Biomedical subjects

A H Galy

Publications and source records attributed to A H Galy.

12 recordsLinked to original sources

PubMed1998

Bone marrow repopulation by human marrow stem cells after long-term expansion culture on a porcine endothelial cell line.

In vitro exposure of murine hematopoietic stem cells (HSCs) to cell cycle-inducing cytokines has been shown to result in a defect in the ability of these cells to engraft. We used a porcine microvascular endothelial cell (PMVEC) line in conjunction with exogenous interleukin (IL)-3, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) to expand human HSCs that express the CD34 and Thy-1 antigens but lack lineage-associated markers (CD34+Thy-1+Lin- cells). Ex vivo expansion of hematopoietic cells was evaluated in comparison to stromal cell-free, cytokine-supplemented cultures. Cells expressing the CD34+Thy-1+Lin- phenotype were detectable in both culture systems for up to 3 weeks. These cells were reisolated from the cultures and their ability to engraft human fetal bones implanted into SCID mice (SCID-hu bone) was tested. HSCs expanded in PMVEC coculture were consistently capable of competitive marrow repopulation with multilineage (CD19+ B lymphoid, CD33+ myeloid, and CD34+ cells) progeny present 8 weeks postengraftment. In contrast, grafts composed of cells expanded in stroma-free cultures did not lead to multilineage SCID-hu bone repopulation. Proliferation analysis revealed that by 1 week of culture more than 80% of the cells in the PMVEC cocultures expressing the primitive CD34+CD38- phenotype had undergone cell division. Fewer than 1% of the cells that proliferated in the absence of stromal cells remained CD34+CD38-. These data suggest that the proliferation of HSCs in the presence of IL-3, IL-6, GM-CSF, and SCF without stromal cell support may result in impairment of engraftment capacity, which may be overcome by coculture with PMVECs.

Animals↗

PubMed1995

Delineation of T-progenitor cell activity within the CD34+ compartment of adult bone marrow.

T-cell production is largely dependent on the presence of a thymus gland where CD34+ precursors mature into T lymphocytes. Prethymic stages of T-cell development are less defined. Therefore, this study aims to delineate T-progenitor cell potential within the CD34+ Lineage--(Lin-) cell compartment of adult bone marrow (ABM). Fractionation of CD34+ Lin- ABM cells with CD45RA, Thy-1, CD38, and HLA-DR failed to absolutely segregate T-cell reconstituting ability, indicating broad distribution of T-progenitor cell potential. Titration experiments showed that low numbers of CD34+ Lin- CD45RA+ (RA+) cells had greater thymus repopulating ability than CD34+ Lin- CD45RA- cells (RA-). The great majority (> 95%) of RA+ cells expressed CD38, HLA-DR and 70% to 90% of RA+ cells lacked Thy-1 surface expression. RA+ cells contained colony-forming unit granulocyte-macrophage (CFU-GM) progenitor cells but were depleted of erythroid potential, did not provide hematopoietic reconstitution of human bone fragments implanted into SCID mice, and did not efficiently maintain CD34+ cells with secondary clonogenic potential in bone marrow cultures. Thus, RA+ cells are oligopotent (nonprimitive) CD34+ progenitors with T-cell reconstituting ability. In contrast, these same assays indicated that CD34+ Lin- CD45RA- cells (RA- cells) comprised hematopoietic stem cells (HSC) with primitive multilineage (T, B, myeloid, and erythroid) hematopoietic potential. It was confirmed that HSC-containing populations, such as CD34+ Lin- CD45RA- Thy-1+ cells had thymus repopulating ability. Culture of RA- cells on murine bone marrow stromal cells in the presence of interleukin (IL)-3, IL-6, and leukemia inhibitory factor (LIF) generated CD34+ CD45RA+ progeny engrafting in a secondary severe combined immunodeficiency (SCID)-hu thymus assay. Altogether, our results underscore the fact that T-cell reconstituting potential can be dissociated from HSC activity. Furthermore, we speculate that HSC might develop into the T lineage indirectly, via differentiation into an intermediate oligopotent CD34+ CD45RA+ stage. Finally, T-progenitor cells can be cultured in vitro.

