Liver Graft-versus-host Disease Research Articles

Superior Survival of Combined Gastrointestinal and Skin GvHD Compared to Isolated Gastrointestinal GvHD in Children Post Allogeneic Stem Cell Transplantation

Abstract 4535 Objective:To compare the prognosis of isolated gastrointestinal (GI) graft versus host disease (GvHD) vs combined (GI and skin) GvHD in children post allogeneic stem cell transplantation (SCT). Methods:Allogeneic SCT recipients from January 2000-Dec 2009 were included and patients who were diagnosed with skin and GI GVHD were identified. Demographics and outcome data were collected and analyzed. Results:Four hundred and fifty children (0–18 years) underwent allogeneic SCT in the study period. Seventy-nine (17.5%) patient underwent endoscopy and GI biopsy for GI GvHD. Of the seventy-nine patients, 49 had isolated GI GvHD and 30 had combined GvHD. Fifty-one patients received SCT for a malignant indication and 28 for a non-malignant cause. Donor sources were; isolated GI group, related 10, unrelated 20: combined group, related 20, unrelated 29). All patients received myeloablative conditioning regimens and the majority received cyclosporine A and methotrexate for GvHD prophylaxis. Sixty-seven percent of patients with combined GvHD had grade III-IV while only 31% had grade III-IV in patients with isolated GI GvHD. To ensure there was no bias in a quicker initiation of anti GvHD therapy in children with skin involvement, we included only children who had a GI scope and GI biopsy looking for GI GvHD and also we compared if GvHD therapy initiation was different in patients with or without skin involvement [In the combined group 16 patients (53%), therapy was started prior to GI biopsy versus 16 patients (33%) in the isolated GIT group P=0.2]. Interestingly, 40% of children with combined GvHD had also liver GvHD while only 6% of patients with isolated GI GvHD had liver involvement (p≤0.0001). Overall response to GvHD therapy was similar in both groups (83% vs 87%). However, despite having less patients in the isolated GI group with severe (grade III-IV GvHD), there was a significant increase in non-relapse mortality (NRM) in the isolated GI group compared to the combined group (37% vs 16%) resulting in a statistically significant superior overall survival for those children with combined GvHD (79% vs 49% P= 0.02). Disease relapse was similar in both groups (8% and 6% for combined and isolated GIT group, respectively). Conclusion:Children with GI GvHD has better prognosis when there is skin involvement. Further research is warranted to examine if there is certain biological features that make GI GvHD extends to skin and other organs and why isolated GI GVHD is associated with worse prognosis? Disclosures:No relevant conflicts of interest to declare.

Hematopoietic Cell Transplantation After Reduced Intensity Conditioning for Severe Paroxysmal Nocturnal Hemoglobinuria:

Abstract 2047 Objectives:Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare acquired clonal hematopoietic stem cell disease. Patients may present with intermittent hemolysis, thrombotic events or cytopenias. The manifestations can be life threatening. Hematopoietic Cell Transplantation (HCT) is still the only curative therapy. Hegenbart et al reported early results in 7 patients with PNH with high risk features who received transplants from matched related (n=2) or unrelated donors (n=5) after conditioning with a fludarabine / total body irradiation (TBI) based regimen (Biol Blood Marrow Transplant 2003; 9: 689–697). Here we update the data and report outcomes in 12 additional patients. Patients:Nineteen patients diagnosed with severe PNH underwent allogeneic HCT after a reduced intensity conditioning (RIC) regimen. Patients were treated at 9 centers. Eighteen patients had at least 1 high-risk feature (Table), 1 patient had none but underwent transplant because of severe hemolysis prior to the era of eculizumab. Conditioning consisted of fludarabine (30 mg/m2/d) on days −4 to −2 before HCT and 2 Gy (n=16) or 4 Gy (n=3) of TBI on day 0 (n=16) followed by treatment with mycophenolate mofetil and cyclosporine or tacrolimus. All patients received peripheral blood stem cells (PBSC). Donors were HLA-matched related (n=3), HLA-matched unrelated (n=12) or HLA-mismatched unrelated (n=4). Results:The follow-up period ranges between 2 months and almost 10 years (median 27.5 months) after HCT. Neutropenia (absolute neutrophil count<500/μl) developed in 15 of 19 patients after transplantation. The median time to neutrophil recovery in patients who engrafted was 16 days (range 0–29) with a median duration of neutropenia of 12 days (range 0–17, n=12). The median duration of thrombocytopenia (platelet count <20,000/μl) was 3 days (range, 0 to 10 days, n=12). All but 2 patients (89.5%) had a sustained engraftment. One patient experienced primary graft failure and remained neutropenic. He received a second nonmyeloablative HLA-matched unrelated PBSC transplant, 47 days after the first transplant, from a different donor, with 3 Gy TBI and 90mg/m2 fludarabine, and had sustained engraftment. The second patient experienced after engraftment, late graft failure 6 months after HCT. She underwent, 7 months after the first transplant, a second nonmyeloablative, HLA- matched unrelated PBSC transplant, from a different donor, with 4 Gy TBI and 150mg/m2 fludarabine, and had sustained engraftment. Eighteen patients were assessable for acute graft-versus-host disease (GvHD). The incidence of grades 2, 3, and 4 acute GvHD were 22% (4/18), 17% (3/18), and 5% (1/18), respectively. Chronic GvHD developed in 78% (14/18) of evaluable patients. Fifteen patients (78.9%) are alive at the time of last follow-up (Fig.). None of the 15 patients still alive has any evidence of relapse, clinically nor by flow cytometry. Four patients died (21.1%) of complications (necrotizing pancreatitis of unknown cause, pseudomonas infection during treatment of chronic GvHD, hemorrhage after liver biopsy with subacute/chronic liver GvHD, intracerebral bleed). All 15 surviving patients have an ECOG performance status of 0 or 1. [Display omitted] Conclusion:The RIC regimen reported here is well tolerated and leads to a high rate of successful engraftment. The graft-versus-host effect seems to be sufficient to eradicate the PNH clone. This regimen may be equally effective in medically fit PNH patients to eradicate the PNH clone while reducing treatment related complications of higher intensity conditioning regimens. Note:1st 3 authors contributed equally.TablePatient and disease characteristicsCharacteristicNumber (n=19)PercentageSexMale526Female1474Age, yearsMedian34Range19–67Number of high risk features1015110632726315ThrombosisYes1047No953Treatment prior to transplantSteroids1684CSP842ATG2516MMF15Azathioprine15Danazol15IVIG315Eculizumab210MyelodysplasiaYes626No1374HCT-CI40-16233+6Not available4[1]High risk features are occurrence of thrombosis (relative risk [RR] 10.2), evolution to pancytopenia (RR 5.5), myelodysplastic syndrome or acute leukemia (RR 19.1), age over 55 years at diagnosis (RR 4), more than one treatment (RR 2.1), and thrombocytopenia at diagnosis (RR 2.2).2Antithymocyte globulin.3Intravenous immunoglobulin.4Hematopoietic Cell Transplant-Comorbidity Index score. Disclosures:No relevant conflicts of interest to declare.

