Immunoglobulin (Ig) VSTM2A (V-set and transmembrane domain containing 2A) is a top-ranked secretory protein frequently silenced during colorectal carcinogenesis; however, its role in immune modulation remains largely unknown. Bioinformatic and immunohistochemistry analysis of human colorectal specimens and Vstm2a+/− knockout mice indicated that VSTM2A positively correlated with CD8a and immune infiltration in both physiological and pathological conditions. We then utilized liquid chromatography-mass spectrometry to pinpoint programmed death ligand 1 (PD-L1) as a membrane receptor of VSTM2A. A series of in vitro biochemistry assays further revealed the binding pattern and kinetics between VSTM2A and PD-L1 proteins through their IgV domains at a dissociation constant of 0.7–2.5 nM. Recombinant VSTM2A protein inhibited the PD-1/PD-L1 interaction and induced NFAT response element (RE) luciferase activity dose dependently. Furthermore, interleukin (IL)-2 production from DO11.10 T cells upon co-culture with mouse non-T splenocytes was upregulated in the presence of VSTM2A conditioned medium. Finally, tumor killing assay and ex vivo data from human peripheral blood mononuclear cells and autologous dendritic cell-T cell co-culture demonstrated that VSTM2A significantly enhanced immune activation via the release of granzyme B and interferon (IFN)-γ cytokines. In conclusion, our study demonstrates the tumor-extrinsic role of VSTM2A in sterically blocking the PD-L1/PD-1 interaction at a picomole to nanomole affinity, which leads to the enhanced anti-tumor effect of cytotoxic T cells.

中文翻译：

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VSTM2A 通过拮抗 PD-L1/PD-1 相互作用来逆转结直肠癌中的免疫抑制


免疫球蛋白 （Ig） VSTM2A（V 组和包含 2A 的跨膜结构域）是一种排名靠前的分泌蛋白，在结直肠癌发生过程中经常沉默;然而，它在免疫调节中的作用在很大程度上仍然未知。人结直肠标本和 Vstm2a+/- 敲除小鼠的生物信息学和免疫组化分析表明，VSTM2A 在生理和病理条件下与 CD8a 和免疫浸润呈正相关。然后，我们利用液相色谱-质谱法确定程序性死亡配体 1 （PD-L1） 是 VSTM2A 的膜受体。一系列 体外生化分析进一步揭示了 VSTM2A 和 PD-L1 蛋白之间通过其 IgV 结构域（解离常数为 0.7–2.5 nM）的结合模式和动力学。重组 VSTM2A 蛋白抑制 PD-1/PD-L1 相互作用，并剂量依赖性地诱导 NFAT 反应元件 （RE） 荧光素酶活性。此外，在 VSTM2A 条件培养基存在下，与小鼠非 T 脾细胞共培养后 DO11.10 T 细胞产生的白细胞介素 （IL）-2 上调。最后，来自人外周血单核细胞和自体树突状细胞-T 细胞共培养的肿瘤杀伤测定和离 体数据表明，VSTM2A 通过释放颗粒酶 B 和干扰素 （IFN） γ细胞因子显着增强免疫激活。总之，我们的研究表明 VSTM2A 在以皮摩尔到纳摩尔亲和力空间阻断 PD-L1/PD-1 相互作用中的肿瘤外源性作用，这导致细胞毒性 T 细胞的抗肿瘤作用增强。