活化T细胞核因子（NFAT）诱导PD-1/TIM-3调控淋巴瘤T细胞耗竭的机制研究

T-cells exhaustion in tumor environment is one of the mechanisms which responsible for the progression of lymphoma. In previous studies, we have shown that PD-1 and TIM-3 are closely related to T cell exhaustion in lymphoma patients;IL-12, IL-10, TGF-beta were involved in T cell exhaustion through different mechanisms; Activation of T cell nucleus factor (NFAT) is highly expressed on T cells of patients with refractory follicular lymphoma, and are positively correlated with PD-1 and TIM-3 expression. Therefore, we speculate that NFAT signal pathway may be involved in T cell exhaustion via inducing PD-1 and TIM-3 expression. This program will continue to study the relationship of NFAT expression with PD-1 and TIM-3 expression in follicular lymphoma patients; We will further downregulate the expression of NFAT by siRNA transfection, then to evaluate the phenotype and function changes of T cells. We further use gene chip and biology technologies to explore the differences of genes expression of NFAT signaling pathway after NFAT downregulation.We will also testify the effects of blockade of NFAT on tumor growth and T cell exhaustion in BALB-c-nu follicular lymphoma mouse model. This study will help to elucidate whether NFAT could be the potential target in follicular lymphoma.

肿瘤微环境的T细胞耗竭是淋巴瘤疾病进展的机制之一。我们前期研究显示淋巴瘤中PD-1及TIM-3是T细胞衰竭的关键信号，IL-12、IL-10、TGF-β通过不同机制参与T细胞耗竭；活化T细胞核因子（NFAT）在复发难治性滤泡淋巴瘤患者中表达升高，且与PD-1及TIM-3表达呈正相关。因此，我们推测：NFAT通过诱导PD-1和TIM-3的表达调控T细胞耗竭，可能成为难治性滤泡淋巴瘤治疗的潜在靶点。本课题将在前期研究的基础上，检测滤泡淋巴瘤患者体内T细胞的NFAT表达与T细胞表型及功能的关系；通过RNA干扰技术下调NFAT基因表达，比较干扰前后T细胞表型及功能变化；利用基因芯片技术及生物信息学分析NFAT干扰前后基因表达差异；通过裸鼠滤泡淋巴瘤动物模型，评估抑制NFAT能否逆转体内的T细胞耗竭。本课题将为阐明NFAT能否成为滤泡淋巴瘤治疗的潜在靶点提供理论依据。

肿瘤微环境的T细胞耗竭是淋巴瘤疾病进展的机制之一。我们前期研究显示淋巴瘤中PD-1及TIM-3是T细胞衰竭的关键信号；活化T细胞核因子（NFAT）在复发难治性滤泡淋巴瘤患者中表达升高，且与PD-1及TIM-3 表达呈正相关。本课题在前期研究的基础上，通过流式细胞学技术检测滤泡淋巴瘤患者体内T细胞的NFAT与T细胞耗竭标志物PD-1、TIM-3的共表达情况，结果显示FL患者存在NFATc1+PD-1+Tim-3+高表达的T细胞，这群细胞的增殖能力和活化能力明显低于对照组，提示NFATc1+PD-1+Tim-3+高表达的T细胞可能是FL的耗竭T细胞，这群耗竭T细胞与患者的临床表现侵袭成正相关。随后，我们采用IL-12体外诱导出NFATc1+Tim-3+双表达的耗竭T细胞，采用IL-12受体抗体可阻断耗竭T细胞NFATc1和Tim-3的表达，提示IL-12抗体可以阻断T细胞耗竭。采用shRNA干扰技术沉默NFATc1基因，能引起PD-1和Tim-3的表达下调及功能上调，提示PD-1/TIM-3的表达涉及NFATc1的调控。免疫组化技术显示部分淋巴瘤患者肿瘤组织内高表达PD-1、PD-L1和TP63，PD-1的表达与患者总生存率延长有关，是一个独立的预测标记物；PD-L1的表达与患者预后差相关，TP63的表达与患者的总生存率无关。患者血清炎性因子，PET-CT上的SUV最大摄取值和肿瘤病灶的Ki67可作为预测淋巴瘤患者疗效的参考指标。患者体内炎性因子越高，NFAT/PD-1/TIM-3相关信号通路越激活，患者T细胞耗竭越明显，患者预后越差。结果显示：滤泡淋巴瘤患者体内的NFAT通过调控PD-1/TIM-3的表达参与T细胞耗竭，而NFAT/PD-1/TIM-3的活化受到体内多种炎性因子调控，IL-12能够诱导NFAT/PD-1/TIM-3通路激活，阻断IL-12有望逆转该通路活化。炎性因子可通过参与NFAT/PD-1/TIM-3相关信号通路的激活引起T细胞耗竭，从而引起不良预后。本课题阐明了NFAT有希望成为滤泡淋巴瘤治疗的潜在治疗靶点。