Acute Graft-versus-host Disease In Recipients Research Articles

Model-Based Antithymocyte Globulin in αβhaplo-Hematopoietic Stem Cell Transplantation Facilitates Engraftment, Expedites T Cell Recovery, and Mitigates the Risk of Acute Graft-versus-Host Disease

In αβ T-cell/CD19 B-cell depleted hematopoietic stem cell transplantation (αβhaplo-HSCT) recipients, antithymocyte globulin (ATG; Thymoglobulin) is used for preventing graft rejection and graft-versus-host disease (GVHD). The optimal dosing remains to be established, however. Here we present the first comparative analysis of 3 different ATG dosing strategies and their impact on immune reconstitution and GVHD. Our study aimed to evaluate the effects of 3 distinct dosing strategies of ATG on engraftment success, αβ+ and γδ+ T cell immune reconstitution, and the incidence and severity of acute GVHD in recipients of αβhaplo-HSCT. This comparative analysis included 3 cohorts of pediatric patients with malignant (n = 36) or nonmalignant (n = 8) disease. Cohorts 1 and 2 were given fixed ATG doses, whereas cohort 3 received doses via a new nomogram, based on absolute lymphocyte count (ALC) and body weight (BW). Cohort 3 showed a 0% incidence of day 100 grade II-IV acute GVHD, compared to 48% in cohort 1 and 27% in cohort 2. Furthermore, cohort 3 (the ALC/BW-based cohort) had a significant increase in CD4+ and CD8+ naïve T cells by day 90 (P = .04 and .03, respectively). Additionally, we found that the reconstitution and maturation of γδ+ T cells post-HSCT was not impacted across all 3 cohorts. Cumulative ATG exposure in all cohorts was lower than previously reported in T cell-replete settings, with a lower pre-HSCT exposure (<40 AU*day/mL) correlating with engraftment failure (P = .007). Conversely, a post-HSCT ATG exposure of 10 to 15 AU*day/mL was optimal for improving day 100 CD4+ (P = .058) and CD8+ (P = .03) immune reconstitution without increasing the risk of relapse or nonrelapse mortality. This study represents the first comparative analysis of ATG exposure in αβhaplo-HSCT recipients. Our findings indicate that (1) a 1- to 2-fold ATG to ATLG bioequivalence is more effective than previously established standards, and (2) ATG exposure post-HSCT does not adversely affect γδ+ T cell immune reconstitution. Furthermore, a model-based ATG dosing strategy effectively reduces graft rejection and day 100 acute GVHD while also promoting early CD4+/CD8+ immune reconstitution. These insights suggest that further optimization, including more distal administration of higher ATG doses within an ALC/BW-based strategy, will yield even greater improvements in outcomes.

Myeloid-Derived Suppressor Cells (HLA-DR-/lowCD16-CD33+) Expanded By Granulocyte Colony-Stimulating Factor Can Prevent Acute Graft-Versus-Host Disease in Humanized Mouse and Patients Following HSCT

