Supplementary Material for: Differential Expression of Immune Checkpoints TIM-3, LAG3, TIGIT, and Siglec-7 on Circulating NK Cells: Insights from Healthy Donors compared to Gastric Cancer Patients

Due to the multifactorial nature of cancer, the development of reliable immunotherapies poses a significant challenge for medical research. In this context, targeting immune checkpoints holds promising potential. Natural killer (NK) cells are crucial in the primary defense against viral infections, tumor development, and metastasis. The cytotoxic function of NK cells is finely regulated by a complex array of activating and inhibitory receptors, including checkpoint receptors. Malignantly transformed cells can impair NK cell activity by expressing soluble or membrane-bound checkpoint ligands, thereby modulating immune responses to support tumor progression. To investigate this dilemma, we simulated in vitro activation by NK cell co-incubation with K562 cells and analyzed expression of TIM-3, LAG-3, TIGIT and Siglec-7. After that, we analyzed the checkpoint expression on circulating NK cells from 35 healthy donors and compared it to their expression in patients with gastric cancer (n =21) using flow cytometry. In healthy donors, we observed that 25% to 97% of all circulating NK cells expressed TIM-3, TIGIT, and Siglec-7, while only a small fraction of 0.6% expressed LAG-3. Co-incubation of peripheral blood mononuclear cells (PBMCs) from healthy donors with K562 cells resulted in heightened expression levels of TIM-3 and TIGIT on NK cells. Especially targeting the sialic acid-Siglec-7 axis may offer promising therapeutic strategies for various cancer types in the future. Conversely, NK cells in patients with gastric cancer showed an increased LAG-3 and reduced Siglec-7 expression. Our findings suggest the potential of LAG-3 as a next-generation checkpoint molecule, alongside Siglec-7. Especially targeting the sialic acid-Siglec-7 axis may offer promising therapeutic strategies for various cancer types in the future.