Graft-Versus-Host Disease and Graft-Versus-Leukemia Responses

C HAPTER

108 

GRAFT-VERSUS-HOST DISEASE AND GRAFT-VERSUS-LEUKEMIA RESPONSES Pavan Reddy and James L.M. Ferrara

The ability of allogeneic hematopoietic cell transplantation (HCT) to cure certain hematologic malignancies is widely recognized. An important therapeutic aspect of HCT in eradicating malignant cells is the graft-versus-leukemia (GVL) effect. The importance of the GVL effect in allogeneic HCT has been recognized since the earliest experiments in stem cell transplantation. Forty years ago, Barnes and colleagues noted that leukemic mice treated with a subtherapeutic dose of radiation and a syngeneic (identical twin) graft transplant were more likely to relapse than mice given an allogeneic stem cell transplant.1,2 They hypothesized that the allogeneic graft contained cells with immune reactivity necessary for eradicating residual leukemia cells. They also noted that recipients of allogeneic grafts, though less likely to relapse, died of a “wasting syndrome” now recognized as graft-versus-host disease (GVHD). Thus in addition to describing GVL, these experiments highlighted for the first time the intricate relationship between GVL and GVHD. Since these early experiments, both GVHD and the GVL effect have been studied extensively.3 This chapter reviews the pathophysiology, clinical features, and treatment of GVHD and summarizes current understanding of the relationships between GVHD and the GVL effect.

GRAFT-VERSUS-HOST DISEASE: CLINICAL AND PATHOLOGIC ASPECTS Ten years after the work of Barnes and Loutit, Billingham formulated the requirements for the development of GVHD: the graft must contain immunologically competent cells, the recipient must express tissue antigens that are not present in the transplant donor, and the recipient must be incapable of mounting an effective response to destroy the transplanted cells.4 According to these criteria, GVHD can develop in various clinical settings when tissues containing immunocompetent cells (blood products, bone marrow, and some solid organs) are transferred between persons. The most common setting for the development of GVHD is following allogeneic HCT; without prophylactic immunosuppression, most allogeneic HCTs will be complicated by GVHD. GVHD is induced by mismatches between histocompatibility antigens between the donor and recipient. Matching of the major histocompatibility complex (MHC) antigens hastens engraftment and reduces the severity of GVHD.5 In humans, the MHC region lies on the short arm of chromosome 6 and is called the HLA (human leukocyte antigen) region.6 The HLA region is divided into two classes, class I and class II, each containing numerous gene loci that encode a large number of polymorphic alleles. MHC class I molecules are involved in the presentation of peptides to CD8+ T cells, and class II molecules present peptides to CD4+ T cells.6,7 The determination of HLA types has become much more accurate with molecular techniques that replace earlier serologic or cellular methods. In patients whose ancestry involves extensive interracial mixing, the chances of identifying an HLA identical donor are diminished.8 Despite HLA identity between a patient and donor, substantial numbers of patients still develop GVHD because of differences in minor histocompatibility antigens (MiHAs) that lie outside the HLA loci. Most minor antigens are expressed on the cell surface as degraded peptides bound to specific HLA molecules, but the precise elucidation of many human minor antigens is yet to be accomplished.9 In 1650

the United States, the average patient has a 25% chance of having an HLA match within his or her immediate family.8 Patients who lack an HLA-identical family member donor must seek unrelated donor volunteers or cord blood donations.

Acute Graft-Versus-Host Disease Acute GVHD can occur within days (in recipients who are not HLA-matched with the donor or in patients not given any prophylaxis) or as late as 6 months after transplantation. The incidence ranges from less than 10% to more than 80%, depending on the degree of histoincompatibility between donor and recipient, the number of T cells in the graft, the patient’s age, and the GVHD prophylactic regimen.10 The principal target organs include the immune system, skin, liver, and intestine. GVHD occurs first and most commonly in the skin as a pruritic maculopapular rash, often involving the palms, soles, and ears; it can progress to total-body erythroderma, with bullae formation, rupture along the epidermal-dermal border, and desquamation in severe cases.10 Gastrointestinal (GI) and liver manifestations often appear later and rarely represent the first and only findings. Intestinal symptoms include anorexia, nausea, diarrhea (sometimes bloody), abdominal pain, and paralytic ileus.10 Liver dysfunction includes hyperbilirubinemia and increased serum alkaline phosphatase and aminotransferase values. Coagulation studies may become abnormal, and hepatic failure with ascites and encephalopathy may develop in severe cases.10–12 Hepatic GVHD can be distinguished from hepatic venoocclusive disease by weight gain or pain in the right upper quadrant in the latter.12 Acute GVHD also results in the delayed recovery of immunocompetence.10 The clinical result is profound immunodeficiency and susceptibility to infections, often further accentuated by the immunosuppressive agents used to treat GVHD.10 Pathologically, the sine qua non of acute GVHD is selective epithelial damage of target organs.13,14 The epidermis and hair follicles are damaged and sometimes destroyed. Small bile ducts are profoundly affected, with segmental disruption. The destruction of intestinal crypts results in mucosal ulcerations that may be either patchy or diffuse. Other epithelial surfaces, such as the conjunctivae, vagina, and esophagus, are less commonly involved. A peculiarity of GVHD histology is the early paucity of mononuclear cell infiltrates; however, as the disease progresses, the inflammatory component may be substantial. Studies that identified inflammatory cytokines as soluble mediators of GVHD have suggested that direct contact between target cells and lymphocytes is not always required (see following sections). GVHD lesions are not evenly distributed: in the skin, damage is prominent at the tip of rete ridges; in the intestine, at the base of the crypts; and in the liver, in the periductular epithelium. These areas all contain a high proportion of stem cells, giving rise to the idea that GVHD targets may be undifferentiated epithelial cells with primitive surface antigens.15 The histologic severity of GVHD is at best semiquantitative, and consequently pathologic scores are not used to grade GVHD. Because it is often difficult to obtain an adequate tissue biopsy, and because it can be very difficult to distinguish GVHD from other post-HCT complications such as drug eruptions or infectious complications, the physician is left to use clinical judgment.

Chapter 108  Graft-Versus-Host Disease and Graft-Versus-Leukemia Responses

An independent committee of a multicenter phase III trial that assessed the presence and severity of GVHD was unable to confirm a high incidence of GVHD16,17 Standard grading systems generally include clinical changes in the skin, GI tract, liver, and performance status (Table 108.1).18 Although the severity of GVHD is often difficult to quantify, the overall maximal grade correlates with disease outcome: mild GVHD (grade I or II) is associated with little mortality, whereas higher grades are associated with significantly decreased survival.18,19 Recent advances in the use of biomarkers at the onset of disease may soon be sufficiently accurate to guide therapy.19

Clinical Features of Acute Graft-Versus-Host Disease The clinical features, staging, and grading of acute GVHD are summarized in Tables 108.1 and 108.2. In a comprehensive review of patients receiving therapy for acute GVHD, Martin and colleagues20 found that 81% had skin involvement, 54% had GI involvement, and 50% had liver involvement at the initiation of therapy. After high-intensity (myeloablative) conditioning, acute GVHD generally occurs within 14–35 days of stem cell infusion. The time of onset may depend on the degree of histocompatibility, the number of donor T cells infused, and the prophylactic regimen for GVHD. A TABLE 108.1 

Clinical Manifestations and Staging of Acute   Graft-Versus-Host Disease

Organ

Clinical Manifestations

Staging

Skin

Erythematous, maculopapular rash involving palms and soles; may become confluent Severe disease: bullae

Stage 1: <25% rash Stage 2: 25%–50% rash Stage 3: generalized erythroderma Stage 4: bullae

Liver

Painless jaundice with conjugated hyperbilirubinemia and increased alkaline phosphatase

Stage Stage Stage Stage

Gastrointestinal Upper: nausea, tract vomiting, anorexia Lower: diarrhea, abdominal cramps, distension, ileus, bleeding

1: 2: 3: 4:

Stage 1: day Stage 2: day Stage 3: day Stage 4:

bili bili bili bili

2–3 mg/dL 3.1–6 mg/dL 6.1–15 mg/dL >15 mg/dL

diarrhea >500 mL/ diarrhea >1000 mL/

rapid and severe form of GVHD may occur in patients with severe HLA mismatches and in patients who receive T-cell replete transplants without or with inadequate in vivo GVHD prophylaxis.21 Although such GVHD is sometimes called “hyperacute,” this term is misleading because it is pathophysiologically distinct from hyperacute rejection after solid organ allografting, which is caused by preformed antibodies. This form of GVHD, which is manifested by fever, generalized erythroderma and desquamation, and often edema, typically occurs about 1 week after stem cell infusion and may be rapidly fatal. In patients receiving standard (in vivo) GVHD prophylaxis such as a combination of cyclosporine and methotrexate, the median onset of GVHD is typically 21–25 days after transplantation; however, after in vitro T-cell depletion of the graft, the onset of GVHD symptoms may be much later.21 Thus the findings of rash and diarrhea by 1 week after transplantation would very likely be because of ineffective prophylaxis and would be very unlikely with the use of calcineurin inhibitors or in vitro T-cell depletion of the stem cell inoculum. A less ominous syndrome of fever, rash, and fluid retention occurring in the first 1–2 weeks after stem cell infusion is the “engraftment syndrome.” These manifestations may be seen with either allogeneic or autologous transplantation. Although this syndrome’s pathophysiology is poorly understood, it is thought to be caused by a wave of cytokine production as the graft starts to recover. These symptoms are related to, but distinct from, the “cytokine storm”22 of acute GVHD because there is no concomitant T-cell– mediated attack. This syndrome responds immediately to steroids in most patients, and it typically presents earlier than acute GVHD.15 Skin is the most commonly affected organ (Fig. 108.1). In patients receiving transplants after myeloablative conditioning, the skin is usually the first organ involved, and GVHD often coincides with engraftment. However, the presentation of GVHD is more varied following nonmyeloablative transplants or donor lymphocyte infusions.23 The characteristic maculopapular rash can spread throughout

Glucksberg Criteria for Staging of Acute   Graft-Versus-Host Diseasea

TABLE 108.2  Overall Grade

Skin

Liver

I

1–2

0

II

1–3

1

and/or

1

III

2–3

2–4

and/or

2–3

IV

2–4

2–4

and/or

2–4

a

diarrhea >1500 mL/ ileus, bleeding

A

B

Gut 0

See Table 108.1 for individual organ staging. Traditionally, individual organs are staged without regard to attribution. The overall grade of graft-versus-host disease, however, reflects the actual extent of graft-versus-host disease. To achieve each overall grade, skin disease, liver and/or gut involvement are required.

C

Fig. 108.1  GRAFT-VERSUS-HOST DISEASE, SKIN BIOPSY. This 40-year-old man with a history of relapsed Hodgkin lymphoma was status-postallogeneic stem cell transplant with donor lymphocyte infusion. He developed painful oral ulcers and a macular-papular rash on the arms, hand, and chest. The skin biopsy is from the palmar surface of the hand (A). It shows a scant lymphoid infiltrate in the dermis with a developing subepithelial blister (right). There is basal vacuolar change with single lymphocytes in the epithelium, as well as apoptotic keratinocytes accompanied by lymphocytes (B, and detail, C). (Courtesy Vesna Petronic-Rosic and Mark Racz, University of Chicago.)

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Part X  Transplantation

the rest of the body but usually spares the scalp; it is often described as feeling like a sunburn, tight or pruritic. In severe cases the skin may blister and ulcerate.24 Histologic confirmation is critical to rule out drug reactions, viral infections, etc. Apoptosis at the base of dermal crypts is characteristic. Other features include dyskeratosis, exocytosis of lymphocytes, satellite lymphocytes adjacent to dyskeratotic epidermal keratinocytes, and dermal perivascular lymphocytic infiltration.25 GI tract involvement of GVHD may present as nausea, vomiting, anorexia, diarrhea, and/or abdominal pain.26 It is a panintestinal process, often with differences in severity between the upper and lower GI tracts. Gastric involvement gives rise to postprandial vomiting that is not always preceded by nausea. Although gastroparesis is seen after bone marrow transplant, it is usually not associated with GVHD. The diarrhea of GVHD is secretory; significant GI blood loss may occur as a result of mucosal ulceration and is associated with a poor prognosis.27 In advanced disease, diffuse, severe abdominal pain, and distension is accompanied by voluminous diarrhea (>2 liters/day).19,28 Radiologic findings of the GI tract include luminal dilatation with thickening of the wall of the small bowel and air/fluid levels suggestive of an ileus on abdominal flat plates or small bowel series. Abdominal computed tomography may show the “ribbon” sign of diffuse thickening of the small bowel wall.24 Little correlation exists between the extent of disease and the appearance of mucosa on endoscopy, but mucosal sloughing is pathognomonic for severe disease.29 Nevertheless, some studies have shown that antral biopsies correlate well with the severity of GVHD in the duodenum and in the colon even when the presenting symptom is diarrhea.29 Histologic analysis of tissue is imperative to establish the diagnosis. The histologic features of GI GVHD are the presence of apoptotic bodies in the base of crypts, crypt abscesses, crypt loss, loss of Paneth cells, and flattening of the surface epithelium.28,30,31 Liver function test abnormalities are common after bone marrow transplant and occur secondary to venoocclusive disease, drug toxicity, viral infection, sepsis, iron overload, and other causes of extrahepatic biliary obstruction.12 The exact incidence of hepatic GVHD is unknown because many patients do not undergo liver biopsies. The development of jaundice or an increase in the alkaline phosphatase and bilirubin may be the initial features of acute GVHD of the liver. The histologic features of hepatic GVHD are endothelialitis, lymphocytic infiltration of the portal areas, pericholangitis, and bile duct destruction and loss.19,32

Other Organs

Whether GVHD affects organs other than the classic triad of skin, liver, and gut has remained a matter of debate, although numerous reports suggest additional organ manifestations. The most likely candidate is the lung. Lung toxicity, including interstitial pneumonitis and diffuse alveolar hemorrhage, may occur in 20% to 60% of allogeneic transplant recipients but in fewer autologous transplant recipients. Causes of pulmonary damage other than GVHD include engraftment syndrome (see earlier), infection, radiation pneumonitis, and chemotherapy-related toxicity (e.g., methotrexate, busulfan).21,33 One retrospective analysis failed to link severe pulmonary complications to clinical acute GVHD per se.34 The mortality caused by pneumonia increases with the severity of GVHD, but this association may be related to increased immunosuppressive therapy.21 A histopathologic signature of lymphocytic bronchitis has been associated with GVHD,33 although not always. Despite the ability of kidneys and hearts to serve as targets of transplant rejection, there is no convincing evidence for direct renal or cardiac damage from acute GVHD that is not secondary to drugs or infection. Similarly, neurologic complications are also common after transplantation but most can be attributed to drug toxicity, infection, or vascular insults.

Differential Diagnosis

Acute GVHD ought to be distinguished from any process that causes a constellation of fever, erythematous skin rash, and pulmonary edema that may occur during neutrophil recovery and has been

termed engraftment or capillary leak syndrome.35,36 In allogeneic transplant recipients distinction from acute GVHD is difficult. Engraftment syndrome is thought to reflect cellular and cytokine activities during early recovery of (donor-derived) blood cell counts and/or homeostatic proliferation of lymphocytes, but a precise delineation of the activated cells and mechanisms has not been demonstrated. Engraftment syndrome may be associated with increased mortality, primarily but not exclusively from pulmonary failure. Corticosteroid therapy may be effective particularly for the treatment of pulmonary manifestations.37 Skin rashes may reflect delayed reactions to the conditioning regimen, antibiotics, or infections; furthermore, histopathologic skin changes consistent with acute GVHD can be mimicked by chemoradiotherapy and drug reactions.21,38 Diarrhea can be a consequence of total-body irradiation (TBI), viral infection (especially with cytomegalovirus and other herpes viruses), parasitic infection, Clostridium difficile infection, nonspecific gastritis, narcotic withdrawal, and drug reactions: all of which mimic GVHD of the gut. Liver dysfunction can be caused by parenteral nutrition, venoocclusive disease, and viral or drug-induced hepatitis.

