Cytotherapy, 2014; 16: 1528e1536

Expanded umbilical cord blood T cells used as donor lymphocyte infusions after umbilical cord blood transplantation

SOFIA BERGLUND1,2, JENS GERTOW2, MICHAEL UHLIN1,2 & JONAS MATTSSON1,2 1 2

Center for Allogeneic Stem Cell Transplantation, Karolinska University Hospital, Stockholm, Sweden, and Department of Laboratory Medicine, Division of Therapeutic Immunology, Karolinska Institutet, Stockholm, Sweden

Abstract Background. Umbilical cord blood (UCB) is an alternative graft source for hematopoietic stem cell transplantation and has been shown to give results comparable to transplantation with other stem cell sources. Donor lymphocyte infusion (DLI) is an effective treatment for relapsed malignancies after hematopoietic stem cell transplantation. However, DLI is not available after UCB transplantation. Methods. In this study, in vitroecultured T cells from the UCB graft were explored as an alternative to conventional DLI. The main aim was to study the safety of the cultured UCB T cells used as DLI because such cell preparations have not been used in this context previously. We also assessed potential benefits of the treatment. Results. The cultured UCB T cells (UCB DLI) were given to 4 patients with mixed chimerism (n ¼ 2), minimal residual disease (n ¼ 1) and graft failure (n ¼ 1). No adverse reactions were seen at transfusion. Three of the patients did not show any signs of graft-versus-host disease (GVHD) after UCB DLI, but GVHD could not be excluded in the last patient. In the patient with minimal residual disease treated with UCB DLI, the malignant cell clone was detectable shortly before infusion but undetectable at treatment and for 3 months after infusion. In 1 patient with mixed chimerism, the percentage of recipient cells decreased in temporal association with UCB DLI treatment. Conclusions. We saw no certain adverse effects of treatment with UCB DLI. Events that could indicate possible benefits were seen but with no certain causal association with the treatment. Key Words: cell culture, DLI, T-cell expansion, umbilical cord blood

Introduction Allogeneic hematopoietic stem cell transplantation (HSCT) with umbilical cord blood used as a stem cell source has been shown to give results comparable to bone marrow or peripheral blood stem cells [1e3]. Umbilical cord blood transplantation (UCBT) is associated with fewer incidences of graftversus-host disease (GVHD) and permissiveness for greater human leukocyte antigen (HLA) disparity between donor and recipient [3,4]. However, the smaller cell dose combined with the naivety of the immune cells results in delayed engraftment and immune reconstitution [5e8]. Donor lymphocyte infusion (DLI) is a treatment for relapsed or residual malignancies after HSCT. DLI has also been successfully used to treat threatening graft rejection and mixed donor-recipient chimerism [9e11]. Patients receiving UCBT lack the possibility of receiving DLI [12]. In vitroecultured cord blood DLI (UCB DLI) presents an attractive option for patients with complications after UCBT [13e16]. Sufficient numbers of T cells for DLI can

be obtained with the use of an aliquot of a clinical UCB graft by means of Good Manufacturing Practiceegrade in vitro culture through cluster of differentiation (CD)3/CD28 cross-linking and clinical-grade interleukin-2 (IL-2) [13,16]. The cultured T cells have an activated phenotype and respond functionally to re-stimulation with non-specific mitogenic stimuli in vitro [13,16,17]. Addition of clinical-grade interleukin-7 (IL-7) to the culture protocol has been explored and was found to enhance proliferation and reduce apoptosis [18,19]. The present study was performed with the aim of evaluating the safety of the in vitroeproduced UCB DLI as an alternative to conventional DLI. To our knowledge, cell infusions prepared in this manner have not been administered to patients in this context previously. We also studied potentially beneficial effects of the infusions. Methods We performed a preliminary safety study of treatment with cultured umbilical cord blood T cells, in