IntroductionMyeloid-derived suppressor cells (MDSCs) are proposed to control graft-versus-host disease (GVHD) in allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the definition of human MDSCs has not yet reached consensus. Granulocyte colony-stimulating factor (G-CSF) has been routinely used to mobilize stem cells to peripheral blood in healthy donors. It was also recognized as a novel mediator of T-cell tolerance. However, the effects of G-CSF administration on donor-derived MDSCs and the further regulatory effects of these MDSCs on GVHD remained unclear.AmisThe aim of this study is to evaluate the in vitro and in vivo effects of G-CSF expanded, donor-derived MDSCs (HLA-DR-/lowCD16-CD33+) in preventing acute GVHD after allo-HSCT.MethodsThe frequency and cell numbers of different kinds of MDSCs in peripheral blood before and after G-CSF administration from 10 healthy donors were analyzed by flow cytometry. Cells morphological features were detected by May-Grünwald-Giemsa cytospin. Secondly, the suppressive and regulatory functions of HLA-DR-/lowCD16-CD33+ population on CD3+ T cells were assessed via in vitro experiments. A humanized xenogeneic acute GVHD model was established to determine whether this population could prevent acute GVHD in vivo. Furthermore, a clinical prospective cohort study enrolled one hundred consecutive transplant recipients was performed to assess the effects of HLA-DR-/lowCD16-CD33+ contained in HSC grafts on the occurrence of acute GVHD.ResultsThe findings of this study include: First, a novel phenotype of HLA-DR-/lowCD16-CD33+ MDSCs with suppressive function and morphological features similar to those of immature monocyte was identified. The median of percentages of this subset were significantly increased both in peripheral blood (PB, 6.5% vs. 4.6%, P=0.0122) and peripheral blood stem cells harvest (PBSCs, 15.5% vs. 4.6%, P<0.0001) after treating healthy donors with G-CSF than those of PB before mobilization. The median of percentage of interleukin-10 (IL-10) and transforming growth factor beta (TGF-β) positive cells among HLA-DR-/lowCD16-CD33+ population were both significantly higher than those of PB before mobilization. (IL-10+, 10.1% vs.0.62%, P=0.002; TGF-β+, 60.24% vs. 10.39%, P=0.0003). Donor-derived HLA-DR-/lowCD16-CD33+population inhibited the proliferation of T cells in a TGF-β-dependent manner. In vitro cell co-culture experiments also showed that this MDSCs subset could promote regulatory T cells (Treg, CD4+CD25+Foxp3+) expansion and induce T helper 2(Th2, CD4+IL-4+) differentiation (The median of percentage of Treg in co-culture with MDSCs group, 21.4% vs. without MSDCs group, 8.35%, P=0.0048. The median of fold change of Th2/Th1 in co-culture with MDSCs group, 1.35 vs. without MSDCs group 0.98, P=0.0159. The median of fold change of Th2/(Th1+Th17) in co-culture with MDSCs group, 1.28 vs. without MSDCs group 1.00, P=0.0095. Th1 (CD4+IFNγ+), Th17 (CD4+IL-17A+)). Second, we demonstrated that these cells could prevent acute GVHD in a humanized mouse model. Adoptive transfer human G-CSF-mobilized HLA-DR-/lowCD16-CD33+ cells significantly prolonged the survival and ameliorated the weight loss and tissue damage in GVHD mice. Third, clinical cohort results showed that the number of HLA-DR-/lowCD33+CD16- cells in the donor graft was the only independent risk factor inversely correlated with the incidence II-IV acute GVHD in recipients (HR 0.388, 95% CI: 0.158-0.954, P=0.039).ConclusionOur results suggest that MSDCs with HLA-DR-/lowCD16-CD33+ phenotype in G-CSF-mobilized PBSCs have monocytic features and immune-regulatory properties, which could alleviate acute GVHD in the allo-HSCT settings.Key words: Myeloid-derived suppressor cells; granulocyte colony-stimulating factor; graft-versus-host disease DisclosuresNo relevant conflicts of interest to declare.

No Survival Advantage After Double Umbilical Cord Blood (UCB) Compared to Single UCB Transplant in Children with Hematological Malignancy: Results of the Blood and Marrow Transplant Clinical Trials Network (BMT CTN 0501) Randomized Trial