Genetic Basis of Graft-Versus-Host Disease The graft-versus-host (GVH) reaction was first noted when irradiated mice were infused with allogeneic marrow and spleen cells.39 Although mice recovered from radiation-induced injury and marrow aplasia, they subsequently died with “secondary disease,”39 a phenomenon subsequently recognized as acute GVHD. Three requirements for the development of GVHD were formulated by Billingham.4 First, the graft must contain immunologically competent cells, now recognized as mature T cells. In both experimental and clinical allogeneic HCT, the severity of GVHD correlates with the number of donor T cells transfused.40,41 The precise nature of these cells and the mechanisms they use are now understood in greater detail (see later). Second, the recipient must be incapable of rejecting the transplanted cells (i.e., immunocompromised). After allogeneic HCT, the recipient is typically immunosuppressed by chemotherapy and/or radiotherapy before the hematopoietic cell infusion.42 Third, the recipient must express tissue antigens that are not present in the transplant donor. Thus Billingham’s third postulate stipulates that the GVH reaction occurs when donor immune cells recognize disparate host antigens.4 These differences are governed by the genetic polymorphisms.42

HLA Matching

Recognition of alloantigens depends on the match with the presenting major histocompatibility molecule.43–45 In humans, the MHC is governed by the HLA antigens that are encoded by the MHC gene complex on the short arm of chromosome 6 and can be categorized as class I, II, and III. Class I antigens (HLA-A, HLA-B, and HLA-C) are expressed on almost all cells of the body.46 Class II antigens (DR, DQ, and DP) are primarily expressed on hematopoietic cells, although their expression can also be induced on other cell types following inflammation.46 The incidence of acute GVHD is directly related to the degree of MHC mismatch.42 The role of HLA mismatching of cord blood (CB) donors is more difficult to analyze compared with unrelated donor HCT, because allele typing of CB units for HLA-A, HLA-B, HLA-C, DRB1, and DQB1 is not routinely performed.47 Nonetheless, the total number of HLA disparities between the recipient and the CB unit has been shown to correlate with risk for acute GVHD as the frequency of severe acute GVHD is lower in patients transplanted with HLA-matched (6/6) CB units.47–49

Minor Histocompatibility Antigens

In most clinical allogeneic transplants where MHC of donor and recipient are matched, donor T cells recognize MHC-bound peptides derived from the protein products of polymorphic genes (MiHAs) that are present in the host but not in the donor.9,50–55 Substantial numbers (50%) of patients will develop acute GVHD despite receiving HLA-identical grafts as well as optimal

Chapter 108  Graft-Versus-Host Disease and Graft-Versus-Leukemia Responses

postgrafting immune suppression.9,42,56 MiHAs are widely but variably expressed in different tissue,51,56 which is one possible explanation for the unique target organ distribution in GVHD. Many MiHAs such as HA-1 and HA-2 are expressed on hematopoietic cells, which may be one reason for the host immune system to be a primary target for the GVH response, and helps explain the critical role of direct presentation by professional recipient antigenpresenting cells (APCs) in the GVH response.57 By contrast, other MiHAs such as H-Y and HA-3 are expressed ubiquitously.56 MiHAs do not all equally induce lethal GVHD but show hierarchic immunodominance.58,59 Furthermore, the difference in a single immunodominant MiHA is insufficient to elicit GVHD in murine models, even though a single MiHA can elicit T-cell–mediated damage in a skin explant model.60,61 However, the role of specific MiHAs that are able to induce clinical GVHD has not been systematically evaluated in large groups of patients.62

Other Non-HLA Genes

Genetic polymorphisms in several non-HLA genes such as in killer-cell immunoglobulin-like receptors (KIRs), cytokines, and nucleotidebinding oligomerization domain containing 2 (NOD2) genes have recently been shown to modulate the severity and incidence of GVHD. KIRs on natural killer (NK) cells that bind to the HLA class I gene products are encoded on chromosome 19. Polymorphisms in the transmembrane and cytoplasmic domains of KIRs govern whether the receptor has inhibitory (such as KIR2DL1, 2DL2, 2DL3, and 3DL1) or activating potential. Two competing models have been proposed for HLA-KIR allorecognition by donor NK cells following allogeneic HCT: the “mismatched ligand” and the “missing ligand” models.5,63–66 Both models are supported by several clinical observations, albeit in patients receiving very different transplant and immunosuppressive regimens (see Chapters 20 and 102).64,67–69 Proinflammatory cytokines involved in the classic cytokine storm of GVHD cause pathologic damage to target organs, such as the skin, liver, and GI tract (see later).22 Several cytokine gene polymorphisms in both recipients and donors have been implicated. Specifically, tumor necrosis factor (TNF) polymorphisms (TNFd3/d3 in the recipient, TNF863 and TNF857 in donors and/or recipients and TNFd4, TNF-α-1031C, and tumor necrosis factor receptor (TNFR) II-196R in the donors) have been associated with an increased risk for acute GVHD and transplant-related mortality (TRM).70,71 The three common haplotypes of the interleukin (IL)-10 gene promoter region in recipients, representing high, intermediate, and low production of IL-10, have been associated with severity of acute GVHD following HLA-matched sibling donor allogeneic HCT.72 By contrast, smaller studies have found neither IL-10 nor TNF-α polymorphisms to be associated with GVHD following HLA-mismatched cord blood transplantation.71,73 Interferon-gamma (IFN-γ) polymorphisms of the 2/2 genotype (high IFN-γ production) and 3/3 genotype (low IFN-γ production) have been associated with decreased or increased acute GVHD, respectively.71,74 NOD2/caspase-activating recruitment domain 15 (CARD15) gene polymorphisms in both the donors and recipients were recently shown to have a striking association between GI GVHD and overall mortality following related and unrelated donor allogeneic HCT.75 Several of the associations with non-HLA polymorphisms will need to be confirmed in larger and more diverse populations. Furthermore, it is likely that the importance of non-HLA gene polymorphisms in GVHD will differ depending on the donor source (related versus unrelated), HLA disparity (matched versus mismatched), graft source (CB versus bone marrow [BM] versus peripheral blood stem cells), and the intensity of the conditioning.

PATHOPHYSIOLOGY OF ACUTE   GRAFT-VERSUS-HOST DISEASE It is helpful to remember two important principles when considering the pathophysiology of acute GVHD. First, acute GVHD represents exaggerated but normal inflammatory responses against foreign

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antigens (alloantigens) that are ubiquitously expressed in a setting where they are undesirable. The donor lymphocytes that have been infused into the recipient function appropriately, given the foreign environment they encounter. Second, donor lymphocytes encounter tissues in the recipient that have been often profoundly damaged. The effects of the underlying disease, prior infections, and the intensity of conditioning regimen all result in substantial changes not only in the immune cells but also in the endothelial and epithelial cells. Thus the allogeneic donor cells rapidly encounter not simply a foreign environment, but one that has been altered to promote the activation and proliferation of inflammatory cells. Therefore the pathophysiology of acute GVHD may be considered a distortion of the normal inflammatory cellular responses that, in addition to the absolute requirement of donor T cells, involves multiple other innate and adaptive cells and mediators.76 The development and evolution of acute GVHD can be conceptualized in three sequential phases (Fig. 108.2) to provide a unified perspective on the complex cellular interactions and inflammatory cascades that lead to acute GVHD: (1) activation of the APCs; (2) donor T-cell activation, differentiation, and migration; and (3) effector phase.76 It is important to note that this three-phase description permits a unified perspective on GVHD biology but it is not meant to suggest that all three phases are of equal importance or that GVHD occurs in a stepwise and sequential manner. The spatiotemporal relationships among these biologic processes, depending on the context, are likely to vary and their relevance to the induction, severity, and maintenance of GVHD may depend on the factors cited earlier.

Phase 1: Activation of Antigen-Presenting Cells The earliest phase of acute GVHD is initiated by the profound damage caused by the underlying disease and infections and further exacerbated by bone marrow transplantation (BMT) conditioning regimens (which include TBI and chemotherapy) that are administered even before the infusion of donor cells.77–81 This first step results in activation of the APCs.7 Specifically, damaged host tissues respond with multiple changes, including the secretion of proinflammatory cytokines, such as TNF-α, IL-1 and IL-6 described as the cytokine storm.79,80,82,83 Such changes increase expression of adhesion molecules, costimulatory molecules, MHC antigens, and chemokine gradients that alert the residual host and the infused donor immune cells.80 These “danger signals” activate host APCs.84,85 Damage to the GI tract from the conditioning is particularly important in this process because it allows for systemic translocation of immunostimulatory microbial products such as lipopolysaccharide (LPS) that further enhance the activation of host APCs, and the secondary lymphoid tissue in the GI tract is likely the initial site of interaction between activated APCs and donor T cells.80,86,87 This scenario accords with the observation that an increased risk for GVHD is associated with intensive conditioning regimens that cause extensive injury to epithelial and endothelial surfaces with a subsequent release of inflammatory cytokines and increases in expression of cell surface adhesion molecules.80,81 The relationship among conditioning intensity, inflammatory cytokine, and GVHD severity has been supported by elegant murine studies.82 Furthermore, the observations from these experimental studies have led to two recent clinical innovations to reduce clinical acute GVHD: (1) reduced intensity conditioning to decrease the damage to host tissues and thus limit activation of host APC and (2) KIR mismatches between donor and recipients to eliminate the host APCs by the alloreactive NK cells.65,88 Host-type APCs that are present and have been primed by conditioning are critical for the induction of this phase; recent evidence suggests that donor-type APCs exacerbate GVHD, but in certain experimental models, donor-type APC chimeras also induce GVHD.85,89–91 In clinical situations, if donor-type APCs are present in sufficient quantity and have been appropriately primed, they too might play a role in the initiation and exacerbation of GVHD.92–94 Among the cells with antigen-presenting capability, dendritic cells

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Part X  Transplantation (II) Conditioning Tissue damage

Host tissues

Small intestine

TNF-α IL-1 LPS

LPS

Mφ Host APC

TNF-α IL-1

IFN-γ Donor T cell

Th

CD4 CTL

(II) Donor T-cell activation

Target cell apoptosis TNF-α IL-1 CD8 CTL

(III) Cellular and inflammatory effectors

Fig. 108.2  PATHOPHYSIOLOGY OF GRAFT-VERSUS-HOST DISEASE. During step 1, irradiation and chemotherapy both damage and activate host tissues, including intestinal mucosa, liver, and the skin. Activated cell hosts then secrete inflammatory cytokines (e.g., TNF-α and IL-1), which can be measured in the systemic circulation. The cytokine release has important effects on APCs of the host, including increased expression of adhesion molecules (e.g., ICAM-1, VCAM-1) and of MHC class II antigens. These changes in the APCs enhance the recognition of host MHC and/or minor H antigens by mature donor T cells. During step 2, donor T-cell activation is characterized by proliferation of GVHD T cells and secretion of the Th1 cytokines IL-2 and IFN-γ. Both of these cytokines play central roles in clonal T-cell expansion, induction of CTL and NK cell responses, and the priming of mononuclear phagocytes. In step 3, mononuclear phagocytes primed by IFN-γ are triggered by a second signal such as endotoxin LPS to secrete cytopathic amounts of IL-I and TNF-α. LPS can leak through the intestinal mucosa damaged by the conditioning regimen to stimulate gut-associated lymphoid tissue or Kupffer cells in the liver; LPS that penetrates the epidermis may stimulate keratinocytes, dermal fibroblasts, and macrophages to produce similar cytokines in the skin. This mechanism results in the amplification of local tissue injury and further production of inflammatory effectors such as nitric oxide, which, together with CTL and NK effectors, leads to the observed target tissue destruction in the stem cell transplant host. CTL effectors use Fas/FasL, perforin/granzyme B, and membrane-bound cytokines to lyse target cells. APC, Antigen-presenting cell; CTL, cytotoxic T lymphocyte; GVHD, graft-versus-host disease; ICAM, intercellular adhesion molecule; IFN, interferon; IL, interleukin; LPS, lipopolysaccharide; MHC, major histocompatibility complex; NK, natural killer; TNF, tumor necrosis factor; VCAM, vascular cell adhesion molecule.

(DCs) are the most potent and play an important role in the induction of GVHD.95 Experimental data suggest that GVHD can be regulated by qualitatively or quantitatively modulating distinct DC subsets.96–101 Langerhans cells were also shown to be sufficient for the induction of GVHD when all other APCs were unable to prime donor T cells, although the role for Langerhans cells when all APCs are intact is dispensable.102,103 Studies have yet to define roles for other DC subsets. In one clinical study persistence of host DC after day 100 correlated with the severity of acute GVHD, whereas elimination of host DCs was associated with reduced severity of acute GVHD.93 The allostimulatory capacity of mature monocyte-derived DCs (mDCs) after reduced-intensity transplants was lower for up to 6 months compared with the mDCs from myeloablative transplant recipients, thus suggesting a role for host DCs and the reduction in danger signals secondary to less intense conditioning in acute

GVHD.104 Nonetheless, this concept of enhanced host APC activation explains a number of clinical observations such as increased risks for acute GVHD associated with advanced-stage malignancy, conditioning intensity, and histories of viral infections. However, recent data suggest that even in the absence of all host hematopoietic derived APCs, GVHD can still be initiated by host nonhematopoietic cells.105 The exact nature of the host nonhematopoietic cells that can initiate GVHD and the context under which they may play a more dominant role remains to be understood. Moreover when all of host CD11c+ DCs are eliminated, the severity of GVHD was found to be enhanced demonstrating a role for host DCs in mitigating GVHD severity.106,107 Furthermore, a specific subset of host DCs, the CD8+ DCs might mitigate GVHD severity.108,109 By contrast donor-derived DCs, specifically, CD103+CD11b- DCs migrate from the colon and markedly enhance alloantigen presentation within the mesenteric lymph nodes

Chapter 108  Graft-Versus-Host Disease and Graft-Versus-Leukemia Responses

(mLNs).110 Critically, alloantigen presentation in the mLNs imprints gut-homing integrin signatures on donor T cells, leading to their emigration into the GI tract where they mediate fulminant disease. Thus anatomically distinct, donor DC subsets amplify GVHD. GVHD once initiated by host hematopoietic and/ or nonhematopoietic recipient APCs, generates a profound, localized, and lethal feed-forward cascade of donor DC-mediated indirect alloantigen presentation and cytokine secretion within the GI tract and aggravates its severity. Other professional APCs such as monocytes/macrophages or semiprofessional APCs might also play a role in this phase.7 For example, recent data suggest that host-type B cells might play a regulatory role under certain contexts,111 whereas in other contexts, they were dispensable for GVHD induction. Similarly, host basophils have been shown not to affect the induction or severity of acute GVHD.112,113 A small subset of radioresistant host-derived neutrophils infiltrate intestines after allo-HCT and the infiltration levels are dependent on the local microbial flora while such infiltration is not seen in germ-free conditions. Depletion of these neutrophils has been shown to reduce GVHD mortality.114 Data suggest that radiosensitive hematopoietic-derived APCs may not be obligatory for induction of APCs. Also, host or donor-type nonhematopoietic stem cells (such as endothelial cells, epithelial cells, or stromal cells) can function as APCs in the context of inflammation.105 The role of these cells in the presence and absence of professional hematopoietic-derived APCs remains to be elucidated. Alloantigen presentation by the host APCs has been shown to be modulated by the local microflora and by the release of PAMPs and also by the release of damage associated molecular patterns (DAMP) molecules from condition-mediated damage. Classically, tissue damage releases exogenous and endogenous damage/pathogen-associated molecules termed DAMPs/PAMPs that are detected by pattern recognition receptors. Examples of pattern recognition receptors include Toll-like receptors (TLR) and NOD-like receptors that result in the activation of APCs.115 The purine nucleoside adenosine triphosphate (ATP) is one DAMP whose release has been implicated as an early danger signal following tissue damage after allogeneic HCT. Elevated levels of ATP are found in peritoneal fluids of humans and mice following GVHD or irradiation. ATP interaction with the purinergic receptor P2X7 on host APCs resulted in increased expression of costimulatory molecules CD80/CD86 and production of inflammatory cytokines. Interrupting this interaction reduced GVHD in experimental models.116 More recently, a multiprotein complex termed the Nlrp3 inflammasome was shown to respond to DAMPs/PAMPs such as uric acid.117 Mice deficient in critical components of Nlrp3 or adaptor protein for caspase-1 cleavage demonstrated less severe GVHD. Emerging data have demonstrated a role for negative regulators of DAMP responses in controlling the severity of GVHD. Siglecs are a family of sialic acid binding Ig-like lectins, which function as counter regulators to immune activation and Siglec-G−/− animals have increased GVHD, an effect confined to radiosensitive host APCs. In contrast, the enhanced Siglec-G signaling with CD24 in wild-type animals was protective.118 Likewise inhibition of endogenous DAMPs such as heparan sulfate by endogenous protease inhibitors such as alpha-1 antitrypsin also mitigated GVHD in mice.119,120 These and other data suggest that DAMPs such as ATP, heparin sulfate, and uric acid may have nonredundant roles in the aggravation of GVHD. In addition to the role of LPS, recent observations have brought back renewed interest in the role of the quantitative and qualitative contributions of microbiota-driven inflammatory signals on GVHD. These signals are influenced by the variety and pathogenicity of organisms present, and have been demonstrated to affect the severity of GVHD.121–123 It is apparent that GVHD mediates a loss of Paneth cell-derived antimicrobial peptides that play an important role in shaping the diversity of microbiota, in addition to the use of pharmaceutic antimicrobials, nonetheless our mechanistic understanding of the changes in microflora is currently limited. Understanding these mechanisms may offer manipulable targets to alter this primary, inflammation-mediated, initiation phase of GVHD.

