James L.M. Ferrara

Graft-versus-host disease

Haemopoietic-cell transplantation (HCT) is an intensive therapy used to treat high-risk haematological malignant disorders and other life-threatening haematological and genetic diseases. The main complication of HCT is graft-versus-host disease (GVHD), an immunological disorder that affects many organ systems, including the gastrointestinal tract, liver, skin, and lungs. The number of patients with this complication continues to grow, and many return home from transplant centres after HCT requiring continued treatment with immunosuppressive drugs that increases their risks for serious infections and other complications. In this Seminar, we review our understanding of the risk factors and causes of GHVD, the cellular and cytokine networks implicated in its pathophysiology, and current strategies to prevent and treat the disease. We also summarise supportive-care measures that are essential for management of this medically fragile population.

Acute graft-versus-host disease does not require alloantigen expression on host epithelium

Alloantigen expression on host antigen-presenting cells (APCs) is essential to initiate graft-versus-host disease (GvHD); therefore, alloantigen expression on host target epithelium is also thought to be essential for tissue damage. We tested this hypothesis in mouse models of GvHD using bone-marrow chimeras in which either major histocompatibility complex class I or class II alloantigen was expressed only on APCs. We found that acute GvHD does not require alloantigen expression on host target epithelium and that neutralization of tumor necrosis factor-α and interleukin-1 prevents acute GvHD. These results pertain particularly to CD4-mediated GvHD but also apply, at least in part, to CD8-mediated GvHD. These results challenge current paradigms about the antigen specificity of GvHD effector mechanisms and confirm the central roles of both host APCs and inflammatory cytokines in acute GvHD.

Induction of graft-versus-host disease as immunotherapy for relapsed chronic myeloid leukemia.

BACKGROUND The ability of allogeneic bone marrow transplantation to cure chronic myeloid leukemia (CML) is due to both the conditioning regimen and the antileukemic effects of the lymphocytes in the grafted marrow. We studied the ability of interferon alfa-2b and infusions of mononuclear cells from the marrow donor to induce a graft-versus-leukemia reaction in patients with CML in relapse after bone marrow transplantation. METHODS Eleven patients with relapsed CML after allogeneic bone marrow transplantation were treated with interferon alfa-2b and infusions of mononuclear cells. The patients were monitored for toxic effects, for hematologic and cytogenetic responses, and, with use of the polymerase chain reaction, for elimination of cells containing the bcr/abl messenger RNA transcript characteristic of the leukemic cells. RESULTS Six of the eight patients with stable CML after relapse had complete remissions according to molecular genetic criteria, since no cells with bcr/abl messenger RNA transcripts were detected (the method can identify 1 leukemic cell among 1 million normal cells). The three patients with accelerated CML after relapse did not enter remission. Myelosuppression was prominent in eight patients. Grade I acute graft-versus-host disease (GVHD) occurred in six patients, and grade III acute GVHD occurred in three. Limited chronic GVHD developed in five patients. CONCLUSIONS The induction of a graft-versus-leukemia reaction with interferon alfa-2b and infusions of donor mononuclear cells in patients with CML in relapse after bone marrow transplantation is an effective antileukemic therapy that may offer an alternative to a second marrow transplantation.

Pathophysiologic mechanisms of acute graft-vs.-host disease

Graft-vs.-host disease (GVHD) remains the major toxicity of allogeneic bone marrow transplantation. Mechanistic studies in experimental animal models provide a better understanding of the complex relationships and cascade of events mediated by cellular and inflammatory factors. Also, advances in basic immunology have cleared the way for a more precise view of allogeneic reactions between donor and host. In addition, the use of mutant mice lacking critical cytolytic proteins has helped map out the molecular pathways by which GVHD targets organ damage. In this article, these mechanisms are reviewed and synthesized into a coherent conceptual framework, providing a state-of-the-art summary of the pathophysiology of acute GVHD.

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.