Influence of glutathione S-transferase T1 donor/recipient mismatch and anti-GSTT1 antibodies in hepatic graft-versus-host-disease

B cell responses to minor histocompatibility antigens (mHags) have not been extensively studied after allogeneic hematopoietic stem cell transplantation (HSCT). Glutathione S-transferase T1 (GSTT1) is a drug metabolizing enzyme encoded by a single gene that is highly expressed in liver and kidney. Anti-GSTT1 antibodies have been described in the context of antibody-mediated rejection in kidney and liver transplantation, due to a mismatch between donor and recipient. The aim of the present study was to investigate the specific immune response against GSTT1 in HSCT with production of antibodies and their influence in the development of hepatic graft-versus-host-disease (GVHD). Forty patients and their respective donors were included in the study. The median follow-up time was 35.6months (range 0.6–76months) and a total of 349 serum samples were tested for the presence of anti-GSTT1 antibodies by ELISA test. Statistical analysis was performed by defining the GSTT1 null donor/positive recipient as mismatch compared with the other three genetic combinations regarded as GSTT1-matched. Antibodies were found in three patients within the group of null donor/positive recipient and one within the null/null group. Development of liver GVHD, particularly its acute form, was highly associated with the GSTT1-mismatch (P=0.0178) and with the presence of post-transplant anti-GSTT1 antibodies (P=0.0076). We conclude that GSTT1 could be considered as a new mHag in hepatic GVHD. The fact that three donors were parous females and the rapid production of antibodies after HSCT suggests the existence in the graft of memory B-cells specific for the GSTT1 antigen.

Hyperbilirubinemia after hematopoietic stem cell transplantation: comparison of clinical and pathologic findings in 41 autopsied cases

Hyperbilirubinemia is often associated with morbidity and mortality after hematopoietic stem cell transplantation (HSCT). Diagnosis of its etiology is usually made clinically among various possible causes, and analysis of histological findings as compared with the clinical diagnosis has not been performed sufficiently. We retrospectively analyzed clinical and pathological findings in 41 autopsied patients who died with hyperbilirubinemia (>2 mg/dL). Overall, liver graft-versus-host disease (GVHD) showed the most prominent discordance between clinical and pathological diagnoses. Only 11 of the 22 patients, considered to have liver GVHD clinically, had GVHD findings at autopsy. Serum gamma-glutamyl transpeptidase (GGT), GGT/aspartate aminotransferase (AST) ratio, and alkaline phosphatase (ALP)/AST ratio in GVHD patients were significantly higher compared with those without GVHD (p = 0.02, <0.01, and 0.03, respectively), which was useful in clinical diagnosis of liver GVHD. Other major findings include liver invasion of the primary malignancies in 8 patients, post-transplant lymphoproliferative disorder of the liver in two patients, and disseminated liver invasion by fungus or varicella-zoster virus in one patient, respectively. Although analysis of clinical data is useful for narrowing diagnosis, histological analysis by liver biopsy is crucially important, especially in cases suspected of having GVHD.

Rituximab in bronchiolitis obliterans after haematopoietic stem cell transplantation

To the Editors: Bronchiolitis obliterans (BO) following allogeneic haematopoietic stem cell transplantation (HSCT) is recognised as a pulmonary manifestation of chronic graft- versus -host disease (GVHD) [1, 2]. As lung biopsy is rarely performed in these patients, the diagnosis of BO syndrome (BOS) usually relies on pulmonary function testing (PFT) and computed tomography (CT) lung scans [1, 2]. Very little retrospective data exist regarding the treatment of BOS, which currently consists of increasing a patient’s baseline dose of systemic immunosuppressive therapy; however, this treatment strategy has been shown to have poor efficacy [2, 3]. The efficacy of new drugs for the treatment of chronic GVHD, including the anti-CD20 monoclonal antibody rituximab, must be specifically evaluated for BOS. Here, we report three cases in which patients received rituximab for BO after allogeneic HSCT. Patient 1 was a 15-yr-old male who received bone marrow from a sibling human leukocyte antigen (HLA)-matched donor for acute myeloid leukaemia after a myeloablative conditioning regimen that included 12 Gy fractionated total body irradiation (FTBI) and cyclophosphamide. 5 months after transplantation, he was diagnosed with chronic liver GVHD that led to the initiation of prednisone at a dosage of 1 mg·kg−1·day−1 as well as tacrolimus. 8 months after transplantation, as his liver GVHD was coming under control, he presented with dyspnoea, dry cough and chest pain. At this time, he was receiving tacrolimus and prednisone at a dosage of 20 mg·day−1. PFT revealed a severe obstructive pattern based on a forced expiratory volume in 1 s (FEV1) of 1.13 L (33% predicted), a forced vital capacity (FVC) of 2.79 L (65% pred) and FEV1/FVC ratio of 41%. A lung CT scan demonstrated the presence of air trapping and a …

Organ-Specific Protection from CD8+ T Cell-Mediated Acute Skin GVHD by Bortezomib Administration Correlates with Decreased IL-6.

