Barbara Davis Center for Childhood Diabetes, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.
Immunol Res. 2013 Mar;55(1-3):91-9. doi: 10.1007/s12026-012-8368-5.
G6PC2, also known as islet-specific glucose 6-phosphatase catalytic subunit-related protein (IGRP), is a major target of autoreactive CD8(+) T cells in both diabetic human subjects and the non-obese diabetic (NOD) mouse. However, in contrast to the abundant literature regarding the CD8(+) response to this antigen, much less is known about the potential involvement of IGRP-reactive CD4(+) T cells in diabetogenesis. The single previous study that examined this question in NOD mice was based upon a candidate epitope approach and identified three I-A(g7)-restricted epitopes that each elicited spontaneous responses in these animals. However, given the known inaccuracies of MHC class II epitope prediction algorithms, we hypothesized that additional specificities might also be targeted. To address this issue, we immunized NOD mice with membranes from insect cells overexpressing full-length recombinant mouse IGRP and measured recall responses of purified CD4(+) T cells using a library of overlapping peptides encompassing the entire 355-aa primary sequence. Nine peptides representing 8 epitopes gave recall responses, only 1 of which corresponded to any of the previously reported sequences. In each case proliferation was blocked by a monoclonal antibody to I-A(g7), but not the appropriate isotype control. Consistent with a role in diabetogenesis, proliferative responses to 4 of the 9 peptides (3 epitopes) were also detected in CD4(+) T cells purified from the pancreatic draining lymph nodes of pre-diabetic female animals, but not from peripheral lymph nodes or spleens of the same animals. Intriguingly, one of the newly identified spontaneously reactive epitopes (P8 [IGRP(55-72)]) is highly conserved between mice and man, suggesting that it might also be a target of HLA-DQ8-restricted T cells in diabetic human subjects, an hypothesis that we are currently testing.
G6PC2，也称为胰岛特异性葡萄糖 6-磷酸酶催化亚基相关蛋白（IGRP），是糖尿病患者和非肥胖型糖尿病（NOD）小鼠中自身反应性 CD8+T 细胞的主要靶标。然而，与大量关于该抗原的 CD8+反应的文献相比，关于 IGRP 反应性 CD4+T 细胞在糖尿病发病机制中的潜在作用知之甚少。唯一一项在 NOD 小鼠中研究此问题的先前研究基于候选表位方法，鉴定了三个 I-A（g7）限制的表位，每个表位在这些动物中都引发了自发反应。然而，鉴于 MHC Ⅱ类表位预测算法的已知不准确性，我们假设也可能针对其他特异性。为了解决这个问题，我们用昆虫细胞过表达全长重组小鼠 IGRP 的膜免疫 NOD 小鼠，并使用涵盖整个 355-aa 原始序列的重叠肽文库测量纯化的 CD4+T 细胞的回忆反应。代表 8 个表位的 9 个肽引起回忆反应，只有 1 个与以前报道的序列相对应。在每种情况下，增殖均被针对 I-A（g7）的单克隆抗体阻断，但不是适当的同种型对照。与糖尿病发病机制相关，在来自于前糖尿病雌性动物的胰腺引流淋巴结中纯化的 CD4+T 细胞中也检测到对 9 个肽（3 个表位）中的 4 个的增殖反应，但不在同一动物的外周淋巴结或脾脏中检测到。有趣的是，新鉴定的自发反应性表位之一（P8[IGRP（55-72）]）在小鼠和人中高度保守，这表明它也可能是糖尿病患者 HLA-DQ8 限制的 T 细胞的靶标，我们目前正在测试这一假设。
