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W van Ewijk

Publications and source records attributed to W van Ewijk.

At least 19 recordsLinked to original sources
PubMed1996

Disrupted splenic architecture, but normal lymph node development in mice expressing a soluble lymphotoxin-beta receptor-IgG1 fusion protein.

Early in ontogeny, the secondary lymphoid organs become populated with numerous cells of mesodermal origin which forms both the lymphoid and stromal elements. The critical receptor/ligand interactions necessary for lymphoid organogenesis to occur are for the most part unknown. Although lymphotoxin-alpha (LT alpha) has been shown to be required for normal lymph node, Peyer's patch, and splenic development, it is unclear if soluble LT alpha 3, and/or cell-bound lymphotoxin-alpha beta (LT alpha beta) mediate these developmental events. Here we report that blocking LT alpha beta/lymphotoxin-beta receptor (LT beta R) interaction in vivo by generating mice which express a soluble LT beta R-Fc fusion protein driven by the human cytomegalovirus promoter results in an array of anatomic abnormalities affecting both the spleen and Peyer's patches, but not the lymph nodes. These results demonstrate that surface LT alpha beta ligand plays a critical role in normal lymphoid organ development.

Aging↗
PubMed1996

Cross-linking of T-cell receptors on double-positive thymocytes induces a cytokine-mediated stromal activation process linked to cell death.

To investigate molecular events associated with the intrathymic process of negative selection, we established an in vivo system using an anti-CD3 epsilon monoclonal antibody to induce synchronous apoptosis in the thymus of AND T-cell receptor (TCR) transgenic RAG-2-/- mice in a non-selecting haplotype. This model eliminates endogenous negative selection as well as gene activation in the mature thymocyte compartment, offering an ideal source of tester (anti-CD3 epsilon-treated) and driver (untreated) thymus RNA for representational difference analysis (RDA). Fourteen mRNA sequences that are up-regulated in the thymuses of such mice 2-6 h after anti-CD3 epsilon treatment were identified. Surprisingly, the majority of these transcripts were derived from stromal cells rather than the TCR-cross-linked CD4+CD8+TCRlow thymocytes including the macrophage products IL-1, the chemokine Mig and the transcription factor LRG-21. IFN-gamma secretion from the CD4+CD8+TCRlow thymocytes regulates macrophage Mig production. Three other cytokines (IL-4, GM-CSF and TNF-alpha), known to activate a variety of stromal cells, are also induced in the same thymocyte population undergoing apoptosis. Expression of a TNF-alpha-inducible gene, B94, in stromal cells after TCR ligation further supports the notion of cross-talk between thymocytes and stroma. Thus, TCR-triggered immature thymocytes elaborate cytokines which may regulate the delivery of further signals from stromal cells required for apoptosis.

Animals↗
PubMed1996

CD27 cooperates with the pre-T cell receptor in the regulation of murine T cell development.

CD27 is a lymphocyte-specific member of the TNF receptor family and has a TNF-related transmembrane ligand, CD70. The CD27/CD70 receptor-ligand pair cooperates with the TCR in the regulation of the peripheral T cell response. The study presented here reveals that CD27 may play a similar role in thymic pre-T cell development. We have previously cloned the cDNA encoding murine CD27, prepared specific mAbs and observed that murine CD27 is expressed on virtually all thymocytes, with the exception of a subpopulation of CD4-8- precursor T cells. It is shown here that induction of murine CD27 expression occurs at the transition from the CD4-8-25+ to the CD4-8-25- precursor T cell stage and is regulated by the pre-TCR. Therefore, we investigated whether CD27 contributes to pre-TCR-mediated thymocyte development. Pre-TCR function was mimicked by the induction of CD3 signaling in thymocytes of recombination activating gene (RAG)-deficient mice. This in vivo anti-CD3 epsilon mAb treatment induces an about fifty fold numerical expansion of CD4-8-25+ thymocytes and their differentiation to the CD4+8+25- stage. Co-injection of anti-CD27 mAb inhibited the CD3-mediated expansion and differentiation of the CD4-8-25+ precursor population. Also, injection of anti-CD27 mAb in TCR alpha-/- mutant mice led to a reduction in the absolute number of CD4+8+25- thymocytes. We present evidence that in these in vivo systems, anti-CD27 mAb inhibits CD27-ligand interaction. Therefore, we conclude that CD27 may contribute to normal murine T cell development by synergizing with the pre-TCR-mediated signal.

Animals↗
PubMed1996

Mice lacking the MHC class II transactivator (CIITA) show tissue-specific impairment of MHC class II expression.