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Phase 2: Donor T-Cell Activation, Differentiation,   and Migration The infused donor T cells interact with the primed APCs leading to the initiation of the second phase of acute GVHD. This phase includes antigen presentation by primed APCs and the subsequent activation, proliferation, differentiation, and migration of alloreactive donor T cells. After allogeneic hematopoietic stem cell transplants, both host and donor-derived APCs are present in secondary lymphoid organs.124,125 The T-cell receptor (TCR) of the donor T cells can recognize alloantigens either on host APCs (direct presentation) or donor APCs (indirect presentation).126,127 In direct presentation, donor T cells recognize either the peptide bound to allogeneic MHC molecules or allogeneic MHC molecules without peptide.127,128 During indirect presentation, T cells respond to the peptide generated by degradation of the allogeneic MHC molecules presented on selfMHC.128 Experimental studies demonstrated that APCs derived from the host, rather than from the donor, are critical in inducing GVHD across MiHA mismatch.7,126 Recent data suggest that presentation of distinct target antigens by the host-type and donor-type APCs might play a differential role in mediating target organ damage.7,129,130 In humans, most cases of acute GVHD developed when both host DCs and donor DCs were present in peripheral blood after BMT.93

Costimulation The interaction of donor lymphocyte TCR with the host allopeptide presented on the MHC of APCs alone is insufficient to induce T-cell activation.7,131 Both TCR ligation and costimulation via a “second” signal through interaction between the T-cell costimulatory molecules and their ligands on APCs are required to achieve T-cell proliferation, differentiation, and survival.132 The danger signals generated in phase 1 augment these interactions and significant progress has been made on the nature and impact of these second signals.133,134 Costimulatory pathways are now known to deliver both positive and negative signals and molecules from two major families: the B7 family and the TNF receptor (TNFR) family play pivotal roles in GVHD.135 Interruption of the second signal by the blockade of various positive costimulatory molecules (CD28, ICOS, CD40, CD30, 4-1BB, and OX40) reduces acute GVHD in several murine models, whereas antagonism of the inhibitory signals (programmed death (PD)-1 and cytotoxic T lymphocyte antigen (CTLA)-4) exacerbates the severity of acute GVHD.136–142 The role of adhesion molecule DNAX accessory molecule-1 (DNAM-1) was recently explored in GVHD.143,144 Splenocytes deficient in DNAM-1 and prophylactic treatment with anti–DNAM-1 antibody prevented and treated GVHD. The various T-cell and APC costimulatory molecules and the impact on acute GVHD are summarized in Table 108.3. The specific context and the hierarchy in which each of these signals plays a dominant role in the modulation of GVHD remain to be determined.

T-Cell Subsets T cells consist of several subsets whose responses differ based on antigenic stimuli, activation thresholds, and effector functions. The alloantigen composition of the host determines which donor T-cell subsets proliferate and differentiate.

CD4+, CD8+ Cells and Naive/Memory T-Cell Subsets

CD4 and CD8 proteins are coreceptors for constant portions of MHC class II and class I molecules, respectively.145 Therefore MHC class I (HLA-A, HLA-B, HLA-C) differences stimulate CD8+ T cells and MHC class II (HLA-DR HLA-DP, HLA-DQ) differences stimulate CD4+ T cells.145–148 In the majority of HLA-identical BMT, acute GVHD can be induced by either or both CD4+ and CD8+ subsets in response to MiHAs.149 Several independent groups have found that although the naive (CD62L+) T cells were alloreactive and caused acute GVHD, this was

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TABLE 108.3 

T Cell–Antigen-Presenting Cell Interactions

T Cell

APC

Adhesion ICAMs LFA-1 CD2 (LFA-2) CD 226 (DNAM-1)

LFA-1 ICAMs LFA-3 CD155, CD112

Recognition TCR/CD4 TCR/CD8

MHC II MHC I

Costimulation CD28 CD152 (CTLA-4) ICOS PD-1

CD80/86 CD80/86 B7H/B7RP-1 PD-L1, PD-L2

Unknown CD154 (CD40L) CD134 (OX 40) CD137 (4-1BB) HVEM

B7-H3 CD40 CD134L (OX40L) CD137L (4-1BBL) LIGHT

APC,Antigen-presenting cell; CTLA-4, cytotoxic T lymphocyte antigen 4; DNAM-1, DNAX accessory molecule-1; HVEM, HSV glycoprotein D for herpesvirus entry mediator; ICAM, intercellular adhesion molecule; L, ligand; LFA, leukocyte function–associated antigen; LIGHT, homologous to lymphotoxins, shows inducible expression, and competes with HVEM, a receptor expressed by T lymphocytes; MHC, major histocompatibility complex; PD, programmed death; TCR, T-cell receptor.

not the case for the memory (CD62L–) T cells across different donor/ recipient strain combinations.150–153 Furthermore, expression of naive T-cell marker CD62L was also found to be critical for regulation of GVHD by donor natural regulatory T cells.154 By contrast, another recent study demonstrated that alloreactive memory T cells and their precursor cells (memory stem cells) caused robust GVHD.155,156

Regulatory T Cells

Recent advances indicate that distinct subsets of regulatory CD4+CD25+, CD4+CD25–IL10+ Tr cells, γδT cells, DN– T cells, NKT cells, and regulatory DCs control immune responses by induction of anergy or active suppression of alloreactive T cells.97,98,157–165 Several studies have demonstrated a critical role for the natural donor CD4+CD25+ Foxp3+ regulatory T cells (Treg), obtained from naive animals or generated ex vivo, in the outcome of acute GVHD. Some studies have demonstrated that donor CD4+CD25+ T cells suppressed the early expansion of alloreactive donor T cells and their capacity to induce acute GVHD without abrogating GVL effector functions, while others have shown that depending on the tumors used and the context, GVL may be reduced as well.166–168 The mechanisms by which donor Tregs suppress GVHD are being better understood. The presence of signal transducer and activation of transcription (STAT)1 signaling has been shown to enhance Treg-mediated suppression of GVHD.169 A key role for host APCs in the induction of GVHD protection, and for donor APCs in the sustenance of the protection by the infused mature Tregs has been demonstrated.170 Several small clinical trials that either include expanding Tregs in vivo with IL-2, or by epigenetic targeting of histone acetylation at the Foxp3 locus, or by preferential expansion of Tregs by cyclophosphamide have shown clinical benefit in early phase II trials.171–174 Direct infusion of ex vivo expanded donor Tregs has also demonstrated potential clinical benefit in small early phase trials and several clinical trials are underway in the United States and Europe with attempts to substantially expand these cells ex vivo and use for prevention of GVHD.175,176 Host NK1.1+ T cells are another T-cell subset that has been shown to suppress acute GVHD in an IL-4 dependent manner.164,165,177 By contrast, donor NKT cells were found to reduce GVHD and enhance

perforin-mediated GVL in an IFN-γ dependent manner.178–180 Recent clinical data suggest that enhancing recipient NKT cells by repeated TLI conditioning promoted Th2 polarization and dramatically reduced GVHD.165 Experimental data also show that activated donor NK cells can reduce GVHD through the elimination of host APCs or by secretion of transforming growth factor-β (TGF-β).179 A murine BMT study using mice lacking SH2-containing inositol phosphatase, in which the NK compartment is dominated by cells that express two inhibitory receptors capable of binding either self or allogeneic MHC ligands, suggests that host NK cells may play a role in the initiation of GVHD.181

T-Cell Apoptosis and Signaling Deletional mechanisms of tolerance fall into two categories: (1) central (thymic) deletion and (2) peripheral deletion.182 Central deletion is an effective way to eliminate continued thymic production of alloreactive T cells. To this end, lymphoablative treatments have been used as a condition to create a mixed hematopoietic chimeric state in murine BMT models.183 In this strategy, donor cells seed the thymus and maturing donor-reactive T-cell clones are deleted through intrathymic apoptosis.184,185 The pathways of T-cell apoptosis by which peripheral deletion occurs can be broadly categorized into activation-induced cell death (AICD) and passive cell death (PCD).186 An important mediator of AICD in T cells is the Fas receptor.187 Activated T cells expressing the Fas molecule undergo apoptotic cell death when brought into contact with cells expressing Fas ligand. A critical role for Fas-mediated AICD has been clearly demonstrated in attenuation of acute GVHD by several type 1 T helper (Th1) cytokines.42 PCD, or “death by neglect,” illustrates the exquisite dependence of activated T cells on growth factors (e.g., IL-2, IL-4, IL-7, and/or IL-15) for survival; apoptotic cell death in this instance is largely because of rapid downregulation of B-cell lymphoma 2.188–190 Transplantation of B-cell lymphoma-extra large T cells into nonirradiated recipients significantly exacerbates GVHD; however, no difference in GVHD mortality is observed in animals that have been lethally irradiated.191 Selective elimination of donor T cells in vivo after BMT using transgenic T cells in which a thymidine kinase (TK) suicide gene is targeted to T cells has also been shown to attenuate the severity of acute GVHD.191–194 Another recent approach to prevent GVHD is the selective depletion of alloantigen-specific donor T cells by a photodynamic cell-purging process, wherein donor T cells are treated with photoactive 4,5-dibromorhodamine 123 and subsequently exposed to visible light.195 Targeting alloreactive T-cell bioenergetics has emerged as a newer strategy to mitigate GVHD in mice.196,197 Thus several deletional mechanisms have been shown to reduce acute GVHD but the conditions under which one or another of these deletional mechanisms predominate remain to be determined. APC and T-cell activation results in rapid intracellular biochemical signaling cascades that activate or negatively regulate alloreactive donor T-cell responses. The calcineurin-nuclear factor of activated T cell (NFAT) pathway has been shown to be an effective target for mitigating GVHD and forms the bedrock of clinical care. Specifically, the calcineurin inhibitors cyclosporine and tacrolimus. Cyclosporine binds to the cytosolic protein peptidyl prolyl cis-trans isomerase A (also known as cyclophilin), whereas tacrolimus binds to the peptidylprolyl cis-trans isomerase FK506-binding protein (FKBP)12, and these complexes (cyclosporine–cyclophilin or tacrolimus–FKBP12) inhibit calcineurin, thereby blocking the dephosphorylation of NFAT and its nuclear translocation.198 These events prevent NFAT from exerting its transcriptional function, resulting in the inhibition of transcription of IL-2 and of other cytokines and ultimately leading to a reduced function of T-cells and mitigation of GVHD. Merging data have identified several key T-cell signaling pathways, many of which are being targeted in the clinic. Notably, the mammalian target of rapamycin (mTOR) pathway has been shown to be critical for blocking IL-2–mediated signal transduction and prevents cellcycle progression in naive T cells.199 Inhibition of mTOR with

Chapter 108  Graft-Versus-Host Disease and Graft-Versus-Leukemia Responses

sirolimus is being used clinically to prevent and treat GVHD. Several additional signaling pathways that regulate alloreactive T-cell responses have recently been identified, such as Notch, protein kinase C (PKC) θ, spleen tyrosine kinase (syk), Janus-activated kinase (JAK)2, signal transducer and activator of transcription (STAT)3 and nuclear factor kappa-B (NFκB).115 Emerging data are further defining a role for regulation T-cell proliferation/apoptosis epigenetic regulation through posttranslational modification by ubiquitination200,201 or acetylation,202 or by microRNAs (miRNAs). Specifically miRNAs such as miRNA 412 and miRNA 155 modulate alloimmunity by regulation of T-cell cycling and thus mitigate GVHD.203,204

Cytokines and T-Cell Differentiation Classically the Th1 cytokines (IFN-γ, IL-2), TNF-α and IL-6 have been implicated in the cytokine storm that occurs early after BMT and have been shown to be critical for the pathophysiology of acute GVHD.83,205–208 In addition to IL-2, several other common γ-chain cytokines (IL-21, IL-7, and IL-15) have been shown to play critical and potentially nonredundant roles in GVHD pathogenesis.209 Type 1 or Tc1/Th1 maturation is recognized as the dominant pattern in acute GVHD.210,211 Increased quantities of Th1-associated cytokines, TNF and IFN-γ, in acute GVHD are associated with earlier onset and more severe disease in preclinical models and clinical BMT. Although the dominance of Th1 subsets is well established, Th2 and Th17 subsets are also involved in pathology, and the balance between subsets determines acute GVHD severity, in addition to organ specificity, and the pathogenic or protective effects of any subset cannot be viewed in isolation.212,213 Specifically, Th2 differentiation is often seen as opposing Th1 differentiation; however, this subset is also recognized as causing acute GVHD but with predominant pathology in pulmonary, hepatic, and cutaneous tissues,214 in contrast to the strong GI association with Th1. Cutaneous pathology also may be generated by Th17 cells; although they are more commonly associated with chronic GVHD, they also have been associated with acute pathology.215,216 Recent data have shown that blockade of L-33 binding with the Th2 ST2 receptor during allogeneic-hematopoietic cell transplantation by exogenous ST2-Fc infusions had a marked reduction in GVHD lethality, indicating a role for ST2 as a decoy receptor modulating GVHD.217 Th17 differentiation is initiated by IL-6,218 and RORγt is the defining transcription factor, whereas maintenance and amplification relies on IL-23 and IL-21, respectively. The use of RORC-deficient donor T cells results in attenuated acute GVHD severity and lethality.219 Further studies are needed to better define the role of this subset in late acute GVHD versus early chronic GVHD, as well as the relative contribution of IL-17 from CD4 and CD8 T cells to end-organ pathology.

