Comparison of WT and CD37−/− DC migration 18–20 h after oxazolone

Comparison of WT and CD37−/− DC migration 18–20 h after oxazolone treatment revealed significant reductions in migratory function Sorafenib in vitro and random migration in CD37−/− DCs (see Oxa, Fig. 5A–C). This is further illustrated by comparison of the XY-displacement tracks of DC migration in WT and CD37−/− mice, which show extensive paths of migration in WT mice, in contrast to minimal responses in CD37−/− mice (Fig. 5D).

In addition, a significant proportion of CD37−/− DCs were less motile displaying an increased frequency of cells with <5 μm displacement (Fig. 5E). Videos showing this impaired in vivo directional migration of CD37−/− DCs compared with that of WT controls are included in the Supporting Information (Supporting Information Fig. 3 and 4). Taken together, Figure 4 and 5 demonstrate that CD37 ablation induces a significant impairment in DC migration. Tetraspanins molecularly associate with integrins and regulate outside-in signaling and cytoskeletal rearrangement as evidenced by impaired adhesion strengthening under flow and cell spreading observed in tetraspanin-deficient cells [27-31]. To test if CD37 plays a similar role in DCs, we first measured DC adhesion to ECM substrates under low shear flow conditions. WT DCs adhered efficiently to fibronectin, but poorly

to laminin and collagen (Fig. 6A). However, despite normal expression of the fibronectin receptors CD49d and CD49e integrins (Fig. 6B), the PLX-4720 chemical structure absence of CD37 resulted in significantly RVX-208 reduced BMDC fibronectin adhesion (Fig. 6A). Cell spreading upon adhesion and membrane protrusion formation are dependent on cytoskeletal rearrangement driven by actin polymerization. To assess the role of CD37 in these processes, activated BMDCs were allowed to adhere and spread on fibronectin. Actin-dependent cell spreading was visualized by Phalloidin staining (Fig. 6C and F), bright field imaging (Fig. 6F), and scanning electron microscopy (SEM) (Fig. 6G). The percentage of cells with membrane

protrusions and the area of adhered cells were quantitatively determined (Fig. 6D and E). While WT DC readily spread, formed membrane protrusions and showed a classical dendritic morphology, CD37−/− DCs had a smaller rounded morphology with a relative absence of protrusive membranes (Fig. 6C–G). We conclude that CD37 is essential for cytoskeletal-dependent processes such as adhesion under flow, cell spreading upon adhesion, and the formation of membrane protrusions. CD37−/− mice display poor adaptive cellular responses to live tumors, irradiated tumors, and soluble antigens (Fig. 1 and 2). These findings are difficult to reconcile with exaggerated T-cell proliferative [14] and DC antigen-presenting phenotypes [15] observed when examining CD37-deficient cells in vitro.

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