CIITA activates the expression of multiple genes involved in antigen presentation and it is believed to be required for both constitutive and IFN gamma-inducible expression of these genes. To understand the role of CIITA in vivo, we have used gene targeting to generate mice that lack CIITA. CIITA-deficient (-/-) mice do not express conventional MHC class II molecules on the surface of splenic B cells and dendritic cells. In addition, macrophages resident in the peritoneal cavity do not express MHC class II molecules upon IFN gamma stimulation nor do somatic tissues of mice injected with IFN gamma, in contrast with wild-type mice. The levels of Ii and H-2M gene transcripts are substantially decreased but absent in CIITA (-/-) mice. The transcription of nonconventional MHC class II genes is, however not affected by CIITA deficiency. A subset of thymic epithelial cells express MHC class II molecules. Nonetheless, very few mature CD4 T cells are present in the periphery of CIITA (-/-) mice despite MHC class II expression in the thymus. Consequently, CIITA(-/-) mice are impaired in T-dependent antigen responses and MHC class II-mediated allogeneic responses.

Animals↗
PubMed1996

Expression of tyrosine kinase gene in mouse thymic stromal cells.

Amongst the most important signal transduction molecules involved in regulating growth and differentiation are the protein tyrosine kinases (PTK). Since T cell development is a consequence of interactions between thymic stromal cells (TSC) and thymocytes, identification of the PTK in both compartments is required to dissect the mechanisms that control this process. Here we report a search for PTK in mouse TSC, using RT-PCR to survey the repertoire of PTK mRNAs expressed in a freshly isolated TSC preparation. We identified 10 different PTK cDNAs among the 216 cDNAs sequenced, and demonstrate that transcripts of three of those (ufo, fyn and fer) are widely expressed among a large panel of immortalized thymic epithelial cell lines (TEC) and in primary cultures of TSC. Of the other seven, none were expressed in established TEC lines but, instead, displayed distinct expression patterns in cell types likely to have contaminated the fresh TSC preparation, i.e., macrophages, B cells, T cells and fibroblasts. Among the three PTK expressed in TEC lines, only one, ufo, exhibited expression exclusively in cells of non-hemopoietic origin. Although expression of ufo (also known as tyro 7, axl or ark) is not thymic-specific, in that it is also expressed in cell types of mesodermal origin in other tissues, its presence in TEC suggests a role for ufo in differentiation of the TSC compartment. Consistent with this notion, high-level expression of this receptor PTK at the protein level could be documented in every TEC line investigated, as well as in fresh thymus tissue sections. These data provide the first example of a receptor PTK in TSC and open new approaches to study the regulation of TSC differentiation.

Amino Acid Sequence↗
PubMed1995

Developmental control point in induction of thymic cortex regulated by a subpopulation of prothymocytes.

T lymphocytes of the alpha/beta T-cell receptor (TCR) lineage mature in the thymus, where they undergo a series of differentiation, expansion and selection events. For normal T-cell ontogeny to occur, thymocytes must interact physically with cortical and medullary thymic stroma cells. In parallel, interactions of the thymic stromal cells with TCR-positive thymocytes are necessary for the development of the thymic medulla. Comparable requirements for the differentiation of the cortex have not been defined, however. Here we analyse mutant mouse strains to assess the function of early prothymocytes in the induction of the thymic cortex. We find that animals with a developmental block at the earliest stage of T-lineage commitment lack a functional thymic cortex. This abnormality could be corrected in fetal but not adult animals by transplantation of either fetal or adult wild-type haematopoietic stem cells. Thus a developmentally restricted interaction of fetal stromal cells with early prothymocytes is required for the induction of a cortical microenvironment. In addition, a normal thymic architecture is necessary for sustained T-cell ontogeny.

Aging↗
PubMed1995

Specific immunologic recognition of the tumor-specific E2A-PBX1 fusion-point antigen in t(1;19)-positive pre-B cells.

The t(1;19) translocation is the most commonly observed chromosomal translocation in childhood acute lymphoblastic leukemia (ALL). Its presence among pre-B cell ALL cases, has been associated with a poor prognosis. Two genes, E2A and PBX1, are involved in this t(1;19) translocation. As a consequence, parts of the E2A and PBX1 genes are fused, resulting in a chimeric E2A-PBX1 gene, encoding chimeric E2A-PBX1 proteins. As such, the amino acid sequence at the fusion site represents a unique tumor-specific determinant. We report on the generation of a polyclonal antiserum, termed BP 1/19, raised against the tumor-specific E2A-PBX1 junction of E2A-PBX1 proteins. The specificity of antiserum BP 1/19 for the E2A-PBX1 fusion-point is demonstrated at the peptide and at the protein level. Furthermore, specific binding of antiserum BP 1/19 to t(1;19) positive cells was shown using immunofluorescence techniques. The study shows that: (1) the tumor-specific fusion-point epitope on E2A-PBX1 proteins is presented in an antigenic fashion, and (2) this particular fusion-point epitope can be used in immunological marker analysis using fluorescence microscopy.

