Background: T cells, play a pivotal role in Rheumatoid Arthritis (RA) persistence and disease progression. Polyfunctional T cells correlate with disease progression and are present in the synovial tissue of individuals at risk. Although synovialCD4+ T cells have an exhausted phenotype (PD-1, MHCII) they exhibit increased fitness and resistance to the hypoxic environment of the inflamed joint. The exact mechanism by which T cells can survive and proliferate in the synovium, despite the unfavourable conditions, has not been elucidated. Autophagy, a mechanism usually activated during stress states in cells, such as nutrient and oxygen deprivation, is suspected to contribute to disease pathogenesis. The potential role of autophagy in T cell responses in RA is poorly understood.

Objectives: This study aims to ascertain the role of autophagy in CD4+ T cells and its effect on their metabolism, fitness and function in RA.

Methods: Bioinformatic analysis of single cell RNA sequencing (scRNA-seq) data of synovial tissue from patients with RA and Healthy Controls (HC) was performed to evaluate the difference in the expression of genes related to autophagy pathway. Patient with RA and HC derived CD4 T cells were stimulated in vitro (aCD3/aCD28) in the presence or absence of autophagy inhibitors (BafA1, 3MA) or promoter (Torin) under atmospheric O ₂ or hypoxic conditions that simulate the inflamed joint. Differences in Autophagy were accessed by PCR for (ULK1, MAP1LC3, GABARAP, SQSTM1, BECN1and RCAN1), flow cytometry based LC3b-II staining and Western Blot for (LC3, GABARAP, ULK1, p62/SQSTM1 and Beclin 1). Cell metabolism was evaluated using a flow cytometric approach and metabolic inhibitors. Cytokine production of pro-inflammatory cytokines was assessed by multiplex ELISA.

Results: ScRNA-seq data revealed elevated expression of autophagy genes (MAP1LC3B, SQSTM1 and GABARAP) in the synovial tissue T cells of patients with RA compared to HC. In vitro experiments of stimulated T cells, demonstrated that patients with RA do not exhibit enhanced autophagy pathway activity at baseline level compared to HC. However, patient with RA derived CD4 T cells stimulated in the presence of 3-MA an early-stage autophagy inhibitor or BafA1 a late-stage autophagy inhibitor, showed differential autophagy pathway activity with 3-MA leading to higher expression of ULK1 and p62 and reduced expression of GABARAP and RCAN while BafA1 led to increased expression of ULK1, LC3B, GABARAP and BECN compared to HC derived CD4 T cells. Preliminary WB and Flow cytometric analysis for LC3B-II showed that in vitro stimulated CD4 T cells of patients with RA have increased protein expression of LC3B-II compared to HC. Importantly, T cells from patients with RA adapt metabolically and functionally to the hypoxic conditions due to altered autophagy pathway activity.

Conclusion: Autophagy pathway activity of T cells is dysregulated in patients with RA and may contribute to the increased fitness of synovial T cells and resistance to induction of anergy. These insights give rise to new potential therapeutic targets of T cells for autophagy regulation that may help combat disease progression.

REFERENCES: NIL.

Acknowledgements: Rachel Kenny, and the HRB grant EIA-2022-002.

Disclosure of Interests: None declared.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.