ALBERT

Description: Abstract 4484 Hematopoietic stem cell transplantation (HSCT) is the treatment for an increasing number of inherited diseases. Graft rejection and acute Graft Versus Host Disease (GVHD) are known to be the two major complications of this procedure. A high frequency of graft rejection is observed particularly in patients with inflammatory conditions associated with congenital metabolic diseases, IFN-gR1 deficiency, familial hemophagocytic lymphohistiocytosis. GVHD results from immunological attack on target recipient organs (skin, liver and gut) by donor allogeneic T cells that are transferred along with the allograft. A better understanding of the patients' inflammatory environment in the course of HSCT may allow for the establishment of predictive markers for the occurrence of engraftment or GVHD and the improvement of conditioning regimens and post-transplant immunosuppressive therapy. Methods: All pediatric patients treated in the Department of Immuno-Hematology at the Necker Hospital who received allogeneic HSCT after full intensity conditioning regimen for nonmalignant disorders including inherited immunodeficiencies, metabolic diseases or hemoglobinopathies are eligible for inclusion. Plasma samples are collected before conditioning (day-15), at the time of transplant (day 0), at day+15 and day+30 post-HSCT. Validated markers of GVHD (Elafin, Reg3α, and ST2) as well as pro-inflammatory, anti-inflammatory, and angiogenic cytokines were analyzed, respectively, using sequential enzyme-linked immunosorbent assays and Luminex technology, which enables the determination of large numbers of cytokines in very small amounts of childrens' plasma. Engraftment was assessed on the presence of full donor chimerism, GVHD upon clinical, biological and histopathological data. Results: From April 2011 to March 2012, 15 patients were included (8 males and 7 females with an age ranging from 4 months to 9 years). Five patients presented with severe combined immunodeficiencies, three with chronic granulomatous disease, five with metabolic diseases (three Hurler and two osteopetrosis), one with congenital aplastic anemia and one with sickle cell diseaseConditioning regimen consisted of Busulfan/Fludarabin/ATG for ten patients, Busulfan/Fludarabin/Thiotepa/ATG for four patients and Busulfan/Cyclophosphamid/ATG in one. Donors were matched unrelated (n=6), 4 matched related (n=4), haplo-identical (n=4), and with intrafamilial mismatched donor (n=1). Acute graft rejection was observed in 2 of 15 patients and sustained engraftment in 13 of 15. Acute GVHD was observed in 6 of 15 patient occurring between day+10 and day+45 [grade 1 (n=1), grade 3 (n=3) and grade 4 (n=2)]. At day+30, we found significantly higher plasma concentrations of IFNγ, TNFα, ST2, and VEGF in patients with GVHD as compared to patients without GVHD. TNFα was the only cytokine for which the plasma concentrations was already significantly higher at day+15 in patients with GVHD versus patients without GVHD. No statistically significant differences in plasma concentrations of IL-4, IL-6, TGFβ, IL-7, IL-8, IL-12, IL-13 and IL-17 were observed. The cytokine that appeared to differentiate patients with GVHD and patients without GVHD the most was IFN-α2 which had plasma concentrations that were consistently and significantly higher in patients without GVHD as compared to patients with GVHD from day-15 to day+15. Conclusion: These results confirm the Th1 associated profile of acute GVHD and the importance of endothelial dysfunction in GVHD, as suggested by increased concentrations of ST2 and VEGF in patients with GVHD. The potential forIFN-α2 as a predictive marker of GVHD in our population needs further confirmation through additional inclusions. A larger population is also needed to determine if a specific cytokine milieu is associated with other common HSCT complications seen in our population such as graft rejection and vascular complications such as sinusoidal obstructive syndrome of the liver and pulmonary arterial hypertension. Disclosures: No relevant conflicts of interest to declare.

Description: Patients lacking expression of either RAG-1 or RAG-2 suffer from a Severe Combined Immuno-Deficiency (SCID) disease characterized by an early block in T and B lymphocytes differentiation leading to the absence of both mature lymphocyte subsets. This disease accounts for about 20% of SCID and the only curative treatment is hematopoietic stem cell transplantation, usually successful when an HLA-genoidentical donor is available. In the absence of such a donor, the success rate decreases along with the degree of HLA disparity between donor and recipient. Ex-vivo gene therapy of hematopoietic stem cells can be considered as an alternative treatment as a selective advantage of transgene-expressing cells is expected. Moreover, constitutive expression of only one of the two RAG proteins should not be harmful as concomitant expression of both genes is required for the recombination activity. We used a lentivecteur containing the RAG-1 cDNA transgene as a therapeutic vector to transduce bone marrow CD34+ cells obtained from RAG-1 deficient patients. The transduced cells were injected into N0D-SCID mice previously irradiated (3Gy) and treated with an anti-TMb1 antibody. Ten weeks after transplantation, in all treated mice, 35±15% of the bone-marrow cells express the human CD45 marker. In this population, 24±2% co-express CD19 and IgM demonstrating that B cell differentiation capacity has been restored. We also detected some CD33+ cells attesting the presence of human myeloid progenitors cells. Altogether, these results suggest that both lymphoid and myeloid precursors have been transduced and demonstrate that gene transfer into hematopoietic cells can reconstitute B cell development in vivo. Our data support the hypothesis that gene therapy could represent a possible alternative to bone marrow transplantation in RAG-1 deficient SCID disease.

