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Background: Transfusion-dependent β-thalassaemia (TDT) is a severe disease, resulting in lifelong blood transfusions, iron overload, and associated complications. Betibeglogene autotemcel (beti-cel) gene therapy uses autologous haematopoietic stem and progenitor cells (HSPCs) transduced with BB305 lentiviral vector to enable transfusion independence., Methods: HGB-212 was a non-randomised, multicentre, single-arm, open-label, phase 3 study of beti-cel in patients with TDT conducted at eight centres in France, Germany, Greece, Italy, the UK, and the USA. Patients with β 0 /β 0 , β 0 /β +IVS-I-110 , or β +IVS-I-110 /β +IVS-I-110 genotypes, clinically stable TDT, and a transfusion history of at least 100 mL/kg per year of packed red blood cells (pRBCs) or at least eight transfusions of pRBCs per year in the 2 years before enrolment were eligible for participation. After undergoing HSPC mobilisation and busulfan-based, pharmacokinetic-adjusted myeloablative conditioning, patients were infused with beti-cel and followed up for 24 months. The primary efficacy outcome was transfusion independence, defined as weighted average haemoglobin level of 9 g/dL or above without pRBC transfusions for 12 or more months. The primary outcome was measured in all patients who received an infusion of beti-cel (transplant population); safety was evaluated in all patients who initiated study treatment (intention-to-treat population). Patients were eligible to enrol in the ongoing 13-year long-term follow-up study (for a total of 15 years), LTF-303 (registered at ClinicalTrials.gov, NCT02633943). This trial, HGB-212, was registered at ClinicalTrials.gov (NCT03207009), and is complete., Findings: From June 8, 2017, to March 12, 2020, 20 patients were screened for eligibility. One patient was ineligible and one withdrew consent before HSPC mobilisation and myeloablative conditioning. Of the 18 patients who received beti-cel, ten (56%) were male and eight (44%) were female; 13 (72%) were younger than 18 years at the time of informed consent, and five (28%) were older than 18 years. 12 (67%) patients had β 0 /β 0 genotypes, three (17%) had β 0 / β +IVS-I-110 , and three (17%) had β +IVS-I-110 /β +IVS-I-110 . As of Jan 30, 2023, all patients enrolled in the long-term follow-up study and the median follow-up was 47·9 months (range 23·8-59·0). All 18 patients were evaluable for transfusion independence, with 16 (89%) of 18 reaching and maintaining transfusion independence to last follow=up (estimated effect size 89·9% [95% CI 65·3-98·6]). All patients had at least one adverse event after beti-cel infusion. There were no serious adverse events considered to be related to beti-cel, and no deaths., Interpretation: These data demonstrate that beti-cel can allow patients with genotypes that cause severe β-thalassaemia (β 0 /β 0 , β 0 /β +IVS-I-110 , or β +IVS-I-110 /β +IVS-I-110 ) to reach transfusion independence. Beti-cel offers the potential to attain near-normal haemoglobin levels for those with severe forms of TDT, and a potentially curative option without the risks and limitations of allogeneic HSPC transplantation. Patients are being followed up for a total of 15 years to assess the durability of transfusion independence and long-term safety profile of beti-cel., Funding: Bluebird Bio, Somerville, MA, USA., Competing Interests: Declaration of interests JLK reports research funding from Bluebird Bio, Vertex Pharmaceuticals, Editas, Sangamo, Sanofi, Regeneron, Agios, Forma, and Imara; consultancy fees from Bluebird Bio, Vertex Pharmaceuticals, Forma, and Imara; and advisory board roles at Celgene (Bristol Myers Squibb), Silence Therapeutics, Chiesi, and Pfizer. IT reports advisory board roles from Bluebird Bio, Vertex Pharmaceuticals, Global Blood Therapeutics, and Pfizer. AAT reports research funding from Bluebird Bio, Vertex Pharmaceuticals, Beam, Editas, and Novartis; consultancy fees from Bluebird Bio, Beam, Editas, and Roche; and equity ownership in Global Blood Therapeutics. FL reports consultancy fees from Bluebird Bio and Vertex; and speaker bureau fees from Sanofi, AMGEN, BMS, Miltenyi, and Gilead. MCW reports consultancy from Vertex Pharmaceuticals and an advisory board role from Ensoma. AEK reports royalties and honoraria from Bluebird Bio. JBK reports consultancy fees from Bluebird Bio, Novartis, Vertex Pharmaceuticals, Global Blood Therapeutics, and Pfizer; research funding from Global Blood Therapeutics and Pfizer; advisory board roles at Global Blood Therapeutics and Novartis; and honoraria from Pfizer and Novartis. EY reports clinical research funding from Bluebird Bio. GT, HE, SA, HLT, ALo, JL, and RAC report employment at and stock in Bluebird Bio. All other authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved, including those for text and data mining, AI training, and similar technologies.)

We performed a retrospective analysis on 124 patients with transfusion-dependent thalassemia who were registered in the German pediatric registry for stem cell transplantation. All patients underwent first allogeneic hematopoietic stem cell transplantation (HSCT) between 2011 and 2020 and belonged mainly to Pesaro risk class 1-2. Four-year overall (OS) and thalassemia-free survival (TFS) were 94.5% ± 2.9% and 88.0% ± 3.4% after treosulfan-fludarabine-thiotepa- and 96.9% ± 3.1% (P = 0.763) and 96.9% ± 3.1% (P = 0.155) after busulfan-fludarabine-based conditioning. Mixed chimerism below 75% occurred predominantly in treosulfan-based regimens (27.5% versus 6.2%). OS and TFS did not differ significantly between matched sibling, other matched family and matched unrelated donor (UD) HSCTs (OS: 100.0%, 100.0%, 96.3% ± 3.6%; TFS: 96.5% ± 2.4%, 90.0% ± 9.5%, 88.9% ± 6.0%). However, mismatched UD-HSCTs performed less favorable (OS: 84.7% ± 7.3% (P = 0.029); TFS: 79.9% ± 7.4% (P = 0.082)). We generated a scoring system reflecting the risk to develop mixed chimerism in our cohort. The main risk-reducing factors were a high CD3+ cell count (≥6 × 10 7 /kg) in the graft, busulfan-conditioning, pre-conditioning therapy and low-targeted ciclosporin A trough levels. Acute GvHD grade III-IV in treosulfan-based concepts predominantly occurred in patients with UD and reduced GvHD prophylaxis but not in the context of high CD3+ cell doses. Taken together, this information might be used to develop more risk-adapted HSCT regimens for thalassemia patients., (© 2024. The Author(s).)