Leukocyte Migration Donor T cells migrate to lymphoid tissues, recognize alloantigens on either host or donor APCs, and become activated. They then exit the lymphoid tissues and traffic to the target organs and cause tissue damage.220 The molecular interactions necessary for T-cell migration and the role of lymphoid organs during acute GVHD have recently become the focus of a growing body of research. Chemokines play a critical role in the migration of immune cells to secondary lymphoid organs and target tissues.221 T-lymphocyte production of macrophage inflammatory protein-1-alpha is critical to the recruitment of CD8+ but not CD4+ T cells to the liver, lung, and spleen during acute GVHD.222 Several chemokines such as CCL2-5, CXC-chemokine ligand (CXCL)2, CXCL9-11, CCL17, and CCL27 are overexpressed and might play a critical role in the migration of leukocyte subsets to target organs liver, spleen, skin, and lungs during acute GVHD.220,223 CXC-chemokine receptor (CXCR)3+ T and CCR5+ T cells cause acute GVHD in the liver and intestine.220,224–226 CCR5 expression has also been found to be critical for Treg migration in GVHD.227 Recent

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clinical data from an early phase trial suggests that this may be a viable clinical strategy.228 In addition to chemokines and their receptors, expression of selectins and integrins and their ligands also regulate the migration of inflammatory cells to target organs.221 For example, interaction between α4β7 integrin and its ligand MadCAM-1 are important for homing of donor T cells to Peyer patches and in the initiation of intestinal GVHD.86,229 αLβ2/intercellular adhesion molecule 1(ICAM1), ICAM2, ICAM3, and α4β1/vascular cell adhesion molecule (VCAM)2 interactions are important for homing to the lung and liver after experimental HCT.220 The expression of CD62L on donor Tregs is critical for their regulation of acute GVHD, suggesting that their migration in secondary tissues is critical for their regulatory effects.125 The migratory requirement of donor T cells to specific lymph nodes (e.g., Peyer patches) for the induction of GVHD might depend on other factors such as the conditioning regimen, inflammatory milieu, etc.86,230 Furthermore, FTY720, a pharmacologic sphingosine-1-phosphate receptor agonist, inhibited GVHD in murine but not in canine models of HCT.231,232 Thus significant species differences may also factor in the ability of these molecules to regulate GVHD.

Phase 3: Effector Phase The effector phase that leads to the GVHD target organ damage is a complex cascade of multiple cellular and inflammatory effectors that further modulate each other’s responses either simultaneously or successively. Effector mechanisms of acute GVHD can be grouped into cellular and inflammatory effectors. Inflammatory chemokines expressed in inflamed tissues upon stimulation by proinflammatory effectors, such as cytokines, are specialized for the recruitment of effector cells, such as cytotoxic T lymphocytes (CTLs).233 Furthermore, the spatiotemporal expression of the cytochemokine gradients might determine not only the severity but also the unusual cluster of GVHD target organs (skin, gut, and liver).220,234

Cellular Effectors CTLs are the major cellular effectors of GVHD.235,236 The principle CTL effector pathways that have been evaluated after allogeneic BMT are the Fas-Fas ligand (FasL), the perforin-granzyme (or granule exocytosis), and the TNFR-like death receptors (DR), such as TNFrelated apoptosis-inducing ligand (TRAIL: DR4, 5 ligand) and TNF-like weak inducers of apoptosis (TWEAK: DR3 ligand).236–241 The involvement of each of these molecules in GVHD has been tested by using donor cells that are unable to mediate each pathway. Perforin is stored in cytotoxic granules of CTLs and NK cells, together with granzymes and other proteins. Although the exact mechanisms remain unclear, following the recognition of a target cell through the TCR-MHC interaction, perforin is secreted and inserted into the cell-membrane, forming “perforin pores” that allow granzymes to enter the target cells and induce apoptosis through various downstream effector pathways such as caspases.242 Transplantation of perforin-deficient T cells results in a marked delay in the onset of GVHD in transplants across MiHA disparities only, both MHC and MiHA disparities, and across isolated MHC I or II disparities.236,243–247 However, mortality and clinical and histologic signs of GVHD were still induced even in the absence of perforin-dependent killing in these studies, demonstrating that the perforin-granzyme pathway plays little role in target organ damage. A role for the perforin-granzyme pathway for GVHD induction is also evident in studies using donor T-cell subsets. Perforin-deficient or granzyme B-deficient CD8+ T cells caused less mortality than wild-type T cells in experimental transplants across a single MHC class I mismatch. This pathway, however, seems to be less important compared with the Fas/FasL pathway in CD4-mediated GVHD.246–248 Thus it seems that CD4+ CTLs preferentially use the Fas-FasL pathway, whereas CD8+ CTLs primarily use the perforin-granzyme pathway.

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Fas, a TNF-receptor family member, is expressed by many tissues, including GVHD target organs.249 Its expression can be upregulated by inflammatory cytokines such as IFN-γ and TNF-α during GVHD, and the expression of FasL is also increased on donor T cells, indicating that FasL-mediated cytotoxicity may be a particularly important effector pathway in GVHD.236,250 FasL-defective T cells cause less GVHD in the liver, skin, and lymphoid organs.245,248,250 The Fas-FasL pathway is particularly important in hepatic GVHD, consistent with the keen sensitivity of hepatocytes to Fas-mediated cytotoxicity in experimental models of murine hepatitis.236 Fas-deficient recipients are protected from hepatic GVHD, but not from other organ GVHD, and administration of anti-FasL (but not anti-TNF) monoclonal antibodies (MAbs) significantly blocked hepatic GVHD damage occurring in murine models.236,251,252 Although the use of FasLdeficient donor T cells or the administration of neutralizing FasL MAbs had no effect on the development of intestinal GVHD in several studies, the Fas-FasL pathway may play a role in this target organ, because intestinal epithelial lymphocytes exhibit increased FasL-mediated killing potential.253 Elevated serum levels of soluble FasL and Fas have also been observed in at least some patients with acute GVHD.254,255 The use of a perforin-granzyme and FasL cytotoxic doubledeficient (cdd) T cells, showed that they were unable to induce lethal GVHD across MHC class I and class II disparities after sublethal irradiation.244 However, when recipients were conditioned with a lethal dose of irradiation, cdd CD4+ T cells produced similar mortality to wild-type CD4+ T cells.240 These results were confirmed by a recent study demonstrating that GVHD target damage can occur in mice that lack alloantigen expression on the epithelium, preventing direct interaction between CTLs and target cells.241 Recently, several additional TNF family apoptosis-inducing receptors/ligands have been identified, including TWEAK, TRAIL, and LTβ/LIGHT, all of which have been proposed to play a role in GVHD and GVL responses.136,256–262 However, whether these distinct pathways play a more specific role for GVHD mediated by distinct T-cell subsets in certain situations remains unknown. Taken together, experimental data suggest some distinction between the use of different lytic pathways for the specific GVHD target organs and GVL, but the clinical applicability of these observations is as yet largely unknown.

Inflammatory Effectors Inflammatory cytokines synergize with CTLs resulting in the amplification of local tissue injury and further promotion of an inflammation, which ultimately leads to the observed target tissue destruction in the transplant recipient.263 The cytokines TNF-α, IL-6, and IL-1 are produced by an abundance of cell types during processes of both innate and adaptive immunity; they often have synergistic, pleiotropic, and redundant effects on both activation and effector phases of GVHD.207,219 A critical role for TNF-α in the pathophysiology of acute GVHD was first suggested over 20 years ago because mice transplanted with mixtures of allogeneic BM and T cells developed severe skin, gut, and lung lesions that were associated with high levels of TNF-α messenger RNA (mRNA) in these tissues.264 Target organ damage could be inhibited by infusion of anti–TNF-α MAbs, and mortality could be reduced from 100% to 50% by the administration of the soluble form of the TNF-α receptor (sTNFR), an antagonist of TNF-α.79,82,265 TNF-TNF1 interactions on donor T cells promote alloreactive T-cell responses and TNF-TNFR2 interactions are critical for intestinal GVHD.211,259,266 TNF-α also seems to be an important effector molecule in GVHD in skin and lymphoid tissue.264,267 In addition, TNF-α might also be involved in hepatic GVHD, probably by enhancing effector cell migration to the liver via the induction of inflammatory chemokines.268 The second major proinflammatory cytokine that appears to play an important role in the effector phase of acute GVHD is IL-1.42,263 Secretion of IL-1 appears to occur predominantly during the effector phase of GVHD of the spleen and

skin, two major GVHD target organs.269 A similar increase in mononuclear cell IL-1 mRNA has been shown during clinical acute GVHD. Mice receiving IL-1 displayed a wasting syndrome and increased mortality that appeared to be an accelerated form of disease. By contrast, intraperitoneal administration of IL-1 receptor antagonist (IL-1RA) was able to reverse the development of GVHD in the majority of animals, providing a significant survival advantage to treated animals.270 However, the attempt to use IL-1RA to prevent acute GVHD in a randomized trial was not successful.271 As a result of activation during GVHD, macrophages also produce nitric oxide (NO), which contributes to the deleterious effects on GVHD target tissues, particularly immunosuppression.272 NO also inhibits the repair mechanisms of target tissue destruction by inhibiting proliferation of epithelial stem cells in the gut and skin.273 In humans and rats, the development of GVHD is preceded by an increase in serum levels of NO oxidation products.274–276 IL-6 has also been identified as a critical cytokine that promotes a proinflammatory response during GVHD. Recent data suggest that IL-6 might in fact be the most critical cytokine that increases GVHD severity. It has direct cytopathic effects on the GI tract following allogeneic BMT and likely inhibits the reconstitution of Tregs.208,277 It plays a dominant role in mitigating lung injury after allo-BMT.278 Recent clinical trial demonstrated that targeting IL-6 early after transplant, particularly following high-intensity conditioning could mitigate GVHD.

BIOMARKERS OF ACUTE GRAFT-VERSUS-HOST DISEASE Emerging data from large datasets have identified and validated plasma biomarkers with important prognostic value at the onset of symptoms of acute GVHD. Markers of systemic GVHD include IL-2Rα, TNFR1, IL-8, and hepatocyte growth factor.1,279 Elafin has been identified as a biomarker specific for skin GVHD, and regenerating islet-derived 3-alpha (REG3α) as a biomarker of gastrointestinal GVHD, and ST2 or the soluble IL-33 receptor as a maker of resistance to therapy.2–4,280–282 Recently, the concentrations of three of these plasma biomarkers (TNFR1, ST2, and Reg3α) were used in the creation of an algorithm that computed the probability of nonrelapse mortality (NRM) for individual patients.5,283 The algorithm was developed in a multicenter training set of 328 patients, and two separate multicenter validation sets of 164 and 300 patients, respectively. The investigators identified thresholds that created three distinct Ann Arbor GVHD scores. In all three datasets (training, test, and validation), the cumulative incidence of 6-month NRM significantly increased as the Ann Arbor GVHD score increased: 8% for score 1, 27% for score 2, and 46% for score 3 (P <.0001). Conversely, the response to primary GVHD treatment decreased as the GVHD score increased: 86% for score 1, 67% for score 2, and 46% for score 3, P <.0001). These findings suggest that biomarker-based scores can be used to guide risk-adapted therapy at the onset of acute GVHD. High-risk patients with a score of 3 are candidates for intensive primary therapy, while low-risk patients with a score of 1 are candidates for rapid tapers of systemic steroid therapy. Recently the number of Paneth cells in duodenal biopsies for GVHD has been shown to correlate with response to treatment and with long-term survival.6,31 The plasma concentration of REG3α, the clinical severity of GVHD, and the histologic severity at GVHD diagnosis independently predicted lack of response to GVHD therapy 4 weeks following treatment and TRM. Patients who had all three risk factors experienced significantly greater NRM than those with any two of the risk factors (86% versus 66%, P <.001). The integration of clinical stage, histologic grade, and biomarkers into a single grading system may permit better risk stratification and rapid identification of patients for whom standard treatment is likely to be insufficient.3,281 These biomarkers may also provide new insights into the biology of GVHD. For example, the IL-22-Reg3 axis protects the epithelial barrier function of the intestinal mucosa. Intestinal stem cells (ISCs)

Chapter 108  Graft-Versus-Host Disease and Graft-Versus-Leukemia Responses

are principal cellular targets of GVHD in the GI tract, where intestinal flora are critical for amplification of GVHD damage. ISCs are protected by antibacterial proteins such as REG3α secreted by neighboring Paneth cells. Mucosal barrier disruption caused by stem cell dropout and subsequent lack of mucosal regeneration may preferentially allow Paneth cell proteins, including REG3α, to traverse into the bloodstream. Thus the plasma levels of REG3α may serve as a “liquid biopsy” and surrogate marker for the cumulative area of these breaches to GI mucosal barrier integrity, a parameter impossible to measure by individual tissue biopsies.

Prevention of Acute Graft-Versus-Host Disease Elimination of T cells with monoclonal antibodies, immunotoxins, lectins, CD34 columns, or physical techniques are effective at reducing GVHD. A typical unmanipulated marrow transplant entails the infusion of approximately 107 T cells per kg of recipient weight. A T-cell dose less than or equal to 105 per kg has been associated with complete control of GVHD.41 More recently, the combination of very high stem cell numbers and less than 3 × 104 CD3 cells per kg allowed haploidentical transplantation without GVHD.284 Presumably host immune cells that survive the initial conditioning are responsible for graft rejection. When the stem cell source contains large numbers of T cells, the GVH reaction further reduces the residual population capable of alloreactivity, thus decreasing graft rejection. To some degree, the higher graft failure rates may be controlled by increasing the intensity of the immunosuppression of the conditioning regimen,285,286 or adding back T cells.287 Overall there has been no improvement in survival that can be definitively attributed to T-cell depletion. Treatment of established GVHD with specific T-cell antibodies has produced mixed results. Although antithymocyte globulin has definite activity in established GVHD, the nonspecific clearance of T cells may result in increased opportunistic infections and no improvement in survival. More specific therapy with the humanized anti–IL-2 receptor antibody, daclizumab288,289 or the humanized antiCD3 antibody, visilizumab290,291 are promising, since they offer the potential of selectively removing the activated T cells. However, an increased risk for infection may still be observed.292 The first generally prescribed GVHD preventive regimen was the administration of intermittent low-dose methotrexate as developed in a dog model by Thomas and Storb.293 The principle of this approach was to administer a cell cycle–specific chemotherapeutic agent immediately after the transplant, when the T cells have started to divide after exposure to allogeneic antigens. Subsequently, the addition of antithymocyte globulin, prednisone, or both resulted in incremental improvement in the GVHD rate but no improvement in survival.294,295 Ultimately, the course of methotrexate was abbreviated and combined with a T-cell activation inhibitor, such as cyclosporine or tacrolimus. The introduction of cyclosporine in the late 1970s was a significant advance in GVHD prevention. A similar agent, tacrolimus, has been shown to provide similar control of GVHD.296 As a single agent, cyclosporine was about as effective as methotrexate.297 However, in combination with methotrexate, there was a significant reduction in the incidence of GVHD and an improvement in survival.298 Subsequent trials of tacrolimus and methotrexate compared with cyclosporine and methotrexate showed no advantage for either combination.296 The addition of prednisone to the conventional two-drug regimen resulted in similar rates of GVHD and no improvement in survival.299 Sirolimus (rapamycin) is a macrocyclic lactone immunosuppressant that is similar in structure to tacrolimus and cyclosporine. All three drugs bind to immunophilins; however, sirolimus complexed with FKBP12 inhibits T-cell proliferation by interfering with signal transduction and cell-cycle progression and can prevent GVHD.300 Because Sirolimus acts through a separate mechanism from the tacrolimus-FKBP complex (and cyclosporine-cyclophilin complex), it may be synergistic with both tacrolimus and cyclosporine. More recently, mycophenolate mofetil (MMF) has been studied. It is the

