Modeling the roles of cohesotaxis, cell-intercalation, and tissue geometry in collective cell migration of Xenopus mesendoderm.

Collectively migrating Xenopus mesendoderm cells are arranged into leader and follower rows with distinct adhesive properties and protrusive behaviors. In vivo, leading row mesendoderm cells extend polarized protrusions and migrate along a fibronectin matrix assembled by blastocoel roof cells. Traction stresses generated at the leading row result in the pulling forward of attached follower row cells. Mesendoderm explants removed from embryos provide an experimentally tractable system for characterizing collective cell movements and behaviors, yet the cellular mechanisms responsible for this mode of migration remain elusive. We introduce an agent-based computational model of migrating mesendoderm in the Cellular-Potts computational framework to investigate the relative contributions of multiple parameters specific to the behaviors of leader and follower row cells. Sensitivity analyses identify cohesotaxis, tissue geometry, and cell intercalation as key parameters affecting the migration velocity of collectively migrating cells. The model predicts that cohesotaxis and tissue geometry in combination promote cooperative migration of leader cells resulting in increased migration velocity of the collective. Radial intercalation of cells towards the substrate is an additional mechanism to increase migratory speed of the tissue. Summary Statement: We present a novel Cellular-Potts model of collective cell migration to investigate the relative roles of cohesotaxis, tissue geometry, and cell intercalation on migration velocity of Xenopus mesendoderm.

Characterization of convergent thickening, a major convergence force producing morphogenic movement in amphibians.

The morphogenic process of convergent thickening (CT) was originally described as the mediolateral convergence and radial thickening of the explanted ventral involuting marginal zone (IMZ) of Xenopus gastrulae (Keller and Danilchik, 1988). Here, we show that CT is expressed in all sectors of the pre-involution IMZ, which transitions to expressing convergent extension (CE) after involution. CT occurs without CE and drives symmetric blastopore closure in ventralized embryos. Assays of tissue affinity and tissue surface tension measurements suggest CT is driven by increased interfacial tension between the deep IMZ and the overlying epithelium. The resulting minimization of deep IMZ surface area drives a tendency to shorten the mediolateral (circumblastoporal) aspect of the IMZ, thereby generating tensile force contributing to blastopore closure (Shook et al., 2018). These results establish CT as an independent force-generating process of evolutionary significance and provide the first clear example of an oriented, tensile force generated by an isotropic, Holtfreterian/Steinbergian tissue affinity change.

Progenitor cell therapy for acquired pediatric nervous system injury: Traumatic brain injury and acquired sensorineural hearing loss.

While cell therapies hold remarkable promise for replacing injured cells and repairing damaged tissues, cell replacement is not the only means by which these therapies can achieve therapeutic effect. For example, recent publications show that treatment with varieties of adult, multipotent stem cells can improve outcomes in patients with neurological conditions such as traumatic brain injury and hearing loss without directly replacing damaged or lost cells. As the immune system plays a central role in injury response and tissue repair, we here suggest that multipotent stem cell therapies achieve therapeutic effect by altering the immune response to injury, thereby limiting damage due to inflammation and possibly promoting repair. These findings argue for a broader understanding of the mechanisms by which cell therapies can benefit patients.

Autologous hematopoietic cell transplantation for the treatment of relapsed/refractory pediatric, adolescent, and young adult Hodgkin lymphoma: a single institutional experience.

Pediatric, adolescent, and young adult patients with relapsed or refractory Hodgkin lymphoma receive multimodal therapy, including autologous hematopoietic cell transplantation (AutoHCT). Despite aggressive therapy, historical outcomes for this patient population have been poor. This paper describes a single institutional experience utilizing AutoHCT in 74 patients treated from 1988-2015. Our results demonstrate significantly improved outcomes over time. Compared with patients treated in the earlier era (1988-2001), 5-year overall survival improved from 62.5 ± 9.6% to 91.8 ± 4.4% (p < 0.001) and event free survival improved from 41.7 ± 9.6% to 87.7 ± 5.3% (I < 0.001) for patients treated in a later era (2002-2015). Improvements in survival are multifactorial, including reductions in both relapse and nonrelapse mortality. Further investigation is needed to determine the role of AutoHCT in a modern treatment cohort that includes frequent use of targeted immunotherapies. In addition, as the use and availability of effective novel therapeutics increases for this patient population there may be an opportunity for the reduction of standard cytotoxic therapies, including in AutoHCT preparative regimens, thereby mitigating late effects.

