Identification of shared TCR sequences from T cells in human breast cancer using emulsion RT-PCR
Identification of shared TCR sequences from T cells in human breast cancer using emulsion RT-PCR
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DOI：
10.1073/pnas.1606994113
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发表时间：
2016-07-19
影响因子：
11.1
通讯作者：
Slansky, Jill E.
中科院分区：
文献类型：
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作者：
Munson, Daniel J.;Egelston, Colt A.;Slansky, Jill E.
T细胞在乳腺肿瘤中的渗透与乳腺癌患者存活率的提高有关，尽管这些肿瘤中的突变相对较少。为了确定是否可以利用T细胞特异性来加强乳腺癌的免疫治疗，我们试图确定在多个患者之间共享的肿瘤浸润性淋巴细胞的α-β配对T细胞受体(TCR)。因为TCR是异源二聚体蛋白，所以我们使用了基于乳胶的RT-PCR方法来连接和扩增TCR对。在工程T细胞杂交瘤上使用这种方法，我们观察到接近85%的精确度配对保真度，尽管TCR恢复频率不同。当我们将这项技术应用于患者样本时，我们发现对于任何给定的TCR对，主要的α或β结合伙伴占总结合伙伴的90%。对原发肿瘤的TCR序列分析显示，与无肿瘤的前哨淋巴结相比，有肿瘤的前哨淋巴结重叠约四倍。此外，双侧乳腺癌患者的两个肿瘤的序列比较显示有10%的重叠。最后，我们确定了在患者肿瘤和外周血之间共享的一组独特的TCR，这些TCR在对照组的外周血中没有发现。这些TCR编码α链上的一系列V、J和互补决定区3(CDR3)序列，并显示限制的V-β用途。编码这些共享的TCRCDR3的核苷酸不同，表明这种反应是免疫选择的。利用这些T细胞可能会提供实用的策略，以改善对乳腺癌的共同抗原特异性反应。
Infiltration of T cells in breast tumors correlates with improved survival of patients with breast cancer, despite relatively few mutations in these tumors. To determine if T-cell specificity can be harnessed to augment immunotherapies of breast cancer, we sought to identify the alpha-beta paired T-cell receptors (TCRs) of tumor-infiltrating lymphocytes shared between multiple patients. Because TCRs function as heterodimeric proteins, we used an emulsion-based RT-PCR assay to link and amplify TCR pairs. Using this assay on engineered T-cell hybridomas, we observed similar to 85% accurate pairing fidelity, although TCR recovery frequency varied. When we applied this technique to patient samples, we found that for any given TCR pair, the dominant alpha-or beta-binding partner comprised similar to 90% of the total binding partners. Analysis of TCR sequences from primary tumors showed about fourfold more overlap in tumor-involved relative to tumor-free sentinel lymph nodes. Additionally, comparison of sequences from both tumors of a patient with bilateral breast cancer showed 10% overlap. Finally, we identified a panel of unique TCRs shared between patients' tumors and peripheral blood that were not found in the peripheral blood of controls. These TCRs encoded a range of V, J, and complementarity determining region 3 (CDR3) sequences on the alpha-chain, and displayed restricted V-beta use. The nucleotides encoding these shared TCR CDR3s varied, suggesting immune selection of this response. Harnessing these T cells may provide practical strategies to improve the shared antigen-specific response to breast cancer.