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αβ T cell receptor (TCRαβ) T cells modified to express chimeric antigen receptors (CAR), are now available as authorized therapies for certain B-cell malignancies. However the process of autologous harvest and generation of patient-specific products is costly, with complex logistics and infrastructure requirements. Premanufactured banks of allogeneic donor-derived CAR T cells could help widen applicability if the challenges of HLA-mismatched T-cell therapy can be addressed. Genome editing is being applied to overcome allogeneic barriers, most notably, by disrupting TCRαβ to prevent graft-versus-host disease, and multiple competing editing technologies, including CRISPR/Cas9 and base editing, have reached clinical phase testing. Improvements in accuracy and efficiency have unlocked applications for a wider range of blood malignancies, with multiplexed editing incorporated to target HLA molecules, shared antigens and checkpoint pathways. Clinical trials will help establish safety profiles and determine the durability of responses as well as the role of consolidation with allogeneic transplantation., (© 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.)

Greenhouse vegetable production (GVP) systems in China receive excessive amounts of fertilizers (>1500 kg N ha -1 yr -1 ) and irrigation (>1200 mm yr -1 ), which results in severe soil degradation. Moreover, soil borne diseases are common as the same crop is planted continuously over years. Anaerobic soil disinfestation (ASD) is a method carried out every 3-4 years during the summer fallow period to combat soil-borne diseases and to improve soil health. The standard ASD practice, which is carried out before the cropping season, involves incorporation of organic matter (i.e. rice shells or straw) into the soil, covering of the soil with plastic films and soil irrigation until saturation. However, many farmers incorporate large amounts of organic nitrogen fertilizer for priming ASD. In this study, we investigated if incorporation of rice shells plus chicken manure (ASD+RM; farmers practice) provokes higher environmental N losses (N 2 O emissions and N leaching) during the ASD and the following tomato crop growing period as compared to the standard ASD practice (ASD+R: only rice shells) or a Control (fallow, but with incorporation of organic manure, standard in non-ASD years). Results showed that ASD+RM increased seasonal (ASD/fallow period plus tomato crop growing period) soil N 2 O emissions by a factor of 3 (ASD+RM: 14.1 kg N 2 O-N ha -1 ; ASD+R: 4.7 kg N 2 O-N ha -1 ), with 2/3 of emissions occurring during the 25 days long ASD period. Across all treatments, nitrate (NO 3 - ) leaching dominated total N leaching (75%), with significantly lower rates observed for ASD+R as compared to ASD+RM. For both ASD treatments, total dissolved organic nitrogen (DON) leaching was a factor of two higher than for the Control. Crop productivity was not affected by ASD. Our findings imply that ASD+RM should be abandoned as the additional supply of manure N results in high environmental N losses without further increasing yields., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Greenhouse vegetable production in China mostly involves excessive N fertilization and flood irrigation. This causes serious soil degradation and spreading of soil borne diseases. As a countermeasure against soil borne diseases anaerobic soil disinfestation (ASD) is applied during the summer fallow period. Current practices involve the incorporation of organic C sources, covering of the soil with plastic film and flood irrigation. However, farmers not only apply straw but also organic manure in ASD which may result in significant greenhouse gas emissions and N leaching. A field experiment was conducted in a greenhouse during the summer fallow period to test the impact of three ASD practices on soil GHG (N 2 O, CO 2 and CH 4 ) emissions and N leaching: 1) control (CK), bare soil, no ASD; 2) ASD without straw incorporation (ASD-S); 3) ASD plus straw incorporation (ASD+S) and 4) ASD plus straw and chicken manure incorporation (ASD+SM). Applying any form of ASD resulted in an increase in N 2 O emissions from approximately 1 kg N ha -1 month -1 to 10.7 (ASD)-47.0 (ASD+SM) kg N ha -1 month -1 . Furthermore, N leaching from treatments of ASD ranged from 24.1-54.2 kg N ha -1 month -1 , with highest values in ASD-S. However, while N leaching in ASD-S was solely in the form of NO 3 - , DON leaching was with approximately 12-20% a significant component of total N leaching in ASD+S and ASD+SM. Overall, ASD+SM showed the highest environmental N losses, which were dominated by N 2 O emissions. This highlights the need to advise farmers and policy makers to ban the incorporation of chicken manure instead of straw only during the ASD period and to optimize