Corticosteroid resistance is predetermined by early immune response dynamics at acute graft-versus-host disease onset.

Steroid-resistant acute graft-versus-host disease (SR-aGVHD) is the leading life-threatening complication after allogeneic hematopoietic stem cell transplantation. Therapeutic development is impeded by scarce knowledge of biological pathways leading to steroid resistance after aGVHD diagnosis. To gain insight into circulating immune cell subsets and their functions at the onset of aGVHD, we performed a single-cell deep phenotyping and transcriptome analysis on peripheral blood mononuclear cells from two cohorts of patients (discovery cohort, n = 53; and validation cohort, n = 32) with aGVHD before steroid treatment or without aGVHD. Frequencies of circulating immune subsets were not associated with steroid resistance. However, pathway analysis and inferred ligand-receptor interactions revealed major functional divergences between steroid-sensitive aGVHD (SS-aGVHD) and SR-aGVHD, suggesting that steroid resistance is an intrinsic property of immune cells before any treatment. SR-aGVHD was mainly associated with tumor necrosis factor-α (TNF-α) activation. Steroid resistance resulted from a specific cross-talk characterized by inflammasome and caspase-1 activation in monocyte subsets; by TNF-α/TNF receptor, interleukin-1β (IL-1β), IL-18, CCL3, and CCL4 signaling between myeloid and T cells; and by lower involvement of interferon-α and interferon-γ signaling pathways. Immune trajectories in CD8+ T cells demonstrated a direct transition from an early naïve state to a highly activated one. By contrast, SS-aGVHD involved gene signatures across multiple intermediate differentiation stages during cell-to-cell transitions into CD8+ T subsets. These findings provide evidence that steroid resistance is driven by intrinsic mechanisms already present at the onset of the alloimmune response, which may enable previously unknown therapeutic strategies.