ALL immunobiology and the bone marrow niche

根据本项目的第一个目标，工作分为两个方面。首次探索了同种异体环境对肿瘤T细胞受体定向免疫治疗的调节作用，并发现选择性抑制供体浆细胞样树突状细胞中的STAT1可以在保留抗肿瘤T细胞应答的同时减轻GVHD的严重程度。这项研究已发表在《血液》杂志(Capitini等人)上。该项目的第二部分研究了同种异体抗原表达在异基因HSCT后抑制抗肿瘤免疫反应中的隔室特异性效应，并利用了来自转基因表达E2aPBX1的小鼠的前体B细胞白血病株，E2aPBX1是一种在大约5%的儿童ALL中存在的反复易位(Bijl等人，基因和发展，2005年)，并在我们实验室发展成可移植的模型。这项研究发表在《血液和骨髓移植生物学》(Shand等人)上。利用这个模型，我们已经开始研究白血病在骨髓中的早期进展以及这种进展对T细胞的影响。我们已经发现，在白血病浸润性隔室中，有相当大比例的T细胞表达高水平的T细胞功能的负调节因子--程序性死亡1(PD-1)受体。此外，研究表明，PD-1+T细胞的百分比与白血病的受累程度有关，PD-1+T细胞还表达其他衰老表型的标志，如T细胞免疫球蛋白和粘蛋白结构域3(Tim-3)。有趣的是，仔细评估早期白血病进展中的T细胞表明，PD1的诱导发生得更早(在注射白血病后第5天)，而其他T细胞衰老标记物的获得发生得更晚，如TIM-3和淋巴细胞激活基因3(LAG3)，这表明这些标记物在抗白血病潜力方面可能更具功能相关性。事实上，辐射肿瘤细胞诱导的小鼠的T细胞也表达PD1，但不表达TIM-3或LAG3，并介导抗白血病作用。最后，来自ALL患者的人骨髓样本白血病样本(来自我们的血液恶性肿瘤生物学研究)的初步数据显示，PD1、TIM-3和LAG3在T细胞亚群上表达。综上所述，这些数据提供了关于骨髓微环境中ALL的免疫抑制效应如何作为ALL靶向免疫治疗的佐剂而被逆转的见解。在该项目的目标2下，我们从James Kochenderfer博士那里获得了一种小鼠CD19靶向嵌合抗原受体(CAR)，类似于正在进行的临床试验中用于诱导50-70%的化疗无效或复发ALL患者缓解的嵌合抗原受体(CAR)。这些混合受体(免疫球蛋白抗原结合域和T细胞受体信号组件)是正在南联盟实验室进行的ZIA BC 011565项目的主要重点。利用目标1中描述的CD19和同种异体移植模型，我们研究了异基因HSCT后与CAR治疗相关的免疫生物学。我们已经证明，表达CAR的T细胞可以由内源性同种异体反应性TCR介导的GVHD，但只有在存在CAR抗原的情况下才会发生这种情况，并且CAR T细胞产生细胞因子(主要是IL-6，这是临床上观察到的CD19 CAR治疗后的主要毒性反应的一种细胞因子)会加剧这种情况。这部作品已提交出版。利用这个CAR/ALL模型，结合Shand等人开发的HY T细胞受体转基因系统，我们研究了TCR信号对CAR T细胞活性的影响，前提是在异基因HSCT后利用CAR T细胞时会发生这种情况。我们已经发现TCR信号可以对CAR T细胞功能产生负面影响，特别是在CD8T细胞的情况下。我们目前正在评估这种效果的机制。在目标2下，我们已经开始在实验室开发的同源模型中研究CAR治疗后的晚期所有复发。重要的是，尽管在临床上针对CD19的CAR T细胞具有强大的活性，但大约20%的患者复发并失去CD19的表达。这在异种移植系统中很难模拟，因为在异种移植系统中，异种GVHD阻止了对使用人类CAR T细胞治疗的小鼠的长期监测。使用同基因模型，我们发现持续的汽车压力会导致ALL复发，并伴随着表型和基因表达的全球变化，这表明存在去分化和谱系转换。在本项目的第三个目标下，利用AIM下开发的工具，即ALL对骨髓微环境的影响(衰竭标志物的T细胞表达)，我们已经开始研究骨髓利基因子在ALL进展和治疗耐药中的作用。我们已经开始了基于基因组筛选结果的候选方法，其中发现前B细胞的一个亚群都过度表达胸腺间质受体(TSLPR)，并且该受体的过度表达与较高的复发风险相关。重要的是，TSLP细胞因子与由TSLPR和IL-7受体α链组成的异二聚体受体结合。南联盟实验室过去拥有IL-7免疫生物学方面的专业知识。使用我们的可移植ALL模型，我们证明了通过转导过表达TSLPR会加速白血病的早期进展和对化疗药物的耐药性。我们还表明，TSLP是由骨髓基质细胞产生的，这表明在ALL中TSLPR过表达的生物学效应是配体依赖的。我们目前正在探索中和TSLP是否可以逆转TSLPR过度表达的影响，这具有潜在的治疗意义。

Under the first aim of this project work has been divided into 2 areas. The first explored modulation of cancer T cell receptor directed immunotherapy by the allogeneic environment and identified that selective inhibition of STAT1 in donor plasmacytoid dendritic cells could reduce the severity of GVHD with preservation of anti-tumor T cell responses. This work has been published in Blood (Capitini et al). The second part of the project studied compartment-specific effects of alloantigen expression on inhibition of antitumor immune responses following alloHSCT and utilized 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) and developed in our laboratory into a transplantable model. This work was published in Biology of Blood and Marrow Transplantation (Shand et al). 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 surprisingly 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. 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). Interestingly, careful assessment T cells during early leukemia progression have shown that the induction of PD1 occurs early (by day 5 after injection of leukemia) whereas acquisition of other T cells senescent markers such as Tim-3 and Lymphocyte Activation Gene 3 (LAG3) occur later suggesting that these markers may be more functionally relevant in terms of antileukemic potential. Indeed, T cells from irradiated tumor cell primed mice also express PD1 but not Tim-3 or LAG3 and mediate an antileukemic effect. Finally, preliminary a data from human bone marrow samples leukemia samples from patients with ALL (obtained from our Hematologic Malignancy Biology Study) have shown expression of