Anthony Bryan A, Hadley Gregg A
Department of Surgery, The Ohio State University Medical Center, USA.
J Vis Exp. 2012 Aug 29(66):e3697. doi: 10.3791/3697.
Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al. developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.
移植物抗宿主病（GVHD）是骨髓移植作为多种血液系统缺陷的治愈性疗法广泛应用的限制障碍。GVHD是由注入受体的骨髓移植物中存在的成熟同种异体反应性T细胞引起的，这些T细胞会对宿主器官造成损害。然而，在小鼠中，必须将T细胞添加到骨髓接种物中才能引发GVHD。尽管已经进行了大量工作来表征移植后T细胞的反应，但生物发光成像技术是一种在体内监测T细胞迁移模式的非侵入性方法。在致死性照射后，将受体小鼠移植来自供体小鼠的骨髓细胞和脾细胞。移植中包括来自L2G85.B6（组成性表达荧光素酶的转基因小鼠）的T细胞亚群。通过仅移植某些T细胞亚群，能够在体内追踪特定的T细胞亚群，并根据它们的位置，就特定T细胞亚群在不同时间点促进GVHD中的作用提出假设。在移植后的预定时间间隔，使用Xenogen IVIS CCD相机对受体小鼠进行成像。可以使用Living Image软件对光强度进行量化，以根据光子强度生成伪彩色图像（红色=高强度，紫色=低强度）。移植后4 - 7天之间，受体小鼠开始出现GVHD的临床症状。Cooke等人开发了一种评分系统，根据受体小鼠的皮毛质地﹑皮肤完整性﹑活动情况﹑体重减轻和姿势来量化疾病进展。每天对小鼠进行评分，当它们濒死时实施安乐死。受体小鼠通常在移植后20 - 30天濒死。小鼠模型是研究GVHD免疫学的有价值工具。选择性移植特定的T细胞亚群有助于仔细确定每个亚群所起的作用。在体内非侵入性地追踪T细胞反应为小鼠GVHD模型增添了另一层价值。
