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D Vanhecke

Publications and source records attributed to D Vanhecke.

14 recordsLinked to original sources
PubMed2006

High-throughput RNA interference in functional genomics.

RNA interference (RNAi) refers to post-transcriptional silencing of gene expression as a result of the introduction of double-stranded RNA into cells. The application of RNAi in experimental systems has significantly accelerated elucidation of gene functions. In order to facilitate large-scale functional genomics studies using RNAi, several high-throughput approaches have been developed based on microarray or microwell assays. The recent establishment of large libraries of RNAi reagents combined with a variety of detection assays has further improved the performance of functional genome-wide screens in mammalian cells.

Animals↗
PubMed2005

High-throughput subcellular protein localization using cell arrays.

Accomplishment of the human and mouse genome projects resulted in accumulation of extensive gene sequence information. However, the information about the biological functions of the identified genes remains a bottleneck of the post-genomic era. Hence, assays providing simple functional information, such as localization of the protein within the cell, can be very helpful in the elucidation of its function. Transfected cell arrays offer a robust platform for protein localization studies. Open reading frames of unknown genes can be linked to a His6-tag or GFP (green fluorescent protein) reporter in expression vectors and subsequently transfected using the cell array. Cellular localization of the transfected proteins is detected either by specific anti-His-tag antibodies or directly by fluorescence of the GFP fusion protein and by counterstaining with organelle-specific dyes. The high throughput of the method in terms of information provided for every single experiment makes this approach superior to classical immunohistological methods for protein localization.

Animals↗
PubMed2003

A rapid microbiopsy system to improve the preservation of biological samples prior to high-pressure freezing.

A microbiopsy system for fast excision and transfer of biological specimens from donor to high-pressure freezer was developed. With a modified, commercially available, Promag 1.2 biopsy gun, tissue samples can be excised with a size small enough (0.6 mm x 1.2 mm x 0.3 mm) to be easily transferred into a newly designed specimen platelet. A self-made transfer unit allows fast transfer of the specimen from the needle into the specimen platelet. The platelet is then fixed in a commercially available specimen holder of a high-pressure freezing machine (EM PACT, Leica Microsystems, Vienna, Austria) and frozen therein. The time required by a well-instructed (but not experienced) person to execute all steps is in the range of half a minute. This period is considered short enough to maintain the excised tissue pieces close to their native state. We show that a range of animal tissues (liver, brain, kidney and muscle) are well preserved. To prove the quality of freezing achieved with the system, we show vitrified ivy leaves high-pressure frozen in the new specimen platelet.

Animals↗
PubMed2000

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 alpha-chain of the IL-9 receptor (IL-9Ralpha) in early human T cell development. We report that addition of the mAb AH9R7, which recognizes and blocks selectively the human high affinity alpha-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-9Ralpha signaling is critical in early T lymphoid development.

Adjuvants, Immunologic↗
PubMed2000

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

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) TCR alpha beta, TCR gamma delta lymphocytes, NK cells, and dendritic cells complete their differentiation, (b) IL-7R alpha 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 cells and thymic homing of precursor cells.

Adult↗
PubMed1999

Thymic repopulation by CD34(+) human cord blood cells after expansion in stroma-free culture.

Thymic repopulation by transplanted hematopoietic progenitor cells (HPC) is likely to be important for long-term immune reconstitution and for successful gene therapy of diseases affecting the T-cell lineage. However, the T-cell progenitor potential of HPC, cultured in vitro for cell number expansion and gene transfer remains largely unknown. Here, we cultured highly purified human umbilical cord blood (CB) CD34(+)CD38(-) or CD34(+)CD38(+) cells for up to 5 weeks in stroma-free 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 expanded cells in vitro and in vivo. After up to 5 weeks of culture in IL-3 + SCF + IL-6 or TPO + FL + SCF supplemented medium, the progeny of CD34(+)CD38(-) CB cells generated T cells and natural killer cells in the thymus. Limiting dilution experiments demonstrated increase in the number of T-cell progenitors during culture. After 3 weeks of culture, gene marked CD34(+)CD38(-) CB cells injected in the human thymus fragment transplanted in severe combined immunodeficient (SCID) mice (SCID-hu) generated thymocytes expressing the retroviral encoded marker gene GFP in vivo. Thus, our results show that the progeny of CD34(+)CD38(-) CB cells cultured for extensive periods, harbor thymus-repopulating cells that retain T-cell progenitor potential after expansion and gene transfer.