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prodrug of mycophenolic acid (MPA), a selective inhibitor of inosine monophosphate dehydrogenase, an enzyme critical to the de novo synthesis of guanosine nucleotide. Since T lymphocytes are more dependent on such synthesis than myeloid or mucosal cells, MPA preferentially inhibits proliferative responses of T cells.301 One hypothesis that flows from the three-step model of GVHD posits that reduction of intestinal colonization with bacteria could prevent GVHD. Animal studies in germ-free environments support this notion; GVHD was not observed until mice were colonized with gram-negative organisms.302 Later, gut decontamination and use of a laminar air flow environment was associated with less GVHD and better survival in patients with severe aplastic anemia.303 Similarly, studies of intestinal decontamination in patients with malignancies have shown less GVHD in some,304,305 but not all studies.306 Finally, another recent approach to GVHD prevention has been the use of nonmyeloablative conditioning transplants. Administration of vorinostat in combination with standard GVHD prophylaxis after related-donor reduced-intensity conditioning hemopoietic stem-cell transplantation reduced cumulative incidence of grade II–IV acute GVHD by day 100 to 22%, lower than the expected incidence of severe acute GVHD.172 A less intensive preparative regimen decreases the tissue toxicity and subsequent release of cytokines in animal models.79,82 Patients generally experience mild toxicity in the initial peritransplant period and develop little or no GVHD, although many develop GVHD later, especially after donor lymphocyte infusions. In fact, the rates of GVHD are often higher than with conventional transplants, and GVHD is associated with a significant portion of the GVL effect.307–309 A recent study has shown that addition of one dose of a humanized anti–IL-6 monoclonal antibody (tocilizumab) in addition to standard cyclosporine methotrexate prophylaxis resulted in only 12% Grade II–IV acute GVHD in 48 patients.310,311 An important role for TNF-α in clinical acute GVHD has been suggested by studies demonstrating elevated levels of TNF-α in the serum of patients with acute GVHD and other endothelial complications such as venoocclusive disease.312–315 Therapy of GVHD with humanized anti-TNF-α (infliximab)316,317 or a dimeric fusion protein consisting of the extracellular ligand-binding portion of the human TNF-α receptor (TNFR) linked to the Fc portion of human immunoglobulin G1 (etanercept)318 have shown some promise.319,320 The second major proinflammatory cytokine that appears to play an important role in the effector phase of acute GVHD is IL-1. Secretion of IL-1 appears to occur predominantly during the effector phase of GVHD in the spleen and skin, two major GVHD target organs.269 IL-1RA is a naturally occurring pure competitive inhibitor of IL-1 that is produced by monocytes/macrophages and keratinocytes. Of note, the IL-1RA gene is polymorphic, and the presence in the donor of the allele that is linked to higher secretion of IL-1RA was associated with less acute GVHD.321 Two-phase I/II trials showed promising data that specific inhibition of IL-1 with either the soluble receptor or IL-1RA could result in remissions in 50% to 60% of patients with steroid-resistant GVHD.322,323 However, a subsequent randomized trial of the addition of IL-1RA or placebo to cyclosporine and methotrexate beginning at the time of conditioning and continuing through day 14 after stem cell infusion did not show any protective effect of the drug, despite the attainment of very high plasma levels.271,324 Therefore, at least as administered in this study, IL-1 inhibition was insufficient to prevent GVHD in humans. IL-11 was also able to protect the GI tract in animal models and prevent GVHD, but it did not prevent clinical GVHD.324 Thus not all preclinical strategies successfully translate to new therapies.

Therapy for Acute Graft-Versus-Host Disease Glucocorticoid steroids are the initial therapy for acute GVHD. The mechanisms by which steroids work are multifactorial; they act as lympholytic agents and inhibit the release of inflammatory cytokines such as IL-1, IL-2, IL-6, gamma interferon, and TNF-α. Because of its intravenous availability, methylprednisolone is the steroid most

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commonly given for acute GVHD. Various dosing regimens have been used, none of which is clearly superior. High-bolus doses (10–20 mg/kg or 500 mg/m2) have higher initial response rates, but flares on tapering and opportunistic infections are common. Both the Seattle and Minnesota transplant groups have found that treatment with steroids was as effective as, or more effective than, other therapies or combination of therapies, with 20% to 40% of patients having durable long-term responses.325,326 Long-term salvage rates for patients who did not respond to steroids were 20% or less; most patients eventually died from infection, acute GVHD, and/or chronic GVHD. More recently, a randomized trial demonstrated that topical therapy with oral budesonide can have prednisone sparing effects and is efficacious in treatment of GI GVHD.327 Clinically, two types of failure of corticosteroid treatment of acute GVHD can be distinguished: true steroid resistance (i.e., progression of GVHD symptoms and manifestations while patients are receiving full-dose corticosteroid treatment) and steroid dependence (i.e., reoccurrence [or flare] of GVHD during or after tapering of steroid treatment).328 In general, the prognosis with true steroid-resistant GVHD is worse than the prognosis of steroid-dependent patients.328 A comparison of trials dealing with steroid-resistant GVHD is hampered by variable inclusion of both patient groups in many of these trials. A number of agents have been tested, including chemical immunosuppressants such as MMF, antithymocyte globulin (ATG), anti-CD3, anti-T-cell antibodies, and more specific agents directed against activation or adhesion molecules anti-CD25, anti-CD147, or cytokines or extracorporeal photopheresis.328,329 To date, there are no randomized trials testing one agent versus the other in this clinical situation. Recent data suggested a role for TNF inhibition when added to steroids in treating GVHD, although a randomized trial failed to demonstrate any difference when compared with the addition of pentostatin or anti-IL2 to steroids and was inferior to the addition of MMF.319,330 Targeted elimination of alloreactive T cells has recently been demonstrated to be a safe and efficacious method to mitigate GVHD. Fusion of human caspase 9 to a modified human FK-binding protein that allowed for dimerization when exposed to a synthetic dimerizing drug led to the rapid death of 90% of alloreactive T cells expressing this construct and mitigated acute GVHD in a pilot trial of five patients following haploidentical BMT.331

Other Supportive Approaches Infections are the main cause of death in patients with steroidrefractory acute GVHD, and careful surveillance and control of infections is mandatory in patients with acute GVHD. Fungal infections, especially aspergillosis, are the leading complication. Prophylaxis and early aggressive treatment should be facilitated by the introduction of new azoles (voriconazole, posiconazole) or echinocandins (caspofungin, micafungin), which broaden therapeutic efficacy with acceptable toxicity. Other supplementary approaches have been suggested, such as the use of octreotide332 and oral beclomethasone (or budesonide) to control large volumes of diarrhea.327

CHRONIC GRAFT-VERSUS-HOST DISEASE Chronic GVHD was initially defined as a GVHD syndrome presenting more than 100 days after transplantation; its onset occurred either as an extension of acute GVHD (progressive), after a disease-free interval (quiescent), or with no precedent (de novo).333,334 Chronic GVHD may be limited or extensive (see Table 108.3). Any grade of acute GVHD increases the probability of chronic GVHD, although no singular pathologic feature of the former predicts the development of the latter. Its incidence ranges from 30% to 60% after transplantation with the bone marrow, although it may be higher after peripheral blood progenitor transplants.335 As with acute GVHD, the immune system appears to be affected in all patients, who are highly susceptible to bacterial, viral, fungal, and opportunistic infections. Specific abnormalities of cellular

immunity include decreases in the production of antibodies against specific antigens, defects in the number and function of CD4+ T cells, and increases in the number of nonspecific suppressor cells, which further diminish lymphocyte responses. Skin changes resembling widespread lichen planus with papulosquamous dermatitis, plaques, desquamation, dyspigmentation, and vitiligo occur in 80% of patients.309,336 Destruction of dermal appendages leads to alopecia and onychodysplasia. Severe chronic GVHD of the skin can resemble scleroderma, with induration, joint contractures, atrophy, and chronic skin ulcers. Chronic cholestatic liver disease occurs in 80% of patients and often resembles acute GVHD; it rarely progresses to cirrhosis. Severe mucositis of the mouth and esophagus can result in weight loss and malnutrition. Intestinal involvement, however, is infrequent.309,336 Chronic GVHD also produces a sicca syndrome, with atrophy and dryness of mucosal surfaces caused by lymphocytic destruction of exocrine glands, usually affecting the eyes, mouth, airways, skin, and esophagus.24,336,337 The hematopoietic system may also be affected, and thrombocytopenia is an unfavorable prognostic factor in patients with chronic GVHD.309 Important predictors of unfavorable outcome are progressive onset, lichenoid skin changes, elevated serum bilirubin level, continued thrombocytopenia, and failure to respond to 9 months of therapy.309,338–340 Among patients with none of these risk factors, 70% are expected to survive, compared with less than 20% with two or more of these risk factors.340 Histologic examination of the immune system reveals involution of thymic epithelium, disappearance of Hassall corpuscles, depletion of lymphocytes, and absence of secondary germinal centers in lymph nodes.337 Pathologic skin findings include epidermal atrophy with changes characteristic of lichen planus and striking inflammation around eccrine units. Sclerosis of the dermis and fibrosis of the hypodermis subsequently develop. GI lesions include localized inflammation of the mucosa and stricture formation in the esophagus and small intestine.336 Histologic findings in the liver are often similar to those that occur in acute GVHD but are more intense, with chronic changes such as fibrosis and hyalinization of portal triads, obliteration of bile ducts, and hepatocellular cholestasis.309 The endocrine glands of the eyes, mouth, esophagus, and bronchi show destruction focused on centrally draining ducts, with secondary involvement of alveolar components.337 Findings of bronchiolitis obliterans, similar to those that occur in rejection of lung transplants, are now generally considered a pulmonary manifestation of chronic GVHD, although the pathogenesis of this process remains unclear.337

Clinical Manifestations of Chronic   Graft-Versus-Host Disease Chronic GVHD can present with a plethora of clinical manifestations. Because of its unpredictable pattern and the late onset, when patients are no longer receiving care at their transplant center, the diagnosis is often delayed or not recognized. The staging of chronic GVHD is summarized in Table 108.4. However, consensus criteria recently developed by the National Institutes of Health (NIH) might soon become the standard for diagnosing and evaluating responses for chronic GVHD.341,342

Dermatologic Skin involvement in chronic GVHD presents with varied features. Lichenoid chronic GVHD presents as an erythematous, papular rash that resembles lichen planus with no typical distribution pattern.24 Sclerodermatous GVHD may involve the dermis and/or the muscular fascia and clinically resembles systemic sclerosis. The skin is thickened, tight, and fragile, with very poor wound healing. Either hypo or hyperpigmentation may occur. In severe cases the skin may become blistered and ulcerate. Hair changes can include increased brittleness, premature graying, and alopecia. Fingernails and toenails may also be

Chapter 108  Graft-Versus-Host Disease and Graft-Versus-Leukemia Responses

TABLE 108.4 

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Commonly Administered Drugs for Graft-Versus-Host Disease Prophylaxis and Treatment

Drug

Mechanism

Adverse Effects

Corticosteroids

Direct lymphocyte toxicity; suppress proinflammatory cytokines such as TNF-α

Hyperglycemia, acute psychosis, severe myopathy, neuropathy, osteoporosis, cataract development

Methotrexate (MTX)

Antimetabolite: inhibit T-cell proliferation

Significant renal, hepatic, and gastrointestinal toxicities

Cyclosporine A (CSA)

IL-2 suppressor; blocks Ca2+-dependent signal transduction distal to TCR engagement

Renal and hepatic insufficiency, hypertension, hyperglycemia, headache, nausea and vomiting, hirsutism, gum hypertrophy, seizure with severe toxicity

Tacrolimus (FK506)

IL-2 receptor; blocks Ca2+-dependent signal transduction distal to TCR engagement

Similar to CSA

Mycophenolate mofetil (MMF)

Inhibits de novo purine synthesis

Body aches, abdominal pain, nausea and vomiting, diarrhea, neutropenia

Sirolimus

mTOR inhibitor

Thrombocytopenia, hyperlipidemia, TTP

Antithymocyte globulin (ATG)

Polyclonal immunoglobulin

Anaphylaxis, serum sickness

IL-2, Interleukin-2; mTOR, mammalian target of rapamycin; TCR, T-cell receptor; TNF-α, tumor necrosis factor-α; TTP, thrombotic thrombocytopenic purpura.

affected by chronic GVHD. Destruction of sweat glands can cause hyperthermia.343

Pulmonary

Ocular GVHD usually presents with xerophthalmia or dry eyes. Irreversible destruction of the lacrimal glands results in dryness, photophobia, and burning. Local therapy with preservative-free tears and ointment or the placement of punctal plugs by an ophthalmologist might be required. Conjunctival GVHD, a rare manifestation of severe chronic GVHD, has a poor prognosis.24,343

Bronchiolitis obliterans is a late and serious manifestation of chronic GVHD. Patients typically present with a cough or dyspnea.343 Severe sclerotic disease of the chest wall may also give rise to similar symptoms with no intrinsic pulmonary disease. Pulmonary function tests demonstrate obstructive physiology and a reduction in DLCO. Chest computed tomography results may be normal or may show hyperinflation with a ground-glass appearance. Overall, patients with bronchiolitis obliterans have minimal response to therapy and a very poor prognosis. Patients with chronic GVHD are also at risk for chronic sinopulmonary infections, but symptoms may be minimal.24

Oral

Hematopoietic

Oral GVHD causes xerostomia and/or food sensitivity.343 More advanced disease may cause odynophagia caused by esophageal damage and strictures, although esophageal involvement occurs rarely without oral disease. Physical examination may reveal only erythema with a few white plaques, prompting a misdiagnosis of thrush or herpetic infections. Lichenoid changes in advanced disease can cause extensive plaque formation.24

Cytopenias in chronic GVHD are common. This may be a result of stromal damage, but autoimmune neutropenia, anemia, and/or thrombocytopenia are also seen. Thrombocytopenia at the time of chronic GVHD diagnosis is associated with poor prognosis. However, thrombocytopenia posttransplant is a poor prognostic factor regardless of GVHD, and eosinophilia is occasionally seen with chronic GVHD.

Gastrointestinal

Immunologic

Patients with chronic GVHD have GI complaints that mimic other disease states, including acute GVHD, infection, dysmotility, lactose intolerance, pancreatic insufficiency, and drug-related side effects. In one retrospective review of the intestinal biopsies of patients with chronic GVHD and persistent GI symptoms, a majority of patients had evidence of both acute and chronic GVHD, and only 7% of the patients had isolated chronic GVHD.24,336 Thus although chronic GVHD may involve the GI tract alone, it may be difficult to diagnose in those circumstances without concurrent acute GVHD.

Chronic GVHD is inherently immunosuppressive. Functional asplenia with an increased susceptibility to encapsulated bacteria is common, and circulating Howell-Jolly bodies can be seen on peripheral blood smear. Patients are also at risk for invasive fungal infections and Pneumocystis carinii pneumonia. Hypoglobulinemia is common, and patients with levels below 500 mg/dL should be supplemented with intravenous immunoglobulin.

Ocular

Hepatic Hepatic disease typically presents as cholestasis with elevated serum levels of alkaline phosphatase and bilirubin. Isolated hepatic chronic GVHD has become more common with the increasing use of donor lymphocyte infusions.11 Liver biopsy is required to confirm the diagnosis of chronic hepatic GVHD in patients with no other target organ involvement.

Musculoskeletal Fascial involvement in sclerodermatous GVHD is usually associated with skin changes. Fasciitis in joint areas can cause severe restriction of range of motion. Muscle cramps are a common complaint in patients with chronic GVHD, but myositis with elevated muscle enzymes is rare. Many patients with chronic GVHD are on steroid therapy and have low levels of sex hormone posttransplant. Thus avascular necrosis, osteopenia, and osteoporosis are frequent complications. Although several cases have been described, it is yet to be determined in large studies whether kidneys, which are primary targets in

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some animal models of chronic GVHD, are also involved.344 Among the myriad clinical features of chronic GVHD, three definitive signs appear to be risk factors for increased mortality: (1) extensive skin GVHD involving greater than 50% of the body surface area, (2) platelet count of less than 100,000/µL, and (3) progressive onset and acute GVHD that continues uninterrupted beyond day 100.345 However, chronic GVHD remains, except in cases with obvious features, a difficult diagnosis; response to therapy is even more difficult to assess. Recent criteria established by the NIH consensus conference might prove to be beneficial in establishing uniform guidelines for diagnosis, treatment, and response.342 The NIH consensus criteria are currently being evaluated.