ADP-ribosyl Cyclase↗

PubMed1994

Human fetal bone marrow early progenitors for T, B, and myeloid cells are found exclusively in the population expressing high levels of CD34.

Experimentation on human stem cells is hampered by the relative paucity of this population and by the lack of assays identifying multilineage differentiation, particularly along the lymphoid lineages. In our current study, phenotypic analysis of low-density fetal bone marrow cells showed two distinct populations of CD34+ cells: those expressing a high density of CD34 antigen on their surface (CD34hi) and those expressing an intermediate level of CD34 antigen (CD34lo). Multiple tissues were used to characterize the in vitro and in vivo potential of these subsets and showed that only CD34hi cells support long-term B lymphopoiesis and myelopoiesis in vitro and mediate T, B, and myeloid repopulation of human tissues implanted into SCID mice. CD34lo cells repeatedly failed to provide long-term hematopoietic activity in vivo or in vitro. These results indicate that a simple fractionation based on well-defined CD34 antigen levels can be used to reproducibly isolate cells highly enriched for in vivo long-term repopulating activity and for multipotent progenitors, including T- and B-cell precursors. Additionally, given the limited variability in the results and the high correlation between in vitro and in vivo hematopoietic potential, we propose that the CD34hi population contains virtually all of the stem cell activity in fetal bone marrow and therefore is the population of choice for future studies in hematopoietic stem cell development and gene therapy.

Animals↗

PubMed1994

Lymphoid and myeloid differentiation of fetal liver CD34+lineage- cells in human thymic organ culture.

In this article, we report that the human fetal thymus contains CD34bright cells (< 0.01% of total thymocytes) with a phenotype that resembles that of multipotent hematopoietic progenitors in the fetal bone marrow. CD34bright thymocytes were CD33-/dull and were negative for CD38, CD2, and CD5 as well as for the lineage markers CD3, CD4, and CD8 (T cells), CD19 and CD20 (B cells), CD56 (NK cells), glycophorin (erythrocytes), and CD14 (monocytes). In addition, total CD34+ lineage negative (lin-) thymocytes contained a low number of primitive myeloid progenitor cells, thus suggesting that the different hematopoietic lineages present in the thymus may be derived from primitive hematopoietic progenitor cells seeding the thymus. To investigate whether the thymus is permissive for the development of non-T cells, human fetal organ culture (FTOC) assays were performed by microinjecting sorted CD34+lin- fetal liver cells into fragments of HLA-mismatched fetal thymus. Sequential phenotypic analysis of the FTOC-derived progeny of CD34+lin- cells indicated that the differentiation into T cells was preceded by a wave of myeloid differentiation into CD14+CD11b+CD4dull cells. Donor-derived B cells (CD19+CD20+) were also generated, which produced immunoglobulins (IgG and IgM) when cultured under appropriate conditions, as well as functional CD56+CD3- NK cells, which efficiently killed K562 target cells in cytotoxicity assays. These results demonstrate that the microinjection of fetal liver hematopoietic progenitors into fetal thymic organ fragments results in multilineage differentiation in vitro.

Antigens, CD↗

PubMed1994

Generation of T cells from cytokine-mobilized peripheral blood and adult bone marrow CD34+ cells.

The present study compared the T-cell progenitor content of CD34+ lineage (Lin)- cells isolated from normal adult bone marrow (ABM) and mobilized peripheral blood (MPB). Both cell populations were found to differentiate into T cells when injected into human fetal thymi implanted into severe combined immunodeficient mice. Cytokine-MPB cells were less efficient than ABM cells in engrafting in the fetal human thymus, although both gave rise to thymocytes with identical phenotypes based on the analysis of CD1a, CD3, CD4, and CD8 expression. Thymocytes derived from adult CD34+ Lin- cells were capable of fully differentiating into mature CD3+ T cells expressing either the T-cell receptor (TCR) gamma delta or the TCR alpha beta (the later associated with CD4 or CD8), showing that the T-cell progenies of adult CD34+ cells were polyclonal and functional. Our data indicate that human MPB CD34+ cells are qualitatively identical to their BM counterparts, and demonstrate the existence of T-lymphoid progenitor cell activity in MPB.