Safety of Autologous Umbilical Cord Blood Therapy for Acquired Sensorineural Hearing Loss in Children.

BACKGROUND AND OBJECTIVES: Sensorineural hearing loss (SNHL) in children is associated with neurocognitive morbidity. The cause of SNHL is a loss of hair cells in the organ of Corti. There are currently no reparative treatments for SNHL. Numerous studies suggest that cord blood mononuclear cells (human umbilical cord blood, hUCB) allow at least partial restoration of SNHL by enabling repair of a damaged organ of Corti. Our objective is to determine if hUCB is a safe treatment for moderate to severe acquired SNHL in children. Subjects and. METHODS: Eleven children aged 6 months to 6 years with moderate to severe acquired SNHL were treated with intravenous autologous hUCB. The cell dose ranged from 8 to 30 million cells/kg body weight. Safety was assessed by measuring systemic hemodynamics during hUCB infusion. Infusion-related toxicity was evaluated by measuring neurologic, hepatic, renal and pulmonary function before and after infusion. Auditory function, auditory verbal language assessments and MRI with diffusion tensor imaging (DTI) were obtained before and after treatment. RESULTS: All patients survived, and there were no adverse events. No infusionrelated changes in hemodynamics occurred. No infusion-related toxicity was recorded. Five subjects experienced a reduction in auditory brainstem response (ABR) thresholds. Four of those 5 subjects also experienced an improvement in cochlear nerve latencies. Comparison of MRI with DTI sequences obtained before and after treatment revealed increased fractional anisotropy in the primary auditory cortex in three of five subjects with reduced ABR thresholds. Statistically significant (p<0.05) reductions in ABR thresholds were identified. CONCLUSIONS: TIntravenous hUCB is feasible and safe in children with SNHL.

Large, long range tensile forces drive convergence during Xenopus blastopore closure and body axis elongation.

Indirect evidence suggests that blastopore closure during gastrulation of anamniotes, including amphibians such as Xenopus laevis, depends on circumblastoporal convergence forces generated by the marginal zone (MZ), but direct evidence is lacking. We show that explanted MZs generate tensile convergence forces up to 1.5 µN during gastrulation and over 4 µN thereafter. These forces are generated by convergent thickening (CT) until the midgastrula and increasingly by convergent extension (CE) thereafter. Explants from ventralized embryos, which lack tissues expressing CE but close their blastopores, produce up to 2 µN of tensile force, showing that CT alone generates forces sufficient to close the blastopore. Uniaxial tensile stress relaxation assays show stiffening of mesodermal and ectodermal tissues around the onset of neurulation, potentially enhancing long-range transmission of convergence forces. These results illuminate the mechanobiology of early vertebrate morphogenic mechanisms, aid interpretation of phenotypes, and give insight into the evolution of blastopore closure mechanisms.

Selective T-cell depletion targeting CD45RA reduces viremia and enhances early T-cell recovery compared with CD3-targeted T-cell depletion.

BACKGROUND: T-cell depletion (TCD) effectively reduces severe graft-versus-host disease in recipients of HLA-mismatched allografts. However, TCD is associated with delayed immune recovery and increased infections. We hypothesized that specific depletion of CD45RA+ naive T cells, rather than broad depletion of CD3+ T cells, can preserve memory-immunity in the allografts and confer protection against important viral infections in the early post-transplant period. METHODS: Sixty-seven patients who received TCD haploidentical donor transplantation for hematologic malignancy on 3 consecutive trials were analyzed. RESULTS: Patients receiving CD45RA-depleted donor grafts had 2000-fold more donor T cells infused, significantly higher T-cell counts at Day +30 post transplant (550/µL vs 10/µL; P < .001), and higher T-cell diversity by Vbeta spectratyping at Day +100 (P < .001). Importantly, these recipients experienced a significant reduction in both the incidence (P = .002) and duration (P = .02) of any viremia (cytomegalovirus, Epstein-Barr virus, or adenovirus) in the first 6 months post transplant. Specifically, recipients of CD3-depleted grafts were more likely to experience adenovirus viremia (27% vs 4%, P = .02). CONCLUSION: CD45RA-depletion provided a large number of donor memory T cells to the recipients and was associated with enhanced early T-cell recovery and protection against viremia.