Correspondence: Sofia Berglund, MD, Division of Therapeutic Immunology, F79, Karolinska University Hospital Huddinge, 14186 Stockholm, Sweden. E-mail: sofi[email protected] (Received 11 April 2014; accepted 9 August 2014) http://dx.doi.org/10.1016/j.jcyt.2014.08.001 ISSN 1465-3249 Copyright Ó 2014, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Expanded UCB T cells used as DLI after UCBT accordance with the European hospital exception rules. The regional ethics committee approved the study (2009/4:1), including the extraction of aliquots from clinical cord blood units, the culture procedure and use of the UCB DLI. All patients, or the legal guardians in the case of minors, gave their informed consent before inclusion. Patients and cord blood units Five percent of the volume of every UCB graft was collected at transplantation for T-cell expansion of UCB DLI at the Centre for Allogeneic Stem cell Transplantation (CAST), Karolinska University Hospital Huddinge, Stockholm, Sweden, and for two grafts at the Department of Hematology at Sahlgrenska University Hospital, Gothenburg, Sweden. In total, cultures were performed for 40 CB units (31 patients, 33 UCBTs). The expansions were given consecutive numbers as they were collected. The median total nucleated cell count in the aliquot was 60.5 million (range, 20e154). Cell culture The expansion of UCB-derived T cells has been described previously [16,18]. Briefly, T cells were positively selected and stimulated with anti-CD3/ CD28 paramagnetic beads (Life Technologies [Invitrogen], Grand Island, NY, USA). The median yield of T cells after separation was 2.7 million (range, 0.25e28). T cells were cultured at a concentration of 3  105 cells/mL in complete medium: 1640 Roswell Park Memorial Institute medium (Life Technologies [Gibco]) supplemented with 10% pooled human AB serum (Department of Transfusion Medicine at Karolinska University Hospital), 100 IU/mL of penicillin G, 100 mg/mL of streptomycin, 0.25 mg/mL amphotericin B (Life Technologies [Gibco]) and 2 mmol/L L-glutamine (Sigma Aldrich Inc, St Louis, MO, USA). Recombinant IL-2 (PeproTech, Rocky Hill, NJ, USA) was added at 600 IU/mL until expansion number 33. From expansion number 34, 100 IU/mL of IL-2 was combined with IL-7 (20 ng/mL) (PeproTech). Cell culture was performed at 37 C at 5% CO2. Viable cells were counted with the use of trypan blue exclusion on days 4, 5, 6 and 7, and cell concentration was maintained at 0.5  109/L) up to 3 months and at 6, 9, 12, 18 and 24 months and annually thereafter. Polymerase chain reaction amplification of variable numbers of tandem repeats was used to evaluate donor and recipient chimerism in CD3þ, CD19þ and CD33þ cells enriched from

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Table I. Patient characteristics and UCB DLI composition.

Gender Age Diagnosis Single/Double UCBT HLA-match Conditioning ATG-dose (mg/kg) GVHD prophylaxis Cell dose: TNC (107/kg) CD34þ (105/kg) Engraftment: Neutrophils (>0.2  109/L) Platelets (>30  109/L) Acute GVHD; Chronic GVHD CoD, time of death (days) Time of follow up (days) Relapse (Yes/No) Cause of DLI treatment Day of DLI (CD3þ cells/kg)

Adverse effects; Response Phenotype of DLI: CD4/CD8 ratio Memory T cell stages, (% of CD3þ/CD4þ/CD8þ) Central memory T cells (CD45ROþ CCR7þ) Effector memory T cells (CD45ROþ CCR7-) YdþT cells (% out of CD3þ)

Patient 1

Patient 2

Patient 3

Patient 4

M 43 AML Double 5/6, 5/6 BuþCy 7,8 TacþSir

M 17 Pre-B ALL Single 4/6 CloþThiþMel 5,5 CyAþPred

F 17 PNH/MDS Single 5/6 BuþCy 5,8 CyAþPred

F 52 Pre-B ALL Single 5/6 CyþfTBI 6 CyAþPred

3,8 1,5

2,4 0,5

17 34 No; No þ1825 Yes MC þ 114 (5  103) þ 141 (1  104) þ 174 (1  105) þ202 (5  105) No; No obvious

43 No certain; No Infection, þ116 No Graft failure þ48 (1  105) þ66 (1  106) þ98 (1  106) No; No obvious

3 0,9

3,7 3

16 41 Grade I skin; No 1280 No MC þ59 (1  105)

