Optimizing the graft versus leukemia effect for pediatric ALL

在该项目的第一个目标下，我们从转基因表达E2aPBX1的小鼠中获得了前体B细胞白血病系，E2aPBX1是儿科ALL中约5%的反复易位（Bijl等人，基因与发育，2005）。静脉注射这些细胞可再生地导致白血病的发展，从少至10,000个细胞分布到骨髓、血液、淋巴结、脾脏、肝脏和中枢神经系统。利用同源标记，早在静脉注射后3天就可以在骨髓中识别白血病细胞，灵敏度为骨髓细胞的0.01%。用荧光素酶转染了一个亚系，白血病的发展也可以通过发光成像原位跟踪。该细胞系被证实具有免疫基因性，因为接种辐照白血病细胞可保护其免受随后的E2aPBX1侵袭，但对其他肿瘤无效。使用单克隆抗体来消耗细胞亚群，我们证明免疫小鼠的保护需要CD4和CD8 T细胞，并且在NK细胞消耗后受损。在目的2下，我们进行了同基因骨髓移植实验，在小鼠移植后一周注射E2aPBX1。随后，将供体的T细胞过继转移至经辐照的E2aPBX1全细胞疫苗。虽然与接受未启动小鼠的纯化T细胞的BMT受体相比，白血病的发展延迟，但所有受体最终都死于白血病。综上所述，这些数据表明，全细胞疫苗接种诱导肿瘤特异性T细胞介导的免疫反应，但无法阻止同基因BMT后白血病的最终发展。重要的是，这些实验还表明，ALL可以被T细胞靶向，但需要事先启动白血病抗原。使用同种异体骨髓而不是同基因骨髓引入了次要的组织相容性抗原，作为白血病细胞的潜在靶点。事实上，同种异体移植后，E2aPBX1白血病攻击，随后转移引物的同种异体T细胞，可治愈所有小鼠的白血病。然而，小鼠出现体重减轻和与GVHD一致的组织学变化，导致晚期死亡。有趣的是，用受体（和白血病）非恶性B细胞注入T细胞供体并不能治愈小鼠，这表明在该模型中，次要抗原和白血病相关抗原都能治愈小鼠。接下来，我们试图通过选择T细胞亚群来分离抗白血病GVL和GVHD的作用。单独来自供体的CD4和CD8 T细胞都不足以治愈所有小鼠。利用基于CD44和CD62L （l -选择素）表达的流式分选，我们已经证明，中枢记忆表型T细胞（CD44+/CD62L+）可以在不诱导GVHD的情况下治愈白血病，而中性T细胞（CD44-/CD62L+）可以诱导快速致死性GVHD。总之，我们已经证明，ALL可以通过体内T细胞反应有效靶向，但这种反应需要供体T细胞接种疫苗。其次，同种异体抗原有助于T细胞输注后的治愈，但会导致GVHD。最后，来自供体的T细胞群可以介导选择性移植物对抗白血病反应。利用这个模型，我们已经开始研究骨髓中白血病的早期进展以及这种进展对T细胞的影响。我们已经发现，在白血病浸润的细胞室中，有相当大比例的T细胞表达高水平的T细胞功能负调节因子，程序性死亡1 （PD-1）受体。此外，E2aPBX1细胞在体外和体内均表达该受体的配体（PD-L1）。此外，研究表明，PD-1+ T细胞的百分比与白血病的侵袭程度相关，并且PD-1+ T细胞还表达衰老表型的其他标记物，如T细胞免疫球蛋白和粘蛋白结构域3 （Tim-3）。正在进行的研究正在探索PD-1诱导T细胞的机制。初步研究表明，在体外共培养过程中不会发生这种情况，这表明可能需要骨髓环境中存在的因素。最后，初步研究表明，阻断PD-1可以增强过继T细胞治疗的疗效。实验室研究的另一个领域是了解对正常组织中表达的次要组织相容性抗原（移植物对宿主）的反应对同种异体造血干细胞移植后抗肿瘤免疫反应的影响。在project ZIA BC 011320项目中，我们已经证明并发表了即使是轻微的GVHD也会显著损害针对肿瘤的疫苗的反应（Capitini等人，Blood, 2009）。我们现在已经证明这种疫苗反应的衰减是由于增殖减少和细胞凋亡增加的结果。在这些研究中使用的肿瘤抗原复合物是HY系统，其中将自然表达Y染色体衍生抗原的实体肿瘤注射到接受雌性同种异体骨髓和T细胞的雌性小鼠中（其中小鼠株特异性次要同种异体抗原与HY衍生的肿瘤抗原不同）。目前正在进行的研究正在评估产生该肿瘤的同一品系雄性小鼠的HY肿瘤反应。重要的是，在这个模型中，肿瘤抗原与肿瘤抗原完全重叠，这是一种临床相关的情况，肿瘤特异性抗原可能很弱或不存在。使用CD4和CD8 HY特异性T细胞受体转基因供体可以仔细跟踪靶向共同抗原的T细胞。初步研究表明，尽管HY特异性T细胞介导轻度GVHD，并且在雄性宿主中的扩张程度远高于雌性宿主（尽管接种了HY疫苗），但这些对正常组织和肿瘤都具有特异性的T细胞在诱导肿瘤消退方面的效力较弱。正在进行的工作是探索这种T细胞功能障碍的机制。我们目前正在培育E2aPBX1转基因小鼠，从中可以产生表达hy的前b细胞白血病系。因此，我们将能够研究肿瘤抗原的正常组织表达是否会对血液系统恶性肿瘤的免疫反应产生类似的影响。Aim 3正在进行中，涉及将Aim 2下开展的工作扩展到临床相关的白血病靶点。我们已经确定E2aPBX1过表达Wilm's Tumor 1基因，该基因在大约70-80%的人类白血病中也过表达，并被证实为患者的靶标。为了获得大量WT-1特异性T细胞，我们培养了表达WT-1显性I类表位特异性T细胞受体的小鼠（称为Db126）。这些小鼠的T细胞在体外表现出对Db126肽的强大扩增，从而使T细胞在体外具有靶向肽脉冲靶标的能力。虽然E2aPBX1细胞也可以作为靶标，但结果却不尽相同。我们正在优化这些T细胞在体内对抗过表达WT-1的E2aPBX1和其他血液学恶性肿瘤的使用。儿科肿瘤科的一项开放协议（Alan Wayne博士，首席研究员）正在利用针对WT-1肽的树突状细胞疫苗接种作为同种异体造血干细胞移植后复发患者的治疗干预措施。我们评估同种异体反应性对疫苗反应影响的临床前研究结果将用于开发移植后疫苗干预试验，以降低适当选择的高危人群的复发风险。我们希望与儿科血液和骨髓移植协会合作进行这些试验。

Under the first aim of this project we have generated a precursor B cell leukemia line derived from mice with transgenic expression of E2aPBX1, a recurring translocation present in approximately 5% of pediatric ALL (Bijl et al, Genes and Development, 2005). 