Bone marrow transplants, like the high-profile match between the son of heavyweight fighter Evander Holyfield and 19-year-old Nashville native Darian Craig, profiled in People magazine earlier this month, run the risk of graft-versus-host disease (GVHD), a life-threatening complication where the donor cells attack the patient’s own cells.
BMT procedures replace damaged or destroyed bone marrow with healthy bone marrow stem cells. The marrow is the soft tissue at the heart of bones where immature cells that give rise to all blood cells – which include immune cells - are made. During the repopulation of the immune cells after a BMT, different populations of T cells mature quicker compared to others, and this difference contributes to attack by the newly reconstituting immune cells on the body in GVHD.
Specifically, CD4 and CD8 T cells mature faster, so they repopulate the body sooner after a BMT. However, T regulatory cells - Tregs for short - are much slower to develop and their relative shortage is a key factor in development of graft-versus-host disease.
“The idea is to one day engineer Tregs so they can be harvested from a donor, grown up, and then given to a recipient at the same time as the BMT, as a sort-of mature Tregs booster while the immature Tregs in the marrow gradually develop,” says Wayne Hancock, MD, PhD, professor of Pathology and Laboratory Medicine. “Or, the Tregs could be given shortly before a transplant, to introduce the BMT recipient to the donor Tregs, and then also given at time of transplant, as a booster.”
Earlier this year, Hancock and colleagues published a paper in Blood that describes the Foxp3 transcription factor in Tregs, and how to use them for better transplant outcomes. Transcription factors are molecules that bind directly to DNA to control the transcription of DNA into messenger RNA.
Protocols to expand and use Tregs for cellular therapy, especially after unrelated, or allogeneic, stem cell transplantation, are currently being developed and tested by various groups. Inhibitors of DNA methyltransferase (Dnmt) enzymes have been advocated as a means to promote and stabilize Foxp3 expression in Tregs undergoing expansion, prior to their injection into a transplant recipient. If a gene is heavily methylated, that is, has many methyl chemical groups attached to it, it is usually silenced and not available to be transcribed into a protein. For example, CD4 and CD8 T cells are heavily methylated, which keeps them from expressing Foxp3, via this epigenetic control.
The Hancock team investigated the effects of conditionally deleting two Dnmt enzymes that work on Foxp3 in Tregs. Deletion of Dnmt1, but not Dnmt3a, decreased the numbers and function of Tregs and impaired the conversion of conventional T cells into Foxp3-expressing Tregs.
The team found that if CD4 T cells are grown with the cytokine IL2 and TGF beta, they express Foxp3 as a result of making the Fox3 locus become partially methylated, so it can be transcribed. If they added an inhibitor of the DNA methyltransferase enzyme, the locus became fully demethylated, so that full transcription of the Fox3p locus occurred.
But Hancock and colleagues saw that this could have undesired effects on Foxp3 positive Tregs. Specifically, when Dnmt1 was completely deleted just within Tregs by genetic means, theTregs lost their suppressive function completely and mice died from autoimmunity, unless they were rescued by an injection of wild-type Treg cells. See image above.
Right now, the only use of Dnmt1 inhibitors is in pre-clinical research, not yet in patients, but Foxp3 expression may have potential utility in efforts to develop Foxp3-expressing Tregs for cell therapy.
“We determined that Dnmt1 is necessary for maintaining the core gene program for the development and function of Tregs, and that its deletion leads to lethal autoimmunity,” says Hancock. “Hence, our research certainly shows that caution is warranted when considering using DNMT inhibitors for developing T reg-based therapies for people, since too much demethylation is as bad, or worse, than too little demethylation.” 
Wang, L., Liu, Y., Beier, U., Han, R., Bhatti, T., Akimova, T., & Hancock, W. (2013). Foxp3+ T-regulatory cells require DNA methyltransferase 1 expression to prevent development of lethal autoimmunity Blood, 121 (18), 3631-3639 DOI: 10.1182/blood-2012-08-451765