New immunotherapeutic advances for common epithelial cancers relies on our ability to stimulate T lymphocytes against specific tumour antigens (Ag). Next-generation sequencing now allows rapid identification of somatic cancer mutations that can lead to the expression of mutated Ag. We hypothesize that gastrointestinal cancer metastases are infiltrated by T cells recognizing tumour mutated Ag. Our aim is to set up an experimental platform to screen for and study the frequency and function of mutation-reactive T cells, while characterizing novel tumour mutated Ag.
To do this, we used two cancer cell lines generated from a liver metastasis of a gastric cancer patient. The cell line A was recognized by an autologous CD8+ T cell clone infiltrating the patient's metastasis, restricted by HLA C*0701, while cell line B was not recognized by the same clone despite its expression of the HLA C*0701. The CD8+ T cell clone was not reactive to a large panel of HLA-C*0701 expressing gastrointestinal cancer cell lines, reinforcing the hypothesis that the Ag recognized was unique to the autologous gastric cancer cell line. Exome and transcriptome sequencing was performed to compare the mutated genes differentially expressed by cancer cell line A and B. A total of 27 mutated Ag were selected as candidates: 26 Ag only expressed by cell line A, and one Ag overexpressed by cell line A. To screen for reactivity of the CD8+ T cell clone to mutated Ag, 25 amino acid (aa) mini-genes containing the mutation flanked upstream and downstream by normal aa were synthesised and cloned in tandem into 3 expression plasmids. A control sequence from the MAGE-A12 gene containing an epitope restricted by HLA-C*0701was included in each tandem minigene (TMG) construct. The mRNA from these 3 TMG constructs were in vitro transcribed and electroporated into CD40-activated B cells expressing HLA C*0701, used as antigen presenting cells. Co-culture assays are ongoing, using the CD8+ T cell clones as effectors and T cells transduced with a MAGE-A12-specific TCR as control. For the detection of mutation-reactive T cells, the sensitivity of TMG expression is compared to pulsing mutated peptides on B cells.
Taking advantage of the differential recognition of two cancer cell lines by an autologous CD8+ T cell clone, we have established a bioinformatics approach based on next-generation sequencing to obtain a list of candidate mutated Ag and we have designed an experimental system to assess their recognition by T cells. This platform will allow us to study the function of T cells reactive against metastatic gastrointestinal cancers and should lead to the discovery of new tumour Ag. Gaining a better understanding of T cells reactive to gastrointestinal cancers should ultimately contribute to the development of immunotherapies for these common malignancies.
Citation Format: Mélissa Mathieu, Sandy Pelletier, David Laperrière, Sylvie Mader, Simon Turcotte. Identification of mutation-reactive T cells in patients with gastrointestinal cancers. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A084.
- ©2016 American Association for Cancer Research.