Adult↗

PubMed1993

Phenotypic and functional analysis of T-cell precursors in the human fetal liver and thymus: CD7 expression in the early stages of T- and myeloid-cell development.

It has been proposed that the CD7 molecule is the first antigen expressed on the membrane of cells committed to the T-cell lineage during human fetal T-cell ontogeny. To further identify the pre-T cell subpopulation that migrates to the thymus early in ontogeny, we analyzed the phenotypic and functional characteristics of the fetal liver populations separated on the basis of CD7 expression. Three populations expressing different levels of CD7 were observed: CD7bright, CD7dull, and CD7-. A CD7bright population depleted of mature T, B, and myeloid cells (lineage negative, lin-) and mostly composed of CD56+ CD34- natural killer cells did not mature into T cells in a fetal thymic organ culture (FTOC) assay and was devoid of myeloid progenitors in a clonal colony-forming cell assay. In contrast, the CD7-/dull CD34+ lin- populations were capable of differentiating into phenotypically mature T cells after injection into FTOC and contained early myeloid progenitors. Here we phenotypically compared the fetal liver CD7 populations with the most immature fetal thymic subset that differentiated in the FTOC assay, namely the triple negative (TN, CD3-CD4-CD8-) thymocytes. Fetal TN lin- expressed high levels of CD34 marker and were further subdivided by their expression of CD1 antigen, because CD1- TN thymocytes express higher levels of CD34 antigen compared with CD1+ TN cells. CD1- lin -TN thymocytes are characterized by expressing high levels of CD2, CD7, and CD34 markers and dull levels of CD5, CD10, and CD28 molecules. We could not find fetal liver pre-T cells with a phenotype equivalent to that of TN thymocytes. Our data show that CD7 does not necessarily identify T-cell precursors during fetal T-cell development and strongly support the hypothesis that the acquisition of early T-cell markers as CD2, CD28, and CD5 molecules on the cell surface of T-cell progenitors takes place intrathymically.

Antigens, CD↗

PubMed1993

Untransfected and SV40-transfected fetal and postnatal human thymic stromal cells. Analysis of phenotype, cytokine gene expression and cytokine production.

The thymic stromal network is complex and heterogeneous, containing thymic epithelial cells which are thought to play an important role during T-cell development and thymic fibroblasts which role is less defined. We herein present a phenotypic and functional comparison between defined thymic stromal cell populations. We transfected SV40 ori- into fetal and postnatal thymic stromal cell cultures and obtained SV40-immortalized clones of epithelial and fibroblastic nature as demonstrated by expression of intracellular keratin. These various clones were characterized in detail and compared to their untransfected bulk culture counterparts for phenotype, cytokine gene expression and cytokine production. All the different thymic stromal cells examined, constitutively expressed ICAM-1, LFA-3, MHC class I antigens, CD44, and the genes coding for IL-7, SCF and TGF-beta, but not TNF-alpha. After IL-1 stimulation, epithelial cells seemed to produce more GM-CSF than fibroblasts, and that trend was also seen for IL-6 secretion. SV40 cells were also regulated by IFN-gamma which induced MHC class II antigens and inhibited the IL-1 induced GM-CSF production. SV40 cells differed from their untransfected counterparts by an atypical expression of CD40 and lacked constitutive IL-1 alpha gene expression. We isolated clones with distinct properties, 24SV48, a highly proliferative CD34 positive TEC secreting low levels of GM-CSF and lacking constitutive IL-1 alpha and beta gene expression, and CT1SV93, an epithelial clone of postnatal origin with a high IL-1-induced cytokine production. In spite of differences with untransfected bulk cultures, the various SV40 immortalized clones may represent useful tools to further study the human thymic stroma.

