Figure 5: Antigen specific CD8+ CTL assay using recombinant DCs expressing an artificial HLA-B*44:03 or B*44:35 molecules as target. Antigen-specific CD8+ CTL activity against the allogeneic HLA-B*44/156 variants was determined by flow cytometry. Target DCs transduced with lentiviral constructs to express additional HLA-B*44/ 156 variant were labelled with CFSE and lysis of these cells by CTLs was determined by staining with 7-AAD. 5a, b, c) neg. controls for antigen specific CTL response. Freshly isolated CD3+ T cells from an HLA-B*44:02 expressing donor were incubated with autologous DCs expressing additional HLA-B*44/156 molecule – i.e B*44:02 or B*44:03 or B*44:35. 5d, e, f) CD8+ T cells stimulated with autologous DCs expressing additional B*44:02 or B*44:03 or B*44:35 allele were used as effector cells against recombinant B*44:02 or B*44:03 or B*44:35 expressing target DCs respectively. A high percentage of B*44:03 (21 %) and B*44:35 (30 %) expressing target DCs were found to be lysed by autologous CD8+ T cells from the B*44:02 donor.