CD1d-restricted invariant natural killer T (iNKT) cells actively patrol the liver and possess valuable antitumor potential. However, clinical trials evaluating administration of iNKT cell–specific agonist α-galactosylceramide (α-GalCer) have failed to achieve obvious tumor regression. Improving the efficacy of iNKT cell–based immunotherapy requires a better understanding of the factors restraining the clinical benefits. In the context of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC), we found circulating and hepatic iNKT cells were hyperactivated but demonstrated imbalances in ratio and defective α-GalCer responsiveness. Exogenous IL2 helped to expand residual α-GalCer–responsive clones with reduced T-cell receptor diversity. However, transcriptome-wide analysis revealed activation of the senescence-associated secretory phenotype and dampened cytotoxicity in iNKT cells, weakening their immune surveillance capacity. The senescent status of iNKT cells from the patients was further illustrated by cell-cycle arrest, impaired telomere maintenance, perturbed calcium transport-related biological processes, and altered metabolism. Lipidomic profiling revealed the accumulation of long-chain acylcarnitines (LCAC) and aberrant lipid metabolism in HCC tissue. Exogenous LCACs, especially palmitoyl-carnitine and stearoyl-carnitine, inhibited iNKT cell expansion and promoted senescence. Collectively, our results provide deeper insights into iNKT cell dysregulation and identify a cell senescence–associated challenge for iNKT cell–based immunotherapy in HBV-related HCC. The mechanistic links between iNKT cell senescence and accumulated LCACs suggest new targets for anti-HCC immunotherapies. Significance: Patients with HBV-related HCC exhibit a cell senescence–associated dysregulation of invariant natural killer cells that is related to altered lipid metabolism and accumulated LCACs in tumor tissue.

Activation of invariant NKT (iNKT) cells manifests antiviral immune responses in vivo. However, clinical trials have failed to show consistent hepatitis B virus (HBV) DNA reduction postadministration of iNKT cell–specific agonist α-galactosylceramide (α-GalCer). In this study, we aimed to investigate HBV infection–related iNKT cell defects and explore iNKT cell–based therapeutic potential for chronic hepatitis B (CHB). Liver specimens from 30 HBV-infected hepatocellular carcinoma patients were collected for CD1d/hepatitis B surface Ag (HBsAg) staining and/or intrahepatic iNKT cell assay. Two hundred and six chronic HBV-infected patients (including 130 CHB patients) were enrolled in the study of circulating iNKT cell frequency and function. We found that liver and hepatoma tissue that positively stained for HBsAg had higher CD1d expression as compared with HBsAg negatively stained counterparts. The elevated CD1d expression in infected tissue is supposed to facilitate the iNKT cell–based antiviral effects locally. However, iNKT cell defects that related with disease progression suggested iNKT cells attenuated their effects during chronic HBV infection. The residual iNKT cells in CHB patients showed aberrant activation and hyporesponsiveness to α-GalCer. Exogenous IL-2 fully rescued α-GalCer–induced expansion of iNKT cells from CHB patients, and synergistic effects of IL-2 and IL-15 helped to recover the CD1d-dependent IFN-γ production. In conclusion, our results highlight the increased CD1d expression in HBV-infected liver and differential iNKT cell defects associated with disease progression during chronic HBV infection. The reversibility of iNKT cell defects suggests protective immune responses could be partially recovered in CHB.

As the implantation and long-term existence of tumor-specific T cells in host are the prerequisite for adoptive immunotherapy, memory stem T cells (TSCM) with self-renewal and differentiation capacity show the greatest potential to implant and long-term exhibit function in vivo, compared with other T cells of differentiation stages. Hence, tumor-specific TSCM have become potential candidate for adoptive T cell therapy of cancer. Here, we reported a protocol to generate allogeneic antigen-specific CD8+ TSCM cells from human PBLs. To prepare allogeneic antigen-specific CD8+ TSCM, we used an LCL named E007 of defined HLA allotyping as simulator, a co-culture of E007 and allogeneic PBLs was carried out in the presence of differentiation inhibitor TWS119 for 7 days. Sorting of proliferation cells ensured the E007-specificity of the prepared TSCM cells. The sorted lymphocytes underwent further expansion by cytokines IL-7 and IL-15 for further 7 days, making the E007-specific CD8 + TSCM expanded in number. The stem cell and T memory cell properties of the prepared CD8+ TSCM were observed in NOD-SCID mice. Our protocol began with 1 × 107 PBLs and resulted in 2 × 107 E007-specific CD8+ TSCM cells in 2 weeks of preparation. The prepared TSCM cells exhibited a proliferative history and rapid differentiation into effector cells upon the E007 re-stimulation. Importantly, the prepared TSCM cells were able to exist long and reconstitute other T cell subsets in vivo, eradicating the E007 cells effectively after transferred into the LCL burden mice. This protocol was able to prepare allogeneic antigen-specific CD8+ TSCM cells from human PBLs. The prepared TSCM showed the properties of stem cells and T memory cells. This study provided a reference method for generation of antigen-specific TSCM for T cell adoptive immunotherapy.