Background: Circulatory CD4 ⁺ HLADR ⁺ effector and regulatory T cells have been previously shown to possess strong overlap in immunological phenotypes and TCRs with synovium counterparts[1,2]. In particular, systemic CD4 ⁺ HLADR ⁺ T effectors were significantly increased in arthritic patients that are non-responsive to TNFi treatment. Yet, despite of the presence of regulatory T cells within the synovium, why CD4 ⁺ HLADR ⁺ T effectors continue to perpetuate inflammation and its relationship with CD4 ⁺ HLADR ⁺ regulatory counterparts remains unknown.

Objectives: This study aims to employ immunomics platforms (CyToF, deep RNA sequencing) coupled with regulatory and antigenic T cell assays, to provide critical insights in understanding the functional and pathogenic properties of CD4 ⁺ HLADR ⁺ T cells in juvenile idiopathic arthritis (JIA). Importantly, we seek to dissect the disease mechanisms for which CD4 ⁺ HLADR ⁺ T cells contribute to autoimmunity in JIA.

Methods: CyToF analysis of JIA patients (PBMC=7, SFMC=7). FACS sorting and deep RNA sequencing of CD4 ⁺ HLADR ⁺ or CD4 ⁺ HLADR ^- effectors or regulatory T cells in (a) JIA patients (PBMC=8, paired PBMC/SFMC=8). CpG methylation profiling in JIA patients (paired PBMC/SFMC=3) and healthy controls (n=6). Micro-suppression assay in JIA patients (paired PBMC/SFMC=8) and healthy controls (n=7). CD4 ⁺ HLADR ⁺ Treg destabilisation assay (n=6). MHC-II pathway flow cytometry analysis (JIA PBMC=5, Healthy controls=11). Antigenic memory recall response of JIA (SFMC=7).

Results: We performed CyToF analysis of the CD4 memory landscape in JIA patients and detected a significant increase (p < 0.001) of CD4 ⁺ HLADR ⁺ T effectors in the synovium as compared with circulation. The synovium CD4 ⁺ HLADR ⁺ T effectors were inflamed, expressing higher levels of TNFa, IFNg and IL-21 as compared with HLADR ^- counterparts. CD4 ⁺ HLADR ⁺ T effectors strongly correlated (r= 0.74, p < 0.01) with levels of CD4 ⁺ HLADR ⁺ T regulatory cells across the circulatory and synovium compartments. Next, we sorted CD4 ⁺ HLADR ^± subsets from JIA patients and performed deep RNA sequencing, seeking to understand their functional properties and relationships. Phylogenetic tree analysis reveal that circulatory CD4 ⁺ HLADR ⁺ T effectors and regulatory cells were uncoupled from their respective compartments and displayed strong convergence. This transcriptomic convergence in circulatory CD4 ⁺ HLADR ⁺ subsets was further supported by pathway/transcription factor network analysis, indicating similar T cell activation pathways and transcriptomic drivers. Importantly, synovium CD4 ⁺ HLADR ⁺ T cells were enriched for 7 key pathways (MHC-II, IFNg signalling, viral defence, chemotaxis, apoptosis, T cell co-stimulation and LPS/Bacteria defence), and displayed stronger TCR clonatype sharing (p <0.01) as compared with HLADR ^- counterparts. JIA CD4 ⁺ HLADR ⁺ Tregs displayed stable CpG methylation profiles in the FoxP3 promoter sites, and were capable of suppressing disease T effectors. Yet, synovium CD4 ⁺ HLADR ⁺ T effectors were able to resist regulatory control (p < 0.05). As CD4 ⁺ HLADR ⁺ T effectors expressed high levels of IFNg and exhibited pronounced IFNg signalling, we destabilised Tregs in the presence of IFNg and mitotic agents. CD4 ⁺ HLADR ⁺ regulatory cells demonstrated higher instability and decreased FoxP3 levels (p < 0.05) as compared with HLADR- counterparts. Additionally, CD4 ⁺ HLADR ⁺ T effectors demonstrated memory recall response to disease relevant peptide (Hsp60) in the absence of APCs.

Conclusion: We have shown that CD4 ⁺ HLADR ⁺ T cells are expanded in systemic and synovium compartments of JIA patients, whilst displaying strong transcriptomic convergence with shared TCR clonatypes, indicating a strong ontogenic disease origin. Though CD4 ⁺ HLADR ⁺ Tregs are poised to suppress, CD4 ⁺ HLADR ⁺ T effectors could resist regulatory control and respond to disease relevant antigens independently, thereby amplifying disease activity. Specific targeting of CD4 ⁺ HLADR ⁺ T effectors in JIA may present as a potential therapeutic avenue.

REFERENCES: [1] Spreafico R, et al. Ann Rheum Dis. 2016 Feb;75(2):459-65.

[2] Rossetti M, et al. Ann Rheum Dis. Feb 2017; 76(2): 435-441.

Acknowledgements: NIL.

Disclosure of Interests: None declared.