Lymph node stromal cells negatively regulate antigen-specific CD4+ T cell responses

Lymph node (LN) stromal cells (LNSCs) form the functional structure of LNs and play an important role in lymphocyte survival and the maintenance of immune tolerance. Despite their broad spectrum of function, little is known about LNSC responses during microbial infection. In this study, we demonstrate that LNSC subsets display distinct kinetics following vaccinia virus infection. In particular, compared with the expansion of other LNSC subsets and the total LN cell population, the expansion of fibroblastic reticular cells (FRCs) was delayed and sustained by noncirculating progenitor cells. Notably, newly generated FRCs were preferentially located in perivascular areas. Viral clearance in reactive LNs preceded the onset of FRC expansion, raising the possibility that viral infection in LNs may have a negative impact on the differentiation of FRCs. We also found that MHC class II expression was upregulated in all LNSC subsets until day 10 postinfection. Genetic ablation of radioresistant stromal cell-mediated Ag presentation resulted in slower contraction of Ag-specific CD4(+) T cells. We propose that activated LNSCs acquire enhanced Ag-presentation capacity, serving as an extrinsic brake system for CD4(+) T cell responses. Disrupted function and homeostasis of LNSCs may contribute to immune deregulation in the context of chronic viral infection, autoimmunity, and graft-versus-host disease.

Drug Hypersensitivity: How Drugs Stimulate T Cells via Pharmacological Interaction with Immune Receptors.

Small chemicals like drugs tend to bind to proteins via noncovalent bonds, e.g. hydrogen bonds, salt bridges or electrostatic interactions. Some chemicals interact with other molecules than the actual target ligand, representing so-called 'off-target' activities of drugs. Such interactions are a main cause of adverse side effects to drugs and are normally classified as predictable type A reactions. Detailed analysis of drug-induced immune reactions revealed that off-target activities also affect immune receptors, such as highly polymorphic human leukocyte antigens (HLA) or T cell receptors (TCR). Such drug interactions with immune receptors may lead to T cell stimulation, resulting in clinical symptoms of delayed-type hypersensitivity. They are assigned the 'pharmacological interaction with immune receptors' (p-i) concept. Analysis of p-i has revealed that drugs bind preferentially or exclusively to distinct HLA molecules (p-i HLA) or to distinct TCR (p-i TCR). P-i reactions differ from 'conventional' off-target drug reactions as the outcome is not due to the effect on the drug-modified cells themselves, but is the consequence of reactive T cells. Hence, the complex and diverse clinical manifestations of delayed-type hypersensitivity are caused by the functional heterogeneity of T cells. In the abacavir model of p-i HLA, the drug binding to HLA may result in alteration of the presenting peptides. More importantly, the drug binding to HLA generates a drug-modified HLA, which stimulates T cells directly, like an allo-HLA. In the sulfamethoxazole model of p-i TCR, responsive T cells likely require costimulation for full T cell activation. These findings may explain the similarity of delayed-type hypersensitivity reactions to graft-versus-host disease, and how systemic viral infections increase the risk of delayed-type hypersensitivity reactions.

Sexual health in hematopoietic stem cell transplant recipients

Hematopoietic stem cell transplantation (HSCT) plays a central role in patients with malignant and, increasingly, nonmalignant conditions. As the number of transplants increases and the survival rate improves, long-term complications are important to recognize and treat to maintain quality of life. Sexual dysfunction is a commonly described but relatively often underestimated complication after HSCT. Conditioning regimens, generalized or genital graft-versus-host disease, medications, and cardiovascular complications as well as psychosocial problems are known to contribute significantly to physical and psychological sexual dysfunction. Moreover, it is often a difficult topic for patients, their significant others, and health care providers to discuss. Early recognition and management of sexual dysfunction after HSCT can lead to improved quality of life and outcomes for patients and their partners. This review focuses on the risk factors for and treatment of sexual dysfunction after transplantation and provides guidance concerning how to approach and manage a patient with sexual dysfunction after HSCT.

