Abstract：
Mesenchymal stromal cells (MSCs) have been extensively used in the clinic due to their exquisite tissue repair capacity. However, they also hold promise in the field of cellular vaccination as they can behave as conditional antigen presenting cells in response to interferon (IFN)-gamma treatment under a specific treatment regimen. This suggests that the immune function of MSCs can be pharmacologically modulated. Given the capacity of the agonist pyrimido-indole derivative UM171a to trigger the expression of various antigen presentation-related genes in human hematopoietic progenitor cells, we explored the potential use of UM171a as a means to pharmacologically instill and/or promote antigen presentation by MSCs.
Besides completing a series of flow-cytometry-based phenotypic analyses, several functional antigen presentation assays were conducted using the SIINFEKL-specific T-cell clone B3Z. Anti-oxidants and electron transport chain inhibitors were also used to decipher UM171a\'s mode of action in MSCs. Finally, the potency of UM171a-treated MSCs was evaluated in the context of therapeutic vaccination using immunocompetent C57BL/6 mice with pre-established syngeneic EG.7T-cell lymphoma.
Treatment of MSCs with UM171a triggered potent increase in H2-Kb cell surface levels along with the acquisition of antigen cross-presentation abilities. Mechanistically, such effects occurred in response to UM171a-mediated production of mitochondrial-derived reactive oxygen species as their neutralization using anti-oxidants or Antimycin-A mitigated MSCs\' ability to cross-present antigens. Processing and presentation of the immunogenic ovalbumin-derived SIINFEKL peptide was caused by de novo expression of the Psmb8 gene in response to UM171a-triggered oxidative stress. When evaluated for their anti-tumoral properties in the context of therapeutic vaccination, UM171a-treated MSC administration to immunocompetent mice with pre-established T-cell lymphoma controlled tumor growth resulting in 40% survival without the need of additional supportive therapy and/or standard-of-care.
Altogether, our findings reveal a new immune-related function for UM171a and clearly allude to a direct link between UM171a-mediated ROS induction and antigen cross-presentation by MSCs. The fact that UM171a treatment modulates MSCs to become antigen-presenting cells without the use of IFN-gamma opens-up a new line of investigation to search for additional agents capable of converting immune-suppressive MSCs to a cellular tool easily adaptable to vaccination.
Besides completing a series of flow-cytometry-based phenotypic analyses, several functional antigen presentation assays were conducted using the SIINFEKL-specific T-cell clone B3Z. Anti-oxidants and electron transport chain inhibitors were also used to decipher UM171a\'s mode of action in MSCs. Finally, the potency of UM171a-treated MSCs was evaluated in the context of therapeutic vaccination using immunocompetent C57BL/6 mice with pre-established syngeneic EG.7T-cell lymphoma.
Treatment of MSCs with UM171a triggered potent increase in H2-Kb cell surface levels along with the acquisition of antigen cross-presentation abilities. Mechanistically, such effects occurred in response to UM171a-mediated production of mitochondrial-derived reactive oxygen species as their neutralization using anti-oxidants or Antimycin-A mitigated MSCs\' ability to cross-present antigens. Processing and presentation of the immunogenic ovalbumin-derived SIINFEKL peptide was caused by de novo expression of the Psmb8 gene in response to UM171a-triggered oxidative stress. When evaluated for their anti-tumoral properties in the context of therapeutic vaccination, UM171a-treated MSC administration to immunocompetent mice with pre-established T-cell lymphoma controlled tumor growth resulting in 40% survival without the need of additional supportive therapy and/or standard-of-care.
Altogether, our findings reveal a new immune-related function for UM171a and clearly allude to a direct link between UM171a-mediated ROS induction and antigen cross-presentation by MSCs. The fact that UM171a treatment modulates MSCs to become antigen-presenting cells without the use of IFN-gamma opens-up a new line of investigation to search for additional agents capable of converting immune-suppressive MSCs to a cellular tool easily adaptable to vaccination.