AbstractAbstract 3735We have previously demonstrated that while early administration of bortezomib after murine allogeneic bone marrow transplantation (allo-BMT) can prevent acute graft versus host disease (GVHD), delayed administration of bortezomib results in increased GVHD-dependent gastrointestinal toxicity (PNAS 2004, Blood 2005). This was primarily mediated by donor CD4+ T cells in fully MHC-mismatched allo-BMT (Blood 2008). In the current study, we assessed continuous bortezomib administration on CD8+ T cell-mediated acute GVHD. Using an MHC-matched, mHAg-mismatched model (C3.SW donor cells into lethally irradiated C57BL/6 recipients) in which acute GVHD is primarily induced by CD8+ T cells, we confirmed that significant skin and liver GVHD was observed after CD8+ T cell transfer. Histological evaluation of acute liver GVHD displayed severe lymphocyte infiltrates and severe subacute bile duct hepatitis consistent with hepatic GVHD. Severe acute skin GVHD was noted by persistent fur loss and skin ulcerations. Cutaneous histopathological assessment exhibited severe inflammatory infiltrates, epidermal hypertrophy, loss of hair follicles, satellite cell necrosis and dermal fibrosis. Notably, after allo-BMT, 6 weeks or thereafter, a significant increase in serum IL-6 levels was observed in the mice exhibiting marked skin damage, suggesting organ-specific correlation of higher serum IL-6 expression with acute cutaneous GVHD associated injury. We next investigated the effects of continuous treatment with bortezomib in this GVHD model. Surprisingly, we found that continuous bortezomib administration at the conventional dose of 0.75–1 mg/kg twice per week resulted in fractional GVHD- dependent lethal toxicity, exacerbating lymphocyte infiltration in liver, gut and lung as evidenced by pathological examination. However, skin GVHD pathology was markedly improved. Due to the limitations of conventional doses of bortezomib for treatment of acute GVHD, we then performed a low-dose bortezomib treatment at 0.125 mg/kg, twice per week. We found that mice receiving low-dose bortezomib were protected from lethal toxicity and displayed significant amelioration of skin GVHD pathology. Significant decreases in serum IL-6 levels were also consistently observed consistent with the amelioration of skin GVHD. Interestingly, while skin GVHD was markedly improved, there was no difference in liver, gut and lung pathology compared to control GVHD recipients indicating an organ-specific protection. These results demonstrate that continuous bortezomib can be applied in CD8+ T cell-mediated acute GVHD but necessitates lower dosing and organ-specific protection is observed. Disclosures:No relevant conflicts of interest to declare.

Third Party Umbilical Cord Blood Regulatory T Cells Prevents Graft Versus Host Disease In a Xenogenic Murine Model.

AbstractAbstract 3737 Background:Umbilical Cord Blood (UUCB) Regulatory T cells (Tregs) co-expressing CD4/CD25 have been shown to inhibit alloreactive T cell function. We hypothesized that prophylactic infusion of 3rd party UCB Tregs can abrogate Graft versus Host Disease (GvHD). Methods:(a) Treg generation: Naturally occurring CD25+ Tregs, purified from thawed UCB units by magnetic activated cell sorting (MACS) using Miltenyi CD25 reagents (as per manufacturer's instructions), were cultured at 1×106 cells/ml in ExVivo medium (Gibco) containing interleukin (IL)-2 (100 IU/ml) with irradiated K562 cells engineered to express CD86, CD69, CD187c, IL-15, CD64 and OKT3 (provided by Dr. Laurence Cooper, MDACC) or with microbeads bearing CD3 and CD28 antibodies on their surface (T-Cell Expander, Invitrogen). Cultures were initiated at a ratio of 1 Treg: 4 engineered K562 cells or Invitogen beads and maintained at 1×106 cells/ml over a 14 day period by the addition of fresh medium. Fresh IL-2 was added to maintain 100 IU/ml on an every-other-day basis. Flow cytometry, Western blot analysis and demonstration of ability to reduce in vitro MLR reactions confirmed the Treg phenotype of the expanded cells. (b) Xenogeneic graft vs. host model: On day -1, NOD-SCID IL2Rgnull (NSG) mice were sublethally-irradiated (320 cGy) and received 107 Treg cells intravenously. On day 0, 107 human apheresis PBMCs were injected intravenously. Mice were regularly monitored, weighed and graded according to a GvHD scale developed by Dr. James Ferrara (University of Michigan Comprehensive Cancer Center). Results:(a) Ex vivo Treg expansion of up to 700 fold was achieved yielding approximately 2.5×108 Treg cells from an average frozen UCB unit. Analysis of the ex vivo expanded product after 14 days revealed: (i) 95% co-expressed FOXP3/CD4/CD25 (flow cytometry), and (ii) expression of peptidase inhibitor 16, known to be uniquely associated with FOXP3 expression (western blot, Figure 1). Further, addition of UCB Tregs showed suppression of two-way allo-MLR by 93% (measured by tritiated thymidine and CFSE studies). (b) In the NSG GvHD model, mice receiving PBMC showed: (i) a significant (P=0.0002, student's paired t-test) 30% reduction in body weight, (ii) gross evidence of GvHD (average Ferrara score of 6) and (iii) reduced survival, when compared to mice receiving intravenous injection of Tregs on day -1. By comparison, mice receiving Tregs on day -1: (i) did not lose weight, (ii) scored consistently lower on the Ferrara scale (average score of 3) and (iii) had significantly improved survival (70% survival vs. 15%; P < 10-4, figure 2). Xenogenic transplanted mice showed clear histopathological evidence of GvHD in skin, gut, liver, spleen, lung, esophagus, and bone marrow whereas histopathology remained comparable to controls in the Treg rescued mice. [Display omitted] [Display omitted] Conclusion:Infusion of 3rd party UCB Tregs markedly reduced the severity of xenoGVHD in a mouse transplant model. These preclinical data support the initiation of a phase I/II clinical trial to assess the efficacy of third party allogenic UCB Tregs in a traditional transplant setting. Disclosures:No relevant conflicts of interest to declare.

