M De Smedt

T-, B- and NK-lymphoid, but not myeloid cells arise from human CD34 + CD38 − CD7 + common lymphoid progenitors expressing lymphoid-specific genes

Hematopoietic stem cells in the bone marrow (BM) give rise to all blood cells. According to the classic model of hematopoiesis, the differentiation paths leading to the myeloid and lymphoid lineages segregate early. A candidate ‘common lymphoid progenitor’ (CLP) has been isolated from CD34+CD38− human cord blood cells based on CD7 expression. Here, we confirm the B- and NK-differentiation potential of CD34+CD38−CD7+ cells and show in addition that this population has strong capacity to differentiate into T cells. As CD34+CD38−CD7+ cells are virtually devoid of myeloid differentiation potential, these cells represent true CLPs. To unravel the molecular mechanisms underlying lymphoid commitment, we performed genome-wide gene expression profiling on sorted CD34+CD38−CD7+ and CD34+CD38−CD7− cells. Interestingly, lymphoid-affiliated genes were mainly upregulated in the CD7+ population, while myeloid-specific genes were downregulated. This supports the hypothesis that lineage commitment is accompanied by the shutdown of inappropriate gene expression and the upregulation of lineage-specific genes. In addition, we identified several highly expressed genes that have not been described in hematopoiesis before.

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.

Homeobox gene expression profile in human hematopoietic multipotent stem cells and T-cell progenitors: implications for human T-cell development

Class I homeobox (HOX) genes comprise a large family of transcription factors that have been implicated in normal and malignant hematopoiesis. However, data on their expression or function during T-cell development is limited. Using degenerated RT-PCR and Affymetrix microarray analysis, we analyzed the expression pattern of this gene family in human multipotent stem cells from fetal liver (FL) and adult bone marrow (ABM), and in T-cell progenitors from child thymus. We show that FL and ABM stem cells are similar in terms of HOX gene expression, but significant differences were observed between these two cell types and child thymocytes. As the most immature thymocytes are derived from immigrated FL and ABM stem cells, this indicates a drastic change in HOX gene expression upon entry into the thymus. Further analysis of HOX-A7, HOX-A9, HOX-A10, and HOX-A11 expression with specific RT-PCR in all thymocyte differentiation stages showed a sequential loss of 3′ region HOX-A cluster genes during intrathymic T-cell development and an unexpected expression of HOX-A11, previously not recognized to play a role in hematopoiesis. Also HOX-B3 and HOX-C4 were expressed throughout thymocyte development. Overall, these data provide novel evidence for an important role of certain HOX genes in human T-cell development.

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.

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.

Human intrathymic development: a selective approach

Human T lymphocytes can be generated from CD34 progenitor cells from different sources. This can be obtained in an in vivo model wherein human thymic tissue and fetal liver is transplanted in an immunodeficient mouse. However, human T cells are also generated in immunodeficient mice without co-transplantation of human thymus or in in vitro hybrid human–mouse fetal thymus organ culture. This shows that xenogeneic mouse thymus tissue supports human T cell differentiation. Finally, human T cells are generated on co-culture with murine stromal cells that express the Delta-like1 ligand for the Notch receptor. How these different environments influence the human T cell repertoire is reviewed and discussed.

Developmentally regulated responsiveness to transforming growth factor-β is correlated with functional differences between human adult and fetal primitive hematopoietic progenitor cells

Important functional differences exist between primitive CD34++CD38− hematopoietic progenitor cells derived from human fetal liver (FL) and adult bone marrow (ABM). FL progenitors are known to have higher proliferative capacities and lower cytokine requirements than their ABM counterparts. In this study, we isolated FL and ABM CD34++CD38− cells and used a two-stage culture system to investigate the effects of transforming growth factor-β (TGF-β) and blocking anti-TGF-β antibodies (anti-TGF-β) on these cells. First, we demonstrate that FL progenitors are significantly less sensitive to the inhibitory effects of TGF-β than ABM cells. Second, whereas ABM cells are significantly stimulated by anti-TGF-β, only very limited effects are seen on FL cells. Third, we show that the effect of anti-TGF-β is mainly situated at the level of the initial cell cycles of very primitive progenitor cells with a high proliferation potential. Fourth, we demonstrate that blocking the effects of endogenous TGF-β reduces the growth factor requirements of ABM cells in order to proliferate and differentiate. Based on these data, we hypothesize that at least part of the functional differences that exist between adult and fetal stem cells can be accounted for by a developmental different responsiveness to TGF-β.

