DataSheet_2_Mapping T Cell Responses to Native and Neo-Islet Antigen Epitopes in at Risk and Type 1 Diabetes Subjects.pdf

AimsRecent studies highlight the potentially important role of neoepitopes in breaking immune tolerance in type 1 diabetes. T cell reactivity to these neoepitopes has been reported, but how this response compares quantitatively and phenotypically with previous reports on native epitopes is not known. Thus, an understanding of the relationship between native and neoepitopes and their role as tolerance breakers or disease drivers in type 1 diabetes is required. We set out to compare T cell reactivity and phenotype against a panel of neo- and native islet autoantigenic epitopes to examine how this relates to stages of type 1 diabetes development. MethodsFifty-four subjects comprising patients with T1D, and autoantibody-positive unaffected family members were tested against a panel of neo- and native epitopes by ELISPOT (IFN-γ, IL-10, and IL-17). A further subset of two patients was analyzed by Single Cell Immune Profiling (RNAseq and TCR α/β) after stimulation with pools of native and neoepitope peptides. ResultsT cell responses to native and neoepitopes were present in patients with type 1 diabetes and at-risk subjects, and overall, there were no significant differences in the frequency, magnitude, or phenotype between the two sets of peptide stimuli. Single cell RNAseq on responder T cells revealed a similar profile in T1D patients stimulated with either neo- or native epitopes. A pro-inflammatory gene expression profile (TNF-α, IFN-γ) was dominant in both native and neoepitope stimulated T cells. TCRs with identical clonotypes were found in T cell responding to both native and neoepitopes. Conclusion/InterpretationThese data suggest that in peripheral blood, T cell responses to both native and neoepitopes are similar in terms of frequency and phenotype in patients with type 1 diabetes and high-risk unaffected family members. Furthermore, using a combination of transcriptomic and clonotypic analyses, albeit using a limited panel of peptides, we show that neoepitopes are comparable to native epitopes currently in use for immune-monitoring studies.## 研究目的 近期研究显示，新表位（neoepitope）在打破1型糖尿病的免疫耐受过程中可能发挥关键作用。已有研究报道了T细胞对这类新表位的反应性，但目前尚不明确该反应与既往针对天然表位（native epitope）的研究在定量特征与表型层面存在何种差异。因此，亟需阐明天然表位与新表位之间的关联，以及二者作为免疫耐受破坏因子或疾病驱动因子在1型糖尿病中的作用。本研究旨在通过一组胰岛自身抗原表位，对比T细胞对新表位与天然表位的反应性及表型特征，进而探究其与1型糖尿病发病进程的关联。 ## 研究方法 本研究纳入54名受试者，包括1型糖尿病患者与自身抗体阳性的未患病家族成员。采用酶联免疫斑点试验（ELISPOT，检测干扰素-γ（IFN-γ）、白细胞介素-10（IL-10）与白细胞介素-17（IL-17）），对受试者样本进行新表位与天然表位的检测。此外，选取2名患者的亚组样本，在经天然表位与新表位肽库刺激后，通过单细胞免疫分析（Single Cell Immune Profiling，包含RNA测序（RNA-seq）与T细胞受体α/β链（TCR α/β）分析）进行进一步研究。 ## 研究结果 在1型糖尿病患者与高危受试者中，均可检测到T细胞对天然表位与新表位的应答；整体而言，两类肽刺激下的T细胞应答频率、强度与表型均无显著差异。对应答性T细胞的单细胞RNA测序分析显示，经新表位或天然表位刺激的1型糖尿病患者T细胞具有相似的转录谱。两类表位刺激的T细胞均以促炎基因表达谱（肿瘤坏死因子-α（TNF-α）、干扰素-γ（IFN-γ））为主。同时，在针对天然表位与新表位的应答T细胞中，均发现了具有相同克隆型（clonotype）的T细胞受体。 ## 结论与解读 本研究数据表明，在外周血中，1型糖尿病患者与高危未患病家族成员的T细胞对天然表位与新表位的应答在频率与表型层面均较为相似。此外，尽管本研究使用的肽组范围有限，但结合转录组学与克隆型分析，我们证实新表位与当前用于免疫监测研究的天然表位具有可比性。