irrigation schemes instead of flood irrigation to reduce environmental N losses. Putting in more environmental sound ASD practices will certainly help to improve the sustainability of greenhouse vegetable production., Competing Interests: Declaration of competing interest None., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Genome-edited donor-derived allogeneic anti-CD19 chimeric antigen receptor (CAR) T cells offer a novel form of CAR-T-cell product that is available for immediate clinical use, thereby broadening access and applicability. UCART19 is one such product investigated in children and adults with relapsed or refractory B-cell acute lymphoblastic leukaemia. Two multicentre phase 1 studies aimed to investigate the feasibility, safety, and antileukaemic activity of UCART19 in children and adults with relapsed or refractory B-cell acute lymphoblastic leukaemia., Methods: We enrolled paediatric or adult patients in two ongoing, multicentre, phase 1 clinical trials to evaluate the safety and antileukaemic activity of UCART19. All patients underwent lymphodepletion with fludarabine and cyclophosphamide with or without alemtuzumab, then children received UCART19 at 1·1-2·3 × 10 6 cells per kg and adults received UCART19 doses of 6 × 10 6 cells, 6-8 × 10 7 cells, or 1·8-2·4 × 10 8 cells in a dose-escalation study. The primary outcome measure was adverse events in the period between first infusion and data cutoff. These studies were registered at ClinicalTrials.gov, NCT02808442 and NCT02746952., Findings: Between June 3, 2016, and Oct 23, 2018, seven children and 14 adults were enrolled in the two studies and received UCART19. Cytokine release syndrome was the most common adverse event and was observed in 19 patients (91%); three (14%) had grade 3-4 cytokine release syndrome. Other adverse events were grade 1 or 2 neurotoxicity in eight patients (38%), grade 1 acute skin graft-versus-host disease in two patients (10%), and grade 4 prolonged cytopenia in six patients (32%). Two treatment-related deaths occurred; one caused by neutropenic sepsis in a patient with concurrent cytokine release syndrome and one from pulmonary haemorrhage in a patient with persistent cytopenia. 14 (67%) of 21 patients had a complete response or complete response with incomplete haematological recovery 28 days after infusion. Patients not receiving alemtuzumab (n=4) showed no UCART19 expansion or antileukaemic activity. The median duration of response was 4·1 months with ten (71%) of 14 responders proceeding to a subsequent allogeneic stem-cell transplant. Progression-free survival at 6 months was 27%, and overall survival was 55%., Interpretation: These two studies show, for the first time, the feasibility of using allogeneic, genome-edited CAR T cells to treat patients with aggressive leukaemia. UCART19 exhibited in-vivo expansion and antileukaemic activity with a manageable safety profile in heavily pretreated paediatric and adult patients with relapsed or refractory B-cell acute lymphoblastic leukaemia. The results this study are an encouraging step forward for the field of allogeneic CAR T cells, and UCART19 offers the opportunity to treat patients with rapidly progressive disease and where autologous CAR-T-cell therapy is unavailable., Funding: Servier., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Recessive dystrophic epidermolysis bullosa (RDEB) is a debilitating genodermatosis caused by loss-of-function mutations in COL7A1 encoding type VII collagen (C7), the main component of anchoring fibrils at the dermal-epidermal junction. With no curative treatments presently available, retrovirally transduced autologous epidermal grafts and intradermal lentivirally engineered fibroblast injections are being investigated. Alternative approaches aim to infuse allogeneic mesenchymal stromal cells (MSCs) to provide a more generalized treatment for RDEB. We investigated whether healthy human MSCs could be engineered to overexpress C7 and correct RDEB in a human:murine chimeric model. Initially, engineered MSCs incorporated ex vivo into RDEB grafts, their presence confirmed by fluorescence in situ hybridization, revealed recovery of function of the dermal-epidermal junction with no signs of blister formation. Importantly, the detection of anchoring fibrils by transmission electron microscopy corroborated structural recovery. Next, MSCs cotransduced to express C7 and luciferase were delivered intradermally into grafted RDEB skin, resulting in localized MSC persistence with deposition of de novo C7 at the site. Notably, C7 expression was sufficient to restore anchoring fibril density to normal levels. In contrast, intravenously injected engineered MSCs were undetectable within grafts and lacked anchoring fibril reconstitution. Our data suggest that although localized correction may be achievable using engineered MSCs, strategies for