Differential Diagnosis The distinction between chronic and acute GVHD has been traditionally based on the time of onset. However, with the advent of low-intensity HCT, that distinction has become less relevant. The NIH working group has, in addition to the two main categories of GVHD, added two subcategories. The broad category of acute GVHD includes (1) classic acute GVHD (maculopapular rash, nausea, vomiting, anorexia, profuse diarrhea, ileus, or cholestatic hepatitis), occurring within 100 days after transplantation or donor leukocyte infusion (DLI), (without diagnostic or distinctive signs of chronic GVHD), and (2) persistent, recurrent, or late acute GVHD: features of classic acute GVHD without diagnostic or distinctive manifestations of chronic GVHD occurring beyond 100 days of transplantation or DLI (often seen after withdrawal of immune suppression). The broad category of chronic GVHD includes (1) classic chronic GVHD without features characteristic of acute GVHD and (2) an overlap syndrome in which features of chronic and acute GVHD appear together. In the absence of histologic or clinical signs or symptoms of chronic GVHD, the persistence, recurrence, or new onset of characteristic skin, GI tract, or liver abnormalities should be classified as acute GVHD regardless of the time after transplantation. With appropriate stratification, patients with persistent, recurrent, or late acute GVHD or overlap syndrome can be included in clinical trials with patients who have chronic GVHD.

CHRONIC GRAFT-VERSUS-HOST   DISEASE: PATHOPHYSIOLOGY The pathophysiology of chronic GVHD is generally much less well understood than that of acute GVHD and has undergone less intensive experimental modeling.24 It is important to recognize that chronic GVHD was originally defined as a temporal rather than a clinical or pathophysiologic entity. The initial clinical reports of chronic GVHD described abnormalities that occurred at least 150 days after stem cell infusion.346,347 By convention, day 100 after stem cell infusion is used as an arbitrary divider between acute and chronic GVHD. But some manifestations of acute GVHD occur after day 100, and some manifestations of chronic GVHD may occur before day 100. Thus it is preferable to consider the clinical symptoms and signs per se rather than their timing of onset. Relatively little is known about the pathophysiology of chronic GVHD. This is in part because of the absence of appropriate animal models that can capture the kinetics and the protean manifestation of chronic GVHD.348 However, recent studies using multiple models that collectively mimic many of the chronic GVHD manifestations have begun to shed light on the complex biology. T cells: Based on certain clinical features chronic GVHD has been considered to be an autoimmune disease, with some experimental data suggesting that chronic GVHD results from defective central negative selection, which leads to the generation of autoreactive clones that escape tolerogenic mechanisms operating in the periphery.349,350 The autoreactive cells of chronic GVHD are associated with a damaged thymus, which can be injured by several mechanisms,

including acute GVHD, the conditioning regimen, or age-related involution and atrophy. In chronic GVHD the ability of the thymus to delete autoreactive T cells (negative selection) and to induce tolerance is impaired.24,351,352 Chronic GVHD could also be a product of T cells that have undergone relatively chronic antigen stimulation as a result of the presence of inexhaustible and ubiquitous MiHA antigens. Allo-T cells under circumstances of chronic MiHA antigen stimulation can induce syndromes resembling those induced by the chronic antigen stimulation in autoimmune diseases. This concept is also consistent with the proposal of acute GVHD as a risk factor for chronic GVHD. The antigens targeted in chronic GVHD could be the same dominant ones targeted in acute GVHD, but the reactive T cells could be different; for example, they may secrete TGF-β. Recent data have shown that the balance between Treg and conventional T-cells is critical for chronic GVHD.171 Cytokines: TGF-β has been implicated in the development of fibrosis and chronic GVHD.180 IL-17 and subsequent T-cell differentiation along the Th17 pathway have recently been strongly associated with cGVHD. IL-17 was shown more recently to result in colony stimulating factor (CSF)1-dependent macrophage accumulation in skin and lung, which drives tissue fibrosis.353 Systemic IL-17 levels increase late after clinical BMT, at a time when chronic GVHD develops.311 Inhibition of Th17 differentiation and CSF1 appear to be relevant to the development of chronic GVHD. IL-2 is critical for Treg homeostasis. Recent data have shown that Treg: conventional T-cell balance is critical for chronic GVHD.171 In addition, inhibition of terminal cytokines involved in fibrosis, such as TGF-β and IL-13, represent additional targets; however, TGF-β inhibition may be problematic given its important role in Treg homeostasis. B cells: In some patient subsets, responses to rituximab, presence of MiHA-specific antibodies, and the presence of chronic GVHD after T-cell depletion (TCD) allo-BMT would indicate that in addition to donor T cells, donor B cells might be a direct effector or might have a role in priming T cells as APCs.354,355 Murine models demonstrated a pathogenic role for donor B cells and alloantibody production in causing experimental chronic GVHD. It is also clear that T follicular helper (TFH) cells and IL-21 play important roles in the development of chronic GVHD via the stimulation of germinal center B cells and alloantibody generation.356 Two tyrosine kinases expressed in the hepatocellular carcinoma (TEC) family of kinases, IL-2–inducible kinase (ITK) and Bruton tyrosine kinase (BTK), share close homology and play critical roles in both T-cell and B-cell function. ITK helps to drive T-cell activation and differentiation while BTK is essential for B-cell receptor signaling. In mouse studies treatment with ibrutinib, an ITK and BTK inhibitor, reversed lung pathology and pulmonary dysfunction in mice with established chronic GVHD in a model dependent on cooperation between TFH and germinal center B cells; additionally, ibrutinib reduced the progression of sclerodermatous chronic GVHD in mice.357 Targeting syk in B cells has been shown to mitigate chronic GVHD in several models. Syk deletion in vivo was effective in treating established chronic GVHD, as was a small-molecule inhibitor of Syk, fostamatinib, which normalized germinal center formation and decreased activated CD80/86(+) dendritic cells.358 In multiple distinct models of sclerodermatous chronic GVHD, clinical and pathologic disease manifestations were not eliminated when mice were therapeutically treated with fostamatinib, though both clinical and immunologic effects could be observed in one of these scleroderma models.

BIOMARKERS OF ACUTE GRAFT-VERSUS-HOST DISEASE Less progress has been made with biomarkers for chronic GVHD than for acute GVHD; nevertheless, several are beginning to emerge. CXCL9 has been identified and validated in several hundred patients from at least two HCT centers.7,359 Other biomarkers with potential utility include soluble B-cell activation factor (sBAFF), anti-dsDNA antibody, soluble IL-2 receptor alpha (sIL-2Rα), and soluble CD13

Chapter 108  Graft-Versus-Host Disease and Graft-Versus-Leukemia Responses

(sCD13); sBAFF and anti-dsDNA also may be elevated in patients with late-onset chronic GVHD, but these biomarkers need to be validated in much larger cohorts before definitive conclusions can be drawn.

THERAPY FOR CHRONIC GRAFT-VERSUS-HOST DISEASE Chronic GVHD has a major impact on both quality of life and survival, frequently involves multiple organs, and necessitates prolonged immunosuppressive therapy.360 One report noted that 15% of cancerfree patients were still receiving immunosuppressive therapy after 7 years.361 The more severe forms of chronic GVHD are clearly associated with a lower disease-free survival. Thus the potential benefit of a GVL effect is shadowed by significant treatment-related mortality.361 Current therapies for chronic GVHD are of limited efficacy, and there is no long-term satisfactory regimen for patients who do not respond to front-line steroid-based therapy. Indeed, no medication has been approved by the Food and Drug Administration for use in chronic GVHD. The lack of standardized response criteria to measure therapeutic efficacy poses a major obstacle to pursuing therapeutic trials in chronic GVHD. Overall survival and/or discontinuation of systemic immunosuppression are accepted long-term endpoints in chronic GVHD trials. The recent NIH-sponsored consensus project provided, for the first time, a set of standardized measures and definitions to use as response criteria in chronic GVHD.341,342 Nonetheless, these recommendations are yet to be tested and validated in prospective studies. The NIH consensus conference has defined response measures that are classified in two main groups: clinician-assessed and patient-reported (Table 108.5).348 The prevention of acute GVHD has not consistently resulted in a lower incidence of chronic GVHD. A clear example is the use of reduced-intensity transplants, consistently associated with a lower incidence of acute GVHD but with no major impact on chronic GVHD.362,363 The extended use of GVHD prophylaxis with cyclosporine, or variations in the cyclosporine dosage used, showed no beneficial effects on the incidence of chronic GVHD.360,364 The addition of thalidomide to cyclosporine and methotrexate prophylaxis, the administration of intravenous immunoglobulin, and early treatment based on biopsy findings of subclinical GVHD in an attempt to preemptively treat chronic GVHD were unsuccessful.360 A randomized placebo- controlled study in Europe showed that the addition of ATG Fresenius resulted in a significant reduction in the severity and incidence of chronic GVHD.8,365 The most commonly used therapies to treat chronic GVHD are cyclosporine A (CSA) and prednisone. Sullivan and colleagues366 reported that prednisone alone is superior to prednisone plus azathioprine for primary treatment of patients with chronic GVHD.

TABLE 108.5 

National Institutes of Health Chronic Graft-VersusHost Disease Measures

Measure

Clinician-Assessed

Patient-Reported

Chronic GVHD Specific Core Measures Signs Organ-specific measures

Not applicable

Symptoms

Clinician-assessed symptoms

Patient-reported symptoms

Global rating

Mild, moderate, or severe

Mild, moderate, or severe

0–10 severity scale 0–10 severity scale 7-point change scale 7-point change scale Chronic GVHD Nonspecific Ancillary Measures Function Grip strength Patient-reported function 2-minute walk time Quality of life

—

GVHD, Graft-versus-host disease.

Patient-reported healthrelated quality of life

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However, in patients classified as high-risk on the basis of platelet counts below 100,000/µL, treatment with prednisone alone resulted in only 26% 5-year survival. When a similar group of patients was treated with alternating-day CSA and prednisone, 5-year survival exceeded 50%.367 A randomized study of patients with extensive GVHD found no difference when prednisone alone was compared with prednisone plus CSA.24 For chronic GVHD that recurs or fails to respond to initial therapy, there is no standard treatment. A small recent study has shown that an 8 week administration of low dose, subcutaneous IL-2 ameliorated several manifestations of chronic GVHD that was resistant to steroids, particularly in the skin.9,368 This improvement was associated with an increase in Tregs. This increase was caused by enhanced, proliferation, thymic export and resistance to apoptosis.10,171 Randomized clinical trials of this approach are currently being conducted. Other experimental therapies include psoralen plus ultraviolet light A, MMF, thalidomide, total lymphoid irradiation, Plaquenil, extracorporeal photopheresis, pentostatin, and acetretin.338 (see Table 108.4, for list of the commonly used GVHD drugs and their side effects.)

Transfusion-Associated Graft-Versus-Host Disease Most blood products administered to immunocompromised patients are now irradiated or at least leukocyte-depleted to avoid the transfusion of viable alloreactive T cells. With most homologous blood products, the MHC incompatibility between donor and recipient results in rapid clearance of transfused T cells by the recipient’s immune system. However, occasionally, transfusions from donors who are homozygous for one of the recipient’s MHC haplotypes are not recognized as foreign by the recipient.369–371 These cells can survive, “engraft,” and mount an immunologic attack against the unshared haplotype in the patient, resulting in transfusion-induced GVHD.371 Transfusion-associated GVHD differs from GVHD occurring after transplantation in terms of kinetics and manifestations (i.e., with transfusion-associated GVHD, the recipient marrow is a major target).370 Since the number of stem cells in the offending blood product is inadequate, there is no hemopoietic recovery from donor cells. This syndrome is generally fatal as a result of refractory pancytopenia and/or other organ involvement.

GRAFT-VERSUS-LEUKEMIA RESPONSES The GVL response after allogeneic HCT results from the immunologic attack of the host tissue and, by extension, the leukemia (i.e., the tumor). This response represents a potent form of immunotherapy that circumvents some of the “immunoediting” mechanisms used by tumor cells to develop in the hosts. The power of the alloimmune response to eliminate malignancy was first reported more than 50 years ago in experimental models by Barnes et al.1 However, GVL as its own entity and its close association with GVHD were not established until another 15 years later.372 GVL responses demonstrate the clearest and arguably to date, the most potent demonstration of the power to harness the immune system to eradicate malignant diseases. The critical and direct evidence of GVL effect in clinical transplantation has been provided by the use of DLI to treat relapses after allogeneic HCT.373–375 Kolb and colleagues first reported three patients with relapsed chronic myeloid leukemia (CML) who achieved complete cytogenetic remission after treatment with IFN-α and DLI from the original donors.376 Subsequently, these findings have been confirmed in several reports.377–379 The most recent and dramatic effect of the GVL effect has been demonstrated using genetic engineering to create tumor antigenspecific T cells (CAR T-cell therapy). Most discussion below is geared towards biology of GVL responses after allo-BMT. The mechanisms of GVL responses after CAR T-cell therapy while seemingly evident (antigen-specific T cells eliminating leukemia and other cells expressing the antigen), the mechanisms for failure and complications are just being explored.

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Clinical Features of Graft-Versus-Leukemia Clinical evidence that the donor graft mediates an important antileukemic effect comes from higher relapse rates for recipients of syngeneic stem cells than for recipients of HLA-matched sibling grafts.380 These findings have also been confirmed in a multicenter analysis of HCT recipients with acute myelogenous leukemia (AML) in first remission and subsequent retrospective analyses by the International Bone Marrow Transplant Registry (IBMTR).381–383 The second IBMTR analysis also showed that the magnitude of this GVL effect is greater for patients with CML and AML and not statistically significant for patients with acute lymphoblastic leukemia (ALL) in first remission.384,385 Several case reports of patients with relapse of leukemia after allogeneic HCT noted remissions of the malignancy either after abrupt withdrawal of immunosuppression or during a flare of acute GVHD.386–389 Patients who develop GVHD after allogeneic HCT experience relapse less frequently than similar patients who do not develop clinical disease. GVHD is protective against relapse both for HCT recipients with advanced leukemia390–392 and for patients who receive transplants in earlier stages of malignancy.393 Additional analyses also suggest that the magnitude of the GVL effect appears to be disease and stage-specific.392–394 Initial reports suggested that chronic GVHD was most protective against relapse,392 but other analyses demonstrate that acute GVHD is also protective.393 Based on these reports, newer trials of immunotherapy are designed to include cessation of immunosuppressive therapy (without taper) to induce a GVL reaction for patients whose malignancy has relapsed after HCT. Furthermore, Childs and colleagues demonstrated that the graft-versustumor (GVT) effect also plays an important role in inducing remissions from a nonhematologic malignancy, renal cell carcinoma.395 Another line of clinical evidence regarding the GVL effect of allogeneic HCT and its tight linkage to GVHD comes from the studies using T-cell depletion of the donor graft. Donor T cells included in the stem cell graft are critical for acute GVHD, and T-cell depletion by various strategies is one of the most successful means of reducing the incidence and severity of GVHD after allogeneic HCT.26,396–401 Unfortunately, although T-cell depletion results in less treatment-related morbidity and mortality, improved overall survival rates have not been reliably demonstrated. This failure is caused in large part by a reciprocal increase in the subsequent relapse rate after T-cell depletion, as well as to graft failure and other complications.394,402,403 T-cell depletion increases relapse rates particularly in CML.393,394,404 This observation provides further strong, albeit indirect, evidence that allogeneic donor T cells are important mediators not only of GVHD, but also of the GVL properties of the allogeneic stem cell graft. Finally, the most compelling evidence of donor T cells in mediating GVL comes from the observations made from donor lymphocyte infusions (discussed later). The induction of GVL is a complex process.

