JAK2_STAT3 as a therapeutic target and marker of graft-versus-host disease

描述（由申请方提供）：移植物抗宿主病（GvHD）是异基因造血干细胞移植（HSCT）后的一种潜在危及生命的并发症。JAK 2抑制诱导同种异体耐受，同时保留Treg发育。初步数据支持JAK 2抑制与IL-2组合提供了抑制同种异体反应性的协同效应。此外，下游STAT 3的阻断消除了作为单一药剂的同种异体反应。这些策略保留了Treg依赖性IL-2/STAT 5信号传导，增加了STAT 5与STAT 3磷酸化的比率。拟议的实验将研究JAK 2抑制与IL-2或单独STAT 3阻断配对的影响;对同种过敏的控制。这项工作与NHLBI的使命高度相关，因为它将为消除GVHD的严重发病率和死亡率提供新的见解。所提出的实验将评估以下具体目的：目的1）检查JAK 2抑制与IL-2配对，或单独的STAT 3阻断，作为控制同种异体反应性和优化Treg体外扩增的平台。目的2）探讨在GVHD和GVL小鼠模型中改变STAT 5/STAT 3磷酸化比例的可行性、生物学影响和治疗效果。目的3）确定第+21天的CD 4+或CD 8 + T细胞中的pSTAT 5/pSTAT 3比率或ERK 1/2磷酸化是否与第+100天之前的急性GvHD的发展相关。树突状细胞（DC）-同种异体刺激的T细胞将用于研究经由JAK 2抑制与IL-2组合或单独STAT 3抑制的pSTAT 5/pSTAT 3极化的影响，作为抑制同种异体反应性和促进Treg扩增的手段。机制研究将评估对Treg抑制、STAT信号传导和辅助性T细胞亚群和相关功能的偏移的次要影响。所得数据将通过用siRNA分子靶向STAT 3来证实。将在MHC错配小鼠模型（C57 BL/6 => Balb/c）中用JAK 2和IL-2或单独的STAT 3抑制剂的GVHD预防方案在体内测试该概念。评估小鼠的GVHD临床评分、存活率和Treg/Th 1/Th 17亚群的改变。STAT 5/STAT 3和ERK 1/2磷酸化作为即将发生的急性GvHD的生物标志物的概念将在110例患者样本研究中进行研究。JAK 2介导的STAT 3和平行的ERK 1/2磷酸化将在同种异体HSCT后第+21天在患者的T细胞中测量。将研究STAT 5/STAT 3磷酸化的比率对GvHD的任何保护性影响。这项工作的逻辑扩展将集中在翻译JAK 2与IL-2配对的概念，或单独的STAT 3阻断，在GVHD预防和治疗的新临床试验中。如果目标3提供的信息数据为阳性，则将在更大的患者群体中验证，以确定STAT 3、STAT 5和ERK 1/2磷酸化是否预测急性GvHD的风险。

DESCRIPTION (provided by applicant): Graft-versus-host disease (GvHD) is a potentially life threatening complication following allogeneic hematopoietic stem cell transplantation (HSCT). JAK2 inhibition induces allotolerance, while preserving Treg development. Preliminary data supports that JAK2 inhibition combined with IL-2 offers a synergistic effect on suppressing alloreactivity. Moreover, blockade of downstream STAT3 abrogates alloresponses as a single agent. These strategies preserve Treg-dependent IL-2/STAT5 signaling, increasing the ratio of STAT5 to STAT3 phosphorylation. The proposed experiments will investigate the influence of JAK2 inhibition paired with IL-2, or STAT3 blockade alone; on the control of allosensitization. This work is highly relevant to the mission of the NHLBI, as it will offer new insights on eliminating the profound morbidity and mortality of GvHD. The proposed experiments will evaluate the following specific aims: Aim 1) Examine JAK2 inhibition paired with IL-2, or STAT3 blockade alone, as a platform to control alloreactivity and optimize Treg expansion in vitro. Aim 2) Investigate the feasibility, biologic influence, and therapeutic effect of skewing the STAT5/STAT3 phosphorylation ratio in a mouse model of GVHD and GVL. Aim 3) Determine if the pSTAT5/pSTAT3 ratio or ERK1/2 phosphorylation in CD4+ or CD8+ T cells on day +21 associate with the development of acute GvHD before day +100. Dendritic cell (DC)-allostimulated T cells will be used to investigate the influence of pSTAT5/pSTAT3 polarization via JAK2 inhibition combined with IL-2, or STAT3 inhibition alone, as a means to suppress alloreactivity and promote Treg expansion. Mechanistic studies will evaluate secondary effects on Treg suppression, STAT signaling, and skewing of T helper subsets and related functions. The resultant data will be confirmed by molecularly targeting STAT3 with siRNA. This concept will be tested in vivo with a GVHD prophylaxis regimen of a JAK2 and IL-2, or STAT3 inhibitor alone, in an MHC- mismatched mouse model (C57BL/6 => Balb/c). Mice will be assessed for GVHD clinical scores, survival, and alterations in Treg/Th1/Th17 subsets. The concept of STAT5/STAT3 and ERK1/2 phosphorylation as biomarkers of impending acute GvHD will be investigated in a 110 patient sample study. JAK2-mediated STAT3 and parallel ERK1/2 phosphorylation will be measured in patients' T cells on day +21 after allogeneic HSCT. The ratio of STAT5/STAT3 phosphorylation will be studied for any protective influence on GvHD. Logical extensions of this work will focus on translating the concept of JAK2 paired with IL-2, or STAT3 blockade alone, in novel clinical trials for GVHD prevention and therapy. The informative data provided by aim 3, if positive, will be validated in a larger patient population to determine f the STAT3, STAT5, and ERK1/2 phosphorylation predict the risk of acute GvHD.