BACKGROUNDAdoptive transfer of EBV-specific T cells can restore specific immunity in immunocompromised patients with EBV-associated complications.METHODSWe provide results of a personalized T cell manufacturing program evaluating donor, patient, T cell product, and outcome data. Patient-tailored clinical-grade EBV-specific cytotoxic T lymphocyte (EBV-CTL) products from stem cell donors (SCDs), related third-party donors (TPDs), or unrelated TPDs from the allogeneic T cell donor registry (alloCELL) at Hannover Medical School were manufactured by immunomagnetic selection using a CliniMACS Plus or Prodigy device and the EBV PepTivators EBNA-1 and Select. Consecutive manufacturing processes were evaluated, and patient outcome and side effects were retrieved by retrospective chart analysis.RESULTSForty clinical-grade EBV-CTL products from SCDs, related TPDs, or unrelated TPDs were generated for 37 patients with refractory EBV infections or EBV-associated malignancies with and without a history of transplantation, within 5 days (median) after donor identification. Thirty-four patients received 1-14 EBV-CTL products (fresh and cryopreserved). EBV-CTL transfer led to a complete response in 20 of 29 patients who were evaluated for clinical response. No infusion-related toxicity was reported. EBV-specific T cells in patients' blood were detectable in 16 of 18 monitored patients (89%) after transfer, and their presence correlated with clinical response.CONCLUSIONPersonalized clinical-grade manufacture of EBV-CTL products via immunomagnetic selection from SCDs, related TPDs, or unrelated TPDs in a timely manner is feasible. Overall, EBV-CTLs were clinically effective and well tolerated. Our data suggest EBV-CTL transfer as a promising therapeutic approach for immunocompromised patients with refractory EBV-associated diseases beyond HSCT, as well as patients with preexisting organ dysfunction.TRIAL REGISTRATIONNot applicable.FUNDINGThis study was funded in part by the German Research Foundation (DFG, 158989968/SFB 900), the Deutsche Kinderkrebsstiftung (DKS 2013.09), Wilhelm-Sander-Stiftung (reference 2015.097.1), Ellen-Schmidt-Program of Hannover Medical School, and German Federal Ministry of Education and Research (reference 01EO0802).

Patients with precursor B-cell acute lymphoblastic leukemia (pB-ALL) who have relapsed after allogeneic hematopoietic stem cell transplantation (allo-HSCT), have relapsed more than once, or are resistant upfront have a dismal prognosis. CD19-targeted chimeric antigen receptor (CAR) T cells have evolved as potent immune therapies. Tisagenlecleucel (Tisa-cel) is a commercially available autologous CD19-directed CAR T-cell product. We performed a retrospective study inviting all CAR T-cell centers in Germany to participate. Eighty-one patients with pB-ALL were included. Twenty-eight days after CAR T-cell infusion, 71 patients (87.7%) were in complete response, and 8 (9.9%) were in nonremission. At 2 years, the probabilities of event-free survival (pEFS), relapse-free survival (pRFS), and overall survival (pOS) were 45.3%, 51.7%, and 53.2%, respectively. pEFS was not different in patients without (n = 16, 55.0%) vs with prior allo-HSCT (n = 65, 43.4%). In patients treated after allo-HSCT, the time to relapse after allo-HSCT was a strong predictor of outcome. Patients relapsing within 6 months of allo-HSCT had a disappointing pEFS of 18.4% (pOS = 16.0%); the pEFS for those relapsing later was 55.5% (pOS = 74.8%). Our study provides real-world experience in pediatric, adolescent, and young adult patients with ALL treated with Tisa-cel, where most patients were treated after having relapsed after allo-HSCT. A total of 45.3% were rescued with a single dose of Tisa-cel. Our novel finding that patients with ALL after allo-HSCT had by far a better pEFS if relapse occurred beyond 6 months might be helpful in clinical decision-making and motivates studies to uncover the reasons., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Total body irradiation (TBI)-based conditioning is associated with superior leukemia-free survival in children with ALL undergoing HSCT. However, the risk for subsequent malignant neoplasms (SMN) remains a significant concern. We analyzed 705 pediatric patients enrolled in the prospective ALL-SCT-BFM-2003 trial and its subsequent registry. Patients >2 years received conditioning with TBI 12 Gy/etoposide (n = 558) and children ≤2 years of age or with contraindications for TBI received busulfan/cyclophosphamide/etoposide (n = 110). The 5- and 10-year cumulative incidence of SMN was 0.02 ± 0.01 and 0.13 ± 0.03, respectively. In total, 39 SMN (34 solid tumors, 5 MDS/AML) were diagnosed in 33 patients at a median of 5.8 years (1.7-13.4), exclusively in the TBI group. Of 33 affected patients, 21 (64%) are alive at a median follow-up of 5.1 years (0-9.9) after diagnosis of their first SMN. In univariate analysis, neither age at HSCT, donor type, acute GVHD, chronic GVHD, nor CMV constituted a significant risk factor for SMN. The only significant risk factor was TBI versus non-TBI based conditioning. This analysis confirms and quantifies the increased risk of SMN in children with ALL after conditioning with TBI. Future strategies to avoid TBI will need careful tailoring within prospective, controlled studies to prevent unfavorable outcomes., (© 2022. The Author(s).)

Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative treatment for patients affected by Wiskott-Aldrich syndrome (WAS). Reported HSCT outcomes have improved over time with respect to overall survival, but some studies have identified older age and HSCT from alternative donors as risk factors predicting poorer outcome. We analyzed 197 patients undergoing transplant at European Society for Blood and Marrow Transplantation centers between 2006 and 2017 who received conditioning as recommended by the Inborn Errors Working Party (IEWP): either busulfan (n = 103) or treosulfan (n = 94) combined with fludarabine ± thiotepa. After a median follow-up post-HSCT of 44.9 months, 176 patients were alive, resulting in a 3-year overall survival of 88.7% and chronic graft-versus-host disease (GVHD)-free survival (events include death, graft failure, and severe chronic GVHD) of 81.7%. Overall survival and chronic GVHD-free survival were not significantly affected by conditioning regimen (busulfan- vs treosulfan-based), donor type (matched sibling donor/matched family donor vs matched unrelated donor/mismatched unrelated donor vs mismatched family donor), or period of HSCT (2006-2013 vs 2014-2017). Patients aged <5 years at HSCT had a significantly better overall survival. The overall cumulative incidences of grade III to IV acute GVHD and extensive/moderate/severe chronic GVHD were 6.6% and 2.1%, respectively. Patients receiving treosulfan-based conditioning had a higher incidence of graft failure and mixed donor chimerism and more frequently underwent secondary procedures (second HSCT, unconditioned stem cell boost, donor lymphocyte infusion, or splenectomy). In summary, HSCT for WAS with conditioning regimens currently recommended by IEWP results in excellent survival and low rates of GVHD, regardless of donor or stem cell source, but age ≥5 years remains a risk factor for overall survival., (© 2022 by The American Society of Hematology.)

Background: Betibeglogene autotemcel (beti-cel) gene therapy for transfusion-dependent β-thalassemia contains autologous CD34+ hematopoietic stem cells and progenitor cells transduced with the BB305 lentiviral vector encoding the β-globin (β A-T87Q ) gene., Methods: In this open-label, phase 3 study, we evaluated the efficacy and safety of beti-cel in adult and pediatric patients with transfusion-dependent β-thalassemia and a non-β 0 /β 0 genotype. Patients underwent myeloablation with busulfan (with doses adjusted on the basis of pharmacokinetic analysis) and received beti-cel intravenously. The primary end point was transfusion independence (i.e., a weighted average hemoglobin level of ≥9 g per deciliter without red-cell transfusions for ≥12 months)., Results: A total of 23 patients were enrolled and received treatment, with a median follow-up of 29.5 months (range, 13.0 to 48.2). Transfusion independence occurred in 20 of 22 patients who could be evaluated (91%), including 6 of 7 patients (86%) who were younger than 12 years of age. The average hemoglobin level during transfusion independence was 11.7 g per deciliter (range, 9.5 to 12.8). Twelve months after beti-cel infusion, the median level of gene therapy-derived adult hemoglobin (HbA) with a T87Q amino acid substitution (HbA T87Q ) was 8.7 g per deciliter (range, 5.2 to 10.6) in patients who had transfusion independence. The safety profile of beti-cel was consistent with that of busulfan-based myeloablation. Four patients had at least one adverse event that was considered by the investigators to be related or possibly related to beti-cel; all events were nonserious except for thrombocytopenia (in 1 patient). No cases of cancer were observed., Conclusions: Treatment with beti-cel resulted in a sustained HbA T87Q level and a total hemoglobin level that was high enough to enable transfusion independence in most patients with a non-β 0 /β 0 genotype, including those younger than 12 years of age. (Funded by Bluebird Bio; HGB-207 ClinicalTrials.gov number, NCT02906202.)., (Copyright © 2021 Massachusetts Medical Society.)

GATA2 deficiency is a heterogeneous multi-system disorder characterized by a high risk of developing myelodysplastic syndrome (MDS) and myeloid leukemia. We analyzed the outcome of 65 patients reported to the registry of the European Working Group (EWOG) of MDS in childhood carrying a germline GATA2 mutation (GATA2 mut ) who had undergone hematopoietic stem cell transplantation (HSCT). At 5 years the probability of overall survival and disease-free survival (DFS) was 75% and 70%, respectively. Non-relapse mortality and relapse equally contributed to treatment failure. There was no evidence of increased incidence of graft-versus-host-disease or excessive rates of infections or organ toxicities. Advanced disease and monosomy 7 (-7) were associated with worse outcome. Patients with refractory cytopenia of childhood (RCC) and normal karyotype showed an excellent outcome (DFS 90%) compared to RCC and -7 (DFS 67%). Comparing outcome of GATA2 mut with GATA2 wt patients, there was no difference in DFS in patients with RCC and normal karyotype. The same was true for patients with -7 across morphological subtypes. We demonstrate that HSCT outcome is independent of GATA2 germline mutations in pediatric MDS suggesting the application of standard MDS algorithms and protocols. Our data support considering HSCT early in the course of GATA2 deficiency in young individuals., (© 2021. The Author(s).)

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Outcome of 333 children with acute myeloid leukaemia relapsing after a first allogeneic haematopoietic stem cell transplantation was analyzed. Four-year probability of overall survival (4y-pOS) was 14%. 4y-pOS for 122 children receiving a second haematopoietic stem cell transplantation was 31% and 3% for those that did not (P = <0·0001). Achievement of a subsequent remission impacted survival (P = <0·0001). For patients receiving a second transplant survival with or without achieving a subsequent remission was comparable. Graft source (bone marrow vs. peripheral blood stem cells, P = 0·046) and donor choice (matched family vs. matched unrelated donor, P = 0·029) positively impacted survival after relapse. Disease recurrence and non-relapse mortality at four years reached 45% and 22%., (© 2020 British Society for Haematology and John Wiley & Sons Ltd.)

