The cleaved molecules were incubated overnight at 37C in a pH 4.3 citrate phosphate buffer in the presence or absence of various amounts of soluble DM (as indicated in figures) and, in some experiments, non-cleaved molecules (0-7 M) as well, along with the125I-labeled indicator peptides and protease inhibitors. == Solid-phase peptide exchange assay == HLA class II molecules captured from cell lysates were used for a peptide exchange assay (13). DQ2 with Ii and DM may provide a basis for the known disease associations of DQ2. Keywords:Antigen Presentation/Processing MHC class II, invariant chain, HLA-DM == Introduction == MHC class II molecules are dimeric membrane glycoproteins expressed on the surface of antigen presenting cells of the immune system. Their function is to present peptide antigens derived from endosomal proteins to CD4+T cells. To ensure that newly synthesized class II molecules intersect with and bind endocytosed peptides, the dimer associates with the chaperone molecule invariant chain (Ii), which both prevents premature binding of ligands and promotes the entrance of the class II-Ii complexes into the endocytic pathway (1). Ii is progressively degraded by proteases in the endocytic pathway, leaving CLIP (class II-associated Ii peptides) bound to the binding groove of all previously studied MHC class II molecules. The subsequent release GSK598809 of CLIP in late endosomes is required for antigenic peptide binding, a process known to be accelerated by HLA-DM (DM). The MHC GSK598809 II-peptide complex is then transported to the cell surface for inspection by CD4+T cells (2). Previous data on peptides eluted from HLA-DQ2 (DQ2) molecules (DQA1*0501, DQB1*0201) that were affinity purified from B lymphoblastoid cell lines (B-LCL), revealed the presence of large amounts of Ii-derived peptides (54% of total) (3,4). Intriguingly, the population of Ii peptides was dominated by a unique peptide species, here termed CLIP2, which had lower IC50values/higher affinity for DQ2 when compared to the conventional CLIP (CLIP1) peptides (3,4). DQ2 is part of the autoimmune HLA-DR3/-DQ2 haplotype associated with multiple autoimmune diseases (5,6). Motivated by this fact, we investigated the binding of CLIP2 to DQ2 and asked whether the presence of substantial amounts of CLIP peptides reflected altered interaction of DQ2 with DM, in comparison to DM interaction with HLA-DR3 (DR3) and HLA-DQ1 (DQ1). == Material and Methods == == Cell lines == The following Epstein Barr-virus transformed B lymphoblastoid cell lines (B-LCL) were used in this study: CD114 (from a celiac disease patient) expressing DQ2, DR3; 2.2.93 expressing DP4, DQ1, DR1, DR3, and no DM (7); 8.1.6 expressing DP4, DQ2, DR3 (8); 9.5.3 expressing DP4, DQ2, DR3 and no DM (8); 3.1.3 expressing DP4 and DQ1 (9); 9.22.3 expressing DP4 and DQ2 (8). The DR3 expressing cells also express DR52a. The DM transfectants of 2.2.93 and 9.5.3 (2.2.93-DM and 9.5.3-DM, respectively) were generated by retroviral transduction as described (7). == Antibodies == Antibodies used in this study were B8.11 (IgG2b, anti-DR, kind gift of B.Malissen), L243 (IgG2a, anti-DR), ISCR3 (IgG2b, anti-DR), SPV-L3 (IgG2a, anti-DQ, kind gift of H.Spits), Ia3 (IgG2a, anti-DQ, Biodesign), 2.12.E11 (IgG1, anti-DQ2), B7/21.2 (IgG3, anti-DP), XD5.A11 (IgG1, anti-class II), 5C1 (IgG1, anti-DM), 16.23 (IgG3, anti-DR3), anti-HLA-DM conjugated with PE (IgG1, BD Pharmingen), CerCLIP (IgG1, anti-human CLIP, kind gift of P.Cresswell) == Flow cytometry == For cell surface staining, cells were incubated (40 min, on ice) with primary antibodies and washed. Bound antibody was detected by incubation (40 min, on ice) with goat F(ab’)2 anti-mouse IgG (H+L) conjugated with FITC or PE (CalTag). For intracellular staining, cells were fixed and permeabilized using the Cytofix/Cytoperm kit (BD Pharmingen) and then stained using PE-conjugated antibody. Cells were analyzed using a FACScan flow cytometer (Becton Dickinson) and data were analyzed using FlowJo software (Tree Star, Inc). == Pulse-chase immunoprecipitation == Cells were washed three times and starved for 1-2 hours in Met/Cys-free RPMI containing 10% dialyzed FBS (Invitrogen). Cells were pulsed with 0.1 Ci/ml ExpreSS VEGFA [35S] labeling mix (Perkin Elmer) for the indicated times, then washed and chased in complete RPMI containing 10% FBS and 2mM L-glutamine (37C, 5% CO2). Aliquots of cells were collected and washed at the indicated time points and lysed in buffer (Tris-HCl, pH 8.0 with MgCl2, 1% NP-40 GSK598809 and complete protease inhibitors (Roche Diagnostics)) at 4C for 12 hrs. Lysates were pre-cleared three times with normal mouse serum and Pansorbin (Calbiochem) and once with protein A sepharose beads (Amersham), then normalized based on total radioactivity, measured by beta-counter (Wallac). Immunoprecipitations were.