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CONCISE REVIEW |
Division of Pediatric Oncology, Dana-Farber Cancer Institute and Children's Hospital; Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA
Key Words. Graft-versus-host-disease • Cytokines • Bone marrow transplantation • Th1/Th2 cells • Review
Correspondence: Dr. Werner Krenger, Division of Pediatric Oncology, D1638, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA.
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Abstract |
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 Top Abstract Definition and EtiologyPathologyRole of Cytokines in...Therapeutic InterventionsConclusionsAcknowledgementsReferences |
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) or by T helper 2 (Th2) cells (interleukin 4, interleukin 10) after allogeneic BMT is hypothesized to govern the extent of the systemic inflammatory response. Because Th2 cytokines can inhibit the production of proinflammatory cytokines such as interleukin 1 and tumor necrosis factor-
, a Th1
Th2 shift in the initial response of donor T cells may interrupt the cytokine cascade and thus offer a new approach to the prevention and treatment of acute GVHD. Successful interventions to modify the response of donor T cells may obviate the need for T cell depletion and thereby avoid the increased risk of relapse of malignancy and impairment of donor cell engraftment.
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Definition and Etiology |
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TopAbstractDefinition and EtiologyPathologyRole of Cytokines in...Therapeutic InterventionsConclusionsAcknowledgementsReferences |
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In 1966, these observations led Billingham to formulate the requirements for the development of GVHD. First, the graft must contain immunologically competent cells, Second, the recipient must be incapable of mounting an effective response to destroy the transplanted cells, and third, the recipient must express tissue antigens that are not present in the transplant donor [2]. According to these criteria, GVHD can develop in various clinical settings when tissues containing immunocompetent cells (blood products, bone marrow, solid organs) are transferred between individuals (Table 1
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The second requirement, the expression of recipient tissue antigens not present in the donor, became the focus of intensive research with the discovery of the major histocompatibility complex (MHC). MHC antigenic differences between donor and recipient are the most important risk factors for the induction of GVHD. In addition, there are minor histocompatibility antigens encoded for by genetic loci, one of which has been recently identified [5]. GVH reactions can also occur between genetically identical strains and individuals [6]. These observations have necessitated a revision of Billingham's second postulate to include, in addition to the recognition of foreign host antigens, the inappropriate recognition of host self antigens, i.e., an autoimmune process.
Billingham's third requirement stipulates that the recipient of immunocompetent T cells must be immunocompromised. A patient with a normal immune system will usually reject T cells from a foreign donor, thus preventing GVHD. This third requirement is most commonly met in allogeneic BMT, where recipients have usually received highly immunosuppressive doses of chemotherapy and radiation before marrow infusion, but may also be met in other situations (Table 1
).
Recipients of solid organ grafts are treated with immunosuppressive drugs to prevent rejection of the transplanted organ and thereby become susceptible to the attack of T cells which are present in the donor graft (e.g., small bowel). GVHD can also occur in an immunocompetent recipient if genetic factors are permissive. Normal recipients, who are heterozygous for HLA proteins, will not reject lymphocytes that are transfused from a donor who is homozygous for one of the recipient's haplotypes (since the donor HLA antigens are already codominantly expressed on the recipient's cells). On this basis, patients undergoing surgery that receive directed blood transfusions (e.g., transfusion of blood from an HLA homozygous parent to a heterozygous child) may develop GVHD, even though they are not immunocompromised [7].
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Pathology |
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TopAbstractDefinition and EtiologyPathologyRole of Cytokines in...Therapeutic InterventionsConclusionsAcknowledgementsReferences |
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The principal target organs of acute GVHD include the immune system, skin, liver, and intestine. In addition, experimental observations suggest that lung tissues may also be affected by consequences of the alloreaction [9, 10]. In cases of transfusion-associated GVHD, bone marrow aplasia is often observed because HLA-incompatible hematopoietic cells become targets. GVHD pathology characteristically includes epithelial damage of target organs, which is usually apoptotic in nature [11]. In the skin, the epidermis and hair follicles are often destroyed. In the liver, small bile ducts are profoundly affected and segmental disruption is common. Intestinal crypt destruction results in mucosal ulcerations which may be either patchy or diffuse. Lung injury consists of a dense mononuclear cell infiltrate around both pulmonary vessels and bronchioles [12]. An acute pneumonitis, composed of a mixed inflammatory infiltrate within a fibrin matrix, has also been observed involving the interstitium and alveolar spaces [9, 10].
