Tu11 - High-Dimensional Immunophenotyping Reveals Altered Regulatory T Cell Fitness Between Inactive and Active Disease in Juvenile Idiopathic Arthritis

Abstract Text: Juvenile Idiopathic Arthritis (JIA), a prevalent autoimmune condition in children, is characterised by unpredictable, T-cell rich inflammatory flares of joints. Despite increased CD4+FoxP3+ regulatory T cell (Treg) numbers within the synovial fluid (SF) of active joints, these fail to suppress autoimmunity. Here, we utilise in-depth phenotyping by 5-laser full spectrum flow cytometry and unbiased high dimensional analysis to identify and distinguish unfit Treg sub-populations present in JIA SF (n=18), clinically active peripheral blood (PB, n=29) compared to inactive disease (n=17) and healthy control PBMCs (n=18). We designed and verified a 33-parameter panel to assess cellular composition (monocyte, B, NK, dendritic and T cell subsets) and comprehensive Treg phenotype (functional markers, activation, memory, co-receptors).
SF saw most changes with 12/18 cell composition and 8/10 Treg clusters changed in frequency. SF CD4-, CD4+ T cells and Tregs showed increased expression of GITR, HLA-DR, CD71 and CD69, with SF Tregs expressing high CTLA-4 and PD-1 levels. However, despite a more active state, SF Tregs were not more proliferative than blood Tregs. The co-stimulatory receptor CD226 was increased on SF Tregs, mostly co-expressed with TIGIT while blood Tregs rarely co-expressed both co-receptors. Moreover, we identified a sub-population of CD137low ID2intermediate Tregs in PB of inactive individuals, with reversed expression pattern in PB Tregs from active disease.
High dimensional flow cytometry revealed subsets of Tregs within the inflammatory joint of JIA and in blood of active compared to clinically inactive patients. These may have biomarker potential for predicting flare-ups and offer potential new mechanistic targets.