AML SCT-BFM 2007 was the first hematopoietic stem cell transplantation (HCT) trial in Germany to comply with the European Clinical Trials Directive, and aimed to standardize pediatric HCT for acute myeloid leukemia (AML) across centers in Germany, Austria, and the Czech Republic. Children with high-risk features and a good early response achieving a complete first remission (CR-1) and those in CR-2 after a first relapse were stratified to receive HCT from a matched donor after myeloablative conditioning consisting of busulfan, cyclophosphamide, and melphalan. Four-year EFS and OS were 61 and 70%. Cumulative incidence of relapse (CIR) was 22%. TRM was 15% and correlated with age reaching 9% (SE 3%) in children younger than 12 years and 31% (SE 9%) in older children and adolescents. Children with poorly responding primary disease or relapse were allocated to receive early HCT after a cytoreductive regimen with fludarabine, amsacrine, and cytarabine, followed by reduced intensity conditioning and prophylactic donor lymphocyte infusions. Four-year EFS and OS were 49 and 53%. CIR was 38% and TRM 11%. For patients with primary poor response disease, early use of RIC HCT followed by prophylactic DLI can induce long-term remissions in more than 50% (EFS 46% (SE 9%)).

Conditional immortalization of hematopoietic progenitors through lentiviral expression of selected transcription factors in hematopoietic stem and progenitor cells provides a promising tool to study stem cell and leukemia biology. In this study, to generate conditionally immortalized lymphoid progenitor (ciLP) cell lines, murine hematopoietic progenitor cells were transduced with an inducible lentiviral "all-in-one" vector expressing LMO2 under doxycycline (DOX) stimulation and the reverse tetracycline-regulated transactivator ( rtTA3) . For selection of LMO2 -expressing ciLPs ( LMO2 -ciLPs) and longitudinal manipulation in T cell differentiation lymphoid conditions, we developed a robust approach based on coculture with OP9-DL1 stromal cells and improved cytokine conditions allowing a controlled balance between cell proliferation and differentiation in vitro . LMO2 -ciLP cell lines with the highest proliferation, vector copy number, and similar insertion pattern were selected for LMO2 "on/off" in vitro study. LMO2 expression under DOX induction resulted in a double negative (DN) 2 differentiation arrest and a propagation of CD44 + CD25 - myeloid cell population characterized by lymphoid and myeloid phenotypes, respectively. Both DN2 and CD44 + CD25 - myeloid cell subpopulations expressed c-KIT, suggesting that LMO2 -ciLPs were similar to uncommitted progenitors under DOX supplementation. DOX removal resulted in cessation of ectopic LMO2 expression and LMO2 -ciLPs continued T cell lymphoid differentiation accompanied by c-KIT downregulation and interleukin 7 receptor expression. Switching off LMO2 expression was accompanied by increased Notch signaling and significant reduction of the CD44 + CD25 - myeloid cell population under T cell differentiation lymphoid conditions. Although vector insertions in cooperation with LMO2 expression could influence the fate of LMO2 -ciLPs and additional experiments are required to evaluate it, our approach provides a promising tool to investigate mechanisms underlying stem cell, leukemia, and lymphocyte biology, leading to novel approaches for disease modeling and therapy evaluation.

The transcription factor B cell CLL/lymphoma 11B (BCL11B) is indispensable for T lineage development of lymphoid progenitors. Here, we show that chimeric antigen receptor (CAR) expression during early phases of ex vivo generation of lymphoid progenitors suppressed BCL11B, leading to suppression of T cell-associated gene expression and acquisition of NK cell-like properties. Upon adoptive transfer into hematopoietic stem cell transplant recipients, CAR-expressing lymphoid progenitors differentiated into CAR-induced killer (CARiK) cells that mediated potent antigen-directed antileukemic activity even across MHC barriers. CD28 and active immunoreceptor tyrosine-based activation motifs were critical for a functional CARiK phenotype. These results give important insights into differentiation of murine and human lymphoid progenitors driven by synthetic CAR transgene expression and encourage further evaluation of ex vivo-generated CARiK cells for targeted immunotherapy.

Background: Immunodysregulation polyendocrinopathy enteropathy x-linked (IPEX) syndrome is a monogenic autoimmune disease caused by FOXP3 mutations. Because it is a rare disease, the natural history and response to treatments, including allogeneic hematopoietic stem cell transplantation (HSCT) and immunosuppression (IS), have not been thoroughly examined., Objective: This analysis sought to evaluate disease onset, progression, and long-term outcome of the 2 main treatments in long-term IPEX survivors., Methods: Clinical histories of 96 patients with a genetically proven IPEX syndrome were collected from 38 institutions worldwide and retrospectively analyzed. To investigate possible factors suitable to predict the outcome, an organ involvement (OI) scoring system was developed., Results: We confirm neonatal onset with enteropathy, type 1 diabetes, and eczema. In addition, we found less common manifestations in delayed onset patients or during disease evolution. There is no correlation between the site of mutation and the disease course or outcome, and the same genotype can present with variable phenotypes. HSCT patients (n = 58) had a median follow-up of 2.7 years (range, 1 week-15 years). Patients receiving chronic IS (n = 34) had a median follow-up of 4 years (range, 2 months-25 years). The overall survival after HSCT was 73.2% (95% CI, 59.4-83.0) and after IS was 65.1% (95% CI, 62.8-95.8). The pretreatment OI score was the only significant predictor of overall survival after transplant (P = .035) but not under IS., Conclusions: Patients receiving chronic IS were hampered by disease recurrence or complications, impacting long-term disease-free survival. When performed in patients with a low OI score, HSCT resulted in disease resolution with better quality of life, independent of age, donor source, or conditioning regimen., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Trials with second generation CD19 chimeric antigen receptors (CAR) T-cells report unprecedented responses but are associated with risk of cytokine release syndrome (CRS). Instead, we studied the use of donor Epstein-Barr virus-specific T-cells (EBV CTL) transduced with a first generation CD19CAR, relying on the endogenous T-cell receptor for proliferation. We conducted a multi-center phase I/II study of donor CD19CAR transduced EBV CTL in pediatric acute lymphoblastic leukaemia (ALL). Patients were eligible pre-emptively if they developed molecular relapse (>5 × 10 -4 ) post first stem cell transplant (SCT), or prophylactically post second SCT. An initial cohort showed poor expansion/persistence. We therefore investigated EBV-directed vaccination to enhance expansion/persistence. Eleven patients were treated. No CRS, neurotoxicity or graft versus host disease (GVHD) was observed. At 1 month, 5 patients were in CR (4 continuing, 1 de novo), 1 PR, 3 had stable disease and 3 no response. At a median follow-up of 12 months, 10 of 11 have relapsed, 2 are alive with disease and 1 alive in CR 3 years. Although CD19CAR CTL expansion was poor, persistence was enhanced by vaccination. Median persistence was 0 (range: 0-28) days without vaccination compared to 56 (range: 0-221) days with vaccination (P=0.06). This study demonstrates the feasibility of multi-center studies of CAR T cell therapy and the potential for enhancing persistence with vaccination.

