Rao Chaitra, Cater Daniel T, Roy Saptarshi, Xu Jerry, De Oliveira Andre G, Evans-Molina Carmella, Piganelli Jon D, Eizirik Decio L, Mirmira Raghavendra G, Sims Emily K
Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN, USA.
Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, Indianapolis, IN, USA.
Diabetologia. 2025 Feb;68(2):382-396. doi: 10.1007/s00125-024-06313-2. Epub 2024 Nov 7.
AIMS/HYPOTHESIS: Surviving beta cells in type 1 diabetes respond to inflammation by upregulating programmed death-ligand 1 (PD-L1) to engage immune cell programmed death protein 1 (PD-1) and limit destruction by self-reactive immune cells. Extracellular vesicles (EVs) and their cargo can serve as biomarkers of beta cell health and contribute to islet intercellular communication. We hypothesised that the inflammatory milieu of type 1 diabetes increases PD-L1 in beta cell EV cargo and that EV PD-L1 may protect beta cells against immune-mediated cell death.
Beta cell lines and human islets were treated with proinflammatory cytokines to model the proinflammatory type 1 diabetes microenvironment. EVs were isolated using ultracentrifugation or size exclusion chromatography and analysed via immunoblot, flow cytometry and ELISA. EV PD-L1 binding to PD-1 was assessed using a competitive binding assay and in vitro functional assays testing the ability of EV PD-L1 to inhibit NOD CD8 T cells. Plasma EV and soluble PD-L1 were assayed in the plasma of islet autoantibody-positive (Ab) individuals or individuals with recent-onset type 1 diabetes and compared with levels in non-diabetic control individuals.
PD-L1 protein co-localised with tetraspanin-associated proteins intracellularly and was detected on the surface of beta cell EVs. Treatment with IFN-α or IFN-γ for 24 h induced a twofold increase in EV PD-L1 cargo without a corresponding increase in the number of EVs. IFN exposure predominantly increased PD-L1 expression on the surface of beta cell EVs and beta cell EV PD-L1 showed a dose-dependent capacity to bind PD-1. Functional experiments demonstrated specific effects of beta cell EV PD-L1 to suppress proliferation and cytotoxicity of murine CD8 T cells. Plasma EV PD-L1 levels were increased in Abindividuals, particularly in those positive for a single autoantibody. Additionally, in Ab individuals or those who had type 1 diabetes, but not in control individuals, plasma EV PD-L1 positively correlated with circulating C-peptide, suggesting that higher EV PD-L1 could be protective for residual beta cell function.
CONCLUSIONS/INTERPRETATION: IFN exposure increases PD-L1 on the beta cell EV surface. Beta cell EV PD-L1 binds PD1 and inhibits CD8 T cell proliferation and cytotoxicity. Circulating EV PD-L1 is higher in Ab individuals than in control individuals. Circulating EV PD-L1 levels correlate with residual C-peptide at different stages in type 1 diabetes progression. These findings suggest that EV PD-L1 could contribute to heterogeneity in type 1 diabetes progression and residual beta cell function and raise the possibility that EV PD-L1 could be exploited as a means to inhibit immune-mediated beta cell death.
目的/假设：1型糖尿病中存活的β细胞通过上调程序性死亡配体1（PD-L1）来响应炎症，从而与免疫细胞程序性死亡蛋白1（PD-1）结合，限制自身反应性免疫细胞的破坏。细胞外囊泡（EVs）及其所载物质可作为β细胞健康的生物标志物，并有助于胰岛细胞间通讯。我们推测，1型糖尿病的炎症环境会增加β细胞EV所载物质中的PD-L1，且EV PD-L1可能保护β细胞免受免疫介导的细胞死亡。
用促炎细胞因子处理β细胞系和人胰岛，以模拟1型糖尿病的促炎微环境。通过超速离心或尺寸排阻色谱法分离EVs，并通过免疫印迹、流式细胞术和酶联免疫吸附测定法进行分析。使用竞争性结合试验评估EV PD-L1与PD-1的结合，并通过体外功能试验检测EV PD-L1抑制非肥胖糖尿病（NOD）CD8 T细胞的能力。检测胰岛自身抗体阳性（Ab）个体或近期发病的1型糖尿病个体血浆中的EV和可溶性PD-L1，并与非糖尿病对照个体的水平进行比较。
PD-L1蛋白在细胞内与四跨膜蛋白相关蛋白共定位，并在β细胞EV表面被检测到。用干扰素-α（IFN-α）或干扰素-γ（IFN-γ）处理24小时可使EV PD-L1所载物质增加两倍，而EV数量没有相应增加。IFN暴露主要增加β细胞EV表面的PD-L1表达，且β细胞EV PD-L1显示出与PD-1结合的剂量依赖性能力。功能实验证明β细胞EV PD-L1对抑制小鼠CD8 T细胞增殖和细胞毒性具有特异性作用。Ab个体的血浆EV PD-L1水平升高，尤其是那些单一自身抗体呈阳性的个体。此外，在Ab个体或患有1型糖尿病的个体中，但在对照个体中未发现，血浆EV PD-L1与循环C肽呈正相关，这表明较高的EV PD-L1可能对残余β细胞功能具有保护作用。
结论/解读：IFN暴露会增加β细胞EV表面的PD-L1。β细胞EV PD-L1与PD1结合并抑制CD8 T细胞增殖和细胞毒性。Ab个体的循环EV PD-L1高于对照个体。在1型糖尿病进展的不同阶段，循环EV PD-L1水平与残余C肽相关。这些发现表明，EV PD-L1可能导致1型糖尿病进展和残余β细胞功能的异质性，并增加了EV PD-L1可被用作抑制免疫介导的β细胞死亡手段的可能性。