Yes MRD þ 137 (1  105)

No; Possible

No; Possible

3

12 66 No; No Infection, þ288

1,1

1,1

13/21/11

2/3/2

2/6/9

5/2/14

86/79/89

91/91/90

90/92/72

92/96/73

0,2

1,5

1,4

4,7

2

Patient characteristics and information regarding CD DLI doses for the 4 patients are listed.ALL, acute lymphatic leukemia; AML, acute myeloid leukemia; ATG, anti-thymocyte globulin; Bu, busulfan; CB, cord blood; Clo, clofarabine; CR, complete remission; CoD, cause of death; Cy, cyclophosphamide; CyA, cyclosporine A; fTBI, fractionated total body irradiation; MC, mixed chimerism; MDS, myelodysplastic syndrome; Mel, melphalan; NA, not assessable; PNH, paroxysmal nocturnal hemoglobinuria; Pred, prednisone; Sir, sirolimus; Tac, tacrolimus; TCR, T-cell receptor; Thi, thiotepa; TNC, total nucleated cells.

peripheral blood with the use of immunomagnetic beads (Dynal, Oslo, Norway) [28]. Two methods were used: one was based on mini-satellites [28] and a real-time polymerase chain reaction approach that was based on single-nucleotide polymorphism [29]. Complete donor chimerism was defined as >95% donor cells. Mixed chimerism was defined as 5% donor cells. Morphological analysis and flow cytometry were performed on bone marrow samples for assessment of the underlying hematological disease. Eligibility and treatment with UCB DLI UCB DLI treatment was opted for in cases of a high or increasing proportion of recipient chimerism, in the case of detection of minimal residual disease (MRD) and once in primary graft failure. UCB DLI, like unmanipulated DLI, could also be used for

relapse of hematological malignancy, but this did not occur in the present study. Signs of active GVHD would have been contra-indicative to treatment with UCB DLI. However, no patient eligible for UCB DLI showed any signs of active GVHD at treatment. The dosing scheme for UCB DLI was designed through the use of previous experience of conventional DLI, with focus on DLI after HLA-mismatched HSCT [11,30,31]. The starting dose was set comparatively low, 5  103/kg patient weight, in consideration of (i) the activated phenotype of the cells [16] and (ii) the presence of HLA mismatches. Both these factors could be speculated to increase the risk of GVHD. Dose escalation by 0.5e1 logs was performed in the case of insufficient response and absence of adverse effects. The minimum interval between doses was set at 3 weeks. No maximum number of UCB DLI infusions was specified, but the number of doses was limited by the available

Expanded UCB T cells used as DLI after UCBT cryopreserved cells; the largest dose was 5  105/kg to 1  106/kg patient weight. The starting dose was increased to 1  105/kg patient weight after the first patient because of a lack of either positive or negative effects of the infused UCB DLI at any given dose. Processing of UCB DLI units at infusion The frozen UCB DLI units were thawed and washed twice in a 9-mg/mL sodium chloride solution with 5% human albumin added (Baxter Medical AB, Kista, Sweden) before injection. Samples were taken for microbiological testing at (i) the end of culture and (ii) at thawing before infusion to exclude contamination. The tests had to be confirmed as negative before the release of a UCB DLI unit. Results Four patients were treated with UCB DLI for increasing recipient chimerism (n ¼ 2), MRD (n ¼ 1) or graft failure (n ¼ 1). Patient characteristics and UCB DLI composition are listed in Table I. Patient 1 The first patient was a 43-year-old man with acute myeloid leukemia. He underwent a double cord blood transplantation in first complete remission (Table I). From early after transplantation, there was a high proportion of recipient cells in the CD3þ cell lineage in peripheral blood, as measured by chimerism analysis. At day þ108 after UCBT, a majority of cells were of recipient origin also in the CD33þ cell lineage (Figure 1A). The immunosuppressive treatment is shown in Figure 1B. To prevent a threatening rejection in the absence of other treatment alternatives, the patient received UCB DLI from the dominating transplanted CB unit on day þ114 (5  103 CD3þ cells/kg patient weight), day þ141 (1  104 CD3þ cells/kg), day þ174 (1  105 CD3þ cells/kg) and day þ202 (5  105 CD3þ cells/kg) (Table I). The initial doses were lower than in the standard DLI protocol because this was the first treatment of cells produced in this manner ever administered and in consideration of the activated state of the UCB DLI. No adverse reaction occurred at UCB DLI treatment. No clear effects on the chimerism development could be seen after treatment. Patient 2 Patient 2 was a 17-year-old male with Pre-B acute lymphatic leukemia. He was diagnosed at 2 years of age and received standard chemotherapy resulting in remission. However, he relapsed at the age of 14 years and underwent UCBT. A second relapse 2.5 years after transplantation led to re-transplantation