AbstractAbstract ▪359▪This icon denotes a clinically relevant abstract Background:On the basis of pilot data in adults with acute leukemia suggesting that the co-infusion of two UCB units was safe and associated with survival rates comparable to that in children, a randomized trial was proposed to determine whether the transplantation of two units might confer a survival advantage since cell dose is a critical determinant of hematopoietic recovery, non relapse mortality (NRM) and survival. Because adults are not as likely to have an adequate single unit, the study was performed in children. Patients and Methods:The BMT CTN, sponsored by NHLBI and NCI, conducted a multi-center, randomized, phase III trial with a primary objective of comparing one-year overall survival in the two study arms using an intent-to-treat analysis. Secondary objectives included comparisons of engraftment, acute and chronic graft-versus-host disease (GVHD), NRM, relapse and disease-free survival (DFS). Thirty-eight BMT CTN, PBMTC or COG centers enrolled patients between December 2006 and February 2012. All patients had to have available two UCB units; unit 1 was the best available HLA-matched unit to the patient with ≥2.5 × 107 total nucleated cell/kg patient weight; unit 2 was the next best HLA-matched unit with ≥1.5 × 107 total nucleated cell/kg. Each unit had to be at least 4/6 HLA-matched to the patient and 3/6 matched between units (at intermediate resolution for HLA-A, -B and allele-level for HLA-DRB1). Patients were randomized to receive a conventional single (n=113) or double (n=111) UCB transplant with randomization stratified by transplant center and patient age. Compliance was >95% in both treatment groups. Of those randomized to a single UCB transplant, 2.7% crossed over to a double and of those randomized to a double UCB transplant, 1.0% crossed over to single; 1.8% in both groups were not transplanted. Patient primary disease (AML, ALL, MDS, CML), disease risk, gender, age, race, ethnicity, CMV serology, performance score and HLA-match were balanced between the two study groups; ALL was the predominant diagnosis (half) in both study groups. All patients received a uniform conditioning regimen (fludarabine 75mg/m2, TBI 1320 cGy, cyclophosphamide 120 mg/kg) and GVHD prophylaxis (cyclosporine and mycophenolate mofetil). Results:The median follow-up of surviving patients is 25 months (both study arms). Overall, 92% patients were in remission with 60% of these in second or subsequent remission. Most units were mismatched at one or two HLA-loci (45% and 40% of single UCB and 42% and 45% of double UCB transplants, respectively). The median pre-cryopreserved total nucleated cell dose was 4.8 and 8.9 × 107/kg for recipients of single and double UCB transplants, respectively. Except for a higher risk of grade III-IV acute GVHD in recipients of a double UCB transplant, all outcomes were similar between the two groups (Table 1). The primary causes of death on both arms were similar with most due to relapse and GVHD. Conclusion:In children with hematological malignancies, outcomes were similar with no survival advantage in recipients of a double UCB transplant as compared to those transplanted with an adequately dosed single UCB unit. While recipients of two units had a higher incidence of acute GVHD, relapse risk was unchanged. Therefore, on the basis of these results, single UCB transplant should be the standard approach in children for whom a single unit containing ≥2.5 × 107nucleated cells/kg matched at zero, one or two HLA-loci is available. Double UCB transplant however remains a suitable alternative in the absence of an adequately dosed single UCB unit in children with hematologic malignancies.Table 1Single UCB TransplantDouble UCB TransplantP-valueIntent-to-treatOverall survival at 1-year71% (62–79)%65% (55–73)%0.13TransplantedOverall survival at 1-year71% (61–79)%66% (56–74)%0.12DFS at 1-year68% (58–76)%64% (54–73)%0.20Relapse at 1-year12% (6–18)%14% (7–20)%0.37NRM at 1-year20% (12–27)%22% (14–30)%0.45Neutrophil recovery at day-4289% (83–95)%87% (80–93)%0.08Platelet recovery at day-18080% (72–88)%72% (63-82)%0.06Acute grade II-IV GVHD at day-10057% (48–67)%57% (48–67)%0.94Acute grade III-IV GVHD at day-10014% (7–20)%23% (15–32)%0.03Any chronic GVHD at 1-year32% (23–41)%30% (20–39)%0.64 Disclosures:No relevant conflicts of interest to declare.

Granulocyte Colony-Stimulating Factor Induced Acute and Chronic Graft-Versus-Host Disease

A recent experimental study in mice shed new light on the controversy as to whether granulocyte colony-stimulating factor (G-CSF) increases graft-versus-host disease (GVHD). Total body irradiation and bone marrow were found to be prerequisites for acute GVHD. This study encouraged us to perform a retrospective clinical study. We compared 260 patients given G-CSF prophylaxis after allogeneic hematopoietic stem-cell transplantation with 205 controls transplanted between 1993 and 2003. G-CSF hastened the engraftment of neutrophils, but that of platelets was delayed (P<0.0001). The proportion of acute GVHD of grades II to IV was 29% in the G-CSF group and 19% in the controls (P<0.01) and that of chronic GVHD was 54% and 43%, respectively (P=0.019). G-CSF increased acute and chronic GVHD in patients preferentially conditioned with chemotherapy. Unexpectedly, it exacerbated acute GVHD in recipients of peripheral blood stem cells and enhanced chronic GVHD in bone marrow recipients. A multivariable analysis showed that acute GVHD (hazards ratio=1.52, P=0.03) and chronic GVHD (hazards ratio=1.51, P=0.004) were associated with G-CSF. There was no significant difference between study groups regarding nonrelapse mortality, relapse, or survival. G-CSF increased acute and chronic GVHD in patients treated with chemotherapy but did not affect relapse or survival.

