Alistair L. J. Don

Recipient nonhematopoietic antigen-presenting cells are sufficient to induce lethal acute graft-versus-host disease

The presentation pathways by which allogeneic peptides induce graft-versus-host disease (GVHD) are unclear. We developed a bone marrow transplant (BMT) system in mice whereby presentation of a processed recipient peptide within major histocompatibility complex (MHC) class II molecules could be spatially and temporally quantified. Whereas donor antigen presenting cells (APCs) could induce lethal acute GVHD via MHC class II, recipient APCs were 100–1,000 times more potent in this regard. After myeloablative irradiation, T cell activation and memory differentiation occurred in lymphoid organs independently of alloantigen. Unexpectedly, professional hematopoietic-derived recipient APCs within lymphoid organs had only a limited capacity to induce GVHD, and dendritic cells were not required. In contrast, nonhematopoietic recipient APCs within target organs induced universal GVHD mortality and promoted marked alloreactive donor T cell expansion within the gastrointestinal tract and inflammatory cytokine generation. These data challenge current paradigms, suggesting that experimental lethal acute GVHD can be induced by nonhematopoietic recipient APCs.

NKT cell–dependent leukemia eradication following stem cell mobilization with potent G-CSF analogs

NKT cells have pivotal roles in immune regulation and tumor immunosurveillance. We report that the G-CSF and FMS-like tyrosine kinase 3 ligand (Flt-3L) chimeric cytokine, progenipoietin-1, markedly expands the splenic and hepatic NKT cell population and enhances functional responses to alpha-galactosylceramide. In a murine model of allogeneic stem cell transplantation, donor NKT cells promoted host DC activation and enhanced perforin-restricted CD8+ T cell cytotoxicity against host-type antigens. Following leukemic challenge, donor treatment with progenipoietin-1 significantly improved overall survival when compared with G-CSF or control, attributable to reduced graft-versus-host disease mortality and paradoxical augmentation of graft-versus-leukemia (GVL) effects. Enhanced cellular cytotoxicity was dependent on donor NKT cells, and leukemia clearance was profoundly impaired in recipients of NKT cell-deficient grafts. Enhanced cytotoxicity and GVL effects were not associated with Flt-3L signaling or effects on DCs but were reproduced by prolonged G-CSF receptor engagement with pegylated G-CSF. Thus, modified G-CSF signaling during stem cell mobilization augments NKT cell-dependent CD8+ cytotoxicity, effectively separating graft-versus-host disease and GVL and greatly expanding the potential applicability of allogeneic stem cell transplantation for the therapy of malignant disease.

Stem cell mobilization with G-CSF induces type 17 differentiation and promotes scleroderma

The recent shift to the use of stem cells mobilized by granulocyte colony-stimulating factor (G-CSF) for hematopoietic transplantation has increased chronic graftversus-host disease (GVHD), although the mechanisms of this are unclear. We have found that G-CSF invokes potent type 17 rather than type 1 or type 2 differentiation. The amplification of interleukin-17 (IL-17) production by G-CSF occurs in both CD4 and CD8 conventional T cells and is dependent on, and downstream of, G-CSF-induced IL-21 signaling. Importantly, donor IL-17A controls the infiltration of macrophages into skin and cutaneous fibrosis, manifesting late after transplantation as scleroderma. Interestingly, donor CD8 T cells were the predominant source of IL-17A after transplantation and could mediate scleroderma independently of CD4 T cells. This study provides a logical explanation for the propensity of allogeneic stem cell transplantation to invoke sclerodermatous GVHD and suggests a therapeutic strategy for intervention.