Clinical Benefit of Intermediate-Dose of Steroid In Chronic Graft-Versus-Host-Disease of the Liver Presented with the Elevation of Serum Aminotransferases

AbstractAbstract 2331 Introduction:Chronic graft-versus-host disease (GVHD) of the liver is generally known as cholestatic jaundice. Although there are many aetiologies of elevated hepatic enzyme after hematopoietic stem cell transplantation (HSCT), such as iron overload and hepatitis B and/or C, acute hepatic enzyme elevation has been recently considered as a manifestation of chronic liver GVHD. Here we compare the incidence and the therapeutic response of chronic liver GVHD between two phenotypes; hepatic enzyme elevation type and hyperbilirubinemia type. Patients and methods:We reviewed the data of 402 patients who underwent allogeneic HSCT at our institute from 1995 to 2009, and searched the cases that satisfied the following criteria: the serum aminotransferase (aspartate aminotransferase (AST) and/or alanine transaminase (ALT)) levels were over 3 times above their normal ranges while the bilirubin level remained within the double of its normal range (hepatic enzyme elevation type), or the bilirubin level was over twice above its normal range while the serum aminotransferase levels remained within 3 times of their normal ranges (hyperbilirubinemia type). We interpreted that the treatment was successful when AST, ALT and bilirubin satisfied neither of the criteria above. Results:Of the 402 patients, 76 patients (18.9%) had chronic GVHD of the liver (hepatic enzyme elevation type in 65 (16.2%) cases and hyperbilirubinemia type in 11 (2.7%) cases). One-year cumulative incidence of chronic liver GVHD were 16.3±1.9% in hepatic enzyme elevation type, and 3.0±0.9% in hyperbilirubinemia type. Among the patients with hepatic enzyme elevation type, median time of the onset was at 161 days (range, 60–510 days) after transplantation. Median peaks of serum AST, ALT and bilirubin were 260 IU/L (74-3,492 IU/L), 422 IU/L (89-2,236 U/L), and 1.7 mg/dL (0.5-38.4 mg/dL), respectively. Thirty patients had recently ceased all immunosuppressive drugs and 35 patients had been tapering in line with standard clinical practice. Liver biopsies were undergone in 10 patients and showed damaged and degenerative interlobular bile ducts, one of characteristic features of GVHD. Other features, such as lobular hepatitis and many acidophilic bodies scattered throughout the lobules, were also detected. Regarding viral infection, CMV immunohistochemistry and EBER in situ hybridization were negative. Several treatments were performed for 65 patients; increased dosage of calcineurin inhibitor (CI; Cyclosporine or Tacrolimus) for 25 patients, prednisolone (PSL) alone for 18 patients, CI plus PSL for 20 patients, and only two patients were taken a wait-and-see approach. Next we evaluated the therapeutic response in 60 patients, since five died soon after onset. Median time until treatment success were 31 days (3-85 days) and 63 days (7-259 days) in patients with or without PSL, respectively (n=35 vs 25, p<0.0001). Further, we analyzed the dose-effect of PSL. Median time until treatment success were 25 days (3-85 days) in patients received 1 mg/kg or more PSL (n=25), and 34.5 days (19-72 days) in patients received under 1 mg/kg PSL (n=10) (p=0.62). We also compared the difference of response to steroid between hepatic enzyme elevation type and hyperbilirubinemia type. Three of 38 (7.9%) patients resulted in treatment failure in hepatic type, while four of eight (50%) patients in hyperbilirubinemia type (P=0.012). Conclusions:In our study, hepatic enzyme elevation type is more frequent than hyperbilirubinemia type in chronic liver GVHD. Intermediate-dose steroid can prevent progressive hepatocellular injury and bile duct destruction in hepatic enzyme elevation type, resulting in good outcome compared with in hyperbilirubinemia type. Disclosures:No relevant conflicts of interest to declare.

Clofarabine ± Fludarabine with Once Daily i.v. Busulfan as Pretransplant Conditioning Therapy for Advanced Myeloid Leukemia and MDS

Although a combination of i.v. busulfan (Bu) and fludarabine (Flu) is a safe, reduced-toxicity conditioning program for acute myelogenous leukemia/myelodysplastic syndromes (AML/MDS), recurrent leukemia posttransplantation remains a problem. To enhance the conditioning regimen's antileukemic effect, we decided to supplant Flu with clofarabine (Clo), and assayed the interactions of these nucleoside analogs alone and in combination with Bu in Bu-resistant human cell lines in vitro. We found pronounced synergy between each nucleoside and the alkylator but even more enhanced cytotoxic synergy when the nucleoside analogs were combined prior to exposing the cells to Bu. We then designed a 4-arm clinical trial in patients with myeloid leukemia undergoing allogeneic stem cell transplantation (allo-SCT). Patients were adaptively randomized as follows: Arm I-Clo:Flu 10:30 mg/m(2), Arm II-20:20 mg/m(2), Arm III-30:10 mg/m(2), and Arm IV-single-agent Clo at 40 mg/m(2). The nucleoside analog(s) were/was infused over 1 hour once daily for 4 days, followed on each day by Bu, infused over 3 hours to a pharmacokinetically targeted daily area under the curve (AUC) of 6000 μMol-min ± 10%. Fifty-one patients have been enrolled with a minimum follow-up exceeding 100 days. There were 32 males and 19 females, with a median age of 45 years (range: 6-59). Nine patients had chronic myeloid leukemia (CML) (BC: 2, second AP: 3, and tyrosine-kinase inhibitor refractory first chronic phase [CP]: 4). Forty-two patients had AML: 14 were induction failures, 8 in first chemotherapy-refractory relapse, 7 in untreated relapse, 3 in second or subsequent relapse, 4 were in second complete remission (CR), and 3 in second CR without platelet recovery (CRp), 2 were in high-risk CR1. Finally, 1 patient was in first CRp. Graft-versus-host disease (GVHD) prophylaxis was tacrolimus and mini-methorexate (MTX), and those who had an unrelated or 1 antigen-mismatched donor received low-dose rabbit-ATG (Thymoglobulin™). All patients engrafted. Forty-one patients had active leukemia at the time of transplant, and 35 achieved CR (85%). Twenty of the 42 AML patients and 5 of 9 CML patients are alive with a projected median overall survival (OS) of 23 months. Marrow and blood (T cell) chimerism studies at day +100 revealed that both in the lower-dose Clo groups (groups 1+2) and the higher-dose Clo groups (groups 3+4), the patients had a median of 100% donor (T cell)-derived DNA. There has been no secondary graft failure. In the first 100 days, 1 patient died of pneumonia, and 1 of liver GVHD. We conclude that (1) Clo ± Flu with i.v. Bu as pretransplant conditioning is safe in high-risk myeloid leukemia patients; (2) clofarabine is sufficiently immunosuppressive to support allo-SCT in myeloid leukemia; and (3) the median OS of 23 months in this high-risk patient population is encouraging. Additional studies to evaluate the antileukemic efficacy of Clo ± Flu with i.v. Bu as pretransplant conditioning therapy are warranted.

