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

描述（由申请人提供）：移植物抗宿主病（GvHD）是同种异体造血干细胞移植（HSCT）后可能危及生命的并发症。JAK2抑制诱导异体耐受性，同时保持Treg的发育。初步数据支持JAK2抑制与IL-2联合在抑制异体反应性方面具有协同作用。此外，阻断下游STAT3可使异位反应作为单一药物失效。这些策略保留treg依赖性IL-2/STAT5信号，增加STAT5与STAT3磷酸化的比例。拟议的实验将研究JAK2抑制与IL-2配对或单独阻断STAT3的影响；异敏化的控制。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。该实验将评估以下具体目标：目的1)检测JAK2抑制与IL-2配对，或单独阻断STAT3，作为控制异体反应性和优化体外Treg扩增的平台。目的2)探讨扭曲STAT5/STAT3磷酸化比例在GVHD和GVL小鼠模型中的可行性、生物学影响和治疗效果。目的3)确定第21天CD4+或CD8+ T细胞中pSTAT5/pSTAT3比值或ERK1/2磷酸化是否与第100天前急性GvHD的发生有关。树突状细胞（DC）异体刺激的T细胞将通过JAK2抑制联合IL-2或单独抑制STAT3来研究pSTAT5/pSTAT3极化的影响，作为抑制异体反应性和促进Treg扩增的手段。机制研究将评估对Treg抑制、STAT信号和辅助T亚群及相关功能的次要影响。所得数据将通过siRNA分子靶向STAT3得到证实。这一概念将在MHC错配小鼠模型（C57BL/6 => Balb/c）中，通过JAK2和IL-2或STAT3抑制剂单独的GVHD预防方案进行体内测试。小鼠将被评估GVHD临床评分、生存率和Treg/Th1/Th17亚群的改变。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例患者样本研究中进行研究。同种异体造血干细胞移植后第21天，将在患者T细胞中测量jak2介导的STAT3和平行ERK1/2磷酸化。我们将研究STAT5/STAT3磷酸化比例对GvHD的保护作用。这项工作的逻辑扩展将集中在翻译JAK2与IL-2配对的概念，或单独阻断STAT3，用于GVHD预防和治疗的新型临床试验。目的3提供的信息数据，如果阳性，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.