A prominent pathologic feature of acute GVHD is the disparity between the severity of tissue destruction and the paucity of the lymphocytic infiltrate. Recent studies of soluble mediators of GVHD such as interleukin (IL)-1 and tumor necrosis factor (TNF)- have suggested that direct contact between target cell and lymphocyte is not required for target cell destruction.
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Role of Cytokines in the Immunopathophysiology of Acute GVHD |
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TopAbstractDefinition and EtiologyPathologyRole of Cytokines in...Therapeutic InterventionsConclusionsAcknowledgementsReferences |
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). First, recognition of host tissues activates donor T lymphocytes (afferent phase). Activated T cells subsequently attack target tissues in transplanted recipients (efferent phase) or recruit effector cells which contribute to the development of tissue damage in GVHD target organs. Recent findings have further increased our understanding of this process. There is now substantial evidence to implicate the inappropriate production of cytokines, which are the central regulatory molecules of the immune system, as a primary cause for the induction and progression of experimental and clinical GVHD [13-15].
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and IL-1 [16]. The presence of inflammatory cytokines during this phase may upregulate adhesion molecules and MHC antigens [17, 18], thereby enhancing the recognition of host MHC and/or minor histocompatibility antigens by mature donor T cells after allogeneic BMT. This process is in accordance with the observation that enhanced risk of GVHD after clinical BMT is associated with certain intensive conditioning regimens that cause extensive injury to epithelial and endothelial surfaces and the subsequent release of inflammatory cytokines and enhanced expression of cell-surface adhesion molecules [19, 20]. Donor T cell activation (antigen recognition, proliferation, and differentiation) occurs during the second step of the afferent phase of acute GVHD. These processes are increasingly understood at the subcellular and molecular level [21]. During antigen presentation, T cells recognize MHC/peptide complexes via antigen-specific T cell receptors. In allogeneic interactions leading to GVHD, mature donor T cells recognize foreign (host) MHC molecules containing foreign peptides. Host antigen-presenting cells (APCs) provide costimulatory activation signals by B7/CD28 interaction and IL-1 [22, 23]. In addition to the T cell receptor, accessory molecules such as CD4, CD8, LFA-1, LFA-2 and CD44 participate in cell-cell interactions by enhancing cellular contact and communication. Alloantigen composition of the host determines which subset of T cells proliferates and differentiates. MHC class II (HLA-DR, DP, DQ) differences stimulate CD4+ T cells; MHC class I (HLA-A, B, C) differences stimulate CD8+ T cells. CD4 and CD8 proteins are coreceptors for constant portions of MHC class II and MHC class I molecules, respectively. In mouse models of GVHD, where genetic differences between multiple strain combinations can be controlled, CD4+ cells induce GVHD to MHC class II molecules, and CD8+ cells induce GVHD to MHC class I molecules [3]. Thus, both CD4+ and CD8+ T cell subsets can initiate the afferent phase of GVHD. In the majority of clinical marrow HLA identical transplants, GVHD may be induced by either subset or simultaneously by both.
Antigen presentation induces the activation of individual T cells. This involves multiple, rapidly occurring intracellular biochemical changes, including the rise of cytoplasmic free calcium and the activation of protein kinase C and tyrosine kinases [24, 25]. These pathways in turn activate transcription of genes for cytokines, such as IL-2, interferon (IFN)-, and their receptors. These cytokines are preferentially produced by the T helper (Th) 1 subset of T cells [26, 27]. Both IL-2 and IFN- have long been implicated in the pathophysiology of acute GVHD, and play central roles in further T cell activation, induction of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cell responses. They also prime additional donor and residual host mononuclear phagocytes to produce IL-1 and TNF-. IL-2 also induces the expression of its own receptor (autocrine effect) and stimulates proliferation of other cells expressing the receptor (paracrine effect). The T cell activation phase is followed by clonal expansion and differentiation. Functional differentiation then occurs as cells produce proteins required for specific effector functions, such as the protein esterases that are required for CTLs [28]. The expression of many cell surface molecules is altered; receptors for lymphocyte homing and migration may be downregulated, and other adhesion molecules (e.g., LFA-1) may be upregulated, thus altering the T cells' ability to traffic in vivo [29].
