Longitudinal analysis identifies bystander-activated innate cytotoxic CD8⁺ T cells as predictors and therapeutic biomarkers for acute graft-versus-host disease

Accurate prediction and monitoring of acute graft-versus-host disease (aGVHD) remain challenging in allogeneic hematopoietic stem cell transplantation (allo-HSCT) as current diagnostic approaches rely on symptomatic presentation. Therefore, this study sought to identify predictive biomarkers and therapeutic targets for aGVHD through longitudinal immune monitoring and mechanistic investigations. In this study, peripheral blood samples were collected weekly for 100 days from a group of 115 allo-HSCT recipients. CD38⁺HLA-DR⁺CD8⁺ T (activated CD8⁺ T) cells were analyzed using the t-distributed stochastic neighbor embedding (t-SNE) algorithm for high-dimensional data visualization and population identification. Clinical data integration was used to assess biomarker utility. Mechanistic studies included interleukin-15 (IL-15) stimulation, signaling pathway inhibition, cytotoxicity assays, and xenogeneic GVHD modeling with anti-CD38 (daratumumab) intervention. Our results revealed that sustained elevation of activated CD8⁺ T cells (> 36.6%) within the first month post-transplantation predicted aGVHD onset with high accuracy (AUC = 0.84, P < 0.001). Cell frequency dynamically correlated with treatment outcome, decreasing substantially in responders. Mechanistically, IL-15 drove T-cell receptor (TCR)-independent cytotoxicity via PI3K/mTOR activation, mediated by natural killer group 2D (NKG2D) and major histocompatibility complex class Ⅰ chain related proteins A (MIC-α) interactions, validated by reduced K562 cell lysis following antibody blockade. In an 8–10-week-old NSG mouse model for xenogeneic transplantation, treatment with daratumumab (5 mg/kg) effectively lowered histopathological damage and increased survival. In conclusion, activated CD8⁺ T cells can serve as dual-purpose biomarkers for early aGVHD prediction and treatment monitoring. Their IL-15-driven cytotoxicity represents a targetable pathway, with daratumumab demonstrating therapeutic efficacy.