The prognosis of hepatocellular carcinoma (HCC) is extremely poor, of which hepatitis B virus-related hepatocellular carcinoma (HBV-HCC) accounts for the majority in China. Immune checkpoint inhibitors have become an effective immunotherapy method for the treatment of HCC, but they are mainly used for T cells. NK cells play a vital role as the first line of defense against tumors. Therefore, we explored the characteristic expression pattern of immune checkpoints on NK cells of HBV-HCC patients. We analyzed the correlation between the co-expression of TIGIT and TIM-3 and the clinical progress of patients with HBV-HCC. The co-expression of TIGIT and TIM-3 on NK cells is elevated in patients with HBV-HCC. TIGIT⁺TIM-3⁺NK cells showed exhausted phenotypic characteristics and displayed dysfunction manifested as weakened killing function, reduced cytokine production, and proliferation function. TIGIT⁺TIM-3⁺NK cells participate in NK cells function exhaustion and are closely related to the disease progression of patients with HBV-HCC, suggesting a new target for future immunotherapy.

Background: Alpha-fetoprotein-negative hepatocellular carcinoma (AFP-NHCC) (< 8.78 ng/mL) have special clinicopathologic characteristics and prognosis. The aim of this study was to apply a new method to establish and validate a new model for predicting the prognosis of patients with AFP-NHCC. Methods: A total of 410 AFP-negative patients with clinical diagnosed with HCC following non-surgical therapy as a primary cohort; 148 patients with AFP-NHCC following non-surgical therapy as an independent validation cohort. In primary cohort, independent factors for overall survival (OS) by LASSO Cox regression were all contained into the nomogram1; by Forward Stepwise Cox regression were all contained into the nomogram2. Nomograms performance and discriminative power were assessed with concordance index (C-index) values, area under curve (AUC), Calibration curve and decision curve analyses (DCA). The results were validated in the validation cohort. Results: The C-index of nomogram1was 0.708 (95%CI: 0.673–0.743), which was superior to nomogram2 (0.706) and traditional modes (0.606–0.629). The AUC of nomogram1 was 0.736 (95%CI: 0.690–0.778). In the validation cohort, the nomogram1 still gave good discrimination (C-index: 0.752, 95%CI: 0.691–0.813; AUC: 0.784, 95%CI: 0.709–0.847). The calibration curve for probability of OS showed good homogeneity between prediction by nomogram1 and actual observation. DCA demonstrated that nomogram1 was clinically useful. Moreover, patients were divided into three distinct risk groups for OS by the nomogram1: low-risk group, middle-risk group and high-risk group, respectively. Conclusions: Novel nomogram based on LASSO Cox regression presents more accurate and useful prognostic prediction for patients with AFP-NHCC following non-surgical therapy. This model could help patients with AFP-NHCC following non-surgical therapy facilitate a personalized prognostic evaluation.

Yangyin Fuzheng Jiedu Prescription (YFJP), a formulated Chinese herbal medicine, has been used for several decades in the treatment of hepatocellular carcinoma (HCC). Previous studies have demonstrated its anti-tumor efficacy, but the mechanism of action remains uncharacterized. This study aims to evaluate the therapeutic effect of YFJP on H22 tumor-bearing mice. This study aimed to evaluate the therapeutic effect of YFJP on H22 tumor-bearing mice. A total of 50 male H22 tumor-bearing mice were randomly divided into 6 groups and continuous administered either different doses of YFJP or cyclophosphamide (CTX) or normal saline. for 2 weeks. The tumor appearance was observed by taking photos, and the tumor volume, weight, spleen and thymus index were calculated. Morphology of tumor infiltrating lymphocytes and the CD8⁺ T lymphocytes were detected through HE staining immunohistochemistry respectively. The frequency of CD3⁺, CD8⁺ T cell subsets and co-inhibitory receptors PD-1, TIGIT, Tim-3 on CD8⁺ T cell in spleen, peripheral blood and tumor tissue was performed by flow cytometry. Meanwhile, the killing and apoptotic functions of CD8⁺ T cells in tumor tissues were also detected by the same method. The levels of cytokines in peripheral blood were detected by Milliplex map mouse highs sensitivity T Cell kit. The expression of T cell transcription factor T-bet and Eomes in tumor tissues were observed by Western blot. We found that YFJP could effectively inhibit the solid tumor growth and spleen indexes, but showed little effect on the body weight in the established mouse model of HCC. Furthermore, we investigated the effect of YFJP on the phenotypic and functional changes of T cells. The results showed that YFJP could maintain the high ratio of CD3⁺ and CD8⁺ T cells in the peripheral blood, spleen, and tumor tissues while decreasing the expression of programmed cell death-1 (PD-1), T cell immunoglobulin and ITIM domain (TIGIT), T cell immunoglobulin domain and mucin domain-3 (Tim-3) in CD8⁺ T cells, respectively. Surprisingly, PD-1/Tim-3 double-positive T cells in the peripheral blood and tumor tissues were significantly decreased. Additionally, YFJP restored the cytotoxicity of tumor-infiltrating T cells and delayed their apoptosis in H22 tumor-bearing mice. In addition, treatment with YFJP significantly decreased the expression of inflammatory and immunosuppressive cytokines (including IL-1β, IL-6, and IL-10) in the serum and tumor tissues whereas enhancing that of effector cytokines TNF-α, and IFN-γ. Moreover, T cell transcription factors T-bet increased and Eomes degraded in the tumor tissues upon YFJP treatment. In conclusion, these results demonstrated that YFJP could simultaneously exert anti-tumor immune response in H22 tumor-bearing mice by alleviating T cell exhaustion and immunosuppression.