Perturbation of iNKT differentiation during clonal hematopoiesis from rewiring of inflammation and lipid presentation

Abstract

Abstract Invariant Natural Killer T (iNKT) cells are a rare, non-conventional T cell subset that recognize lipid antigens presented by the MHC class I-like molecule CD1d. Upon activation, iNKTs rapidly produce a wide range of cytokines, enabling them to serve as first responders and modulators of both innate and adaptive immunity. Their roles in inflammation, infection, tumor surveillance, and tissue homeostasis are well documented, but how iNKTs behave in the context of early hematologic transformation remains unclear. Clonal Hematopoiesis of Indeterminate Potential (CHIP) is a common, age-associated premalignant condition characterized by somatic mutations in hematopoietic stem/progenitor cells, most frequently in genes involved in epigenetic regulation such as TET2. CHIP not only predisposes to myeloid malignancies but also promotes systemic inflammation, a hallmark of early disease evolution. While TET2 loss has been shown to promote myeloid skewing and inflammatory signaling, its impact on iNKT biology remains unexplored. We hypothesized that TET2-deficient clonal hematopoiesis alters peripheral iNKT cell homeostasis and function through inflammation and disruption of lipid antigen presentation. To model CHIP, we used a non-irradiated bone marrow chimera system in which wild-type recipients were transplanted with Tet2-deficient (Tet2KO) bone marrow cells. This setup preserves a functional thymus, allowing us to study peripheral effects of clonal hematopoiesis on immune cells in the absence of irradiation-induced artifacts. Tet2KO murine chimeras exhibited a significant and progressive loss of peripheral non-mutated iNKT cells, correlating inversely with the extent of Tet2-deficient hematopoiesis. Thymic iNKT development remained intact, suggesting that peripheral environmental factors, rather than developmental defects, are responsible for the observed phenotype. Single-cell RNA sequencing revealed that Tet2-exposed iNKT cells displayed transcriptional signatures consistent with chronic activation, enhanced TCR signaling, and skewing toward the iNKT17 lineage, while iNKT1-defining transcriptional programs were suppressed. Flow cytometry confirmed a marked reduction in iNKT1 cells and a reciprocal expansion of iNKT17 cells in the spleens of Tet2KO chimeras. The cytokine known to promote iNKT17 differentiation, IL-6, was significantly elevated in the plasma of Tet2KO mice. Transcriptomic data revealed robust enrichment of IL-6–STAT3 target genes in iNKTs exposed to the Tet2-deficient microenvironment, further supporting a mechanistic link. In parallel, ATAC-seq profiling of iNKTs revealed increased chromatin accessibility at NFκB-associated transcription factor motifs and inflammatory response elements, indicating sustained activation.Given that iNKT activation depends on recognition of lipid antigens presented by CD1d, we next examined whether Tet2 loss alters this axis. Tet2KO hematopoietic cells exhibited significantly increased CD1d surface expression on multiple immune cell populations. To assess whether Tet2 deficiency perturbs the composition of lipid antigens available for CD1d presentation, we performed liquid chromatography–mass spectrometry LC-MS lipidomic profiling on Tet2-deficient and WT K562 cells. Notably, Tet2 loss resulted in significant alterations in endogenous glycolipid composition, including the enrichment of several lipid species known to be presented by CD1d to iNKT cells. These findings suggest that Tet2 regulates not only immune signaling but also the antigenic landscape that shapes iNKT activation and differentiation. Our study reveals a novel mechanism by which TET2 mutations in hematopoietic cells reprogram the immune microenvironment and impair the function of iNKT cells. We demonstrate that TET2 loss promotes a dual-hit effect: it generates a pro-inflammatory environment that skews iNKT differentiation toward an iNKT17 fate, and it alters both CD1d expression and lipid antigen composition, potentially reshaping the antigenic context that governs iNKT activation. These findings uncover a previously unrecognized role for TET2 in regulating lipid antigen presentation and iNKT homeostasis. By linking epigenetic dysregulation to innate-like T cell dysfunction, our work provides a mechanistic framework for understanding immune evasion in early clonal hematopoiesis and offers novel immunometabolic targets for therapeutic intervention in CHIP and preleukemic states.