While inflammatory effector functions of mononuclear phagocytes are stimulated by the IL-2 and IFN- that are secreted by activated Th1 T cell subsets, effector functions of mononuclear phagocytes are inhibited by IL-4 and IL-10, cytokines produced by activated Th2 T cells. Both CD4+ and CD8+ subsets can differentiate into "type 1" and "type 2" cells [30-33]. There is now considerable evidence that the preferential expansion of Th2 T cells after allogeneic transplantation is associated with the development of a "chronic" GVHD syndrome which shows decreased lethality and antibody formation [34-37].
Efferent Phase
The efferent phase, which encompasses step 3 of the pathophysiology of acute GVHD, is complex and probably the least understood. The initial hypothesis that the cytolytic function of CTL directly causes the majority of tissue damage and necrosis has been largely displaced [38]. Large granular lymphocytes or NK cells appear to be prominent in the effector arm of GVHD and may contribute to the pathologic damage, i.e., induce the changes of GVH disease following the T cell-mediated GVH reaction [39]. Large granular lymphocytes do not recognize MHC proteins as targets, but they are probably recruited by cytokines released by T cells. The precise relationship between cytokines induced during step 2 and the mediators of tissue damage during step 3 is an area of active investigation.
Increased secretion of the inflammatory cytokines TNF- and IL-1 by mononuclear phagocytes which had been primed with Th1 cytokines during step 2 is thought to be triggered by a secondary signal. This stimulus may be provided by lipopolysaccharide (endotoxin, LPS), which can leak through the intestinal mucosa damaged by the conditioning regimen. LPS subsequently may stimulate gut-associated lymphocytes and macrophages [40]. LPS reaching skin tissues via the circulation may also stimulate keratinocytes, dermal fibroblasts and macrophages to produce similar cytokines in the dermis and epidermis. TNF- can cause direct tissue damage by inducing necrosis of target cells. Another mechanism by which TNF- may induce tissue destruction during GVHD is apoptosis, or "programmed" cell death. Apoptosis may be characteristic of disease in the large intestine after allogeneic BMT [41]. In addition to these proinflammatory cytokines, excess nitric oxide produced by activated macrophages may contribute to the deleterious effects on GVHD target tissues, particularly immunosuppression [42, 43]. Induction of inflammatory molecules may thus synergize with the lytic component provided by CTL and NK cells [38, 44], amplify local tissue injury and further promote an inflammatory response which ultimately leads to the observed target tissue destruction in the BMT host.
This conceptual framework helps to explain a number of unique and seemingly unrelated aspects of GVHD. For example, a number of clinical studies have noted increased risks of GVHD associated with advanced-stage leukemia, certain intensive conditioning regimens, and histories of viral infections [19, 20, 45]. Similarly, the importance of bowel decontamination in the prevention of GVHD may be explained by the ability of LPS to leak through damaged intestinal mucosal surfaces and stimulate the gut-associated lymphocytes and macrophages to produce inflammatory cytokines [40, 46, 47].
With this cytokine hypothesis as background, we will review individual cytokines as they relate to acute GVHD. There will also be references to some experimental systems that are particularly relevant to cytokine dysregulation even though there is as yet no direct connection to acute GVHD.
Th1 Cytokines
Th1 cells producing IL-2 have a pivotal role in controlling and amplifying the immune response against alloantigens, representing step 2 of the cytokine cascade that initiates acute GVHD (Fig. 1). Experimental data show that maximal levels of IL-2 are produced by donor CD4+ T cells in the first two days after GVHD induction, with lower levels observed in splenocytes from GVHD mice until 7-10 days post-transplant [48]. A similar early and transient increased expression of IL-2 during GVHD was demonstrated in murine GVHD models using reverse-transcriptase-polymerase chain reaction (PCR) techniques for detection of IL-2 mRNA [37]. The addition of low doses of IL-2 during the first week after allogeneic BMT enhanced the severity and mortality of GVHD, but not when GVHD was induced to MHC class II antigens [49-51]. Unlike a mixed lymphocyte culture where bulk IL-2 production of donor cells to host antigens is analyzed, the precursor frequency of host-specific IL-2-producing T cells (pHTLs) predicts the occurrence of GVHD after transplantation between HLA-identical siblings [52, 53]. The critical precursor frequency of host-specific pHTLs seems to be approximately 1/100,000. Patients whose donor bone marrow contained fewer host-specific pHTLs later developed only grade 0-1 GVHD, whereas patients whose donor bone marrow contained greater frequencies all developed significant grade 2 or 3 GVHD. pHTL cells were detectable as early as day 20 after transplant, often preceding the onset of acute GVHD by approximately two weeks, and persisting until the GVHD resolved.
