Isolation of neoantigen-specific T cells from tumor and peripheral lymphocytes
Cyrille J. Cohen, … , Steven A. Rosenberg, Paul F. Robbins
Cyrille J. Cohen, … , Steven A. Rosenberg, Paul F. Robbins
Published September 21, 2015
Citation Information: J Clin Invest. 2015;125(10):3981-3991. https://doi.org/10.1172/JCI82416.
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Isolation of neoantigen-specific T cells from tumor and peripheral lymphocytes

Abstract

Adoptively transferred tumor-infiltrating T lymphocytes (TILs) that mediate complete regression of metastatic melanoma have been shown to recognize mutated epitopes expressed by autologous tumors. Here, in an attempt to develop a strategy for facilitating the isolation, expansion, and study of mutated antigen–specific T cells, we performed whole-exome sequencing on matched tumor and normal DNA isolated from 8 patients with metastatic melanoma. Candidate mutated epitopes were identified using a peptide-MHC–binding algorithm, and these epitopes were synthesized and used to generate panels of MHC tetramers that were evaluated for binding to tumor digests and cultured TILs used for the treatment of patients. This strategy resulted in the identification of 9 mutated epitopes from 5 of the 8 patients tested. Cells reactive with 8 of the 9 epitopes could be isolated from autologous peripheral blood, where they were detected at frequencies that were estimated to range between 0.4% and 0.002%. To the best of our knowledge, this represents the first demonstration of the successful isolation of mutation-reactive T cells from patients’ peripheral blood prior to immune therapy, potentially providing the basis for designing personalized immunotherapies to treat patients with advanced cancer.

Authors

Cyrille J. Cohen, Jared J. Gartner, Miryam Horovitz-Fried, Katerina Shamalov, Kasia Trebska-McGowan, Valery V. Bliskovsky, Maria R. Parkhurst, Chen Ankri, Todd. D. Prickett, Jessica S. Crystal, Yong F. Li, Mona El-Gamil, Steven A. Rosenberg, Paul F. Robbins

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Figure 1

Analysis of FTDs.

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Analysis of FTDs. (A) Schematic representation of the experimental strat...
(A) Schematic representation of the experimental strategy. (B) Approximately 3 × 104 to 5 × 104 cells from FTDs were incubated for 3 to 4 days in lymphocyte medium and then stained with panels of mutated epitope MHC tetramers. The cells were analyzed by flow cytometry (gated on the lymphocyte population). For each patient, the frequency of tetramer-positive cells is indicated (y axis) for each peptide screened (x axis). Patients 3713, 3466, and 3879 were screened for binding to HLA-A*02:01 tetramers and patient 3919 for binding to HLA-A1 tetramers. (C) Tetramer (Tet) staining for selected mutated epitopes identified for each patient. The percentage of positive cells is indicated. (D) Cultured FTD cells were incubated with T2 cells pulsed with the predicted mutated epitopes or incubated with T2 cells pulsed with irrelevant peptide (Ctrl, see Methods) for 16 hours, and IFN-γ secretion was measured by ELISA. Pt., patient.