Isolation of high avidity melanoma-reactive CTL from heterogeneous populations using peptide-MHC tetramers.
Isolation of high avidity melanoma-reactive CTL from heterogeneous populations using peptide-MHC tetramers.
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DOI：
10.4049/jimmunol.162.4.2227
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发表时间：
1999-02
影响因子：
4.4
通讯作者：
C. Yee;P. Savage;P. Lee;Mark M. Davis;P. Greenberg
中科院分区：
文献类型：
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作者：
C. Yee;P. Savage;P. Lee;Mark M. Davis;P. Greenberg
人肿瘤Ag的免疫原性肽已被用于产生抗原特异性CTL。然而，绝大多数这些肽特异性CTL克隆是低贪婪和肽，而不是肿瘤，反应性。肽- mhc四聚体已被证明能以足够的亲和力结合特定的tcr，成为流式细胞术的有用试剂。在本文中，我们证明了肽- mhc四聚体也可以用来选择性地识别高活性的肿瘤反应性CTL，并从异质群体中富集可以裂解肿瘤靶点的肽反应性T细胞亚群。使用黑色素瘤蛋白MART-1和gp100诱导潜在的肿瘤反应性T细胞，并通过TCR结合特定肽- mhc四聚体来评估T细胞染色的强度。检测到荧光强度范围，四聚体结合程度与T细胞贪婪度相关。肽反应性T细胞群体在TCR和粘附分子的表达方面具有表型上的相似性，这表明肿瘤细胞的这种差异亲和性反映了TCR对其肽- mhc配体的差异亲和力。从多肽刺激的PBMCs异质群体中对四聚体高CTL进行分类、克隆和扩增，可以快速选择高亲和性肿瘤反应性CTL克隆，这些克隆在重新扩增时保留其功能和四聚体高表型。这些结果表明，T细胞对其肿瘤靶点的亲和性是由于TCR对其肽- mhc配体的特异性亲和力，这种相互作用可以用肽- mhc四聚体来描述，并用于分离高亲和性的肿瘤反应性CTL。
Immunogenic peptides of human tumor Ag have been used to generate antigen-specific CTL. However, the vast majority of these peptide-specific CTL clones are of low avidity and are peptide, but not tumor, reactive. Peptide-MHC tetramers have been shown to bind specific TCRs with sufficient affinity to be useful reagents for flow cytometry. In this paper we demonstrate that peptide-MHC tetramers can also be used to selectively identify high avidity tumor-reactive CTL and enrich, from a heterogeneous population, the subpopulation of peptide-reactive T cells that can lyse tumor targets. The melanoma proteins, MART-1 and gp100, were used to induce potentially tumor-reactive T cells, and the intensity of T cell staining by TCR binding of specific peptide-MHC tetramers was assessed. A range of fluorescence intensity was detected, and the magnitude of tetramer binding was correlated with T cell avidity. The population of peptide-reactive T cells was phenotypically similar with regard to expression of TCR and adhesion molecules, suggesting that this differential avidity for tumor cells reflected differential affinity of the TCR for its peptide-MHC ligand. Sorting, cloning, and expansion of tetramerhigh CTL from a heterogeneous population of peptide-stimulated PBMCs enabled rapid selection of high avidity tumor-reactive CTL clones, which retained their functional and tetramerhigh phenotype on re-expansion. These results demonstrate that the avidity of a T cell for its tumor target is due to the specific affinity of the TCR for its peptide-MHC ligand, that this interaction can be described using peptide-MHC tetramers and used to isolate high avidity tumor-reactive CTL.