Background Information

Two distinctive forms of GVHD occur: acute and chronic GVHD. These disorders differ in that acute GVHD (AGVHD) predominately involves a T cell-mediated attack on the host, with subsequent inflammatory cytokine-induced systemic effects. Chronic GVHD, in contrast, is an autoimmune disorder characterized by chronic B cell stimulation, and autoantibody production. The basic experimental methods utilized for establishing both acute and chronic GVHD models are similar; variables such as donor/host strain combination and transplant preparative regimen can be individualized to induce the different disease states.

Models of AGVHD

The main model for induction of AGVHD (see Basic Protocol 1) involves transplantation of donor bone marrow and lymphocytes into lethally irradiated allogeneic hosts. This method results in the complete expression of the clinical syndrome, including inflammatory pathology in the liver, skin, and gut, marked weight loss, and significant mortality. Transplantation of donor cells into hosts differing at major or minor histocompatibility loci can produce severe, lethal AGVHD by this method (see Table 4.3.1).

An alternative model (P ^ F1 AGVHD), extensively used to study mechanisms of T cell dysfunction, involves injection of spleen cells from parental strain (P) donors into unirradi-ated immune-competent adult F1 hosts. Unlike in irradiated models, the donor must differ from the host at both class I and class II major histocompatibility (MHC) loci to initiate a reaction, and the parental inocula must include both CD4+ and CD8+ T cells. In the F1 hosts, parental donor T cells expand and produce cytotoxic effectors, resulting in the elimination of the semiallogeneic host lymphohematopoie-tic system (Hakim et al., 1991). Subsequently, the donor/host chimera is repopulated by donor cells derived from stem cells in the original splenic inoculum. A severe deficiency in both T and B cell function is observed for several weeks. In contrast to irradiated AGVHD models, however, skin pathology and systemic weight loss are minimal, and death is uncommon. Hence, the main parameters studied are the generation of anti-host cytotoxic effectors, level of donor/host chimerism, and immune deficiency.

In irradiated AGVHD models, hosts receive both donor bone marrow (as a source of stem cells for hematopoietic reconstitution), and lymphocytes from donor spleen or lymph nodes (as a source of T cells for induction of GHVD). In severe cases, such transplants result in rapid weight loss and death; in less severe GVHD, transient weight loss, delayed immune reconstitution, and prolonged immune deficiency are the only observed abnormalities. The severity of AGVHD in any given model is dependent upon the genetic disparity between donor and host and the T cell subsets present in the allogeneic inocula (Korngold and Sprent,

1991). Donor CD4+ T cells are necessary for lethal GVHD in MHC-disparate or class II antigen-disparate combinations, whereas CD8+ T cells are both necessary and sufficient to generate lethality in class I antigen-disparate combinations. In most MHC-matched, minor antigen-mismatched combinations, CD8+ cells are sufficient to induce lethality, although CD4+ cells can increase the severity in many combinations and can produce lethality in some (Korngold, 1992). Table 4.3.1 outlines the common strain combinations utilized for AGVHD models.

Much of the pathogenesis of AGVHD has been attributed to a "cytokine storm" (Ferrara et al., 1993). The irradiation utilized in acute GVHD models damages the gut and skin, resulting in systemic exposure to bacterial-derived toxins, and subsequent inflammatory cy-tokine release (primarily TNF-a and IL-1) and generalized immune activation. Donor T cells reacting to host alloantigens produce TH1 cytokines, especially IFN-y (Troutt and Kelso,

1992), which activate proinflammatory macrophages; these activated macrophages are then triggered by bacterial products such as LPS to release high levels of IL-1p, TNF-a, and nitric oxide, resulting in weight loss and death by toxic shock (Nestel et al., 1992). Thus, three primary factors influence the severity of AGVHD: the conditioning regimen (radiation dose), donor T cell response, and environmental pathogens (as a source for LPS endo-toxin). Many laboratories have observed that as animal colony conditions have become cleaner or colonies have been converted to a specific-pathogen-free status, the dose of radiation and/or donor T cells needed to induce lethal AGVHD has increased. The importance of LPS in triggering the cytokine storm has also been demonstrated in P ^ F1, unirradiated AGVHD models: injection of low, normally nonlethal doses of LPS to AGVHD recipients results in acute lethality 8 to 12 hr post LPS administration (Nestel et al., 1992).

Models of CGVHD

Whereas AGVHD models emphasize cellmediated immune processes, chronic GVHD (CGVHD) models focus upon the development of chronic, B cell-stimulatory autoimmune disorders (Goldman et al., 1991); AGVHD appears to be an inflammatory process mediated by TH1 cytokines, whereas CGVHD is mediated by Th2 cytokines. CGVHD is characterized by prolonged splenomegaly and lym-phadenopathy. Mice develop symptoms of systemic autoimmune disorders, including immune complex glomerulonephritis (Ito et al., 1992), primary biliary cirrhosis (Saitoh et al., 1991), and Sj0gren-syndrome- or scleroderma-like lesions (Fujiwara et al., 1991). Elevated levels of immunoglobulins, particularly IgG1 and IgE, and a variety of autoantibodies (including anti-DNA) are present in the serum. In general, the autoimmune syndrome of CGVHD is lethal within 6 months, due to development of immune complex-mediated glomerular nephritis. Murine CGVHD models share many of the pathologic features of the clinical disorder of chronic graft-versus-host disease that occurs after allogeneic bone marrow transplantation in man. It is important to note, however, that the human syndrome develops several months after marrow transplant, whereas murine CGVHD develops within weeks. Additionally, human CGVHD occurs after an acute suppressive GVHD, and may require ongoing donor antihost reactivity and aberrant T cell maturation in the post-transplant thymic environment. The murine disorder, in contrast, is usually based on an immediate and continuing donor T cell reaction to allogeneic antigens in a mixed chimeric host.

Murine modeling of clinical CGVHD by use of irradiated hosts has generally been ineffec

In Vivo Assays for



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