G-CSF modulates cytokine profile of dendritic cells and decreases acute graft-versus-host disease through effects on the donor rather than the recipient.

Allogeneic peripheral blood stem cell transplantation (PBSCT) is increasingly used instead of bone marrow transplantation, particularly in HLA identical sibling pairs. Despite the presence of significantly increased numbers of T cells in the PBSC graft, acute graft-versus-host disease (GVHD) is not increased. We have investigated whether granulocyte-colony stimulating factor (G-CSF) administration to PBSCT recipients, both with and without donor G-CSF pretreatment, further modulates acute GVHD in a murine model of PBSCT. Recipients of G-CSF mobilized splenocytes showed a significantly improved survival (P<0.001) and a reduction in GVHD score and serum LPS levels compared with control recipients. G-CSF treatment of donors, rather than recipients, had the most significant effect on reducing levels of tumor necrosis factor (TNFalpha) 7 days after transplantation. As a potential mechanism of the reduction in TNFalpha, we demonstrate G-CSF decreased dendritic cells TNFalpha, and interleukin-12 production to lipopolysaccharide. In conclusion, G-CSF modulates GVHD predominantly by its effects on donor cells, reducing the production of TNFalpha. G-CSF treatment of bone marrow transplantation recipients, without pretreatment of the donor, does not have an impact on acute GVHD.

The p55 TNF-alpha receptor plays a critical role in T cell alloreactivity.

TNF-alpha is known to be an important mediator of tissue damage during allograft rejection and graft-vs-host disease (GVHD), but its role in supporting T cell responses to allogeneic Ags is unclear. We have studied this question by comparing normal mice with those lacking the p55 (p55 TNFR-/-) or p75 (p75 TNFR-/-) TNF-alpha receptors as donors in well-defined bone marrow transplant (BMT) models. Recipients of p55 TNFR-/- cells had significantly reduced mortality and morbidity from GVHD compared with the other two sources of T cells. In vitro, T cells lacking the p55 (but not the p75) TNF-alpha receptor exhibited decreased proliferation and production of Th1 cytokines in MLC. This defect was only partially restored by exogenous IL-2 and affected both CD4+ and CD8+ populations. CD8+ p55 TNFR-/- proliferation was impaired independently of IL-2 whereas CTL effector function was impaired in an IL-2-dependent fashion. Inhibition of TNF-alpha with TNFR:Fc in primary MLC also impaired the proliferation and Th1 differentiation of wild-type T cells. BMT mixing experiments demonstrated that the reduced ability of p55 TNFR-/- donor cells to induce GVHD was due to the absence of the p55 TNFR on T cells rather than bone marrow cells. These data highlight the importance of TNF-alpha in alloreactive T cell responses and suggest that inhibition of the T cell p55 TNF-alpha receptor may provide an additional useful therapeutic maneuver to inhibit alloreactive T cell responses following bone marrow and solid organ transplantation.

IL-11 separates graft-versus-leukemia effects from graft-versus-host disease after bone marrow transplantation.

We recently showed that IL-11 prevents lethal graft-versus-host disease (GVHD) in a murine bone marrow transplantation (BMT) model of GVHD directed against MHC and minor antigens. In this study, we have investigated whether IL-11 can maintain a graft-versus-leukemia (GVL) effect. Lethally irradiated B6D2F1 mice were transplanted with either T cell-depleted (TCD) bone marrow (BM) alone or with BM and splenic T cells from allogeneic B6 donors. Animals also received host-type P815 mastocytoma cells at the time of BMT. Recipients were injected subcutaneously with recombinant human IL-11 or control diluent twice daily, from 2 days before BMT to 7 days after BMT. TCD recipients all died from leukemia by day 23. All control- and IL-11-treated allogeneic animals effectively rejected their leukemia, but IL-11 also reduced GVHD-related mortality. Examination of the cellular mechanisms of GVL and GVHD in this system showed that IL-11 selectively inhibited CD4-mediated GVHD, while retaining both CD4- and CD8-mediated GVL. In addition, IL-11 treatment did not affect cytolytic effector functions of T cells after BMT either in vivo or in vitro. Studies with perforin-deficient donor T cells demonstrated that the GVL effect was perforin dependent. These data demonstrated that IL-11 can significantly reduce CD4-dependent GVHD without impairing cytolytic function or subsequent GVL activity of CD8(+) T cells. Brief treatment with IL-11 shortly after BMT may therefore represent a novel strategy for separating GVHD and GVL.

Differential roles of IL-1 and TNF-alpha on graft-versus-host disease and graft versus leukemia.