Synergistic Effect of Anti-CD3 and Vorinostat in the Prevention of Acute Gvhd and Retention of GVL in a Radiation-Free Conditioning Regimen.

Abstract 3562Poster Board III-499A radiation-free conditioning regimen with anti-T cell globulin (ATG) + Busulfan (B) + Fludarabin (F) (ATG + BF) could replace sublethal total body irradiation (TBI) in clinic, but this regimen did not prevent induction of severe acute graft versus host disease (GVHD). We recently reported that anti-CD3 preconditioning markedly ameliorated acute GVHD in recipients conditioned with sublethal TBI in a mouse model of MHC-mismatched C57BL/6 (H-2b) donor to BALB/c (H-2d) host. In the current studies, we test whether replacing ATG with anti-CD3 prevented acute GVHD. We found that, when spleen and BM cells (50×106, each) from donors were transplanted, the recipients conditioned with ATG + BF developed severe acute GVHD and all (12/12) of them died by 30 days after transplantation, in contrast, the recipients conditioned with anti-CD3 + BF developed only moderate acute GVHD and 50% (6/12) of the recipients survived for more than 100 days. When the donor cells were titrated down to 25, 12.5, or 6.25 ×106, we found that none (0/8) of the recipients conditioned with ATG + BF developed chimerism, in contrast, all (8/8) of the recipients conditioned with anti-CD3 + BF developed complete chimerism, although the recipients showed mild to moderate clinical GVHD in a dose-dependent manner. Next, we tested whether depletion of donor CD4+ T cells could completely prevented GVHD in recipients conditioned with anti-CD3 + BF, and we found that, although all (8/8) of the recipients given 12.5 or 6.25×106 CD4+ T-depleted spleen (CD4−-SPL) cells developed complete chimerism and all survived for more than 100 days, the recipients still showed mild to moderate clinical GVHD (i.e. hair loss). Because vorinostat (V), a histone deacetylase inhibitor was previously reported to inhibit tissue release of proinflammatory cytokines, we tested whether replacing F with V could further ameliorated GVHD. We found when donor CD4−-SPL cells and BM cells (12.5 ×106 each) were transplanted, the recipients conditioned with anti-CD3 + BV showed no clinical GVHD, although the recipients conditioned with anti-CD3 + BF showed mild to moderate clinical GVHD. Furthermore, we found that donor CD4−-SPL and BM cells eliminated luciferase transfected BCL1 leukemia/lymphoma cells much more efficiently in the recipients conditioned with anti-CD3 + BV than in the recipients conditioned with anti-CD3 + BF, as revealed by in vivo bioluminescent imaging. In addition, we observed that the separation of GVL from GVHD was associated with prevention of donor T cell migration into GVHD target tissues, which resulted from reduction of CCR7+ DCs that induce donor T cell expression of tissue homing and chemokine receptors (i.e. a4b7, CCR9, E-Ligand, P-Ligand, CCR4, CCR10) in the recipients conditioned with anti-CD3 + BV. Taken together, anti-CD3 and vorinostat synergistically promote prevention of GVHD and retention of GVL in a clinically applicable radiation-free conditioning regimen. This work was supported by Marcus foundation. Disclosures:No relevant conflicts of interest to declare.

Acute graft-versus-host disease after unrelated donor umbilical cord blood transplantation: analysis of risk factors

Acute graft-versus-host disease (GVHD) occurs less frequently after umbilical cord blood transplantation (UCBT). More recent investigations include the use of 2 partially human leukocyte antigen (HLA)-matched UCB units, or double UCB graft, to meet the minimum cell-dose requirement. The purpose of this analysis was to assess the relative risk of acute GVHD in 265 consecutive patients receiving transplants with UCB graft composed of 1 (n = 80) or 2 (n = 185) units. The incidence of grade III-IV acute GVHD was similar between cohorts. However, the incidence of grade II-IV acute GVHD was higher among double UCBT recipients (58 vs 39%, P < .01). Three risk factors for grade II-IV acute GVHD were identified in multiple regression analysis: use of 2 UCB units, use of nonmyeloablative conditioning, and absence of antithymocyte globulin in the conditioning regimen. Transplantation-related mortality (TRM) at 1 year, however, was significantly lower after double UCBT (24 vs 39%, P = .02) even if recipients had grade II-IV acute GVHD (20 vs 39%, P = .05). These data suggest that, despite a higher incidence of grade II acute GVHD in recipients of 2 partially HLA-matched UCB units, there is no adverse effect on TRM. This study is registered at (http://www.clinicaltrials.gov) under the identifiers NCT00305682 and NCT00309842.

