Tessa Kerre

Generation of T cells from human embryonic stem cell-derived hematopoietic zones

Human embryonic stem cells (hESC) are pluripotent stem cells. A major challenge in the field of hESC is the establishment of specific differentiation protocols that drives hESC down a particular lineage fate. So far, attempts to generate T cells from hESC in vitro were unsuccessful. In this study, we show that T cells can be generated in vitro from hESC-derived hematopoietic precursor cells present in hematopoietic zones (HZs). These zones are morphologically similar to blood islands during embryonic development, and are formed when hESC are cultured on OP9 stromal cells. Upon subsequent transfer of these HZs on OP9 cells expressing high levels of Delta-like 1 and in the presence of growth factors, cells expand and differentiate to T cells. Furthermore, we show that T cells derive exclusively from a CD34highCD43low population, further substantiating the notion that hESC-derived CD34highCD43low cells are formed in HZs and are the only population containing multipotent hematopoietic precursor cells. Differentiation to T cells sequentially passes through the physiological intermediates: CD34+CD7+ T/NK committed, CD7+CD4+CD8− immature single positive, CD4+CD8+ double positive, and finally CD3+CD1−CD27+ mature T cell stages. TCRαβ+ and TCRγδ+ T cells are generated. Mature T cells are polyclonal, proliferate, and secrete cytokines in response to mitogens. This protocol for the de novo generation of T cells from hESC could be clinically and scientifically relevant.

Active Form of Notch Imposes T Cell Fate in Human Progenitor Cells

The crucial role of Notch signaling in cell fate decisions in hematopoietic lineage and T lymphocyte development has been well established in mice. Overexpression of the intracellular domain of Notch mediates signal transduction of the protein. By retroviral transduction of this constitutively active truncated intracellular domain in human CD34+ umbilical cord blood progenitor cells, we were able to show that, in coculture with the stromal MS-5 cell line, depending on the cytokines added, the differentiation toward CD19+ B lymphocytes was blocked, the differentiation toward CD14+ monocytes was inhibited, and the differentiation toward CD56+ NK cells was favored. The number of CD7+cyCD3+ cells, a phenotype similar to T/NK progenitor cells, was also markedly increased. In fetal thymus organ culture, transduced CD34+ progenitor cells from umbilical cord blood cells or from thymus consistently generated more TCR-γδ T cells, whereas the other T cell subpopulations were largely unaffected. Interestingly, when injected in vivo in SCID-nonobese diabetic mice, the transduced cells generated ectopically human CD4+CD8+ TCR-αβ cells in the bone marrow, cells that are normally only present in the thymus, and lacked B cell differentiation potential. Our results show unequivocally that, in human, Notch signaling inhibits the monocyte and B cell fate, promotes the T cell fate, and alters the normal T cell differentiation pathway compatible with a pretumoral state.

Both CD34+38+ and CD34+38- cells home specifically to the bone marrow of NOD/LtSZ scid/scid mice but show different kinetics in expansion.

Human hemopoietic stem cells (HSC) have been shown to engraft, differentiate, and proliferate in the hemopoietic tissues of sublethally irradiated NOD/LtSZ scid/scid (NOD/SCID) mice. We used this model to study homing, survival, and expansion of human HSC populations from different sources or phenotype. We observed that CD34+ cells homed specifically to bone marrow (BM) and spleen, but by 3 days after injection, survived only in the BM. These BM-homed CD34+ cells proliferated intensively and gave rise to a 12-fold, 5.5-fold, and 4-fold expansion in 3 days for umbilical cord blood, adult mobilized peripheral blood, and adult BM-derived cells, respectively. By injection of purified subpopulations, it was demonstrated that both CD34+38+ and CD34+38− umbilical cord blood HSC homed to the BM and expanded. Importantly, kinetics of expansion were different: CD34+38+ cells started to increase in cell number from day 3 onwards, and by 4 wk after injection, virtually all CD34+ cells had disappeared. In contrast, CD34+38− cells remained quiescent during the first week and started to expand intensively from the third week on. In this paper, we have shown that homing, survival, and expansion of stem cells are three independent phenomena important in the early phase of BM engraftment and that kinetics of engraftment differ between CD34+38+ and CD34+38− cells.