We demonstrate an increase in graft-versus-host disease (GVHD) after experimental bone marrow transplant (BMT) when cyclophosphamide (Cy) is added to an otherwise well-tolerated dose (900 cGy) of total body irradiation (TBI). Donor T cell expansion on day +13 was increased after conditioning with Cy/TBI compared with Cy or TBI alone, although cytotoxic T lymphocyte (CTL) function was not altered. Histological analysis of the gastrointestinal tract demonstrated synergistic damage by Cy/TBI and allogeneic donor cells, which permitted increased translocation of LPS into the systemic circulation. TNF-alpha and IL-1 production in response to LPS was increased in BMT recipients after Cy/TBI conditioning. Neutralization of IL-1 significantly reduced serum LPS levels and GVHD mortality, but it did not affect donor CTL activity. By contrast, neutralization of TNF-alpha did not prevent GVHD mortality but did impair CTL activity after BMT. When P815 leukemia cells were added to the bone marrow inoculum, allogeneic BMT recipients given the TNF-alpha inhibitor relapsed at a significantly faster rate than those given the IL-1 inhibitor. To confirm that the role of TNF-alpha in graft versus leukemia (GVL) was due to effects on donor T cells, cohorts of animals were transplanted with T cells from either wild-type mice or p55 TNF-alpha receptor-deficient mice. Recipients of TNF-alpha p55 receptor-deficient T cells demonstrated a significant impairment in donor CTL activity after BMT and an increased rate of leukemic relapse compared with recipients of wild-type T cells. These data highlight the importance of conditioning in GVHD pathophysiology, and demonstrate that TNF-alpha is critical to GVL mediated by donor T cells, whereas IL-1 is not.

Keratinocyte growth factor separates graft-versus-leukemia effects from graft-versus-host disease.

The major obstacles to successful outcome after allogeneic bone marrow transplantation (BMT) for leukemia remain graft-versus-host disease (GVHD) and leukemic relapse. Improved survival after BMT therefore requires more effective GVHD prophylaxis that does not impair graft-versus-leukemia (GVL) effects. We studied the administration of human recombinant keratinocyte growth factor (KGF) in a well- characterized murine BMT model for its effects on GVHD. KGF administration from day -3 to +7 significantly reduced GVHD mortality and the severity of GVHD in the gastrointestinal (GI) tract, reducing serum lipopolysaccharide (LPS) and tumor necrosis factor (TNF)alpha levels, but preserving donor T-cell responses (cytotoxic T lymphocyte [CTL] activity, proliferation, and interleukin [IL]-2 production) to host antigens. When mice received lethal doses of P815 leukemia cells at the time of BMT, KGF treatment significantly decreased acute GVHD compared with control-treated allogeneic mice and resulted in a significantly improved leukemia-free survival (42% v 4%, P <.001). KGF administration thus offers a novel approach to the separation of GVL effects from GVHD.

Pretransplant chemotherapy reduces inflammatory cytokine production and acute graft-versus-host disease after allogeneic bone marrow transplantation.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) is known to be a critical effector molecule in the pathogenesis of graft-versus-host disease (GVHD), and elevated levels during bone marrow transplantation (BMT) conditioning are associated with more severe GVHD. Many patients receive chemotherapy prior to BMT, but its effect on subsequent toxicities is controversial. METHODS: We studied the effect of prior chemotherapy on GVHD severity and inflammatory cytokine generation in a well-established murine model of allogeneic BMT (B6-->B6D2F1). RESULTS: Three weeks after a single dose of cyclophosphamide, bone marrow and splenic cellularity was reduced by 50% and the production of TNF-alpha to LPS stimulation by macrophages was also markedly impaired (both before and after total body irradiation). Allogeneic BMT recipients previously treated with cyclophosphamide had significantly less GVHD and improved survival relative to recipients previously pretreated with diluent only. This survival advantage was associated with reduced systemic levels of both TNF-alpha and interleukin-1beta 7 days after BMT. This reduction occurred despite equivalent serum levels of lipopolysaccharide, consistent with the reductions in TNF-alpha and interleukin-1beta production by host macrophages after cyclophosphamide pretreatment. CONCLUSIONS: These data support the notion that patients entering BMT conditioning without prior cytotoxic treatment (e.g., patients with chronic myeloid leukemia) may be at increased risk of posttransplant complications associated with excessive inflammatory cytokine production.

Granulocyte colony-stimulating factor-mobilized allogeneic stem cell transplantation maintains graft-versus-leukemia effects through a perforin-dependent pathway while preventing graft-versus-host disease.

Minimization of graft-versus-host disease (GVHD) with preservation of the graft-versus-leukemia (GVL) effect is a crucial step to improve the overall survival of allogeneic bone marrow transplantation (BMT) for patients with hematological malignancies. We and other investigators have shown that granulocyte colony-stimulating factor (G-CSF)-mobilized allogeneic peripheral stem cell transplantation (PBSCT) reduces the severity of acute GVHD in murine models. In this study, we investigated whether G-CSF-mobilized PBSC maintain their GVL effect in a murine allogeneic transplant model (B6 --> B6D2F1). B6 mice (H-2(b)) were injected subcutaneously with human G-CSF (100 micrograms/kg/d) for 6 days and their splenocytes were harvested on day 7 as a source of PBSC. G-CSF mobilization dramatically improved transplant survival compared with nonmobilized controls (95% v 0%, P <.001). Systemic levels of lipopolysaccharide and tumor necrosis factor-alpha were markedly reduced in recipients of allogeneic G-CSF-mobilized donors, but cytolytic T lymphocyte (CTL) activity against host tumor target cells p815 was retained in those recipients. When leukemia was induced in recipients by coinjection of p815 tumor cells (H-2(d)) at the time of transplantation, all surviving recipients of G-CSF-mobilized B6 donors were leukemia-free at day 70 after transplant, whereas all mice who received T-cell-depleted (TCD) splenocytes from G-CSF-mobilized B6 donors died of leukemia. When splenocytes from G-CSF-mobilized perforin-deficient (pfp-/-) mice were used for transplantation, 90% of recipients died of leukemia, demonstrating that perforin is a crucial pathway mediating GVL effects after G-CSF-mobilized PBSCT. These data illustrate that G-CSF-mobilized allogeneic PBSCT separate GVL from GVHD by preserving perforin-dependent donor CTL activity while reducing systemic inflammation.