Antigens, Surface↗

PubMed1993

Regulation of M-CSF production by cultured human thymic epithelial cells.

We have studied the regulation of M-CSF production by human thymic epithelial cells (TEC) in a continuing effort to better understand the contribution of TEC to the intrathymic cytokine network. The levels of M-CSF were measured by radioimmunoassay. Five different TEC cultures were studied and we found that all cells examined produced M-CSF constitutively. We also studied the effects of cytokines on the regulation of the M-CSF secretion profile. IL-1, which strongly induces the secretion of a number of cytokines in TEC, was found to up-regulate M-CSF levels. The effects of IL-1 were dose and time dependent suggesting a direct effect on TEC. IFN-gamma is known to up-regulate cell surface antigens, and to modulate the IL-1-induced cytokine response in TEC. IFN-gamma could induce M-CSF by itself, but the effects were less pronounced than those of IL-1. IFN-gamma did not modify the IL-1-induced M-CSF. IL-4, which has been shown to partially modulate the IL-1-induced GM-CSF in TEC, had no effect on constitutive or induced M-CSF levels. These data are discussed in the context of the regulation of myelopoietic growth factor production by thymic stromal cells.

Abortion, Induced↗

PubMed1992

CD40 is functionally expressed on human thymic epithelial cells.

CD40 is a prominent B cell Ag also found on certain epithelial cells and on carcinomas. In this report, we analyzed CD40 distribution in the human thymus. CD40 was not found on the majority of CD45-positive thymocytes, but was present in a CD45-negative stromal cell population. Immunohistology showed CD40 expression on cortical thymic epithelial cells (TEC), medullary TEC, thymic interdigitating cells, and thymic B cells. CD40 was not found on thymocytes, endothelial cells, or on the fibroblasts of the septae. Expression of CD40 was specifically maintained on cultured TEC and not found on cultured thymic fibroblasts. IL-1 alpha, TNF-alpha, IFN-gamma, but not IL-4, significantly up-regulated the membrane expression of CD40 on cultured TEC. The regulation of CD40 was similar to that of ICAM-1, and contrasted with that of MHC class II Ag, which could only be induced by IFN-gamma but not by IL-1, TNF, or IL-4. Northern blot analysis showed the presence of a 1.4-kb mRNA transcript for CD40 in TEC, which was up-regulated by IL-1 and IFN-gamma. Cross-linking of CD40 at the surface of human TEC in the absence of IL-1 stimulation failed to induce cytokine secretion. Triggering of TEC with anti-CD40 mAb in conjunction with IFN-gamma and IL-1 stimulation increased granulocyte-macrophage CSF secretion in a dose-dependent manner. The effect was visible as early as 24 h after triggering, occurred in the absence of cellular proliferation, and was specific for CD40 since triggering of other TEC membrane Ag such as ICAM-1 or MHC class I molecules had no effect to increase cytokine production in TEC. These data demonstrate that CD40 is expressed and is a functional molecule at the surface of the epithelial cells of the thymus.

Antigens, CD↗

PubMed1991

IL-1, IL-4, and IFN-gamma differentially regulate cytokine production and cell surface molecule expression in cultured human thymic epithelial cells.