Specific Notch receptor-ligand interactions control human TCR-αβ/γδ development by inducing differential Notch signal strength.

Jagged2 preferentially signals through Notch3 to promote γδ T cell development.

Bone marrow CD34 ˛ cells generate fewer T cells in vitro with increasing age and following chemotherapy

To assess the influence of high‐dose chemotherapy and age on the intrinsic capacity of stem cells to generate T cells, CD34+ cells derived from bone marrow used in clinical transplantation were evaluated in an in vitro T‐cell assay using a mouse thymic microenvironment. CD34+ cells were sorted from healthy donor and autologous back‐up bone marrow after density gradient centrifugation and depletion for CD1, 3, 4, 7, 8, 19 and glycophorin A using magnetic beads. CD34+ cells were then introduced in day 14–15 fetal SCID mouse thymus lobes by incubation in hanging drops for 48 h. After transfer to gelfoam rafts they were cultured for variable time periods. The lobes were then homogenized in a tissue grinder for flow cytometric analysis gating on human cells. These were evaluated for CD4, CD8, CD3 and HLA‐DR surface expression. 51 samples were analysed and three patterns of T‐cell precursor development could be observed. In pattern A no human cells could be recovered, in pattern B maturation stopped at the CD4+CD8−CD3− pre‐T‐cell stage, and in pattern C maturation to double‐positive CD4+CD8+ thymocytes was reached. In 25 healthy donors under age 40 three showed pattern A, 12 pattern B and 10 pattern C, whereas in 16 healthy donors over the age 40 there were respectively four with A, seven with B and only five with C (P=0.01). In 10 patients who had previously received chemotherapy, none developed pattern C, five pattern B and five pattern A, in contrast to 15/41 pattern C, 19/41 pattern B and 7/41 pattern A in healthy donors. These data suggest an intrinsic loss of T‐cell generation capacity from adult bone marrow stem cells in comparison to reports on stem cells of fetal origin. This loss correlated weakly with age, irrespective of thymic involution, and may be further reduced by prior chemotherapy.

Functionally Mature CD4 and CD8 TCRαβ Cells Are Generated in OP9-DL1 Cultures from Human CD34+ Hematopoietic Cells

Human CD34+ hematopoietic precursor cells cultured on delta-like ligand 1 expressing OP9 (OP9-DL1) stromal cells differentiate to T lineage cells. The nature of the T cells generated in these cultures has not been studied in detail. Since these cultures do not contain thymic epithelial cells which are the main cell type mediating positive selection in vivo, generation of conventional helper CD4+ and cytotoxic CD8+ TCRαβ cells is not expected. Phenotypically mature CD27+CD1− TCRγδ as well as TCRαβ cells were generated in OP9-DL1 cultures. CD8 and few mature CD4 single-positive TCRαβ cells were observed. Mature CD8 single-positive cells consisted of two subpopulations: one expressing mainly CD8αβ and one expressing CD8αα dimers. TCRαβ CD8αα and TCRγδ cells both expressed the IL2Rβ receptor constitutively and proliferated on IL-15, a characteristic of unconventional T cells. CD8αβ+ and CD4+ TCRαβ cells were unresponsive to IL-15, but could be expanded upon TCR stimulation as mature CD8αβ+ and CD4+ T cells. These T cells had the characteristics of conventional T cells: CD4+ cells expressed ThPOK, CD40L, and high levels of IL-2 and IL-4; CD8+ cells expressed Eomes, Runx3, and high levels of granzyme, perforin, and IFN-γ. Induction of murine or human MHC class I expression on OP9-DL1 cells had no influence on the differentiation of mature CD8+ cells. Similarly, the presence of dendritic cells was not required for the generation of mature CD4+ or CD8+ T cells. These data suggest that positive selection of these cells is induced by interaction between T precursor cells.