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with a second UCBT. He then developed graft failure. The majority of the peripheral blood cells were of donor origin, as assessed by chimerism analysis (Figure 1C). The immunosuppressive treatment is depicted in Figure 1D. The graft failure caused a severe immune deficiency: the patient had development of an acyclovir-resistant herpes simplex virus infection and a severe adenovirus infection. The treatment alternatives were few: a stem cell booster was not possible after UCBT, and re-transplantation was not considered an option. To support the patient’s immune system, 3 doses of UCB DLI were given on day þ48 (1  105 CD3þ cells/kg), day þ66 (1  106 CD3þ/kg) and day þ98 (1  106 CD3þ/kg) (Table I). No adverse reaction was seen at infusion. The patient had diarrhea and skin rash 2 days before the second infusion, which resolved after 7 days. Similar symptoms recurred 4 days before the third UCB DLI dose and became persistent. Bilirubin levels increased at day þ86. These symptoms probably were signs of the adenovirus infection, but GVHD could not be wholly excluded. Invasive diagnostics were impossible because of his poor physical status. The patient had multiple infections and died of septicemia on day þ116. Patient 3 The third patient was a 17-year-old female with paroxysmal nocturnal hemoglobinuria and suspected development of myelodysplastic syndrome. At diagnosis, the paroxysmal nocturnal hemoglobinuria clone was >50%, and the patient had anemia and leukopenia with marked neutropenia, interpreted as myelodysplastic syndrome. The patient was transplanted with a single UCB unit. Soon after transplantation, she had development of acute GVHD grade I of the skin that was successfully treated with an increase of the prednisone dosage for a short period of time (Figure 2B). After transplantation, chimerism analysis showed an increasing percentage of CD3þ cells of recipient origin. In the absence of other effective treatment alternatives, the patient received 1 dose of UCB DLI on day þ59 (1  105 CD3þ cells/kg). There was no adverse reaction to the infusion (Table I). Shortly after this point, there was a decrease in the fraction of recipient cells in the CD19þ cell lineage and in the CD3þ cell lineage, after an initial increase, until plateauing on the current level approximately 10% (Figure 2A). The patient was doing well 42 months after UCBT. Patient 4 This patient was a 52-year-old woman with Pre-B acute lymphatic leukemia. She was initially treated

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Figure 1. Chimerism development and immunosuppressive treatment shown for patient 1 and patient 2. (A, C) Results of chimerism analysis over time, presented as percentage of recipient cells, performed on peripheral blood for CD19þ, CD3þ and CD33þ cells. Patient 1 received double cord blood transplantation, and the percentage of non-recipient cells (not plotted) was made up by the summed proportions of both donors until 180 days after transplantation, after which only 1 donor remained. (B, D) Immunosuppressive treatment over time, with prednisone, combined with tacrolimus and sirolimus for patient 1 and cyclosporine A for patient 2. Arrows show when cultured cord blood donor lymphocyte infusions were given.

according to standard protocol with intravenous and intrathecal chemotherapy. After 22 months, she had a relapse and underwent UCBT. An MRD of 0.05% was present in the last bone marrow aspirate before transplantation. The patient had an early cytomegalovirus re-activation at day þ6 and then had development of infections with respiratory syncytial virus, parainfluenza virus and human herpes virus 6. All four infections persisted for several months. A computer

tomography of the chest performed at day þ180 showed infiltrates of suspected fungal origin. Chimerism analysis showed a majority of donor cells in all cell lineages in peripheral blood (Figure 2C) during the whole post-transplantation period. An MRD of 0.05% was detectable at day þ100 after UCBT (previous analyses after UCBT had not detected any MRD), and, because no other practicable treatment alternatives were available, UCB DLI was given on day þ137