PD1, Tim-3 and LAG3 on a subset of T cells. In summary, this data provides insights into how immunosuppressive effects of ALL in the bone marrow microenvironment may be reversed as an adjuvant to ALL-targeted immunotherapy. Under Aim 2 of this project we have obtained a murine CD19-targeted chimeric antigen receptor (CAR) from Dr. James Kochenderfer analogous to those used in the clinical setting to induce remission in 50-70% of patients with chemotherapy-refractory or recurrent ALL in ongoing clinical trials. These hybrid receptors (Immunoglobulin antigen binding domain and T cell receptor signaling components) are the primary focus of project ZIA BC 011565 being conducted in the Fry laboratory. Using the CD19 and the allogeneic transplant models described in Aim 1 we have study the immunobiology associated with CAR therapy after alloHSCT. We have demonstrated that CAR expressing T cells can cause GVHD mediated by the endogenous alloreactive TCR but that this occurs only when there CAR antigen present and that this is exacerbated by CAR T cell production of cytokines (mainly IL-6, a cytokine implicated in major toxicity observed after CD19 CAR therapy in the clinic). This work has been submitted for publication. Using this CAR/ALL model and incorporating the HY T cell receptor transgenic system developed for Shand et al publication we have studied the impact of TCR signaling on the activity of CAR T cells based on the premise that this situation would occur when CAR T cells are utilized after alloHSCT. We have identified that TCR signaling can negatively impact CAR T cell function, particularly in the case of CD8 T cells. We are currently evaluating the mechanism for this effect. Under Aim 2 we have begun studying late ALL relapses afterCAR treatment in the syngeneic model developed in the lab. Importantly, despite the potent activity of CAR T cells targeting CD19 in the clinic approximately 20% of patients relapse with ALL that loses expression of CD19. This cannot be easily modeled in xenograft systems where xenogeneic GVHD prevents long-term monitoring of mice treated with human CAR T cells. Using the syngeneic model we have found that persistent CAR pressure induces relapse of ALL with global changes in phenotype angd gene expression that suggest de-diffierentiaion and lineage switch. Under the third Aim of this project and utilizing tools developed under Aim where the effect of ALL on the bone marrow microenvironment (T cell expression of exhaustion markers) we have begun studying the role of bone marrow niche factors on the progression and therapeutic resistance of ALL. We have started with a candidate approach based on the results of genomic screening where it was found that a subset of pre B cell ALL overexpresses the thymic stromal stromal receptor (TSLPR) and that overexpression of this receptor is associated with a higher risk of relapse. Importantly, the TSLP cytokine binds to a heterodimeric receptor comprised of TSLPR and the IL-7 receptor alpha chain. The Fry laboratory has past expertise in the immunobiology of IL-7. Using our transplantable ALL model we have demonstrated that overexpression of TSLPR by transduction results in accelerated early progression of leukemia and resistance to chemotherapeutic agents. We have also shown that TSLP is produced by bone marrow stromal cells suggesting that the biologic effect of TSLPR overexpression in ALL is ligand-dependent. We are currently exploring whether neutralization of TSLP can reverse the impact of TSLPR overexpression which has potential therapeutic implications.