The clinical potential of transplantation is often reduced by T cell-mediated alloresponses that cause graft rejection or graft-versus-host disease. Integrin-mediated adhesion between alloreactive T cells and antigen-presenting cells is essential for allorejection. The identity of the signaling events needed for the activation of integrins such as LFA-1 is poorly understood. Here, we identified a novel role of the protein tyrosine phosphatase SHP-1 in the regulation of murine LFA-1-mediated adhesion in an allograft setting. Upon alloactivation, SHP-1 activity is reduced, resulting in an increase in LFA-1 adhesion compared to that for syngeneically activated T cells. The importance of these differential activation properties was further indicated by small interfering RNA (siRNA) knockdown of SHP-1 in syngeneically and allogeneically stimulated T cells. Mechanistically, SHP-1 modulated the binding of SLP-76 to ADAP by dephosphorylation of the YDGI tyrosine motif of ADAP, a known docking site for the Src family kinase Fyn. This novel key role of SHP-1 in the regulation of LFA-1-mediated adhesion may provide a new insight into T cell-mediated alloresponses and may pave the way to the development of new immunosuppressive pharmaceutical agents., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Retroviral engineering of hematopoietic stem cell-derived precursor T-cells (preTs) opens the possibility of targeted T-cell transfer across human leukocyte antigen (HLA)-barriers. Alpharetroviral vectors exhibit a more neutral integration pattern thereby reducing the risk of insertional mutagenesis. Cord blood-derived CD34+ cells were transduced and differentiated into preTs in vitro. Two promoters, elongation-factor-1-short-form, and a myeloproliferative sarcoma virus variant in combination with two commonly used envelopes were comparatively assessed choosing enhanced green fluorescent protein or a third-generation chimeric antigen receptor (CAR) against CD123 as gene of interest. Furthermore, the inducible suicide gene iCaspase 9 has been validated. Combining the sarcoma virus-derived promoter with a modified feline endogenous retrovirus envelope glycoprotein yielded in superior transgene expression and transduction rates. Fresh and previously frozen CD34+ cells showed similar transduction and expansion rates. Transgene-positive cells did neither show proliferative impairment nor alteration in their lymphoid differentiation profile. The sarcoma virus-derived promoter only could express sufficient levels of iCaspase 9 to mediate dimerizer-induced apoptosis. Finally, the CD123 CAR was efficiently expressed in CD34+ cells and proved to be functional when expressed on differentiated T-cells. Therefore, the transduction of CD34+ cells with alpharetroviral vectors represents a feasible and potentially safer approach for stem cell-based immunotherapies for cancer.

Co-transplantation of hematopoietic stem cells with those engineered to express leukemia-reactive T-cell receptors (TCRs) and differentiated ex vivo into precursor T cells (preTs) may reduce the risk of leukemia relapse. As expression of potentially self-(leukemia-) reactive TCRs will lead to negative selection or provoke autoimmunity upon thymic maturation, we investigated a novel concept whereby TCR expression set under the control of an inducible promoter would allow timely controlled TCR expression. After in vivo maturation and gene induction, preTs developed potent anti-leukemia effects. Engineered preTs provided protection even after repeated leukemia challenges by giving rise to effector and central memory cells. Importantly, adoptive transfer of TCR-transduced allogeneic preTs mediated anti-leukemia effect without evoking graft-versus-host disease (GVHD). Earlier transgene induction forced CD8(+) T-cell development was required to obtain a mature T-cell subset of targeted specificity, allowed engineered T cells to efficiently pass positive selection and abrogated the endogenous T-cell repertoire. Later induction favored CD4 differentiation and failed to produce a leukemia-reactive population emphasizing the dominant role of positive selection. Taken together, we provide new functional insights for the employment of TCR-engineered precursor cells as a controllable immunotherapeutic modality with significant anti-leukemia activity.

Background: Over the last decades, molecular cloning has transformed biological sciences. Having profoundly impacted various areas such as basic science, clinical, pharmaceutical, and environmental fields, the use of recombinant DNA has successfully started to enter the field of cellular engineering. Here, the polymerase chain reaction (PCR) represents one of the most essential tools. Due to the emergence of novel and efficient PCR reagents, cloning kits, and software, there is a need for a concise and comprehensive protocol that explains all steps of PCR cloning starting from the primer design, performing PCR, sequencing PCR products, analysis of the sequencing data, and finally the assessment of gene expression. It is the aim of this methodology paper to provide a comprehensive protocol with a viable example for applying PCR in gene cloning., Results: Exemplarily the sequence of the tdTomato fluorescent gene was amplified with PCR primers wherein proper restriction enzyme sites were embedded. Practical criteria for the selection of restriction enzymes and the design of PCR primers are explained. Efficient cloning of PCR products into a plasmid for sequencing and free web-based software for the consecutive analysis of sequencing data is introduced. Finally, confirmation of successful cloning is explained using a fluorescent gene of interest and murine target cells., Conclusions: Using a practical example, comprehensive PCR-based protocol with important tips was introduced. This methodology paper can serve as a roadmap for researchers who want to quickly exploit the power of PCR-cloning but have their main focus on functional in vitro and in vivo aspects of cellular engineering.