Consolidation Therapy for Newly Diagnosed Pediatric Patients with High-Risk Neuroblastoma Using Busulfan/Melphalan, Autologous Hematopoietic Cell Transplantation, Anti-GD2 Antibody, Granulocyte-Macrophage Colony-Stimulating Factor, Interleukin-2, and Haploidentical Natural Killer Cells.

The treatment of pediatric high-risk neuroblastoma is intensive and multimodal. Despite the introduction of immunotherapy for minimal residual disease, survival rates remain suboptimal and new therapies are needed. As part of a phase 2 trial, we are using a consolidation therapy regimen that combines a busulfan/melphalan conditioning schema, autologous hematopoietic cell transplantation (AHCT), and experimental immunotherapy with hu14.18K322A (a humanized anti-GD2 monoclonal antibody), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-2, with or without the adoptive transfer of haploidentical natural killer cells (NKs). Here we report on 30 patients who have undergone AHCT with this experimental immunotherapy regimen, 21 of whom received haploidentical NKs. The median time to neutrophil engraftment was 13 days (range, 10 to 28 days) and to platelet engraftment of at least 20 × 103/mm3 was 36.5 days (range, 0 to 102 days); no clinical difference was seen in those who did or did not receive NKs. Eight patients developed veno-occlusive disease, with 3 having multiorgan dysfunction. Toxicities were similar for patients who did or did not receive NKs. We conclude that this consolidation regimen is feasible and has an acceptable acute toxicity profile.

Molecular model for force production and transmission during vertebrate gastrulation.

Vertebrate embryos undergo dramatic shape changes at gastrulation that require locally produced and anisotropically applied forces, yet how these forces are produced and transmitted across tissues remains unclear. We show that depletion of myosin regulatory light chain (RLC) levels in the embryo blocks force generation at gastrulation through two distinct mechanisms: destabilizing the myosin II (MII) hexameric complex and inhibiting MII contractility. Molecular dissection of these two mechanisms demonstrates that normal convergence force generation requires MII contractility and we identify a set of molecular phenotypes correlated with both this failure of convergence force generation in explants and of blastopore closure in whole embryos. These include reduced rates of actin movement, alterations in C-cadherin dynamics and a reduction in the number of polarized lamellipodia on intercalating cells. By examining the spatial relationship between C-cadherin and actomyosin we also find evidence for formation of transcellular linear arrays incorporating these proteins that could transmit mediolaterally oriented tensional forces. These data combine to suggest a multistep model to explain how cell intercalation can occur against a force gradient to generate axial extension forces. First, polarized lamellipodia extend mediolaterally and make new C-cadherin-based contacts with neighboring mesodermal cell bodies. Second, lamellipodial flow of actin coalesces into a tension-bearing, MII-contractility-dependent node-and-cable actin network in the cell body cortex. And third, this actomyosin network contracts to generate mediolateral convergence forces in the context of these transcellular arrays.

Mechanical strain determines the axis of planar polarity in ciliated epithelia.

Epithelia containing multiciliated cells align beating cilia along a common planar axis specified by the conserved planar cell polarity (PCP) pathway. Specification of the planar axis is also thought to require a long-range cue to align the axis globally, but the nature of this cue in ciliated and other epithelia remains poorly understood. We examined this issue using the Xenopus larval skin, where ciliary flow aligns to the anterior-posterior (A-P) axis. We first show that a planar axis initially arises in the developing skin during gastrulation, based on the appearance of polarized apical microtubules and cell junctions with increased levels of stable PCP components. This axis also arises in severely ventralized embryos, despite their deficient embryonic patterning. Because ventralized embryos still gastrulate, producing a mechanical force that strains the developing skin along the A-P axis, we asked whether this strain alone drives global planar patterning. Isolated skin explanted before gastrulation lacks strain and fails to acquire a global planar axis but responds to exogenous strain by undergoing cell elongation, forming polarized apical microtubules, and aligning stable components of the PCP pathway orthogonal to the axis of strain. The planar axis in embryos can be redirected by applying exogenous strain during a critical period around gastrulation. Finally, we provide evidence that apical microtubules and the PCP pathway interact to align the planar axis. These results indicate that oriented tissue strain generated by the gastrulating mesoderm plays a major role in determining the global axis of planar polarity of the developing skin.

Monitoring Central Venous Catheter Resistance to Predict Imminent Occlusion: A Prospective Pilot Study.

