Format

Send to

Choose Destination
Proc Natl Acad Sci U S A. 2016 Jul 19;113(29):8272-7. doi: 10.1073/pnas.1606994113. Epub 2016 Jun 15.

Identification of shared TCR sequences from T cells in human breast cancer using emulsion RT-PCR.

Author information

1
Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, CO 80045;
2
Department of Immuno-Oncology, Beckman Research Institute, City of Hope, Duarte, CA 91010;
3
Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR 97201;
4
Department of Surgery, Beckman Research Institute, City of Hope, Duarte, CA 91010;
5
Department of Medicine, University of Colorado School of Medicine, Aurora, CO 80045;
6
Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, CO 80045; Department of Medicine, University of Colorado School of Medicine, Aurora, CO 80045;
7
Department of Pediatrics, National Jewish Health, Denver, CO 80206;
8
Center for Genes, Environment and Health, National Jewish Health, Denver, CO 80206;
9
Division of Medical Oncology, University of Colorado School of Medicine, Aurora, CO 80045;
10
Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, CO 80045; Howard Hughes Medical Institute, National Jewish Health, Denver, CO 80206; Department of Biomedical Research, National Jewish Health, Denver, CO 80206; Barbara Davis Center for Childhood Diabetes, University of Colorado, Aurora, CO 80045 kapplerj@njhealth.org jill.slansky@ucdenver.edu.
11
Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, CO 80045; kapplerj@njhealth.org jill.slansky@ucdenver.edu.

Abstract

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 ∼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 ∼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.

KEYWORDS:

T-cell receptors; T-cell repertoire profiling; breast cancer; emulsion RT-PCR; high-throughput sequencing

PMID:
27307436
PMCID:
PMC4961128
DOI:
10.1073/pnas.1606994113
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center