Description: Identification of a thymus-seeding progenitor originating from human bone marrow constitutes a key milestone in understanding and correcting defects in T-cell development. Here, we report the characterization of a novel human bone marrow lymphoid-restricted subpopulation which is part of the lineage-negative CD34+CD10+ progenitor population and which can be distinguished from B-cell-committed precursors by the absence of CD24 expression. We demonstrated that these Lin-CD34+CD10+CD24- progenitors lack myeloid and erythroid potential but can generate B, T and NK lymphocytes following culture on MS5 or OP9-hDelta1 stroma. The gene expression profile of this population, analyzed by a multiplex RT-PCR assay, revealed co-expression of RAG1, TdT, PAX5, CD3ε and IL-7Rα. These progenitors are not only present in the bone marrow but also in the blood throughout life, suggesting an ability to circulate. Moreover we showed that the Lin-CD34+CD10+CD24- cells also correspond to the most immature population of the thymus which gives rise to Lin-CD34+CD7+ T-cell precursors. Taken as a whole these findings unravel for the first time the existence of a postnatal lymphoid-restricted population which is capable of migrating from the bone marrow to the thymus.

Description: Abstract 3532 Poster Board III-469 Prolonged posttransplant immune deficiency is a major complication following hematopoietic stem cell transplantation, particularly in the T-lymphoid compartment. Accelerating T-cell development by injecting donor derived T-cell precursors has been proposed as a novel strategy to shorten the immune deficient phase. Several research groups have successfully generated T-cell precursors from murine and human HSC in vitro by transitory exposure to the Notch-ligand presenting murine OP9DL1-cell line. Transfer of the in vitro generated murine T-cell precursors into irradiated NOD/SCID/γcnull-mice accelerated T-cellular reconstitution. However, the clinical application of the OP9DL1-system is limited. Recent studies have demonstrated that short exposure of cord blood CD34+ cells to Notch-ligand Delta-like 4 is sufficient to promote human T-cell differentiation in vitro. Here, we modified this technique to better characterize and ameliorate T-cell development in vitro, with the objective of eventually transferring this method to a clinical phase. Towards this aim, we exposed human CD34+ HSC derived from any available source to immobilized Notch-ligand Delta-like 4 in the presence of different cytokine combinations implicated in human haematopoiesis (IL-7, SCF, Flt3-ligand and TPO). Within 7 days a population of CD34+CD7+ and CD34-CD7++ T-cell precursors emerged in the presence of Delta-like 4, but not under control conditions. After 7 days the CD34+CD7+ population subsequently declined while further amplification of the CD34-CD7++ population was observed. Two distinct progenitor subsets emerged within the CD34-CD7++ population, namely CD34-CD7++CD5+ and CD34-CD7++CD5-. The CD34-CD7++CD5+ subset further acquired CD1a and, thus, adopted a pre-T-cell phenotype. Between days 7 and 14 the CD34-CD7++CD5- acquired a NK-cell phenotype, as indicated by CD16 and CD56 expression. Beyond 14 days no further expansion of the pre-T-cell fraction was observed, while the NK-cell fraction continued proliferating. More advanced stages T-cell development, such as immature single positive CD4+ cells as observable in OP9DL1 co-cultures, did not arise after exposing cells only to immobilized Delta-like4. Intermittent emergence of a CD13+CD14+CD7- myeloid population was observed within the first 14 days of culture on Delta-like 4; however, this population disappeared spontaneously and did not preserve its common myeloid progenitor. Selecting a more immature CD34+CD38- population resulted in a two-fold increase of the frequency of CD34+CD7+ and CD34-CD7++ cells as compared to the whole CD34+ population, while myeloid differentiation was inhibited. A further increase was obtained by replanting cultured cells to freshly coated plates with Delta-like 4 every 3 days of culture. T-cell precursors cells derived after 7 days of culture were injected into NOD/SCID/γcnull mice. The in vivo-experiments are ongoing and results are pending. Our results provide further evidence that human T-cell precursors can be generated in vitro, not only in co-culture with murine OP9DL1-cells but also by short exposure to immobilized Notch-ligand Delta-like 4. These ongoing experiments are an important prerequisite for the potential clinical application of this method. Disclosures: No relevant conflicts of interest to declare.