Amino Acid Sequence↗
PubMed1994

Markers of mouse macrophage development detected by monoclonal antibodies.

In this review, we present and discuss a selected panel of antibody-defined markers expressed during different stages of mouse macrophage development. We distinguish four categories of markers, which are characteristic of: (1) macrophage precursors and immature macrophages (ER-MP12, ER-MP20, ER-MP54, ER-MP58); (2) mature macrophages in general (F4/80, BM8, Mac-1, Mac-2, ER-BMDM1); (3) macrophage subsets (ER-HR3, ER-MP23, ER-TR9, Forssman antigen, MOMA-1, MOMA-2, Monts-4, SER-4), and (4) IFN-gamma-stimulated macrophages (H-2Ia, LFA-1, ICAM-1, 158.2, MBR-2, TM-2, TM-4, and TM-5). It should be noted that many of the markers in this last category are inducible by other stimuli as well. The rigid classification of markers into four separate groups should be regarded as a digitalization of a continuum, thus inevitably implicating a simplification of the complex phenotypic changes that occur during mononuclear phagocyte development. Nevertheless, the current selection of antibodies against markers for different developmental stages of macrophages constitutes an important tool for characterization of mouse macrophages which participate in various biological processes.

Animals↗
PubMed1994

New monoclonal antibodies against the putative immunosuppressive site of retroviral p15E.

Both retroviral infections as well as human tumors may cause immunosuppression. One of the factors involved in immunosuppression in patients with squamous cell carcinoma of the head and neck (SCC-HN) is a protein related to the retroviral protein p15E. A conserved, 17-amino acid sequence represents the immunosuppressive epitope of retroviral p15E. In order to study the relationship between SCC-HN associated immunosuppression and retroviral p15E, we produced three new monoclonal antibodies (MAbs; ER-IS1, ER-IS2, and ER-IS5) directed against the immunosuppressive synthetic CKS-17 peptide. These MAbs react with the immunosuppressive peptide (in enzyme-linked immunosorbent assay), with human tumor cell lines (in FACScan analysis), with retroviral p15E (on Western blot), and with cryostat sections of SCC-HN tumor tissue. In addition, the MAbs neutralize the immunosuppressive low molecular weight factors present in sera of patients with SCC-HN. These results show that retroviral p15E and the immunosuppressive factors associated with SCC-HN share a conserved immunosuppressive epitope and that MAbs against this epitope can be used for detection and neutralization of the tumor-associated immunosuppressive protein(s).

Amino Acid Sequence↗
PubMed1994

Distinct mouse bone marrow macrophage precursors identified by differential expression of ER-MP12 and ER-MP20 antigens.

The characterization of early branch points in the differentiation of leukocytes requires identification of precursor cells in the bone marrow. Recently, we produced two monoclonal antibodies, ER-MP12 and ER-MP20, which in two-color flow-cytometric analysis divide the murine bone marrow into six defined subsets. Here we show, using fluorescence-activated cell sorting followed by macrophage colony-stimulating factor-stimulated culture in soft agar, that precursors of the mononuclear phagocyte system reside only within the ER-MP12hi20-, ER-MP12+20+ and ER-MP12-20hi bone marrow subsets. Together, these subsets comprise 15% of nucleated bone marrow cells. Furthermore, we provide evidence that the macrophage precursors present in these subsets represent successive stages in a maturation sequence where the most immature ER-MP12hi20- cells develop via the ER-MP12+20+ stage into ER-MP12-20hi monocytes.

Animals↗
PubMed1994

Inhibition of proliferation and differentiation during early T cell development by anti-transferrin receptor antibody.

