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 4

Loss of DNMT3a expression results in a trafficking advantage for donor T cells to SLOs.

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Loss of DNMT3a expression results in a trafficking advantage for donor T...
Purified WT and KO B6 T cells were stained with CSFE and e450, respectively (and vice versa in replicate experiments) and were coadoptively transferred at a 1:1 ratio (3 × 106 to 5 × 106 cells each) into lethally irradiated allogeneic F1 animals. Spleen (A) and lymph node (not shown) flow cytometry was performed 24 and 48 hours later. Data from 2 experiments; n = 6 per time point per group. (B) On day +4, most cells lost the proliferation dye. WT and KO populations were distinguished via allelic differences between CD45.1/2 and CD90.1/2. Data from 2 experiments; n = 8 per group (except MLNs n = 5). (C) Serum chemokine levels by multiplex bead assay in the B6→F1 model on day +7. Data from 3 experiments; Syn n = 8, Allo n = 17 each. *P < 0.05, **P < 0.01, ***P < 0.001, Mann-Whitney U test or Mantel-Cox log-rank test for survival data.