Glazyrin Alexey L, Kan-Mitchell June, Mitchell Malcolm L S
Karmanos Cancer Institute, 110 East Warren Avenue, Detroit, Michigan 48201-1379, USA.
Cancer Immunol Immunother. 2003 Mar;52(3):171-8. doi: 10.1007/s00262-002-0339-6. Epub 2003 Feb 11.
In this study we compared several protocols for in vitro induction of cytotoxic T lymphocytes (CTL) from naïve HLA-A*0201(+) peripheral blood mononuclear cells (PBMC) against allogeneic melanoma cells. As immunization material we compared: (1) the lysate of apoptotic or live melanoma tumor cells [MSM-M14 (M14) and MSM-M3 (M3)]; and (2) total RNA extracted from the same melanoma cell lines, either unconjugated or conjugated with a charged carrier (DMRIE-C). Overall killing activity was very similar in CTL induced by tumor lysate or RNA. CTL induced by both methods preferentially killed an HLA class I-matched M14 melanoma cell line rather than HLA class I-unmatched M3 cells. Cytotoxicity could be partially blocked by anti-HLA class I antibodies. There were no significant differences in cytotoxicity and in other clonal characteristics in CTL lines induced by a lysate of apoptotic bodies as compared to lines induced by lysate of viable cells. However, CTL induced by DMRIE-C-bound total RNA demonstrated superior cytotoxicity when compared with CTL induced by unconjugated total RNA. Polyclonal CTL induced by tumor lysate contained a substantial percentage of tyrosinase(368-376 370N) tetramer-positive cells and demonstrated specific killing activity against tyrosinase(368-376 370N) peptide-labeled T2 cells, comparable to cytotoxicity of the CTL developed against this peptide alone. In contrast, there were no detectable tyrosinase(368-376 370N)-tetramer positive cells and no specific anti-tyrosinase peptide(368-376 370N) response in polyclonal CTL induced by immunization with tumor RNA. These data demonstrate that both total tumor RNA and tumor lysate are effective for inducing of cytotoxic anti-melanoma CTL, but tyrosinase(368-376 370N) specific cells were detected only in lysate-induced CTL cultures. This suggests that nature of the antigens present in tumor lysate might be different from those in tumor RNA.
在本研究中，我们比较了几种从初始HLA - A*0201(+)外周血单核细胞(PBMC)体外诱导细胞毒性T淋巴细胞(CTL)以对抗同种异体黑色素瘤细胞的方案。作为免疫材料，我们比较了：(1)凋亡或活的黑色素瘤肿瘤细胞[MSM - M14 (M14)和MSM - M3 (M3)]的裂解物；以及(2)从未经偶联或与带电载体(DMRIE - C)偶联的相同黑色素瘤细胞系中提取的总RNA。肿瘤裂解物或RNA诱导的CTL的总体杀伤活性非常相似。两种方法诱导的CTL均优先杀伤HLA I类匹配的M14黑色素瘤细胞系，而非HLA I类不匹配的M3细胞。细胞毒性可被抗HLA I类抗体部分阻断。与活细胞裂解物诱导的细胞系相比，凋亡小体裂解物诱导的CTL系在细胞毒性和其他克隆特征方面无显著差异。然而，与未偶联的总RNA诱导的CTL相比，DMRIE - C偶联的总RNA诱导的CTL表现出更高的细胞毒性。肿瘤裂解物诱导的多克隆CTL含有相当比例的酪氨酸酶(368 - 376 370N)四聚体阳性细胞，并表现出对酪氨酸酶(368 - 376 370N)肽标记的T2细胞的特异性杀伤活性，与单独针对该肽产生的CTL的细胞毒性相当。相反，用肿瘤RNA免疫诱导的多克隆CTL中未检测到酪氨酸酶(368 - 376 370N) - 四聚体阳性细胞，也没有特异性抗酪氨酸酶肽(368 - 376 370N)反应。这些数据表明，肿瘤总RNA和肿瘤裂解物均能有效诱导细胞毒性抗黑色素瘤CTL，但仅在裂解物诱导的CTL培养物中检测到酪氨酸酶(368 - 376 370N)特异性细胞。这表明肿瘤裂解物中存在的抗原性质可能与肿瘤RNA中的不同。
