A115: Deep Immunophenotyping in the Identification of Clinically Meaningful Immune Signatures in Juvenile Idiopathic Arthritis

Abstract

Background/Purpose:The evolution of molecular immunology has made available high‐throughput tools which can provide an entirely new, comprehensive and multidimensional picture of the immune system. We have applied a combination of deep phenotyping by cytometry, multiplex expression and functional assays, to identify immunological signatures which could associate with, and perhaps contribute to, lack of responsiveness to treatment in juvenile idiopathic arthritis (JIA).Methods:We developed a platform of deep immunophenotyping to identify immune signatures on a variety of blood cell types with special focus given to CD4+ effector and regulatory T cells (Teff and Treg, respectively).Results:Analysis of samples from the NIH funded TREAT JIA study showed there were no significant changes between R and NR when Teff and Treg functions were analyzed as a whole. However, in‐depth characterization of the two cell subsets led to the identification of specific phenotypical differences at both time points. Strikingly, the combination of a restricted set of CD4+ T cell markers was able to segregate R and NR with up to 87.5% accuracy. Importantly, our analysis identified a population of cells significantly more represented in NR and characterized by an activated phenotype, expressing pro‐inflammatory markers and chemokine receptors targeting cells to inflammatory sites, and bearing signs of recent antigen recognition, possibly indicating their direct involvement in autoimmunity. Our approach will be greatly benefitted by the addition of recent advances in flow cytometry, with the development of the mass cytometer CyTOF, which dramatically improves single cell multiplexing. These methods are currently being applied to the analysis of samples from 137 subjects enrolled from 16 sites in a prospective multicenter NIH funded study who had Polyarticular forms of JIA (extended oligoarticular, Poly RF+, Poly RF/‐) who had reached clinical inactive disease (CID) for /G6 months at the time the anti‐TNF agent was withdrawn and followed for at least /G8 months to determine if they do or do not meet a validated definition of disease “flare”. Samples for immunophenotyping were obtained at baseline, time of stopping anti‐TNF therapy and last study visit (i.e. time of flare or at least 8 months of CID off anti‐TNF therapy). Duration of CID at baseline was mean/median/range of 1.2/0.5/1 day‐12.1 yrs. 106 (77%) subjects maintained CID for the first 6 months and stopped anti‐TNF as per protocol. 67/106 (63%) maintained CID for ≥8 mos off anti‐TNF while 39 (37%) flared. The mean/median/range for time to flare was 108/105/7–271 days.Conclusion:Immunophenotyping, when performed as an integrated analysis of immune signatures, has demonstrated effectiveness in identifying immune signatures that have excellent accuracy in segregating and predicting clinical disease states and responses. These methods are currently being analyzed in these study populations.