Role of janus tyrosine kinase -3 (JAK3) and signal transducer and activator of transcription (STAT5) pathway in T lymphocyte activation

T cell activation and expansion is essential for immune response against foreign antigens. However, uncontrolled T cell activity can be manifested as a number of lymphoid derived diseases such as autoimmunity, graft versus host disease, and lymphoma. The purpose of this research was to test the central hypothesis that the Jak3/Stat5 pathway is critical for T cell function. To accomplish this objective, two novel Jak3 inhibitors, AG490 and PNU156804, were identified and their effects characterized on Jak3/Stat5 activation and T cell growth. Inhibition of Jak3 selectively disrupted primary human T lymphocyte growth in response to Interleukin-2 (IL-2), as well as other γ c cytokine family members including IL-4, IL-7, IL-9, and IL-15. Inhibition of Jak3 ablated IL-2 induced Stat5 but not TNF-α mediated NF-κβ DNA binding. Loss of Jak3 activity did not affect T cell receptor mediated signals including activation of p56Lck and Zap70, or IL-2 receptor a chain expression. To examine the effects of Jak3/Stat5 inhibition within a mature immune system, we employed a rat heart allograft model of Lewis (RT1 1) to ACI (RT1a). Heart allograft survival was significantly prolonged following Jak3/Stat5 inhibition when rats were treated with AG490 (20mg/kg) or PNU156804 (80mg/kg) compared to non-treated control animals. This effect was synergistically potentiated when Jak3 inhibitors were used in combination with a signal 1/2 disrupter, cyclosporine, but only additively potentiated with another signal 3 inhibitor, rapamycin. This suggested that sequential inhibition of T cell function is more effective. To specifically address the role of Stat5 in maintaining T cell activity, novel Stat5 antisense oligonucleotides were synthesized and characterized in vitro. Primary human T cells and T-cell tumor lines treated with Stat5 antisense oligonucleotide (7.5 μM) rapidly underwent apoptosis, while no changes in cell cycle were observed as measured by FACS analysis utilizing Annexin-V-Fluorescein and Propidium iodide staining. Evidence is provided to suggest that caspase 8 and 9 pathways mediate this event. Thus, Stat5 may act rather as a negative regulator of apoptotic signals and not as a positive regulator of cell cycle as previously proposed. We conclude that the Jak3/Stat5 pathway is critical for γc cytokine mediated gene expression necessary for T cell expansion and normal immune function and represents an therapeutically relevant effector pathway to combat T cell derived disease. ^

Definition of HLA-DQ as a transplantation antigen

Recent studies have demonstrated the importance of recipient HLA-DRB1 allele disparity in the development of acute graft-versus-host disease (GVHD) after unrelated donor marrow transplantation. The role of HLA-DQB1 allele disparity in this clinical setting is unknown. To elucidate the biological importance of HLA-DQB1, we conducted a retrospective analysis of 449 HLA-A, -B, and -DR serologically matched unrelated donor transplants. Molecular typing of HLA-DRB1 and HLA-DQB1 alleles revealed 335 DRB1 and DQB1 matched pairs; 41 DRB1 matched and DQB1 mismatched pairs; 48 DRB1 mismatched and DQB1 matched pairs; and 25 DRB1 and DQB1 mismatched pairs. The conditional probabilities of grades III-IV acute GVHD were 0.42, 0.61, 0.55, and 0.71, respectively. The relative risk of acute GVHD associated with a single locus HLA-DQB1 mismatch was 1.8 (1.1, 2.7; P = 0.01), and the risk associated with any HLA-DQB1 and/or HLA-DRB1 mismatch was 1.6 (1.2, 2.2; P = 0.003). These results provide evidence that HLA-DQ is a transplant antigen and suggest that evaluation of both HLA-DQB1 and HLA-DRB1 is necessary in selecting potential donors.

Binding of thalidomide to alpha1-acid glycoprotein may be involved in its inhibition of tumor necrosis factor alpha production.

In addition to its well known sedative and teratogenic effects, thalidomide also possesses potent immunomodulatory and antiinflammatory activities, being most effective against leprosy and chronic graft-versus-host disease. The immunomodulatory activity of thalidomide has been ascribed to the selective inhibition of tumor necrosis factor alpha from monocytes. The molecular mechanism for the immunomodulatory effect of thalidomide remains unknown. To elucidate this mechanism, we synthesized an active photoaffinity label of thalidomide as a probe to identify the molecular target of the drug. Using the probe, we specifically labeled a pair of proteins of 43-45 kDa with high acidity from bovine thymus extract. Purification of these proteins and partial peptide sequence determination revealed them to be alpha1-acid glycoprotein (AGP). We show that the binding of thalidomide photoaffinity label to authentic human AGP is competed with both thalidomide and the nonradioactive photoaffinity label at concentrations comparable to those required for inhibition of production of tumor necrosis factor alpha from human monocytes, suggesting that AGP may be involved in the immunomodulatory activity of thalidomide.