systemic administration require further modeling., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: In this era of life highly comminuted and multi planar tibial plateau fractures involving the posterior corners are more commonly seen and addressed in the literature than before. Among these several types have not been described in the currently used classification systems. In fact simple classification systems ignore several fracture types and leniently grouped the fractures with different mechanism, morphology, treatment modalities and prognosis in same category. On the other hand, more extensive nature classifications with detailed subdivisions are difficult to remember for clinicians. The clinical reliability of these classifications is another problem. All these issues demand the potential need of a new classification. The aim of this study was to describe a quadrant specific two column classification of tibial plateau fractures and to analyse its inter-observer and intra-observer reliability, clinical assessment and application., Materials and Methods: From January 2009 to December 2015, 44 patients with tibial plateau fractures were studied retrospectively. The antero-posterior (AP), lateral X-rays and computed tomography (CT) with axial transverse, sagittal, coronal and three dimensional (3D) reconstruction images were performed for all the patients. All of the fractures were categorized according to quadrant specific two column classification and the traditional Schatzker's classification. The comparative analysis for inter-observer and intra-observer reliability of the new classification and the Schatzker's classification was conducted by four observers., Results: Three cases didn't match any type in the Schatzker's classification. While on the other hand, all cases were classified by two column classification. The mean kappa values for inter-observer reliability by using the Schatzker's classification was 0.723 (range, 0.674-0.823), representing substantial agreement, whereas the mean kappa value was 0.939 (range: 0.897-0.974), representing almost perfect agreement according to two column classification. The mean kappa values for intra-observer reliability using the Schatzker's classification and two column classification were 0.789 (range: 0.590-0.864) and 0.955 (range:0.923-0.948) showing substantial agreement and almost perfect agreement., Conclusion: The quadrant specific two column classification is anatomically oriented, CT based and clinically valid. The different fracture types according to anatomic location are represented alphanumerically so that treatment matched to specific fracture type (quadrant specific anatomic fixation) for optimal outcomes. Furthermore, it demonstrates higher inter-observer and intra-observer reliability. This classification can be adopted to strengthen the traditional Schatzker's classification, particularly in the multi planar and posteriorly extended plateau fractures. It can be used as a reliable research tool. The database can be used to distinguish different fracture types, individual type incidences, specific treatment and also prognosis. Authors suggest a large multi-centre study., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Adenovirus (ADV) reactivation can cause significant morbidity and mortality in children after allogeneic stem cell transplantation. Antiviral drugs can control viremia, but viral clearance requires recovery of cell-mediated immunity., Method: This study was an open-label phase 1/2 study to investigate the feasibility of generating donor-derived ADV-specific T cells (Cytovir ADV, Cell Medica) and to assess the safety of pre-emptive administration of ADV-specific T cells in high-risk pediatric patients after allogeneic hematopoietic stem cell transplantation (HSCT) to treat adenoviremia. Primary safety endpoints included graft-versus-host disease (GvHD), and secondary endpoints determined antiviral responses and use of antiviral drugs., Results: Between January 2013 and May 2016, 92 donors were enrolled for the production of ADV T cells at three centers in the United Kingdom (UK), and 83 products were generated from 72 mobilized peripheral blood harvests and 20 steady-state whole blood donations. Eight children received Cytovir ADV T cells after standard therapy and all resolved ADV viremia between 15 and 127 days later. ADV-specific T cells were detectable using enzyme-linked immunospot assay (ELISpot) in the peripheral blood of all patients analyzed. Serious adverse events included Grade II GvHD, Astrovirus encephalitis and pancreatitis., Conclusion: The study demonstrates the safety and feasibility of pre-emptively manufacturing peptide pulsed ADV-specific cells for high-risk pediatric patients after transplantation and provides early evidence of clinical efficacy., (Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.)