Efforts to limit GVHD mediated by alloreactive donor T cells after allogeneic bone marrow transplantation are limited by a concomitant decrease in graft-versus-tumor (GVT) activity and increased possibilities of tumor relapse. Using a novel approach, we adoptively transferred conventional T cells expressing the transcription factor promyelocytic leukemia zinc finger (PLZF), which confers effector properties resembling invariant natural killer T cells, such as copious production of cytokines under suboptimal stimulation. PLZF expression in T-cell allografts attenuates expansion of alloreactive T cells, leading to lower GVHD. Intact alloreactivity-driven antitumor cytokine responses result in preserved GVT effects, leading to improved survival. Our findings suggest that therapy with PLZF-overexpressing T cells would result in overall improved outcomes due to less GVHD and intact GVT effects., (©2013 AACR.)

Current strategies to suppress graft-versus-host disease (GVHD) also compromise graft-versus-tumor (GVT) responses. Furthermore, most experimental strategies to separate GVHD and GVT responses merely spare GVT function without actually enhancing it. We have previously shown that endogenously expressed TNF-related apoptosis-inducing ligand (TRAIL) is required for optimal GVT activity against certain malignancies in recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT). In order to model a donor-derived cellular therapy, we genetically engineered T cells to overexpress TRAIL and adoptively transferred donor-type unsorted TRAIL+ T cells into mouse models of allo-HSCT. We found that murine TRAIL+ T cells induced apoptosis of alloreactive T cells, thereby reducing GVHD in a DR5-dependent manner. Furthermore, murine TRAIL+ T cells mediated enhanced in vitro and in vivo antilymphoma GVT response. Moreover, human TRAIL+ T cells mediated enhanced in vitro cytotoxicity against both human leukemia cell lines and against freshly isolated chronic lymphocytic leukemia (CLL) cells. Finally, as a model of off-the-shelf, donor-unrestricted antitumor cellular therapy, in vitro-generated TRAIL+ precursor T cells from third-party donors also mediated enhanced GVT response in the absence of GVHD. These data indicate that TRAIL-overexpressing donor T cells could potentially enhance the curative potential of allo-HSCT by increasing GVT response and suppressing GVHD.

Based on the results from the AML-BFM 98 trial, hematopoietic SCT (HSCT) is recommended for children with AML in second CR only. Here, we retrospectively analyze interphase data of children who underwent HSCT after myeloablative conditioning with BU, CY, and melphalan (BuCyMel) for AML in second remission (CR2) between 1998 and 2009. Out of 152 children, transplant data were available on 109 individuals. Sixty out of 109 children (55%) received BuCyMel. Median age at HSCT was 12.2 years (range 3.0; 18.3). GVHD prophylaxis mostly consisted of CsA and short term MTX with or without antithymocyte globulin. Matched-sibling donors were used for 6/60 analyzed recipients, the remainder either received grafts from matched unrelated (30/60) or mismatched donors. OS after 5 years was 62% (s.e. 6%), relapse incidence 35% (18/60 children) and treatment-related mortality accounted for 12% (7/60) of fatal events. In conclusion, even taking into account possible selection bias in this retrospective analysis, HSCT in CR2 using BuCyMel resulted in a respectable OS. Based on this data the prospective, controlled and centrally monitored AML SCT-BFM 2007 trial has started to recruit patients in January 2010 aiming to generate valid outcome data for further strategy decisions.

The surface of spherical, nonporous silica nanoparticles (SiO2-NPs) was modified with gadolinium (Gd) complexes, fluorophores, and cell-penetrating peptides to achieve multifunctionality on a single particle. The Gd surface concentrations were 9-16 μmol/g resulting in nanomaterials with high local longitudinal and transversal relaxivities (~1×10(5) and ~5×10(5) /mm/s/NP, respectively). Rapid cellular uptake was observed in vitro; however, larger extracellular agglomerates were also formed. In vivo administration revealed a fast distribution throughout the body followed by a nearly complete disappearance of fluorescence in all organs except the lungs, liver, and spleen after 24 h. Such NPs have the potential to serve as efficient multimodal probes in molecular imaging.

Inflammatory cell recruitment after myocardial infarction needs to be tightly controlled to permit infarct healing while avoiding fatal complications such as cardiac rupture. Growth differentiation factor-15 (GDF-15), a transforming growth factor-β (TGF-β)-related cytokine, is induced in the infarcted heart of mice and humans. We show that coronary artery ligation in Gdf15-deficient mice led to enhanced recruitment of polymorphonuclear leukocytes (PMNs) into the infarcted myocardium and an increased incidence of cardiac rupture. Conversely, infusion of recombinant GDF-15 repressed PMN recruitment after myocardial infarction. In vitro, GDF-15 inhibited PMN adhesion, arrest under flow and transendothelial migration. Mechanistically, GDF-15 counteracted chemokine-triggered conformational activation and clustering of β(2) integrins on PMNs by activating the small GTPase Cdc42 and inhibiting activation of the small GTPase Rap1. Intravital microscopy in vivo in Gdf15-deficient mice showed that Gdf-15 is required to prevent excessive chemokine-activated leukocyte arrest on the endothelium. Genetic ablation of β(2) integrins in myeloid cells rescued the mortality of Gdf15-deficient mice after myocardial infarction. To our knowledge, GDF-15 is the first cytokine identified as an inhibitor of PMN recruitment by direct interference with chemokine signaling and integrin activation. Loss of this anti-inflammatory mechanism leads to fatal cardiac rupture after myocardial infarction.