LPS antagonism reduces graft-versus-host disease and preserves graft-versus-leukemia activity after experimental bone marrow transplantation

Acute graft-versus-host disease (GVHD) and leukemic relapse remain the two major obstacles to successful outcomes after allogeneic bone marrow transplantation (BMT). Recent studies have demonstrated that the loss of gastrointestinal tract integrity, and specifically the translocation of LPS into the systemic circulation, is critical to the induction of cytokine dysregulation that contributes to GVHD. Using a mouse BMT model, we studied the effects of direct LPS antagonism on GVHD severity and graft-versus-leukemia (GVL) activity. Administration of B975, a synthetic lipid-A analogue from day 0 to day +6, reduced serum TNF-alpha levels, decreased intestinal histopathology, and resulted in significantly improved survival and a reduction in clinical GVHD, compared with control-treated animals. Importantly, B975 had no effect on donor T cell responses to host antigens in vivo or in vitro. When mice received lethal doses of P815 tumor cells at the time of BMT, administration of B975 did not impair GVL activity and resulted in significantly improved leukemia-free survival. These findings reveal a critical role for LPS in the early inflammatory events contributing to GVHD and suggest that a new class of pharmacologic agents, LPS antagonists, may help to prevent GVHD while preserving T cell responses to host antigens and GVL activity.

A biomarker panel for acute graft-versus-host disease.

No validated biomarkers exist for acute graft-versus-host disease (GVHD). We screened plasma with antibody microarrays for 120 proteins in a discovery set of 42 patients who underwent transplantation that revealed 8 potential biomarkers for diagnostic of GVHD. We then measured by enzyme-linked immunosorbent assay (ELISA) the levels of these biomarkers in samples from 424 patients who underwent transplantation randomly divided into training (n = 282) and validation (n = 142) sets. Logistic regression analysis of these 8 proteins determined a composite biomarker panel of 4 proteins (interleukin-2-receptor-alpha, tumor-necrosis-factor-receptor-1, interleukin-8, and hepatocyte growth factor) that optimally discriminated patients with and without GVHD. The area under the receiver operating characteristic curve distinguishing these 2 groups in the training set was 0.91 (95% confidence interval, 0.87-0.94) and 0.86 (95% confidence interval, 0.79-0.92) in the validation set. In patients with GVHD, Cox regression analysis revealed that the biomarker panel predicted survival independently of GVHD severity. A panel of 4 biomarkers can confirm the diagnosis of GVHD in patients at onset of clinical symptoms of GVHD and provide prognostic information independent of GVHD severity.

Differential roles of IL-1 and TNF-α 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-α 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-α 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-α inhibitor relapsed at a significantly faster rate than those given the IL-1 inhibitor. To confirm that the role of TNF-α 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-α receptor–deficient mice. Recipients of TNF-α 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-α is critical to GVL mediated by donor T cells, whereas IL-1 is not. J. Clin. Invest. 104:459-467 (1999).

Immunobiology of acute graft-versus-host disease

Graft-versus-host disease (GVHD) has been the primary limitation to the wider application of allogeneic bone marrow transplantation (BMT). The immunobiology of acute GVHD is complex and can be conceptualized to be a three-step process. In step 1, the conditioning regimen (irradiation and/or chemotherapy) leads to the damage and activation of host tissues and induces the secretion of inflammatory cytokines TNF-alpha and IL-1. As a consequence expression of MHC antigens and adhesion molecules is increased, thus enhancing the recognition of host alloantigens by donor T cells. Donor T-cell activation in step 2 is characterized by donor T-cell interaction with host APCs and subsequent proliferation, differentiation, and secretion of cytokines. Cytokines such as IL-2 and IFN-gamma enhance T-cell expansion, induce cytotoxic T cells (CTL) and natural killer (NK) cell responses, and prime additional mononuclear phagocytes to produce TNF-alpha and IL-1. These inflammatory cytokines in turn stimulate production of inflammatory chemokines, thus recruiting effector cells into target organs. In step 3, effector functions of mononuclear phagocytes are triggered via a secondary signal provided by lipopolysaccharide (LPS) that leaks through the intestinal mucosa damaged during step 1. This mechanism may result in the amplification of local tissue injury and further promotion of an inflammatory response, which, together with the CTL and NK components, leads to target tissue destruction in the transplant host.