van der Kraan, P. | Search bioRxiv

bioRxiv2026

Imaging guided single-cell multiomics unveils shared autoreactive CD4+ T-cell responses in blood, locoregional lymph node and affected tissues of patients with systemic autoimmunity

Systemic autoimmune connective tissue diseases (CTDs) are characterized by anti-nuclear antibodies, shared HLA-associated genetic risk, and frequent disease overlap, suggesting a central role for CD4+ T cells in pathogenesis. However, defining disease-driving CD4+ T-cell responses remains challenging due to their localization within lymphoid and affected tissues and the lack of approaches linking these responses to circulating counterparts. We combined [18F]-labeled thymidine PET/CT-guided tissue sampling, ex vivo antigen stimulation, and single-cell multiomics to characterize CD4+ T-cell responses in blood, PET-avid locoregional lymph nodes (LNs), and disease-affected tissues from patients with the immunologically distinct CTDs systemic sclerosis and Sjogren's disease. PET-avid LNs from both diseases exhibited enhanced adaptive immune activity and contained an expanded population of interferon-stimulated gene (ISG)-expressing TRAIL+ CD4+ T cells. In Sjogren's disease, active LNs and affected tissues harbored diverse effector CD4+ T-cell populations, including follicular and peripheral helper T cells and Th2/Th17 cells. In contrast, systemic sclerosis tissues lacked effector CD4+ T cells, while active LNs were enriched for naive, regulatory, and TRAIL+ ISG CD4+ T cells. Antigen stimulation of peripheral blood mononuclear cells enriched for expanded effector CD4+ T-cell populations that shared activation profiles and clonal relationships with cells in LNs and affected tissues, many representing autoreactive antigen-specific T cells. TRAIL+ CD4+ T cells suppressed effector T-cell differentiation, autoreactive plasma cell generation, and autoantibody production in vitro, identifying a previously unrecognized immunoregulatory population. Together, this workflow enables comprehensive characterization of pathogenic and regulatory CD4+ T-cell responses across CTDs.

immunology↗

bioRxiv2025

CD8+ T cells drive myofibroblast activation and contraction via JAK/STAT3 and TGFβ signaling

BackgroundFibrosis is a major cause of morbidity and mortality in rheumatic connective tissue diseases. In these conditions, fibrotic tissues are characterized by the infiltration of CD8+ T cells and pro-fibrotic myofibroblasts. However, the role of CD8+ T cells in driving myofibroblast activity remains unexplored. Our aim was to investigate this interaction between CD8+ T cells and skin myofibroblasts and to elucidate its underlying mechanisms. MethodsPrimary skin-derived myofibroblasts were co-cultured with peripheral blood mononuclear cells (PBMCs) or sorted T cells in a 3D collagen type I hydrogel. To model autoimmunity, allogeneic mismatching was applied. T cell activation and cytokine expression were assessed using flow cytometry and Luminex. Myofibroblast activation was analysed through immunohistochemistry (IHC) and quantitative polymerase chain reaction (qPCR), while activation-induced contraction was measured macroscopically. Intracellular signalling pathway activation in myofibroblasts was evaluated using luciferase reporter cell lines. ResultsCo-culture of myofibroblasts with PBMCs strongly induced hydrogel contraction and expression of activation-related markers such as podoplanin, fibroblast activation protein, pSTAT3 and IL-6 in myofibroblasts. CD4+ T cells and CD8+ T cells were also activated by co-culture as identified by increased CD25 and CD69 expression and elevated IL-2 and IFN-{gamma} production. Upon co-culture with either sorted CD4+ or CD8+ T cells, CD8+ T cells more strongly induced myofibroblast contraction and activation than CD4+ T cells. This was not associated with cytotoxicity but with increased IL-6 production by CD8+ T cells compared to CD4+ T cells and STAT3/TGF{beta}-induced signaling in myofibroblasts. Use of either the JAK/STAT3-inhibitor tofacitinib or the TGF{beta} receptor inhibitor SB-505124 blocked the activated myofibroblast phenotype, and combined use of both inhibitors had an additive effect on myofibroblast activation and contraction. ConclusionsCD8+ T cells drive primary skin derived myofibroblast contraction and activation not via cytotoxicity-related program but via cytokine release. This sheds light on novel mechanisms of immune cell mediated tissue fibrosis. Furthermore, our results suggest that combining JAK/STAT inhibition with an anti-fibrotic agent that blocks matrix synthesis might be promising in mitigating immune cell mediated tissue fibrosis in connective tissue rheumatic disorders and has added value over blocking these pathways individually.

immunology↗