X-linked recessive ectodermal dysplasia with immunodeficiency is a rare primary immunodeficiency caused by hypomorphic mutations of the IKBKG gene encoding the nuclear factor κB essential modulator (NEMO) protein. This condition displays enormous allelic, immunological, and clinical heterogeneity, and therapeutic decisions are difficult because NEMO operates in both hematopoietic and nonhematopoietic cells. Hematopoietic stem cell transplantation (HSCT) is potentially life-saving, but the small number of case reports available suggests it has been reserved for only the most severe cases. Here, we report the health status before HSCT, transplantation outcome, and clinical follow-up for a series of 29 patients from unrelated kindreds from 11 countries. Between them, these patients carry 23 different hypomorphic IKBKG mutations. HSCT was performed from HLA-identical related donors (n = 7), HLA-matched unrelated donors (n = 12), HLA-mismatched unrelated donors (n = 8), and HLA-haploidentical related donors (n = 2). Engraftment was documented in 24 patients, and graft-versus-host disease in 13 patients. Up to 7 patients died 0.2 to 12 months after HSCT. The global survival rate after HSCT among NEMO-deficient children was 74% at a median follow-up after HSCT of 57 months (range, 4-108 months). Preexisting mycobacterial infection and colitis were associated with poor HSCT outcome. The underlying mutation does not appear to have any influence, as patients with the same mutation had different outcomes. Transplantation did not appear to cure colitis, possibly as a result of cell-intrinsic disorders of the epithelial barrier. Overall, HSCT can cure most clinical features of patients with a variety of IKBKG mutations.

Purpose: Appropriate fixation method for the posterior malleolar fractures (PMF) according to the fracture size is still not clear. Aim of this study was to evaluate the outcomes of the different fixation methods used for fixation of PMF by finite element analysis (FEA) and to compare the effect of fixation constructs on the size of the fracture computationally., Materials and Methods: Three dimensional model of the tibia was reconstructed from computed tomography (CT) images. PMF of 30%, 40% and 50% fragment sizes were simulated through computational processing. Two antero-posterior (AP) lag screws, two postero-anterior (PA) lag screws and posterior buttress plate were analysed for three different fracture volumes. The simulated loads of 350N and 700N were applied to the proximal tibial end. Models were fixed distally in all degrees of freedom., Results: In single limb standing condition, the posterior plate group produced the lowest relative displacement (RD) among all the groups (0.01, 0.03 and 0.06mm). Further nodal analysis of the highest RD fracture group showed a higher mean displacement of 4.77mm and 4.23mm in AP and PA lag screws model (p=0.000). The amounts of stress subjected to these implants, 134.36MPa and 140.75MPa were also significantly lower (p=0.000). There was a negative correlation (p=0.021) between implant stress and the displacement which signifies a less stable fixation using AP and PA lag screws., Conclusion: Progressively increasing fracture size demands more stable fixation construct because RD increases significantly. Posterior buttress plate produces superior stability and lowest RD in PMF models irrespective of the fragment size., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Heterozygous CTLA-4 deficiency has been reported as a monogenic cause of common variable immune deficiency with features of immune dysregulation. Direct mutation in CTLA-4 leads to defective regulatory T-cell (Treg) function associated with impaired ability to control levels of the CTLA-4 ligands, CD80 and CD86. However, additional mutations affecting the CTLA-4 pathway, such as those recently reported for LRBA, indirectly affect CTLA-4 expression, resulting in clinically similar disorders. Robust phenotyping approaches sensitive to defects in the CTLA-4 pathway are therefore required to inform understanding of such immune dysregulation syndromes. Here, we describe assays capable of distinguishing a variety of defects in the CTLA-4 pathway. Assessing total CTLA-4 expression levels was found to be optimal when restricting analysis to the CD45RA - Foxp3 + fraction. CTLA-4 induction following stimulation, and the use of lysosomal-blocking compounds, distinguished CTLA-4 from LRBA mutations. Short-term T-cell stimulation improved the capacity for discriminating the Foxp3 + Treg compartment, clearly revealing Treg expansions in these disorders. Finally, we developed a functionally orientated assay to measure ligand uptake by CTLA-4, which is sensitive to ligand-binding or -trafficking mutations, that would otherwise be difficult to detect and that is appropriate for testing novel mutations in CTLA-4 pathway genes. These approaches are likely to be of value in interpreting the functional significance of mutations in the CTLA-4 pathway identified by gene-sequencing approaches., (© 2017 by The American Society of Hematology.)

