Reticular dysgenesis is a human severe combined immunodeficiency that is primarily characterized by profound neutropenia and lymphopenia. The condition is caused by mutations in the adenylate kinase 2 (AK2) gene, resulting in the loss of mitochondrial AK2 protein expression. AK2 regulates the homeostasis of mitochondrial adenine nucleotides (ADP, ATP and AMP) by catalyzing the transfer of high-energy phosphate. Our present results demonstrate that AK2-knocked-down progenitor cells have poor proliferative and survival capacities and are blocked in their differentiation toward lymphoid and granulocyte lineages. We also observed that AK2 deficiency impaired mitochondrial function in general and oxidative phosphorylation in particular – showing that AK2 is critical in the control of energy metabolism. Loss of AK2 disrupts this regulation and leads to a profound block in lymphoid and myeloid cell differentiation.
Slow T-cell reconstitution is a major clinical concern after transplantation of cord blood (CB)-derived hematopoietic stem cells. Adoptive transfer of in vitro-generated T-cell progenitors has emerged as a promising strategy for promoting de novo thymopoiesis and thus accelerating T-cell reconstitution. Here, we describe the development of a new culture system based on the immobilized Notch ligand Delta-like-4 (DL-4). Culture of human CD34+ CB cells in this new DL-4 system enabled the in vitro generation of large amounts of T-cell progenitor cells that (a) displayed the phenotypic and molecular signatures of early thymic progenitors and (b) had high T lymphopoietic potential. When transferred into NOD/SCID/γc−/− (NSG) mice, DL-4 primed T-cell progenitors migrated to the thymus and developed into functional, mature, polyclonal αβ T cells that subsequently left the thymus and accelerated T-cell reconstitution. T-cell reconstitution was even faster and more robust when ex vivo-manipulated and nonmanipulated CB samples were simultaneously injected into NSG mice (i.e., a situation reminiscent of the double CB transplant setting). This work provides further evidence of the ability of in vitro-generated human T-cell progenitors to accelerate T-cell reconstitution and also introduces a feeder-cell-free culture technique with the potential for rapid, safe transfer to a clinical setting.
Several obstacles to the production, expansion and genetic modification of immunotherapeutic T cells in vitro have restricted the widespread use of T-cell immunotherapy. In the context of HSCT, delayed naïve T-cell recovery contributes to poor outcomes. A novel approach to overcome the major limitations of both T-cell immunotherapy and HSCT would be to transplant human T-lymphoid progenitors (HTLPs), allowing reconstitution of a fully functional naïve T-cell pool in the patient thymus. However, it is challenging to produce HTLPs in the high numbers required to meet clinical needs. Here, we found that adding tumor necrosis factor alpha (TNFα) to a DL-4-based culture system led to the generation of a large number of nonmodified or genetically modified HTLPs possessing highly efficient in vitro and in vivo T-cell potential from either CB HSPCs or mPB HSPCs through accelerated T-cell differentiation and enhanced HTLP cell cycling and survival. This study provides a clinically suitable cell culture platform to generate high numbers of clinically potent nonmodified or genetically modified HTLPs for accelerating immune recovery after HSCT and for T-cell-based immunotherapy (including CAR T-cell therapy).
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.
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.
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