A cell line (ISO-HAS) has been established from tumor tissue of a human hemangiosarcoma arising on the scalp by the use of conditioned medium from a murine-phenotypic angiosarcoma cell line (ISOS-1). Cells have been cultured for more than 2 years with up to 100 passages. The cells retained endothelial-cell properties, such as a characteristic cobblestone appearance at confluency, contact-inhibited growth, active uptake of acetylated low-density lipoprotein labeled with 1,1-dioctadecyl 1,3,3,3,3-tetramethyl-indocarbocyanine perchlorate (Dil-Ac-LDL) and CD31 expression. However, they were weakly positive for von-Willebrand-factor (vWf) antigen and for binding of Ulex europaeus agglutinin-I (UEA-1) lectin, and lacked tube-formation activity. These findings indicate that ISO-HAS is a poorly differentiated endothelial cell line. ISO-HAS cells showed accumulation of p53 protein in the nuclei, and a new-typed p53-gene point mutation was found in exon 7 at codon 240. When inoculated s.c. into severe-combined-immunodeficiency (SCID) mice, the cells showed solid-tumor growth that caused death. These properties suggest that ISO-HAS is a malignant endothelial cell line with high tumorigenicity.

SUMMARYCD44 is a multifunctional adhesion molecule that has been shown to be a costimulatory factor for Tcell activation in vitro and in vivo. The aim of the present study was to expand these ®ndings by characterizing the role of CD44 during dendritic cell (DC) antigen presentation to naive, resting T cells. Certain monoclonal antibodies (mAbs) directed against all CD44 isoforms (pan CD44), or against the epitope encoded by the alternatively spliced exon v4 (CD44v4), dose-dependently inhibited the capacity of murine DC to induce proliferation of naive alloreactive T cells. Preincubation of the T cells or DC with these CD44 mAbs revealed that the effect was dependent upon mAb binding to DC, but not to T cells. DC treated with anti-pan CD44 and anti-CD44v4 mAbs induced CD4T-cell apoptosis, as shown by annexin V staining and TdT-mediated biotin±dUTP nick-end labelling (TUNEL) assays. However, CD4T-cell apoptosis was not dependent on the Fas/Fas ligand (Fas/FasL) system, as DC from FasL-de®cient (Gld) mice and T cells from Fasde®cient (Lpr) mice were still susceptible to apoptosis induced by CD44-treated DC. To investigate whether CD44 treatment of DC affects early T-cell/DC interactions, time-lapse video microscopy was performed using peptide-speci®c T cells from T-cell receptor (TCR) transgenic mice. Interestingly, calcium signalling in CD4T cells was signi®cantly diminished following interaction with CD44 mAb-treated DC, but this was not observed in CD8 T cells. Taken together, we found that perturbation of distinct epitopes of CD44 on DC interfere with early Ca 2 signalling events during the activation of CD4T cells, resulting in T-cell apoptosis.

Low dose UVB irradiation of dendritic cells (DC) dose-dependently decreases their allostimulatory capacity and inhibits alloreactive T cell proliferation. The reduction of the stimulatory capacity is not associated with a perturbation of CD28 costimulation. To examine the underlying mechanism, cell cycle analysis of T cells from cocultures with UVB-irradiated DC (UVB-DC) was performed, revealing no cell cycle arrest, but an increased number of apoptotic T cells in sub-G0 phase. We confirmed T cells to undergo apoptosis after coincubation with UVB-DC by TUNEL staining and DNA laddering. To analyze whether T cell apoptosis requires the Fas/Fas ligand (FasL) pathway, MLRs were performed with Fas-, FasL-deficient, and wild-type DC and T cells. No differences were found on comparison of wild-type DC with Fas-/FasL-deficient DC or T cells. Likewise, addition of a neutralizing anti-TNF-α mAb to cocultures could not overcome inhibition of T cell proliferation by UVB-DC, excluding involvement of the TNF-α/TNF-αR pathway. FACS analysis of CD69 and CD25 revealed no up-regulation on T cells cocultured with UVB-DC, suggesting a perturbation of early T cell activation. Analysis of UVB-DC by confocal microscopy demonstrated impaired filamentous actin bundling, a process critical for T cell stimulation. To investigate the functional relevance of these observations, time lapse video microscopy was performed. Indeed, calcium signaling in CD4+ T cells was significantly diminished after interaction with UVB-DC. In conclusion, UVBR of DC impairs their cytoskeletal rearrangement and induces apoptosis in CD4+ T cells by disruption of early DC-T cell interaction, resulting in a reduced Ca2+ influx in T cells.

We have shown that low-dose UVB radiation converts Langerhans cells(LC) from immunogenic to tolerogenic APC. Therefore, we questionedwhether low-dose UVB irradiation of bone marrow-derived dendritic cells(DC) alters their APC function, thereby inducing tolerance in T cells.To address this issue, cocultures of DC; and naïve, allogeneicT cells; naïve, OVA-specific TCR-transgenic T cells fromDO11.10 mice; or primed, antigen-specific T cells using the Th1 cloneAE7 were analyzed. First, we found low-dose UVB-irradiated DC(UVB-DC) to dose-dependently (50–200 J/m2) inhibit T-cellproliferation of naive and primed T cells. In addition, supernatantsharvested from cocultures of UVB-DC and naive T cells showed markedlyreduced levels of IL-2 and IFN-γ and to a lesser degree of IL-4 andIL-10, suggesting a preferential down-regulation of Th1 responses byUVB-DC. FACS analysis of UVB-DC revealed no changes in surfaceexpression of MHC, costimulatory, and adhesion molecules. To testtolerance induction, allo- or antigen-specific T cells isolated fromcocultures with unirradiated DC and UVB-DC were restimulated withunirradiated DC or IL-2. It is interesting that UVB-DC inducedantigen-specific tolerance in the Th1 clone AE7. In contrast, UVB-DCinduced a partial inhibition of allogeneic T-cell proliferation but notolerance with similar unresponsiveness to restimulation with IL-2 andunirradiated DC irrespective of their haplotype. Similar observationswere made when naïve, TCR-transgenic T cells from DO11.10 micewere used. In conclusion, UVB-DC are impaired in their APC function andtolerize the primed antigen-specific Th1 clone AE7 but not naive allo-or OVA-specific T cells.