Center for Transplantation Sciences, Department of Surgery, Massachusetts General Hospital, Boston, MA, USA.
Immune Tolerance Network, Bethesda, MD, USA.
J Immunol Methods. 2021 Apr;491:112987. doi: 10.1016/j.jim.2021.112987. Epub 2021 Feb 6.
Precise analyses of alloreactive T cell phenotype and function can inform both the nature and intensity of adaptive responses to transplant antigens. However, alloreactive T cells are sparse and difficult to detect, particularly in cryopreserved peripheral blood mononuclear cells (PBMCs) and from hypo-responsive individuals. An assay to identify and phenotype alloreactive cells would be particularly valuable, especially for multi-center clinical trials that often store frozen samples for batch analysis. Herein we demonstrate consistent and reproducible alloreactive T cell detection in cryopreserved PBMC following a short-term mixed lymphocyte reaction (MLR). The inherent background expression levels of activation markers on responder T cells were minimized by including a resting period prior to the assay. Stimulator cells were activated before inclusion in the MLR by addition of CD40L and IL-4. The time frame and markers to identify and phenotype alloreactive T cells following stimulation were optimized using short term co-cultures. We defined subsets of CD4+ and CD8+ T cells co-expressing CD69 and either CD154 or CD137 following allostimulation as alloreactive, and further phenotyped these cells with a variety of surface markers such as PD-1, LAG-3, and TIM-3. This assay may allow for the monitoring of donor-specific T cells in transplant recipients with longitudinally collected and cryopreserved PBMCs and provide a useful tool to identify biomarkers associated with tolerance. These biomarkers may add to mechanistic insights in immune recognition of transplanted tissues and/or cells.
精确分析同种反应性 T 细胞的表型和功能可以为对移植抗原的适应性反应的性质和强度提供信息。然而，同种反应性 T 细胞数量稀少且难以检测，尤其是在冷冻保存的外周血单核细胞（PBMC）和低反应个体中。鉴定和表型同种反应性细胞的测定将特别有价值，特别是对于经常储存冷冻样本进行批量分析的多中心临床试验。在此，我们证明了在短期混合淋巴细胞反应（MLR）后，冷冻保存的 PBMC 中同种反应性 T 细胞的检测具有一致性和可重复性。通过在测定前包括休息期，最小化了反应性 T 细胞上激活标志物的固有背景表达水平。在将刺激细胞纳入 MLR 之前，通过添加 CD40L 和 IL-4 对其进行激活。使用短期共培养优化了鉴定和表型同种反应性 T 细胞的时间框架和标记物。我们将 CD4+和 CD8+T 细胞中共同表达 CD69 和 CD154 或 CD137 的亚群定义为同种反应性，并用各种表面标志物（如 PD-1、LAG-3 和 TIM-3）对这些细胞进行进一步表型分析。该测定方法可用于监测移植受者中使用纵向收集和冷冻保存的 PBMC 进行的供体特异性 T 细胞，并提供一种有用的工具来鉴定与耐受相关的生物标志物。这些生物标志物可能会增加对移植组织和/或细胞免疫识别的机制见解。