Decreased risk of acute graft-versus-host disease following allogeneic hematopoietic stem cell transplantation in patients with the 5,10-methylenetetrahydrofolate reductase 677TT genotype

Polymorphism in 5,10-methylenetetrahydrofolate reductase (MTHFR), a central enzyme in folate metabolism, has been shown to affect the sensitivity of patients to folate-based drugs such as methotrexate. In this study, we investigated whether a common single nucleotide polymorphism at position 677 in the donor or recipient's MTHFR gene affects the risk for acute graft-versus-host disease (GVHD) following allogeneic hematopoietic stem cell transplantation (HSCT) from HLA-identical sibling donors when the recipient receives prophylactic treatment with methotrexate for GVHD. MTHFR genotypes were determined in 159 recipients with a hematological disease and their donors using polymerase chain reaction-restriction fragment length polymorphism analysis of genomic DNA. The 677TT genotype, which encodes an enzyme with approximately 30% of the activity of the wild-type (677CC), was observed in 13% of patients and in 8% of normal donors. Multivariate analyses demonstrated a significant association between 677TT genotype in patients and a lower incidence of grade I-IV acute GVHD (relative risk, 0.35; 95% confidence interval, 0.13-0.95; P = 0.040). There was no association between the incidence of acute GVHD and the donor MTHFR genotypes. These results suggest that greater immunosuppression by methotrexate due to low MTHFR enzyme activity decreases the risk of acute GVHD in recipients of allogeneic HSCT.

Relation of an interleukin-23 receptor gene polymorphism to graft-versus-host disease after hematopoietic-cell transplantation

Polymorphisms in cytokine genes can influence immune responses and inflammation and thereby affecting the outcome of hematopoietic stem-cell transplantation. We analyzed a single-nucleotide polymorphism in the gene for the interleukin-23 receptor (IL-23R) (1142G>A) in a cohort of 221 transplant recipients and their human leukocyte antigen (HLA)-identical sibling donors and in a second cohort of 186 transplant recipients and their HLA-identical unrelated donors. Genotypes were tested for an association with graft-versus-host disease (GVHD) by multivariate analysis. The donor's IL-23R genotype was significantly associated with a reduced risk of acute GVHD in both cohorts for patients after transplant. Analysis of all 407 transplant recipients showed that IL-23R (1142G>A, Arg381Gln) genotype of the donor was associated with a decreased risk of grades 2-4 acute GVHD (31.6 compared to 51.0%, P=0.02) and grades 3-4 severe acute GVHD (3.9 compared to 23.4%, P=0.003). Death in remission was significantly lower in patients transplanted from donors with variant IL23-R (11.7 versus 27.7%, P=0.028), whereas overall survival or relapse rates were not influenced significantly by the IL-23R genotype. Among recipients of hematopoietic cells from HLA-identical donors, the IL-23R (Arg381Gln) gene variant on the donor side has a protective effect on the occurrence of acute GVHD in recipients after transplantation.

Single Versus Double Umbilical Cord Blood Transplantation (UCBT): Higher Risk of Acute Graft-Versus-Host Disease (GVHD) but Lower Transplant Related Mortality (TRM) in Recipients of Double UCBT.