Due to their apparent importance in initiating acute GVHD, IL-2-producing donor T cells have been the target of many experimental designs to control GVHD. The administration of cyclosporine, a powerful inhibitor of IL-2 production, is an effective prophylactic agent against GVHD [54, 55]. The importance of IL-2 is further underscored by experiments showing that monoclonal antibodies (mAbs) against the IL-2 receptor are efficient in preventing GVHD in animals or in clinical GVHD when administered shortly after the infusion of T cells [56, 57]. It should be noted that in two clinical trials, the addition of an anti-IL-2 receptor antibody was only moderately successful in reducing the incidence of severe GVHD [58, 59]. Moreover, a brief administration of high doses of exogenous IL-2 early after BMT protects animals from GVHD mortality [60]. This protective effect, which is augmented by the cotransplantation of syngeneic bone marrow, is not associated with a decrease in either alloengraftment or a graft-versus-leukemia (GVL) effect [61, 62]. It has been suggested that IL-2 mediates its protective effect via inhibition of other cytokines, e.g., IFN- [63], and that it might also involve the in vivo activation of NK cells and/or lymphocyte-activated killer cells [64].
IFN- is another crucial cytokine that can be implicated in the second step of the pathophysiology of acute GVHD. Using a method to enumerate cytokine mRNA-containing cells that combined limiting dilution analysis and PCR amplification of cDNA, IFN- secretion was found to be greatly increased during experimental GVHD [65]. Transcription of IFN- mRNA occurred in 2% of unstimulated splenocytes from mice with GVHD, a 40-fold increase above controls. Stimulation of these cells via T cell receptor ligation increased the number of cells containing IFN- mRNA to 67%, a 70-fold increase over non-GVHD controls. A less quantitative approach has also demonstrated that IFN- is significantly increased 14 days after induction of acute GVHD in a nonirradiated GVHD model [35]. The release of IFN- is an early event in the cascade leading to GVHD because IFN- production in animals with GVHD peaks at day 7 post-BMT, before clinical manifestations are apparent. In several models of experimental acute GVHD, lymphocytes produce large amounts of IFN- when restimulated in vitro with mitogen or other stimuli involving T cell receptor ligation, in contrast to lymphocytes from hosts receiving syngeneic or T cell-depleted BMT [66-71]. In clinical BMT, a large proportion of T cell clones isolated from GVHD patients also produce IFN- [72]. In a small number of patients studied, a modest increase in serum IFN- levels has been found [73].
Experimental data suggest that IFN- is involved in several aspects of the pathophysiology of acute GVHD. First, it may be directly involved in producing cytopathic effects of GVHD in a skin explant model, where high levels of both IFN- and TNF- correlate with the most intense cellular damage [74]. Second, IFN- can mediate the development of pathological processes in the gastrointestinal tract during GVHD, and the addition of an anti-IFN- mAb prevents this damage [75]. Because the expression of MHC class I and II molecules is induced by IFN-, it is possible that an increase of these molecules during GVHD [76] might make target cells in gut and epithelium more susceptible to lysis by cytotoxic T cells [77]. Third, IFN- appears to mediate the suppression of lymphocyte proliferation in response to both mitogens and alloantigens in several experimental GVHD systems [42, 78-81].
The synergy of IFN- with LPS in the activation of mononuclear phagocytes to produce proinflammatory cytokines [82, 83] may also play a role during acute GVHD; a now classic study showed that IFN- is sufficient to produce macrophage priming during a PF1 GVHD model [40]. The subsequent administration of LPS to host mice caused death due to the increased systemic production of TNF- by macrophages. Recent data have confirmed these observations and demonstrated that the inhibition of IFN- production after MHC class I or II disparate BMT by injection of polarized donor T cells (which secrete IL-4 but not IFN-) results in the downregulation of LPS-triggered TNF- production and inhibits GVHD-related mortality [69].
