IL-17 as a targetable pathway to reverse early NK cell dysregulation in high-risk MDS with late relapse

Abstract Myelodysplastic syndrome (MDS) is a clonal hematopoietic stem cell disorder characterized by ineffective hematopoiesis, high risk of leukemic transformation, and poor survival in high-risk patients. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the only potentially curative treatment. However, relapse remains the major cause of treatment failure, with relapse-related mortality exceeding 40% and 2-year disease-free survival (DFS) often falling below 30%. Improvements in donor matching, conditioning, and graft-versus-host disease (GVHD) prophylaxis have not substantially reduced relapse rates, highlighting an urgent need to understand and target early post-transplant immune reconstitution to mitigate relapse risk. Natural killer (NK) cells are the first lymphocyte subset to recover after allo-HSCT and play a crucial role in mediating early graft-versus-leukemia (GVL) effects. While delayed or defective NK reconstitution has been associated with adverse outcomes, the prognostic significance of quantitative NK recovery and the molecular characteristics of post-transplant NK subsets remain poorly defined, especially in MDS patients. Furthermore, the contribution of the bone marrow microenvironment and key cytokine signaling pathways in shaping early NK cell dynamics is largely unexplored. To address these gaps, we comprehensively assessed early NK cell recovery in a cohort of 329 allo-HSCT recipients with high-risk MDS, with a median follow-up of 45 months. Using P-spline modeling, maximally selected rank statistics, and ROC analysis, we identified a consistent NK30 threshold at day +30 after transplantation of 82 cells/μl. Higher NK30 counts were associated with significantly improved overall survival (OS) and reduced cumulative incidence of relapse (CIR), without impacting non-relapse mortality (NRM). Multivariate analysis using LASSO-penalized Cox regression confirmed NK30 as the most robust independent predictor of relapse (HR=0.25, P=0.001). NK30 positively correlated with graft CD34⁺ cell dose (r=0.711, P<0.001) but was unaffected by grafted T cell numbers or early post-transplant T cell recovery, suggesting a hematopoietic reconstitution-driven mechanism. To further investigate the underlying biological processes, we performed single-cell RNA sequencing (scRNA-seq) on 17 bone marrow samples from 12 high-risk MDS/MDS-AML patients with relapse or continuous-CR at diagnosis, day +30 post-HSCT, and relapse for relapse group. We enriched CD34⁺ hematopoietic progenitors and Lin⁻CD7⁺ NK cells for transcriptomic profiling. In NK cells, dimensionality reduction and unsupervised clustering identified five transcriptionally distinct subsets (bri1, bri2, dim1, dim2, dim3), representing a continuum from cytokine-primed precursors to mature effector populations. NK cells from day +30 post-HSCT from patients destined to relapse (who initially had CR after allo-HSCT and then had disease relapse) displayed an altered subset composition, with decreased cytotoxic dim1 cells and expansion of dysfunctional bri2 and dim3 subsets. Differential expression and pathway enrichment analyses revealed reduced expression of cytotoxicity-related genes (e.g., GZMB, PRF1), impaired cell cycle activity, increased stress and apoptotic signaling, and marked enrichment of IL-17-mediated inflammatory pathways in NK cells from patients with poor outcomes. Integrated scRNA-seq data analysis with bone marrow cytokine profiling showed elevated IL-17 levels in relapsed patients. In murine allo-HSCT models (C57BL/6J→C3H), exogenous IL-17A/F suppressed NK maturation and cytotoxic activity, while blockade of IL-17A/F signaling promoted NK development, enhanced effector function, and improved leukemia control. In conclusion, we establish early NK30 count as a robust and independent biomarker for relapse risk in MDS patients undergoing allo-HSCT. Single-cell transcriptomics revealed profound defects in NK maturation and cytotoxic programming in relapse-prone patients, mechanistically linked to IL-17-driven suppression. Targeting IL-17 signaling may offer a novel approach to enhance NK-mediated GVL effects while reduce post-transplant relapse in high-risk MDS.