CD28+ CD8+ Tem cells with a STAT1-dependent glucocorticoid receptor deficit contribute to steroid-refractory acute GVHD.

Steroid-refractory (SR) disease develops in a substantial fraction of patients with grade II to IV acute graft-versus-host disease (aGVHD) and is associated with poor long-term survival. Improved mechanistic insight is needed to identify reliable predictors of steroid resistance. We retrospectively profiled peripheral blood collected before glucocorticoid treatment from allogeneic hematopoietic cell transplantation recipients without aGVHD, with steroid-sensitive aGVHD, and with SR-aGVHD using an integrated multiomics approach, and validated our findings in an independent multicenter cohort. Mass cytometry revealed an expansion of activated CD28+ CD8+ effector-memory T (Tem) cells in SR-aGVHD. Absolute counts of these cells at neutrophil engraftment predicted subsequent steroid resistance in the multicenter cohort and performed comparably to established clinical classifiers. This phenotype was associated with a proinflammatory milieu enriched in interleukin-2 (IL-2), IL-27, and interferon gamma. Single-cell RNA sequencing and functional assays implicated a STAT1-glucocorticoid receptor (GR) regulatory axis, in which inflammatory cytokines induce STAT1 phosphorylation and suppress GR expression, consistent with intrinsic glucocorticoid resistance. JAK inhibition rescued cytokine-induced steroid resistance in vitro, whereas in patients with SR-aGVHD, clinical response to ruxolitinib was accompanied by reduced STAT1 activation, restoration of GR expression, and contraction of the expanded CD8+ Tem cell pool. These findings identify immune dysregulation in SR-aGVHD centered on CD8+ Tem cells with a STAT1-dependent GR deficit and support a mechanistic link to steroid refractoriness. CD28+ CD8+ Tem cell counts may serve as a biomarker of SR-aGVHD and inform the development of preemptive, pathway-targeted therapeutic strategies.