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. Using congenic markers, leukemia cells can be identified in the bone marrow as early as 3 days after intravenous injection with a sensitivity of 0.01% of bone marrow cells. A subline has been transfected with luciferase and leukemia development can also be tracked in situ by luminescent 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 have 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 whole-cell 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 ultimate 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 as potential targets on leukemia cells. Indeed, allogeneic transplantation followed by E2aPBX1 leukemia challenge and subsequent transfer of primed allogeneic T cells results in cure of leukemia in all mice. However, the mice develop weight loss and histologic changes consistent with GVHD that results in late mortality. 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 nor CD8 T cells from primed donors alone were sufficient to cure all of the mice. Using flow sorting based on expression of CD44 and CD62L (L-selectin) we have demonstrated that central memory phenotype T cells (CD44+/CD62L+) can cure leukemia without the induction of GVHD whereas nave T cells (CD44-/CD62L+) induce rapidly lethal GVHD. In summary, we have demonstrated that ALL can be effectively targeted by a T cell response in vivo but that this response requires vaccination of the donor T Cell inocula. Second, allogeneic antigens contribute to the cure following T cell infusion but results in GVHD. Finally, sorted populations of T cells from primed donors can mediate selective graft versus leukemia responses. Using this model we have begun studying the early progression of the leukemia in bone marrow and the impact of this progression on T cells. We have identified that a surprising large percentage of T cells in leukemia-infiltrated compartments express high levels of the negative regulator of T cell function, programmed death 1 (PD-1) receptor. In addition, E2aPBX1 cells express the ligand for this receptor (PD-L1) in vitro and in vivo. Addition studies have shown that the percentage of PD-1+ T cells correlates with the extent of leukemic involvement and that PD-1+ T cells also express other markers of a senescent phenotype such as T cell immunoglobulin and mucin domain 3 (Tim-3). Ongoing studies are exploring the mechanism of PD-1 induction on T cells. Initial studies indicate that this does not happen during coculture in vitro suggesting that factors present in the bone marrow environment may be required Finally, preliminary studies have indicated that blockade of PD-1 can enhance the therapeutic efficacy of adoptive T cell therapy. Another area of investigation in the laboratory is focused on understanding the impact of the response to minor histocompatibility antigens expressed in normal tissues (graft versus host) on the anti-tumor immune response following allogeneic HSCT. Under project Project ZIA BC 011320, we have demonstrated and published that even mild GVHD can significantly impair responses to vaccines