Immunogenicity of somatic mutations in human gastrointestinal cancers

Abstract

It is unknown whether the human immune system frequently mounts a T cell response against mutations expressed by common epithelial cancers. Using a next-generation sequencing approach combined with high-throughput immunologic screening, we demonstrated that tumor-infiltrating lymphocytes (TILs) from 9 out of 10 patients with metastatic gastrointestinal cancers contained CD4(+) and/or CD8(+) T cells that recognized one to three neo-epitopes derived from somatic mutations expressed by the patient's own tumor. There were no immunogenic epitopes shared between these patients. However, we identified in one patient a human leukocyte antigen-C*08:02-restricted T cell receptor from CD8(+) TILs that targeted the KRAS(G12D) hotspot driver mutation found in many human cancers. Thus, a high frequency of patients with common gastrointestinal cancers harbor immunogenic mutations that can potentially be exploited for the development of highly personalized immunotherapies.

Copyright © 2015, American Association for the Advancement of Science.

Figures

Fig. 1.. Identification of personalized mutation-specific T cells in a patient with metastatic colon cancer (4007).

(A) Twenty-three different TIL cultures were cocultured with autologous DCs transfected with an irrelevant TMG RNA, or the indicated TMG construct encoding the various putative mutations identified with whole-exomic sequencing. T cell responses were measured the next day by means of IFN-γ ELISPOT assay (top) and flow cytometric analysis for 4-1BB up-regulation on CD8+ T cells (bottom). (B) TIL cultures 8, 16, and 23 were cocultured with DCs transfected with an irrelevant TMG RNA, or TMG-7 or TMG-14 RNA, and CD8+ T cells that up-regulated 4-1BB were purified by means of FACS and expanded. (C) IFN-γ ELISPOT assay (left axis) and flow cytometric analysis of 4-1BB expression (right axis) on TMG-7– and TMG-14–reactive CD8+ T cells isolated in (B) after an overnight coculture with DCs pulsed with long peptides encoded by either TMG-7 or TMG-14, or transfected with TMG-7 or TMG-14 RNA. (D) Autologous open-repertoire peripheral blood T cells were genetically modified with the TCRs derived from the SKIV2L and H3F3B mutation-reactive T cells identified in (B) and (C) and then cocultured with DCs pulsed with the indicated wild-type (wt) and mutated (mut) long peptides. Flow cytometric analysis is gated on live CD8+ T cells, and TCR transduction efficiencies ranged between 60 and 80%. Plate-bound antibody to CD3 (OKT3) was used as a positive control in all coculture assays. “>” ELISPOT assay is not accurate above ~500 spots. Data from (B) to (D) are representative of at least two independent experiments.

Fig. 2.. Identification of KRASG12D-mutation-specific T cells in a patient with colorectal cancer (3995).

(A) IFN-γ ELISPOT assay of P3W5+6 TILs cocultured overnight with DCs transfected with irrelevant (Irrel.) TMG or TMG-3 RNA (which encodes the KRASG12D minigene), or DCs pulsed with wt or KRASG12D 24–amino acid (AA)–long peptides. Numbers are spots per 3 × 104 TILs. (B) P3W5+6 TILs were stimulated with DCs pulsed with wt or KRASG12D 24-AA-long peptides, and 4-1BB+ CD8+ T cells were purified by means of FACS and expanded. (C) IFN-γ ELISPOT assay and flow cytometric analysis of 4-1BB expression on the KRASG12D-enriched CD8+ T cells from (B) cocultured overnight with DCs pulsed with wt or KRASG12D 24-AA-long peptides, or DCs transfected with full-length wt or KRASG12D RNA. (D) IFN-γ enzyme-linked immunosorbent assay (ELISA) of T cells genetically modified with the KRASG12D-reactive TCR cocultured with COS-7 cells cotransfected with nothing (Mock) or the indicated KRAS gene along with nothing (No HLA), or the HLA-B and -C alleles expressed by the patient. (E) IFN-γ ELISA of T cells genetically modified with the KRASG12D-reactive TCR cocultured with various pancreatic cancer cell lines transduced with nothing (Mock) or the HLA-C*08:02 allele. The presence or absence of endogenously expressed KRASG12D is shown. AS, ASPC-1; MD, MDA-Panc48; PK, PK-45p; FA, FA6-2; HP, HPAC; Bx, BxPC-3 (KRAS-wt); A8, A818.8 (KRASG12R); SK, SK-PC3 (KRASG12V); MI, MIA PaCa-2 (KRASG12C). (F) KRASG12D-reactive TILs enriched from (B) were cocultured for 6 hours with pancreatic cancer cell lines transduced with nothing (Mock) or the HLA-C*08:02 allele, and flow cytometry was used to assess CD107a expression and TNF production by means of intracellular cytokine staining. Autologous APCs (peripheral blood mononuclear cells) pulsed overnight with wt or KRASG12D 24-AA-long peptides were used as control target cells. Data are gated on CD8+ T cells expressing the KRASG12D-reactive TCR Vβ5.2. “>” ELISPOT assay is not accurate above ~500 spots. Error bars are ±SD. All data are representative of at least two independent experiments.