Noninvasive monitoring of intracellular targets such as enzymes, receptors, or mRNA by means of magnetic resonance imaging (MRI) is increasingly gaining relevance in various research areas. A vital prerequisite for their visualization is the development of cell-permeable imaging probes, which can specifically interact with the target that characterizes the cellular or molecular process of interest. Here, we describe a dual-labeled probe, Gd-DOTA-k(FR)-Gal-CPP, designed to report the presence of intracellular β-galactosidase (β-gal) enzyme by MRI. This conjugate consists of a galactose based core serving as cleavable spacer, incorporated between the cell-penetrating peptide D-Tat(49-57) and reporter moieties (Gd-DOTA, fluorescein (FR)). We employed a facile building block approach to obtain our bimodal probe, Gd-DOTA-k(FR)-Gal-CPP. This strategy involved the preparation of the building blocks and their subsequent assembly using Fmoc-mediated solid phase synthesis, followed by the complexation of ligand 14 with GdCl(3). Gd-DOTA-k(FR)-Gal-CPP showed a considerably higher relaxivity enhancement (16.8±0.6 mM(-1)s(-1), 123 MHz, ∼21°C) relative to the commercial Gd-DOTA (4.0±0.12 mM(-1)s(-1), 123MHz, ∼21 °C). The activation of Gd-DOTA-k(FR)-Gal-CPP was based on a cellular retention strategy that required enzymatic cleavage of the delivery vector from galactose moiety following the cell internalization to achieve a prolonged accumulation of the reporter components (Gd-DOTA/FR) in the β-gal expressing cells. Cellular uptake of Gd-DOTA-k(FR)-Gal-CPP in β-gal expressing C6/LacZ and enzyme deficient parental C6 rat glioma cells was confirmed by fluorescence spectroscopy, MR imaging and ICP-AES measurements. All methods showed higher accumulation of measured reporters in C6/LacZ cells compared to enzyme deficient parental C6 cells. Fluorescence microscopy of cells labeled with Gd-DOTA-k(FR)-Gal-CPP indicated a predominantly vesicular localization of the green fluorescent conjugate around cell nuclei. This cellular distribution was most likely responsible for the observed non-specific background signal in the enzyme deficient C6 cells. Even though the specific accumulation of our bimodal probe has to be further improved, it could be already used for cell imaging by MRI and optical modalities., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Adoptive transfer (AT) of T cells forced to express tumor-reactive T-cell receptor (TCR) genes is an attractive strategy to direct autologous T-cell immunity against tumor-associated antigens. However, clinical effectiveness has been hampered by limited in vivo persistence. We investigated whether the use of major histocompatibility complex-mismatched T cells would prolong the in vivo persistence of tumor-reactive TCR gene expressing T cells by continuous antigen-driven proliferation via the endogenous potentially alloreactive receptor. Donor-derived CD8(+) T cells engineered to express a TCR against a leukemia-associated antigen mediated strong graft-versus-leukemia (GVL) effects with reduced graft-versus-host disease (GVHD) severity when given early after transplantation. AT later after transplantation resulted in a complete loss of GVL. Loss of function was associated with reduced expansion of TCR-transduced T cells as assessed by CDR3 spectratyping analysis and PD-1 up-regulation on T cells in leukemia-bearing recipients. PD-L1 blockade in allogeneic transplant recipients largely restored the GVL efficacy without triggering GVHD, whereas no significant antileukemia effects of PD-L1 blockade were observed in syngeneic controls. These data suggest a clinical approach in which the AT of gene-modified allogeneic T cells early after transplantation can provide a potent GVL effect without GVHD, whereas later AT is effective only with concurrent PD-L1 blockade.

Molecular imaging of cells and cellular processes can be achieved by tagging intracellular targets such as receptors, enzymes, or mRNA. Seeking to visualize the presence of specific mRNAs by magnetic resonance (MR) imaging, we coupled peptide nucleic acids (PNA) with gadolinium-based MR contrast agents using cell-penetrating peptides for intracellular delivery. Antisense to mRNA of DsRed2 protein was used as proof of principle. The conjugates were produced by continuous solid-phase synthesis followed by chelation with gadolinium. Their cellular uptake was confirmed by fluorescence microscopy and spectroscopy as well as by MR imaging of labeled cells. The cell-penetrating peptide D-Tat(57-49) was selected over two other derivatives of HIV-1 Tat peptide, based on its superior intracellular delivery of the gadolinium-based contrast agents. Further improved delivery of conjugates was achieved upon coupling peptide nucleic acids (antisense to mRNA of DsRed2 protein and nonsense with no natural counterpart). Significant enhancement in MR contrast was obtained in cells labeled with concentrations as low as 2.5 μM of these agents. Specific binding of the targeting PNA containing conjugate to its complementary oligonucleotide sequence was proven by in vitro cell-free assay. In contrast, a lack of specific enrichment was observed in transgenic cells containing the target due to nonspecific vesicular entrapment of contrast agents. Preliminary biodistribution studies showed conjugate-related fluorescence in several organs, especially the liver and bladder, indicating high mobility of the agent in spite of its high molecular weight. No conjugate related toxicity was observed. These results are encouraging, as they warrant further molecular optimization and consecutive specificity studies in vivo of this new generation of contrast agents.