We investigated the response of purified and cloned human thymic epithelial cells (TEC) to IL-1, IL-4, and IFN-gamma stimulation in vitro. IL-1 alpha strongly up-regulated the production of granulocyte-macrophage CSF (GM-CSF), granulocyte CSF (G-CSF), IL-6, and IL-8, as measured by specific immunoenzymetric assays and by increased steady state mRNA levels. IL-4 or IFN-gamma did not induce these cytokines in TEC but in a sustained and dose-dependent manner down-regulated the IL-1-induced GM-CSF protein and mRNA levels. Only IFN-gamma, and not IL-4, suppressed the IL-1-induced G-CSF and IL-8 production, as shown at both the protein and mRNA levels. The inhibition was dose dependent, sustained for at least 96 h, and more pronounced for G-CSF than for IL-8. In contrast, both IL-4 and IFN-gamma enhanced the IL-1-induced IL-6 production. IL-4 and IFN-gamma had additive effects to increase IL-6 secretion and to more completely suppress the IL-1-induced GM-CSF. Analyses of cell surface molecules showed that intercellular adhesion molecule 1 (ICAM-1) expression on TEC was increased by IL-1 or IFN-gamma. IL-4 slightly down-regulated constitutive ICAM-1 levels but did not significantly modify the levels of expression induced by either IL-1 or IFN-gamma. MHC class II expression was induced by IFN-gamma but not by IL-1 or IL-4. The combination of IL-1 and IL-4 with IFN-gamma did not alter the levels of class II MHC Ag induced by IFN-gamma. In conclusion, TEC cytokine production and cell surface molecule expression are differentially regulated via a complex cytokine network. Our data suggest that developing T cells provide, in part, the signals controlling the function of their supporting stroma.

Blotting, Northern↗

PubMed1990

Effects of cytokines on human thymic epithelial cells in culture. II. Recombinant IL 1 stimulates thymic epithelial cells to produce IL6 and GM-CSF.

Our earlier study reported the ability of interleukin 1 (IL1) to promote proliferation and to induce morphological changes of human thymic epithelial cells (TEC) in culture. The present study was undertaken to examine the effects of IL1 on the secretory function of TEC. Both human recombinant IL1 alpha and IL1 beta induced TEC to produce molecules in the culture supernatant fluids (TES) which displayed marked thymocyte proliferative capacities. This activity was specifically induced by IL1 since other TEC growth factors such as epidermal growth factor and a bovine pituitary extract had no effect on promoting secretion of T cell-activating molecules by TEC. Using specific radioimmunoassays for both forms of IL1, we found that unstimulated TEC produced negligible amounts of IL1 alpha and IL1 beta in TES, which were not increased by IL1 stimulation, and we concluded that the IL1-induced TES molecules were not IL1. IL1 induced TEC to produce IL6, as detected by the hybridoma growth factor biological activity. Neutralizing anti-IL6 antibodies completely blocked the thymocyte activating capacities of the IL1-induced TES thus implying a major role for IL6 in TEC-derived T cell activation. IL1 also induced TEC to produce GM-CSF as measured by bioassay and confirmed by an immunoenzymetric assay. Our results confirm that TEC are a source of cytokines and show that TEC respond to IL1 by producing cytokines with consequences on the thymic lymphoid population. This further emphasizes the importance and complexity of paracrine molecular interactions involved in intrathymic development.

Antibodies↗

PubMed1989

Effects of cytokines on human thymic epithelial cells in culture: IL1 induces thymic epithelial cell proliferation and change in morphology.

The role of thymic epithelium in T cell development has given rise to a number of studies, but less information is available concerning the factors regulating thymic epithelial cells (TEC) themselves. Several cytokines, natural or recombinant, were investigated for their effects on human TEC proliferation. This study presents evidence for the first time that human recombinant interleukin 1 (IL1) and IL1-containing mixed cytokine preparations induced DNA synthesis of TEC as measured in a 48-hr stimulation assay. The effects of IL1 were dose dependent and sustained in time. The following recombinant cytokines, IL2, IL3, IL4, interferon-gamma (IFN-gamma), IFN-alpha, tumor necrosis factor-alpha (TNF alpha), and TNF beta, as well as thymosin fraction 5 and Escherichia coli lipopolysaccharide (LPS), were not found to modify TEC proliferation but IFN-gamma and TNF alpha enhanced the effects of IL1. We also report that IL1 induced a profound change in the morphology of TEC. Our observations suggest that TEC are targets for the action of cytokines and emphasize the important role played by IL1 within the thymus.

Cell Division↗