The JAK1/2 inhibitor baricitinib revealed unique immune biomarkers in multirefractory immune thrombocytopenia patients

Abstract Introduction Multirefractory immune thrombocytopenia (ITP), defined by resistance to both corticosteroids and thrombopoietin receptor agonists (TPO-RAs), represents a major clinical challenge with limited therapeutic options. The mechanisms of this dual resistance are poorly understood. Emerging evidence suggests that multirefractory patients harbor unique immune dysregulation patterns involving unconventional cell subsets that maintain chronic inflammatory states. We investigated that a distinct immune signature, driven by specific unconventional T cells, underpins this refractory state and can be targeted therapeutically. Methods We performed 41-marker mass cytometry (CyTOF) analysis on peripheral blood mononuclear cells from 27 individuals, comprising 9 healthy controls (HC), 9 non-multirefractory ITP patients (Non-MR ITP), and 9 multirefractory ITP patients (MR ITP). The 9 MR ITP patients subsequently received baricitinib, with samples collected at baseline and 12 weeks post-treatment. Unsupervised clustering algorithms, t-SNE dimensionality reduction, and statistical modeling were used to dissect the peripheral immune landscape and identify cell populations associated with multirefractory status and treatment response. Results High-dimensional analysis revealed a distinct immune signature in multirefractory (MR) patients compared to both healthy controls (HC) and non-multirefractory (Non-MR) ITP patients. This signature was centrally characterized by a profound 3.8-fold expansion of effector memory γδT cells re-expressing CD45RA (γδT EMRA). These cells displayed a hyper-cytotoxic phenotype, co-expressing high levels of CD57 and the chemokine receptor CXCR3, and were further defined by elevated intracellular granzyme B and the pro-inflammatory transcription factor T-bet. Concurrently, this pro-inflammatory state was associated with a severely compromised regulatory capacity. Myeloid-derived suppressor cells (MDSCs) were markedly reduced by 70% in MR patients, a loss primarily driven by the monocytic (CD14⁺HLA-DR⁻/low) subset. Furthermore, the cytotoxic skew extended to the innate immune compartment, with natural killer (NK) cell analysis revealing an extreme shift toward the CD56dim cytotoxic phenotype, characterized by maximal CD16 expression and minimal levels of the pro-survival receptor CD127 (IL-7Rα). Crucially, intervention with baricitinib in the MR cohort induced a significant remodeling of this aberrant immune signature. After 12 weeks of treatment, the pathogenic γδT EMRA population decreased by 64%, a reduction that was strongly correlated with platelet count recovery (r = -0.82, p < 0.01). This therapeutic effect was mirrored by a significant 3.2-fold restoration of the M-MDSC frequency and a rebalancing of the NK-cell compartment toward a more regulatory CD56bright phenotype, indicating a broad re-establishment of immune homeostasis. Conclusions This study reveals a distinct immune signature specifically associated with multirefractory ITP, which is dominated by the expansion of a cytotoxic γδT cell population. These findings implicate cytotoxic γδT cells as key players in the pathophysiology of dual treatment resistance in ITP. Furthermore, the reversal of this signature by a JAK inhibitor provides a mechanistic rationale for its use. High-dimensional immunological profiling may therefore guide precision therapeutic strategies for this challenging patient population.