Proliferating cells require iron and, therefore, express the transferrin receptor (CD71) that mediates cellular iron uptake. Cycling thymocytes, which have the CD4-8-3-, CD4-8+3-, or CD4+8+3- phenotypes, also express CD71. The importance of CD71-mediated iron uptake for proliferation and maturation of thymocytes was studied using fetal thymus organ cultures at day 14 of gestation and treating them for 7 days with a CD71 monoclonal antibody (mAb). The intracellular iron deficiency caused by this treatment, inhibits both proliferation and maturation of the thymocytes. Cell recovery was reduced by 60%, but cells still expanded tenfold during the culture. Remarkably, the final maturation of alpha beta T cells was completely blocked as no thymocytes with low or high CD3/alpha beta TcR expression developed. Moreover, only few cells reached the CD4+8+3- stage of T cell development. CD4-8-3- thymocytes, however, as well as its CD44-25+ subset developed in normal numbers, suggesting that CD44-25+ CD4-8-3- cells, or their immediate progeny, were most vulnerable to CD71 mAb treatment. The development of gamma delta T cells, which also express CD71, was not affected in these cultures. This suggests that gamma delta T cells are either less iron-dependent or possess alternative iron-uptake mechanisms. Thus, our observations indicate that CD71 treatment, causing decreased intracellular iron levels, severely inhibits the major proliferation phase from the CD44-25+ CD4-8-3- to the CD4+8+3- cells, and completely abrogates the final maturation of CD4+8+3- cells into alpha beta TcR-expressing cells. In contrast, proliferation and differentiation of the earliest thymic precursors into CD44-25+ CD4-8-3- cells is not affected by CD71 treatment.

Animals↗
PubMed1994

Transferrin receptor expression as a marker of immature cycling thymocytes in the mouse.

Dividing cells require iron and, therefore, express the transferrin receptor (CD71) on the cell surface to enable internalization of transferrin-bound iron. Since early T cell development is marked by intense proliferation, we questioned whether CD71 might serve as a marker of immature T cells. Therefore, we analyzed the expression of CD71 on fetal, neonatal, and adult thymocytes in correlation with cell size, cell cycle status, and expression of CD3, CD4, CD8, alpha beta TcR, and gamma delta TcR. Phenotypic analysis showed that only the large, immature CD4-8-3-, CD4-8+3-, and CD4+8+3- cells in fetal, neonatal, and adult thymus expressed CD71. In addition, DNA analysis showed that all CD71+ large adult thymocytes were cycling. Downregulation of CD71 occurs when proliferation ceases, i.e., within the CD4+8+3- thymocyte subpopulation. The gradual changes in size and CD71 expression suggest a sequential development within this CD4+8+3- subpopulation from large CD71+ via small CD71+/- to small CD71- cells. As a consequence, CD71 expression is downregulated, in adult T cell development as well as in ontogeny, before the alpha beta TcR appears on the cell surface of the thymocyte. Together, our findings show that CD71 is a marker of immature, proliferating T cells.

Animals↗
PubMed1994

The ER-TR4 monoclonal antibody recognizes murine thymic epithelial cells (type 1) and inhibits their capacity to interact with immature thymocytes: immuno-electron microscopic and functional studies.

The thymic stroma is heterogeneous with regard to cellular morphology and cellular function. In this study, we employed the monoclonal antibody ER-TR4 to characterize stromal cells at the ultrastructural level. To identify the labelled cell type, we used two techniques: immunogold labelling on ultrathin frozen sections and immunoperoxidase staining on thick "vibratome" sections. ER-TR4 reacted with thymic Type 1 epithelial cells (according to our classification). A dense labelling appears in the cytoplasm of cortical cells using the two techniques. Immunogold labelling identified small cytoplasmic vesicles whereas the cytoplasm and the cell membrane seem to be labelled with the immunoperoxidase technique. ER-TR4 also identified isolated thymic nurse cells (TNC), and was observed in vitro to inhibit the capacity of some type 1 epithelial cells to establish interactions with immature thymocytes. This finding supports the hypothesis that the factor is involved in the formation of lymphoepithelial interactions within thymic nurse cells, and thus in the relations that immature thymocytes establish with the thymic microenvironment.

Animals↗
PubMed1994

Crosstalk in the mouse thymus.

The development of mature T cells within the thymus is dependent upon intact cortical and medullary microenvironments. In turn, thymic microenvironments themselves are dependent on lymphoid cells to maintain their integrity. Here, Willem van Ewijk and colleagues discuss experiments that have established the phenomenon of 'crosstalk' within the mouse thymus and suggest a mechanism whereby lymphoid and stromal cells influence each other in a consecutive manner during T-cell development.

Animals↗
PubMed1994

Recognition of the ALL-specific BCR-ABL junction in P190bcr-abl by monoclonal antibody ER-FP1.

