NF-Y cooperates with USF1/2 to induce the hematopoietic expression of HOXB4.

The transcription factor homeobox B4 (HOXB4) is preferentially expressed in immature hematopoietic cells and implicated in the transition from primitive hematopoiesis to definitive hematopoiesis as well as in immature hematopoietic cell proliferation and differentiation. We previously identified Hox response element 1 (HxRE-1) and HxRE-2/E-box as 2 critical DNA-binding sites of the HOXB4 promoter active in hematopoietic cells and demonstrated that upstream stimulating factor 1 and 2 (USF1/2) activate HOXB4 transcription through their binding to the E-box site. Here we report that the trimeric regulatory complex nuclear factor Y (NF-Y) is the factor that recognizes HxRE-1 and activates the HOXB4 promoter in hematopoietic cells. We further show that NF-Y interacts biochemically with USF1/2 on the HOXB4 promoter, and that the formation of this NF-Y/USF1/2 complex is required for the full activity of the HOXB4 promoter. Most important, NF-Ya subunit protein levels are found to be lower in c-Kit-Gr-1+ granulocytic bone marrow (BM) cells than in c-Kit+ immature BM cells, in parallel with a reduction of NF-Y occupancy on the HOXB4 promoter as shown by chromatin immunoprecipitation (ChIP) assay. These results suggest that NF-Y is a developmentally regulated inducer of the HOXB4 gene in hematopoietic cells.

APCs in the liver and spleen recruit activated allogeneic CD8+ T cells to elicit hepatic graft-versus-host disease.

Host APCs are required for initiating T cell-dependent acute graft-vs-host disease (GVHD), but the role of APCs in the effector phase of acute GVHD is not known. To measure the effect of tissue-resident APCs on the local development of acute GVHD, we selectively depleted host macrophages and DCs from the livers and spleens, but not from the skin, peripheral lymph nodes (PLN), or mesenteric lymph nodes (MLN), of C57BL/6 (B6) mice by i.v. administration of liposomal clodronate before allogeneic bone marrow transplantation. Depletion of host hepatic and splenic macrophages and DCs significantly inhibited the proliferation of donor C3H.SW CD8(+) T cells in the spleen, but not in the PLN or MLN, of B6 mice. Such organ-selective depletion of host tissue APCs also markedly reduced the trafficking of allogeneic CD8(+) T cells into the livers and spleens, but not PLN and MLN, of B6 recipients compared with that of the control mice. Acute hepatic, but not cutaneous, GVHD was inhibited as well, resulting in improved survival of liposomal clodronate-treated B6 recipients. When C3H.SW CD8(+) T cells were activated in normal B6 recipients, recovered, and adoptively transferred into secondary B6 recipients, activated donor CD8(+) T cells rapidly migrated into the livers and spleens of control B6 recipients but were markedly decreased in B6 mice that were depleted of hepatic and splenic macrophages and DCs. Thus, tissue-resident APCs control the local recruitment of allo-reactive donor T cells and the subsequent development of acute GVHD.

Preterminal host dendritic cells in irradiated mice prime CD8+ T cell-mediated acute graft-versus-host disease.

To understand the relationship between host antigen-presenting cells (APCs) and donor T cells in initiating graft-versus-host disease (GVHD), we followed the fate of host dendritic cells (DCs) in irradiated C57BL/6 (B6) recipient mice and the interaction of these cells with minor histocompatibility antigen- (miHA-) mismatched CD8+ T cells from C3H.SW donors. Host CD11c+ DCs were rapidly activated and aggregated in the T cell areas of the spleen within 6 hours of lethal irradiation. By 5 days after irradiation, <1% of host DCs were detectable, but the activated donor CD8+ T cells had already undergone as many as seven divisions. Thus, proliferation of donor CD8+ T cells preceded the disappearance of host DCs. When C3H.SW donor CD8+ T cells were primed in vivo in irradiated B6 mice or ex vivo by host CD11c+ DCs for 24-36 hours, they were able to proliferate and differentiate into IFN-gamma-producing cells in beta(2)-microglobulin-deficient (beta(2)m(-/-)) B6 recipients and to mediate acute GVHD in beta(2)m(-/-) --> B6 chimeric mice. These results indicate that, although host DCs disappear rapidly after allogeneic bone marrow transplantation, they prime donor T cells before their disappearance and play a critical role in triggering donor CD8+ T cell-mediated GVHD.