Poelchen Juliane, Pardo Sandra, Novak Daniel, Sun Qian, Steinfass Tamara, Vierthaler Marlene, Cicek Sener Özge, Granados Blanco Karol, Wang Yiman, Nicolay Jan Peter, Guermonprez Pierre, Harbottle Richard, Umansky Viktor, Utikal Jochen
Skin Cancer Unit, German Cancer Research Center (DKFZ), Heidelberg, Germany.
Department of Dermatology, Venereology and Allergology, University Medical Center Mannheim, Ruprecht Karl University of Heidelberg, Mannheim, Germany.
Int J Cancer. 2025 Nov 1;157(9):1876-1887. doi: 10.1002/ijc.35524. Epub 2025 Jun 12.
Melanoma accounts for the majority of all skin cancer-related deaths with rising incidence rates. Adoptive cell therapies (ACT) with tumor antigen-specific CD8+ T cells derived from human-induced pluripotent stem cells (hiPSCs) might offer a promising treatment strategy for advanced malignant melanoma patients. In this study, we investigated two strategies for the generation of CD8+ T cells from hiPSCs expressing a T cell receptor (TCR) specific for the melanoma-associated antigen recognized by T cells (MART-1) or a chimeric antigen receptor (CAR) specific for the melanoma-associated chondroitin sulfate proteoglycan (MCSP), respectively. While the long-term co-culture of bioengineered OP9 stromal cells with CD34+ hematopoietic stem/progenitor cells (HSPCs) facilitated the generation of CD4 + CD8+ double-positive (DP) T cells, we encountered difficulties in obtaining high percentages of CD8+ single-positive (SP) T cells using this method. However, the replacement of the OP9 cells with a T cell differentiation kit enabled the generation of CD8+ SP T cells after 47 days. Despite a low expression of the T cell marker CD3 on the surface of the generated CD8+ SP T cells, we detected intracellular IFN-γ and surface CD107a expression upon stimulation. Moreover, the generated CD8+ SP T cells exhibited cytotoxic effects when co-cultured with melanoma cell lines. The use of scaffold/matrix attachment region (S/MAR) DNA vectors ensured persistent expression of the TCR or the CAR during differentiation of T cells. Hence, these findings demonstrate the potential as well as the challenges associated with using S/MAR DNA vector-transfected hiPSCs for the generation of melanoma antigen-specific CD8+ T cells.
黑色素瘤导致了大多数与皮肤癌相关的死亡，且发病率不断上升。采用源自人诱导多能干细胞（hiPSC）的肿瘤抗原特异性CD8+T细胞进行过继性细胞疗法（ACT），可能为晚期恶性黑色素瘤患者提供一种有前景的治疗策略。在本研究中，我们分别研究了两种从hiPSC生成CD8+T细胞的策略，这些hiPSC表达针对T细胞识别的黑色素瘤相关抗原（MART-1）的T细胞受体（TCR）或针对黑色素瘤相关硫酸软骨素蛋白聚糖（MCSP）的嵌合抗原受体（CAR）。虽然生物工程化的OP9基质细胞与CD34+造血干/祖细胞（HSPC）的长期共培养促进了CD4+CD8+双阳性（DP）T细胞的生成，但我们在用这种方法获得高比例的CD8+单阳性（SP）T细胞时遇到了困难。然而，用T细胞分化试剂盒替代OP9细胞，能够在47天后生成CD8+SP T细胞。尽管所生成的CD8+SP T细胞表面的T细胞标志物CD3表达较低，但我们在刺激后检测到细胞内IFN-γ和表面CD107a表达。此外，所生成的CD8+SP T细胞与黑色素瘤细胞系共培养时表现出细胞毒性作用。支架/基质附着区域（S/MAR）DNA载体的使用确保了TCR或CAR在T细胞分化过程中的持续表达。因此，这些发现证明了使用S/MAR DNA载体转染的hiPSC生成黑色素瘤抗原特异性CD8+T细胞的潜力以及相关挑战。