Animals↗
PubMed1999

Human immunodeficiency virus nef gene expression affects generation and function of human T cells, but not dendritic cells.

Human immunodeficiency virus (HIV)-infected individuals develop an acquired immune deficiency syndrome (AIDS) due to loss in their lymphocyte numbers and cellular defects in T cells and antigen-presenting cells (APC). HIV infection of the thymus results in deficient replenishment of the peripheral naive T-cell pool. The HIV nef gene was shown to be important for progression towards AIDS and cellular depletion of the infected thymus. Here, we demonstrate by retroviral gene transfer that nef expression, in the absence of other HIV genes, impaired human thymic T-cell development. Thymocytes were generated in reduced numbers and downmodulated CD4 and CD8beta cell surface expression. T cells grown from nef-expressing thymocytes were hyperproliferative in vitro upon T-cell receptor triggering. Mature dendritic cells (DC) were functional and had normal surface CD4 levels despite nef expression. Thus, nef expression alone may contribute to AIDS development by reduced T-cell generation and T-cell hyperresponsiveness.

Animals↗
PubMed1999

In vitro intrathymic differentiation kinetics of human fetal liver CD34+CD38- progenitors reveals a phenotypically defined dendritic/T-NK precursor split.

Human CD34+CD38- hematopoietic precursor cells from fetal liver are able to develop into T, NK, and dendritic cells in a hybrid human/mouse fetal thymic organ culture (FTOC). In this report, we pay particular attention to the early events in differentiation of these precursor cells. We show that the CD34+CD38- precursor cells, which are CD4-CD7-cyCD3-HLA-DR-/++ (cy, cytoplasmatic), differentiate into a CD4+ population that remained CD7-cyCD3-HLA-DR++ and a CD4- population that expressed CD7 and cyCD3. The CD4+CD7-cyCD3- cells differentiate into phenotypically and functionally mature dendritic cells, but do not differentiate into T or NK cells. The CD4-CD7+cyCD3+ population later differentiates into a CD4+CD7+cyCD3+HLA-DR- population, which has no potential to differentiate into dendritic cells but is able to differentiate into NK cells and gammadelta and alphabeta T lymphocytes. These findings support the notion that the T/NK split occurs downstream of the NK/dendritic split.

ADP-ribosyl Cyclase↗
PubMed1997

Human thymocytes become lineage committed at an early postselection CD69+ stage, before the onset of functional maturation.

Mature functional CD4 or CD8 single positive (SP) thymocytes differentiate from immature CD4+ 8+ double positive (DP) precursors through a process of positive selection and terminal differentiation. To study CD4/CD8 lineage commitment, human postselection CD69+ thymocytes were separated into distinct subpopulations based on the differential expression of CD27, CD1, and CD45RA/RO. We demonstrate that these CD69+ subpopulations represent transitional stages of a common differentiation pathway during which CD69+ thymocytes that are initially CD27- CD1+ CD45RA- will sequentially up-regulate CD27, down-regulate CD1, and eventually acquire CD45RA upon maturation. Examination of CD4 and CD8 expression on these CD69+ subsets identified an early postselection CD69+ CD27- CD4SP population that gives rise to both CD4SP and CD8SP mature T cells when cultured in mouse thymus organs. In addition, a CD4+ 8+ DP population was identified that is CD69+ and CD27+, which only gives rise to CD8SP progeny upon culture. Although these results suggest that development of CD4SP and CD8SP cells may proceed through distinct intermediates, examination of active biosynthesis of CD4 and CD8 by the various subsets demonstrated that cells that have selectively terminated CD4 synthesis are already present in the CD27- CD4SP and CD27+ DP populations before culture. These data support a model of thymocyte differentiation whereby the decision of thymocytes to differentiate into one or the other lineage occurs concomitantly with, or very soon after, acquisition of CD69 and before the cells acquire CD27, down-regulate CD1, or acquire functional properties.

