Session: 503. Clonal Hematopoiesis, Aging, and Inflammation: Poster I
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Hematopoiesis, Immune mechanism, Biological Processes, Technology and Procedures, Omics technologies
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 DNMT3Amut 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 DNMT3Amut and wild-type cells. Differential gene- and surface protein expression analysis (CITEseq) revealed downregulated MHC-II molecules in DNMT3AR882mut compared to wild-type HSCs. This observation was restricted to DNMT3AR882mut comparted to DNMT3ANon-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-DRlow vs. HLA-DRhigh FACS-sorted CD34+ HSPCs by digital-droplet PCR, thereby confirming an up to 2.5-fold higher DNMT3AR882mut fraction in HLA-DRlow 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 DNMT3AR882mut HSCs can alter the activation of CD4+ T cells, which can functionally impact the development of CH over time. To explore mechanisms linking DNMT3AR882 mutations to the MHC-II phenotype in a model system, we studied interactions between DNMT3AR882Hmut 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+:DNMT3AWT/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 DNMT3AR882H 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 (OVA329-339 peptide), when presented via MHC-II. Our preliminary analysis revealed that despite lower MHC-II expression, DNMT3AR882H 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 DNMT3AR882Hmut CH that HSCs carrying DNMT3AR882 hotspot mutations in CH down-regulate MHC-II molecules, which potentially leads to altered immunosurveillance mechanisms in DNMT3AR882 mutant CH.
Disclosures: Vassiliou: AstraZeneca: Research Funding; STRM.BIO: Consultancy.
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