Conventional dendritic cells are the critical donor APC presenting alloantigen after experimental bone marrow transplantation

We have quantified the relative contribution of donor antigen-presenting cell populations to alloantigen presentation after bone marrow transplantation (BMT) by using transgenic T cells that can respond to host-derived alloantigen presented within the donor major histocompatibility complex. We also used additional transgenic/knockout donor mice and/or monoclonal antibodies that allowed conditional depletion of conventional dendritic cells (cDCs), plasmacytoid DC (pDCs), macrophages, or B cells. Using these systems, we demonstrate that donor cDCs are the critical population presenting alloantigen after BMT, whereas pDCs and macrophages do not make a significant contribution in isolation. In addition, alloantigen presentation was significantly enhanced in the absence of donor B cells, confirming a regulatory role for these cells early after transplantation. These data have major implications for the design of therapeutic strategies post-BMT, and suggest that cDC depletion and the promotion of B-cell reconstitution may be beneficial tools for the control of alloreactivity.

Induction of natural killer T cell–dependent alloreactivity by administration of granulocyte colony–stimulating factor after bone marrow transplantation

Granulocyte colony–stimulating factor (G-CSF) is often used to hasten neutrophil recovery after allogeneic bone marrow transplantation (BMT), but the clinical and immunological consequences evoked remain unclear. We examined the effect of G-CSF administration after transplantation in mouse models and found that exposure to either standard G-CSF or pegylated-G-CSF soon after BMT substantially increased graft-versus-host disease (GVHD). This effect was dependent on total body irradiation (TBI) rendering host dendritic cells (DCs) responsive to G-CSF by upregulating their expression of the G-CSF receptor. Stimulation of host DCs by G-CSF subsequently unleashed a cascade of events characterized by donor natural killer T cell (NKT cell) activation, interferon-γ secretion and CD40-dependent amplification of donor cytotoxic T lymphocyte function during the effector phase of GVHD. Crucially, the detrimental effects of G-CSF were only present when it was administered after TBI conditioning and at a time when residual host antigen presenting cells were still present, perhaps explaining the conflicting and somewhat controversial clinical studies from the large European and North American BMT registries. These data have major implications for the use of G-CSF in disease states where NKT cell activation may have effects on outcome.

Graft-versus-Host Disease Prevents the Maturation of Plasmacytoid Dendritic Cells

The role of Ag presenting cell subsets in graft-versus-host disease (GVHD) remains unclear. We have thus examined the ability of plasmacytoid dendritic cells (pDC) to modulate transplant outcome. Surprisingly, host pDC were exquisitely sensitive to total body irradiation and were depleted before transplantation, thus allowing us to focus on donor pDC. The depletion of all pDC from bone marrow grafts resulted in an acceleration of GVHD mortality while the depletion of mature pDC from G-CSF mobilized splenic grafts had no effect. Thus, donor bone marrow pDC, but not mature pDC contained within stem cell grafts attenuate acute GVHD. In the presence of GVHD, donor pDC completely failed to reconstitute although a CD11clow120G8+ precursor DC reconstituted in an exaggerated and transient manner. These cells expressed Flt-3, the macrophage colony stimulating factor receptor and, consistent with a common dendritic cell (DC) precursor, were capable of differentiation into pDC and conventional DC in vivo in the absence of GVHD. These precursors were MHC class II+ and CD80/86+ but lacked CD40, were actively presenting host Ag and inhibited GVHD and T cell proliferation in a contact-dependent fashion. These data demonstrate that GVHD prevents the maturation of pDC and instead promotes the generation of a suppressive precursor DC, further contributing to the state of immune paralysis after transplantation.

Type I-IFNs control GVHD and GVL responses after transplantation

Although the effects of type II-IFN (IFN-γ) on GVHD and leukemia relapse are well studied, the effects of type I-interferon (type I-IFN, IFN-α/β) remain unclear. We investigated this using type I-IFN receptor-deficient mice and exogenous IFN-α administration in established models of GVHD and GVL. Type I-IFN signaling in host tissue prevented severe colon-targeted GVHD in CD4-dependent models of GVHD directed toward either major histocompatibility antigens or multiple minor histocompatibility antigens. This protection was the result of suppression of donor CD4(+) T-cell proliferation and differentiation. Studies in chimeric recipients demonstrated this was due to type I-IFN signaling in hematopoietic tissue. Consistent with this finding, administration of IFN-α during conditioning inhibited donor CD4(+) proliferation and differentiation. In contrast, CD8-dependent GVHD and GVL effects were enhanced when type I-IFN signaling was intact in the host or donor, respectively. This finding reflected the ability of type I-IFN to both sensitize host target tissue/leukemia to cell-mediated cytotoxicity and augment donor CTL function. These data confirm that type I-IFN plays an important role in defining the balance of GVHD and GVL responses and suggests that administration of the cytokine after BM transplantation could be studied prospectively in patients at high risk of relapse.