Expanded UCB T cells used as DLI after UCBT

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Figure 2. Chimerism development and immunosuppressive treatment of patient 3 and patient 4. (A, C) Results of chimerism analysis over time, presented as percentage of recipient cells, performed for the CD19þ, CD3þ and CD33þ cell lineages in peripheral blood. (C) Development of MRD, as assessed by flow cytometry, is shown for patient 4. Inset in (C) shows the time span between day þ75 and day þ180 to illustrate the variations of recipient chimerism and MRD. (B, D) Immunosuppressive treatment over time, with prednisone and cyclosporine A. Arrows show when the cultured cord blood donor lymphocyte infusions were given.

(1  105 CD3þ cells/kg, Table I). At the time of infusion, MRD was undetectable, and it continued to be undetectable for 5 months until day þ274, when the patient had a morphological relapse (Figure 2C). She died of fungal pneumonia 3 weeks later. The immunosuppressive treatment is shown in Figure 2D. Discussion DLI is an important treatment in relapse and rejection after HSCT. However, conventional DLI

is not available after UCBT. As the number of UCBTs performed rises, it becomes increasingly important to explore possible alternatives. One such option is UCB lymphocytes cultured in vitro. We and others have shown that the use of antieCD3-/ CD28-coated paramagnetic beads and IL-2 [13,16], or IL-2 in combination with IL-7 [18], it is possible to produce adequate numbers of T cells for DLI. However, to our knowledge, in vitroecultured UCB T cells produced in this manner have not been used for clinical DLI treatment previously. The only

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previously reported case of treatment with cultured UCB cells after UCBT concerns CD4þ T cells for mixed chimerism in a patient with Omenn syndrome [32]. In vitro stimulation of conventional DLI has been explored previously with the aim of improving the outcome in patients with a relapse of hematological malignancies known to have a low response rate to conventional DLI [17]. In that study, 8 of 18 patients achieved a complete response, which indicates that the treatment was feasible and even beneficial. However, UCB lymphocytes pose additional challenges. The number of lymphocytes available is limited: the few millions of T cells present in 5% of the graft constitute the available material in the present study. Cord blood lymphocytes appear to be inefficient cytokine producers [33,34] and have reduced cytolytic activity [35]. The culture process alleviates some of these deficiencies: the number of cells attained is feasible for DLI use, and the cultured T cells have the capacity for cytokine production and re-activation to stimuli [16,18]. The in vivo effects are, however, difficult to predict because the in vitro culture environment is markedly different from the in vivo conditions. Cytokine deprivation could induce apoptosis in the transferred cells [36,37], and in vitro expansion may cause exhaustion of proliferative capacity [38]. We have tried to counteract this by lowering the concentrations of IL-2 in culture media and adding IL-7 to the culture [18]. IL-7 is known to have anti-apoptotic effects in T cells [39]. However, because only 1 patient (patient 4) has been treated with cells cultured according to the modified protocol, no comparison is possible. There were no adverse reactions observed at infusion of UCB DLI. Positive clinical events that may indicate benefit of the treatment could be seen in 2 of the patients. Patient 3, who was treated with UCB DLI because of mixed chimerism, had decreased levels of recipient cells in the CD19þ cell lineage and, after an initial rise, also in the CD3þ cell lineage, after the infusion (Figure 2A,B). Patient 4 (Figure 2C,D) received UCB DLI because of the recurrence of detectable MRD. After a single UCB DLI dose, no MRD was detectable for several months. It should be noted that the MRD was already negative just before infusion. Unfortunately, the patient had a hematological relapse later without any preceding molecular signs of disease recurrence. It is arguable whether patient 4 could have benefited from more UCB DLI infusions. However, there is no certain causal association between those positive developments and the treatment. No signs indicating possible benefit of the treatment were seen in the 2 remaining patients.