targeting tumors (Capitini et al, Blood, 2009). We have now shown that this attenuation of vaccine responses results from both diminished proliferation and increased apoptosis. The tumor antigenic complex used in these studies is the HY system in which a solid tumor that naturally expresses Y chromosome-derived antigens was injected into female mice receiving female allogeneic bone marrow and T cells (where the mouse strain-specific minor allogeneic antigens are distinct from the HY-derived tumor antigens). Ongoing studies are now assessing HY tumor responses in male mice of the same strain from which the tumor is derived. Importantly, in this model, the tumor antigens completely overlap with tumor antigens, a clinically relevant scenario in which tumor-specific antigens may be weak or absent. The use of CD4 and CD8 HY specific T cell receptor transgenic donors allows careful tracking of T cells targeting shared antigens. Preliminary studies indicated that, while HY-specific T cells mediate mild GVHD and expand to a much larger extent in males than in female hosts (despite HY vaccination), these T cells with specificity to both normal tissues and tumors are less potent at inducing tumor regression. Ongoing work is exploring the mechanism of this T cell dysfunction. We are currently breeding E2aPBX1 transgenic mice from which HY-expressing pre-B cell leukemia lines can be generated. Thus, we will be able to study whether the impact of normal tissue expression of tumor antigens will have a similar impact on immune responses to hematologic malignancies. Aim 3 is ongoing and involves the extension of work conducted under aim 2 to clinically relevant leukemia 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 a target in patients. In order to obtain large numbers of WT-1 specific T cells, we have generated mice that express a T cell receptor specific for the dominant class I epitope derived from WT-1 (called Db126). T cells from these mice show robust expansion to Db126 peptide in vitro resulting T cells with the capability of targeting peptide pulsed targets in vitro. While E2aPBX1 cells can also be targeted, the results have been variable. We are in the process of optimizing the use of these T cells in vivo against E2aPBX1 and other hematologic malignancies that overexpress WT-1. An open protocol in the Pediatric Oncology Branch (Dr. Alan Wayne, Principal Investigator) is utilizing dendritic cell vaccination targeting WT-1 peptides as a therapeutic intervention in patients relapsing following allogeneic HSCT. Results obtained from our preclinical studies assessing the impact of alloreactivity on vaccine responses will be used to develop subsequent post-transplant vaccine intervention trials to reduce the risk of relapse in appropriately selected high-risk groups. We hope to conduct these trials in collaboration with the Pediatric Blood and Marrow Transplant Consortium.