Clonal hematopoiesis and immune dysregulation in classic PNH

Abstract Introduction Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder originating from somatic mutations in PIGA within hematopoietic stem cells (HSCs). PNH appears to be related to aplastic anemia (AA). Immune evasion has been proposed as a key contributor to the clonal advantage of PNH cells. However, the intrinsic and extrinsic stress on HSCs, and the regulatory impact of wild-type (WT) or mutant mature blood cells on hematopoietic stem and progenitor cell (HSPC) homeostasis in PNH, remain to be fully elucidated. Methods We performed multiparameter immunophenotyping on fresh bone marrow samples from individuals with PNH, AA/PNH, AA, and healthy donors (HD). Single-cell RNA sequencing (scRNA-seq) using the 10x Genomics platform was conducted on sorted mutant HSPCs and WT/mutant mature blood cells from four classic PNH patients. Additionally, peripheral blood (PB) samples from three classic PNH patients before and after 2 months of complement inhibitor treatment were analyzed via 10x scRNA-seq. Results Immunophenotyping revealed an increased T cell proportion across patients with classic PNH, AA/PNH, and AA. Despite shared features of reduced HSPCs in all patient groups, lineage composition differed across diseases. In AA and AA/PNH, both WT and mutant hematopoietic stem cell / multipotent progenitor (HSC/MPP) subsets were decreased. In classic PNH and AA/PNH, lymphoid progenitors were markedly reduced regardless of genotype, while they remained largely preserved in AA. WT megakaryocyte-erythroid progenitors (MEPs) decreased in PNH and AA, whereas mutant MEPs increased in classic PNH, suggesting a shift towards erythro-myeloid differentiation. In vitro assays showed impaired expansion and differentiation potential of both WT and mutant HSPCs from PNH patients. Nonetheless, colony-forming cell assays showed higher erythroid/myeloid colony numbers in PNH, indicating a compensatory erythro-myeloid bias. Conversely, B/NK colony formation was significantly reduced, suggesting lymphoid impairment. scRNA-seq confirmed increased myeloid gene expression and reduced lymphoid genes in mutant MLPs. Mutant HSC/MPPs displayed diminished stemness, enhanced inflammatory signaling, and enrichment of erythro-myeloid-skewed subsets, indicating early lineage bias. WT T cells in PNH exhibited elevated expression of genes involved in activation, proliferation, and cytokine responses across multiple subsets, along with reduced co-inhibitory molecule expression in Tregs. Functional assays confirmed broad activation of WT T cells. Mutant neutrophils also showed pro-inflammatory activation. Correlation analysis of flow cytometry data revealed negative associations between T cell frequency and both WT and mutant HSC/MPP proportions, suggesting immune-mediated suppression. ScRNA-seq further showed up-regulation of pro-inflammatory ligands (e.g., TNF, IFNG) in activated T cells, implicating them in targeting mutant HSPCs. To test this hypothesis, CSA rescue experiments were conducted. CSA treatment improved HSPC viability and differentiation while suppressing T cell activation and proliferation. A small retrospective clinical analysis also demonstrated higher HSPC proportions in classic PNH patients who received CSA, supporting its role in mitigating hematopoietic exhaustion. Furthermore, scRNA-seq of PB samples before and after complement inhibition revealed down-regulation of inflammatory and cytokine pathways in myeloid cells, decreased T cell activation/proliferation scores, and restored expression of co-inhibitory markers in Tregs. These findings suggest that complement blockade may help alleviate immune activation and indirectly stabilize HSPC function. Conclusions This study reveals early lineage bias and functional exhaustion of HSPCs in classic PNH. WT T cells are aberrantly activated and may contribute to the suppression of mutant HSPCs. CSA restores HSPC number and function by dampening T cell activation, and complement inhibitors also alleviate immune activation, suggesting that immune microenvironment remodeling may represent a viable strategy to alleviate hematopoietic impairment in PNH.