Alexandre Potocnik

Fetal and Adult Hematopoietic Stem Cells Require β1 Integrin Function for Colonizing Fetal Liver, Spleen, and Bone Marrow

Homing of hematopoietic stem cells (HSCs) into hematopoietic organs is a prerequisite for the establishment of hematopoiesis during embryogenesis and after bone marrow transplantation. We show that beta1 integrin-deficient HSCs from the para-aortic splanchnopleura and the fetal blood had hematolymphoid differentiation potential in vitro and in fetal organ cultures but were unable to seed fetal and adult hematopoietic tissues. Adult beta1 integrin null HSCs isolated from mice carrying loxP-tagged beta1 integrin alleles and ablated for beta1 integrin expression by retroviral cre transduction failed to engraft irradiated recipient mice. Moreover, absence of beta1 integrin resulted in sequestration of HSCs in the circulation and their reduced adhesion to endothelioma cells. These findings define beta1 integrin as an essential adhesion receptor for the homing of HSCs.

Expression of Activation Antigens on T Cells in Rheumatoid Arthritis Patients

The aim of our study was to identify differences in cell surface marker expression between T cells taken from the peripheral blood (PB) of healthy individuals and T cells recovered from inflamed joints of rheumatoid arthritis (RA) patients. Out of 118 monoclonal antibodies (MoAbs) directed against activation antigens on haematopoietic cells, 12 MoAbs recognizing nine distinct surface molecules were selected after a sereening procedure to study the expression of the corresponding antigens on T cells from the PB, synovial fluid and synovial tissue of RA patienls, and also on T cells from PB and spleens of controls. Using two‐colour flow cytometry and immunohistology we found the molecules B‐C5, CD39. CD40, CD45 R0, CD54, CD76 and potentially 1D11 to be substantially up‐regulated on T cells from various body compartments in RA patients. We thus could determine that the cell surface of T cells in RA patients not only differs in MHC class II expression, but also in a number of other activation‐associated cell surface molecules from T cells in healthy individuals.

Oligoclonal T Cells in Rheumatoid Arthritis: Identification Strategy and Molecular Characterization of a Clonal T-Cell Receptor

Immunodominant antigens in rheumatoid arthritis (RA) should induce an expansion of T cells bearing a corresponding T‐cell receptor (TCR). We therefore analysed the TCR repertoire at the site of inflammation using two fundamentally different strategies. The total TCR repertoire was examined by generating‘representative’T‐cell clone panels, which were subsequently tested for clonality by restriction mapping of the TCRβ gene locus. No clonality was detected in large T‐cell clone panels generated with cells from three patients. However, when we selectively analysed the TCR repertoire of in vivo pre‐activated, interleukin‐2 (IL‐2)‐responsive T cells, significant T‐cell/TCR clonality was found in 2 out or4 patients. The clonal T cells represented a minority of the total T‐cell population with an estimated frequency of 1 in 300 to 1 in 1000 cells. Molecular characterization of a clonal TCR and the use of a specific TCR Vβ MoAb ruled out an over representation of T cells bearing the same Vβlelement in the total T‐cell population, rendering the involvement of superantigens in the induction of T‐cell clonality in this case unlikely.

Reconstitution of B cell subsets in Rag deficient mice by transplantation of in vitro differentiated embryonic stem cells

In vitro differentiated embryonic stem (ES) cells contain a population which is similar to fetal liver pro/pre-B cells on the basis of cell surface antigens and cytoplasmic expression of immunoglobin heavy chain. This population was purified and transplanted into Rag-1 deficient recipients to characterize its developmental potential in vivo. Following intravenous transfer, these cells rapidly reconstituted the splenic B but not the T cell compartment. Reconstitution was transient, indicating the lack of long-term reconstituting capacity. Similar to fetal liver, B-1 type as well as conventional B cells were generated, accompanied by high serum IgM levels. Intraperitoneal injection generated high numbers of peritoneal B cells, predominately of the B-1a phenotype, with poor splenic repopulation and low serum IgM levels. These observations suggest the emergence of two different B lineage precursor populations during in vitro ES cell differentiation and define a possible role of the microenvironment in directing lymphoid development.

Early expression of endomucin on endothelium of the mouse embryo and on putative hematopoietic clusters in the dorsal aorta: Endomucin in Mouse Embryo Development

Endomucin is a recently identified sialomucin that is specifically expressed on endothelium of the adult mouse. Here, we have analysed the expression of endomucin during development of the vascular system by immunohistochemistry by using three monoclonal antibodies (mAb). We demonstrate that two of the mAb, V.5C7 and V.1A7, recognize epitopes on the nonglycosylated protein, because they recognize the antigen when it is synthesized as a bacterial fusion protein and when it is in vitro translated in a membrane‐free reticulocyte lysate. During in vitro differentiation of embryonic stem cells to endothelial cells, endomucin is expressed at day 6 after onset of differentiation, 1 day later than PECAM‐1. During differentiation of the mouse embryo, endomucin is first detected at E8.0 in all embryonic blood vessels detectable at this stage but is absent in blood islands of the yolk sac. Analysing the paraaortic‐splanchnopleura (P‐SP) region and the aorta‐gonad‐mesonephros (AGM) region as sites of intraembryonic hematopoiesis, we found that endothelium of the dorsal aorta is brightly positive for endomucin at E8.5–9.0 and at E11.5. At later stages and in the adult aorta, endothelial staining is strongly reduced and confined to focal areas. Cell clusters associated with the luminal surface of the endothelium of the dorsal aorta could be stained for endomucin and for CD34. At a later stage (E15.5) single leukocytes in the lumen of large venules were stained for endomucin. We conclude that endomucin is an early endothelial‐specific antigen that is also expressed on putative hematopoietic progenitor cells.