J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL, United States.
Barbara Davis Center for Diabetes, Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, CO, United States.
Front Endocrinol (Lausanne). 2022 Nov 25;13:989815. doi: 10.3389/fendo.2022.989815. eCollection 2022.
There is a critical need for therapeutic approaches that combine renewable sources of replacement beta cells with localized immunomodulation to counter recurrence of autoimmunity in type 1 diabetes (T1D). However, there are few examples of animal models to study such approaches that incorporate spontaneous autoimmunity directed against human beta cells rather than allogenic rejection. Here, we address this critical limitation by demonstrating rejection and survival of transplanted human stem cell-derived beta-like cells clusters (sBCs) in a fully immune competent mouse model with matching human HLA class I and spontaneous diabetes development. We engineered localized immune tolerance toward transplanted sBCs inducible cell surface overexpression of PD-L1 (iP-sBCs) with and without deletion of all HLA class I surface molecules beta-2 microglobulin knockout (iP-BKO sBCs). NOD.HLA-A2.1 mice, which lack classical murine MHC I and instead express human HLA-A02:01, underwent transplantation of 1,000 human HLA-A02:01 sBCs under the kidney capsule and were separated into HLA-A2 positive iP-sBC and HLA-class I negative iP-BKO sBC groups, each with +/- doxycycline (DOX) induced PD-L1 expression. IVIS imaging showed significantly improved graft survival in mice transplanted with PD-L1 expressing iP-sBC at day 3 post transplantation compared to controls. However, luciferase signal dropped below detection limits by day 14 for all groups in this aggressive immune competent diabetes model. Nonetheless, histological examination revealed significant numbers of surviving insulin/PD-L1 sBCs cells for DOX-treated mice at day 16 post-transplant despite extensive infiltration with high numbers of CD3 and CD45 immune cells. These results show that T cells rapidly infiltrate and attack sBC grafts in this model but that significant numbers of PD-L1 expressing sBCs manage to survive in this harsh immunological environment. This investigation represents one of the first studies recapitulating key aspects of human autoimmune diabetes to test immune tolerance approaches with renewable sources of beta cells.
治疗 1 型糖尿病（T1D）需要将可再生的β细胞替代物与局部免疫调节相结合的方法，但目前很少有动物模型能够研究针对人类β细胞的自发性自身免疫而不是同种异体排斥的此类方法。在这里，我们通过在具有匹配的人 HLA Ⅰ类和自发性糖尿病发展的完全免疫功能正常的小鼠模型中证明移植的人干细胞衍生的β样细胞簇（sBC）的排斥和存活，解决了这一关键限制。我们通过在移植的 sBC 上诱导性细胞表面过表达 PD-L1（iP-sBC），并在不删除所有 HLA Ⅰ类表面分子β-2 微球蛋白敲除（iP-BKO sBC）的情况下，实现了对移植的 sBC 的局部免疫耐受。缺乏经典的鼠 MHC I 而表达人 HLA-A02:01 的 NOD.HLA-A2.1 小鼠在肾包膜下接受了 1000 个 HLA-A02:01 sBC 的移植，并分为 HLA-A2 阳性 iP-sBC 和 HLA Ⅰ类阴性 iP-BKO sBC 组，每组均用 +/- 强力霉素（DOX）诱导 PD-L1 表达。IVIS 成像显示，与对照组相比，在移植后第 3 天移植 PD-L1 表达的 iP-sBC 的小鼠中，移植物的存活率显著提高。然而，在这种具有侵袭性免疫能力的糖尿病模型中，所有组的荧光素酶信号在第 14 天均降至检测限以下。尽管如此，在移植后第 16 天，经 DOX 处理的小鼠的组织学检查显示仍有大量存活的胰岛素/PD-L1 sBC 细胞，尽管有大量的 CD3 和 CD45 免疫细胞浸润。这些结果表明，T 细胞在该模型中迅速浸润并攻击 sBC 移植物，但大量表达 PD-L1 的 sBC 设法在这种恶劣的免疫环境中存活下来。这项研究代表了首次重现人类自身免疫性糖尿病的关键方面以测试可再生β细胞来源的免疫耐受方法的研究之一。