Gas6 deficiency in recipient mice of allogeneic transplantation alleviates hepatic graft-versus-host disease

AbstractGrowth arrest-specific gene 6 (Gas6) is expressed in antigen-presenting cells and endothelial cells (ECs) but not in T cells. When wild-type (WT) or Gas6−/− mice received allogeneic non–T cell–depleted bone marrow cells, hepatic graft-versus-host disease (GVHD) was alleviated in Gas6−/− recipients regardless of donor genotype, but not in WT recipients. T-cell infiltration was more prominent and diffuse in WT than in Gas6−/− recipients' liver. When mice received 0.5 × 106 allogeneic T cells with T cell–depleted allogeneic bone marrow, clinical signs indicated that GVHD was less severe in Gas6−/− than in WT recipients, as shown by a significant improvement of the survival and reduced liver GVHD. These data demonstrate that donor cells were not involved in the protection mechanism. In addition, lack of Gas6 in antigen-presenting cells did not affect WT or Gas6−/− T-cell proliferation. We therefore assessed the response of WT or Gas6−/− ECs to tumor necrosis factor-α. Lymphocyte transmigration was less extensive through Gas6−/− than WT ECs and was not accompanied by increases in adhesion molecule levels. Thus, the lack of Gas6 in ECs impaired donor T-cell transmigration into the liver, providing a rationale for considering Gas6 pathway as a potential nonimmunosuppressive target to minimize GVHD in patients receiving allogeneic hematopoietic stem cell transplantation.

Treatment of Severe Acute Graft-Versus-Host Disease with Mesenchymal Stromal Cells.

Abstract 2249Poster Board II-226 BACKGROUND.Allogeneic hematopoietic stem cell transplantation (HSCT) is being used to treat a range malignant and nonmalignant conditions. However, it often causes potentially lethal Graft-versus-Host Disease (GVHD). Several studies revealed that mesenchymal stromal cells (MSCs) from human bone marrow can down regulate GVHD after HSCT. METHODS.MSCs were obtained from BM, expanded and characterized by their morphology, immunophenotype, immunosuppressive potential for autologous, partially and fully mismatched lymphocytes. Twenty five patients (pts) got 39 (range, 1 to 4) MSCs infusions for 27 episodes of acute GVHD, which was defined as steroid resistant grade IV aGVHD in all but one patient. RESULTS.GvHD characteristics. Acute GVHD started from day +6 to d +46 from HSCT (median d+18) excluding single pt in whom it occurred on d+150. In 2 pts disease manifested in a hyper acute form before the engraftment (on d+6 and d+8, respectively). In all but one pt GI GVHD was defined as grade IV with full clinical picture of it. Skin GVHD accompanied GI symptoms in 17 pts and 13 was > grade II, in 4 grade IV. Liver symptoms presented in 13 pts, one pt was determinate as VOD. In 3 pts liver involvement was defined as grade IV. In all 24 of 25 pts had GVHD grade IV 4 and one grade III. Previous conventional anti GVHD therapies included: MP in all pts, MTX, various calcineurin inhibitors, MMF, ECP, serotherapy. Only 3 pts showed temporary limited partial response.Treatment with MSCs. The 1st infusion with MSC was given on day +32.5 (range, 8 to 74); d+50 (range, +28 to +180) post diagnosis of aGVHD and HSCT HSCT, respectively. In 22 evaluable pts we treated 24 separate episodes of GVHD: 22 first episodes and in 2 pts relapse of GVHD. In 24 of 39 cases treatment was performed with frozen MSC and in 15 with fresh cultured cells. In 37/39 cases passages 1 to 3 were used. Median number of infused cells was 1 (range, 0.3 to 3.1) x10 6 per kg of pt body weight in each treatment. Initial response was seen in 17/24 episodes of aGVHD (70.8%), in 8 partial (PR) and in 9 complete (CR). Two pts experienced GVHD relapse after achieving CR and very good PR: one case was treated successfully while another was resistant to MSCs. The latest pt with grade IV aggressive liver GVHD received MSCs injections intra hepatic arteries under radiological control following IRB approval with no anti GVHD effect. The procedure was uneventful with no evidence of microembolisation, no changes of blood flow or deterioration of liver tests (cytolysis). We observed a trend for better effect with higher MSC cell dose: total and per first infusion MSCs dose (1.93±1.28 vs. 1.23±0.50, p=0.078; 1.28±0.79 vs. 0.88±0.28, p=0.086). In all cases effect was seen after the first procedure. No difference was noted in the anti GVHD activity between fresh vs. frozen MSCs. There were no immediate or late toxicity or side effects.Overall survival. 14/25 patients are alive up to 20 months follow up. 8/11 pts, died from GVHD (4 within 1-10 d from MSCs infusion), and 3 from other unrelated causes including TTP-1, DAH-1 and disease progression-1. CONCLUSION.Treatment with MSCs seems to be a new novel type of therapy for steroids resistant GVHD with promising preliminary results and low toxicity. Obviously multicenter 2 arm randomized study is in need to confirm these encouraging. Disclosures:No relevant conflicts of interest to declare.

Should Methotrexate plus Calcineurin Inhibitors Be Considered Standard of Care for Prophylaxis of acute Graft-versus-Host Disease?

In a rapidly developing field, one can always anticipate that different interpretations of similar data will coexist. Stem cell transplanters can be a contentious lot, especially in the absence of controlled randomized trials. Thus, although improvements in the basic understanding of acute graft-versus-host disease (aGVHD) has led to many testable hypotheses in the management of GVHD, there remains little consensus regarding the most effective and least toxic approach to GVHD prevention. In the 1980s, the comparison would have been between cyclosporine-based regimens and ex vivo T cell depletion (TCD). Although ex vivo TCD is still used in some settings, pharmacologic-based therapy and in vivo TCD with serotherapy now predominate. This review is meant to highlight the advantages and disadvantages of the "standard of care" and assess the prospects for future regimens that may be more effective.

