Optimizing the graft versus leukemia effect for pediatric ALL

在该项目的第一个目标下，我们利用了来自表达E2 aPBX 1转基因的小鼠的前体B细胞白血病。来自单个小鼠的E2aPBX1白血病细胞最初在体内传代，随后调节以在体外生长。静脉内注射这些细胞可重复地导致白血病从少至10，000个细胞发展，并分布到骨髓、血液、淋巴结、脾、肝和中枢神经系统。用荧光素酶转染亚系，可以使用成像原位跟踪白血病的发展。该细胞系被证实是免疫原性的，因为用辐射的白血病细胞接种疫苗可以保护随后的E2aPBX1攻击，但不能保护其他肿瘤。使用单克隆抗体耗尽细胞亚群，我们证明了免疫小鼠的保护需要CD4和CD8 T细胞，并在NK细胞耗尽后受损。在目标2下，我们接下来进行同基因骨髓移植实验，其中在移植后一周向小鼠注射E2aPBX1。随后过继转移来自用辐照的E2aPBX1疫苗致敏的供体的T细胞。虽然与接受来自未致敏小鼠的纯化T细胞的BMT受体相比，白血病的发展被延迟，但所有受体最终都死于白血病。总之，这些数据表明，全细胞疫苗接种诱导肿瘤特异性T细胞介导的免疫应答，其不能预防同基因BMT后白血病的发展。重要的是，这些实验还证明ALL可以被T细胞靶向，但需要预先引发白血病抗原。使用同种异体骨髓而不是同基因骨髓在白血病细胞上引入了次要的组织相容性抗原潜在靶点。事实上，同种异体移植后E2aPBX1白血病攻击和随后转移致敏的同种异体T细胞导致所有小鼠的白血病治愈，但体重减轻和组织学变化与中度GVHD一致。有趣的是，用受体（和白血病）株非恶性B细胞致敏T细胞供体并不能治愈小鼠，表明次要抗原和白血病相关抗原都是该模型中治愈的原因。我们接下来试图通过选择T细胞亚群来将抗白血病GVL效应与GVHD分开。无论是CD4还是CD8 T细胞都不足以治愈所有小鼠。然而，对CD62L（l-选择素）阳性T细胞（与致敏供体中的中枢记忆亚型一致）的选择能够介导选择性的、治愈性的GVL效应而没有GVHD。目标3正在进行中，涉及白血病相关靶点的鉴定。我们已经确定E2aPBX1过表达Wilm's肿瘤1基因，该基因也在大约70 - 80%的人白血病中过表达，并被证实为患者中的潜在靶点。为了获得大量WT-1特异性T细胞，我们正在产生表达对WT-1衍生的显性表位特异性的T细胞受体的小鼠。T细胞受体已经被克隆，并且已经产生了表达大量WT-1特异性T细胞的多个创始者。一旦扩增了鼠系，将在E2aPBX1模型中测试来自这些小鼠的T细胞。如果成功，异基因移植后的后续实验将有助于验证和表征白血病定向T细胞作为治疗或预防复发的方式的使用。

Under the first aim of this project we have utilized a precursor B cell leukemia derived from mice that express an E2aPBX1 transgene. E2aPBX1 leukemia cells from a single mouse were initially passaged in vivo and subsequently conditioned to grow in vitro. Injection of these cells intravenously reproducibly results in leukemia development from as few as 10,000 cells with distribution to bone marrow, blood, lymph nodes, spleen, liver and central nervous system. A subline has been transfected with luciferase and leukemia development can be tracked in situ using imaging. The cell line was confirmed to be immunogeneic as vaccination with irradiated leukemia cells protects against subsequent challenge with E2aPBX1 but not other tumors. Using monoclonal antibodies to deplete cell subsets, we demonstrated that protection in immunized mice requires both CD4 and CD8 T cells and is impaired following NK cell depletion. Under aim 2 we next performed syngeneic bone marrow transplantation experiments in which mice were injected with E2aPBX1 one week following transplant. This was followed by adoptive transfer of T cells from donors primed with an irradiated E2aPBX1 vaccine. While leukemia development was delayed compared to BMT recipients receiving purified T cells from unprimed mice, all recipients eventually succumbed to leukemia. Together, this data indicates that whole-cell vaccination induces a tumor-specific, T cell mediated immune response that is unable to prevent the develop of leukemia following syngeneic BMT. Importantly, these experiments also demonstrate that ALL can be targeted by T cells but requires prior priming to leukemia antigens The use of allogeneic bone marrow rather than syngeneic bone marrow introduces minor histocomaptibility antigens potential targets on leukemia cells. Indeed, allogeneic transplantation followed by E2aPBX1 leukemia challenge and subsequent transfer of primed allogeneic T cells resulted in cure of leukemia in all mice but weight loss and histologic changes consistent with moderate GVHD. Interestingly, priming T cell donors with recipient (and leukemia) strain non-malignant B cells did not cure the mice indicating that both minor antigens and leukemia-associated antigens are responsible for cure in this model. We next sought to separate the anti-leukemic GVL effect from GVHD by selecting for T cells subsets. Neither CD4 or CD8 T cells from primed donors alone were sufficient to cure all of the mice. However, selection for CD62L (l-selectin) positive T cells (consistent with a central memory subtype in primed donors) was capable of mediating a selective, curative GVL effect without GVHD. Aim 3 is ongoing and involves identification of leukemia-associated targets. we have established that E2aPBX1 overexpresses the Wilm's Tumor 1 gene, also overexpressed on approximately 70-80% of human leukemias and validated as potential target in patients. In order to obtain large numbers of WT-1 specific T cells, we are in the process of generating mice that express a T cell receptor specific for the dominant epitope derived from WT-1. The T cell receptor has been cloned and multiple founders have been generated that express large numbers of WT-1 specific T cells. Once the murine lines have been expanded, T cells from these mice will be tested in the E2aPBX1 model. If, successful, subsequent experiments following allogeneic transplantion will help validate and characterize the use of leukemia directed T cells as a modality to treat or prevent relapse.