The role of the transcriptional regulator Lmo4 in CD8+ T cell stemness, memory formation and antitumor immunity

与其他器官系统相似，CD8+T细胞包括处于不同分化阶段的细胞。在最初的免疫反应中，幼稚T细胞(TN)进入T记忆干细胞(TSCM)、中央记忆T细胞(Tcm)和效应记忆T细胞(TEM)，最终形成短暂的效应T细胞(TEFF)。一些证据表明，TSCM具有干细胞样的特征，比任何其他记忆T细胞群都多。与此同时，记忆T细胞诱导肿瘤缩小的能力从TSCM到TSCM和TEM逐渐减弱。因此，发现产生和扩展TSCM的策略对于开发下一代基于T细胞的免疫疗法至关重要。T细胞使用各种转录网络来促进TSCM的产生和维持，并限制末端效应器的分化。一个潜在的候选基因是转录调节因子LIM结构域仅4(LMO4)，这将是拟议项目的主题。宿主实验室发现，在有利于诱导TSCM的培养条件下，LMO4过表达。此外，他们还证明，通过加强CD8+T细胞中LMO4的表达，病毒感染小鼠的T细胞扩张和记忆形成得到了增强。根据初步数据，我建议剖析转录调控因子LMO4在CD8+T细胞干性、记忆形成和抗肿瘤免疫中的作用。首先，用编码同源抗原gp100的重组痘苗病毒(gp100-Vv)感染受体小鼠，观察LMO4对转移的抗原特异性CD8+T细胞增殖和凋亡的影响。此外，还将分析在分化过程中不同表达的记忆前体和急性免疫反应高峰期Teff的频率以及各种细胞因子的表达。此外，还将测试LMO4表达对T细胞代谢的影响，并探索是否可以通过扩增LMO4表达来增加TSCM的生成。之后，计划阐明LMO4的下游效应，并评估在CD8+T细胞分化的背景下，LMO4的遗传操作如何影响这些基因的表达。接下来，我将评估增强一代的TSCM在用编码gp100抗原的腺病毒(gp100-adv)再次攻击小鼠后是否会促进更强的召回反应，并将确定过继转移到患有皮下黑色素瘤的小鼠体内的LMO4过表达细胞的疗效。为了评估LMO4过表达的队列是否会产生持久的抗肿瘤免疫反应，这些动物将再次受到肿瘤的挑战。

Similar to other organ systems, the CD8+ T cell compartment includes cells, which are at different phases of differentiation. In a primary immune response, naive T cells (TN) proceed to T memory stem cells (TSCM), central memory T cells (TCM) and effector memory T cells (TEM), culminating in the genesis of short-lived effector T cells (TEFF). Several pieces of evidence suggest that TSCM have stem cell-like features, more than any other memory T cell population. At the same time, the capability of memory T cells to induce tumor reduction progressively diminishes from TSCM to TCM and TEM. Therefore, discovering strategies that generate and expand TSCM is crucial for the development of the next generation of T cell-based immunotherapies. Various transcriptional networks are used by T cells to facilitate the generation and maintenance of TSCM and to restrict terminal effector differentiation. A potential candidate gene is the transcriptional regulator LIM domain only 4 (Lmo4), which will be the topic of the proposed project. The host laboratory has found that Lmo4 is overexpressed in culture conditions favoring the induction of TSCM. Moreover, they demonstrated that by enforcing the expression of Lmo4 in CD8+ T cells, T cell expansion and memory formation in virus-infected mice was enhanced. Based on the preliminary data, I propose to dissect the role of the transcriptional regulator Lmo4 in CD8+ T cell stemness, memory formation and antitumor immunity. First, the effect of Lmo4 on proliferation and apoptosis of transferred antigen-specific CD8+ T cells will be evaluated following infection of recipient mice with a recombinant strain of vaccinia virus encoding the cognate antigen gp100 (gp100-VV). Furthermore, the frequency of memory precursors and TEFF at peak of the acute immune response and the expression of various cytokines, which are differently expressed in the course of differentiation, will be analyzed. Moreover, the impact of Lmo4 expression on T cell metabolism will be tested and it will be explored if the generation of TSCM could be increased by amplifying Lmo4 expression. Afterwards, the plan is to elucidate downstream effectors of Lmo4 and to evaluate how the genetic manipulation of Lmo4 would affect expression of these genes in the context of CD8+ T cell differentiation. Next, I will evaluate if the enhanced generation of TSCM will promote stronger recall responses after re-challenging mice with an adenovirus encoding gp100 antigen (gp100-Adv) and will determine the efficacy of Lmo4-overexpressing cells adoptively transferred into mice bearing subcutaneous melanomas. To evaluate if the Lmo4-overexpression cohort develop long-lasting antitumor immune responses the animals will be re-challenged with tumors.