Antigen-presenting cells (APCs) of host origin drive graft-versus-leukemia (GVL) effects but can also trigger life-threatening graft-versus-host disease (GVHD) after hematopoietic cell transplantation (HCT) across major histocompatibility complex (MHC) barriers. We show that in vitro priming of donor lymphocytes can circumvent the need of recipient-derived APCs in vivo for mediating robust GVL effects and significantly diminishes the risk of severe GVHD. In vitro, generated and expanded T cells (ETCs) mediate anti-leukemia effects only when primed on recipient-derived APCs. Loading of APCs in vitro with leukemia cell lysate, chimerism status of the recipient, and timing of adoptive transfer after HCT are important factors determining the outcome. Delayed transfer of ETCs resulted in strong GVL effects in leukemia-bearing full chimera (FC) and mixed chimera (MC) recipients, which were comparable with the GVL/GVHD rates observed after the transfer of naive donor lymphocyte infusion (DLI). Upon early transfer, GVL effects were more pronounced with ETCs but at the expense of significant GVHD. The degree of GVHD was most severe in MCs after transfer of ETCs that had been in vitro primed either on nonpulsed recipient-derived APCs or with donor-derived APCs.

Identification of dominant leukemia-associated neoantigens and favoring specific priming and subsequent expansion of T cells reactive to these antigens might harbor therapeutic potential. Here, a new strategy combines a specific T-cell activation step using tumor lysate-pulsed bone marrow-derived dendritic cells with a nonspecific large-scale expansion method. The leukemia cell line C1498 transduced with a potentially immunodominant antigen (ovalbumin) was used to track expansion and functionality of antigen-specific cytotoxic T cell (CTL), both in vitro and in vivo. Three times more leukemia-specific CTL could be generated when compared with the respective controls. CTL generated after increasing the antigen-specific T-cell precursor frequency in vitro cured up to 80% of mice bearing leukemia with the respective antigen (P < 0.005, as compared with controls). Alternatively, the yield of CTL reactive to a immunodominant neoantigen increased by factor 2 to 6 when T-cell donors were immunized with dendritic cell presenting the respective antigen. However, increasing the leukemia-reactive precursor frequency to a clinically exploitable level will be the key for the design of successful T-cell therapy trials.

When bacterial cells are stressed by a change in the environment, they respond by changing the activity of enzymes at both the transcriptional and post-transcriptional levels. The UVA component (400-315 nm) of solar radiation reaching the Earth's surface is one of the most common stresses encountered by bacteria in their environment. Bacteria have evolved various antioxidant defense systems to increase survival when subjected to the deleterious effects of UVA irradiation. Recently, UVA-induced cytotoxicity and oxidative damage have been shown to be dependent on radiation intensity and dose distribution, not just total energy dose. We now report that when Escherichia coli is subjected to continuous sublethal, low-fluence UVA irradiation (7.4 W/m(2)) while growing to stationary phase, it responds by changing the activity levels of hydroperoxidases (HPI, HPII), glutathione reductase and manganese superoxide dismutase. This leads to an attenuation of the growth-delay response and an increase resistance to lethal UVA irradiation. When E. coli is given a UVA dose of 135 kJ/m(2) delivered at a fluence rate of 50 W/m(2), extensive protein oxidation occurs which may contribute to the inhibition of key cellular enzymes, leading to cellular dysfunction, DNA damage and eventually death. Changes in antioxidant enzymes induced by low-fluence UVA irradiation do not confer greater protection from protein oxidation after a challenge dose of UVA irradiation delivered at a fluence rate of 50 W/m(2).

Autosomal recessive osteopetrosis (OP) is a rare, lethal disorder in which osteoclasts are absent or nonfunctional, resulting in a bone marrow cavity insufficient to support hematopoiesis. Because osteoclasts are derived from hematopoietic precursors, allogeneic hematopoietic cell transplantation can cure the bony manifestations of the disorder. However, high rates of graft failure have been observed in this population. It is not possible to harvest bone marrow from these patients for reinfusion should graft failure be observed. We report that 8 of 10 patients with OP had high numbers of circulating CD34(+) cells (3% +/- 0.9%). This increased proportion of peripheral CD34(+) cells made it possible to harvest 2 x 10(6) CD34(+) cells per kilogram with a total volume of blood ranging from 8.3 to 83.7 mL (1.3-11.6 mL/kg). In addition, colony-forming assays documented significantly more colony-forming unit-granulocyte-macrophage and burst-forming unit-erythroid in the blood of osteopetrotic patients compared with controls; the numbers of colony-forming units approximated those found in control marrow. We conclude that OP patients with high levels of circulating CD34(+) are candidates for peripheral blood autologous harvest by limited exchange transfusion. These cells are then available for reinfusion should graft failure be observed in patients for whom retransplantation is impractical.

To determine the mechanisms by which adoptive immunotherapy could reduce lethality to acute myelogenous leukemia (AML), a novel technique was developed to track both leukemic blasts and adoptively transferred cytotoxic T cells (CTLs) independently and simultaneously in mice. To follow the fate of ex vivo generated anti-AML-reactive CTLs, splenocytes obtained from enhanced green fluorescent protein transgenic mice were cocultured with AML lysate-pulsed dendritic cells, which subsequently were expanded by exposure to anti-CD3/CD28 monoclonal antibody-coated magnetic microspheres. To track AML cells, stable transfectants of C1498 expressing DsRed2, a red fluorescent protein, were generated. Three factors related to CTLs correlated with disease-free survival: (a). CTL L-selectin expression. L-Selectin high fractions resulted in 70% disease-free survival, whereas L-selectin low-expressing CTLs resulted in only 30% disease-free survival. (b). Duration of ex vivo expansion (9 versus 16 days). Short-term expanded CTLs could be found at high frequency in lymphoid organs for longer than 4 weeks after transfer, whereas long-term expanded CTLs were cleared from the system after 2 weeks. Duration of expansion correlated inversely with L-selectin expression. (c). CTL dose. A higher dose (40 versus 5 x 10(6)) resulted in superior disease-free survival. This survival advantage was achieved with short-term expanded CTLs only. The site of treatment failure was mainly the central nervous system where no CTLs could be identified at AML sites.