Unraveling the tolerogenic potential of lymph node fibroblastic reticular networks in autoimmune diabetes

项目摘要： 中枢和外周耐受性受损导致1型糖尿病（T1 D）; b细胞抗原反应性T细胞逃逸 胸腺中的负选择和淋巴结中的调节/缺失。专业抗原的作用- 已经证明了提呈细胞和调节性T细胞在维持外周耐受中的作用， 目前正在评估用于治疗自身免疫性疾病，包括T1 D。相反，非专业人士 抗原呈递细胞如淋巴结基质细胞是更稳定的致耐受性细胞，但它们的免疫耐受性的潜力是， T1 D中的治疗耐受性诱导尚未被测试。 成纤维细胞网状细胞是淋巴结基质细胞的一种亚型，支持和重塑淋巴 节点和FRC网状结构的完整性对于适应性免疫至关重要。在转基因小鼠中， 通过FRC向特异性T细胞呈递模型抗原诱导其增殖，然后缺失， 淋巴结基质细胞上MHC-II的缺乏与自身免疫的进行性发展相关。 因此，针对组织特异性抗原的外周过表达的FRC工程化，包括那些不表达的抗原， 在胸腺中，可用于自身免疫治疗中的耐受诱导。我们展示 胰岛素的表达，一种主要的b细胞抗原，相对FRC频率和FRC网状重塑 在T1 D中性能下降。因此，为了利用FRC促进抗原特异性耐受的能力， 对于T1 D的治疗性治疗，FRC需要被工程化以（i）通过治疗性治疗增加它们的频率， 移植和（ii）过表达疾病相关抗原。 我们最近开发了基因工程方法来过表达T1 D相关抗原，包括杂交 肽，在FRC和组织工程方法中制造三维FRC网状结构， 在副皮质淋巴结中重现FRC组织。过表达b细胞的工程化FRC网状细胞 抗原和缺乏共刺激分子表达的小鼠移植物以募集抗原特异性T细胞， 在体外，工程化的FRC促进特异性T细胞的接合、无反应性和调节性T细胞扩增， 其可促进体内抗原特异性耐受。因此，在目标1.1中，我们将测试治疗应用 我们的工程化FRC网状物在小鼠T1 D中的体外和体内耐受性诱导和机制 模型与选定的疾病相关抗原。对于临床翻译，在目标1.2中，我们将评估是否 用来自人类淋巴结的表达b细胞抗原的FRC工程化的网状物可以接合人类T细胞 针对抗原特异性进行工程化，这对于我们的方法的临床转化很重要。外围 淋巴结基质细胞向自身反应性T细胞表达和呈递组织特异性抗原 淋巴结中的逃逸胸腺缺失可能有助于外周耐受。在目标2中，我们将测试 淋巴结FRC的b细胞抗原表达和呈递对b细胞抗原耐受性的贡献 T1 D小鼠模型中的自身反应性CD 8 T细胞。

Project Summary: Impaired central and peripheral tolerance cause type-1 diabetes (T1D); b-cell antigen-reactive T cells escape negative selection in the thymus and regulation/deletion in lymph nodes. The role of professional antigen- presenting cells and regulatory T cells in maintaining peripheral tolerance has been demonstrated and is currently being evaluated for therapy of autoimmune diseases, including T1D. Conversely, non-professional antigen-presenting cells like lymph node stromal cells are more stable tolerogenic cells but their potential for therapeutic tolerance induction in T1D has not been tested yet. Fibroblastic reticular cells (FRCs) are a subtype of lymph node stromal cells that support and remodel the lymph node and the integrity of FRC reticula is critical for adaptive immunity. In transgenic mice, expression and presentation of model antigens by FRCs to specific T cells induced their proliferation followed by deletion and absence of MHC-II on lymph node stromal cells was associated with progressive development of autoimmunity. Thus, FRC engineering for peripheral overexpression of tissue-specific antigens, including those not expressed in the thymus, may be exploited for tolerance induction in the therapeutic treatment of autoimmunity. We showed that expression of insulin, a major b-cell antigen, the relative FRC frequency and the FRC reticular remodeling properties are decreased in T1D. Thus, to exploit the capability of FRCs to promote antigen-specific tolerance for therapeutic treatment of T1D, FRCs need to be engineered to (i) increase their frequency by therapeutic transplantation and (ii) overexpress disease-relevant antigens. We recently developed genetic engineering approaches to overexpress T1D-relevant antigens, including hybrid peptides, in FRCs and tissue engineering approaches to fabricate three-dimensional FRC reticula that recapitulate FRC organization in the lymph node paracortex. Engineered FRC reticula overexpressing b-cell antigens and lacking expression of costimulatory molecules engraft in mice to recruit antigen-specific T cells and in vitro, engineered FRCs promote engagement of specific T cells, anergy, and regulatory T cell expansion, which could promote antigen-specific tolerance in vivo. Thus, in aim 1.1, we will test the therapeutic application of our engineered FRC reticula for tolerance induction and mechanisms in vitro and in vivo in a murine T1D model with selected disease-relevant antigens. For clinical translation, in aim 1.2, we will evaluate whether reticula engineered with b-cell antigen-expressing FRCs from human lymph nodes can engage human T cells engineered for antigen specificity, which is important for clinical translation of our approach. Peripheral expression and presentation of tissue-specific antigens by lymph node stromal cells to autoreactive T cells escaping thymic deletion in lymph nodes could contribute to peripheral tolerance. In aim 2, we will test the contribution of b-cell antigen expression and presentation by lymph node FRCs on tolerization of b-cell antigen autoreactive CD8 T cells in a mouse model of T1D.