Highlights d Human fetuses in 2 nd trimester show T cell diversity with effector-memory phenotype d Fetal organs show diverse bacterial genera that can be cultured and propagated d Bacterial structures with mucin-like threads are visualized in 14-weeks EGA fetal gut d Fetal bacteria induce syngeneic memory T cell activation in fetal mLN T cells

ObjectivesBiologics treatment with antitumour necrosis factor alpha (TNFα) is efficacious in patients with juvenile idiopathic arthritis (JIA). Despite displaying clinical inactivity during treatment, many patients will flare on cessation of therapy. The inability to definitively discriminate patients who will relapse or continue to remain in remission after therapy withdrawal is currently a major unmet medical need. CD4 T cells have been implicated in active disease, yet how they contribute to disease persistence despite treatment is unknown.MethodsWe interrogated the circulatory reservoir of CD4+ immune subsets at the single-cell resolution with mass cytometry (cytometry by time of flight) of patients with JIA (n=20) who displayed continuous clinical inactivity for at least 6 months with anti-TNFα and were subsequently withdrawn from therapy for 8 months, and scored as relapse or remission. These patients were examined prior to therapy withdrawal for putative subsets that could discriminate relapse from remission. We verified on a separate JIA cohort (n=16) the dysregulation of these circulatory subsets 8 months into therapy withdrawal. The immunological transcriptomic signature of CD4 memory in relapse/remission patients was examined with NanoString.ResultsAn inflammatory memory subset of CD3+CD4+CD45RA−TNFα+ T cells deficient in immune checkpoints (PD1−CD152−) was present in relapse patients prior to therapy withdrawal. Transcriptomic profiling reveals divergence between relapse and remission patients in disease-centric pathways involving (1) T-cell receptor activation, (2) apoptosis, (3) TNFα, (4) nuclear factor-kappa B and (5) mitogen-activated protein kinase signalling.ConclusionsA unique discriminatory immunomic and transcriptomic signature is associated with relapse patients and may explain how relapse occurs.

Autoimmune inflammation is characterized by tissue infiltration and expansion of antigen-specific T cells. Although this inflammation is often limited to specific target tissues, it remains yet to be explored whether distinct affected sites are infiltrated with the same, persistent T cell clones. Here we performed CyTOF analysis and T cell receptor (TCR) sequencing to study immune cell composition and (hyper-)expansion of circulating and joint-derived Tregs and non-Tregs in Juvenile Idiopathic Arthritis (JIA). We studied different joints affected at the same time, as well as over the course of relapsing-remitting disease. We found that the composition and functional characteristics of immune infiltrates are strikingly similar between joints within one patient, and observed a strong overlap between dominant T cell clones, especially Treg, of which some could also be detected in circulation and persisted over the course of relapsing remitting disease. Moreover, these T cell clones were characterized by a high degree of sequence similarity, indicating the presence of TCR clusters responding to the same antigens. These data suggest that in localized autoimmune disease there is auto-antigen driven expansion of both Teffector and Treg clones, that are highly persistent and are (re)circulating. These dominant clones might represent interesting therapeutic targets.

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