1 Department of Nephrology, Leiden University Medical Center (LUMC), Leiden, the Netherlands. 2 Department of Immunohematology and Blood Transfusion, LUMC, Leiden, the Netherlands. 3 Department of Hematology, LUMC, Leiden, The Netherlands. 4 Address correspondence to: Cees van Kooten, MD, PhD, Albinusdreef 2, C07-35 2333ZA Leiden, The Netherlands.
Transplantation. 2014 Jun 15;97(11):1119-27. doi: 10.1097/TP.0000000000000113.
Recognition of donor antigens can occur through two separate pathways: the direct pathway (non-self HLA on donor cells) and the indirect pathway (self-restricted presentation of donor derived peptides on recipient cells). Indirect allorecognition is important in the development of humoral rejection; therefore, there is an increasing interest in the monitoring of indirect alloreactive T-cells. We have used an in vitro model to determine the optimal requirements for indirect presentation and assessed the risk for semidirect presentation in this system.
HLA-typed monocyte-derived dendritic cells (moDCs) were incubated with cellular fragments or necrotic cells and incubated with either indirect or direct alloreactive T-cell clones. T-cell reactivity was measured through proliferation or cytokine secretion. HLA-typed moDC, monocytes, or PBMCs were incubated with HLA class I monomers, in combination with either direct/indirect T-cell clones.
Although both were efficiently taken up, alloreactivity was limited to the semi-direct pathway, as measured by allospecific CD4 (indirect) and CD8 T-cell clones (direct) when cells were used. In contrast, HLA-A2 monomers were not only efficiently taken up but also processed and presented by HLA-typed moDC, monocytes, and PBMCs. Activation was shown by a dose-dependent induction of IFN-γ production and proliferation by the CD4 T-cell clone. Antigen presentation was most efficient when the monomers were cultured for longer periods (24-48 hr) in the presence of the T-cells. Using this method, no reactivity was observed by the CD8 T-cell clone, confirming no semidirect alloreactivity.
We have developed a system that could be used to monitor indirect alloreactive T-cells.
供体抗原的识别可以通过两条独立的途径发生：直接途径（供体细胞上的非自身 HLA）和间接途径（受体内源性呈递供体衍生肽）。间接同种异体识别在体液性排斥反应的发展中很重要；因此，人们越来越关注间接同种反应性 T 细胞的监测。我们使用体外模型来确定间接呈递的最佳要求，并在该系统中评估半间接呈递的风险。
将 HLA 分型的单核细胞衍生树突状细胞 (moDC) 与细胞碎片或坏死细胞孵育，并与间接或直接同种反应性 T 细胞克隆孵育。通过增殖或细胞因子分泌来测量 T 细胞反应性。将 HLA 分型的 moDC、单核细胞或 PBMC 与 HLA 类 I 单体孵育，同时与直接/间接 T 细胞克隆孵育。
尽管两种方法都能有效摄取，但只有在使用细胞时，半间接途径才有限制性的同种反应性，这可以通过同种反应性 CD4（间接）和 CD8 T 细胞克隆（直接）来测量。相比之下，HLA-A2 单体不仅能有效摄取，而且能被 HLA 分型的 moDC、单核细胞和 PBMC 加工和呈递。CD4 T 细胞克隆的剂量依赖性诱导 IFN-γ产生和增殖证明了激活。当单体在 T 细胞存在的情况下培养更长时间（24-48 小时）时，抗原呈递效率最高。使用这种方法，CD8 T 细胞克隆没有观察到反应性，这证实了没有半间接同种异体反应性。
我们已经开发出一种可用于监测间接同种反应性 T 细胞的系统。
