Inhibition of Cdk2 Inactivates EZH2 and Induces Epigenetic Regulation of FoxP3 Leading to the Generation of CD8+ Treg and Protection from GvHD

In spite of intense efforts, control of graft versus host disease (GvHD) remains incomplete and novel therapeutic approaches are required. Cdk2 has a central role in cell cycle re-entry of mature T lymphocytes and inhibition of Cdk2 is mandatory for induction of T cell anergy in vitro and tolerance in vivo. To determine the effects of Cdk2 inhibition on GvHD, we used the B6D2F1 mouse model of allogeneic BMT and two different Cdk2 inhibitors (Cdk2i), CYC202 and CYC205. Lethally irradiated B6D2F1(K^d) recipients were infused with bone marrow from C57BL/6(K^b) donors with (BMT) or without splenocytes and were subsequently treated with each Cdk2i for three weeks. Treatment was administered daily during week 1, every other day on week 2, and twice a week on week 3 followed by assessment of GvHD during a 70-day period. BMT recipients treated with Cdk2i displayed a transient weight loss and subsequently regained weight to levels comparable to controls. Treated BMT recipients also displayed delayed GvHD mortality (p=0.0054). Treg have a central role in mediating protection from GvHD. To examine whether Cdk2i induced Treg, we used GFP^- T cells from Foxp3.GFP-KI mice as a source of T cells. Assessment of peripheral blood lymphocytes, splenocytes, lymph nodes and intestinal lymphoid cells (ILC) in treated and control BMT recipients revealed no differences in CD4⁺GFP⁺ Treg. In contrast, CD8⁺GFP⁺ Treg were increased in the treated group, predominantly in ILC, which displayed a 5-fold increase of CD8⁺ Treg (p=0.05). To investigate the mechanisms via which Cdk2i had a selective effect on CD8⁺ Treg, we isolated CD4⁺GFP^- and CD8⁺GFP^- T cells from Foxp3.GFP-KI mice and subjected them to in vitro Treg polarization. Cdk2i had almost no effect on CD4⁺GFP⁺ cells but induced a 2-4 fold increase of CD8⁺GFP⁺ cells. Culture of CD8⁺GFP^- cells with stable concentrations of Cdk2i and decreasing concentrations of TGF-β revealed that Cdk2i induced CD8⁺ Treg differentiation in the presence of TGF-β concentrations that failed to induce CD8⁺ Treg cells when used alone. Expression of FOX family genes is regulated by transcriptional and epigenetic mechanisms. A critical epigenetic regulator of FOX transcription factors in cancer cells is the Polycomb group (PcG) protein, enhancer of zeste homologue 2 (EZH2), which promotes histone H3 lysine 27 trimethylation (H3K27me3) and induces epigenetic gene silencing. Cdk1 and Cdk2 phosphorylate EZH2 at Thr350 in an evolutionarily conserved motif. Phosphorylation of Thr350 is important for EZH2 recruitment and maintenance of H3K27me3 levels at EZH2-target loci. Upon polarizing CD8⁺ T cell culture, EZH2 displayed robust phosphorylation on Thr350, which was blocked by Cdk2i. This event temporally coincided with a 44-fold increase in Foxp3 mRNA expression compared to control T cells. These results reveal an unexpected mechanism via which Cdk2 inhibitors induce CD8⁺ Treg and protection from GvHD.