摘要：
间充质基质细胞（MSCs）由于其良好的组织修复能力，已被广泛用于临床。然而,它们在细胞疫苗接种领域也有希望,因为在特定的治疗方案下,它们可以表现为响应干扰素(IFN)-γ治疗的条件抗原呈递细胞.这表明MSC的免疫功能可以在药理学上调节。鉴于激动剂嘧啶吲哚衍生物UM171a在人造血祖细胞中触发各种抗原呈递相关基因表达的能力，我们探讨了UM171a作为一种药理学方法在MSCs中导入和/或促进抗原呈递的潜在用途.
除了完成一系列基于流式细胞术的表型分析，使用SIINFEKL特异性T细胞克隆B3Z进行了几种功能性抗原呈递测定.抗氧化剂和电子传递链抑制剂也用于破译UM171a在MSCs中的作用模式。最后,使用免疫活性C57BL/6小鼠预先建立的同基因EG.7T细胞淋巴瘤,在治疗性疫苗接种的背景下评估了UM171a治疗的MSCs的效力.
用UM171a处理MSC引发H2-Kb细胞表面水平的有效增加以及抗原交叉呈递能力的获得。机械上,这种效应发生在对UM171a介导的线粒体衍生活性氧产生的反应中,因为它们使用抗氧化剂或抗霉素-A中和作用减轻了MSCs交叉呈递抗原的能力.免疫原性卵清蛋白来源的SIINFEKL肽的加工和呈递是由响应于UM171a触发的氧化应激的Psmb8基因的从头表达引起的。当在治疗性疫苗接种的背景下评估它们的抗肿瘤特性时，UM171a处理的MSC施用至具有预先建立的T细胞淋巴瘤的免疫活性小鼠,控制肿瘤生长,导致40%存活率,而不需要额外的支持疗法和/或标准护理。
总之,我们的发现揭示了UM171a的一种新的免疫相关功能,并清楚地暗示了UM171a介导的ROS诱导与MSCs的抗原交叉呈递之间的直接联系.UM171a处理在不使用IFN-γ的情况下调节MSC成为抗原呈递细胞的事实开辟了一条新的研究路线,以寻找能够将免疫抑制MSC转化为易于适应疫苗接种的细胞工具的其他试剂。
除了完成一系列基于流式细胞术的表型分析，使用SIINFEKL特异性T细胞克隆B3Z进行了几种功能性抗原呈递测定.抗氧化剂和电子传递链抑制剂也用于破译UM171a在MSCs中的作用模式。最后,使用免疫活性C57BL/6小鼠预先建立的同基因EG.7T细胞淋巴瘤,在治疗性疫苗接种的背景下评估了UM171a治疗的MSCs的效力.
用UM171a处理MSC引发H2-Kb细胞表面水平的有效增加以及抗原交叉呈递能力的获得。机械上,这种效应发生在对UM171a介导的线粒体衍生活性氧产生的反应中,因为它们使用抗氧化剂或抗霉素-A中和作用减轻了MSCs交叉呈递抗原的能力.免疫原性卵清蛋白来源的SIINFEKL肽的加工和呈递是由响应于UM171a触发的氧化应激的Psmb8基因的从头表达引起的。当在治疗性疫苗接种的背景下评估它们的抗肿瘤特性时，UM171a处理的MSC施用至具有预先建立的T细胞淋巴瘤的免疫活性小鼠,控制肿瘤生长,导致40%存活率,而不需要额外的支持疗法和/或标准护理。
总之,我们的发现揭示了UM171a的一种新的免疫相关功能,并清楚地暗示了UM171a介导的ROS诱导与MSCs的抗原交叉呈递之间的直接联系.UM171a处理在不使用IFN-γ的情况下调节MSC成为抗原呈递细胞的事实开辟了一条新的研究路线,以寻找能够将免疫抑制MSC转化为易于适应疫苗接种的细胞工具的其他试剂。
