van Lummel Menno, van Veelen Peter A, de Ru Arnoud H, Janssen George M C, Pool Jos, Laban Sandra, Joosten Antoinette M, Nikolic Tatjana, Drijfhout Jan W, Mearin M Luisa, Aanstoot Henk J, Peakman Mark, Roep Bart O
Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, 2300 RC Leiden, the Netherlands;
Department of Pediatrics, Leiden University Medical Center, 2300 RC Leiden, the Netherlands;
J Immunol. 2016 Apr 15;196(8):3253-63. doi: 10.4049/jimmunol.1501282. Epub 2016 Mar 4.
Identifying T cell epitopes of islet autoantigens is important for understanding type 1 diabetes (T1D) immunopathogenesis and to design immune monitoring and intervention strategies in relationship to disease progression. Naturally processed T cell epitopes have been discovered by elution from HLA-DR4 of pulsed B lymphocytes. The designated professional APC directing immune responses is the dendritic cell (DC). To identify naturally processed epitopes, monocyte-derived DC were pulsed with preproinsulin (PPI), glutamic acid decarboxylase (65-kDa isoform; GAD65), and insulinoma-associated Ag-2 (IA-2), and peptides were eluted of HLA-DR3 and -DR4, which are associated with highest risk for T1D development. Proteome analysis confirmed uptake and processing of islet Ags by DC. PPI peptides generated by DC differed from those processed by B lymphocytes; PPI signal-sequence peptides were eluted from HLA-DR4 and -DR3/4 that proved completely identical to a primary target epitope of diabetogenic HLA-A2-restricted CD8 T cells. HLA-DR4 binding was confirmed. GAD65 peptides, eluted from HLA-DR3 and -DR4, encompassed two core regions overlapping the two most immunodominant and frequently studied CD4 T cell targets. GAD65 peptides bound to HLA-DR3. Strikingly, the IA-2 ligandome of HLA-DR was exclusively generated from the extracellular part of IA-2, whereas most previous immune studies have focused on intracellular IA-2 epitopes. The newly identified IA-2 peptides bound to HLA-DR3 and -DR4. Differential T cell responses were detected against the newly identified IA-2 epitopes in blood from T1D patients. The core regions to which DC may draw attention from autoreactive T cells are largely distinct and more restricted than are those of B cells. GAD65 peptides presented by DC focus on highly immunogenic T cell targets, whereas HLA-DR-binding peptides derived from IA-2 are distinct from the target regions of IA-2 autoantibodies.
鉴定胰岛自身抗原的T细胞表位对于理解1型糖尿病（T1D）的免疫发病机制以及设计与疾病进展相关的免疫监测和干预策略至关重要。通过从脉冲B淋巴细胞的HLA-DR4上洗脱已发现了天然加工的T细胞表位。指导免疫反应的指定专业抗原呈递细胞（APC）是树突状细胞（DC）。为了鉴定天然加工的表位，用前胰岛素原（PPI）、谷氨酸脱羧酶（65 kDa异构体；GAD65）和胰岛瘤相关抗原2（IA-2）对单核细胞衍生的DC进行脉冲处理，然后从与T1D发生风险最高相关的HLA-DR3和-DR4上洗脱肽段。蛋白质组分析证实了DC对胰岛抗原的摄取和加工。DC产生的PPI肽段与B淋巴细胞加工产生的不同；从HLA-DR4和-DR3/4上洗脱的PPI信号序列肽段被证明与致糖尿病的HLA-A2限制性CD8 T细胞的主要靶表位完全相同。证实了其与HLA-DR4的结合。从HLA-DR3和-DR4上洗脱的GAD65肽段包含两个核心区域，与两个最具免疫优势且研究频繁的CD4 T细胞靶标重叠。GAD65肽段与HLA-DR3结合。引人注目的是，HLA-DR的IA-2配体组完全由IA-2的细胞外部分产生，而之前大多数免疫研究都集中在细胞内IA-2表位上。新鉴定的IA-2肽段与HLA-DR3和-DR4结合。在T1D患者血液中检测到针对新鉴定的IA-2表位的不同T细胞反应。DC可能引起自身反应性T细胞关注的核心区域与B细胞的核心区域在很大程度上不同且更具局限性。DC呈递的GAD65肽段聚焦于高度免疫原性的T细胞靶标，而源自IA-2的HLA-DR结合肽段与IA-2自身抗体的靶区域不同。
