Abstract
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 AND 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.