great interest the work by Watad et al, 1 which the authors demonstrated the characterisation of enthesisresident T cells and their corresponding cytokine responses upon stimulation. This commendable work mimicked enthesitis-involved inflammatory pathogenesis of spondyloarthritis, for instance, psoriatic arthritis (PsA).…”

“…This commendable work mimicked enthesitis-involved inflammatory pathogenesis of spondyloarthritis, for instance, psoriatic arthritis (PsA). Particularly, the authors proposed that entheseal T cells may secrete interleukin (IL)-17 and much more tumour necrosis factor α (TNF-α) in response to anti-CD3 and anti-CD28 (as suggested in figure 3 by Watad et al 1 ); furthermore, phosphodiesterase 4 (PDE4) inhibitors suppressed the expression of the above mentioned inflammatory cytokines (as suggested in figure 5 by Watad et al 1 ). As PDE4 inhibitors have been used to treat autoimmune diseases and advanced malignancies, 2 we are highly interested in whether infliximab, a neutralised antibody for TNF-α and a widely prescribed biologic disease-modifying antirheumatic drugs for a number of autoimmune diseases, would as well modulate the immunity of entheseal or synovial T cells in patients with PsA in clinical settings.…”