Identifying high TRAIL-expressing liver NK cells from deceased and living donors for effective immunotherapy

Introduction: Natural killer (NK) cells play a crucial role as the frontline of the immune defense against circulating tumor cells. The mechanisms underlying NK cell cytotoxicity against tumors are well-established, with liver NK cells in particular expressing TNF-related apoptosis-inducing ligand (TRAIL) and exerting direct killing via the TRAIL death receptor pathway. Several clinical trials have utilized liver NK cells in liver transplant recipients. However, TRAIL expression varies among individuals. This study aims to identify the donor characteristics of liver NK cells with high TRAIL expression and understand the characteristics of liver NK cells from deceased donors (DDs) to enhance the efficacy of immunotherapy using liver NK cells. Methods: The study enrolled 18 DDs, 40 living donors (LD), and 5 healthy volunteers (HV). Lymphocytes were cultured with human recombinant IL-2 (100 IU/ml) for three days. All analyses, including the activation receptors and cytotoxicity of NK cells against K562 cell lines stained with CFSE, were conducted using a flow cytometer. Results: (1) The proportion of NK cells from DDs was significantly higher among liver NK cells compared to peripheral NK cells from HVs (38.3 % vs. 1.8%, p<0.01). The expression of CD69 (94.0% vs. 86.0%, p<0.01) and NKG2D (97.5% vs. 82.0%, p<0.01) was higher on liver NK cells.However, the expression of TRAIL (24.2% vs. 27.9%, N.S.), NKp44 (22.7% vs. 22.2%, N.S.), and NKp46 (87.2% vs. 86.0%, N.S.) was similar between both NK cells. The cytotoxicity of liver NK cells against K562 cells was significantly higher (Tumor cells: NK cells =1:40, N=5, 62.0% vs. 25.0%, p<0.01). (2) Compared to LDs, DDs showed significantly higher expression of NKp46 (93.2% vs. 79.2%, p<0.01) and significantly lower expression of TRAIL (24.2% vs. 36.9%, p<0.05).(3) The study included a total of 40 LDs. After IL-2 stimulation, TRAIL expression on liver NK cells was 42.3% [9.9%-97%]. In multivariate analyses, higher levels of albumin +0.1mg/ml (OR: 1.86, 95%CI 1.08-3.20, p<0.01), lower Fib-4 index+0.1 (OR:0.74, 95%CI 0.57-0.98, p=0.02), and higher BMI (OR:1.65, 95%CI 1.00-2.72, p=0.02) were associated with higher TRAIL expression on liver NK cells.However, this trend was not observed in DDs. Conclusion: Liver NK cells exhibit potent cytotoxicity against tumors compared to peripheral NK cells. However, this study reveals individual differences in TRAIL expression on liver NK cells from DDs, and further cases need to be accumulated to analyze the donor characteristics factors associated with higher TRAIL expression on liver NK cells. By focusing on the donor characteristics of high TRAIL-expressing liver NK cells, effective immunotherapy using liver NK cells from DDs can be developed.