Genetic Basis The immunotherapeutic effect that occurs in the allo-BMT setting is primarily mediated by allogeneic donor T or NK cells directed against the alloantigens shared by the recipient tumor and target tissues and/or tumor-specific antigens (TSAs) that have the advantage of not being subjected to tolerance mechanisms by the host tumor.56,405,406 Understanding of the exquisite specificity of T-cell responses led to attempts to identify specific antigens that are responsible for the GVL effect. Much of the focus has been on the identification of (1) certain oncogenic viral proteins (because these are absent in normal cells but expressed by transformed tumor cells [certain Epstein-Barr virus peptides such as Epstein-Barr virus nuclear antigen-1, latent membrane protein {LMP}-1, LMP, LCL]), (2) antigens that are expressed in a tissue-specific fashion (melanoma specific proteins), and (3) proteins that are overexpressed in tumors (WT1, proteinase 3, survivin, telomerase reverse transcriptase, CYPB1, and human epidermal growth factor receptor 2/neu).53,56

Although these antigens are specific, most T-cell responses to these antigens are limited because of the poor immunogenicity of these proteins, expression on normal cells, defects in the processing or presentation of tumor antigens, or production of factors that disable T-cell responses. Thus clinical attempts to obtain high specificity of T-cell responses have been offset with difficulties in obtaining enough sensitivity and vice versa. Furthermore, given the current concepts of stem cell origins of leukemia and cancers, identification of the immunogenic proteins that are specifically expressed in the malignant stem cells and harnessing T-cell responses to those antigens will be needed for the optimal GVL effect to cure malignancy.407,408 In contrast to the TSAs or tumor-associated antigens (TAAs) discussed earlier, alloantigens are not subjected to tolerance mechanisms. Vaccination strategies with autologous T cells using TAAs or TSAs have yielded disappointing clinical antitumor responses.409 By contrast, allogeneic HCT has met with remarkable GVL responses perhaps owing to recognition of minor alloantigens in addition to the TAAs.410 This concept has been demonstrated by recent murine studies, which showed that alloantigen on the tumor cells is required for GVL responses and that the principal targets of GVL are the immunodominant allogeneic MiHAs rather than the TAAs.60,91 Thus T cells specific for MiHA antigens could provide for a potent GVL effect. Significant progress has been made in the identification of MiHAs that are specifically expressed in the host hematopoietic tissues and therefore might allow for a GVL response without causing GVHD.53 Together, these results suggest that in addition to tumorspecific proteins, expression of alloantigens and cognate interactions between donor T cells and the tumor tissues are required for the effective induction of the majority of GVL responses. However, T cells specific for some MiHAs are also responsible for GVHD, and a means of consistently separating the beneficial GVL effect from GVHD has not yet been clinically achieved.

Killer-Cell Immunoglobulin-Like   Receptor Polymorphisms The two competing models described earlier, the “mismatched ligand” and the “missing ligand” models for HLA-KIR allorecognition, have been supported by clinical observations of GVL responses in different patient and transplant populations.65,66 The former model has been shown to separate GVL and GVHD responses in the context of TCD haploidentical HCT for AML.46,67 Even though this model is supported by elegant laboratory studies, it was found to be invalid for ALL and also for AML after unrelated donor HCT with immunosuppression.67 Recent retrospective clinical data suggest that GVHD and GVL can be separated by the “missing ligand” model in CML/AML and myelodysplastic syndrome patients after TCD HLA identical sibling HCT.46,66,69 Further validation of either models by clinical prospective studies and a better understanding of the balance between the inhibitory and activating receptor-ligand interaction of the NK cells are needed to adequately exploit the interface between HLA-KIR genetics to separate GVHD from GVL (see Chapter 102).64,411

Chimeric Antigen Receptor T Cells and Cytokine Release Syndrome Infusion of unselected CD19 specific CAR T cells is associated with a massive cytokine storm that causes severe toxicity, known as cytokine release syndrome (CRS). CRS is a nonantigen-specific toxicity that occurs as a result of high-level immune activation.412,413 The magnitude of immune activation typically required to mediate clinical benefit using modern immunotherapies exceeds the levels of immune activation that occur in more natural settings. The symptomatology and severity associated with CRS varies greatly and many features of CRS mimic infection. Thus fever is a hallmark, at times temperatures exceeding 40.0°C. Other features, some potentially life

Chapter 108  Graft-Versus-Host Disease and Graft-Versus-Leukemia Responses

threatening, include adult respiratory distress syndrome, fluid retention, neurologic toxicity, cardiac, renal and/or hepatic failure, and disseminated intravascular coagulation. Some of these, particularly lung and cardiac dysfunction, can be rapid onset and severe, but are typically reversible. Neurologic symptoms occurring in the context of CRS are varied and may occur coincident with other symptoms of CRS or may arise when the other symptoms of CRS are resolving.414 Magnetic resonance imaging often reveals no abnormalities. IL-6 appears to be the most relevant cytokine in CRS, although likely not the only cytokine involved. CRS may also be associated with findings of macrophage activation syndrome/hemophagocytic lymphohistiocytosis.414 In addition, rapid turnover of underlying leukemia/tumor might lead to tumor lysis syndrome that may coincide or contribute to the severity of CRS. Appropriate supportive care, steroids, and anti–IL-6, tociluzimab remain the current mainstay for management of CRS. Furthermore, whether CRS severity is dependent on the type of T cell being engineered (central versus effector versus naive versus bulk),or the type of vector and/or antigen being targeted remains unknown.

Immunobiology of Graft-Versus-Leukemia Responses Given the tight association of clinical GVHD and GVL, as well as the common biologic principles governing these responses after allogeneic HCT, it is important to discuss the similarities and distinctions between them in the context of the three cellular phases of GVHD.415

Phase 1: Activation of Antigen-Presenting Cells The concept that tumor eradication after allogeneic HCT might not require toxic chemoradiotherapy and could be achieved primarily by the immunotherapeutic effect from the GVL responses has led to the clinical development of nonmyeloablative HCT for hematologic and nonhematologic malignancies.416 Phase 1 is characterized by the development of the cytokine storm-generated danger signals from the conditioning regimen and the subsequent activation of APCs.415 Experimental data suggested that the reduction in conditioning would attenuate the cytokine storm, lead to the development of mixed donor-host chimerism, and confine the GVH response primarily to secondary lymphoid organs, thus cause less severe GVHD without impairing GVL responses.417–419 However, nonmyeloablative HCT has delayed the kinetics but did not reduce the overall incidence of GVHD and a significant number of patients either failed to respond or relapsed.21 Furthermore, recent murine and human studies have suggested that homeostatic expansion of T cells in a lymphopenic environment induced by conditioning (as opposed to mere immunosuppression) improves the antitumor efficacy of adoptively transferred syngeneic or autologous T cells by increasing the availability of space, enhancing the memory responses, and reducing the competition for homeostatic cytokines (such as IL-7 and IL-15) for transferred T cells while eliminating regulatory T cells.420–422 Thus low-intensity HCT clearly demonstrates the principle of the GVL effect, but the roles of the cytokine storm and homeostatic expansion of allogeneic T cells in shaping the intensity of GVL responses are not known. Host and donor APCs are critical for the induction and severity of GVHD.7 Activation of APCs is the key step in phase 1 of GVHD.415 Significant progress has been made in understanding the role of APCs in GVL. Recent experimental evidence has demonstrated a crucial role for professional host APCs in the induction of GVL responses mediated by donor T cells, even when the tumor cells showed some features of APCs.91,423 Tumors that merely express costimulatory molecules may still be unable to stimulate an effective immune response because of their various “immunoediting” processes that cause ineffective antigen presentation.424 However, when the tumor cell itself functions as a professional APC, as with CML, it can generate an effective GVL response.425,426 By contrast, cancers such as acute leukemias that seldom differentiate into APCs generate poor GVL

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responses. Data also demonstrated that given sufficient time and a low tumor burden, cross-presentation of TAAs and/or alloantigens by professional donor APCs can occur and may promote or sustain GVL responses by maintaining or expanding alloreactive T cells after initial priming on host APCs.91,426 This concept is consistent with clinical GVL responses in CML in which the final stage of a GVL response to CML may be the result of donor T cells responding not directly to the small number of CML stem cells or progenitors (which would be undifferentiated and therefore poor APCs) but to tumor antigens cross-presented on professional donor APCs. Emerging data suggest that enhancing such cross-presentation is sufficient to elicit effective GVL responses against acute or advanced leukemia. These data, however, suggest that GVL responses generated after low-intensity conditioning may not be as robust as those after full intensity HCT and highlight the need for a clearer understanding of the effects of the cytokine storm and lymphopenia generated danger signals on the activation of APCs in mediating GVL. Recent data showed that APC subsets could potentially be modulated to enhance the GVL effect without aggravating GVHD. Host-derived CD8α+DCs were shown to be required for the induction of optimal GVT responses when Batf3 deficient mice were used as recipients in experimental allo-HCT.109 TLR3 stimulation via poly I:C in host CD8α+DCs, enhanced GVL responses without exacerbating GVHD. However, cellular processes of regulating GVL responses in host APCs still remain unclear. The molecular mechanisms underpinning the role of host APC subsets in GVL are just now being deciphered. It has been observed that absence of Ikaros in host hematopoietic APCs exacerbates GVHD, but without concomitantly enhancing GVT responses in multiple models.427 The role of donor-derived DCs in mediating GVL is also being explored. Initial reports regarding this association demonstrated that donor APCs are not required for GVL responses, but play an indispensable role in GVHD in a MHC-matched, MiHA-mismatched BMT model. In order to present host TSAs, via donor APCs, to donor CD8+T cells, donor APCs must have the capacity for crosspresentation as they do not express both endogenous alloantigens and TSAs.428 Furthermore, additional studies are needed to determine which specific subsets of donor APCs play a critical role in enhancing GVT responses. Reports suggest that donor CD11b− APCs within the BM grafts consist mostly of pDC progenitors (pre-pDCs) and enhance the GVL activity of donor T cells by promoting differentiation into Th1/type 1 CTLs. Pre-pDCs also regulate GVH and GVT responses altering the balance between donor Tregs and inflammatory T cells by inducing indoleamine 2,3-dioxygenase synthesis.428

Phase 2: Donor T-Cell Activation The core of GVL responses, as with GVHD, is also dependent on the activation of appropriate numbers of T cells. The “second” signals from professional APCs (or certain tumor cells that function as effective APCs) are critical for generating an effective GVL response.230 Several of the costimulatory pathways that modulate GVHD have also been evaluated in mediating GVL responses. Blockade of CD28 costimulation preserved GVL responses but reduced GVHD in murine studies.429 However, when the tumor cells also expressed B7 molecules, such blockade reduced the GVL responses.425 Ex vivo blockade of CD40-CD40L interaction has been shown to reduce GVHD by generating Tregs but still preserve GVL. By contrast, blockade of the 4-1BB pathway reduced both GVHD and GVL.140 The other costimulatory molecules (OX40 and ICOS) and the inhibitory molecules (CTLA-4 and PD-1) also modulate antitumor responses.141,142 A better understanding of the context (i.e., low intensity or DLI) and the hierarchy of timing, duration, and extent of costimulatory requirements of donor T-cell subsets might allow for balancing the intensity of GVL and GVHD responses. Clinical and experimental evidence suggest that donor T-cell numbers correlate with the severity of GVHD and GVL responses. TCD grafts had reduced GVHD but increased disease relapse, suggesting a role for

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T-cell numbers in GVL responses as well.430 Clinical attempts to separate GVHD and GVL by regulating allogeneic T-cell dose have met with limited success. For example, administration of 1 × 105 T cells/kg after HLA-matched sibling transplantation did not mediate GVL effects and yet was associated with a measurable incidence of GVHD. Thus infusion of the correct numbers of donor T-cell effectors is crucial for GVL responses.430 This has been demonstrated by durable responses that are observed in CML and other malignancies after DLI (see later), despite the experimental evidence that host APCs stimulate a stronger GVL response than do donor APCs.91,426 This could be because, clinically, DLI is almost always given without immunosuppression to an individual who has not developed GVHD either from the chemical immunosuppression or physical removal of donor T cells from the allograft. This lack of immunosuppression after DLI increases the likelihood of a GVH response, and DLI is almost always associated with clinical GVHD. The delivery of additional allogeneic effector cells in DLI also increases the effector: target ratio compared with the time of initial HCT. The latter is also clinically demonstrated by a more effective GVL response to DLI against minimal residual disease (BCR-ABL positivity by PCR) compared with the response against high leukemic burden (e.g., blast crisis) in CML patients.426 Thus DLI provides the proof, in principle, for the concepts that sufficient T-cell numbers and appropriate antigen presentation are required for both GVHD and an effective GVL response.

T-Cell Subsets

Most experimental studies have implicated donor CD8+ T cells as the primary mediators of GVL, but there are no clinical data for CD8+mediated GVL responses in the absence of CD4 T cells.56,236,426 Moreover, some clinical data suggest a role for greater CD4-mediated GVL responses without an increase in GVHD after allogeneic HCT and DLI.423,431–434 But it is unclear whether CD4+ T-cell initiated GVL responses occur in the absence of generation of MiHA specific CD8+ T cells. Given the critical requirement of alloantigens for most GVL responses, the specific requirement of CD4 and/or CD8 T cells for GVL and GVHD is likely to be determined by the expression of the relevant immunodominant MiHAs and/or TAAs. Therefore it is unlikely that GVHD and GVL responses can be separated under all circumstances merely by depletion of either subset of alloreactive T cells. However, experimental data suggest it might be possible to separate GVHD and GVL when certain donor T-cell subsets are either depleted or infused (DLI) at an appropriate interval after transplant.423 But the optimal time interval, if any, after clinical HCT is yet to be determined. Because of recent identification and understanding of the role of various T-cell subsets in mediating immune responses, depletion of specific T-cell subsets to separate GVHD and GVL remains an area of active investigation. For example, recent experimental data suggest that CD62L expressing naive T cells home to secondary lymph nodes and are critical for initiating GVHD.151 By contrast, CD62L-negative effector memory T cells with enhanced reactivity to recall antigens mediated GVL responses with minimal GVHD.151 An important caveat to these data is the fact that the lack of a priori knowledge of the repertoire of human memory T cells would make it difficult to predict whether these cells might cross-react only with TAAs or with the recipient’s alloantigens. Using CD62L status alone as a determinant of GVHD potential can also have other unintended consequences; recent studies have demonstrated that its expression is critical for the regulation of GVHD by Tregs (see later). Moreover, it is not known whether the behavior of human memory T cells parallels that of murine memory T cells in their migratory, functional, and cytolytic capabilities. Although Tregs reduce antitumor immunity in murine models and in human subjects, experimental data suggest that administration of donor-type Tregs, either at the time of HCT or when delayed, reduced GVHD but preserved CD8+-mediated perforin-dependent GVL responses.435,436 Similar preservation of experimental GVL was also observed by harnessing donor NKT cell function with granulocyte colony stimulating factor analogues.178,179 However, it remains unclear whether these observations are valid after

clinical HCT when the GVL responses might not be entirely dependent on CD8 T cells.

T-Cell Migration

It is conceivable that manipulation of these interactions to focus the alloimmune response to lymphohematopoietic tissues would enhance GVL responses but not GVHD. For example, blockade of the CCR9 ligand TECK or CCR5 and CCL17 may prevent the migration of donor T cells to GI tract and skin respectively, but preserve GVL.220 Pharmacologic manipulation with the immunosuppressive agent FTY720 has recently provided the proof in principle for this approach.231 Given the redundancy, strategies to modulate the chemokine biology for separation of GVHD and GVL will require greater understanding of these networks in modulating the migration not only of specific T-cell subsets but also of the other immune cells in the context of different conditioning regimens.