These data illustrate that activation of Th1 T cells secreting IFN- is an important event in the induction of acute GVHD. The injection of IFN-, however, can actually prevent the development of experimental GVHD [84]. These data were surprising because, according to the findings described above, one would expect IFN- to promote rather than to counteract GVHD. These results may be due to the diametrically opposed effects of local versus systemic actions of IFN- [85]. The mechanism by which IFN- prevents GVHD may involve a reduction in the number of IFN--secreting donor T cells through activation of host NK by IFN- [86, 87]. However, further experiments are required to prove this hypothesis.
Inflammatory Cytokines
Murine BMT models have provided the strongest evidence of the link between excessive production of inflammatory cytokines and clinical acute GVHD. During the course of this disease, mononuclear phagocytes become activated, as demonstrated by an increase in their phagocytic and bactericidal activity. Secretion of inflammatory cytokines by activated macrophages plays a key role in causing tissue damage during step 3 of the cytokine hypothesis [88]. TNF- is an inflammatory cytokine which causes an extremely wide variety of biological effects, including the induction of direct tissue injury and of metabolic alterations in muscle and fat tissues leading to cachexia (characterized by anorexia and weight loss) which ultimately leads to death [89].
A critical role for TNF- in the pathophysiology of acute GVHD was first suggested almost 10 years ago because mice transplanted with mixtures of allogeneic bone marrow and T cells developed severe skin, gut, and lung lesions that were associated with high levels of TNF- mRNA in these tissues [90]. Subsequently, the presence of TNF- mRNA was also demonstrated in the skin of mice with GVHD induced to minor histocompatibility antigens [91]. Target organ damage could be inhibited by infusion of anti-TNF- antibodies and mortality could be reduced from 100% to 50% by administration of the soluble form of the TNF- receptor [16], an antagonist of TNF-. The mechanisms by which TNF- may induce tissue destruction during GVHD include necrosis and apoptosis.
The role of TNF- in GVHD was further elucidated using an experimental murine GVHD model with nonirradiated recipients, where macrophages in animals with GVHD are primed to release TNF- after stimulation with LPS [40]. Injection of small, normally non-lethal amounts of LPS caused elevated TNF- serum levels and death in animals with GVHD; this mortality could be prevented with antiserum against TNF-. These experiments strongly supported the role of mononuclear cells, or macrophages, as sources of inflammatory cytokines during the effector phase of acute GVHD. It appears that injury to the gastrointestinal tract due to the conditioning regimen enables bacterial breakdown products like LPS to enter the circulation and to reach immune compartments and ultimately to trigger release of cytokines from mononuclear phagocytes. It has long been known that pathogen-free mice are protected from GVHD after allogeneic BMT and that recolonization of gram-negative bacteria in the gut leads to increased GVHD severity [46]. The anatomic localization of macrophages to gut-associated lymphoid tissue and the ability of endotoxin to trigger inflammatory cytokine release together provide the most persuasive explanation for the ability of gut decontamination to prevent systemic GVHD.
Recently, we have explored the roles of TNF- and LPS in the development of acute lung injury after transplantation. Idiopathic pneumonia syndrome (IPS) refers to diffuse, noninfectious pneumonia that occurs after allogeneic BMT and constitutes approximately 50% of all pulmonary insults after clinical BMT [92]. We have developed a model of IPS using a well-characterized murine BMT system (B10.BRCBA) in which lung injury can be induced by minor H antigens [9]. Evaluation of lung pathology and bronchoalveolar lavage (BAL) fluid from recipients of syngeneic and allogeneic BMT six weeks after transplant demonstrated both parenchymal pneumonitis and periluminal infiltrates around vessels and bronchioles (as described earlier) only in the allogeneic group. In this model, the development of significant lung injury correlated with the presence but not severity of systemic GVHD in affected animals. This pulmonary injury was also associated with elevated BAL fluid levels of LPS, neutrophils, and TNF-, and serum levels of LPS. No pathogenic organisms were isolated from the respiratory tract of any animal. When the role of LPS in the development of this injury was tested directly, injection of LPS six weeks after transplant caused profound lung injury only in mice with moderate GVHD. Dramatic increases in BAL neutrophils and TNF- were also observed, with alveolar hemorrhage occurring in 4/12 of these mice but in no other group.
