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

描述(申请人提供)：移植物抗宿主病(GvHD)是异基因造血干细胞移植(HSCT)后潜在的威胁生命的并发症。抑制JAK2诱导同种异体耐受，同时保护Treg的发育。初步数据支持JAK2抑制与IL-2结合在抑制同种异体反应性方面具有协同作用。此外，阻断下游的STAT3使同种异体反应不再是单一的药物。这些策略保护了Treg依赖的IL-2/STAT5信号，增加了STAT5和STAT3的磷酸化比率。拟议的实验将研究JAK2抑制与IL-2或STAT3单独阻断联合使用对同种异体致敏控制的影响。这项工作与NHLBI的使命高度相关，因为它将为消除GvHD的深刻发病率和死亡率提供新的见解。拟议的实验将评估以下特定目标：目的1)研究JAK2抑制与IL-2或STAT3阻断联合使用，作为控制同种异体反应和优化Treg体外扩增的平台。目的2)探讨在移植物抗宿主病(GVHD)和移植物抗宿主病(GVL)小鼠模型中改变STAT5/STAT3磷酸化比例的可行性、生物学影响和治疗效果。目的3)探讨+21天CD4+或CD8+T细胞pSTAT5/pSTAT3比值或ERK1/2磷酸化水平与+100天前急性移植物抗宿主病(GvHD)发病的关系。树突状细胞(DC)刺激的T细胞将被用来研究JAK2抑制联合IL-2或单独抑制STAT3对pSTAT5/pSTAT3极化的影响，作为抑制同种异体反应和促进Treg扩张的一种手段。机制研究将评估对Treg抑制、STAT信号和T辅助细胞亚群及其相关功能的扭曲的次要影响。所得到的数据将通过用siRNA分子靶向STAT3来确认。这一概念将通过JAK2和IL-2的GVHD预防方案或单独使用STAT3抑制剂在MHC不匹配的小鼠模型(C57BL/6=&gt；Balb/c)中进行体内测试。将评估小鼠的GVHD临床评分、存活率和Treg/Th1/Th17亚群的变化。STAT5/STAT3和ERK1/2磷酸化作为即将到来的急性GvHD的生物标志物的概念将在110名患者样本研究中进行。在异基因造血干细胞移植后+21天，患者的T细胞中将检测到JAK2介导的STAT3和平行的ERK1/2磷酸化。STAT5/STAT3的磷酸化比例将被研究是否对GvHD有任何保护作用。这项工作的合理扩展将集中于在预防和治疗移植物抗宿主病的新临床试验中将JAK2与IL-2配对或单独使用STAT3阻滞剂的概念转化。如果AIM 3提供的信息数据为阳性，将在更大的患者群体中进行验证，以确定STAT3、STAT5和ERK1/2磷酸化预测急性移植物抗宿主病的风险。

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.