Patient 1, who received treatment because of mixed chimerism, continued to have high levels of recipient chimerism after UCB DLI (Figure 1A,B). However, it is known that the chance of success is small with conventional DLI treatment of mixed chimerism with high levels of recipient cells [10,11]. In patient 2 (Figure 1C,D), treated for graft failure and severe infections, there were no obvious effects of the UCB DLI treatment on the status of his infections. However, the patient’s critical situation would have been difficult to ameliorate with any means. The cultured T cells of the UCB DLI had an activated phenotype. The memory subset markers shifted from a naive phenotype toward a memory phenotype with a predominance of effector memory T cells expressing CD45RO but not CCR7 (Table I) [16,18,20]. The CD4/CD8 ratio was higher in the units given to patients 3 and 4 (Table I). However, with only 4 cases, no certain conclusion can be drawn regarding the in vivo effects of phenotypical differences between units. Conventional DLI has long been known to induce GVHD [10,40] [at our center, 34% of DLI recipients have development of acute GVHD [11]]. It can be speculated that this risk increases with activated lymphocyte treatment. Consequently, the risk of GVHD development was a concern in this study. However, this was not the case in a study regarding activated conventional DLI [17]. In the present study, 1 patient (patient 2) had symptoms consistent with acute GVHD, but these symptoms predated the UCB DLI infusion and could have been caused by adenovirus infection. Unfortunately, because of the patient’s poor status, the diagnosis could not be clarified. The cost-effectiveness of UCB DLI requires consideration in the event of a transfer into clinical routine. The cost of culturing cells at every UCBT should be weighed against potential benefits. Currently, approximately 10% of patients transplanted with graft sources other than UCB receive DLI at our center. A similar or larger fraction [considering the selection of high-risk patients in this group [5]] can be expected to need DLI after UCBT. If UCB DLI is effective, we believe that the cost is justified by the lives that could be saved. To conclude, we have seen no certain adverse effects of the UCB DLI. There were some positive clinical events in 2 of the patients that could indicate benefits of the treatment, but with no certain causal association with the UCB DLI infusions. The material of this study is small, and the results should be viewed as preliminary and interpreted with caution.

Expanded UCB T cells used as DLI after UCBT Acknowledgments The authors thank the staff at CAST, the respective staff at the departments of Hematology, Pediatrics and Stem cell Laboratory at Karolinska University Hospital Huddinge, and the staff at the Hematology Department at Sahlgrenska University Hospital, in particular Dr Mats Brune, for their cooperation, compassionate care of the patients and optimal handling of the umbilical cord blood units. This study was supported by grants from the Swedish Research Council (K2012e64X-22020e01e3 and 2011e2377), the Swedish Childhood Cancer Foundation (PROJ10/052 and PROJ12/006) and E och KG Lennanders stipendiestiftelse. Disclosure of interests: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article. References [1] Hwang WY, Samuel M, Tan D, Koh LP, Lim W, Linn YC. A meta-analysis of unrelated donor umbilical cord blood transplantation versus unrelated donor bone marrow transplantation in adult and pediatric patients. Biol Blood Marrow Transplant 2007;13:444e53. [2] Majhail NS, Brunstein CG, Wagner JE. Double umbilical cord blood transplantation. Curr Opin Immunol 2006;18: 571e5. [3] McKenna DH, Brunstein CG. Umbilical cord blood: current status and future directions. Vox Sang 2011;100:150e62. [4] Zhong XY, Zhang B, Asadollahi R, Low SH, Holzgreve W. Umbilical cord blood stem cells: what to expect. Ann N Y Acad Sci 2010;1205:17e22. [5] Berglund S, Le Blanc K, Remberger M, Gertow J, Uzunel M, Svenberg P, et al. Factors with an impact on chimerism development and long-term survival after umbilical cord blood transplantation. Transplantation 2012;94:1066e74. [6] Escalon MP, Komanduri KV. Cord blood transplantation: evolving strategies to improve engraftment and immune reconstitution. Curr Opin Oncol 2010;22:122e9. [7] Barker JN, Rocha V, Scaradavou A. Optimizing unrelated donor cord blood transplantation. Biol Blood Marrow Transplant 2009;15(Suppl. 1):154e61. [8] Chan KW, Grimley MS, Taylor C, Wall DA. Early identification and management of graft failure after unrelated cord blood transplantation. Bone Marrow Transplant 2008;42: 35e41. [9] Bader P, Willasch A, Klingebiel T. Monitoring of posttransplant remission of childhood malignancies: is there a standard? Bone Marrow Transplantation 2008;42(Suppl. 2): S31e4. [10] Kolb HJ, Schattenberg A, Goldman JM, Hertenstein B, Jacobsen N, Arcese W, et al. Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients. Blood 1995;86:2041e50. [11] Sairafi D, Remberger M, Uhlin M, Ljungman P, Ringden O, Mattsson J. Leukemia lineage-specific chimerism analysis and molecular monitoring improve outcome of donor lymphocyte infusions. Biol Blood Marrow Transplant 2010; 16:1728e37.

