Figure 4

Cytotoxicity in vitro and in vivo monitoring of UTA2-1-directed T-cell response with tetramer staining correlated to clinical GvT. (a) Clone 503A1 effectively kills HLA-A*02⁺ mHag⁺ MM cell line U266 in various E:T ratios after 24 h incubation in a bioluminescence cytotoxicity assay. Either mHag⁻ (L363) or HLA-A*02⁻ (UM9) MM cell lines are not lysed by 503A1, but after transduction of genotypically mHag⁺ UM9 with HLA-A*0201 this cell line is also recognized. U266 survives unhindered in the presence of an irrelevant mHag-directed T-cell clone (3AB11). (b) Also, primary MM cells from the original mHag⁺ SCT-recipient (pt A) were specifically lysed by 503A1 in a FACS cytotoxicity assay after incubation together for 18 h. Other primary MM cells were also killed in an antigen-specific and dose-dependent manner, although with lower efficacy than pt A. (c) Various HLA-A*0201⁺ mHag⁺ primary CLL cells were lysed in a dose-dependent manner by CTL 503A1 likewise in a FACS cytotoxicity assay, whereas HLA-A*0201⁻ mHag⁻ CLL cells were not (left graph). Moreover, we measured IFN-γ responses of 503A1 to some of the CLL samples (right graph). (d) Gene C12orf35 is highly expressed on both benign and malignant hematopoietic cells. All values are relative to a calibrator PBMC sample and C12orf35 gene expression is normalized to the housekeeping gene GUS. Median values are depicted. (e) The time course from diagnosis of MM until present is depicted, with the solid line representing the course of the disease, measured by M-protein level in the peripheral blood (left y axis). The dotted line indicates the percentage of CD8⁺ T cells that stained positive with the UTA2-1 tetramer (right y axis). After the SCT and after both DLIs the relative amount of tetramer-positive T cells increased, which coincided with clinical responses leading to long-lasting responses after the SCT and after the second DLI. After the SCT and the first DLI also short episodes of mild GvHD of the skin occurred, for which the patient was treated with topical steroids.