Signals from the IL-9 Receptor Are Critical for the Early Stages of Human Intrathymic T Cell Development

Highly purified human CD34+ hemopoietic precursor cells differentiate into mature T cells when seeded in vitro in isolated fetal thymic lobes of SCID mice followed by fetal thymus organ culture (FTOC). Here, this chimeric human-mouse FTOC was used to address the role of IL-9 and of the α-chain of the IL-9 receptor (IL-9Rα) in early human T cell development. We report that addition of the mAb AH9R7, which recognizes and blocks selectively the human high affinity α-chain of the IL-9R, results in a profound reduction of the number of human thymocytes. Analysis of lymphoid subpopulations indicates that a highly reduced number of cells undergo maturation from CD34+ precursor cells toward CD4+CD3−CD8−CD1+ progenitor cells and subsequently toward CD4+CD8+ double positive (DP) thymocytes. Addition of IL-9 to the FTOC resulted in an increase in cell number, without disturbing the frequencies of the different subsets. These data suggest that IL-9Rα signaling is critical in early T lymphoid development.

Outcome in critically ill patients with allogeneic BM or peripheral haematopoietic SCT: a single-centre experience.

Outcome in haematological patients who develop critical illness has significantly improved over the last two decades, but less so in allogeneic BMT recipients. We prospectively investigated the outcome of 44 haematological patients with allogeneic BM or haematopoietic SCT (ABMT/AHSCT) requiring admission to the intensive care unit (ICU) of Ghent University Hospital between January 2000 and December 2007. We related outcome to the cause of critical illness, which was categorized as documented or clinically suspected bacterial infection, non-bacterial infection and non-infectious disease. Mechanical ventilation was required in 32 patients, and 12 patients received renal replacement therapy. Overall ICU-mortality, in-hospital mortality and 6-month mortality rates were 61, 75 and 80%, respectively. Hospital mortality rates in patients with bacterial infection (n=14), non-bacterial infection (n=13) and non-infectious disease (n=17) were 43, 85 and 94% (P=0.003). After adjustment for severity of illness sequential organ failure assessment (SOFA) score, bacterial infection (odds ratio 0.06, 0.01–0.36, P=0.002) was associated with significantly lower odds for hospital mortality. On the basis of our experience, ICU referral of ABMT/AHSCT patients is justifiable, as an acceptable fraction of these patients have longer-term survival. Documented or clinically suspected bacterial infection as the cause of critical illness is associated with better prognosis in comparison with other causes.

Human T lymphopoiesis. In vitro and in vivo study models.

Abstract: Successive Steps In T lymphocyte differentiation and T potential of human stem cells (HSC) can be tested in the following models: (a) the infusion of cells in NOD‐SCID MICE, (b) the injection of cells in renconstituted SCID/hu mice, (c) the differentiation of cells in fetal thymus organ culture (FTOC), and (d) on thymic stromal layers. using mixed human‐murine FTOC, we showed (a) TCRαβ, TCRγδ lymphocytes, NK cells, and dendritic cells complete their differentiation, (b) IL‐7Rα signaling and IL‐7 are essential, (c) a detailed phenotypic and functional analysis of discrete successive steps of positively selected thymocytes, (d) an efficient transduction of genes in HSC with persistent gene expression throughout the T‐lymphocyte differentiation, and (e) adaptation to submerging high oxygen culture increases the test sensitivity to a clonal assay. Other approaches are the in vivo SCID/hu reconstitution model. With this method small fragments of human fetal liver and thymus are implanted under the kidney capsule of an adult SCID mouse with result in an impressive human thymus organ, six months after transplantation. We use this model to study thymus T‐cell developmental kinetics, development of gene‐marked precursor cellsand thymic homing of precursor cells.

Gallstone recurrence after successful shock wave therapy: the magnitude of the problem and the predictive factors.

Gallstone recurrence after successful shock wave therapy: the magnitude of the problem and the predictive factors