Donor T cell DNMT3a regulates alloreactivity in mouse models of hematopoietic stem cell transplantation
Yiouli P. Ktena, Michael A. Koldobskiy, Michael I. Barbato, Han-Hsuan Fu, Leo Luznik, Nicolas J. Llosa, Azeb Haile, Orly R. Klein, Chen Liu, Christopher J. Gamper, Kenneth R. Cooke
Yiouli P. Ktena, Michael A. Koldobskiy, Michael I. Barbato, Han-Hsuan Fu, Leo Luznik, Nicolas J. Llosa, Azeb Haile, Orly R. Klein, Chen Liu, Christopher J. Gamper, Kenneth R. Cooke
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Research Article

Donor T cell DNMT3a regulates alloreactivity in mouse models of hematopoietic stem cell transplantation

Abstract

DNA methyltransferase 3a (DNMT3a) is an important part of the epigenetic machinery that stabilizes patterns of activated T cell responses. We hypothesized that donor T cell DNMT3a regulates alloreactivity after allogeneic blood and marrow transplantation (allo-BMT). T cell conditional Dnmt3a KO mice were used as donors in allo-BMT models. Mice receiving allo-BMT from KO donors developed severe acute graft-versus-host disease (aGVHD), with increases in inflammatory cytokine levels and organ histopathology scores. KO T cells migrated and proliferated in secondary lymphoid organs earlier and demonstrated an advantage in trafficking to the small intestine. Donor T cell subsets were purified after BMT for whole-genome bisulfite sequencing (WGBS) and RNA-Seq. KO T cells had global methylation similar to that of WT cells, with distinct, localized areas of hypomethylation. Using a highly sensitive computational method, we produced a comprehensive profile of the altered epigenome landscape. Hypomethylation corresponded with changes in gene expression in several pathways of T cell signaling and differentiation. Additionally, Dnmt3a-KO T cells resulted in superior graft-versus-tumor activity. Our findings demonstrate a critical role for DNMT3a in regulating T cell alloreactivity and reveal pathways that control T cell tolerance. These results also provide a platform for deciphering clinical data that associate donor DNMT3a mutations with increased GVHD, decreased relapse, and improved survival.

Authors

Yiouli P. Ktena, Michael A. Koldobskiy, Michael I. Barbato, Han-Hsuan Fu, Leo Luznik, Nicolas J. Llosa, Azeb Haile, Orly R. Klein, Chen Liu, Christopher J. Gamper, Kenneth R. Cooke

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Figure 5

Loss of DNMT3a results in distinct areas of localized genomic hypomethylation.

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Loss of DNMT3a results in distinct areas of localized genomic hypomethyl...
Splenic WT and KO CD4+, CD8+, and CD4+CD8+ T cells were isolated by flow cytometry on day +10 in the B6→F1 model, and underwent DNA and RNA extraction for WGBS and RNA-Seq. (A) Similar distribution of MMLs across all purified subsets. (B) Box plots of the JSD showing the comparison of KO and WT CD8+ T cell genomic features annotated by chromatin state and gene-regulatory function as an example. The JSD captures methylation discordance, whether due to dMMLs, methylation entropy, or other statistical factors (66). Differences localize over enhancer elements and promoters bearing bivalent marks. EnrichR analysis (90) of pathways enriched in the genes differentially methylated between experimental groups using the Mouse Gene Atlas (C) and KEGG 2021 Human databases (D). SP, single-positive. (E) The Ccr9 gene promoter as an example of a top-ranked differentially methylated region between DNMT3a WT and KO CD8+ T cells. The peak in JSD indicates differential methylation, and the negative peak in dMML indicates hypomethylation in the KO cells.