Mapping Genotype to Phenotype in Clonal Hematopoiesis Uncovers Downregulated MHC-II Molecules in DNMT3A^R882 - Mutant Hematopoietic Stem Cells

Somatic mosaicism in blood cells, referred to as clonal hematopoiesis (CH), arises when hematopoietic stem cells (HSCs) acquire somatic mutations that provide a substrate for clonal selection and subsequent clonal outgrowth over time. This condition becomes common with aging and is associated with adverse clinical outcomes related to mutated progenitor cells and increased inflammation. It has been shown that mutated clones have growth rates ranging from 5% to more than 50% per year and that they are acquired decades before they reach a substantial clone size. In CH, the most frequently mutated gene is DNMT3A encoding a de novo methyltransferase enzyme and the mutations are predominantly heterozygous, scattered throughout the three functional domains. In contrast, more than half of DNMT3A mutations in acute myeloid leukemia are missense alterations within the catalytic domain of the enzyme at residue R882. The high frequency of mutations at this specifc site suggests a gain-of-function activity. Beyond haploinsufficiency, the common heterozygous R882H allele creates an altered protein with dominant negative activity. Despite the increasing knowledge of how CH develops over time, the question regarding which mechanisms lead to clonal selection of mutant clones remains unresolved.

Here, we performed single-cell multi-omics profiling of FACS-enriched CD34⁺ hematopoietic stem and progenitor cells (HSPCs) and CD34^- mature blood cells from n=9 DNMT3A^mut peripheral blood samples with G-CSF mobilized HSPCs (autologous stem cell grafts) collected from multiple myeloma patients in remission. In total, 140,000 single cells were sequenced. Simultaneous mutation analysis enabled intra-sample comparison between DNMT3A^mut and wild-type cells. Differential gene- and surface protein expression analysis (CITEseq) revealed downregulated MHC-II molecules in DNMT3A^R882mut compared to wild-type HSCs. This observation was restricted to DNMT3A^R882mut comparted to DNMT3A^Non-R882mut samples. To validate this phenotype, we established a FACS-sorting strategy using HLA-DR, an MHC-II cell surface receptor involved in presenting peptides to CD4⁺ T cells, and compared mutant fractions (VAF) in HLA-DR^low vs. HLA-DR^high FACS-sorted CD34⁺ HSPCs by digital-droplet PCR, thereby confirming an up to 2.5-fold higher DNMT3A^R882mut fraction in HLA-DR^low sorted HSPCs.

We have previously shown that HSPCs are capable of activating CD4⁺ T cells upon presentation of both, endogenous and exogenous antigens via MHC-II and that the presentation of immunogenic antigens via MHC-II by HSPCs mediates bidirectional interactions with antigen-specific CD4⁺ T cells. Therefore, we hypothesize that the observed downregulation of MHC-II expression in DNMT3A^R882mut HSCs can alter the activation of CD4⁺ T cells, which can functionally impact the development of CH over time. To explore mechanisms linking DNMT3A^R882 mutations to the MHC-II phenotype in a model system, we studied interactions between DNMT3A^R882Hmut Lin^-Sca1⁺c-Kit⁺ cells and CD4⁺ T cells using an (pI:pC)-inducible humanized mouse model for CH that conditionally expresses human DNMT3A cDNA carrying the R882H hotspot mutation (Mx1-Cre⁺:DNMT3A^WT/R882H). We performed multi-parameter flow cytometry to characterize MHC-II expression on HSPCs and progenitor cell populations. Consistent with our human single-cell data, we observed that MHC-II (I-A/I-E) expression on HSPC subpopulations collected from mice with monoallelic DNMT3A^R882H expression was downregulated compared to wild-type mice. In contrast, there was no difference at the progenitor cell level. To understand whether the observed downregulation of MHC-II molecules impacts the antigen-specific activation of CD4⁺ T cells, we co-cultured FACS-sorted HSPCs with CD4⁺ T cells from OT-II mice that express transgenic T cell receptors specifically recognizing the chicken ovalbumin (OVA_329-339 peptide), when presented via MHC-II. Our preliminary analysis revealed that despite lower MHC-II expression, DNMT3A^R882H HSPCs are still capable of activating CD4⁺ T cells in vitro.

Together, our data provide evidence in primary human CH samples and in an engineered mouse model for DNMT3A^R882Hmut CH that HSCs carrying DNMT3A^R882 hotspot mutations in CH down-regulate MHC-II molecules, which potentially leads to altered immunosurveillance mechanisms in DNMT3A^R882 mutant CH.

Vassiliou:AstraZeneca: Research Funding; STRM.BIO: Consultancy.