We found that RAP did not influence DBP AF488 uptake in the T cells. Furthermore, blocking antibodies against megalin inhibitor Oligomycin A and calcium deprivation, known to in hibit megalin mediated endocytosis, did Inhibitors,Modulators,Libraries not affect DBP uptake. In line with this, high concentrations of DBP did not affect the up take of DBP AF488. Taken together these experiments indicated that DBP is not taken up by megalin mediated endocytosis in T cells. In addition to receptor mediated endocytosis, cells can take up extracellular molecules by macropinocytosis. Macropinocytosis is characterized by its augmentation by cell activation and that it can be inhibited by amilor ide.

We Inhibitors,Modulators,Libraries found that treatment of the cells with the amiloride analogue 5 amiloride significantly inhibited uptake of DBP AF488 arachidonic acid to DBP affected DBP mediated inhib ition of 25 D3 induced T cell responses, we stimu lated CD4 T cells in the absence or presence of DBP plus 25 D3 and increasing Inhibitors,Modulators,Libraries concentrations of actin or arachidonic acid. We found that neither actin nor ara chidonic acid affected 25 D3 induced T cell re sponses as measured by CD38 expression. Since albumin binds 25 D3 with lower affin ity than DBP, the ratio between DBP and albumin could in theory also affect the bioavailability of 25 D3. however, as for actin and arachidonic acid we found that. To ensure that EIPA only blocked macropi nocytosis and not receptor mediated endocytosis, we measured the effect of EIPA on T cell receptor in ternalization in parallel. TCR internalization is dependent on binding of the CD3�� di leucine based internalization motif to adaptor molecules of clathrin coated pits and hence should not be influenced by EIPA.

We found that EIPA did not affect TCR internalization. Thus, we could conclude that DBP uptake by CD4 T cells is not mediated by megalin mediated endocytosis but most likely by macropinocytosis. Whereas Inhibitors,Modulators,Libraries megalin mediated endocytosis of DBP facili tates uptake and conversion of 25 D3 to 1,25 2D3 in kidney and mammary cells, macropino cytosis of DBP 25 D3 complexes did certainly not facilitate conversion of 25 D3 to 1,25 2D3 in T cells. To study whether DBP inhibited the ef fects of 1,25 2D3 in the same way as it inhibited the effects of 25 D3 on T cells, we stimulated CD4 T cells for 3 days in the presence of either 25 D3 or 1,25 2D3 and increasing concentrations of DBP. After 3 days we measured CD38 expression and IFN production.

We found that Inhibitors,Modulators,Libraries in the absence of DBP both 25 D3 and 1,25 2D3 up regulated CD38 ex pression and down regulated IFN production. How ever, whereas addition of DBP clearly inhibited the effect of 25 D3 it did not influence the effect of 1,25 2D3 on T cell responses. Neither actin, arachidonic acid nor albumin affect the DBP mediated inhibition scientific assays of 25 D3 In addition to 25 D3 and 1,25 2D3, DBP can bind actin and fatty acids.