Transplantation of 2 partially HLA matched UCB units has been shown to be a safe and effective means to overcome the cell-dose barrier in adolescents and adults. In addition, preliminary data suggest that double UCBT is associated with a greater graft-versus-leukemia effect (Blood 2005: 106:93a). To date, there has been no comparative analysis on the risk of acute GVHD in this population of patients. Therefore, we compared rates of acute GVHD in recipients of single (n=210) versus double (n=169) UCBT. Double UCB recipients were more likely to be older, transplanted in recent years, treated with a non myeloablative therapy, received mycophenolate mofetil as GVHD prophylaxis, and less likely to receive antithymocyte globulin (ATG) as part of the conditioning. Incidences of grades II–IV and III–IV acute GVHD were higher in recipients of double than single UCBT recipients (60% vs 33%, p.01) and (21% vs 11%, p=.01), respectively. Adjusting for differences between groups, two factors were associated with the development of grade II–IV acute GVHD in Cox regression: use of 2 UCB units (RR 2.0 vs 1.0 [95% CI, 1.3–3.2, p.01]) and absence of ATG in the preparative regimen (RR 1.0 vs 0.5 [95% CI, 0.3–0.8, p.01]). Other factors that were tested in the model but not determined to be significantly associated with acute GVHD were recipient age, gender, weight, diagnosis, time from diagnosis to UCBT, HLA disparity, total and CD3 cell dose, CMV serostatus, conditioning regimen, and GVHD prophylaxis. Despite increased risk of acute GVHD, TRM 1 year was significantly lower in recipients of double UCBT (17%, 95% CI, 5–29%) as compared to recipients of a single UCBT (47%, 95% CI, 26–68%; p =.02) (Figure 1) among those that developed grade III–IV acute GVHD. Survival at 1 year among those with grade III–IV acute GVHD was significantly higher after double UCBT (67%, 95% CI, 51–83%) than after single UCBT (41%, 95% CI 21–61%; p=.04) (Figure 2). [Display omitted] [Display omitted] In conclusion, risk of acute GVHD is significantly higher in recipients of two partially HLA matched UCB units, yet it does not appear to adversely effect TRM or survival. ATG in the preparative therapy, however, does not appear to reduce TRM despite its favorable association with reduced GVHD. Impact of GVHD on immune recovery and infection risk in recipients of single and double UCBT is under investigation.

Methylenetetrahydrofolate Reductase and Thymidylate Synthase Genotypes and Risk of Acute Graft-versus-Host Disease Following Hematopoietic Cell Transplantation for Chronic Myelogenous Leukemia

Methylenetetrahydrofolate reductase (MTHFR) and thymidylate synthase (TS) play key roles in intracellular folate metabolism. Polymorphisms in these enzymes have been shown to modify toxicity of methotrexate (MTX) after hematopoietic cell transplantation. In this study, we evaluated the risk of acute graft-versus-host disease (GVHD) associated with genetic variation in recipient and donor MTHFR and TS genotypes to assess whether genotype alters the efficacy of MTX in acute GVHD prophylaxis. Data on the transplantation course were abstracted from medical records for 304 adults who received allogeneic hematopoietic cell transplants. MTHFR ( C677T and A1298C) and TS (enhancer-region 28-base pair repeat, TSER, and 1494del6) genotypes were determined using polymerase chain reaction/restriction fragment length polymorphism and TaqMan assays. Multivariable logistic regression was used to assess the associations between genotypes and risk of acute GVHD. Compared with recipients with the wild-type MTHFR 677CC genotype, those with the variant 677T allele showed a decreased risk of detectable acute GVHD ( 677CT: odds ratio, 0.8; 95% confidence interval, 0.4-1.6; 677TT: odds ratio, 0.4; 95% confidence interval, 0.2-0.8; P for trend = .01). The variant MTHFR 1298C allele in recipients was associated with an increased risk of acute GVHD compared with the wild-type MTHFR 1298AA genotype ( 1298AC: odds ratio, 2.0; 95% confidence interval, 1.1-3.9; 1298CC: odds ratio, 3.6; 95% confidence interval, 1.0-12.7; P for trend < .01). No association with risk of acute GVHD was observed for donor MTHFR genotypes or for recipient or donor TS genotypes, with the exception of an increase in acute GVHD among recipients whose donors had the TSER 3R/2R genotype (odds ratio, 3.0; 95% confidence interval, 1.3-7.2). These findings indicate that host, but not donor, MTHFR genotypes modify the risk of acute GVHD in recipients receiving MTX, in a manner consistent with our previously reported associations between MTHFR genotypes and MTX toxicity. A direct trade-off between drug toxicity and drug efficacy may play a role. Alternatively, the systemic folate environment, regulated by host tissues, might influence donor T-cell growth and activity.

Nuclear Translocation of RelB within Host and Donor Antigen Presenting Cells Is Critical for the Initiation and Maintenance of Acute Graft-Versus-Host Disease.

