Abstract：
OBJECTIVE: Intestinal epithelial cell (IEC) damage is a hallmark of celiac disease (CeD); however, its role in gluten-dependent T-cell activation is unknown. We investigated IEC-gluten-T cell interactions in organoid monolayers expressing human MHC class II (HLA-DQ2.5), which facilitates gluten antigen recognition by CD4+ T cells in CeD.
METHODS: Epithelial MHC class II (MHCII) was determined in active and treated CeD, and in non-immunized and gluten-immunized DR3-DQ2.5 transgenic mice, lacking mouse MHCII molecules. Organoid monolayers from DR3-DQ2.5 mice were treated with or without IFN-γ, and MHCII expression was evaluated by flow cytometry. Organoid monolayers and CD4+ T cell co-cultures were incubated with gluten, pre-digested, or not by elastase-producing Pseudomonas aeruginosa or its lasB mutant. T cell function was assessed based on proliferation, expression of activation markers, and cytokine release in the co-culture supernatants.
RESULTS: Active CeD patients and gluten-immunized DR3-DQ2.5 mice demonstrated epithelial MHCII expression. Organoid monolayers derived from gluten-immunized DR3-DQ2.5 mice expressed MHCII, which was upregulated by IFN-γ. In organoid monolayer-T cell co-cultures, gluten increased the proliferation of CD4+ T cells, expression of T cell activation markers, and the release of IL-2, IFN-γ, and IL-15 in co-culture supernatants. Gluten metabolized by P. aeruginosa, but not the lasB mutant, enhanced CD4+ T cell proliferation and activation.
CONCLUSIONS: Gluten antigens are efficiently presented by MHCII-expressing IECs, resulting in the activation of gluten-specific CD4+ T cells, which is enhanced by gluten pre-digestion with microbial elastase. Therapeutics directed at IECs may offer a novel approach for modulating both adaptive and innate immunity in CeD patients.
METHODS: Epithelial MHC class II (MHCII) was determined in active and treated CeD, and in non-immunized and gluten-immunized DR3-DQ2.5 transgenic mice, lacking mouse MHCII molecules. Organoid monolayers from DR3-DQ2.5 mice were treated with or without IFN-γ, and MHCII expression was evaluated by flow cytometry. Organoid monolayers and CD4+ T cell co-cultures were incubated with gluten, pre-digested, or not by elastase-producing Pseudomonas aeruginosa or its lasB mutant. T cell function was assessed based on proliferation, expression of activation markers, and cytokine release in the co-culture supernatants.
RESULTS: Active CeD patients and gluten-immunized DR3-DQ2.5 mice demonstrated epithelial MHCII expression. Organoid monolayers derived from gluten-immunized DR3-DQ2.5 mice expressed MHCII, which was upregulated by IFN-γ. In organoid monolayer-T cell co-cultures, gluten increased the proliferation of CD4+ T cells, expression of T cell activation markers, and the release of IL-2, IFN-γ, and IL-15 in co-culture supernatants. Gluten metabolized by P. aeruginosa, but not the lasB mutant, enhanced CD4+ T cell proliferation and activation.
CONCLUSIONS: Gluten antigens are efficiently presented by MHCII-expressing IECs, resulting in the activation of gluten-specific CD4+ T cells, which is enhanced by gluten pre-digestion with microbial elastase. Therapeutics directed at IECs may offer a novel approach for modulating both adaptive and innate immunity in CeD patients.
摘要：
目的：肠上皮细胞（IEC）损伤是乳糜泻（CeD）的标志；然而，其在谷蛋白依赖性T细胞活化中的作用尚不清楚.我们研究了表达人MHCII类（HLA-DQ2.5）的类器官单层中的IEC-谷蛋白-T细胞相互作用，这有助于CeD中CD4+T细胞对谷蛋白抗原识别。
方法：在活性和治疗的CeD中测定上皮MHCII类（MHCII），在非免疫和谷蛋白免疫的DR3-DQ2.5转基因小鼠中，缺乏小鼠MHCII分子。用或不用IFN-γ处理DR3-DQ2.5小鼠的类器官单层，和MHCII表达通过流式细胞术评估。类器官单层和CD4+T细胞共培养物与谷蛋白一起孵育，预消化，或不产生弹性蛋白酶的铜绿假单胞菌或其lasB突变体。T细胞功能基于增殖进行评估，激活标记的表达，和共培养上清液中的细胞因子释放。
结果：活性CeD患者和谷蛋白免疫的DR3-DQ2.5小鼠表现出上皮MHCII表达。来自谷蛋白免疫DR3-DQ2.5小鼠的类器官单层表达MHCII，由IFN-γ上调。在类器官单层T细胞共培养中，麸质增加了CD4+T细胞的增殖，T细胞活化标志物的表达，和IL-2，IFN-γ的释放，和IL-15在共培养上清液中。由铜绿假单胞菌代谢的麸质，但不是lasB突变体,增强CD4+T细胞增殖和活化。
结论：表达MHCII的IECs能有效地呈递面筋抗原，导致谷蛋白特异性CD4+T细胞的激活，通过用微生物弹性蛋白酶预消化麸质来增强。针对IECs的治疗学可以提供一种新的方法来调节CeD患者的适应性和先天免疫。
方法：在活性和治疗的CeD中测定上皮MHCII类（MHCII），在非免疫和谷蛋白免疫的DR3-DQ2.5转基因小鼠中，缺乏小鼠MHCII分子。用或不用IFN-γ处理DR3-DQ2.5小鼠的类器官单层，和MHCII表达通过流式细胞术评估。类器官单层和CD4+T细胞共培养物与谷蛋白一起孵育，预消化，或不产生弹性蛋白酶的铜绿假单胞菌或其lasB突变体。T细胞功能基于增殖进行评估，激活标记的表达，和共培养上清液中的细胞因子释放。
结果：活性CeD患者和谷蛋白免疫的DR3-DQ2.5小鼠表现出上皮MHCII表达。来自谷蛋白免疫DR3-DQ2.5小鼠的类器官单层表达MHCII，由IFN-γ上调。在类器官单层T细胞共培养中，麸质增加了CD4+T细胞的增殖，T细胞活化标志物的表达，和IL-2，IFN-γ的释放，和IL-15在共培养上清液中。由铜绿假单胞菌代谢的麸质，但不是lasB突变体,增强CD4+T细胞增殖和活化。
结论：表达MHCII的IECs能有效地呈递面筋抗原，导致谷蛋白特异性CD4+T细胞的激活，通过用微生物弹性蛋白酶预消化麸质来增强。针对IECs的治疗学可以提供一种新的方法来调节CeD患者的适应性和先天免疫。