An important role for TNF- in clinical acute GVHD has been suggested by studies demonstrating elevated levels of TNF- in the serum of patients with acute GVHD and other endothelial complications such as veno-occlusive disease [93, 94]. Significantly, the first appearance of the increased TNF- levels was predictive of the severity of complications and overall survival. Patients with higher serum TNF- levels during the conditioning regimen (pre-BMT) had an incidence of acute GVHD greater than 90% and less than 30% overall survival. More recently, a phase I/II trial using mAbs to the TNF- receptor during the conditioning regimen as prophylaxis in patients at high risk for severe acute GVHD showed reduction in lesions of the intestine, skin, and liver [95]. In the majority of patients, GVHD flared after discontinuation of treatment. In another study, pentoxifylline, an inhibitor of TNF-, was able to prevent many major complications of BMT, including veno-occlusive disease, mucositis, and acute GVHD [96]. These preliminary data, as well as animal and laboratory studies, suggest that approaches to limit TNF- secretion will be a very important area of investigation in BMT.
IL-1 is the second major proinflammatory cytokine which appears to play an important role in the effector phase of acute GVHD. This cytokine is mainly produced by activated mononuclear phagocytes and shares a variety of biological activities with TNF- [97]. Secretion of IL-1 appears to occur predominantly during the effector phase of GVHD in the spleen and skin, two major GVHD target organs [98]. At four weeks post-BMT when the majority of animals had died of GVHD and surviving animals clearly had active disease, a striking increase of IL-1 mRNA by at least 200 times above controls in both organs was observed. A similar increase in mononuclear cell IL-1 mRNA has been shown during clinical acute GVHD [99]. Indirect evidence for a role of IL-1 in GVHD was obtained with administration of this cytokine to hosts in an allogeneic murine BMT model [100]. Mice receiving IL-1 displayed a wasting syndrome and increased mortality that appeared to be an accelerated form of disease.
If IL-1 is a central mediator of GVHD, specific inhibition of IL-1 action should reduce or eliminate it. Investigations of the role of IL-1 in GVHD have intensified after the discovery of IL-1ra [101]. Intraperitoneal administration of IL-1ra starting on day 10 post-BMT was able to reverse the development of GVHD in the majority of animals, providing a significant survival advantage to treated animals (75% versus 28%) [91]. The important role of IL-1ra may also explain the effect of -globulin in reducing GVHD [102]. When IgG is bound to macrophages, IL-1ra production is preferentially increased over that of IL-1 [103]. Exogenously administered IgG may thus directly modulate the cytokine dysregulation during GVHD. These animal data emphasize the role of IL-1 during the effector phase of the cytokine dysregulation and support further examination of IL-1ra for treatment of clinical GVHD.
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Therapeutic Interventions |
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The most effective method of GVHD prevention is the removal of all T cells from the bone marrow [107]. This can be accomplished either by physical separation (lectin agglutination) or by treatment with mAbs directed at T cells. With the latter approach, T cells are either destroyed by a toxin (e.g., ricin-A chain) that has been attached to the antibody, or by incubating the bone marrow first with the antibody and then with complement, which lyses the antibody-coated T cells. Alternatively, antibodies can be linked to magnetic beads, which remove T cells expressing the appropriate specificities by passage over a magnet. These procedures usually achieve 90% to 99.9% reduction in T cell content of the bone marrow, and result in substantial reductions in the incidence and severity of GVHD.
Depletion of T cells from donor bone marrow has been shown to prevent GVHD in numerous experimental models [108-110], and reports from more than 800 cases of T cell-depleted BMT between 1981 to 1986 confirm this finding [111]. Unfortunately, the use of T cell-depleted marrow is associated with a higher rate of graft failure [111]. Failure of engraftment has serious clinical consequences and is frequently fatal. Although the removal of T cells may cause an unintentional loss of marrow stem cells, T cells themselves probably mediate a graft-enhancing effect. This effect may be due to the suppression of radioresistant, alloreactive host immune cells [112], or to the release by T cells of hematopoietic growth factors [113]. Protocols that increase the immunosuppression of the conditioning regimen or remove only subsets of T cells reduce the risk of graft failure.
