作为一种革兰氏阴性厌氧细菌，Akkermansia muciniphila (AKK) 参与许多癌症的免疫反应。我们的研究重点是AKK影响肺腺癌（LUAD）免疫逃逸的因素和分子机制。我们培养AKK细菌，制备AKK外膜蛋白Amuc_1100并构建皮下移植肿瘤小鼠模型。分别用灭活的AKK、Amuc_1100、Ruxolitinib（JAK抑制剂）和RO8191（JAK激活剂）处理A549、NCI-H1395细胞和小鼠。在 Transwell 测定中对穿透膜的 CD8 T 细胞进行计数。通过乳酸脱氢酶测定评估CD8 T细胞的毒性。应用Western blot测定JAK/STAT相关蛋白和PD-L1的表达，同时通过酶联免疫吸附测定（ELISA）评估CCL5、颗粒酶B和INF-γ的表达。通过流式细胞术测定肿瘤浸润CD8 T细胞的比例以及颗粒酶B和INF-γ的水平。 AKK 显着加速 A549 和 NCI-H1395 募集 CD8 T 细胞并增强 CD8 T 细胞毒性。从AKK纯化的Amuc_1100发挥了相同的促进作用。此外，Amuc_1100 显着抑制 PD-L1、p-STAT 和 p-JAK 表达，并增强 CCL5、颗粒酶 B 和 INF-γ 表达。 Ruxolitinib 治疗可加速 A549 和 NCI-H1395 细胞招募 CD8 T 细胞，增强 CD8 T 细胞毒性、CCL5、颗粒酶 B 和 INF-γ 表达，并抑制 PD-L1 表达。相比之下，RO8191 处理减缓了 Amuc_1100 引起的变化。动物实验表明，Amuc_1100可增加肿瘤浸润CD8 T细胞的数量，提高颗粒酶B和INF-γ的水平，并显着抑制PD-L1、p-STAT和p-JAK的表达，从而发挥抗肿瘤作用。体内抗肿瘤作用。总之，AKK外膜蛋白通过抑制JAK/STAT信号通路，促进LUAD中CD8 T细胞的募集，抑制细胞的免疫逃逸。© 2024 作者。约翰·威利 (John Wiley) 出版的《微生物生物技术》

As a Gram-negative anaerobic bacterium, Akkermansia muciniphila (AKK) participates in the immune response in many cancers. Our study focused on the factors and molecular mechanisms of AKK affecting immune escape in lung adenocarcinoma (LUAD). We cultured AKK bacteria, prepared AKK outer membrane protein Amuc_1100 and constructed a subcutaneous graft tumour mouse model. A549, NCI-H1395 cells and mice were respectively treated with inactivated AKK, Amuc_1100, Ruxolitinib (JAK inhibitor) and RO8191 (JAK activator). CD8+ T cells that penetrated the membrane were counted in the Transwell assay. The toxicity of CD8+ T cells was evaluated by lactate dehydrogenase assay. Western blot was applied to determine JAK/STAT-related protein and PD-L1 expression, whilst CCL5, granzyme B and INF-γ expression were assessed through enzyme-linked immunosorbent assay (ELISA). The proportion of tumour-infiltrating CD8+ T cells and the levels of granzyme B and INF-γ were determined by flow cytometry. AKK markedly accelerated A549 and NCI-H1395 recruiting CD8+ T cells and enhanced CD8+ T cell toxicity. Amuc_1100 purified from AKK exerted the same promoting effects. Besides, Amuc_1100 dramatically suppressed PD-L1, p-STAT and p-JAK expression and enhanced CCL5, granzyme B and INF-γ expression. Treatment with Ruxolitinib accelerated A549 and NCI-H1395 cells recruiting CD8+ T cells, enhanced CD8+ T cell toxicity, CCL5, granzyme B and INF-γ expression, and inhibited PD-L1 expression. In contrast, the RO8191 treatment slowed down the changes induced by Amuc_1100. Animal experiments showed that Amuc_1100 was found to increase the number of tumour-infiltrating CD8+ T cells, increase the levels of granzyme B and INF-γ and significantly inhibit the expression of PD-L1, p-STAT and p-JAK, which exerted an antitumour effect in vivo. In conclusion, through inhibiting the JAK/STAT signalling pathway, AKK outer membrane protein facilitated the recruitment of CD8+ T cells in LUAD and suppressed the immune escape of cells.© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.