Phase 3: Effector Phase of Graft-Versus-Leukemia The effector arm of GVL is also characterized primarily by the antigen-specific cellular components and less by the inflammatory components of alloresponse. Experimental data demonstrate that neutralization of IL-1α reduced GVHD but preserved GVL.269 By contrast, donor TNF-α secretion contributes to both GVHD and GVL effects, and in some cases, antagonism of TNF-α reduced GVHD and GVL responses.437–439 Nonetheless, antagonism of nonspecific inflammatory effectors (such as either IL-1 or TNF-α) appears to regulate GVHD to a greater extent than GVL responses after experimental allogeneic HCT.439 Several lines of experimental and clinical data demonstrate that antigen-specific donor T-cell subsets and NK cells are the key effectors of GVL.56 The cytotoxic pathways that are operative in the NK and T cell–mediated antitumor responses have been well characterized.56 Fas ligand-mediated CTL of tumor targets is used by both NK and T (mostly Th1) cells, but most murine experiments with FasLdeficient donor T cells suggested that FasL is a key effector molecule for causing GVHD but not GVL.236 However, one study found that FasL is required for CD4+-mediated GVL against myeloid leukemia.440,441 By contrast, even though perforin-mediated CTL pathways are also used by T (mostly Th2) and NK cells, experimental data with perforin-deficient donor T cells demonstrated a loss of GVL with a diminution in the severity of GVHD.236 In some other experimental models, perforin was required only for GVL but not for GVHD.236 Recent data showed that TRAIL-mediated CTL had no effect on GVHD severity but was required for optimal GVL.257 Therefore strategies that increase donor T cell TRAIL expression or enhance the susceptibility of tumors to TRAIL-mediated CTL (such as histone deacetylase inhibitors) may promote a robust GVL effect without exacerbating GVHD.442–444 Thus significant progress has been made in recent understanding of the CTL pathways used by donor T cells for GVL responses, but the role and context of use of these pathways by donor NK and NKT cells after allogeneic HCT are not known. It is likely that effector cells responsible for the GVL and GVHD effects of HCT will similarly be responsible for the GVL effect associated with DLI, although this assumption has not been formally proven. The administration of select subsets of donor mononuclear cell fractions is the ideal setting in which to dissect the cellular mechanisms responsible for GVL induction and strategies that delay the infusion of these various cellular subsets will help define the mechanisms and enhance the efficacy of DLI.

Immunobiology of Cytokine Release Syndrome After Chimeric Antigen Receptor T-Cell Therapy CRS has been typically reported with mAb infusions, such as antiCD3 (OKT3), and the CD28 superagonist etc.445,446 In recent years it has been increasingly appreciated as the major acute toxicity from infusion of CAR T cells and bispecific antibodies for leukemia.447,448

Chapter 108  Graft-Versus-Host Disease and Graft-Versus-Leukemia Responses

It is characterized by an acute (days, depending on the inducing agent) and intense inflammatory response wherein the majority of the infused CAR T cells along with other immune cells such as NK cells, monocyte-macrophages, and dendritic cells become activated and release inflammatory cytokines.449,450 Much remains to be understood about the biology of CRS following CAR T-cell therapy. However, it appears that the incidence and severity of the syndrome is greater in patients with large tumor burdens, presumably because of higher levels of T-cell expansion and activation.414 However, as yet, no clear relationship between the cell infusion dose and the incidence/severity of CRS has been observed. It is associated with a massive proinflammatory cytokine storm with elevated IFN-γ, IL-6, TNF-α, IL-2, granulocyte-macrophage colony-stimulating factor and IL-5.414 Amongst these cytokines, it appears that in many instances IL-6 may be the most critical cytokine and its effects are likely from trans-signaling. The source of IL-6 and mechanisms remain unknown. The biology behind the incidence of neurotoxicities remains unclear. It is also unknown whether CRS is required for eventual clinical response and/or if mitigating it blunts response rates. Furthermore, whether CRS is reduced when selected T cells (central memory subset) are engineered with CARs instead of using bulk T cells remains an open question. The relationship of CRS with the type of vectors or other methodologic aspects also remains unknown. Several other critical mechanisms for enhancing the efficacy of CAR T cells or for mitigating their toxicities remain to be understood. The data on whether other targets (instead of CD19) can be as effective remain to be explored. The role of costimulatory domains in promoting efficacy, toxicity or exhaustion remains largely unknown. Recent experimental observations suggest that CARs against nonCD19 targets may be more susceptible to exhaustion and in these cells CD28 costimulation augments, whereas 4-1BB costimulation reduces, exhaustion induced by persistent CAR signaling.451 Thus much remains to be understood with regards to the immunobiology of CRS, and CAR T-cell therapy.

FUTURE DIRECTIONS Complications of HCT, particularly GVHD, remain major barriers to the wider application of allogeneic HCT for a variety of diseases. Recent advances in the biology of genetic polymorphisms, the chemocytokine networks, several novel cellular subsets including regulatory T cells, and the direct mediators of cellular cytotoxicity have led to improved understanding of this complex disease process. Animal studies show that modulation of several mediators of the complex GVHD cascade may be able to reduce the undesirable inflammatory aspects of GVHD while preserving the benefits of GVL. However, most of the laboratory observations remain to be studied in wellcontrolled clinical trials. Multiple cellular effectors may be involved in GVL, although donor T-cell recognition of host antigens is an important element of this process. Cellular immunotherapy such as DLI offers a strategy for separating GVHD and the GVL effect. Both experimental and clinical data suggest that posttransplantation cellular immunotherapy can be performed relatively safely and effectively, and optimization of patient selection, cell dose, and timing of administration may all serve to limit toxicity and enhance the potential GVL effects.

SUGGESTED READINGS Alousi AM, Weisdorf DJ, Logan BR, et al: Etanercept, mycophenolate, denileukin or pentostatin plus corticosteroids for acute graft vs. host disease: a randomized phase II trial from the BMT CTN. Blood 114:511, 2009. Anasetti C, Beatty PG, Storb R, et al: Effect of HLA incompatibility on graftversus-host disease, relapse, and survival after marrow transplantation for patients with leukemia or lymphoma. Hum Immunol 29:79, 1990. Billingham RE: The biology of graft-versus-host reactions. Harvey Lect 62:21, 1966-67.

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Blazar BR, Murphy WJ, Abedi M: Advances in graft-versus-host disease biology and therapy. Nat Rev Immunol 12(6):443, 2012. Choi SW, Braun T, Chang L, et al: Vorinostat plus tacrolimus and mycophenolate to prevent graft-versus-host disease after related-donor reducedintensity conditioning allogeneic haemopoietic stem-cell transplantation: a phase 1/2 trial. Lancet Oncol 15(1):87, 2014. Den Haan JM, Sherman NE, Blokland E, et al: Identification of a graft versus host disease-associated human minor histocompatibility antigen. Science 268:1476, 1995. Dickinson AM, Middleton PG, Rocha V, et al: Genetic polymorphisms predicting the outcome of bone marrow transplants. Br J Haematol 127:479, 2004. Edinger M, Hoffmann P, Ermann J, et al: CD4+CD25+ regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versushost disease after bone marrow transplantation. Nat Med 9:1144, 2003. Glucksberg H, Storb R, Fefer A, et al: Clinical manifestations of graft-versushost disease in human recipients of marrow from HL-A-matched sibling donors. Transplantation 18:295, 1974. Goulmy E, Schipper R, Pool J, et al: Mismatches of minor histocompatibility antigens between HLA-identical donors and recipients and the development of graft-versus-host disease after bone marrow transplantation. N Engl J Med 334:281, 1996. Henden AS, Hill GR: Cytokines in Graft-versus-Host Disease. J Immunol 194:4604, 2015. Kolb H, Mittermuller J, Clemm C, et al: Donor leukocyte transfusions for treatment of recurrent chronic myelogenous leukemia in marrow transplant patients. Blood 76:2462, 1990. Korngold R, Sprent J: Negative selection of T cells causing lethal graft-versushost disease across minor histocompatibility barriers. Role of the H-2 complex. J Exp Med 151:1114, 1980. Levine JE, Braun TM, Harris AC, et al: A prognostic score for acute graftversus-host disease based on biomarkers: a multicentre study. Lancet Haematol 2:e21–e29, 2015. Lindemans CA, Calafiore M, Mertelsmann AM, et al: Interleukin-22 promotes intestinal-stem-cell-mediated epithelial regeneration. Nature 528(7583):560–564, 2015. Lowsky R, Takahashi T, Liu YP, et al: Protective conditioning for acute graftversus-host disease. N Engl J Med 353:1321, 2005. Martin PJ, Weisdorf D, Przepiorka D, et al: National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease: VI. Design of Clinical Trials Working Group report. Biol Blood Marrow Transplant 12:491, 2006. Mathewson ND, Jenq R, Mathew AV, et al: Gut microbiome-derived metabolites modulate intestinal epithelial cell damage and mitigate graftversus-host disease. Nat Immunol 17(5):505–513, 2016. Petersdorf EW, Hansen JA, Martin PJ, et al: Major-histocompatibilitycomplex class I alleles and antigens in hematopoietic-cell transplantation. N Engl J Med 345:1794, 2001. Ratanatharathorn V, Nash RA, Przepiorka D, et al: Phase III study comparing methotrexate and tacrolimus (prograf, FK506) with methotrexate and cyclosporine for graft-versus-host disease prophylaxis after HLA-identical sibling bone marrow transplantation. Blood 92:2303, 1998. Reddy P, Maeda Y, Liu C, et al: A crucial role for antigen-presenting cells and alloantigen expression in graft-versus-leukemia responses. Nat Med 11:1244, 2005. Shlomchik WD, Couzens MS, Tang CB, et al: Prevention of graft versus host disease by inactivation of host antigen-presenting cells. Science 285:412, 1999. Shulman HM, Sharma P, Amos D, et al: A coded histologic study of hepatic graft-versus-host disease after human bone marrow transplantation. Hepatology 8:463, 1988. Storb R, Deeg HJ, Whitehead J, et al: Methotrexate and cyclosporine compared with cyclosporine alone for prophylaxis of acute graft versus host disease after marrow transplantation for leukemia. N Engl J Med 314:729, 1986. van Bekkum DW, Roodenburg J, Heidt PJ, et al: Mitigation of secondary disease of allogeneic mouse radiation chimeras by modification of the intestinal microflora. J Natl Cancer Inst 52:401, 1974.

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Vander Lugt MT, Braun TM, Hanash S, et al: ST2 as a marker for risk of therapy-resistant graft-versus-host disease and death. N Engl J Med 369(6):529–539, 2013. Weiden PL, Flournoy N, Thomas ED, et al: Antileukemic effect of graftversus-host disease in human recipients of allogeneic-marrow grafts. N Engl J Med 300:1068, 1979. Wekerle T, Kurtz J, Ito H, et al: Allogeneic bone marrow transplantation with co-stimulatory blockade induces macrochimerism and tolerance without cytoreductive host treatment. Nat Med 6:464, 2000.

Zeiser R, Blazar BR: Preclinical models of acute and chronic graft-versus-host disease: how predictive are they for a successful clinical translation? Blood 127:3117–3126, 2016.

REFERENCES For the complete list of references, log on to www.expertconsult.com.

Chapter 108  Graft-Versus-Host Disease and Graft-Versus-Leukemia Responses

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300. Blazar BR, Taylor PA, Panoskaltsis-Mortari A, et al: Rapamycin inhibits the generation of graft-versus-host disease and graft-versus-leukemiacausing T cells by interfering with the production of Th1 or Th1 cytotoxic cytokines. J Immunol 160:5355, 1998. 301. Allison AC, Eugui EM: Mycophenolate mofetil and its mechanisms of action. Immunopharmacology 47:85, 2000. 302. van Bekkum DW, Roodenburg J, Heidt PJ, et al: Mitigation of secondary disease of allogeneic mouse radiation chimeras by modification of the intestinal microflora. J Natl Cancer Inst 52:401, 1974. 303. Goerner M, Gooley T, Flowers ME, et al: Morbidity and mortality of chronic GVHD after hematopoietic stem cell transplantation from HLA-identical siblings for patients with aplastic or refractory anemias. Biol Blood Marrow Transplant 8:47, 2002. 304. Beelen DW, Haralambie E, Brandt H, et al: Evidence that sustained growth suppression of intestinal anaerobic bacteria reduces the risk of acute graft-versus-host disease after sibling marrow transplantation. Blood 80:2668, 1992. 305. Beelen DW, Elmaagacli A, Muller KD, et al: Influence of intestinal bacterial decontamination using metronidazole and ciprofloxacin or ciprofloxacin alone on the development of acute graft-versus-host disease after marrow transplantation in patients with hematologic malignancies: final results and long-term follow-up of an open-label prospective randomized trial. Blood 93:3267, 1999. 306. Passweg J, Rowlings P, Atkinson K, et al: Influence of protective isolation on outcome of allogeneic bone marrow transplantation for leukemia. Bone Marrow Transplant 21:1231, 1998. 307. Akpek G, Lenz G, Lee SM, et al: Immunologic recovery after autologous blood stem cell transplantation in patients with AL-amyloidosis. Bone Marrow Transplant 28:1105, 2001. 308. Akpek EA, Mutlu H, Kayhan Z: Difficult intubation in pediatric cardiac anesthesia. J Cardiothorac Vasc Anesth 18:610, 2004. 309. Akpek G, Boitnott JK, Lee LA, et al: Hepatitic variant of graft-versushost disease after donor lymphocyte infusion. Blood 100:3903, 2002. 310. Petersdorf EW, Hansen JA, Martin PJ, et al: Major-histocompatibilitycomplex class I alleles and antigens in hematopoietic-cell transplantation. N Engl J Med 345:1794, 2001. 311. Kennedy GA, Varelias A, Vuckovic S, et al: Addition of interleukin-6 inhibition with tocilizumab to standard graft-versus-host disease prophylaxis after allogeneic stem-cell transplantation: a phase 1/2 trial. Lancet Oncol 15(13):1451–1459, 2014. 312. Holler E, Kolb HJ, Hintermeier-Knabe R, et al: Role of tumor necrosis factor alpha in acute graft-versus-host disease and complications following allogeneic bone marrow transplantation. Transplant Proc 25:1234, 1993. 313. Holler E, Kolb HJ, Moller A, et al: Increased serum levels of tumor necrosis factor alpha precede major complications of bone marrow transplantation. Blood 75:1011, 1990. 314. Holler E, Kolb HJ, Wilmanns W: Treatment of GVHD–TNFantibodies and related antagonists. Bone Marrow Transplant 12:S29, 1993. 315. Campbell GL, Grady LJ, Huang C, et al: Laboratory testing for West Nile virus: panel discussion. Ann N Y Acad Sci 951:179, 2001. 316. Kobbe G, Schneider P, Rohr U, et al: Treatment of severe steroid refractory acute graft-versus-host disease with infliximab, a chimeric human/ mouse antiTNFalpha antibody. Bone Marrow Transplant 28:47, 2001. 317. Couriel DR, Hicks K, Giralt S, et al: Role of tumor necrosis factoralpha inhibition with inflixiMAB in cancer therapy and hematopoietic stem cell transplantation. Curr Opin Oncol 12:582, 2000. 318. Chiang KY, Abhyankar S, Bridges K, et al: Recombinant human tumor necrosis factor receptor fusion protein as complementary treatment for chronic graft-versus-host disease. Transplantation 73:665, 2002. 319. Levine JE, Paczesny S, Mineishi S, et al: Etanercept plus methylprednisolone as initial therapy for acute graft-versus-host disease. Blood 111:2470, 2008. 320. Levine JE, Logan B, Wu J, et al: Graft-versus-host disease treatment: predictors of survival. Biol Blood Marrow Transplant 16:1693, 2010. 321. Cullup H, Dickinson A, Jackson G, et al: Donor interleukin 1 receptor antagonist genotype associated with acute graft-versus-host disease in human leucocyte antigen-matched sibling allogeneic transplants. Br J Haematol 113:807, 2000.

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Part X  Transplantation

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