Intact MHC class II antigen presentation in HD-cell lines. (A) Surface expression of MHC class II αβ dimers and MHC class II αβ–CLIP complexes in HD-cell lines. SUPT1 (S), JJHAN (J), HO, (HO), HDLM2 (H), KMH2 (K), L428 (L), BJAB (B), DAUDI (D), and RAJI (R) cells were either incubated with mouse IgG, CerCLIP.1, or BU27 MoAbs. Bound MoAbs were labeled with FITC-conjugated goat anti-mouse secondary antibodies and the samples processed for flow cytometric analysis. Bars indicate MFI values (MFI) of MHC class II αβ–CLIP complexes (αβ-CLIP, white bars) and MHC class II αβ dimers (αβ, black bars) after subtraction of MFI values obtained from fluorescent staining using mouse IgG as a primary reagent. Shown on top are the relative amounts of MHC class II αβ–CLIP complexes expressed as a percentage of the total amount of surface expressed MHC class II αβ dimers (% αβ-CLIP). (B) Formation of SDS-stable MHC class II αβ dimers in HD-cell lines. 10⁶ SUPT1 (lane 1), JJHAN (lane 2), HO (lane 3), L428 (lane 4), BJAB (lane 5), DAUDI (lane 6), RAJI (lane 7), HDLM2 (lane 8), and KMH2 cells (lane 9) were lysed in presence of SDS under nonreducing conditions and lysates analyzed by SDS-PAGE and Western blotting using the anti-MHC class II antibody, BU27, as a probe. This antibody reacts only with MHC class II αβ dimers. It does not react with either free MHC class II α or β chains. The positions of molecular weight markers (expressed as 10^-3 × M_r) are shown on the right. The symbol, αβ, shown on the left, points to the SDS-stable MHC class II αβ dimers (∼60 kD). HDLM2 (lane 8) and KMH2 cell lysates (lane 9) were run on separate gels because the background produced by the MoAb, BU27, was lower in HDLM2 and higher in KMH2 cells than in the other cell lines used. (C) Leupeptin inhibits surface expression of MHC class II αβ–CLIP complexes in L428 cells. L428 cells were cultured in the absence (black bars) or presence (white bars) of 1.5 mmol/L leupeptin for 24 hours. The cells were stained with the same primary (BU27, αβ; CerCLIP.1, αβ-CLIP) and secondary reagents as described in (A). As a negative control for the effect of leupeptin on the surface expression of MHC class II molecules, cells were labeled with a PE-conjugated anti-CD40 antibody or with a PE-conjugated mouse IgG. Shown are MFI values obtained with the PE-conjugated anti-CD40 antibody (CD40) after subtraction of MFI values generated by the PE-conjugated mouse IgG. The relative reductions of cell surface fluorescence after leupeptin treatment are indicated on top (% Reduction).