Description: In humans, little is known about post-natal lymphoid progenitors, especially those able to circulate, colonize the thymus and generate T lymphocytes. On the basis of a previous work published by Anne Galy in 1995, we have detected in the human post-natal bone marrow up to 60 yrs of age a population of progenitors characterized by their CD34+Lin-CD10+ phenotype. Their differentiation potential analysed by culture in methylcellulose medium indicated that in contrast with their CD10− counterparts, CD10+ progenitors have lost erythroid and myeloid potential. On the other hand, CD10+ progenitors cultured on MS5 or OP9/hDL1 stroma demonstrated an enriched capacity to generate B, T and NK lymphocytes as compared to CD10− precursors. In limiting dilution assays, the high lymhoid potential of CD10+ population was confirmed, since 1 out 15 of them gave rise to T cells, 1 out 23 to B cells and 1 out 90 to NK cells. Gene expression profile shows that CD10+ cells express both B and T restricted factors, such as RAG, Gata3, Pax 5 and TdT. In addition, recombination at the IgH locus is already going on, with multiple DJ, but also VDJ recombination products detected. More importantly, CD10+ precursors circulate in the peripheral blood and are detected in the thymus where they are part of the most immature thymocytes CD34+CD1a-CD38-. Altogether, our results demonstrate for the first time the existence of a post-natal lymphoid progenitor population with a broad lymphoid potential and the ability to reach the thymus and generate efficiently T cells. On the long term, their full characterization will pave the way for their enrichment and usage in therapy of primary lymphoid immunodeficiencies.

Description: Non-HLA identical hematopoietic stem cell transplantation (HSCT) provides a corrective therapy for most life-threatening primary immunodeficiencies (PID) and some malignant hemopathies. Despite advances made, severe complications following the treatment such as the prolonged persistence of T cell immunodeficiency still limit the use of this partially incompatible HSCT. After HSCT, the reconstitution of a functional T cell compartment relies on the availability of T cell precursors to rapidly seed the thymus and differentiate into mature T cells. We have previously demonstrated that an in vitro culture system based on the use of a modified Delta-like-4 (DLL4) Notch ligand and T cell cytokines allows for the effective generation of human T cell precursors from cord blood within 7 days. Moreover, once injected into NOD/SCID/gcko mice, T cell precursors generated in this system were able to colonize the thymus and generate a diversified and functional T-cell compartment. Here, we aimed at testing the capacity of adult HSPCs in this reconstitution system. We found that, like their CB- derived counterparts, T cell precursors generated from adult HPSCs phenotypically resembled thymic CD34+CD7+ cells with high in vitro T-cell differentiation potential. Interestingly, the peak of T cell progenitors for adult HSPCs occurred around day 3, compared to day 7 in CB. At this timepoint, T cell precursors derived from adult HSPC already expressed all critical genes for T cell lineage development, as well as the major chemokine receptors implicated in thymus homing. The introduction of retronectin further improved differentiation and proliferation of T cell progenitors from both HPSC sources in our in vitro system. Comparative molecular analysis of adult- and CB- derived progenitors suggested, that differential requirements for Notch receptor/ligand interactions may explain the differences in kinetics observed during the culture of the two types of HSPC. It remains to be further evaluated, whether targeted modifications of the Notch signaling pathway can improve the outcome of this in vitro T cell differentiation system for adult HPSCs. Overall our results suggest that adult HSPCs, like their CB- derived counterparts, provide an effective source of in vitro cultured T cell progenitors harboring all the necessary requirements for the in vivo -reconstitution of a functional T cell compartment. This is particularly important in the context of future clinical applications in HSCT where adult HSPCs are more available and more frequently used than CB HSPCs. Based on our results, we propose that upon injection into a patient, DLL4- cultured T cell precursors from both HSPC sources could significantly accelerate the reconstitution of the adaptive immune system after a partially HLA-incompatible HSCT. Currently, we are translating these results into a phase I clinical trial including adult and pediatric patients transplanted for malignant hemopathies or PIDs requiring an allogeneic HSCT from a HLA-partially mismatched donors. Disclosures No relevant conflicts of interest to declare.

Description: To generate T cells throughout adult life, the thymus must import hematopoietic progenitor cells from the bone marrow via the blood. The cellular and molecular mechanisms governing the circulation of thymus-seeding progenitor cells are well characterized in mice but not in humans. The aim of the present study was to characterize the molecular mechanisms and cellular components involved in thymus colonization by lymphoid progenitors (CD34+/CD10+/CD7-/CD24-) and the early steps of thymopoiesis under physiological conditions in humans. Our results demonstrate that circulating lymphoid progenitor cells express CCR9 and CXCR4 chemokine receptors, VLA-4, VLA-5 and VLA-6 integrins and PSGL-1 and CD44 adhesion molecules. We used in vitro migration and adhesion assays to validate the functional status of these markers. As in the mouse, human circulating progenitor cells enter the thymus at the corticomedullary junction (CMJ). Once in the thymus, crosstalk with thymic epithelial cells causes the circulating progenitors to commit to the T-cell differentiation pathway. In order to characterize thymic niches and interactions between circulating progenitors and the thymic stroma, we undertook a chemokine/chemokine-receptor-focused gene expression analysis of sorted lymphoid progenitor cells and CMJ epithelial cells (based on the expression of EpCAM and Delta-like-4). We observed an unexpected gene expression profile for chemokines and chemokine regulators in thymus-seeding CD34+/CD10+/CD7-/CD24- cells and epithelial cells at the CMJ. The present results should help us to highlight candidate genes involved in the early steps of human thymopoiesis. Disclosures No relevant conflicts of interest to declare.