Identification of shared TCR sequences from T cells in human breast cancer using emulsion RT-PCR.
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.
Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, CO 80045;
Daniel J
DJ
Munson
Department of Immuno-Oncology, Beckman Research Institute, City of Hope, Duarte, CA 91010;
Colt A
CA
Egelston
Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR 97201;
Kami E
KE
Chiotti
Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, CO 80045;
Zuly E
ZE
Parra
Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, CO 80045;
Tullia C
TC
Bruno
Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, CO 80045;
Brandon L
BL
Moore
Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, CO 80045;
Taizo A
TA
Nakano
Department of Immuno-Oncology, Beckman Research Institute, City of Hope, Duarte, CA 91010;
Diana L
DL
Simons
Department of Immuno-Oncology, Beckman Research Institute, City of Hope, Duarte, CA 91010;
Grecia
G
Jimenez
Department of Surgery, Beckman Research Institute, City of Hope, Duarte, CA 91010;
John H
JH
Yim
Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR 97201;
Dmitri V
DV
Rozanov
Department of Medicine, University of Colorado School of Medicine, Aurora, CO 80045;
Michael T
MT
Falta
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;
Andrew P
AP
Fontenot
Department of Pediatrics, National Jewish Health, Denver, CO 80206;
Paul R
PR
Reynolds
Center for Genes, Environment and Health, National Jewish Health, Denver, CO 80206;
Sonia M
SM
Leach
Division of Medical Oncology, University of Colorado School of Medicine, Aurora, CO 80045;
Virginia F
VF
Borges
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.
John W
JW
Kappler
Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR 97201;
Paul T
PT
Spellman
Department of Immuno-Oncology, Beckman Research Institute, City of Hope, Duarte, CA 91010;
Peter P
0000-0002-2660-4377
PP
Lee
Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, CO 80045; kapplerj@njhealth.org jill.slansky@ucdenver.edu.
Jill E
JE
Slansky
D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics*; D002460:Cell Line; D004655:Emulsions; D005260:Female; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D013601:T-Lymphocytes / Q000378:metabolism*
D004655:Emulsions; D011948:Receptors, Antigen, T-Cell
T-cell receptors; T-cell repertoire profiling; breast cancer; emulsion RT-PCR; high-throughput sequencing