A human postnatal lymphoid progenitor capable of circulating and seeding the thymus

Identification of a thymus-seeding progenitor originating from human bone marrow (BM) constitutes a key milestone in understanding the mechanisms of T cell development and provides new potential for correcting T cell deficiencies. We report the characterization of a novel lymphoid-restricted subset, which is part of the lineage-negative CD34+CD10+ progenitor population and which is distinct from B cell–committed precursors (in view of the absence of CD24 expression). We demonstrate that these Lin−CD34+CD10+CD24− progenitors have a very low myeloid potential but can generate B, T, and natural killer lymphocytes and coexpress recombination activating gene 1, terminal deoxynucleotide transferase, PAX5, interleukin 7 receptor α, and CD3ε. These progenitors are present in the cord blood and in the BM but can also be found in the blood throughout life. Moreover, they belong to the most immature thymocyte population. Collectively, these findings unravel the existence of a postnatal lymphoid-polarized population that is capable of migrating from the BM to the thymus

A method to sequence and quantify DNA integration for monitoring outcome in gene therapy

Human genetic diseases have been successfully corrected by integration of functional copies of the defective genes into human cells, but in some cases integration of therapeutic vectors has activated proto-oncogenes and contributed to leukemia. For this reason, extensive efforts have focused on analyzing integration site populations from patient samples, but the most commonly used methods for recovering newly integrated DNA suffer from severe recovery biases. Here, we show that a new method based on phage Mu transposition in vitro allows convenient and consistent recovery of integration site sequences in a form that can be analyzed directly using DNA barcoding and pyrosequencing. The method also allows simple estimation of the relative abundance of gene-modified cells from human gene therapy subjects, which has previously been lacking but is crucial for detecting expansion of cell clones that may be a prelude to adverse events

Disease Evolution and Response to Rapamycin in Activated Phosphoinositide 3-Kinase δ Syndrome: The European Society for Immunodeficiencies-Activated Phosphoinositide 3-Kinase δ Syndrome Registry

Activated phosphoinositide 3-kinase (PI3K) δ Syndrome (APDS), caused by autosomal dominant mutations in PIK3CD (APDS1) or PIK3R1 (APDS2), is a heterogeneous primary immunodeficiency. While initial cohort-descriptions summarized the spectrum of clinical and immunological manifestations, questions about long-term disease evolution and response to therapy remain. The prospective European Society for Immunodeficiencies (ESID)-APDS registry aims to characterize the disease course, identify outcome predictors, and evaluate treatment responses. So far, 77 patients have been recruited (51 APDS1, 26 APDS2). Analysis of disease evolution in the first 68 patients pinpoints the early occurrence of recurrent respiratory infections followed by chronic lymphoproliferation, gastrointestinal manifestations, and cytopenias. Although most manifestations occur by age 15, adult-onset and asymptomatic courses were documented. Bronchiectasis was observed in 24/40 APDS1 patients who received a CT-scan compared with 4/15 APDS2 patients. By age 20, half of the patients had received at least one immunosuppressant, but 2–3 lines of immunosuppressive therapy were not unusual before age 10. Response to rapamycin was rated by physician visual analog scale as good in 10, moderate in 9, and poor in 7. Lymphoproliferation showed the best response (8 complete, 11 partial, 6 no remission), while bowel inflammation (3 complete, 3 partial, 9 no remission) and cytopenia (3 complete, 2 partial, 9 no remission) responded less well. Hence, non-lymphoproliferative manifestations should be a key target for novel therapies. This report from the ESID-APDS registry provides comprehensive baseline documentation for a growing cohort that will be followed prospectively to establish prognostic factors and identify patients for treatment studies

DNA bar coding and pyrosequencing to analyze adverse events in therapeutic gene transfer

Gene transfer has been used to correct inherited immunodeficiencies, but in several patients integration of therapeutic retroviral vectors activated proto-oncogenes and caused leukemia. Here, we describe improved methods for characterizing integration site populations from gene transfer studies using DNA bar coding and pyrosequencing. We characterized 160 232 integration site sequences in 28 tissue samples from eight mice, where Rag1 or Artemis deficiencies were corrected by introducing the missing gene with gamma-retroviral or lentiviral vectors. The integration sites were characterized for their genomic distributions, including proximity to proto-oncogenes. Several mice harbored abnormal lymphoproliferations following therapy—in these cases, comparison of the location and frequency of isolation of integration sites across multiple tissues helped clarify the contribution of specific proviruses to the adverse events. We also took advantage of the large number of pyrosequencing reads to show that recovery of integration sites can be highly biased by the use of restriction enzyme cleavage of genomic DNA, which is a limitation in all widely used methods, but describe improved approaches that take advantage of the power of pyrosequencing to overcome this problem. The methods described here should allow integration site populations from human gene therapy to be deeply characterized with spatial and temporal resolution

Risk Factor Analysis of Outcomes after Unrelated Cord Blood Transplantation in Patients with Hurler Syndrome

AbstractAllogeneic stem cell transplantation (SCT) is considered effective in preventing disease progression in patients with Hurler syndrome (HS). Unrelated umbilical cord blood (UCB) grafts are suggested as an alternative to bone marrow (BM) or peripheral blood stem cells (PBSC). We studied 93 HS patients receiving an UCB graft to analyze risk factors for outcomes. The median time from diagnosis to transplant was 4.6 months, median follow-up was 29 months, and median number of nucleated CB cells infused was 7.6 × 107/kg. Most of the patients received 1 or 2 HLA disparate grafts, and the most frequently used conditioning regimen was cyclophosphamide + busulfan (Bu/Cy). All patients received anti-T cell antibody. At post transplant day +60, the cumulative incidence of neutrophil engraftment was 85%. A younger age at transplant and a higher CD34+ dose at infusion were favorably associated with engraftment. With the exception of 2 patients, all engrafted patients achieved full and sustained donor chimerism. The 3-year event-free survival (EFS) and 3-year overall survival (OS) rates were 70% and 77%, respectively. In a multivariate analyses, use of Bu/Cy and a shorter interval from diagnosis to transplant were predictors for improved EFS rate (82% for patients transplanted within 4.6 months after diagnosis compared to 57% for the rest). Improved outcomes from early transplantation and immediate availability of CB unit lead us to conclude that CB transplantation is a beneficial option, which should be considered expediently for children with HS