In this sequential digest, neither CatL nor CatB could further digest the 15- and 18-kDa fragments (Data S1), although these enzymes were active as reflected in their ability to degrade MBP. Similarly, we were unable to detect further proteolysis of CatG-treated HLA-DR using CatS, D, X, H, or AEP (data not shown). In order to examine whether endogenous CatG might contribute to MHC II proteolysis in living APCs, we first looked for inverse correlations between changes in MHC II levels and CatG levels in human primary APCs following in vitro stimulation. Indeed, CatG, which is expressed by mDC1s,19 was down-regulated upon

lipopolysaccharide (LPS) stimulation (Data S2), a manipulation known to increase surface MHC II levels and shut down MHC II turnover.4 Similarly, decreased levels of

CatG correlated with increased MHC II levels in primary human B cells after stimulation with interleukin 3-MA mw (IL)-4 (Data S2). Next, to test whether CatG is causally involved in MHC II turnover, we examined whether addition of CatG to APCs modulates DR expression. Previously, we demonstrated that B-LCLs do not express CatG, but can acquire CatG added to culture media and target it to endocytic compartments.38 To evaluate the impact of CatG on the steady-state levels of HLA-DR molecules, we incubated B-LCLs with or without CatG for 4·5 hr. The cells were harvested, Linsitinib ic50 and HLA-DR levels were compared by immunoblotting. Levels of both alpha and beta chains of HLA-DR were unchanged when incubated with purified CatG or with CatG and the CatG inhibitor (Data S3). Furthermore, CatG added exogenously to a B-LCL did not alter surface levels of HLA-DR molecules, as quantified by flow cytometry with L243 (Data S4) and the anti-DR antibody Tü36 (data not shown). Importantly, the B-LCL used in these experiments, 9.5.3, is DM-deficient; thus, these negative results were not explained by DM-mediated protection Farnesyltransferase from CatG. In order to examine whether endogenous CatG expression in certain primary human APC types, such as mDC1 and

B cells,19 contributes to MHC II turnover, we tested whether the CatG inhibitor,29 which inhibits intracellular CatG in intact cells,21 causes accumulation of DR in such APCs. No increase in total DR levels in primary B cells was seen by western blot, however, after 4·5, 24 and 72 hr of CatG inhibition (Fig. 6a–c). Similarly, total DR levels were unchanged in mDC1s when CatG was subjected to prolonged inhibition (24 and 72 hr; data not shown). Moreover, analysis of cell surface levels of HLA-DR by flow cytometry demonstrated that the CatG inhibitor did not affect expression of surface HLA-DR in either B cells or mDC1s (Data S5). These results argued against a causal relationship between the inversely correlated levels of endogenous CatG and MHC II expression.