Table_2_In vitro Generation of Cytotoxic T Cells With Potential for Adoptive Tumor Immunotherapy of Multiple Myeloma.DOCX

Multiple myeloma is a life-threatening hematological malignancy, which is rarely curable by conventional therapies. Immunotherapy, using tumor antigen-specific, cytotoxic T-lymphocytes, may represent an alternative or additional treatment for multiple myeloma. In this study, we used hybrid cell lines, generated by fusion of an EBV B-lymphoblastoid cell line (B-LCL) and myeloma cells, to stimulate in vitro peripheral blood lymphocytes (PBLs) from patients with multiple myeloma. We investigated induction of antigen-specific, cytotoxic T-lymphocytes to the well-defined tumor associated antigens (TAAs) hTERT, MUC1, MAGE-C1 and CS1, which have been shown to be expressed in a high proportion of cases of multiple myeloma. HLA-A2-peptide pentamer staining, interferon-γ and perforin ELISpot assays, as well as cytotoxicity assays were used. Following several rounds of in vitro stimulation, the hybrid cell lines induced antigen-specific, cytotoxic T-lymphocytes to four candidate TAAs in PBLs from HLA-A2+ multiple myeloma patients, using known HLA-A2 restricted peptide epitopes of the TAAs. In contrast, the HLA-A2+ myeloma cell line U266 failed to induce antigen-specific, cytotoxic T-lymphocytes in vitro. Our data indicate that B-LCL/myeloma hybrid cell lines induce antigen-specific, cytotoxic T-lymphocytes in PBLs isolated from multiple myeloma patients in vitro and may represent a novel strategy for use in adoptive immunotherapy of multiple myeloma.

多发性骨髓瘤是一种危及生命的血液系统恶性肿瘤，传统治疗手段往往难以治愈。利用肿瘤抗原特异性、细胞毒性T淋巴细胞进行的免疫治疗，可能为多发性骨髓瘤提供一种替代或补充的治疗方法。在本研究中，我们采用融合EBV B淋巴母细胞系（B-LCL）和骨髓瘤细胞的杂交细胞系，以刺激来自多发性骨髓瘤患者的体外外周血淋巴细胞（PBLs）。我们研究了诱导针对预先定义的肿瘤相关抗原（TAAs）hTERT、MUC1、MAGE-C1和CS1的抗原特异性、细胞毒性T淋巴细胞，这些抗原已在多发性骨髓瘤病例的高比例中表达。我们使用了HLA-A2-肽五聚体染色、干扰素-γ和穿孔素ELISpot检测以及细胞毒性检测。经过几轮体外刺激后，杂交细胞系利用已知TAAs的HLA-A2限制性肽表位，在HLA-A2阳性多发性骨髓瘤患者的PBLs中诱导了针对四种候选TAAs的抗原特异性、细胞毒性T淋巴细胞。相比之下，HLA-A2阳性骨髓瘤细胞系U266在体外未能诱导抗原特异性、细胞毒性T淋巴细胞。我们的数据表明，B-LCL/骨髓瘤杂交细胞系能够在体外诱导来自多发性骨髓瘤患者的PBLs中的抗原特异性、细胞毒性T淋巴细胞，这可能代表一种用于多发性骨髓瘤过继免疫治疗的新策略。