Cytolytic T cells induce ceramide-rich platforms in target cell membranes to initiate graft-versus-host disease

AbstractAlloreactive donor cytolytic T lymphocytes play a critical role in pathophysiology of acute graft-versus-host disease (GVHD). As GVHD progression involves tumor necrosis factor superfamily receptor activation, and as apoptotic signaling for some tumor necrosis factor superfamily receptors might involve acid sphingomyelinase (ASMase)–mediated ceramide generation, we hypothesized that ASMase deletion would ameliorate GVHD. Using clinically relevant mouse models of acute GVHD in which allogeneic bone marrow and T cells were transplanted into asmase+/+ and asmase−/− hosts, we identify host ASMase as critical for full-blown GVHD. Lack of host ASMase reduced the acute inflammatory phase of GVHD, attenuating cytokine storm, CD8+ T-cell proliferation/activation, and apoptosis of relevant graft-versus-host target cells (hepatocytes, intestinal, and skin cells). Organ injury was diminished in asmase−/− hosts, and morbidity and mortality improved at 90 days after transplantation. Resistance to cytolytic T lymphocyte–induced apoptosis was found at the target cell membrane if hepatocytes lack ASMase, as hepatocyte apoptosis required target cell ceramide generation for formation of ceramide-rich macrodomains, sites concentrating proapoptotic Fas. These studies indicate a requirement for target cell ASMase in evolution of GVHD in liver, small intestines, and skin and provide potential new targets for disease management.

A new induction protocol for the control of steroid refractory/dependent acute graft versus host disease with alefacept and tacrolimus

We have shown previously that alefacept is effective in acute steroid resistant/dependent and chronic extensive graft versus host disease (GvHD) with a protocol using timings similar to those used for psoriasis treatment. In this study, we describe the use of an alefacept induction (e.g. for 7 consecutive days) followed by a bi-weekly maintenance treatment in combination with tacrolimus for acute steroid resistant/dependent GvHD 1, 3. Sixteen patients were treated in this cohort, most with refractory GvHD. The pre-treatment GvHD grade ranged from 2 to 4 (median 3), involving the skin 16, gut 11 and liver 5. Twelve out of the 16 patients showed a response. As with the first protocol, the response of GvHD in the skin was fastest. In contrast to our previous protocol, however, the gastro-intestinal (GI) GvHD response was faster (P=0.05 compared with the first cohort). A hepatic response was seen in 4/6 patients and was complete in three. All responses were durable, including mucocutaneous, gut and liver GvHD. In all responding patients we were able to decrease the steroid dose significantly and in seven it was completely withdrawn. Alefacept induction is safe in acute steroid resistant/dependent GvHD and may be more effective therapeutically than our previous alefacept protocol. We speculate that alefacept initiates an allo-versus-allo cellular effect through its Fc receptor.

Reactivation of Cured Hepatitis B Virus after Allogeneic Hematopoietic Stem Cell Transplantation and Its Asociation with Liver Graft Versus Host Disease.

Introduction: The prevalence of hepatitis B virus (HBV) in transplanted patients is not negligible and reactivation of latent HBV infection in the setting of allogeneic HSCT is a well-known complication. However, reverse seroconversion (RS) from positive hepatitis B surface antibody (HBsAb), positive hepatitis B core antibody (HBcAb) and negative hepatitis B surface antigen (HBsAg) to negative HBsAb and positive HBsAg has been less reported. The aims of our study were to describe the clinical characteristics of patients with cured hepatitis B and to identify associated conditions with RS after allogeneic HSCT. Secondarily, we have analyzed the behavior of patient's serology for HBV after transplantation in relation with donors' immunity.Materials and methods: From April 1999 to April 2008, we evaluated the outcome regarding HBV serological status and liver function tests abnormalities of patients cured hepatitis B who received allogeneic HSCT. We assessed the presence of liver GVHD, sinusoidal obstruction syndrome, conditioning treatment, subjacent hematological disease and number of infused CD34+ cells as possible variables associated with RS. Data were analyzed with the statistical software SPSS15.0. Chi-Square test, Fisher's exact test and Mann-Whitney test were used for statistical analysis.Results: Of 107 transplanted patients, we found 24 patients (22.4%) with serology consistent with cured hepatitis B previous to transplant. Median age at transplant was 25 years (range, 16–49). The median follow-up time after transplant was 27 months (range. 2–76). The subjacent hematological disease was acute leukemia in 11 patients, lymphoma in 4, myeloproliferative syndromes in 3, myelodysplastic syndromes in 2, severe aplastic anemia in 2 and multiple myeloma in 2. All patients received myeloablative conditioning. Twenty patients were transplanted from siblings, 3 patients were transplanted from matched unrelated donors and 1 patient received umbilical cord blood progenitor cells. Nineteen patients received peripheral stem cells and 4 patients were infused with bone marrow. The mean number of infused CD34 was 5,18x106/Kg. Eight patients developed liver GVHD confirmed by biopsy. Four patients had sinusoidal obstruction syndrome. Six of the 24 patients (25%) developed RS. All the patients with RS had liver GVHD and acute hepatitis appeared after a mean time of 13.5 months (range, 3–34) after immunosuppressive treatment for GVHD was stopped. The most prevalent hematological disease in patients with RS was acute leukemia (5 of the 6 patients with RS, not significant, p=0.2). Four of the 6 patients with RS died of liver failure. The remaining 2 patients are presently on treatment with entecavir. HBV RS was significantly associated with liver GVHD (p=0.007). No other significant association was found. Three donors were naturally immunized, 12 vaccinated, and 4 not-immunized. Data were missing in 5 donors. Loss of HBcAb and HBsAb was seen in all the three patients transplanted from naturally immunized donors within the year of transplantation. Five of twelve patients (41.6%) transplanted from vaccinated donors maintained immunization during a median follow up time of 40 months.Conclusion: RS is a not minor complication after HSCT in patients with previously cured hepatitis B. To our knowledge, this is the first study that establishes an association between liver GVHD and RS. Immunosuppressive treatment for liver GVHD is possibly implicated in the pathophysiology

Redundant Mechanisms for Dendritic Cell Activation in GVHD Induction: Signalings Via TLRs, TNF-α, IL-1 and CD40 Are Not Required

