Immune checkpoints associate with dysfunctional T cells, which have a reduced ability to clear pathogens or cancer cells. 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. could engage with PD-L1 on the CIK surface, thus impairing CIK cytotoxicity to some degree. PD-L2 (B7-DC) expression was reported to be largely restricted to dendritic cells (DCs) and activated macrophages. In the present study, PD-L2 expression was not observed in CIK cells. However, one study found PD-L2 expression in mouse T cells upon T-cell immune response [34]. TIM-3 was initially identified as a specific marker of KX2-391 fully differentiated IFN- producing CD4+ T helper 1 (Th1) and CD8 cytotoxic (Tc1) cells [35]. In addition to T cells, TIM-3 is also highly expressed on monocytes, macrophages, and DCs [36]. The current study first reported that TIM-3 expression was upregulated KX2-391 on CIK cells. Although TIM-3 was rarely expressed on CD4+, CD8+, and NKT cells from PBMC, all subsets of CIK cells strongly expressed TIM-3. Moreover, TIM- 3 expression remained at a high level during long-term culture. Similarly, a drastic elevation of TIM-3 expression has been reported on human na?ve CD4+ T cells activated KX2-391 with plate-bound anti-CD3/anti-CD28 for seven days [37]. Furthermore, the regulation mechanism of Tim-3 expression is involved in Th1-specific transcription factor T-bet in mice [38]. Some reports demonstrated that binding of Gal- 9 to TIM-3 causes an inhibitory signal, resulting in apoptosis of Th1 cells and cytotoxic CD8 T cells [39, 40]. Galectin-9 (Gal-9), acted as one of the TIM-3 ligands, is widely distributed in tissues involved in the immune system, i.e. spleen, thymus and peripheral blood lymphocytes, and in tissues of endodermal origin, i.e. liver, intestine, stomach and lung [41, 42]. Therefore, when CIK cells encountered Gal-9 or (e.g., CD80 or Gal-9). Furthermore, implementing combined treatment LKB1 on CIK cells before transfusion via antibodies that target PD-L1, LAG-3, TIM- 3, and CEACAM-1 might improve the efficiency of CIK therapy for individuals with NSCLC. MATERIALS AND METHODS Patients A total of 16 patients with NSCLC were enrolled, including 3 patients with squamous carcinoma and 13 patients with adenocarcinoma but free of congestive heart failure, severe coronary artery disease, cardiac arrhythmias, HIV infection, chronic active hepatitis, and concomitant corticosteroid therapy. The clinical characteristics of the patients were summarized in Table ?Table1.1. This study was approved by the State Food and Drug Administration of China (2006L01023) and by the Ethical Committee of Cancer Hospital of Tianjin Medical University, Tianjin, China, according to the guidelines of the Declaration of Helsinki. Informed consent was obtained from all subjects before their entry into the study. Table 1 Basic information of patient samples CIK cells preparation CIK cells were prepared as described in our previous studies [14, 16]. Briefly, in large-scale culture system, 40 ml PBMCs were collected from the patients with NSCLC by using a Cobe Spectra Apheresis System (CaridianBCT). The PBMCs were then cultured in AIM-V medium (Invitrogen) containing 50 ng/mL anti-CD3 antibody (e-Bioscience), 100 U/mL recombinant human IL-1 (e-Bioscience), and 1,000 U/mL IFN- (PeproTech), at 37C with 5% CO2 for 24 hours. Thereafter, 300 U/mL recombinant human IL-2 (rhIL-2; Proleukin) was added into the KX2-391 media. The medium was replaced by fresh IL-2, and the IFN–containing medium was replaced every 5 days. At day 15, CIK cells were harvested and transfused into NSCLC patients. At the same time, CIK cells were analyzed for phenotype and cytotoxicity. All products were free of.