Interleukin-2 stimulated T cell receptor Vγ3 positive thymocytes do not migrate to the skin

Abstract T cell receptor (TcR) Vγ3+ thymocytes, which only develop in the fetal thymus, migrate to the skin [1]. IL-2 stimulation of fetal day 18 murine thymocytes results in a cell population of which 45% of the cells express the TcR Vγ3 [2]. In this study, we describe that those IL-2 cultured TcR Vγ3+ thymocytes have the killing capacity of lymphokine activated killer cells: NK-susceptible as well as NK-resistant tumor cell lines were killed in an MHC-unrestricted manner. Because of these findings, IL-2-expanded TcR Vγ3+ thymocytes could have a potential use in adoptive immunotherapy for skin-located tumors. Therefore, we analyzed the migration pattern of IL-2-cultured TcR Vγ3+ thymocytes upon i.v. injection. We describe their initial entrapment in the lungs and subsequent accumulation in the liver. Localization in the skin was practically absent, and did not differ from that of IL-2 cultured adult thymocytes (mainly TcR αβ+). The migration pattern was identical in adult and newborn normal mice, and in adult nude mice. Analysis of the expression of asialo-GM1 revealed that it increased strongly after IL-2 culture. The relevance of this change in asialo-GM1 expression with reference to the migration upon i.v. injection is discussed. This study indicates that an improved understanding of the determinants of in vivo localization of IL-2 cultured cells may lead to improved strategies for adoptive immunotherapy of cancer.

Efficiency of transgenic T cell generation from gene-marked cultured human CD34+ cord blood cells is determined by their maturity and the cytokines present in the culture medium.

Success of gene therapy for diseases affecting the T cell lineage depends on the thymic repopulation by genetically engineered hematopoietic progenitor cells (HPC). Although it has been shown that retrovirally transduced HPC can repopulate the thymus, little information is available on the effect of the culture protocol. Moreover, for expansion of the number of HPC, cytokine supplemented culture is needed. Here, we transduced purified human umbilical cord blood (CB) CD34+ cells in cultures supplemented with various combinations of the cytokines thrombopoietin (TPO), stem cell factor (SCF), flt3/flk-2 ligand (FL), interleukin-3 (IL-3) and IL-6, and investigated thymus-repopulating ability of gene-marked HPC in vitro. Irrespective of the cytokine cocktail used, transduced CD34+CD38− CB cells, expressing the marker green fluorescent protein (GFP) encoded by the MFG-GFP retrovirus, have both superior proliferative and thymus-repopulating potential compared with transduced CD34+CD38+ CB cells. Effectively transduced GFP+CD34+CD38− HPC, cultured for 3 or 17 days, more readily generated T cells than GFP− HPC from the same culture. The reverse was true in the case of CD34+CD38+ HPC cultures. Finally, our results indicate that the number of GFP+ T cell progenitors actually increased during culture of CD34+CD38− HPC, in a magnitude that is determined by the cytokine cocktail used during culture.

LDH isoenzyme distribution in human Tγ lymphocytes

Abstract The lactate dehydrogenase (LDH) isoenzyme distribution was measured in T γ + lymphocytes from normal individuals. T γ + lymphocytes were obtained from purified T lymphocytes by ox-IgG rosette sedimentation. The B:A subunit ratio was clearly lower in the T γ + lymphocytes. Phenotyping of the T γ + lymphocytes showed a vast majority of OKT11 + , OKM1 + , OKT3 − lymphocytes. In one experiment, T γ + lymphocytes were enriched by OKT3 depletion of T lymphocytes. The low B:A ratio was also found in these cells indicating that the LDH pattern is not the consequence of an in vitro activation by immune complexes. As the T γ + lymphocytes were considerably enriched in cells having the characteristics of NK cells according to their phenotyping, morphology and NK cell activity, we may assume that NK cells have a low B:A ratio.