The pH chromosome, resulting from the t(9;22) translocation, is the most frequently observed cytogenetic aberration in acute lymphoblastic leukemia (ALL). Two genes, bcr and abl, are involved in this translocation. As a consequence, parts of the bcr and abl genes are fused, resulting in chimeric bcr-abl genes encoding chimeric BCR-ABL proteins. Three bcr-abl genes and proteins have been identified: e1-a2 P190bcr-abl, b2-a2 P210bcr-abl, and b3-a2 P210bcr-abl. Since these chimeric proteins only occur in Ph-chromosome-positive leukemic cells, they are by definition tumor-specific markers. Ph-chromosome-positive ALL is correlated with a bad prognosis, therefore the detection of chimeric BCR-ABL proteins is of prime importance in ALL diagnosis. In the present study, we report on the generation of a monoclonal antibody termed ER-FP1, raised against the tumor-specific e1-a2 BCR-ABL junction in P190bcr-abl. We show that ER-FP1 reacts highly specifically with e1-a2 P190bcr-abl in different assays. The reactivity of ER-FP1 with e1-a2 P190bcr-abl in soluble form was analyzed in an immunoprecipitation assay; specificity was confirmed by peptide inhibition studies. Binding of ER-FP1 to e1-a2 P190bcr-abl at the single cell level was detected by using immunofluorescence techniques. Immunological double-staining experiments using ER-FP1 and a monoclonal antibody recognizing all BCR-ABL proteins confirmed the specificity of ER-FP1 for the e1-a2 fusion point.

Adult↗
PubMed1993

ER-MP12 antigen, a new cell surface marker on mouse bone marrow cells with thymus-repopulating ability: I. Intrathymic repopulating ability of ER-MP12-positive bone marrow cells.

We searched for new cell surface markers that allow a positive identification of thymus-repopulating cells in the bone marrow (BM) of the mouse. Recently we raised two rat monoclonal antibodies (ER-MP12 and ER-MP20) that recognize cell surface antigens expressed by mouse haematopoietic progenitor cells, among which are progenitor cells of the macrophage lineage. Here we show that the ER-MP12 antigen, but not the ER-MP20 antigen, is also expressed by BM cells with thymus-repopulating ability. Using ER-MP12 and ER-MP20 in two-colour immunofluorescence analysis six subpopulations of BM cells can be identified. The thymus-repopulating ability of each BM subpopulation was assessed after fluorescence-activated cell sorting and subsequent intrathymic injection into sublethally irradiated Thy-1 congenic recipient mice. Thymus-repopulating activity appeared to be exclusively confined to two subsets of BM cells expressing either high or intermediate levels of the ER-MP12 antigen, but lacking ER-MP20 antigen expression. These BM subsets comprised 1-2% and 30% of total nucleated BM cells respectively. The frequency of thymus-repopulating cells was maximal in the minor BM subpopulation with the highest level of ER-MP12 antigen expression. We conclude that ER-MP12 detects a hitherto unknown cell surface marker expressed by BM cells with thymus-repopulating ability.

Animals↗
PubMed1993

ER-MP12 antigen, a new cell surface marker on mouse bone marrow cells with thymus-repopulating ability: II. Thymus-homing ability and phenotypic characterization of ER-MP12-positive bone marrow cells.

In the accompanying paper we showed that six distinct subsets of bone marrow (BM) cells can be identified using the mAb ER-MP12 and ER-MP20 in two-colour immunofluorescence analysis. Upon intrathymic transfer into sublethally irradiated mice thymus-repopulating ability was restricted to ER-MP20- BM cells expressing either high or intermediate levels of the ER-MP12 antigen (1-2% and approximately 30% of BM nucleated cells respectively). The highest frequency of thymus-repopulating cells was found in the minor subset of ER-MP12(+)+20- BM cells. In the present study we demonstrate that upon intravenous transfer, thymus-homing and -repopulating BM cells are exclusively confined to the ER-MP12(+)+20- and ER-MP12+20- subpopulations, the highest frequency being detected among ER-MP12(+)+20- BM cells. Analysis of the peripheral blood leucocytes of reconstituted mice showed that not only prothymocytes but also progenitor cells of the B cell lineage as well as the myeloid lineage were present within both subsets. Three-colour flow cytometric analysis revealed that ER-MP12(+)+20- BM cells in particular were phenotypically heterogeneous with respect to the expression of the cell surface markers Thy-1, Sca-1, CD44, B220 and c-kit. Taken together our data demonstrate that ER-MP12 positively identifies BM cells with the ability to home to and repopulate the thymus. The phenotypic heterogeneity displayed by the ER-MP12(+)+20- BM subset, containing the highest frequency of thymus-homing and -repopulating cells, provides a basis for further separation of prothymocyte activity from other haematopoietic activities in the BM of the mouse.

Animals↗