PI: Yi Zhang Graft-versus-host disease (GVHD) causes reconstitution of donor plasmacytoid dendritic cells (pDCs) to fail. pDCs are critical for immune protection and tolerance. However, little is known about GVHD-associated pDC molecular defects and the precise effect of donor pDCs on GVHD. We used both murine and human systems to uncover the mechanisms by which GVHD induces donor pDC defects. GVHD depleted donor multipotent progenitors (MPPs) that sustain pDCs, leading to impaired pDC generation. MPP loss was associated with decreased numbers of MPP-producing hematopoietic stem cells (HSCs) and oxidative stress-induced death of remaining proliferating MPPs; this correlates with the common clinical observation of low blood counts in the setting of GVHD. In our models, alloreactive T cells produce GM-CSF to inhibit MPP expression of Tcf4, the transcription factor essential for pDC development, subverting MPP production of pDCs. GM-CSF did not affect the maturation of pDC precursors. Notably, enhanced recovery of donor pDCs upon adoptive transfer early after allogeneic HSC transplantation (allo-HSCT) repressed GVHD and restored de novo generation of donor pDCs in recipient mice. pDCs suppress the proliferation and expansion of activated T cells via a type-I IFN signaling- dependent mechanism. They also produce PD-L1 and LILRB4 to inhibit T cell production of IFN-g. Thus, MPPs represent an effective target to bolster pDC reconstitution for GVHD prevention. In spite of these promising preliminary findings, the mechanisms by which MPPs produce pDCs and GVHD impairs MPPs remain unknown. Our continuing studies suggested that Dot1l, which specifically catalyzes H3K79 methylation, is crucial for pDC development from Flt3L-induced HSPCs. Dot1l appears to be critical for HSC and MPP differentiation into pDCs, PD-L1- and LILRB4-expressing pDCs (termed P/L+ pDCs) in particular. Intriguingly, GVHD decreases Dot1l function in MPPs. These data suggest that impaired Dot1l activity may be the major contributor to pDC defects during GVHD. Successful harnessing of pDCs for GVHD prevention will rely on an understanding of how Dot1l regulates MPP generation, maintenance and pDC formation. We will test our central hypothesis that Dot1l promotes the generation and maintenance of MPPs and regulates pDC specification of MPPs. Alloreactive T cell responses can inhibit this Dot1l effect, inducing pDC defects and causing feed-forward GVHD exacerbation. Aim-1 will define the role of Dot1l in pDC reconstitution and the impact of GVHD on Dot1l action in HSPCs. Aim- 2 is designed to determine the molecular mechanisms through which pDCs repress alloreactive T cell responses. Aim-3 focuses on the impact of human pDCs on alloreactive T cell responses and assesses whether donor P/L+ pDCs in the allografts can be utilized to predict GVHD in patients. Our proposal explores a heretofore unrecognized role of Dot1l in controlling pDC production and function. If successful, our work will ...