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. 2022 Dec 2;28:1–14. doi: 10.1016/j.omto.2022.11.007

Figure 1.

Figure 1

Selection of MAGE-reactive T cells

(A) Flowchart describing the four steps for isolation of MAGE-reactive T cell clones. T cell clones were selected based on peptide titration experiment and specific tumor cell line recognition. (B) Six of 18 T cell clones recognizing MAGE-A1 KVLEYVIKV in HLA-A∗02:01 (MAGE-A1 KVL/A2) are depicted in this graph that were overnight co-cultured with KVL peptide loaded Raji cells. These six T cell clones are a representative for T cell clone selection process of all 187 MAGE-specific T cell clones. (C and D) T cell clones were stimulated with tumor cell lines of different origin, including multiple myeloma (RPMI8226, U266, L363, OPM-2, UM9), osteosarcoma (U2-OS, ZK-58, Saos-2), melanoma (88.23, MEL01.14, SK2.3, 518A2, OPM-2), chronic myeloid leukemia k562, cervix carcinoma Caski, and prostate carcinoma cell line PC-3M-PRO4. T cell reactivity was demonstrated by IFN-γ production, as measured by ELISA, after an overnight co-culture. MAGE-gene expression levels, measured by qPCR, are depicted between brackets as percentage relative to HKGs. Targeted HLA was naturally expressed or transduced (+) into the target cell. (C) Four of 18 MAGE-A1 KVL/A2 recognizing T cell clones are depicted as representatives. T cell clones 4A2 and 4F7 strictly recognize the MAGE-expressing tumor cell lines efficiently and therefore selected for further analysis. (D) Results of all selected T cell clones reactive against MAGE-A1 LTQDLVQEKYLEY in HLA-A∗01:01 (MAGE-A1 LTQ/A1), MAGE-A1 KVL/A2, MAGE-A1 SLFRAVITK in HLA-A∗03:01 (MAGE-A1 SLF/A3), MAGE-A1 RVRFFFPSL in HLA-B∗07:02 (MAGE-A1 RVR/B7), MAGE-A1 VRFFFPSL in HLA-C∗07:02 (MAGE-A1 VRF/C7), MAGE-A3/A6 EVDPIGHLY/EVDPIGHVY in HLA-B∗35:01 (MAGE-A3/A6 EVD/B35), and MAGE-A1 YVGKEHMFY in HLA-A∗01:01 (MAGE-A9 YVG/A1). The Burkitt lymphoma Raji, negative for all MAGE antigens, was included as negative control. To confirm proper HLA expression and recognition capacity of the targets allo-HLA reactive T cell clones (white bars) were included for each HLA specificity. Values and error bars represent mean and standard deviations of technical duplicates. Experiments are representative of at least two independent experiments.