Animals↗
PubMed1997

Phenotypic and functional maturation of TCR gammadelta cells in the human thymus.

In contrast to thymic differentiation of TCR alphabeta cells, differentiation stages of TCR gammadelta cells are largely unknown. This report shows that CD1, a known marker of immature TCR alphabeta thymocytes, was expressed on some postnatal TCR gammadelta thymocytes. Only CD1+ TCR gammadelta thymocytes expressed recombination-activating gene-1 mRNA, and they were shown to differentiate into CD1- TCR gammadelta thymocytes. Functionally, sorted CD1- TCR gammadelta thymocytes proliferated in the presence of immobilized anti-CD3 Ab plus exogenous rIL-2 or rIL-15. Interestingly, in contrast to CD1- TCR alphabeta cells, CD1- TCR gammadelta thymocytes also proliferated extensively when cultured with exogenous rIL-2 or rIL-15 alone. FACS analysis as well as reverse transcription-PCR analysis showed that only CD1- TCR gammadelta thymocytes expressed IL-2Rbeta protein and mRNA. The differential expression of maturation markers, such as CD27, CD45RO, and CD45RA, as a function of expression of CD1 was similar in TCR gammadelta and TCR alphabeta thymocytes. An important exception is the expression of CD4 and CD8. Whereas TCR alphabeta thymocytes are mainly CD4-CD8 double positive at the immature CD1+ stage and CD4 or CD8 single positive at the mature CD1- stage, CD1(bright) TCR gammadelta thymocytes all expressed CD4, but only some of them expressed CD8. Some CD1- TCR gammadelta thymocytes also expressed CD8, but were negative for CD4. Collectively, our data clearly show that CD1 is a useful marker to distinguish immature human TCR gammadelta thymocytes from functional mature gammadelta cells based on recombination-activating gene-1 expression, in vitro differentiation, and phenotypic and functional characteristics.

Antigens, CD1↗
PubMed1997

MHC class II molecules are required for initiation of positive selection but not during terminal differentiation of human CD4 single positive thymocytes.

Positive selection of T cell precursors is an MHC dependent, multistep process by which functionally mature CD4+8- helper and CD4-8+ cytotoxic single positive (SP) T cells are generated from immature CD4+8+ double positive (DP) thymocytes. We investigated the requirement for TCR/MHC class II interactions during different stages of positive selection of human CD4 SP thymocytes. We show that sorted CD69- CD4+8+ DP preselection thymocytes cultured in fetal thymus lobes of normal mice were subject to positive selection and differentiated to CD3(high) CD69+, mature CD8 SP, and CD4 SP cells. When cultured in thymus lobes from MHC class II-deficient mice, these precursors failed to develop into mature CD4 SP T cells, indicating that in the hybrid cultures, murine MHC class II molecules are required for the development of mature human CD4 SP T cells. We have previously identified CD4 SP intermediate thymocytes that have received at least some of the signals involved in positive selection, since these cells are CD69+, CD3/TCR(high), and CD8beta- but that are still phenotypically and functionally immature. Here we demonstrate that in contrast to preselection thymocytes, these CD4 SP intermediate thymocytes can give rise to phenotypically mature and functionally CD4 SP progeny both in normal and in MHC class II-deficient thymus lobes. These results suggest that TCR/MHC interactions are required for the initial stages of positive selection, but are not essential during terminal differentiation to functionally mature CD4 SP T cells.

