Although proinflammatory cytokines are key mediators of tissue damage during graft-versus-host disease (GVHD), IFN␥ has previously been attributed with both protective and pathogenic effects. We have resolved this paradox by using wildtype (wt), IFN␥ ؊/؊ , and IFN␥R ؊/؊ mice as donors or recipients in well-described models of allogeneic stem cell transplantation (SCT). We show that donor-derived IFN␥ augments acute GVHD via direct effects on (1) the donor T cell to promote T helper 1 (Th1) differentiation and (2) the gastrointestinal (GI) tract to augment inflammatory cytokine generation. However, these detrimental effects are overwhelmed by a protective role of IFN␥ in preventing the development of idiopathic pneumonia syndrome (IPS). This is the result of direct effects on pulmonary parenchyma to prevent donor cell migration and expansion within the lung. Thus, IFN␥ is the key cytokine differentially controlling the development of IPS and gastrointestinal GVHD after allogeneic SCT. IntroductionAllogeneic bone marrow transplantation (BMT) is a definitive curative therapy for most hematologic malignancies and severe immunodeficiencies. The major complication of allogeneic BMT remains graft-versus-host disease (GVHD) in which the skin, gastrointestinal (GI) tract, liver, and lung are preferentially damaged by the transplanted donor immune system. 1 GVHD occurs in most (50%-70%) recipients and is largely responsible for the high mortality associated with allogeneic BMT. Idiopathic pneumonia syndrome (IPS) is an acute noninfectious lung injury that typically occurs 3 to 4 weeks after BMT, responds poorly to therapy, and is associated with a high mortality. 2 There is thus a pressing need for new treatment approaches to both prevent and treat the full spectrum of GVHD, based on a logical understanding of the underlying disease pathophysiology.Current paradigms suggest that GVHD occurs via a complex cellular network initiated by the interaction of antigen-presenting cells (APCs) and naive donor T cells. [3][4][5] Subsequent T helper 1 (Th1) differentiation leads to the generation of donor cytotoxic T lymphocytes (CTLs) and large amounts of inflammatory cytokines that damage host tissue by both major histocompatibility complex (MHC)-dependent and -independent pathways. 6 Of the Th1 cytokines, IFN␥ is perhaps the most immunologically dominant, influencing a plethora of cell subsets during allograft rejection. 7 However the effects of this cytokine on GVHD are unclear, with a number of contradictory studies [8][9][10][11] suggesting that a clearer understanding of the mechanisms involved are needed. We have re-examined this issue using both IFN␥ Ϫ/Ϫ and IFN␥R Ϫ/Ϫ stem cell transplantation (SCT) donors or recipients following myeloablative conditioning. We demonstrate that donor-derived IFN␥ indeed has both positive and negative effects on GVHD due to differential effects on donor and host tissue, and individual target organs. First, IFN␥ augments acute GVHD via direct affects on the donor T cell to promote Th1 differen...

The lineage of dendritic cells (DC), and in particular their relationship to monocytes and macrophages, remains obscure. Furthermore, the requirement for the macrophage growth factor CSF-1 during DC homeostasis is unclear. Using a transgenic mouse in which the promoter for the CSF-1R (c-fms) directs the expression of enhanced GFP in cells of the myeloid lineage, we determined that although the c-fms promoter is inactive in DC precursors, it is up-regulated in all DC subsets during differentiation. Furthermore, plasmacytoid DC and all CD11c(high) DC subsets are reduced by 50-70% in CSF-1-deficient osteopetrotic mice, confirming that CSF-1 signaling is required for the optimal differentiation of DC in vivo. These data provide additional evidence that the majority of tissue DC is of myeloid origin during steady state and supports a close relationship between DC and macrophage biology in vivo.

