Background: We found a systemic population of CD4 ⁺ HLADR ⁺ T cells that was shown previously to immunologically mimic that of the inflammatory and TCR profiles of synovium T cells[1,2]. These circulatory CD4 ⁺ HLADR ⁺ T cells, composing of both regulatory and effector subsets, were shown to be increased during active disease and elevated in patients non-responsive to TNF inhibitor treatment. The pathogenic interactions of CD4 ⁺ HLADR ⁺ effector and regulatory T cells within the arthritic synovium remain unknown.

Objectives: This study seek to identify the functional and disease properties of CD4 ⁺ HLADR ⁺ T cells in juvenile idiopathic arthritic (JIA) patients, through (a) high parametric assessment of circulatory and synovial immune subsets with the aid of mass cytometry (CyToF), (b) transcriptomic examination of pathogenic pathways with deep RNA sequencing, (c) regulatory, destabilisation and polarisation T cell functional assays to dissect disease mechanisms.

Methods: Mass cytometry interrogation of JIA patients (7 PBMCs, 7 SFMCs) analysed with EPIC analytics suite [3]. FACS sorted for CD4 ⁺ HLADR ^± effectors or regulatory T cells from JIA patients (7 PBMCs, and n=8 paired PBMCs/SFMCs). CpG methylation profiling of FoxP3 promoter site in Tregs and T effectors from JIA patients (3 paired PBMCs/SFMCs) and healthy controls (6 PBMCs). Functional assays; (a) Treg suppression assay in JIA patients (8 paired PBMCs/SFMCs) and healthy controls (7 PBMCs), (b) Treg destabilisation assay (n=6), (c) metabolic assessment of CD36 (9 JIA PBMCs).

Results: Firstly, we performed an unbiased examination of the CD4 ⁺ CD45RO ⁺ memory landscape of JIA patients with CyToF, and detected an increase of CD4 ⁺ HLADR ⁺ T effectors (p <0.001) within the synovium as compared to the circulation. Importantly, synovium CD4 ⁺ HLADR ⁺ T effectors as compared with HLADR- counterparts, demonstrated an inflammatory profile (TNFa, IFNg and IL-21). Notably, CD4 ⁺ HLADR ⁺ T effectors and regulatory T cells positively correlated (r=0.74, p < 0.01) across spatial disease sites (blood versus synovium compartments), suggesting a micro-environmental relation, whereas CD4 ⁺ HLADR ^- subsets did not.

Next, to examine and dissect the functional properties of CD4 ⁺ HLADR ⁺ subsets, we sorted JIA PBMCs/SFMCs and performed deep RNA sequencing. Inspection of CD4 ⁺ HLADR ⁺ subsets within the synovium revealed an enrichment for 7 key pathways (MHC-II, IFNg signalling, viral defence, chemotaxis, apoptosis, T cell co-stimulation and LPS/Bacteria defence). Prominently, HLADR ⁺ effector and regulatory subsets in synovium demonstrated strong TCR clonatype sharing (p < 0.01) as compared with HLADR ^- counterparts, reflecting a common disease ontogenic origin.

While synovium CD4 ⁺ HLADR ⁺ Tregs indicated a stable epigenetic CpG methylation lineage profile in the FoxP3 TSDR promoter site, and were capable of suppression, CD4 ⁺ HLADR ⁺ T effectors were able to resist their regulatory control (p < 0.05). This cognate effector-regulatory T cell interaction is further underscored by the ability of inflammatory IFNg polarisation to destabilise FoxP3 levels (p < 0.05) in CD4 ⁺ HLADR ⁺ Tregs. Furthermore, JIA synovial CD4 ⁺ HLADR ⁺ Tregs had perturbed metabolic transcriptomic profile reflecting diminished capacity for fatty acids utilisation, where IFNG polarisation decreases the expression of the fatty aid uptake receptor CD36 (p < 0.01) critical for Treg functionality [4].

Conclusions: CD4 ⁺ HLADR ⁺ T effectors were inflammatory and uniquely expanded within the synovium compartment. Notably, CD4 ⁺ HLADR ⁺ Tregs demonstrated strong transcriptomic convergence and TCR clonatype sharing with their analogous HLADR ⁺ effectors, indicating a common disease origin. While CD4 ⁺ HLADR ⁺ Tregs were lineage stable and capable of suppression, CD4 ⁺ HLADR ⁺ effectors could circumvent regulatory control through inflammatory IFNg destabilisation of FoxP3 and metabolic reprogramming of Treg counterparts. CD4 ⁺ HLADR ⁺ T effectors may present themselves a possible therapeutic target in autoimmune arthritis.

REFERENCES: [1] Spreafico R and et al. A circulating reservoir of pathogenic-like CD4+ T cells shares a genetic and phenotypic signature with the inflamed synovial micro-environment. Ann Rheum Dis. 2016 Feb;75(2):459-65. doi: 10.1136/annrheumdis-2014-206226.

[2] Rossetti M, and et al. TCR repertoire sequencing identifies synovial Treg cell clonotypes in the bloodstream during active inflammation in human arthritis. Ann Rheum Dis. Feb 2017; 76(2): 435-441. DOI: 10.1136/annrheumdis-2015-208992.

[3] Yeo JG, and et al. The Extended Polydimensional Immunome Characterization (EPIC) web-based reference and discovery tool for cytometry data. Nat Biotechnol. 2020 Jun;38(6):679-684. doi: 10.1038/s41587-020-0532-1. Erratum in: Nat Biotechnol. 2020 Jun;38(6):757. doi: 10.1038/s41587-020-0574-4. PMID: 32440006.

[4] Wang, H., Franco, F., Tsui, YC. et al. CD36-mediated metabolic adaptation supports regulatory T cell survival and function in tumors. Nat Immunol 21, 298–308 (2020). https://doi.org/10.1038/s41590-019-0589-5

Acknowledgments: NIL.

Disclosure of Interests: None declared.