Effect of Demethylating Agents (5-Azacytidine/5-AzaC) On the Immune Response

We have previously shown that 5-azaC inhibits T-cells proliferation and favours the development of Tregs which decreases the risk of GVHD after allogeneic transplantation. This is at least in part due to the effect of the drug on the expression of genes such as FOXP3. Nevertheless, considering the unspecific effect of 5-azaC, it could also favour the overexpression of other genes related to the regulation of the immune response such as TBET, GATA3, IFNg or IL-2, which in turn would favour the development of a Th1, Th2 or Th17 polarization instead of a Treg expansion. In the current study we have evaluated the effect of 5-azaC on these genes in order to know the mechanisms involved in the effect of the drug on the immune response.

We analyzed TBET, GATA3, FOXP3, IFNg and IL-2 mRNA expression of T lymphocytes by RT-PCR after exposure to 1mM 5-azaC during eleven days of culture. These T cells were cultured in medium alone or stimulated with plate-bound anti-CD3 (5 mg/ml) plus soluble anti-CD28 (2.5 mg/ml). Furthermore, we analyzed the methylation status of the promoters of these genes before and after 5-azaC treatment.

The expression of TBET, GATA3 and RORγ is not significantly affected by the exposure to the drug whereas the expression of FOXP3 significantly increases along the culture. Regarding IFNg and IL-2 expression no increased expression was observed after exposure to the drug at different time-points along the 11 days of culture. Upon analyzing the mathylation status of the promoter of these genes, we observed that in steady state the promoter of TBET and GATA is demethylated, which is in contrast to FOXP3 promoter. For this reason, the exposure to the drug decreases the methylation status of the promoter of FOXP3 while there is no effect on the promoters of TBET or RORg, thus justifying the absence of effect on the expression of these genes. By contrat, the promoter of both IFNg and IL-2 is methylated prior to the exposure to 5-azaC and it is demethylated after exposure to the drug, which is in contrast to the absence of increased expression of these genes. Accordingly, other mechanisms in addition to the epigenetic regulation of the promoter of IFNg and IL-2 are responsible for their expression in this model.

In the current study we show by the first time the effect of 5-azaC on the promoters of genes which regulate the immune response. While no effect was observed for TBET, and GATA3 5-azaC induces a strong demethylation in the promoter of IFN or IL-2. In spite of this effect there is no increase in their expression which could be due to the overexpression of FOXP3 or to additional mechanisms involved in their regulation which are currently being evaluated.

Cañizo:Celgene: Membership on an entity's Board of Directors or advisory committees. San Miguel:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Jangssen-cilag: Membership on an entity's Board of Directors or advisory committees; millennium: Membership on an entity's Board of Directors or advisory committees. Off Label Use: The drug used in this study is the demethylating agent 5-azacytidine (5-azaC) and the purpose is the inhibition of graft versus host disease.