The initiation of graft-vs-host disease (GVHD) after stem cell transplantation is dependent on direct Ag presentation by host APCs, whereas the effect of donor APC populations is unclear. We studied the role of indirect Ag presentation in allogenic T cell responses by adding populations of cytokine-expanded donor APC to hemopoietic grafts that would otherwise induce lethal GVHD. Progenipoietin-1 (a synthetic G-CSF/Flt-3 ligand molecule) and G-CSF expanded myeloid dendritic cells (DC), plasmacytoid DC, and a novel granulocyte-monocyte precursor population (GM) that differentiate into class II+,CD80/CD86+,CD40− APC during GVHD. Whereas addition of plasmacytoid and myeloid donor DC augmented GVHD, GM cells promoted transplant tolerance by MHC class II-restricted generation of IL-10-secreting, Ag-specific regulatory T cells. Importantly, although GM cells abrogated GVHD, graft-vs-leukemia effects were preserved. Thus, a population of cytokine-expanded GM precursors function as regulatory APCs, suggesting that G-CSF derivatives may have application in disorders characterized by a loss of self-tolerance.

We investigated whether the protection from graft-versus-host disease (GVHD) afforded by donor treatment with granulocyte colony-stimulating factor (G-CSF) could be enhanced by dose escalation. Donor treatment with human G-CSF prevented GVHD in the B6 → B6D2F1 murine model in a dose-dependent fashion, and murine G-CSF provided equivalent protection from GVHD at 10-fold lower doses. Donor pretreatment with a single dose of pegylated G-CSF (peg-G-CSF) prevented GVHD to a significantly greater extent than standard G-CSF (survival, 75% versus 11%, P < .001). Donor T cells from peg-G-CSF-treated donors failed to proliferate to alloantigen and inhibited the responses of control T cells in an interleukin 10 (IL-10)-dependent fashion in vitro. T cells from peg-G-CSF-treated IL-10-/- donors induced lethal GVHD; T cells from peg-G-CSF-treated wild-type (wt) donors promoted long-term survival. Whereas T cells from peg-G-CSF wt donors were able to regulate GVHD induced by T cells from control-treated donors, T cells from G-CSF-treated wt donors and peg-G-CSF-treated IL-10-/- donors did not prevent mortality. Thus, peg-G-CSF is markedly superior to standard G-CSF for the prevention of GVHD following allogeneic stem cell transplantation (SCT), due to the generation of IL-10-producing regulatory T cells. These data support prospective clinical trials of peg-G-CSF-mobilized allogeneic blood SCT. (Blood. 2004;103:3573-3581)

Donor treatment with granulocyte-colonystimulating factor (G-CSF) attenuates the ability of donor T cells to induce acute graft-versus-host disease (aGVHD) but increases the severity of chronic GVHD (cGVHD). We investigated the role of the regulatory cytokine transforming growth factor ␤ (TGF-␤) in this paradox in wellestablished murine models of aGVHD and cGVHD wherein recipients undergo transplantation with splenocytes from donors treated with G-CSF. Neutralization of TGF-␤ after stem-cell transplantation (SCT) significantly increased the severity of aGVHD, and the concurrent prevention of interleukin-10 (IL-10) production further exaggerated this effect. Early after SCT, donor T cells were the predominant source of TGF-␤ and were able to attenuate aGVHD in a TGF-␤-dependent fashion. Although the neutralization of TGF-␤ augmented the proliferation and expansion of donor T cells after SCT, it paradoxically impaired cellular cytotoxicity to host antigens and associated graft-versus-leukemia (GVL) effects. In cGVHD, neutralization of TGF-␤ from day 14 after SCT attenuated histologic abnormalities, and CD11b ؉ mononuclear cells infiltrating sclerodermatous skin produced 50-fold more TGF-␤ than corresponding T cells. Thus, though the production of TGF-␤ by donor T cells early after transplantation attenuates aGVHD and is required for optimal GVL, the production of TGF-␤ late after SCT is preferentially from mononuclear cells and mediates cGVHD. These data have important implications for the timing of therapeutic TGF-␤ neutralization to prevent cGVHD after allogeneic SCT.