High co-expression of immune checkpoint receptors PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT on tumor-infiltrating lymphocytes in early-stage breast cancer

Abstract

High expression of immune checkpoint receptors (ICRs) in the tumor microenvironment regulates the anti-tumor response. In this study, the differential expressions of ICRs on tumor-infiltrating lymphocytes (TILs) in patients with early-stage breast cancer were investigated.The study included 32 patients who underwent surgery with a diagnosis of early-stage breast cancer between September 2018 and March 2020. TIL isolation was performed using a MACS tumor separation device and tumor separation kit. PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT expression of cytotoxic T and natural killer (NK) cells on TILs and peripheral blood lymphocytes (PBLs) were determined by flow cytometry.Patients with a high Ki-67 index, high TIL density, and HER-2 positivity were more likely to have increased CD16⁺CD56^dim NK cells on TILs. Patients with T2 tumors were more likely to have increased expression of PD-1, LAG-3, and TIGIT on tumor-infiltrating CD8⁺ cytotoxic T cells than those with T1 tumors. PD-1, CTLA-4, TIGIT, LAG-3, and TIM-3 expression of CD8⁺ T and CD16^-CD56^bright NK cells in TILs showed significant positive correlations with each other. PD1⁺CD8⁺, TIGIT⁺CD16⁺, and CTLA-4⁺CD56⁺ cells in PBLs and TILs were found to be negatively correlated, whereas only TIM-3⁺ expression of CD8⁺ T and CD16⁺CD56^dim cells in PBLs and TILs showed positive correlations.Our results suggest that CD16⁺CD56^dim NK cells on TILs may play a major role in the immune response against HER2-positive or highly proliferating breast tumors in patients with early-stage breast cancer. Furthermore, various ICRs were found to be highly co-expressed with each other on TILs, including PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT. These receptors may synergistically suppress the response to the tumor, which may trigger immune escape mechanisms in the early stage of carcinogenesis. However, ICR expressions other than TIM3 on PBLs were not found to accompany their counterparts on TILs.

Keywords: CTLA-4; Early-stage breast cancer; LAG-3; NK; PD-1; TIGIT; TIL; TIM-3.