Supplementary Material for: Compartment-specific NK cell phenotypes reveal distinct maturation and activation states in inflammatory arthritis

Introduction: Synovial natural-killer (NK) cells contribute to inflammation in arthritis by secreting cytokines and modulating synovial fibroblast activation. The aim of this study is to describe systemic versus local inflammatory changes of NK-cell-subsets as well as their physical cell-cell-interactions in arthritis patients.
Methods: Spectral-flow-cytometry was used to compare paired peripheral blood (PB) and synovial fluid (SF) immune cells from patients with active inflammatory arthritis and healthy controls. Physical cell-cell-interactions within tissues were studied by applying a recently developed cellular-interaction-mapping framework.
Results: Our paired approach revealed significant local enrichment of immature and activated NK-cells in SF, characterized by elevated markers of early differentiation, immune-checkpoint-regulation, and tissue-residency, highlighting tightly controlled immune activation at inflamed sites. Single-cell-analysis confirmed heterogeneity within SF-NK-cells, suggesting multiple co-existing activation states and developmental stages. PB-NK-cells from patients differed profoundly compared to healthy controls, showing less immature NK-cell subsets and an enrichment of mature, pro-inflammatory subsets indicative of systemic immune activation. Cellular-interaction-mapping revealed mainly NK/neutrophil-interactions of patients’ NK-cells, while interactions with B-cells, T-cells or monocytes were negligible. T-cells also displayed profound local and systemic alterations. Cellular-interaction-mapping revealed that next to NK-cell/neutrophil interactions, interactions between B-cells with monocytes and T-cells with neutrophils characterize joint inflammation.
Conclusion: This paired high-dimensional-analysis revealed systemic and local alterations in NK-cell-subsets shaped by co-existing developmental stages and immune regulatory mechanisms. Cellular-interaction-mapping indicated that neutrophils are a main interaction-partner of NK-cells in inflamed joints.