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Recognition of human gastrointestinal cancer neoantigens by circulating PD-1+ lymphocytes
Alena Gros, … , James C. Yang, Steven A. Rosenberg
Alena Gros, … , James C. Yang, Steven A. Rosenberg
Published November 1, 2019; First published October 14, 2019
Citation Information: J Clin Invest. 2019;129(11):4992-5004. https://doi.org/10.1172/JCI127967.
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Categories: Research Article Immunology Oncology

Recognition of human gastrointestinal cancer neoantigens by circulating PD-1+ lymphocytes

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Abstract

Tumor-resident lymphocytes can mount a response against neoantigens expressed in microsatellite-stable gastrointestinal (GI) cancers, and adoptive transfer of neoantigen-specific lymphocytes has demonstrated antitumor activity in selected patients. However, whether peripheral blood could be used as an alternative minimally invasive source to identify lymphocytes targeting neoantigens in patients with GI cancer with relatively low mutation burden is unclear. We used a personalized high-throughput screening strategy to investigate whether PD-1 expression in peripheral blood could be used to identify CD8+ or CD4+ lymphocytes recognizing neoantigens identified by whole-exome sequencing in 7 patients with GI cancer. We found that neoantigen-specific lymphocytes were preferentially enriched in the CD8+PD-1+/hi or CD4+PD-1+/hi subsets, but not in the corresponding bulk or PD-1– fractions. In 6 of 7 individuals analyzed we identified circulating CD8+ and CD4+ lymphocytes targeting 6 and 4 neoantigens, respectively. Moreover, neoantigen-reactive T cells and a T cell receptor (TCR) isolated from the CD8+PD-1+ subsets recognized autologous tumor, albeit at reduced levels, in 2 patients with available cell lines. These data demonstrate the existence of circulating T cells targeting neoantigens in GI cancer patients and provide an approach to generate enriched populations of personalized neoantigen-specific lymphocytes and isolate TCRs that could be exploited therapeutically to treat cancer.

Authors

Alena Gros, Eric Tran, Maria R. Parkhurst, Sadia Ilyas, Anna Pasetto, Eric M. Groh, Paul F. Robbins, Rami Yossef, Andrea Garcia-Garijo, Carlos A. Fajardo, Todd D. Prickett, Li Jia, Jared J. Gartner, Satyajit Ray, Lien Ngo, John R. Wunderllich, James C. Yang, Steven A. Rosenberg

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Figure 1

Identification of CD8+ neoantigen-specific lymphocytes in peripheral blood of a patient with gastroesophageal cancer (NCI-4078).

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Identification of CD8+ neoantigen-specific lymphocytes in peripheral blo...
(A) In vitro–expanded peripheral blood CD8+ lymphocyte subsets were cocultured with autologous DCs pulsed with DMSO or with the indicated peptide pools (PPs) containing the 25-mers with the putative mutations identified by WES. T cell reactivity was measured by IFN-γ ELISPOT assay. (B) Reactivity of peripheral blood CD8+PD-1hi cells to DCs pulsed with individual 25-mers from PP1 (left) or PP3 (right). IFN-γ ELISPOT assay and flow cytometric analysis of 4-1BB upregulation on CD8+ lymphocytes are plotted. (C) CD8+PD-1hi T cells were cocultured with DCs pulsed with DMSO, PP1, or PP3, and the CD8+ T cells that upregulated 4-1BB against PP1 or PP3 were FACS purified and expanded. Representative plots show the percentage of 4-1BB+ live CD3+CD8+ lymphocytes after coculture with the PPs specified. (D–G) PP1-reactive (D) or PP3-reactive (E) cells isolated in part C or PBLs transduced with candidate TCR1 (F) or TCR2 (G) were cocultured with autologous DCs pulsed with WT or mutated (MUT) DLATp.G294L or GBASp.E207K 25-mers, respectively. The constructed TCR expressed the mouse constant region, enabling its detection with antibodies specific for the mouse TCRβ constant region (mTCRB). Reactivity was measured by flow cytometric analysis of 4-1BB upregulation on CD8+ cells (D and E) or CD8+mTCRB+ lymphocytes (F and G). The individual neoantigens recognized and the amino acid position and change are noted. “>500” denotes greater than 500 spots. Experiments were performed without technical duplicates. Data from A–G are representative of at least 2 independent experiments.
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ISSN: 0021-9738 (print), 1558-8238 (online)

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