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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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Research Article Oncology Article has an altmetric score of 19

Isolation of neoantigen-specific T cells from tumor and peripheral lymphocytes

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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 3

Treatment TILs recognize mutated antigens.

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Treatment TILs recognize mutated antigens.
(A) Bulk-treatment TIL cultur...
(A) Bulk-treatment TIL cultures were stained with the indicated tetramer and analyzed by flow cytometry. (B) TILs were cocultured with T2 cells pulsed with the indicated mutated epitope at different concentrations (ranging from 10–6 to 10–13 M) or cocultured with T2 cells pulsed with irrelevant peptide (Ctrl, at 10–6 M). Sixteen hours after the start of the coculture, IFN-γ secretion was measured by ELISA. (C) Tetramer-positive cell populations were sorted from infusion TILs and expanded using OKT3. Ten days after the start of the expansion, T cells were assessed for tetramer binding. (D) Bulk-treatment TILs for patient 3703 were analyzed for HLA-A*02:01/mutated epitope tetramer binding. The frequency of tetramer-positive cells (y axis) for each peptide screened (x axis) is indicated (left panel). A positive mutated epitope, NSDHL, was identified (middle panel), and a representative staining plot of 2 tetramer-positive cell populations is shown (right panel). (E) NSDHLhi and NSDHLlo cell populations were sorted and expanded, and 10 days after the start of the expansion, T cells were assessed for NSDHL-tetramer binding (left panels). Additionally, these sorted cell populations were assessed for CD4 and CD8 expression (middle panels). The NSDHLhi cell population, which consisted of 98% CD8+ T cells, and the NSDHLlo cell population, which consisted of 99% CD4+ T cells, were analyzed for peptide reactivity in coculture with T2 cells pulsed with titrated doses of the mutated NSDHL epitope. Sixteen hours after the start of the coculture, IFN-γ secretion was measured in the culture supernatant by ELISA (right panels). Results are representative of 2 independent experiments.

Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)

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