Mécanismes d'action des cellules stromales mésenchymateuses dans le traitement de la réaction du greffon contre l'hôte

La maladie du greffon contre l’hôte (GvHD) est un effet secondaire sérieux de la transplantation de cellules souches hématopoïétiques (HSCT). Cette maladie entraine une haute mortalité et ses symptômes sont dévastateurs. Les traitements actuels de la GvHD comportent plusieurs produits, tels les corticostéroïdes, mais ces derniers sont immunosuppresseurs et leurs effets secondaires sont aussi très dommageables pour les patients et leur guérison. Les cellules stromales mésenchymateuses (MSC) représentent une alternative ou une addition potentielle de traitement pour la GvHD et ces cellules ne semblent pas posséder les effets secondaires des traitements classiques. Un nombre important d’études cliniques faisant l’objet des MSC ont été enregistrées. Malgré cet engouement, le mécanisme de leur immunomodulation reste encore à élucider. Notre objectif est donc de mieux définir ce mécanisme. Nous avons utilisé un modèle simplifié pour simuler la GvHD in vitro. Ce modèle se base sur la stimulation de lymphocytes CD4+ par des cellules dendritiques allogéniques. La mesure de la prolifération de ces cellules stimulées sert d’indicateur de leur réactivité. Selon les résultats obtenus par la technologie CRISPR de génie génétique, les MSC exerceraient leur immunosuppression sur les cellules T CD4+ principalement par la sécrétion de l’enzyme IDO1. Les MSC seraient également capables d’induire certaines cellules CD4+ en cellules régulatrices, un processus indépendant de la sécrétion d’IDO1. Toutefois, ces cellules ne semblent pas correspondre aux cellules Treg conventionnelles.

Donor treatment with pegylated G-CSF augments the generation of IL-10-producing regulatory T cells and promotes transplantation tolerance

We investigated whether the protection from graft-versus-host disease (GVHD) afforded by donor treatment with granulocyte colony-stimulating factor (G-CSF) could be enhanced by dose escalation. Donor treatment with human G-CSIF prevented GVHD in the B6 --> B6D2F1 murine model in a dose-dependent fashion, and murine G-CSF provided equivalent protection from GVHD at 10-fold lower doses. Donor pretreatment with a single dose of pegylated G-CSF (peg-G-CSF) prevented GVHD to a significantly greater extent than standard G-CSIF (survival, 75% versus 11%, P < .001). Donor T cells from peg-G-CSF-treated donors failed to proliferate to alloantigen and inhibited the responses of control T cells in an interleukin 10 (IL-10)-dependent-fashion in vitro. T cells from peg-GCSF-treated IL-10(-/-) donors induced lethal GVHD; T cells from peg-G-CSF-treated wild-type (wt) donors promoted long-term survival. Whereas T cells from peg-G-CSF wt donors were able to regulate GVHD induced by T cells from control-treated donors, T cells from G-CSF-treated wt donors and peg-G-CSF-treated IL-10(-/-) donors did not prevent mortality. Thus, peg-G-CSF is markedly superior to standard G-CSF for the prevention of GVHD following allogeneic stem cell transplantation (SCT), due to the generation of IL-10-producing regulatory T cells. These data support prospective clinical trials of peg-G-CSF-mobilized allogeneic blood SCT. (C) 2004 by The American Society of Hematology.

Outcomes after Unrelated Umbilical Cord Blood Transplantation for Children with Osteopetrosis.

Hematopoietic stem cell transplantation (HSCT) is the only curative treatment for most children with osteopetrosis (OP). Timing of HSCT is critical; therefore, umbilical cord blood transplantation (UCBT) is an attractive option. We analyzed outcomes after UCBT in 51 OP children. Median age at UCBT was 6 months. Seventy-seven percent of the cord blood grafts had 0 or 1 HLA disparity with the recipient. Conditioning regimen was myeloablative (mostly busulfan-based in 84% and treosulfan-based in 10%). Antithymocyte globulin was given to 90% of patients. Median number of total nucleated and CD34(+) cells infused was 14 × 10(7)/kg and 3.4 × 10(5)/kg, respectively. Median follow-up for survivors was 74 months. Cumulative incidence (CI) of neutrophil recovery was 67% with a median time to recovery of 23 days; 33% of patients had graft failure, 81% of engrafted patients had full donor engraftment, and 19% had mixed donor chimerism. Day 100 CI of acute graft-versus-host disease (grades II to IV) was 31% and 6-year CI of chronic graft-versus-host disease was 21%. Mechanical ventilation was required in 28%, and veno-occlusive disease was diagnosed in 16% of cases. Six-year overall survival rate was 46%. Comparative studies with other alternative donors should be performed to evaluate whether UCBT remains a valid alternative for children with OP without an HLA-matched donor.

