The identification and functional analysis of CD8+PD-1+CD161+ T cells in hepatocellular carcinoma

Immunotherapy is a powerful therapeutic strategy for end-stage hepatocellular carcinoma (HCC). It is well known that T cells, including CD8+PD-1+ T cells play important roles involving tumor development. However, their underlying phenotypic and functional differences of T cell subsets remain unclear. We constructed single-cell immune contexture involving approximate 20,000,000 immune cells from 15 pairs of HCC tumor and non-tumor adjacent tissues and 10 blood samples (including 5 of HCCs and 5 of healthy controls) by mass cytometry. scRNA-seq and functional analysis were applied to explore the function of cells. Multi-color fluorescence staining and tissue microarrays were used to identify the pathological distribution of CD8+PD-1+CD161+/- T cells and their potential clinical implication. The differential distribution of CD8+ T cells subgroups was identified in tumor and non-tumor adjacent tissues. The proportion of CD8+PD1+CD161+ T cells was significantly decreased in tumor tissues, whereas the ratio of CD8+PD1+CD161- T cells was much lower in non-tumor adjacent tissues. Diffusion analysis revealed the distinct evolutionary trajectory of CD8+PD1+CD161+ and CD8+PD1+CD161- T cells. scRNA-seq and functional study further revealed the stronger immune activity of CD8+PD1+CD161+ T cells independent of MHC class II molecules expression. Interestingly, similar change of the ratio of CD8+CD161+/ CD8+CD161- T cells was also found in peripheral blood samples collected from HCC cases, indicating their potential usage clinically. We here identified different distribution, function and trajectory of CD8+PD-1+CD161+ and CD8+PD-1+CD161- T cells in tumor lesions, which provided new insights for the heterogeneity of immune environment in HCCs and also shed light on the potential target for immunotherapy.

免疫治疗作为一种强有力的治疗策略，在晚期肝细胞癌（HCC）的治疗中发挥着至关重要的作用。众所周知，T细胞，尤其是CD8+PD-1+ T细胞，在肿瘤的发生发展中扮演着至关重要的角色。然而，T细胞亚群之间的表型与功能差异的深层机制尚不明确。本研究通过流式细胞术构建了包含约2000万免疫细胞的单细胞免疫微环境，这些细胞来源于15对HCC肿瘤及其邻近非肿瘤组织以及10份血液样本（包括5份HCC样本和5份健康对照组）。通过单细胞RNA测序（scRNA-seq）和功能分析，本研究旨在探索细胞的功能。采用多色荧光染色和组织微阵列技术，以识别CD8+PD-1+CD161+/- T细胞在病理组织分布及其潜在的临床意义。在肿瘤和非肿瘤邻近组织中，CD8+ T细胞亚群的差异分布得以识别。肿瘤组织中CD8+PD1+CD161+ T细胞的比率显著降低，而非肿瘤邻近组织中CD8+PD1+CD161- T细胞的比率则相对较低。扩散分析揭示了CD8+PD1+CD161+和CD8+PD1+CD161- T细胞的独特进化轨迹。scRNA-seq和功能研究进一步揭示了CD8+PD1+CD161+ T细胞在MHC II分子表达独立的情况下展现出更强的免疫活性。值得注意的是，外周血样本中CD8+CD161+/CD8+CD161- T细胞比率的相似变化也得以发现，这表明其在临床应用中的潜在价值。本研究揭示了CD8+PD-1+CD161+和CD8+PD-1+CD161- T细胞在肿瘤病变中的不同分布、功能和轨迹，为HCC免疫环境的异质性提供了新的见解，并为免疫治疗提供了潜在的治疗靶点。