Animals↗
PubMed1995

Differentiation to T helper cells in the thymus. Gradual acquisition of T helper cell function by CD3+CD4+ cells.

We investigated at which point during thymocyte differentiation functions were acquired that are characteristic for mature Th cells. Differentiation from CD3+CD69-, CD4+CD8+ double-positive (DP) cells to terminally differentiated CD3+, CD4+CD8- single-positive (SP) cells was broken down into six discrete stages that were purified by four-color sorting: CD69-CD3+DP (stage 0), CD69+CD27-DP (stage 1), CD69+CD27-CD4+SP (stage 2), CD27+CD1+CD4+SP (stage 3), CD1-CD45RO+CD4+SP (stage 4), and CD1-CD45RO-CD4+SP cells (stage 5). Phenotypically, these stages seem to describe consecutive steps in differentiation from immature stage 0 to the terminally matured stage 5. Functionally, the capacity to proliferate on IL-2 after stimulation was absent in CD69- stage 0 cells, but was acquired gradually during stages 1 to 4. Clonal expandability and the capacity to respond to stimulation with the production of cytokines were acquired later and rather abruptly by CD1- stage 4 and 5 cells. Activation markers such as CD69 expression and in vivo IL-2 gene transcription came up simultaneously at the DP stage and peaked at stage 3 to 4. These data suggest that functional maturation of Th cells occurs over an extended period in differentiation, stages 1 to 4, and coincides with a gradual increase in activation markers. After completion of functional differentiation, at stage 5, in vivo IL-2 mRNA transcription and CD69 expression are down-regulated, and the cells become functionally resting naive T cells expressing CD45RA+.

Antigens, CD↗
PubMed1995

Characterization of distinct stages during the differentiation of human CD69+CD3+ thymocytes and identification of thymic emigrants.

During thymocyte development, CD69 expression is induced at an early stage of positive selection. To examine the differentiation of human CD69+CD3+ thymocytes, discrete phenotypes were defined by the relative expression of CD69, CD27, CD1, and CD45RA/RO: CD3+CD69- thymocytes were CD27-CD1+CD45RA-/RO+ (phenotype I), whereas the CD3+ CD69+ population could be subdivided into CD27-CD1+CD45RA-/RA+ (phenotype II), CD27+CD1+CD45RA-/RO+ (phenotype III), CD27+CD1-CD45RA-/dull/RO+ (phenotype IV), and CD27+CD1-CD45RA+/ROdull (phenotype V) thymocytes. Phenotype I thymocytes were CD4+CD8 alpha beta + double positive (DP). Phenotype II thymocytes contained DP and CD4+CD8 alpha dullCD8 beta- cells, whereas phenotype III thymocytes were DP and mostly CD4dullCD8 alpha beta+, indicating that CD27 on DP cells may be a marker for CD8-committed cells. Results obtained with SCID-hu mice, transplanted with human fetal thymus and liver, showed that immature human CD69-CD3+ thymocytes were corticosteroid-sensitive, whereas essentially all CD69+CD3+ cells were resistant. During differentiation of one cohort of corticosteroid-resistant CD69+CD3+ thymocytes, phenotype II and III thymocytes, disappeared within a week, whereas the percentage of phenotype IV and especially V thymocytes increased, suggesting that the latter represent the end stages in differentiation. Recent thymic emigrants in SCID-hu mice were identified as CD69-CD27+CD1-CD45RA-/dull/RO+ or CD45RA+/ROdull cells. Because phenotype IV and phenotype V thymocytes rapidly lose CD69 expression in cell culture, these thymocytes are probably the cells that are exported from the thymus. In conclusion, after the acquisition of CD69, thymocyte differentiation appears to continue in an ordered pattern, and cells that eventually leave the thymus are CD69-CD1-CD45RA+ or CD45RA-/dull.

Animals↗