Traitement de la maladie du greffon contre l’hôte chronique par la photophérèse extra corporelle au TH9402 : m écanisme de régulation de la maladie du greffon contre l’hôte chronique par les cellules T régulatrices

La maladie du greffon contre l’hôte (GVHD) est la principale cause de mortalité et de morbidité suite aux greffes de cellules souches hématopoïétiques. Plusieurs patients demeurent réfractaires aux traitements actuels ce qui rend nécessaire le développement de nouvelles stratégies afin de combattre cette maladie. Dans l’étude qui suit, nous avons utilisé un nouvel agent thérapeutique, le TH9402, une molécule photosensible et démontré qu’elle permet, lorsqu’exposée à la lumière visible (514 nm), d’éliminer sélectivement les cellules T activées in vivo tout en préservant les cellules T au repos et les cellules T régulatrices (Tregs). Les Tregs ainsi préservés peuvent abroger la réponse alloréactive par la sécrétion d’IL-10 ou par contact cellule-cellule via un mécanisme impliquant le CTLA-4. Nous avons découvert que la signalisation du CTLA-4 était associée à une hausse de la population Treg in vitro. Cette hausse est due à la conversion de cellules T CD4+CD25- en Tregs et non à une prolifération sélective des Tregs. Dans la deuxième partie de cette étude, nous avons démontré que la signalisation de CTLA-4 était associée à une augmentation de l’expression de la protéine Indoleamine 2,3 dioxygenase (IDO). Ces effets nécessitent la déplétion du tryptophane ainsi que de la protéine de phase aigue GCN2. Finalement, nous avons observé que l’infusion de cellules traitées au TH9402 chez des patients souffrant de GVHD chronique est associée à une augmentation de la population Treg chez ces patients sans causer de lymphopénie ni de diminution de la réponse immunitaire dirigée contre les antigènes viraux. Ces résultats suggèrent que le traitement au TH9402 pourrait représenter une approche particulièrement intéressante pour le traitement de la GVHD chronique réfractaire aux traitements actuels. De plus, l’augmentation de l’expression d’IDO pourrait être utilisée comme valeur prédictive de la réponse du patient au traitement. Ceci pourrait permettre d’améliorer la qualité de soins ainsi que de la qualité de vie des patients souffrant de GVHD chronique.

Découverte de nouveaux marqueurs pharmacogénomiques de la maladie du greffon contre l'hôte en transplantation de cellules souches hématopoïétiques

La transplantation de cellules souches hématopoïétiques (CSH) constitue une avenue thérapeutique potentiellement curative pour plusieurs cancers hématologiques comme la leucémie. L’utilisation d’une thérapie immunosuppressive pour prévenir la maladie du greffon contre l’hôte (GvHD) est un déterminant majeur du succès de la greffe. Malgré tout, cette complication survient chez 25 à 50% des transplantés et est une cause majeure de mortalité. L’optimisation du régime d’immunosuppression est un facteur facilement modifiable qui pourrait améliorer le pronostic des patients. Particulièrement, les polymorphismes du génome du donneur ou du receveur dans les voies pharmacogénomiques des immunosuppresseurs pourraient influencer l’exposition et l’action de ces médicaments, de même que le pronostic du patient. Le profilage de 20 pharmacogènes prioritaires chez des paires de donneurs-receveurs en greffe de CSH a permis d’identifier des variations génétiques liées au risque de la GvHD aiguë. Principalement, le statut génétique du receveur pour les protéines ABCC1 et ABCC2, impliquées dans le transport du méthotrexate (MTX), ainsi que des cibles moléculaires de ce médicament (ATIC et MTHFR) ont été associées au risque de GvHD aiguë. Similairement, le NFATc1, codant pour une cible moléculaire de la cyclosporine, augmentait lui aussi le risque de la maladie. Les porteurs de deux génotypes à risque et plus étaient particulièrement prédisposés à développer cette complication. Par surcroît, le statut génétique du donneur influençait également le pronostic du receveur après la greffe. Entre autres, des allèles protecteurs ont été identifiés dans les voies liées au transport (SLC19A1) et à l’action du MTX (DHFR). Inversement, NFATc2 a été associé à une augmentation du risque de GvHD aiguë. Afin de mieux comprendre les associations observées entre ces marqueurs génétiques et le risque de GvHD aiguë, une étude prospective innovante est en cours chez des greffés de CSH. Cette étude permettra d’étudier comment la génétique du patient ou du donneur peut influencer la pharmacocinétique et la pharmacodynamie des immunosuppresseurs, de même que leurs liens avec la GvHD aiguë. Ces paramètres sont quantifiés grâce à des approches analytiques que nous avons mises au point afin de répondre aux besoins spécifiques et uniques de cette étude. Les approches proposées dans cette thèse sont complémentaires aux méthodes classiques de suivi des immunosuppresseurs et pourraient aider à optimiser la pharmacothérapie du patient. Une meilleure identification des patients à haut risque de GvHD aiguë avant la greffe, basée sur des marqueurs pharmacogénomiques identitaires, pourrait guider le choix de la prophylaxie immunosuppressive, et ainsi améliorer l’issue clinique de la greffe.