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[12] Szabolcs P, Cairo MS. Unrelated umbilical cord blood transplantation and immune reconstitution. Semin Hematol 2010;47:22e36. [13] Mazur MA, Davis CC, Szabolcs P. Ex vivo expansion and Th1/Tc1 maturation of umbilical cord blood T cells by CD3/ CD28 costimulation. Biol Blood Marrow Transplant 2008; 14:1190e6. [14] Parmar S, Robinson SN, Komanduri K, St John L, Decker W, Xing D, et al. Ex vivo expanded umbilical cord blood T cells maintain naive phenotype and TCR diversity. Cytotherapy 2006;8:149e57. [15] Micklethwaite KP, Savoldo B, Hanley PJ, Leen AM, Demmler-Harrison GJ, Cooper LJ, et al. Derivation of human T lymphocytes from cord blood and peripheral blood with antiviral and antileukemic specificity from a single culture as protection against infection and relapse after stem cell transplantation. Blood 2010;115:2695e703. [16] Okas M, Gertow J, Uzunel M, Karlsson H, Westgren M, Karre K, et al. Clinical expansion of cord blood-derived T cells for use as donor lymphocyte infusion after cord blood transplantation. J Immunother 2010;33:96e105. [17] Porter DL, Levine BL, Bunin N, Stadtmauer EA, Luger SM, Goldstein S, et al. A phase 1 trial of donor lymphocyte infusions expanded and activated ex vivo via CD3/CD28 costimulation. Blood 2006;107:1325e31. [18] Berglund S, Gertow J, Magalhaes I, Mattsson J, Uhlin M. Cord blood T cells cultured with IL-7 in addition to IL-2 exhibit a higher degree of polyfunctionality and superior proliferation potential. J Immunother 2013;36:432e41. [19] Davis CC, Marti LC, Sempowski GD, Jeyaraj DA, Szabolcs P. Interleukin-7 permits Th1/Tc1 maturation and promotes ex vivo expansion of cord blood T cells: a critical step toward adoptive immunotherapy after cord blood transplantation. Cancer Res 2010;70:5249e58. [20] Gertow J, Berglund S, Okas M, Karre K, Remberger M, Mattsson J, et al. Expansion of T-cells from the cord blood graft as a predictive tool for complications and outcome of cord blood transplantation. Clin Immunol 2012;143: 134e44. [21] Abdel-Rehim M, Hassan Z, Blomberg L, Hassan M. On-line derivatization utilizing solid-phase microextraction (SPME) for determination of busulphan in plasma using gas chromatography-mass spectrometry (GC-MS). Therap Drug Monit 2003;25:400e6. [22] Ringden O, Remberger M, Persson U, Ljungman P, Aldener A, Andstrom E, et al. Similar incidence of graftversus-host disease using HLA-A, -B and -DR identical unrelated bone marrow donors as with HLA-identical siblings. Bone Marrow Transplant 1995;15:619e25. [23] Remberger M, Svahn BM, Mattsson J, Ringden O. Dose study of thymoglobulin during conditioning for unrelated donor allogeneic stem-cell transplantation. Transplantation 2004;78:122e7. [24] Glucksberg H, Storb R, Fefer A, Buckner CD, Neiman PE, Clift RA, et al. Clinical manifestations of graft-versus-host disease in human recipients of marrow from HL-A-matched sibling donors. Transplantation 1974;18:295e304. [25] Lee SJ, Klein JP, Barrett AJ, Ringden O, Antin JH, Cahn JY, et al. Severity of chronic graft-versus-host disease: association with treatment-related mortality and relapse. Blood 2002; 100:406e14. [26] Svahn BM, Remberger M, Myrback KE, Holmberg K, Eriksson B, Hentschke P, et al. Home care during the pancytopenic phase after allogeneic hematopoietic stem cell transplantation is advantageous compared with hospital care. Blood 2002;100:4317e24.

