Expression of Lymphocyte-Activation Gene 3 (LAG-3) Immune Checkpoint Receptor Identifies a Tumor-Reactive T Cell Population in the Peripheral Blood of Patients with Colorectal Cancer

BACKGROUND: The use of adoptive T cell therapy has proven to be effective in some advanced malignancies. This study aimed to investigate the effects of lymphocyte-activation gene 3 (LAG-3) immune checkpoint receptor in the enrichment of tumor antigen-specific CD8(+) T lymphocytes derived from peripheral blood mononuclear cells (PBMCs) in patients with colorectal cancer. MATERIAL/METHODS: Peripheral blood samples were obtained from 20 patients with colorectal cancer and apheresis was performed with enrichment and cell sorting to obtain CD8(+)LAG-3(+) T cells, which were expanded using high-dose interleukin-2 (IL-2). T cell subsets were detected using fluorescence-activated cell sorting (FACS), and T cell receptor (TCR) sequencing was used to determine specific clone types. Interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) and cell counting kit-8 (CCK-8) assays were used to measure cell avidity and cytotoxicity. RESULTS: The cultured cells increased in number over time and had the greatest proliferative activity at 15 days, at which time the percentage of CD3(+), CD3(+)CD8(+), and CD8(+)CD28(+) reached maximal levels. High purity CD8(+)LAG-3(+) T cells were isolated by FACS and at 15 days. TCR sequencing showed that CD8(+)LAG-3(+) T cells were oligoclonal, ELISpot identified increased production of tumor-specific IFN-γ, and the CCK-8 assay showed increased cytotoxicity when compared with pre-cultured CD8(+)LAG-3(−) T cells. CONCLUSIONS: In patients with colorectal cancer, CD8(+)LAG-3(+) T cells showed more specific anti-tumor activity following cell culture in vitro, which supported the potential role for the LAG-3 immune checkpoint receptor in enriching tumor-specific T cells in patients with cancer.