Another concern is the increased incidence of relapse among recipients of T cell-depleted marrow grafts with certain types of leukemia, particularly chronic myelocytic leukemia [114]. This appears related to a graft-versus-leukemia effect (GVL) which is associated with GVHD in experimental systems [115]. Although recent data suggest that certain T cells from transplanted patients may recognize tumor-specific antigens [116], it has been difficult to separate GVHD and GVL in humans. In fact, data from Seattle suggest that only clinical GVHD was associated with GVL, whereas subclinical disease showed no benefit [117]. Thus, although T cell depletion reduces mortality from GVHD, problems with engraftment and increased leukemic relapse have resulted in survival rates similar to those of patients who receive conventional prophylaxis.
A further experimental approach to preventing GVHD that has been successful is that of gnotobiosis, i.e., the maintenance of transplant recipients in a germ-free environment [118]. This has allowed for transplantation across MHC barriers in mice without the development of GVHD. Conceptually, this approach prevents T cell activation by eliminating microbial antigens which may cross-react with host alloantigens or which may cause nonspecific activation of macrophages and APCs. The responses of entire families of T cell receptors to highly conserved "superantigens" such as Staphylococcus enterotoxin B may explain the immunogenicity of such microbial antigens. The presence of T cells expressing the 
T cell receptor with tropism for intestinal tissues, the expansion of T cells after BMT and their response to other highly conserved antigens such as heat shock proteins may also be mechanistically involved. Reduction in intestinal flora and the use of protective environments have reduced GVHD in patients transplanted for aplastic anemia, but the benefits in leukemia patients are more equivocal [119-121].
Manipulation of Cytokine Profiles
As detailed above, Th1 cytokines and inflammatory cytokines interact to mediate the deleterious effects of acute GVHD observed after allogeneic BMT. The balance of Th1 (IL-2 and IFN-) and Th2 (IL-4 and IL-10) T cell cytokines in vivo early after BMT may be a critical factor in determining whether a systemic inflammatory response develops. Secretion of Th1 cytokines is generally associated with activation of macrophages, secretion of inflammatory cytokines, activation of NK and CTL cells, and with enhanced production of antibodies of the IgG2a isotype, whereas secretion of Th2 cytokines is generally associated with downregulation of cell-mediated immune responses and enhancement of IgE and IgG1 secretion. The emerging recognition that a Th1Th2 shift of T helper cells can downregulate cell-mediated immune responses and inflammatory processes will intensify research regarding the role of these regulatory cytokines during the course of GVHD.
One potential prophylaxis for GVHD might be the inhibition of Th1 cytokine production by the administration of Th2 cytokines. IL-10 inhibits T cell proliferation and Th1 cytokine production in both human and murine systems [122]. In two recent studies, the therapeutic potential of IL-10 for the prevention of GVHD was tested but the data thus far have been largely negative. The s.c. infusion of recombinant human (rHu) IL-10 for a period of two weeks after murine allogeneic BMT decreased the expansion of donor T cells, and less IFN- was secreted by mitogen-activated splenocytes [68]. Treatment with rHuIL-10, however, was not sufficient to significantly alter the clinical course of GVHD in recipient mice as assessed by survival, weight loss, and splenomegaly. In a second study, the infusion of IL-10 even resulted in an acceleration of lethal GVHD in irradiated recipients of MHC-disparate splenocytes [123].
Because IL-4 is more important than IL-10 in directing precursor T cells toward a Th2 phenotype [124], the administration of IL-4 to prevent GVHD may be more promising. Intraperitoneal injections of rIL-4 led to an increase in the severity of GVHD and to enhanced late mortality in a fully allogeneic BMT model [100]. Recipients of IL-4 showed the same histopathologic changes in skin and liver as mice receiving no cytokine. Thus, direct administration of type 2 cytokines appears to be either ineffective or toxic.
