During embryonic development, lymph sacs form from the cardinal vein, and sprout centrifugally to form mature lymphatic networks. Separation of the lymphatic from the blood circulation by a hitherto unknown mechanism is essential for the homeostatic function of the lymphatic system. O-glycans on the lymphatic endothelium have recently been suggested to be required for establishment and maintenance of distinct blood and lymphatic systems, primarily by mediating proper function of podoplanin. Here, we show that this separation process critically involves platelet activation by podoplanin. We found that platelet aggregates build up in wild-type embryos at the separation zone of podoplanin ؉ lymph sacs and cardinal veins, but not in podoplanin ؊/؊ embryos. Thus, podoplanin ؊/؊ mice develop a "nonseparation" phenotype, characterized by a bloodfilled lymphatic network after approximately embryonic day 13.5, which, however, partially resolves in postnatal mice. The same embryonic phenotype is also induced by treatment of pregnant mice with acetyl salicylic acid, podoplaninblocking antibodies, or by inactivation of the kindlin-3 gene required for platelet aggregation. Therefore, interaction of endothelial podoplanin of the developing lymph sac with circulating platelets from the cardinal vein is critical for separating the lymphatic from the blood vascular system. (Blood. 2010;115(19):3997-4005)

There has been emerging interest whether plasma membrane constituents are moving according to free Brownian motion or hop diffusion. In the latter model, lipids, lipid-anchored proteins, and transmembrane proteins would be transiently confined to periodic corrals in the cell membrane, which are structured by the underlying membrane skeleton. Because this model is based exclusively on results provided by one experimental strategy--high-resolution single particle tracking--we attempted in this study to confirm or amend it using a complementary technique. We developed a novel strategy that employs single molecule fluorescence microscopy to detect confinements to free diffusion of CD59--a GPI-anchored protein--in the plasma membrane of living T24 (ECV) cells. With this method, minimum invasive labeling via fluorescent Fab fragments was sufficient to measure the lateral motion of individual protein molecules on a millisecond timescale, yielding a positional accuracy down to 22 nm. Although no hop diffusion was directly observable, based on a full analytical description our results provide upper boundaries for confinement size and strength.

Transforming growth factor-β (TGF-β), a key modulator of endothelial cell apoptosis, must be activated from the latent form (LTGF-β) to induce biological responses. In the present study, we report activation of TGF-β by functional and physical co-operation of the mannose-6-phosphate/insulin-like-growth-factor-II receptor (CD222) and the urokinase-type plasminogen activator receptor (CD87). We show that endothelial cells express CD222 and CD87 in a membrane complex and demonstrate that the association of these two receptors is essential for the release of active TGF-β in the transduced mouse fibroblast used as model cells. By contrast, smooth-muscle cells, which express CD222 and CD87 at similar density to endothelial cells but not in complexed form, do not activate TGF-β. We also have found that mini-plasminogen is a high-affinity ligand for CD222 and is essential for the activation of TGF-β by the CD87-CD222 complex to induce apoptosis in endothelial cells. This specific mechanism of TGF-β-mediated apoptosis in endothelial cells is thus a potential novel target to be considered for treatment of pathological vascular disorders (e.g. tumor angiogenesis).

The existence of lipid rafts and their importance for immunoreceptor signaling is highly debated. By non-invasive single molecule imaging, we analyzed the dynamics of the T-cell antigen receptor (TCR), the lipid raft-associated glycosylphosphatidylinositol (GPI) proteins CD48 and CD59 and the major leukocyte phosphatase CD45 in living naive T lymphocytes. TCR triggering induced the immobilization of CD45 and CD48 at different positions within the T-cell interface. The second GPI protein, CD59, did not co-immobilize indicating lipid raft heterogeneity in living T lymphocytes. A novel biochemical approach confirmed that lipid raft components are not associated in the plasma membrane of resting cells, and variably associate with specific receptors to distinct lipid rafts upon activation.

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Immune-cell -based approaches using cytotoxic and dendritic cells are under constant scrutiny to design novel therapies for the treatment of tumors. These strategies are hampered by the lack of efficient and economical large-scale production methods for effector cells. Here we describe the propagation of large amounts of a unique population of CD4 + cytotoxic T cells, which we termed tumor killer T cells (TKTC), because of their potent and broad antitumor cell activity. With this cultivation strategy, TKTCs from peripheral blood mononuclear cells are generated within a short period of time using a pulse with a stimulating cell line followed by continuous growth in serum-free medium supplemented with a mixture of interleukin-2 and cyclosporin A. Expression and functional profiling did not allow a classification of TKTCs to any thus far defined subtype of T cells. Cytotoxic assays showed that TKTCs kill a panel of tumor targets of diverse tissue origin while leaving normal cells unaffected. Blocking experiments revealed that TKTC killing was, to a significant extent, mediated by tumor necrosis factor-related apoptosis-inducing ligand and was independent of MHC restriction. These results suggest that TKTCs have a high potential as a novel tool in the adoptive immunotherapy of cancer.