Graft-versus-host disease (GVHD) is initiated by antigen-presenting cells (APCs) that prime alloreactive donor T cells. Amongst APCs, dendritic cells (DCs) are the most potent at priming naïve T cells. A paradigm of DC function holds that instructive maturation signals transform “immature” DCs into “mature” DCs which are optimized for T cell activation. Signaling via Toll-like receptors (TLRs) has emerged as a central mechanism for inducing this maturation in anti-pathogen responses. However, in allogeneic stem cell transplantation, there is no specific pathogen and the signals that induce DC maturation in GVH responses have not been defined. To study potential maturation stimuli we performed GVHD experiments in model systems wherein host APCs are essential for GVHD induction (C3H.SW (H-2b)→B6 and B6bm12→B6). We then compared GVHD in wild type (wt) recipients to that in hosts genetically unable to respond to various DC maturation signals. We first used as hosts mice deficient in TLR4, CD40, IL1-R or TNFR1/R2 and found that in C3H.SW→B6 model, GVHD in each of these was similar. Likewise, GVHD was similar in wt and mice deficient in MyD88, essential for signaling via all TLRs except TLR3 and TLR4. To focus our studies on APCs, we used as hosts radiation bone marrow (BM) chimeras in which wt B6 (CD45.1) mice were reconstituted with BM from CD45.2 wt, MyD88−/−, TRIF−/− (required for signaling via TLR3 and, in-part, TLR4), and MyD88/TRIF−/− mice, in which signaling by all TLRs, IL-1 and IL-18 is completely blocked. When retransplanted with C3H.SW BM and CD8 cells, all chimeras developed similar clinical and histologic GVHD. To exclude that T cells were being primed by residual wt host APCs in BM chimeric recipients, we used as hosts B6bm12 mice that were reconstituted with BM from B6 wt, MyD88−/−, TRIF−/− or MyD88/TRIF−/− mice. These chimeras were then retransplanted with B6bm12 BM and CD4 cells. Because residual host APCs in these BM chimeric hosts were B6bm12 and therefore syngeneic with the donor cells, T cell priming could only occur on B6 background APCs derived from the first transplant. As in the prior experiments, all chimeras developed similar clinical GVHD; pathology scoring is underway. Therefore in contrast to nearly all adaptive T cell responses, GVHD induction in both MHC-matched and MHC-mismatched systems did not require APC maturation signals mediated by TLRs. Absent any phenotype when APC maturation signals were blocked, we used the same approach in the C3H.SW→B6 model to study the roles of IL12 and type I IFNs, which are produced as a consequence of DC maturation. B6 p35−/− and B6 wt mice developed similar GVHD, excluding an essential role for host IL12. Type I IFN receptor-deficient (IFNIR−/−) mice developed similar GVHD as did wt controls, except that IFNIR−/− CD8 recipients lost more weight but had less histologic liver GVHD (P=0.0024). To focus on the activity of Type I IFNs on host hematopoietic cells, we used as recipients IFNIR−/− → B6 chimeras. Both clinical and histologic GVHD were similar in IFNIR−/−→B6 and wt→B6 control chimeras, suggesting that the differences in weight loss and hepatic GVHD observed in IFNIR−/− hosts, were due to parenchymal expression of IFNIR−/−. In sum, these data indicate that DC maturation signals in GVHD are at a minimum redundant, and independent of TLR ligands such as LPS, which are certainly available to the host immune system. This may be a consequence of the ubiquitous expression of alloantigen, such that any maturation signal will create APCs primed to activate donor T cells. If so, strategies for preventing GVHD by blocking APC maturation may need to target final common APC maturation pathways. Alternatively, instructive APC maturation may not be required for alloreactive T cell activation in GVHD.

Donor Single Nucleotide Polymorphism in the CCR9 Gene Affects the Incidence of Acute Skin Graft-Versus-Host Disease

Chemokines are a group of small molecules that regulate trafficking of leukocytes through interactions with a subset of seven-transmembrane, G protein-coupled chemokine receptors. Their interactions may play an important role in initiating acute graft-versushost disease (GVHD) after allogeneic hematopoietic stem cell transplanation. CCR9 is a very unique receptor because, outside of the thymus, it is almost exclusively expressed by epithelial cells and Pyer's patches in the small intestine. We focused on rs12721497 (G926A), one of the single nucleotide polymorphisms (SNPs) in the CCR9 gene, and analyzed the impact of its genotype on the onset of organ-specific acute GVHD and transplant outcome. Included in the study were consecutive patients who had received allogeneic bone marrow or peripheral blood stem cell transplantation from an HLA-identical sibling donor at the Nagoya University Hospital and the Japanese Red Cross Nagoya First Hospital between 1987 and 2006. Of these, a total of 167 patients who received T cell replete transplantation with short-term methotrexate and cyclosporine as a GVHD prophylaxis were analyzed. Informed consent was obtained from all patients and donors, and the study was approved by the ethics committees at Nagoya University Hospital and Japanese Red Cross Nagoya First Hospital. The CCR9 G926A genotype was determined by the polymerase chain reaction-restriction fragment length polymorphism method using genomic DNA obtained from donor cells. The impact of CCR9 genotype on the incidence of acute GVHD in each organ, overall survival rates, relapse rates and nonrelapse mortality rates were analyzed using the Cox proportional hazard regression model. Age, intensity of conditioning, total body irradiation, disease risk, remission state and graft source were used as covariates for adjustment. Median age was 38 years old (range 15–62). Diseases were malignancies (n=148) and non-malignancies (n=19). The incidences of acute GVHD were 22% (grade I), 12% (grade II) and 6.6% (grade III-IV). The frequencies of the AA, AG and GG genotype were 94%, 6% and 0%, respectively. CCR9 926AG genotype was significantly associated with the incidence of stage³2 skin GVHD (hazard ratio 3.2; 95% CI, 1.1–9.1), but not associated with the incidence of liver GVHD, intestinal GVHD, grade II-IV GVHD, overall survival rates, relapse rates and nonrelapse mortality rates. In conclusion, the frequencies of each genotype were compatible with the reports in HapMap database. It was notable that donor CCR9 SNPs affected the incidence of skin GVHD, but did not affect that of intestinal GVHD. Beilhack et al. ( Blood . 2008; 111:2919–2928) recently reported redundancy of secondary lymphoid organs at different anatomical sites in GVHD initiation. They suggested that primed T cells could initiate GVHD at different sites from their original priming sites. Since Pyer's patches are important as sites of antigen presenting, T cell homing to Pyer's patches may be different between each CCR9 926AG genotype, resulting in different incidences of skin GVHD. We are now investigating the functional differences between each genotype.