Recently, new approaches which encompass a Th1Th2 shift in the cytokine profile of donor T cells prior to BMT have emerged to provide a new way to interrupt the amplification cascade after allogeneic transplantation. Fowler and coworkers treated donor mice in vivo with a combination of rHuIL-2 and murine rIL-4 and were able to generate CD4+ enriched splenic populations with a Th2 cytokine pattern [125]. Subsequent transplantation of these cells into nonirradiated F1 recipients inhibited the secretion of the inflammatory cytokine TNF- during the effector phase and protected recipient mice from LPS-induced, TNF--mediated lethality. Cell mixtures of Th2 donor cells with otherwise lethal doses of naive T cells also protected recipient mice from LPS-induced lethality, demonstrating the ability of Th2 cells to modulate Th1 responses after allogeneic BMT [69, 125]. This protocol prevented acute GVHD without the impairment of allogeneic engraftment in sublethally irradiated mice.
Our laboratory recently initiated studies to evaluate whether polarized mature alloreactive donor T cells with a Th2 cytokine phenotype could be generated from naive donor T cells in vitro, and to test whether the polarization of these cells would be sufficient to prevent the development of acute GVHD. Our data showed that the preincubation of donor CD4+ or CD8+ cells in the presence of murine rIL-4 ex vivo during a primary mixed lymphocyte culture to MHC class I or class II disparate antigens was sufficient to polarize both T cell subsets toward a Th2 cytokine phenotype [69]. Transplantation of polarized Th2 T cell populations failed to cause mortality from GVHD when recipients were injected with LPS one to two weeks after transplantation. Reduced mortality correlated with downregulation of the inflammatory cytokines IFN- and TNF- produced by spleen cells isolated from recipients two weeks post-BMT. Long-term mortality (three months post-BMT) and body weights were also substantially improved after transplantation of polarized type 2 donor T cells across minor histocompatibility barriers in a murine GVHD model [126]. Despite this reduction in systemic GVHD, histologic changes of GVHD were evident in skin and liver tissues at seven weeks post-BMT. These data provided further evidence that inflammatory cytokines are pivotal to systemic disease (mortality, weight loss) in the immediate post-transplant period but that they do not correlate with individual target organ damage.
These experiments strongly support the concept that the balance in Th1 and Th2 cytokines is critical for the development (or prevention) of acute GVHD. Further issues need to be addressed, however, before this protocol can be tested in the clinical setting. First, the ability to preserve GVL after transplantation of polarized Th2 cells needs to be investigated. Second, Th2 cells must be evaluated for their ability to support lymphohematopoietic reconstitution.
Mobilization of Donor Cells with G-CSF
Peripheral blood progenitor cells (PBPCs) are an alternative source of bone marrow for allogeneic transplantation. Clinical trials using G-CSF-mobilized PBPCs for allogeneic transplantation in high-risk patients have recently been performed [127-136]. Reports of 73 allogeneic PBPC transplants from HLA-matched donors showed that 45% of the patients developed grade 2 or greater acute GVHD [137]. This incidence is similar to the rate observed after conventional BMT, despite the presence of 10- to 20-fold more T cells in the PBPC infusion [137]. We have recently investigated the possible mechanisms involved in reduced severity of acute GVHD after infusion of G-CSF-mobilized donor cells in a murine transplant model (B6B6D2F1) [138]. Our studies showed that G-CSF-mobilized donor splenocytes substantially reduced the early mortality in acute GVHD after allogeneic transplantation. Unexpectedly, pretreatment of donor mice with G-CSF partially polarized the function of donor T cells toward a Th2 cytokine response (increased IL-4 production, decreased IL-2 and IFN- production) upon stimulation in vitro. This finding was of particular interest since the importance of the balance of Th2 versus Th1 cytokines after allogeneic BMT has now been established. Significantly, long-term engraftment and lymphocyte reconstitution was fully achieved by this regimen and no histologic evidence of GVHD was seen by day 100 after transplantation [139].
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suppresses T-cell proliferation to mitogen via the nitric oxide pathway during experimental acute graft-versus-host disease. Blood 1996;88:1113-1121. in mice with acute graft-versus-host disease. Blood 1996;87:2853-2860. production. J Immunol 1990;144:84-93.[Abstract] is responsible for natural suppressor activity in GVHD spleen and normal bone marrow. Transplantation 1988;45:772-777.[Medline] prevents graft-versus-host disease after allogeneic bone marrow transplantation in mice. J Immunol 1993;151:6451-6459.[Abstract]