Background Aims: In pediatric patients, adenovirus (ADV) reactivation after allogeneic hematopoietic stem cell transplantation (allo HSCT) is a major cause of morbidity and mortality. For patients who do not respond to antiviral drug therapy, a new treatment approach using ADV-specific T cells can present a promising alternative. Here we describe the clinical scale Good Manufacturing Practice (GMP)-compliant manufacture and characterization of 40 ADV-specific T-cell products, Cytovir ADV, which are currently being tested in a multi-center phase I/IIa clinical trial. This process requires minimal intervention, is high yield, and results in a pure T-cell product that is functional., Methods: Mononuclear cells (2 × 10(7)) were cultured in a closed system in the presence of GMP-grade ADV peptide pool and cytokines for 10 days. On day 10, the T-cell product was harvested, washed in a closed system, counted and assessed for purity and potency. Additional characterization was carried out where cell numbers allowed., Results: Thirty-eight of 40 products (95%) met all release criteria. Median purity of the cell product was 88.3% CD3+ cells with a median yield of 2.9 × 10(7) CD3+ cells. Potency analyses showed a median ADV-specific interferon (IFN)γ response of 5.9% of CD3+ and 2345 IFNγ spot-forming cells/million. CD4 and CD8 T cells were capable of proliferating in response to ADV (63.3 and 56.3%, respectively). These virus-specific T cells (VST) were heterogenous, containing both effector memory and central memory T cells. In an exemplar patient with ADV viremia treated in the open ASPIRE trial, ADV-specific T-cell response was detected by IFNγ enzyme-linked immunospot from 13 days post-infusion. ADV DNA levels declined following cellular therapy and were below level of detection from day 64 post-infusion onward., Conclusions: The clinical-scale GMP-compliant One Touch manufacturing system is feasible and yields functional ADV-specific T cells at clinically relevant doses., (Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Novel cell therapies derived from human T lymphocytes are exhibiting enormous potential in early-phase clinical trials in patients with hematologic malignancies. Ex vivo modification of T cells is currently limited to a small number of centers with the required infrastructure and expertise. The process requires isolation, activation, transduction, expansion and cryopreservation steps. To simplify procedures and widen applicability for clinical therapies, automation of these procedures is being developed. The CliniMACS Prodigy (Miltenyi Biotec) has recently been adapted for lentiviral transduction of T cells and here we analyse the feasibility of a clinically compliant T-cell engineering process for the manufacture of T cells encoding chimeric antigen receptors (CAR) for CD19 (CAR19), a widely targeted antigen in B-cell malignancies. Using a closed, single-use tubing set we processed mononuclear cells from fresh or frozen leukapheresis harvests collected from healthy volunteer donors. Cells were phenotyped and subjected to automated processing and activation using TransAct, a polymeric nanomatrix activation reagent incorporating CD3/CD28-specific antibodies. Cells were then transduced and expanded in the CentriCult-Unit of the tubing set, under stabilized culture conditions with automated feeding and media exchange. The process was continuously monitored to determine kinetics of expansion, transduction efficiency and phenotype of the engineered cells in comparison with small-scale transductions run in parallel. We found that transduction efficiencies, phenotype and function of CAR19 T cells were comparable with existing procedures and overall T-cell yields sufficient for anticipated therapeutic dosing. The automation of closed-system T-cell engineering should improve dissemination of emerging immunotherapies and greatly widen applicability., (Copyright © 2016. Published by Elsevier Inc.)

