Clonal hematopoiesis drives T cell dysregulation and immune remodeling revealed by GWAS-based mendelian randomization and single-cell transcriptomics

Abstract Clonal hematopoiesis (CH), driven by somatic mutations in hematopoietic stem and progenitor cells (HSPCs), is associated with increased risk of leukemia, cardiovascular disease, chronic pulmonary conditions, and systemic inflammatory disorders, including autoimmunity. In the context of allogeneic hematopoietic stem cell transplantation (allo-HSCT), CH—particularly involving DNMT3A mutations in donor grafts—has been linked to a higher incidence of graft-versus-host disease (GVHD) and a lower risk of disease relapse. While prior studies have focused on CH-mediated alterations in the myeloid lineage and innate immunity, its role in remodeling the adaptive immune system remains underexplored. Given that CH mutations arise in multipotent HSPCs, we hypothesized that CH reshapes immune homeostasis via effects on lymphoid differentiation and crosstalk between innate and adaptive immunity. To investigate this, we performed Mendelian randomization (MR) analyses to assess causal relationships between CH and 731 immune traits using large-scale genomic and immune phenotyping data from the UK Biobank (n = 200,453; 13.4M SNPs) and the Sardinian cohort (n = 3,757; ~22M SNPs with 731 flow cytometry-defined immunophenotypes). Two MR approaches—MR-PRESSO and weighted mode—were applied to ensure robustness. Of the 731 traits tested, 61 met stringent quality control and significance criteria: (1) no evidence of horizontal pleiotropy (MR-PRESSO global p ≥ 0.05), (2) Bonferroni-adjusted p < 6.84 × 10⁻⁵, and (3) concordant direction of effect across both MR methods. CH was causally associated with increased absolute counts of HSPCs (beta effect size= 7.53, p = 1.60 × 10⁻²⁸) and monocytes (beta= 3.23, p = 1.17 × 10⁻²¹). Strikingly, the frequency of activated regulatory T cells (Tregs) within the CD4⁺ compartment was significantly reduced (beta= –7.33, p = 1.09 × 10⁻⁴³), accompanied by decreased expression of CD25 (beta= –4.45, p = 3.34 × 10⁻²⁴) and CD39 (beta = –3.48, p = 6.78 × 10⁻¹⁹) on activated Tregs. These changes suggest impaired IL-2 responsiveness and reduced suppressive capacity via the ATP–adenosine axis, contributing to a more effector-prone, dysregulated T cell landscape. In terminally differentiated CD8⁺ T cells, expression of HVEM (TNFRSF14)—a coinhibitory receptor—was significantly decreased (beta = –8.75, p = 1.36 × 10⁻⁴⁷), likely disrupting BTLA/CD160-mediated inhibition and promoting unchecked cytotoxic T cell activation and tissue damage, hallmarks of GVHD. In the B cell compartment, CH was associated with increased CD25 expression on B cells (beta = 3.86, p = 8.23 × 10⁻21) and IgD⁺CD38⁻ B cells (beta = 6.11, p = 1.16 × 10⁻⁴³), enriched for antigen-presenting and proinflammatory functions. Conversely, IgD⁺CD38bright B cells—typically representing immature transitional B cells—were markedly reduced (beta = –4.36, p = 1.77 × 10⁻²⁹), suggesting chronic B cell activation and impaired replenishment of naïve subsets. To validate these findings, we performed single-cell RNA sequencing (scRNAseq)of bone marrow mononuclear cells from individuals with CH mutations (DNMT3A-mutated [n = 2] and TET2-mutated [n = 2]) and age-matched healthy controls (n = 4). CH-mutant samples exhibited a reduction in CD4⁺ Tregs and an expansion of CD8⁺ effector and terminal effector cells, with a more exhausted phenotype compared to controls. In the B cell compartment, CH samples were enriched for CD25⁺ MHC-II⁺ activated B cells, while naïve B cells predominated in healthy individuals. These phenotypic shifts were accompanied by transcriptional activation of the TNF-α–NF-κB signaling pathway via TNFR1, particularly in DNMT3A-mutated CH, as determined by GSEA and cell–cell communication modeling using CellChat. This suggests a TNF-driven, proinflammatory milieu promoting persistent activation of T and B lymphocytes. Ongoing single-cell analyses in aged murine models with Dnmt3a knockout in HSPCs are underway to further define mutation-specific adaptive immune changes. Together, our integrative genomic and sc transcriptomic analyses revealed concordant immune remodeling in which CH—particularly DNMT3A-mutated—promotes a persistent, TNFα-driven proinflammatory state marked by expansion of cytotoxic T cells and antigen-presenting B cells with suppressed Tregs. These perturbations may prime the immune system for alloreactivity, providing a mechanistic link between CH and heightened GVHD risk following allo-HSCT.