Either donor or host antigen presenting cells (APC) are sufficient for the initiation of CD4 dependent graft versus host disease (GVHD). However the molecular transcription pathways within APC required for this effect are unknown. The NF-kB/Rel family member RelB is associated with dendritic cell (DC) maturation and is critical for the induction of potent APC function. DC from RelB−/− mice had markedly reduced levels of CD40 and to a lesser extent CD80/CD86 following in vitro activation. Following total body irradiation, the number of residual splenic DC with nuclear RelB was increased 5-fold relative to untreated mice. We therefore examined the role of RelB within donor and host APC in GVHD utilizing two well established bone marrow transplant models of CD4-dependant GVHD. To study the requirement of RelB within host APC we generated chimeric mice by transplanting wild-type (wt) or RelB−/− B6 bone marrow into irradiated wt B6 mice. Following immune reconstitution 4–6 months later, the number and frequency of DC (CD11chi and CD11cdimB220+) was equivalent in RelB−/− and RelB+/+ chimeras, although RelB−/− chimeras were specifically deficient in CD11chiCD4+ DC. Chimeras were subsequently transplanted with allogeneic Balb/c bone marrow and purified T cells. The absence of RelB within host APC significantly improved survival (survival day 60: 83% v 19%, P< .0001) and GVHD clinical scores were significantly reduced in RelB−/− chimeras for the first 4 weeks after transplant but subsequently rose to levels equivalent to those in surviving RelB+/+ chimeras. All RelB−/− and RelB+/+ chimeras that received syngeneic grafts survived without clinical evidence of GVHD. Sera from RelB−/− chimera recipients of allogeneic grafts contained reduced IFNg (117 ± 23 vs 253 ± 45 pg/ml; P< 0.02) and increased IL-5 (358 ± 105 vs 112 ± 20 pg/ml; P<0.05) compared to RelB+/+ chimera recipients (mean ± SE). Furthermore, CD4 T cells purified from the spleens of RelB−/− chimera recipients produced 2.6 fold more IL-4 (451 ± 31 vs 168 ± 17 pg/ml; P=0.01) than those from RelB+/+ chimera recipients. Taken together these data suggest the absence of nuclear RelB translocation within host APC abrogates GVHD and this is associated with the induction of donor Th2 differentiation. To study the role of RelB within donor APC we transplanted wt or RelB−/− B6 bone marrow and wt purified T cells into irradiated B6D2F1 recipients. In this model, GVHD severity was identical for the first 4 weeks after transplant but subsequently GVHD clinical scores in the recipients of RelB−/− donor APC returned toward levels seen in syngeneic recipients (clinical scores at day 49: 1.0 ± 0.6; n=6 vs 3.75 ± 0.4; n=6; RelB−/− vs RelB+/+P=0.01). This attenuation of acute GVHD in recipients of RelB−/− donor-derived APC was associated with the reconstitution of donor DC on day 21. These data suggest the inhibition of the nuclear RelB translocation within APC represents a potential new therapeutic target for the prevention of allograft rejection and GVHD.

Effect of T cell subset dose on outcome of T cell-depleted bone marrow transplantation.

T cell depletion using the murine monoclonal antibody (moAb) T10B9 is unique in that the T cell receptor (TCR)gamma delta bearing subset is relatively spared compared to the TCR alpha beta + subset. We evaluated the probabilities of engraftment, acute and chronic graft-versus-host disease (GVHD), relapse, and survival in 273 recipients of marrow T cell depleted using T10B9. Sixty-two patients received marrow from an HLA-identical sibling, 54 patients received partially matched related donor marrow and 157 patients received unrelated donor marrow. Limiting dilution analysis (LDA) was used to estimate total clonable T cell dose in all patients and a modified LDA using moAb-coated immunomagnetic beads was used to estimate TCR alpha beta +, CD4+, and CD8+ T cells in a subset of patients. TCR gamma delta + cell dose was estimated by flow cytometry. Cox proportional hazards regression models were used to assess the impact of T cell subset dose/kg of body weight on outcome. We found a significant association of TCR gamma delta + T cell dose (P = 0.004), but not TCR alpha beta + T cell dose or total clonable T cell dose, with the probability of engraftment. TCR alpha beta +, CD4+, CD8+ and total clonable T cell dose were significantly associated (P < 0.001) with the risks of grade 2-4 acute GVHD in recipients of marrow from related donors but not in recipients of marrow from unrelated donors. Neither total clonable T cell dose nor any T cell subset dose was found to be significantly associated with chronic GVHD, relapse or survival. The results confirm preclinical studies showing TCR gamma delta + T cells promote engraftment. TCR gamma delta + T cells are not associated with an increased risk of acute GVHD while TCR alpha beta T cells are associated with acute GVHD but not engraftment in recipients of marrow grafts T cell depleted using T10B9. These findings support the hypothesis that T cell subsets differentially contribute to alloengraftment and GVHD.