Cells therapies, engineered to secrete replacement proteins, are being developed to ameliorate otherwise debilitating diseases. Recessive dystrophic epidermolysis bullosa (RDEB) is caused by defects of type VII collagen, a protein essential for anchoring fibril formation at the dermal-epidermal junction. Whereas allogeneic fibroblasts injected directly into the dermis can mediate transient disease modulation, autologous gene-modified fibroblasts should evade immunological rejection and support sustained delivery of type VII collagen at the dermal-epidermal junction. We demonstrate the feasibility of such an approach using a therapeutic grade, self-inactivating-lentiviral vector, encoding codon-optimized COL7A1, to transduce RDEB fibroblasts under conditions suitable for clinical application. Expression and secretion of type VII collagen was confirmed with transduced cells exhibiting supranormal levels of protein expression, and ex vivo migration of fibroblasts was restored in functional assays. Gene-modified RDEB fibroblasts also deposited type VII collagen at the dermal-epidermal junction of human RDEB skin xenografts placed on NOD-scid IL2Rgamma(null) recipients, with reconstruction of human epidermal structure and regeneration of anchoring fibrils at the dermal-epidermal junction. Fibroblast-mediated restoration of protein and structural defects in this RDEB model strongly supports proposed therapeutic applications in man., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Unrelated cord blood transplantation (CBT) without in vivo T-cell depletion is increasingly used to treat high-risk hematologic malignancies. Following T-replete CBT, naïve CB T cells undergo rapid peripheral expansion with memory-effector differentiation. Emerging data suggest that unrelated CBT, particularly in the context of HLA mismatch and a T-replete graft, may reduce leukemic relapse. To study the role of CB T cells in mediating graft-versus-tumor responses and dissect the underlying immune mechanisms for this, we compared the ability of HLA-mismatched CB and adult peripheral blood (PB) T cells to eliminate Epstein-Barr virus (EBV)-driven human B-cell lymphoma in a xenogeneic NOD/SCID/IL2rg(null) mouse model. CB T cells mediated enhanced tumor rejection compared with equal numbers of PB T cells, leading to improved survival in the CB group (P < .0003). Comparison of CB T cells that were autologous vs allogeneic to the lymphoma demonstrated that this antitumor effect was mediated by alloreactive rather than EBV-specific T cells. Analysis of tumor-infiltrating lymphocytes demonstrated that CB T cells mediated this enhanced antitumor effect by rapid infiltration of the tumor with CCR7(+)CD8(+) T cells and prompt induction of cytotoxic CD8(+) and CD4(+) T-helper (Th1) T cells in the tumor microenvironment. In contrast, in the PB group, this antilymphoma effect is impaired because of delayed tumoral infiltration of PB T cells and a relative bias toward suppressive Th2 and T-regulatory cells. Our data suggest that, despite being naturally programmed toward tolerance, reconstituting T cells after unrelated T-replete CBT may provide superior Tc1-Th1 antitumor effects against high-risk hematologic malignancies., (© 2015 by The American Society of Hematology.)

HBV-DNA integration frequently occurs in HBV-related hepatocellular carcinoma (HCC), but whether HBV antigens are expressed in HCC cells and can be targeted by immune therapeutic strategies remains controversial. Here, we first characterized HBV antigen expression in HCC metastases, occurring in a patient who had undergone liver transplantation for HBV-related HCC. We then deployed for the first time in HCC autologous T cells, genetically modified to express an HBsAg specific T cell receptor, as therapy against chemoresistant extrahepatic metastases. We confirmed that HBV antigens were expressed in HCC metastases (but not in the donor liver) and demonstrated that tumour cells were recognized in vivo by lymphocytes, engineered to express an HBV-specific T cell receptor (TCR). Gene-modified T cells survived, expanded and mediated a reduction in HBsAg levels without exacerbation of liver inflammation or other toxicity. Whilst clinical efficacy was not established in this subject with end-stage metastatic disease, we confirm the feasibility of providing autologous TCR-redirected therapy against HCC and advocate this strategy as a novel therapeutic opportunity in hepatitis B-associated malignancies., (Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Severe combined immunodeficiency (SCID) arises from different genetic defects associated with lymphocyte development and function and presents with severe infections. Allogeneic hematopoietic stem cell transplantation is an extremely effective way of restoring immunity in these individuals. Numerous multicenter studies have identified the factors determining successful outcome, and survival for SCID has shown great improvement. Advances in understanding the genetic basis of disease also mean that we increasingly tailor transplant protocols to the specific SCID form. Wherever possible, we attempt to transplant SCID patients without the use of cytoreductive conditioning, but it is clear that this is only successful for specific SCID forms and, although survival is good, in specific patients there are ongoing humoral defects. We aim to use matched related and unrelated donors (including cord blood) whenever possible and have limited the use of mismatched haploidentical donors. The development of autologous hematopoietic stem cell gene therapy provides another treatment of the X-linked and adenosine deaminase-deficient forms of SCID, and we discuss how we have integrated gene therapy into our treatment strategy. These developments together with the advent of universal newborn screening for SCID should allow for a highly favorable outcome for this otherwise lethal condition.