BACKGROUND: Long-term central venous catheters are essential for the management of chronic medical conditions, including childhood cancer. Catheter occlusion is associated with an increased risk of subsequent complications, including bloodstream infection, venous thrombosis, and catheter fracture. Therefore, predicting and pre-emptively treating occlusions should prevent complications, but no method for predicting such occlusions has been developed. METHODS: We conducted a prospective trial to determine the feasibility, acceptability, and efficacy of catheter-resistance monitoring, a novel approach to predicting central venous catheter occlusion in pediatric patients. Participants who had tunneled catheters and were receiving treatment for cancer or undergoing hematopoietic stem cell transplantation underwent weekly catheter-resistance monitoring for up to 12 weeks. Resistance was assessed by measuring the inline pressure at multiple flow-rates via a syringe pump system fitted with a pressure-sensing transducer. When turbulent flow through the device was evident, resistance was not estimated, and the result was noted as "non-laminar." RESULTS: Ten patients attended 113 catheter-resistance monitoring visits. Elevated catheter resistance (>8.8% increase) was strongly associated with the subsequent development of acute catheter occlusion within 10 days (odds ratio = 6.2; 95% confidence interval, 1.8-21.5; p <0.01; sensitivity, 75%; specificity, 67%). A combined prediction model comprising either change in resistance greater than 8.8% or a non-laminar result predicted subsequent occlusion (odds ratio = 6.8; 95% confidence interval, 2.0-22.8; p = 0.002; sensitivity, 80%; specificity, 63%). Participants rated catheter-resistance monitoring as highly acceptable. CONCLUSIONS: In this pediatric hematology and oncology population, catheter-resistance monitoring is feasible, acceptable, and predicts imminent catheter occlusion. Larger studies are required to validate these findings, assess the predictive value for other clinical outcomes, and determine the impact of pre-emptive therapy. TRIAL REGISTRATION: Clinicaltrials.gov NCT01737554.

Natural killer cell therapy in children with relapsed leukemia.

BACKGROUND: Novel therapies are needed for children with relapsed or refractory leukemia. We therefore tested the safety and feasibility of haploidentical natural killer cell therapy in this patient population. PROCEDURE: Twenty-nine children who had relapsed or refractory leukemia were treated with chemotherapy followed by the infusion of haploidentical NK cells. Cohort 1 included 14 children who had not undergone prior allogeneic hematopoietic cell transplantation (HCT), whereas Cohort 2 included 15 children with leukemia that had relapsed after HCT. RESULTS: Twenty-six (90%) NK donors were KIR mismatched (14 with one KIR and 12 with 2 KIRs). The peak NK chimerism levels were >10% donor in 87% of the evaluable recipients. In Cohort 1, 10 had responsive disease and 12 proceeded to HCT thereafter. Currently, 5 (36%) are alive without leukemia. In Cohort 2, 10 had responsive disease after NK therapy and successfully proceeded to second HCT. At present, 4 (27%) are alive and leukemia-free. The NK cell infusions and the IL-2 injections were well-tolerated. CONCLUSIONS: NK cell therapy is safe, feasible, and should be further investigated in patients with chemotherapy-resistant leukemia.

Haploidentical stem cell transplantation augmented by CD45RA negative lymphocytes provides rapid engraftment and excellent tolerability.

BACKGROUND: Haploidentical donors are being increasingly used for allogeneic hematopoietic cell transplantation (HCT). However, the requisite T-cell depletion results in a profound and often long-lasting immunocompromised state, and donor lymphocyte infusions bring a risk of graft-versus-host disease (GVHD). Naïve T-cells are believed to be among the most alloreactive T-cell subset and can be identified by CD45RA expression. Allogeneic HCT using CD45RA depletion has not been previously described for haploidentical donors. PROCEDURE: Eight children with relapsed or refractory solid tumors were transplanted following myeloablative conditioning. Each patient received two cell products, one created by CD3 depletion and the other through CD45RA depletion. RESULTS: Median CD34 recovery was 59.2% with CD45RA depletion, compared to 82.4% using CD3 depletion. Median CD3+ T-cell dose after CD45RA reduction was 99.2 × 10(6) cells/kg, yet depletion of CD3+ CD45RA+ cells exceeded 4.5 log. CD45RA depletion also resulted in substantial depletion of B-cells (median 2.45 log). All eight patients engrafted within 14 days and rapidly achieved 100% donor chimerism. No acute GVHD or secondary graft failure was observed. CONCLUSIONS: CD45RA depletion is a novel approach to haploidentical HCT that offers rapid engraftment with minimal risk of GVHD.