Lack of correlation of MLC reactivity with acute graft-versus-host disease and mortality in unrelated donor bone marrow transplantation

Acute graft-versus-host disease (AGvHD) is a significant cause of morbidity and mortality in patients receiving a bone marrow transplant from an unrelated donor, and in an effort to reduce this problem, donors are selected for the least possible HLA incompatibility with the recipient. Selection criteria have included minimal incompatibility for the HLA-A, -B, and -DR loci and low reactivity in mixed lymphocyte culture (MLC); however, the value of MLC reactivity for prediction of development of AGvHD has been questioned. We therefore examined the correlation of MLC reactivity with AGvHD in recipients of unrelated bone marrow transplants. Reactivity in the GvH direction was assessed as relative response (RR) of donor lymphocytes to recipient stimulator lymphocytes. In 126 transplanted pairs with technically satisfactory MLC tests, the RR was divided into quartiles (0–1, 2–5, 6–16, and 17–117% RR). HLA-DRB1 incompatibilities were more frequent in the highest quartile ( p < 0.001); there were no significant differences among quartiles in donor or recipient age, diagnosis, or frequency of HLA-A or -B incompatibility. Incidence of AGvHD during the first 100 days post-transplant was assessed by Kaplan-Meier analysis. There was no significant difference in incidence of AGvHD among quartiles for the entire group of 126 pairs, for a subset with hematologie. malignancy, for a subset selected by a more stringent standard for “technically satisfactory” MLC, or for a subset matched for A, B, and DRB1. The MLC response of donor lymphocytes to recipient stimulator lymphocytes is thus not predictive of development of AGvHD in our patient population receiving unrelated donor bone marrow. Since there was no difference in mortality related to high and low MLC responses, our data also suggest that MLC results are not predictive of survival in this population. Human Immunology 49, 49–55 (1996)

Unrelated volunteer bone marrow transplantation: initial experience at St Vincent's Hospital, Sydney

Only 30% of patients with leukaemia have an HLA-compatible family member able to act as a marrow donor. The recent development of volunteer bone marrow donor registries has supplied HLA-matched donors for a number of such individuals. To define the problem and outcome of the first cohort of patients given HLA-matched unrelated volunteer bone marrow transplants at St Vincent's Hospital, Sydney. Post transplant outcome of patients with advanced leukaemia given HLA-identical unrelated donor marrow transplants was compared to that of patients transplanted concurrently from HLA-identical sibling donors, in terms of survival, leukaemia-free survival, incidence and severity of acute graft-versus-host disease (GVHD), duration of neutropenia, incidence of infection and duration of transplant hospitalisation. Sixteen patients with advanced leukaemia and without a histocompatible family member donor received unrelated donor bone marrow transplants. Actuarial survival at two years post transplant was 30%. Actuarial survival of 23 recipients of HLA-identical sibling bone marrow transplants with advanced leukaemia transplanted during the same time period was 17% (not significant). Actuarial disease free survival at two years was 30% and 13% respectively. Three of five long term survivors of the unrelated transplants had chronic myeloid leukaemia in blastic transformation at the time of transplant; thus blastic transformation should not preclude consideration of unrelated marrow transplantation. Recipients of unrelated allografts had a higher incidence of acute GVHD which occurred earlier and with greater severity than in recipients of sibling allografts, a longer duration of post transplant neutropenia (24 days to reach 0.5 x 10(9)/L versus 19.5, p = 0.07), a higher frequency of infection in the first 100 days post transplant (p = 0.0004) and a longer duration of transplant hospitalisation (p = 0.04). Transplant-related complications were the commonest cause of death in the unrelated donor recipients, while leukaemic recurrence was the commonest single cause of death in the HLA-identical sibling recipients. Improvements are needed in prophylaxis of infection and in prevention and treatment of acute GVHD in recipients of unrelated donor transplants. Nevertheless, this modality provides curative treatment for patients with otherwise incurable haematological malignancies and should no longer be considered experimental.