Background: Rheumatoid Arthritis (RA) is an autoimmune mediated, chronic inflammatory condition. While remission is an achievable target for many patients, disease flares upon treatment cessation are a common occurrence highlighting the insufficiency of clinical disease activity score to capture the underlying biological heterogeneity of the RA remission state. We previously conducted a high dimensional analysis of RA remission which identified a population of CD8+CD57+ NK cells which were functionally distinct [1].

Objectives: Using single cell transcriptomics and in-vitro functional assays, we aimed to define the mechanism by which this NK cell functionality is modified in the RA remission state

Methods: 10X Genomics single cell RNA sequencing was performed on flow sorted CD56+ NK cells from the blood of patients in stable remission, patients with active RA and healthy controls. Ligand-complex adhesion assays (LC-AA) were developed to study the activation state of LFA-1 across NK cell subsets divided based on expression of CD8 and CD57. Briefly, NK cell activating receptors were cross linked and the active confirmation of LFA-1 was measured using fluorescent Fc-ICAM

Results: Single cell RNA sequencing identified an NK cell transcriptional cluster expanded in stable remission enriched for our previously discovered CD8+CD57+ NK cell subset. Pathway analysis characterised this subset as a highly cytotoxic NK cell subset. Pseudobulk differential gene expression analysis identified upregulation in remission of pathways associated with actin filament organisation (AFO), a key feature of cytotoxic immune synapse formation. Hypergeometric testing identified ITGB2 transcript levels as the strongest signature driving AFO functionality suggesting a strong association with LFA-1 signalling and cytoskeletal rearrangements associated with effective NK cell cytotoxicity. LC-AA assays demonstrated that CD16 cross-linking resulted in enhanced LFA-1 activation within CD57+ NK cells suggesting increased inside-out signalling.

Conclusions: We propose a model in which subset specific differences in the activation dynamics of LFA-1 control downstream pathways involved in effective NK cell cytotoxic functionality. The expansion of a subset with increased LFA-1 activation linked to enhanced AFO controlling cytotoxcity may represent a surrogate measure of the integrity of host defences within RA remission states. Further functional work (including confocal imaging) is ongoing to define the molecular signalling events downstream of LFA-1 controlling NK cell cytotoxcity in remission

REFERENCES: [1] Coyle C, Ma M, Abraham Y, Mahony CB, Steel K, Simpson C, Guerra N, Croft AP, Rapecki S, Cope A, Bowcutt R, Perucha E. NK cell subsets define sustained remission in rheumatoid arthritis. JCI Insight. 2024 Dec 6;9(23):e182390.

Acknowledgments: NIL.

Disclosure of Interests: None declared.