In this retrospective collaborative study, we have analyzed long-term outcome and donor cell engraftment in 194 patients with Wiskott-Aldrich syndrome (WAS) who have been treated by hematopoietic cell transplantation (HCT) in the period 1980- 2009. Overall survival was 84.0% and was even higher (89.1% 5-year survival) for those who received HCT since the year 2000, reflecting recent improvement of outcome after transplantation from mismatched family donors and for patients who received HCT from an unrelated donor at older than 5 years. Patients who went to transplantation in better clinical conditions had a lower rate of post-HCT complications. Retrospective analysis of lineage-specific donor cell engraftment showed that stable full donor chimerism was attained by 72.3% of the patients who survived for at least 1 year after HCT. Mixed chimerism was associated with an increased risk of incomplete reconstitution of lymphocyte count and post-HCT autoimmunity, and myeloid donor cell chimerism < 50% was associated with persistent thrombocytopenia. These observations indicate continuous improvement of outcome after HCT for WAS and may have important implications for the development of novel protocols aiming to obtain full correction of the disease and reduce post-HCT complications.

Children with primary immunodeficiency diseases, particularly those less than 1 year of age, experience significant toxicity after hematopoietic stem cell transplantation, with busulfan- or melphalan-based conditioning. Treosulfan causes less veno-occlusive disease than busulfan and does not require pharmacokinetic monitoring. We report its use in 70 children. Children received 42 g/m(2) or 36 g/m(2) with cyclophosphamide 200 mg/kg (n = 30) or fludarabine 150 mg/m(2) (n = 40), with alemtuzumab in most. Median age at transplantation was 8.5 months (range, 1.2-175 months); 46 (66%) patients were 12 months of age or younger. Donors were as follows: matched sibling donor, 8; matched family donor, 13; haploidentical, 4; and unrelated, 45. Median follow-up was 19 months (range, 1-47 months). Overall survival was 81%, equivalent in those age less or greater than 1 year. Skin toxicity was common. Veno-occlusive disease occurred twice with cyclophosphamide. Eighteen patients (26%) had graft-versus-host disease, and only 7 (10%) greater than grade 2. Two patients rejected; 24 of 42 more than 1 year after transplantation had 100% donor chimerism. The remainder had stable mixed chimerism. T-cell chimerism was significantly better with fludarabine. Long-term follow-up is required, but in combination with fludarabine, treosulfan is a good choice of conditioning for hematopoietic stem cell transplantation in primary immunodeficiency disease.

Cartilage-hair hypoplasia (CHH) is a rare autosomal recessive disease caused by mutations in the RMRP gene. Beside dwarfism, CHH has a wide spectrum of clinical manifestations including variable grades of combined immunodeficiency, autoimmune complications, and malignancies. Previous reports in single CHH patients with significant immunodeficiencies have demonstrated that allogeneic hematopoietic stem cell transplantation (HSCT) is an effective treatment for the severe immunodeficiency, while growth failure remains unaffected. Because long-term experience in larger cohorts of CHH patients after HSCT is currently unreported, we performed a European collaborative survey reporting on 16 patients with CHH and immunodeficiency who underwent HSCT. Immune dysregulation, lymphoid malignancy, and autoimmunity were important features in this cohort. Thirteen patients were transplanted in early childhood ( approximately 2.5 years). The other 3 patients were transplanted at adolescent age. Of 16 patients, 10 (62.5%) were long-term survivors, with a median follow-up of 7 years. T-lymphocyte numbers and function have normalized, and autoimmunity has resolved in all survivors. HSCT should be considered in CHH patients with severe immunodeficiency/autoimmunity, before the development of severe infections, major organ damage, or malignancy might jeopardize the outcome of HSCT and the quality of life in these patients.

Cytotoxic T lymphocytes (CTLs) specific for an HLA-A2-presented peptide epitope of the Wilms tumor antigen-1 (WT1) can selectively kill immature human leukemia progenitor and stem cells in vitro. In this study we have used retroviral gene transfer to introduce a WT1-specific T-cell receptor (TCR) into T lymphocytes obtained from patients with leukemia and from healthy donors. TCR-transduced T cells kill leukemia cells in vitro and display WT1-specific cytokine production. Intravenous injection of TCR-transduced T cells into nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mice harboring human leukemia cells resulted in leukemia elimination, whereas transfer of control T cells transduced with an irrelevant TCR was ineffective. The data suggest that adoptive immunotherapy with WT1-TCR gene-modified patient T cells should be considered for the treatment of leukemia.