Adoptive Immunotherapy Against Allogeneic Kidney Grafts in Dogs with Stable Hematopoietic Trichimerism

Dogs given nonmyeloablative conditioning and marrow grafts from 2 dog leukocyte antigen (DLA)-identical littermate donors developed stable trichimerism and stably accepted a subsequent kidney graft from one of the marrow donors without the need for immunosuppression. In this study, we used trichimeras to evaluate strategies for adoptive immunotherapy to solid tumors, using the kidney as a tumor surrogate. Three DLA-identical trichimeric recipients were established by simultaneously infusing marrow from 2 DLA-identical donor dogs into a DLA-identical recipient conditioned with 2 Gy of total body irradiation (TBI) and given a short course of postgraft immunosuppression. After stable hematopoietic engraftment was confirmed, a kidney was transplanted from 1 of the 2 marrow donors into each respective trichimeric recipient. Peripheral blood lymphocytes from each kidney donor were then used to sensitize the alternate marrow donor. The trichimeric recipients were given donor lymphocyte infusions (DLIs) from the sensitized dogs and monitored for chimerism, graft-versus-host disease (GVHD), and kidney rejection. After DLI, we observed both prompt rejection of the transplanted marrow and donor kidney and disappearance of corresponding hematopoietic chimerism. Presumably due to shared minor histocompatibility antigens, host chimerism also disappeared, and GVHD in skin, gut, and liver developed. The native kidneys, although exhibiting lymphocytic infiltration, remained functionally normal. This study demonstrates that under certain experimental conditions, the kidney—an organ ordinarily not involved in graft-versus-host reactions—can be targeted by sensitized donor lymphocytes.

Catastrophic graft-versus-host disease after lung transplantation proven by PCR-based chimerism analysis

Acute graft-versus-host disease (GvHD) is a rare complication after solid organ transplantation. We describe a 52-year-old female developing neutropenia and fever 48 days after single lung transplantation for chronic obstructive pulmonary disease. Bone marrow (BM) biopsy suggested drug-induced marrow failure, so immunosuppression was reduced. Five days later a maculopapular skin rash was observed, progressing to a generalized erythema with desquamation. Skin biopsy was suspectable for GvHD, so immunosuppression was re-initiated. PCR-based chimerism analysis of BM revealed 78% donor cells. Intensified immunosuppression resulted in temporary improvement, but BM aplasia recurred and the patient experienced severe GvHD of gut and liver. Despite extensive immunosuppression the patient died from multi-organ failure 99 days after transplantation. This report describes the occurrence of neutropenia as an early presenting sign of acute GvHD after lung transplantation. We therefore recommend incorporating GvHD in the differential diagnosis of neutropenia after solid organ transplantation, calling for early chimerism analyses.

Successful Living-Donor Liver Transplantation for Chronic Hepatic Graft-Versus-Host Disease After Bone Marrow Transplantation for Chronic Granulomatous Disease

A 10-year-old boy diagnosed postnatally with chronic granulomatous disease (CGD) underwent human leukocyte antigen-identical bone marrow transplantation (BMT) from an unrelated donor. After engraftment, he developed an erythematous rash, diarrhea, hepatomegaly, and elevated liver enzyme levels and was treated for acute graft-versus-host disease (GVHD) by increased immunosuppressive therapy with tacrolimus and corticosteroids. Although the gut and skin GVHD improved, the hepatic GVHD persisted and was confirmed as chronic hepatic GVHD on liver biopsy. Owing to the persistence of hyperbilirubinemia, elevated liver enzyme levels, thrombocytopenia, and coagulopathy, the chronic GVHD was managed with low-dose corticosteroids. After transplantation, the patient showed complete engraftment of the transplanted bone marrow (99%), and bone marrow biopsy and other investigations showed no evidence of the relapse of CGD. At 3 years after BMT, liver function deteriorated, and the boy underwent living-donor liver transplantation (LDLT) from his mother, a heterozygous donor. The left-lobe graft weighed 510 g, and the graft-to-recipient weight ratio was 1.6%. Both the portal vein and hepatic artery of the graft were flushed with muromonab-CD3 (Orthoclone OKT3) contained in a histidine-tryptophan-ketoglutarate solution to wash out and eliminate donor lymphocytes. Histopathological examination of the explanted liver revealed the presence of ductopenia with foam-cell arteriopathy, which was consistent with chronic hepatic GVHD. Postoperative immunosuppression consisted of low-dose tacrolimus and corticosteroids to prevent GVHD. Acute cellular rejection occurred during the postoperative course, but severity decreased after treatment. The patient was discharged on postoperative day 60 without complications, and has done well on 12 months follow-up with normal liver function. Graft-versus-host disease, a major cause of liver dysfunction after BMT, occurs in 60% to 80% of patients receiving marrow from a partially human leukocyte antigen-matched family donor. Given the host immunosuppression related to BMT, the risk of passenger lymphocyte-associated GVHD in the present case was an additional complication which required additional measures to avoid allogeneic reactions arising from passenger lymphocytes in the graft. To reduce the number of donor-derived passenger lymphocytes, muromonab-CD3 was used for graft perfusion just after harvesting. OX40 (CD134), a member of the tumor necrosis factor receptor superfamily, is expressed on activated T cells, including CD4(+)CD25(+) T regulatory (Treg) cells. We monitored CD4 OX40/CD4 CD25++ ratio as a marker of GVHD before and after LDLT because CD4 OX40/CD4 CD25++ ratio was recently reported to be a reliable indicator of the onset of chronic GVHD donor (1). In the present case, CD4 OX40/CD4 CD25++ ratio remained stable before and after LDLT (Fig. 1), and no evidence of GVHD was obtained.FIGURE 1.: Change in CD4 OX40/CD4 CD25++ ratio after transplantation.Although many cases of liver transplantation for chronic liver GVHD after BMT have been reported, few have involved LDLT, especially the use of liver tissue from the same donor (2–11). Bone marrow transplantation is commonly indicated in leukemia patients. In the case of benign diseases such as CGD, when BMT is successful, the possibility of recurrence of original disease is low. On this basis, liver transplantation is a suitable approach to the treatment of chronic liver GVHD after BMT. Our case was a LDLT to CGD patient after BMT. The liver donor was different to the BMT donor, and heterozygous to the CGD. At least with regard to CGD, no problems derived from the use of a heterozygous donor have occurred. Satoshi Yokoyama Mureo Kasahara Akinari Fukuda Shuichi Sato Department of Transplant Surgery National Center for Child Health and Development Tokyo, Japan Tetsuya Mori Department of Pediatric Oncology National Center for Child Health and Development Tokyo, Japan Atsuko Nakagawa Department of Pathology National Center for Child Health and Development Tokyo, Japan Akira Matsui Department of Transplant Surgery National Center for Child Health and Development Tokyo, Japan