Sensi M, Salvi S, Castelli C, Maccalli C, Mazzocchi A, Mortarini R, Nicolini G, Herlyn M, Parmiani G, Anichini A
Division of Experimental Oncology D, Istituto Nazionale Tumori, Milan, Italy.
J Exp Med. 1993 Oct 1;178(4):1231-46. doi: 10.1084/jem.178.4.1231.
HLA-A2+ melanomas express common melanoma-associated antigens (Ags) recognized in vitro by autologous cytotoxic T lymphocytes (CTL). However, it is not known whether tumor Ags can drive in vivo a selective accumulation/expansion of Ag-specific, tumor-infiltrating T lymphocytes (TIL). Therefore, to evaluate this possibility, 39 CTL clones isolated from several independent mixed lymphocyte tumor cultures (MLTC) of TIL and peripheral blood lymphocytes (PBL) of an HLA-A2+ melanoma patient and selected for T cell receptor (TCR)-dependent, HLA-restricted tumor lysis, were used for analysis of TCR alpha and beta chain structure by the cDNA polymerase chain reaction (PCR) technique with variable gene-specific primers followed by sequencing. Despite absence of oligoclonality in fresh TIL and PBL, as well as in T cells of day 28 MLTC (day of cloning), sequence analysis of TCR alpha and beta chains of TIL clones revealed a dominance of a major category of melanoma-specific, HLA-A2-restricted T cells expressing a V alpha 8.2/J alpha AP511/C alpha and V beta 2.1/D beta 1/J beta 1.1/C beta 1 TCR. The same TCR was also found in 2 out of 14 PBL clones. The other PBL clones employed a V alpha 2.1 gene segment associated with either V beta 13.2, 14, or w22. Clones A81 (V alpha 2.1/J alpha IGRJ alpha 04/C alpha and V beta 14/D beta 1/J beta 1.2/C beta 1) and A21 (V alpha 8.2/J alpha AP511/C alpha and V beta 2.1/D beta 1/J beta 1.1/C beta 1), representative of the two most frequent TCR of PBL and TIL, respectively, expressed different lytic patterns, but both were HLA-A2 restricted and lysed only HLA-A2+ melanomas and normal melanocytes, thus indicating recognition of two distinct HLA-A2-associated and tissue-related Ags. Finally, by the inverse PCR technique, the specific TCR beta chain (V beta 2.1/D beta 1/J beta 1.1/C beta 1) expressed by the dominant TIL clone was found to represent 19 and 18.4% of all V beta 2 sequences expressed in the fresh tumor sample and in the purified TIL, respectively, but < 0.19% of V beta 2+ sequences expressed in PBL. These results are consistent with the hypothesis that a clonal expansion/accumulation of a melanocyte-lineage-specific and HLA-A2-restricted T cell clone occurred in vivo at the site of tumor growth.
HLA - A2阳性的黑色素瘤表达常见的黑色素瘤相关抗原（Ags），这些抗原在体外可被自体细胞毒性T淋巴细胞（CTL）识别。然而，尚不清楚肿瘤抗原能否在体内驱动抗原特异性肿瘤浸润性T淋巴细胞（TIL）的选择性积累/扩增。因此，为评估这种可能性，从一名HLA - A2阳性黑色素瘤患者的TIL和外周血淋巴细胞（PBL）的多个独立混合淋巴细胞肿瘤培养物（MLTC）中分离出39个CTL克隆，并选择这些克隆用于依赖T细胞受体（TCR）的、HLA限制的肿瘤裂解，然后使用可变基因特异性引物通过cDNA聚合酶链反应（PCR）技术分析TCRα和β链结构，随后进行测序。尽管新鲜TIL和PBL以及第28天MLTC（克隆日）的T细胞中不存在寡克隆性，但TIL克隆的TCRα和β链序列分析显示，表达Vα8.2/JαAP511/Cα和Vβ2.1/Dβ1/Jβ1.1/Cβ1 TCR的主要类别的黑色素瘤特异性、HLA - A2限制的T细胞占主导。在14个PBL克隆中的2个中也发现了相同的TCR。其他PBL克隆采用与Vβ13.2、14或w22相关的Vα2.1基因片段。分别代表PBL和TIL中两种最常见TCR的克隆A81（Vα2.1/JαIGRJα04/Cα和Vβ14/Dβ1/Jβ1.2/Cβ1）和A21（Vα8.2/JαAP511/Cα和Vβ2.1/Dβ1/Jβ1.1/Cβ1）表现出不同的裂解模式，但两者均受HLA - A2限制，且仅裂解HLA - A2阳性的黑色素瘤和正常黑素细胞，因此表明识别两种不同的HLA - A2相关且与组织相关的抗原。最后，通过反向PCR技术发现，占主导的TIL克隆表达的特异性TCRβ链（Vβ2.1/Dβ1/Jβ1.1/Cβ1）分别占新鲜肿瘤样本和纯化TIL中表达的所有Vβ2序列的19%和18.4%，但在PBL中表达的Vβ2 +序列中占比<0.19%。这些结果与黑色素细胞谱系特异性且HLA - A2限制的T细胞克隆在肿瘤生长部位发生体内克隆扩增/积累的假设一致。
