Cells Lines and Antibodies

HLA-B27 SCT molecules were generated as described previously (20, 49). The SCT molecule comprised the human β₂m amino-terminal hydrophobic signal sequence, the peptide sequence SRYWAIRTR (residues 383–391 of influenza virus nucleoprotein NP), a GGGGGG(SGG)₃ linker, human β₂m sequence, a second linker (GGGGS)₃ and HLA-B*27:05 class I HC sequence, which was C-terminally V5 tagged. For the HLA-B*35:01 SCT, the EBNA1 Human Papilloma Virus peptide sequence HPVGEADYFEY was employed.

The Invitrogen Flp-In™ system was used to generate isogenic HeLa lines stably expressing two copies of HLA class I constructs driven by the human cytomegalovirus (HCMV) promoter (49). Separate cell lines were generated to express the HLA-B*27:05 or –B*35:01 HCs and an empty vector (referred to subsequently as Empty (E)84). HLA-B27 SCT molecules expressing C67S, C308S, C325S and H114DD116Y (referred to as F pocket) were generated using Quikchange PCR site directed mutagenesis. Sequences were verified using the Dundee University sequencing service. All lines were genetically identical and expressed only two copies of the above constructs. Cells were maintained in DMEM, supplemented with 10% FBS (Globepharm), penicillin/streptomycin (D10 media), hygromycin (150μg/ml) and maintained in a humidified 5% CO₂ 37°C incubator.

Monoclonal antibody ME1 (50), recognising folded HLA-B molecules was kindly provided by Dr. J. Taurog and W6/32 (51) recognising folded HLA-A, B and C molecules was kindly provided by Prof. T. Elliott. Monoclonal antibody HC10 recognises unfolded HLA-B and –C molecules. Anti-V5 antibody was from Serotec, LAMP1-PE antibody (CD107a) (clone H4A3) was purchased from BD-Pharmingen (555801) and CD8-APC antibody (clone RPA-T8) was purchased from BD-Pharmingen (561953). Goat anti-mouse horseradish peroxidase (HRP) and conjugated secondary antibody were purchased from DAKO. Anti-tapasin PASTA antibody was a kind gift from Prof. P. Cresswell and anti-TAP1 monoclonal antibody were obtained from Stressgen.