Identification of Canine Minor Histocompatibility Antigens

项目2：CANINE次要组织相容性抗原的鉴定 项目1在DLA相合的犬造血细胞移植(HCT)中开发了一种方法 导致稳定的供体-宿主混合嵌合体。持久宿主造血可作为实验性的 在方案3和方案4下移植的部分患者可见持续性血液系统恶性肿瘤模型。 混合供体嵌合体转化为全供体嵌合可以通过注射供体淋巴细胞来实现 对外周血单核细胞上表达的次要组织相容性抗原致敏 然而，以往往致命的移植物抗宿主病(GVHD)为代价。针对T细胞的免疫应答 普遍表达的次要抗原被认为是GVHD的原因，而T细胞反应 针对无处不在的和造血特异性的次要抗原的组合有助于消除 残留宿主造血细胞的方式类似于在人类中观察到的移植物抗白血病效应 病人。因此，识别仅限于造血细胞的次要抗原具有很大的前景。 以改善异基因血细胞移植的结局。这一知识将促进敏感化的发展。 以宿主造血细胞为靶点，同时保留GVHD靶组织的策略。然而，虽然这只狗 同种异体血细胞移植模型是临床前开发新的血细胞移植疗法的最佳模型，没有犬科小细胞移植， 到目前为止，已经描述了组织相容性抗原，以及用于次要抗原的现有方法 身份识别效率低下。为了解决这个问题，我们提出了一种新的方法来处理次要抗原。 在狗身上的发现。这种方法利用下一代测序技术来定义蛋白质编码 受体所特有的、在外周血单核细胞中表达的变异体，将用于致敏HCT供体。 然后将致敏的供体T细胞注射到各自的受者体内，目的是将混合的 全供者嵌合体和引起GVHD。转换完成后，T细胞将被 从接受者那里获得，并使用一种新的、高度的- 吞噬T细胞检测。阳性反应将定义真正的次要组织相容性抗原。vbl.使用 ，然后我们将鉴定那些在造血细胞中高表达但不表达的次要抗原。 在GVHD靶组织中。接下来，将使用相关的次要抗原肽来致敏供者T细胞 旨在将混合供体嵌合体转化为无GVHD的全供体嵌合体(项目1)。最终，这一概念将成为 在项目1的急性白血病犬模型上进行了测试。对公共卫生的好处：综合起来， 本项目中提出的研究和项目1中提出的活体研究有可能 开发新的有效方法，使持续/复发的恶性肿瘤患者受益 项目3和4下的同种异体血细胞移植。

PROJECT 2: IDENTIFICATION OFCANINE MINOR HISTOCOMPATIBILITY ANTIGENS Project 1 has developed an approach at DLA-identical canine hematopoietic cell transplantation (HCT) that results in stable mixed donor-host chimerism. Persistent host hematopoiesis can serve as an experimental model of persistent hematologic malignancy seen in some patients transplanted under Projects 3 and 4. Conversion of mixed to all-donor chimerism can be achieved with injection of donor lymphocytes that have been sensitized to host minor histocompatibility antigens expressed on peripheral blood mononuclear cells (PBMC), however at the price of often fatal graft-vs.-host disease (GVHD). T-cell responses directed against ubiquitously expressed minor antigens are thought to be responsible for GVHD, while T-cell responses against a combination of ubiquitous and hematopoietic-specific minor antigens contribute to elimination of residual host hematopoietic cells in a manner analogous to the graft-vs.-leukemia effect observed in human patients. The identification of minor antigens restricted to hematopoietic cells therefore holds great promise for improving allogeneic HCT outcomes. That knowledge would facilitate the development of sensitization strategies that target host hematopoietic cells while sparing GVHD target tissues. However, while the dog model of allogeneic HCT is optimal for preclinical development of novel HCT therapies, no canine minor, histocompatibility antigens have been described to date, and existing methodologies for minor antigen identification are inefficient. To address this problem, we propose a novel approach to minor antigen discovery in the dog. This approach utilizes next generation sequencing technology to define protein coding variations unique to the recipient and expressed in PBMC which will be used for sensitizing the HCT donor. Sensitized donor T-cells will then be injected into the respective recipients with the aim of converting mixed to full-donor chimerism and causing GVHD. After conversion has been accomplished, T-cells will be harvested from recipients and tested for responses against candidate minor antigens using a novel, high- throughput T-cell assay. Positive responses would define genuine minor histocompatibility antigens. Using qRT-PCR, we will then identify those minor antigens that are highly expressed in hematopoietic cells but not in GVHD target tissues. Next, relevant minor antigen peptides will be used to sensitize donor T-cells with the aim of converting mixed to full-donor chimerism without GVHD (Project 1). Eventually, this concept will be tested in a canine model of acute leukemia in Project 1. Benefits to Public Health: Taken together, the studies proposed in this Project and the in vivo studies proposed in Project 1 have the potential of developing new and effective approaches benefiting patients with persisting/relapsing malignancies treated by allogeneic HCT under Projects 3 and 4.