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S. Berglund et al.

[27] Forslow U, Blennow O, LeBlanc K, Ringden O, Gustafsson B, Mattsson J, et al. Treatment with mesenchymal stromal cells is a risk factor for pneumonia-related death after allogeneic hematopoietic stem cell transplantation. Eur J Haematol 2012;89:220e7. [28] Mattsson J, Uzunel M, Tammik L, Aschan J, Ringden O. Leukemia lineage-specific chimerism analysis is a sensitive predictor of relapse in patients with acute myeloid leukemia and myelodysplastic syndrome after allogeneic stem cell transplantation. Leukemia 2001;15:1976e85. [29] Ringden O, Okas M, Uhlin M, Uzunel M, Remberger M, Mattsson J. Unrelated cord blood and mismatched unrelated volunteer donor transplants, two alternatives in patients who lack an HLA-identical donor. Bone Marrow Transplant 2008;42: 643e8. [30] Mattsson J, Uzunel M, Remberger M, Tammik L, Omazic B, Levitsky V, et al. Poor immune reconstitution after four or five major HLA antigens mismatched T celldepleted allogeneic and autologous stem cell transplantation. Clin Exp Immunol 2001;123:162e9. [31] Or R, Hadar E, Bitan M, Resnick IB, Aker M, Ackerstein A, et al. Safety and efficacy of donor lymphocyte infusions following mismatched stem cell transplantation. Biol Blood Marrow Transplant 2006;12:1295e301. [32] Tomizawa D, Aoki Y, Nagasawa M, Morio T, Kajiwara M, Sekine T, et al. Novel adopted immunotherapy for mixed chimerism after unrelated cord blood transplantation in Omenn syndrome. Eur J Haematol 2005;75:441e4.

[33] Chalmers IM, Janossy G, Contreras M, Navarrete C. Intracellular cytokine profile of cord and adult blood lymphocytes. Blood 1998;92:11e8. [34] Cohen SB, Perez-Cruz I, Fallen P, Gluckman E, Madrigal JA. Analysis of the cytokine production by cord and adult blood. Hum Immunol 1999;60:331e6. [35] Risdon G, Gaddy J, Stehman FB, Broxmeyer HE. Proliferative and cytotoxic responses of human cord blood T lymphocytes following allogeneic stimulation. Cell Immunol 1994;154:14e24. [36] Vella AT, Dow S, Potter TA, Kappler J, Marrack P. Cytokine-induced survival of activated T cells in vitro and in vivo. Proc Natl Acad Sci U S A 1998;95:3810e5. [37] Akbar AN, Borthwick NJ, Wickremasinghe RG, Panayoitidis P, Pilling D, Bofill M, et al. Interleukin-2 receptor common gamma-chain signaling cytokines regulate activated T cell apoptosis in response to growth factor withdrawal: selective induction of anti-apoptotic (bcl-2, bcl-xL) but not pro-apoptotic (bax, bcl-xS) gene expression. Eur J Immunol 1996;26:294e9. [38] Jackson SR, Berrien-Elliott MM, Meyer JM, Wherry EJ, Teague RM. CD8þ T cell exhaustion during persistent viral infection is regulated independently of the virus-specific T cell receptor. Immunol Invest 2013;42:204e20. [39] Mackall CL, Fry TJ, Gress RE. Harnessing the biology of IL-7 for therapeutic application. Nat Rev Immunol 2011;11:330e42. [40] Roddie C, Peggs KS. Donor lymphocyte infusion following allogeneic hematopoietic stem cell transplantation. Expert Opin Biol Ther 2011;11:473e87.