SOCS3 regulates graft-versus-host disease

Suppressor of cytokine signaling-3 (SOCS3) is the main intracellular regulator of signaling by granulocyte colony-stimulating factor, an immune-modulatory cytokine used to mobilize stem cells for transplantation. We have therefore studied the contribution of SOCS3 to the spectrum of graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (SCT). Grafts from SOCS3(-/Deltavav) donor mice in which SOCS3 deficiency is restricted to the hematopoietic compartment had an augmented capacity to induce acute GVHD. With the use of SOCS3(-/DeltaLysM) and SOCS3(-/Deltalck) donors in which SOCS3 deficiency was restricted to the myeloid or T-cell lineage, respectively, we confirmed SOCS3 deficiency promoted acute GVHD mortality and histopathology within the gastrointestinal tract by effects solely within the donor T cell. SOCS3(-/Deltalck) donor T cells underwent enhanced alloantigen-dependent proliferation and generation of interleukin-10 (IL-10), IL-17, and interferon-gamma (IFNgamma) after SCT. The enhanced capacity of the SOCS3(-/Deltalck) donor T cell to induce acute GVHD was dependent on IFNgamma but independent of IL-10 or IL-17. Surprisingly, SOCS3(-/Deltalck) donor T cells also induced severe, transforming growth factor beta- and IFNgamma-dependent, sclerodermatous GVHD. Thus, the delivery of small molecule SOCS3 mimetics may prove to be useful for the inhibition of both acute and chronic GVHD.

Immune insufficiency during GVHD is due to defective antigen presentation within dendritic cell subsets

Alloreactivity after transplantation is associated with profound immune suppression, and consequent opportunistic infection results in high morbidity and mortality. This immune suppression is most profound during GVHD after bone marrow transplantation where an inflammatory cytokine storm dominates. Contrary to current dogma, which avers that this is a T-cell defect, we demonstrate that the impairment lies within conventional dendritic cells (cDCs). Significantly, exogenous antigens can only be presented by the CD8(-) cDC subset after bone marrow transplantation, and inflammation during GVHD specifically renders the MHC class II presentation pathway in this population incompetent. In contrast, both classic and cross-presentation within MHC class I remain largely intact. Importantly, this defect in antigen processing can be partially reversed by TNF inhibition or the adoptive transfer of donor cDCs generated in the absence of inflammation.

Invariant natural killer T cell–natural killer cell interactions dictate transplantation outcome after α-galactosylceramide administration

Invariant natural killer T cells (iNKT cells) have pivotal roles in graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effects. iNKT cells are activated through their T-cell receptors by glycolipid moieties (typically the alpha-galactosylceramide [alpha-GalCer] derivative KRN7000) presented within CD1d. We investigated the ability of modified alpha-GalCer molecules to differentially modulate alloreactivity and GVL. KRN7000 and the N-acyl variant, C20:2, were administered in multiple well-established murine models of allogeneic stem cell transplantation. The highly potent and specific activation of all type I NKT cells with C20:2 failed to exacerbate and in most settings inhibited GVHD late after transplantation, whereas effects on GVL were variable. In contrast, the administration of KRN7000 induced hyperacute GVHD and early mortality in all models tested. Administration of KRN7000, but not C20:2, was found to result in downstream interleukin (IL)-12 and dendritic cell (DC)-dependent natural killer (NK)- and conventional T-cell activation. Specific depletion of host DCs, IL-12, or donor NK cells prevented this pathogenic response and the induction of hyperacute GVHD. These data demonstrate the ability of profound iNKT activation to modulate both the innate and adaptive immune response via the DC-NK-cell interaction and raise concern for the use of alpha-GalCer therapeutically to modulate GVHD and GVL effects.