Autonomous growth and increased cytotoxicity of natural killer cells expressing membrane-bound interleukin-15.

Natural killer (NK) cell survival and, hence, cytotoxicity requires cytokine support. We determined whether expression of interleukin-15 (IL-15) in a nonsecretory, membrane-bound form could sustain NK cell growth. We linked the human IL15 gene to that encoding CD8α transmembrane domain (mbIL15). After retroviral transduction, human NK cells expressed mbIL15 on the cell surface; IL-15 secretion was negligible. Survival of mbIL15-NK cells without interleukin-2 (IL-2) after 7-day culture was vastly superior to that of mock-transduced NK cells (P < .001, n = 15) and of NK cells expressing nonmembrane-bound IL-15 (P = .025, n = 9); viable mbIL15-NK cells were detectable for up to 2 months. In immunodeficient mice, mbIL15-NK cells expanded without IL-2 and were detectable in all tissues examined (except brain) in much higher numbers than mock-transduced NK cells (P < .001). Expansion further increased with IL-2. The primary mechanism of mbIL15 stimulation was autocrine; it activated IL-15 signaling and antiapoptotic signaling. NK cells expressing mbIL15 had higher cytotoxicity against leukemia, lymphoma, and solid tumor cells in vitro and against leukemia and sarcoma cells in xenograft models. Thus, mbIL15 confers independent growth to NK cells and enhances their antitumor capacity. Infusion of mbIL15-NK cells would allow NK cell therapy without the potential adverse effects of cytokine administration.

Phase I study of the safety and pharmacokinetics of plerixafor in children undergoing a second allogeneic hematopoietic stem cell transplantation for relapsed or refractory leukemia.

The safety, pharmacokinetics, and biological effect of plerixafor in children as part of a conditioning regimen for chemo-sensitization in allogeneic hematopoietic stem cell transplantation (HSCT) have not been studied. This is a phase I study of plerixafor designed to evaluate its tolerability at dose of .24 mg/kg given intravenously on day -4 (level 1); day -4 and day -3 (level 2); or day -4, day -3, and day -2 (level 3) in combination with fludarabine, thiotepa, melphalan, and rabbit antithymocytic globulin for a second allogeneic HSCT in children with refractory or relapsed leukemia. Immunophenotype analysis was performed on blood and bone marrow before and after plerixafor administration. Twelve patients were enrolled. Plerixafor at all 3 levels was well tolerated without dose-limiting toxicity. Transient gastrointestinal side effects of National Cancer Institute-grade 1 or 2 in severity were the most common adverse events. The area under the concentration-time curve increased proportionally to the dose level. Plerixafor clearance was higher in males and increased linearly with body weight and glomerular filtration rate. The clearance decreased and the elimination half-life increased significantly from dose level 1 to 3 (P < .001). Biologically, the proportion of CXCR4(+) blasts and lymphocytes both in the bone marrow and peripheral blood increased after plerixafor administration.

Long-term outcome and evaluation of organ function in pediatric patients undergoing haploidentical and matched related hematopoietic cell transplantation for sickle cell disease.

HLA-matched related donor (MRD) hematopoietic stem cell transplantation (HSCT) is a well-established therapy for patients with sickle cell disease (SCD); however, experience using alternative donors, including haploidentical donors, in HSCT for SCD is limited. We report the long-term outcomes of 22 pediatric patients who underwent related donor HSCT for SCD at St. Jude Children's Research Hospital, either a myeloablative sibling MRD HSCT (n = 14) or reduced-intensity parental haploidentical donor HSCT (n = 8). The median patient age was 11.0 ± 3.9 years in the MRD graft recipients and 9.0 ± 5.0 years in the haploidentical donor graft recipients. The median follow-up was 9.0 ± 2.3 years, with an overall survival (OS) of 93% and a recurrence/graft failure rate of 0%, for the MRD cohort and 7.4 ± 2.4 years, with an OS of 75%, disease-free survival of 38%, and disease recurrence of 38%, for the haploidentical donor cohort. We report the long-term hematologic response and organ function in patients undergoing MRD or haploidentical donor HSCT for severe SCD. Our data demonstrate long-term hematologic improvements after HSCT with sustained engraftment, and confirm that HSCT offers long-term protection from common complications of SCD, including stroke, pulmonary hypertension, acute chest, and nephropathy, regardless of donor source.