Doença enxerto-versus hospedeiro: determinação da expressão de genes e alterações de vias associadas ao sistema imunológico usando metodos da biofísica

O transplante de medula óssea (TMO) é um procedimento terapêutico importante em casos relacionados à pacientes com leucemia ou linfoma. Em decorrência desse processo, uma reação conhecida como doença enxerto-versus-hospedeiro (GVHD) pode ocorrer em pacientes susceptíveis como conseqüência da presença de células imunocompetentes do doador. Entretanto, não existe um modelo para descrever completamente as ações relacionadas ao mecanismo imunológico da GVHD desde a fase que inicializa a doença até a fase efetora. O Objetivo geral deste estudo é a investigação da resposta imunológica considerando-se o sistema HLA (antígenos leucocitários humano) em pacientes que desenvolveram a GVHD em decorrência do TMO. O National Cancer Institute (NCI) – Pathway interaction Database e Reactome foram usados como bases de dados com o objetivo de se estudar a expressão de genes e vias relacionados às Classes I e II do sistema HLA (antígenos leucocitários humano). O estudo considerou a mudança de expressão de genes relacionados às 17 vias do sistema imunológico com potencialidade para se expressar em pacientes que desenvolveram a GVHD associada à TMO. Dados referentes aos transcriptomas foram obtidos utilizando-se a plataforma GPL570 Affymetrix Genoma Humano U133 Plus. A atividade relativa foi usada para determinar as alterações das vias em amostras de GVHD em relação ao controle. As análises foram realizadas utilizando-se o software Via Complex e Bioconductor. Observou-se aumento significativo da expressão de genes ralacionados às vias do sistema imune adaptativo, antígenos associados às Classe I e II do HLA, fosforilação de CD3 e CD247, sinalização dos receptores de células T em CD4+ nativas e ativação de NF-kapa β nas células B. Também observou-se alterações significativas na mudança de expressão dos genes associados às vias relacionadas à super família de moléculas B7:CD28\CTLA-4 quando comparadas ao controle. Isso pode indicar a necessidade de geração de um segundo sinal co-estimulador em GVHD, acionado pelas moléculas dessa super família. O aumento da expressão do gene CD69 nas amostras experimentais caracteriza a ativação celular e, portanto, a sinalização de estímulos em GVHD. Os achados obtidos neste estudo contribuem para melhor elucidar o mecanismo imunopatogênico associado à GVHD. P

Traitement de la maladie du greffon contre l’hôte chronique par la photophérèse extra corporelle au TH9402 : m écanisme de régulation de la maladie du greffon contre l’hôte chronique par les cellules T régulatrices