Tumor necrosis factor- alpha production to lipopolysaccharide stimulation by donor cells predicts the severity of experimental acute graft-versus-host disease.

Donor T cell responses to host alloantigen are known predictors for graft-versus-host disease (GVHD); however, the effect of donor responsiveness to an inflammatory stimulus such as lipopolysaccharide (LPS) on GVHD severity has not been investigated. To examine this, we used mouse strains that differ in their sensitivity to LPS as donors in an experimental bone marrow transplant (BMT) system. Lethally irradiated (C3FeB6)F1 hosts received BMT from either LPS-sensitive (LPS-s) C3Heb/Fej, or LPS-resistant (LPS-r) C3H/ Hej donors. Mice receiving LPS-r BMT developed significantly less GVHD as measured by mortality and clinical score compared with recipients of LPS-s BMT, a finding that was associated with significant decreases in intestinal histopathology and serum LPS and TNF-alpha levels. When donor T cell responses to host antigens were measured, no differences in proliferation, serum IFN-gamma levels, splenic T cell expansion, or CTL activity were observed after LPS-r or LPS-s BMT. Systemic neutralization of TNF-alpha from day -2 to +6 resulted in decreased intestinal pathology, and serum LPS levels and increased survival after BMT compared with control mice receiving Ig. We conclude that donor resistance to endotoxin reduces the development of acute GVHD by attenuating early intestinal damage mediated by TNFalpha. These data suggest that the responsiveness of donor accessory cells to LPS may be an important risk factor for acute GVHD severity independent of T cell responses to host antigens.

Interleukin-11 promotes T cell polarization and prevents acute graft-versus-host disease after allogeneic bone marrow transplantation.

Administration of IL-11 prevented lethal graft-versus-host disease (GVHD) in a murine bone marrow transplant (BMT) model (B6 --> B6D2F1) across MHC and minor H antigen barriers (survival at day 50: 90 vs 20%, P < 0.001). Surpisingly, IL-11 administration polarized the donor T cell cytokine responses to host antigen after BMT with a 50% reduction in IFNgamma and IL-2 secretion and a 10-fold increase in IL-4. This polarization of T cell responses was associated with reduced IFNgamma serum levels and decreased IL-12 production in mixed lymphocyte cultures (MLC). In addition, IL-11 prevented small bowel damage and reduced serum endotoxin levels by 80%. Treatment with IL-11 also reduced TNFalpha serum levels and suppressed TNFalpha secretion by macrophages to LPS stimulation in vitro. IL-11 thus decreased GVHD morbidity and mortality by three mechanisms: (a) polarization of donor T cells; (b) protection of the small bowel; and (c) suppression of inflammatory cytokines such as TNFalpha. We conclude that brief treatment with IL-11 may represent a novel strategy to prevent T cell-mediated inflammatory processes such as GVHD.

Total body irradiation and acute graft-versus-host disease: the role of gastrointestinal damage and inflammatory cytokines.

The influence of bone marrow transplantation (BMT) conditioning regimens on the incidence and severity of graft-versus-host disease (GVHD) has been suggested in clinical BMT. Using murine BMT models, we show here an increase in GVHD severity in several donor-recipient strain combinations after intensification of the conditioning regimen by increasing the total body irradiation (TBI) dose from 900 cGy to 1,300 cGy. Increased GVHD was mediated by systemic increases in tumor necrosis factor alpha (TNF alpha). Histologic analysis of gastrointestinal tracts showed synergistic damage by increased TBI and allogeneic donor cells that permitted increased translocation of lipopolysacharide (LPS) into the systemic circulation. In vitro, LPS triggered excess TNF alpha from macrophages primed by the GVH reaction. In addition, macrophages isolated within 4 hours of conditioning were primed in proportion to the TBI dose itself to secrete TNF alpha. Thus, the higher TBI dose increased macrophage priming and increased gut damage after allogeneic BMT, causing higher systemic levels of inflammatory cytokines and subsequent severe GVHD. These data highlight the importance of conditioning in GVHD pathophysiology and suggest that interventions to prevent LPS stimulation of primed macrophages may limit the severity of GVHD after intensive conditioning for allogeneic BMT.