Juvet Stephen C, Moshkelgosha Sajad, Sanderson Sharon, Hester Joanna, Wood Kathryn J, Bushell Andrew
Division of Respirology, Departments of Medicine and Immunology, Toronto Lung Transplant Program, Multiorgan Transplant Program, Toronto General Research Institute, University of Toronto and University Health Network;
Latner Thoracic Surgery Laboratories, Toronto General Research Institute, University Health Network.
J Vis Exp. 2017 Apr 19(122):55283. doi: 10.3791/55283.
The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.
测量移植受者对供体抗原的免疫反应性可能对成功减少或停用免疫抑制至关重要。混合淋巴细胞反应（MLR）、有限稀释分析和体内迟发型超敏反应（DTH）分析都已应用于这个问题，但这些方法的预测能力有限和/或存在重大实际限制，降低了它们的实用性。成像流式细胞术是一种将流式细胞术的多参数定量能力与荧光显微镜的成像能力相结合的技术。我们最近利用成像流式细胞术方法来确定能够与供体抗原呈递细胞（APC）形成成熟免疫突触的受者T细胞比例。使用一个特征明确的小鼠心脏移植模型，我们已经表明，在T-APC膜接触事件中体外免疫突触的频率强烈预测了同种异体移植在排斥、耐受以及移植存活取决于诱导调节性T细胞的情况下的结果。在急性排斥期间，T-APC接触的频率随着来自小鼠的T细胞增加，而随着对同种异体抗原无反应的小鼠的T细胞减少。向体外系统中添加调节性T细胞减少了延长的T-APC接触。至关重要的是，在人多克隆扩增的天然存在的调节性T细胞中也观察到了这种效应，已知这些调节性T细胞在人源化小鼠模型中控制人体组织的排斥。这种方法的进一步发展可能允许对移植受者中同种异体反应性T细胞区室进行更深入的表征。未来，使用人类细胞对这种方法进行进一步的开发和评估可能为用于选择免疫抑制最小化患者的检测方法奠定基础，并且它可用于测量临床上致耐受性疗法的影响。