La maladie du greffon contre l’hôte (GVHD) est la principale cause de mortalité et de morbidité suite aux greffes de cellules souches hématopoïétiques. Plusieurs patients demeurent réfractaires aux traitements actuels ce qui rend nécessaire le développement de nouvelles stratégies afin de combattre cette maladie. Dans l’étude qui suit, nous avons utilisé un nouvel agent thérapeutique, le TH9402, une molécule photosensible et démontré qu’elle permet, lorsqu’exposée à la lumière visible (514 nm), d’éliminer sélectivement les cellules T activées in vivo tout en préservant les cellules T au repos et les cellules T régulatrices (Tregs). Les Tregs ainsi préservés peuvent abroger la réponse alloréactive par la sécrétion d’IL-10 ou par contact cellule-cellule via un mécanisme impliquant le CTLA-4. Nous avons découvert que la signalisation du CTLA-4 était associée à une hausse de la population Treg in vitro. Cette hausse est due à la conversion de cellules T CD4+CD25- en Tregs et non à une prolifération sélective des Tregs. Dans la deuxième partie de cette étude, nous avons démontré que la signalisation de CTLA-4 était associée à une augmentation de l’expression de la protéine Indoleamine 2,3 dioxygenase (IDO). Ces effets nécessitent la déplétion du tryptophane ainsi que de la protéine de phase aigue GCN2. Finalement, nous avons observé que l’infusion de cellules traitées au TH9402 chez des patients souffrant de GVHD chronique est associée à une augmentation de la population Treg chez ces patients sans causer de lymphopénie ni de diminution de la réponse immunitaire dirigée contre les antigènes viraux. Ces résultats suggèrent que le traitement au TH9402 pourrait représenter une approche particulièrement intéressante pour le traitement de la GVHD chronique réfractaire aux traitements actuels. De plus, l’augmentation de l’expression d’IDO pourrait être utilisée comme valeur prédictive de la réponse du patient au traitement. Ceci pourrait permettre d’améliorer la qualité de soins ainsi que de la qualité de vie des patients souffrant de GVHD chronique.

Establishing optimal quantitative-polymerase chain reaction assays for routine diagnosis and tracking of minimal residual disease in JAK2-V617F-associated myeloproliferative neoplasms:a joint European LeukemiaNet/MPN&MPNr-EuroNet (COST action BM0902) study

Reliable detection of JAK2-V617F is critical for accurate diagnosis of myeloproliferative neoplasms (MPNs); in addition, sensitive mutation-specific assays can be applied to monitor disease response. However, there has been no consistent approach to JAK2-V617F detection, with assays varying markedly in performance, affecting clinical utility. Therefore, we established a network of 12 laboratories from seven countries to systematically evaluate nine different DNA-based quantitative PCR (qPCR) assays, including those in widespread clinical use. Seven quality control rounds involving over 21,500 qPCR reactions were undertaken using centrally distributed cell line dilutions and plasmid controls. The two best-performing assays were tested on normal blood samples (n=100) to evaluate assay specificity, followed by analysis of serial samples from 28 patients transplanted for JAK2-V617F-positive disease. The most sensitive assay, which performed consistently across a range of qPCR platforms, predicted outcome following transplant, with the mutant allele detected a median of 22 weeks (range 6-85 weeks) before relapse. Four of seven patients achieved molecular remission following donor lymphocyte infusion, indicative of a graft vs MPN effect. This study has established a robust, reliable assay for sensitive JAK2-V617F detection, suitable for assessing response in clinical trials, predicting outcome and guiding management of patients undergoing allogeneic transplant.

Impact of pretransplant minimal residual disease after cord blood transplantation for childhood acute lymphoblastic leukemia in remission: an Eurocord, PDWP-EBMT analysis

To address the prognostic value of minimal residual disease (MRD) before unrelated cord blood transplantation (UCBT) in children with acute lymphoblastic leukemia (ALL), we analyzed 170 ALL children transplanted in complete remission (CR) after myeloablative conditioning regimen. In all, 72 (43%) were in first CR (CR1), 77 (45%) in second CR (CR2) and 21 (12%) in third CR (CR3). The median interval from MRD quantification to UCBT was 18 days. All patients received single-unit UCBT. Median follow-up was 4 years. Cumulative incidence (CI) of day-60 neutrophil engraftment was 85%. CI of 4 years relapse was 30%, incidence being lower in patients with negative MRD before UCBT (hazard ratio (HR) = 0.4, P = 0.01) and for those transplanted in CR1 and CR2 (HR = 0.3, P = 0.002). Probability of 4 years leukemia-free survival (LFS) was 44%, (56, 44 and 14% for patients transplanted in CR1, CR2 and CR3, respectively (P = 0.0001)). Patients with negative MRD before UCBT had better LFS after UCBT compared with those with positive MRD (54% vs 29%; HR = 2, P = 0.003). MRD assessment before UCBT for children with ALL in remission allows identifying patients at higher risk of relapse after transplantation. Approaches that may decrease relapse incidence in children given UCBT with positive MRD should be investigated to improve final outcomes. Leukemia (2012) 26, 2455-2461; doi:10.1038/leu.2012.123