Successful allogeneic hematopoietic cell engraftment after a minimal conditioning regimen in children with relapsed or refractory solid tumors.

Children with relapsed or refractory solid tumors face dismal prognoses, and novel therapies are desperately needed. Allogeneic hematopoietic cell transplantation (HCT) offers potential for cell-based therapy, but the toxicity of myeloablation limits this approach in heavily pretreated patients. We sought to determine the feasibility of HCT in a cohort of 24 children with incurable solid tumors using human leukocyte antigen-matched sibling or unrelated donors and a minimal conditioning regimen. Before stem cell infusion, all patients received 3 daily doses of 30 mg/m(2) fludarabine followed by 2 Gy of total body irradiation. Hematopoietic cell recovery was rapid and reliable. Median time to neutrophil engraftment was 13.5 days for sibling donors and 12 days for unrelated donors. Donor lymphocyte infusions were used safely in 4 patients, all of whom had either improved chimerism or apparent tumor response. Graft-versus-host disease was comparable across donor sources and did not affect survival. Relapse remains a substantial barrier, although objective graft-versus-tumor effect was observed in several patients. Four patients with detectable disease before HCT achieved a complete response for at least 30 days after HCT, and two remain long-term survivors. Three patients were in complete response before HCT and remained in remission for 3, 6, and 74 months after HCT. Early disease response was associated with improved survival. Allogeneic HCT using this conditioning regimen offers a potential platform for novel immunotherapies.

Longitudinal changes in body mass and composition in survivors of childhood hematologic malignancies after allogeneic hematopoietic stem-cell transplantation.

PURPOSE: To measure longitudinal changes in body mass and composition in survivors of childhood hematologic malignancies after allogeneic hematopoietic stem-cell transplantation (HSCT). PATIENTS AND METHODS: Body mass index (BMI) was analyzed in 179 survivors by category (underweight, healthy-weight, overweight, and obese) and by z score. Fat and lean body mass measured by dual-energy x-ray absorptiometry was analyzed as z scores. RESULTS: Over a median 6.6 years of follow-up, BMI z scores diminished significantly (0.32 pre-HSCT v -0.60 at 10 years post-HSCT; P < .001). Mean z scores for fat mass stayed within population norms, but those for lean mass remained below normal levels and diminished significantly over time (P = .018). Pre-HSCT BMI category and/or z score were strongly predictive of post-HSCT BMI (P < .001) and of fat and lean mass z scores (both P < .001). Survivors with extensive chronic graft-versus-host disease were more likely than others to have low BMI (P = .004) and low lean mass (P < .001) post-HSCT. Older age at HSCT (P = .015) and T-cell-depleted graft (P = .018) were predictive of lower post-HSCT BMI. Female patients had higher body fat (P = .002) and lower lean mass (P = .013) z scores than male patients, and black patients had higher fat mass z scores than white patients (P = .026). CONCLUSION: BMI declines significantly after allogeneic HSCT for childhood hematologic malignancies, reflecting primarily a substantial decrease in lean mass but not fat mass. Monitoring and preservation of BMI and lean mass are vital, especially in those with the identified risk factors.

Effects of conditioning regimens and T cell depletion in hematopoietic cell transplantation for primary immune deficiency.

This study analyzes the hematopoietic cell transplantation experience in patients with immune deficiency at a single institution. The objective is to comprehensively evaluate the short-term and long-term outcomes with various preparative regimens, donor grafts, and ex vivo manipulations to identify transplantation approaches that most likely favor early donor immune competency without generating excessive toxicity. Clinical outcomes were evaluated in 52 consecutive patients with immune deficiencies. Thirty-seven of the 52 patients (71%) survived with attenuation of their underlying disease. The use of a melphalan-based reduced-intensity conditioning preparative regimen and immunomagnetic CD3(+) T cell depletion techniques (when T cell depletion was indicated) were associated with improved event-free survival. Survivors who received a preparative regimen other than a melphalan-based reduced-intensity regimen suffered from therapy-related morbidities or chronic/recurrent infections. Our findings indicate that melphalan-based reduced-intensity conditioning regimens and immunomagnetic CD3(+) T cell depletion limit therapy-related toxicity, and demonstrate promising results for the early establishment of donor immune competency.