Immune checkpoints associate with dysfunctional T cells, which have a reduced 
ability to clear pathogens or cancer cells. T-cell checkpoint blockade may 
improve patient survival. However, checkpoint molecules on cytokine-induced 
killer (CIK) cell, a non-specific adoptive immunotherapy, remain unknown. In 
present study, we detected the dynamic expression of eight major checkpoint 
molecules (CTLA-4, PD-1, PD-L1, TIM- 3, CEACAM-1, LAG-3, TIGIT and BTLA) on CIK 
cells from NSCLC patients. The majority of these molecules, except BTLA, were 
sharply elevated during the early stage of CIK cell culture. Thereafter, PD-1 
and TIGIT expressions decreased gradually towards the initial level (day 0). 
Moreover, CTLA-4 faded away during the later stage of CIK culture. LAG-3 
expression decreased but was still significantly higher than the initial level. 
Of note, PD-L1 remained stably upregulated during CIK culture compared with 
PD-1, indicating that PD-L1 might act as an inhibitory molecule on CIK cells 
instead of PD-1. Furthermore, TIM-3 and CEACAM1 were strongly expressed 
simultaneously during long-term CIK culture and showed a significant and 
mutually positive correlation. BTLA displayed a distinct pattern, and its 
expression gradually decreased throughout the CIK culture. These observations 
suggested that CIK cells might be partly exhausted before clinical transfusion, 
characterized by the high expression of PD-L1, LAG-3, TIM- 3, and CEACAM-1 and 
the low expression of TIGIT, BTLA, PD-1, and CTLA-4 compared with initial 
culture. Our results imply that implementing combined treatment on CIK cells 
before transfusion via antibodies targeting PD-L1, LAG-3, TIM-3, and CEACAM-1 
might improve the efficiency of CIK therapy for NSCLC patients.