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

项目总结： 中枢和外周耐受性受损导致1型糖尿病(T1D)；B细胞抗原反应性T细胞逃逸 胸腺的负选择和淋巴结的调节/缺失。专业抗原的作用- 呈递细胞和调节性T细胞在维持外周耐受中的作用已经得到证实 目前正在评估自身免疫性疾病的治疗，包括T1D。相反，非专业人士 抗原提呈细胞，如淋巴结基质细胞，是更稳定的耐受细胞，但其潜在的 T1D的治疗耐受诱导还没有被测试。 成纤维细胞网状细胞(FRC)是支持和重塑淋巴的淋巴结基质细胞的一种亚型 结节和FRC网状结构的完整性是获得性免疫的关键。在转基因小鼠中，表达和 FRC将模型抗原提呈给特定的T细胞，诱导其增殖，然后缺失和 淋巴结基质细胞上MHC-II的缺失与自身免疫的进行性发展有关。 因此，对于组织特异性抗原的外周过度表达，包括那些未表达的抗原，FRC工程 在胸腺中，可用于自身免疫治疗中的耐受诱导。我们展示了 主要b细胞抗原胰岛素的表达、FRC相对频率和FRC网状重塑 在T1D中，性能降低。因此，开发FRCs促进抗原特异性耐受的能力 对于T1D的治疗，FRC需要被设计成：(I)通过治疗增加它们的频率 移植和(Ii)过表达与疾病相关的抗原。 我们最近开发了基因工程方法来过度表达与T1D相关的抗原，包括杂交 FRC中的多肽和组织工程学方法构建三维FRC网状结构， 概述淋巴结旁皮质的FRC组织。高表达b细胞的工程化FRC网状细胞 抗原和共刺激分子表达缺失的小鼠移植以募集抗原特异性T细胞和 在体外，工程化的FRC促进特定T细胞的结合、无能和调节性T细胞的扩张， 在体内可促进抗原特异性耐受。因此，在目标1.1中，我们将测试治疗应用 我们的基因工程FRC网状细胞在小鼠T1D的体内外耐受诱导及其机制 具有选定的疾病相关抗原的模型。对于临床翻译，在Aim 1.2中，我们将评估 人淋巴结中表达b细胞抗原的FRCs构建的网状细胞可与人T细胞结合 为抗原特异性而设计，这对我们的方法的临床翻译很重要。外围 淋巴结基质细胞向自身反应性T细胞表达和递呈组织特异性抗原 避免淋巴结中胸腺的缺失可能有助于外周耐受。在目标2中，我们将测试 B细胞抗原表达和提呈在B细胞抗原耐受性中的作用 T1D小鼠模型中的自身反应性CD8 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.