Post-Transcriptional Regulation of Interleukin-7 Receptor Expression

在这个项目中，我们的主要目标是解决细胞因子受体表达在T细胞发育和分化中的作用，特别是关注gc家族细胞因子受体的作用。在胸腺中，gc表达对于胸腺发育和CD8谱系承诺至关重要，因此IL-7信号提供促生存和促代谢的线索，但也诱导Runx3d的表达，指定CD8谱系分化。除了这些作用外，我们最近发现gc表达在胸腺中产生先天样CD8 T细胞中的新作用。先天样CD8 T细胞对应于一小群终末分化的细胞毒性效应T细胞，它们在表型上表现为抗原经历记忆细胞，表达大量的CD44，在功能上类似于促炎CD8 T细胞，表达大量的IFNg。然而，先天样CD8 T细胞与传统的效应记忆CD8 T细胞不同，因为它们在胸腺生成期间和抗原暴露之前获得表型和功能成熟。此外，先天样CD8 T细胞驻留在胸腺，而传统的效应记忆细胞存在于外周组织。了解驱动先天性样CD8 T细胞胸腺发育和分化的分子机制是T细胞免疫研究的热点。然而，选择配体和促进其产生的环境线索仍然知之甚少。在我们最近的研究中，我们发现细胞因子受体gc是先天CD8 T细胞生成的关键介质，令人惊讶的是，即使在缺乏经典mhc - 1分子H-2 KbDb的情况下，它也能促进先天CD8 T细胞的阳性选择。传统的mhc - 1分子对于胸腺中常规CD8 T细胞的生成至关重要，因此kbdb缺乏导致幼稚CD8 T细胞的严重缺乏。然而，令人惊讶的是，我们发现先天CD8 T细胞的产生是独立于经典MHC-I分子的。具体来说，我们发现gc的过表达导致先天CD8 T细胞急剧增加，不仅在WT小鼠中如此，在kbdb缺陷小鼠中也是如此（Won HY et al., 2023， J. Autoimmunity）。这些结果表明，tcr介导的先天CD8 T细胞的选择是独立于经典mhc - 1分子的，至少在胸腺细胞中gc蛋白丰度增加的驱动下是如此。我们进一步评估并定位了其潜在机制，即产生IL-4的不变性NKT （iNKT）细胞的扩增，因此胸腺内IL-4可用性的增加增加了先天CD8 T细胞的选择。总的来说，这些结果揭示了先天CD8 T细胞的选择是由非经典mhc - 1分子介导的，并由gc细胞因子IL-4的丰度调节。由于我们发现gc表达可以显著改变胸腺iNKT细胞的亚群组成，我们将研究扩展到评估其他细胞因子受体在iNKT细胞分化中的作用。iNKT细胞对应于胸腺生成的具有先天样特征和效应功能的T细胞群。根据其效应细胞因子的产生，iNKT细胞的三个主要亚群已经被确定，即产生ifng的NKT1，产生il -4的NKT2和产生il -17的NKT17细胞。重要的是，NKT17是唯一一个产生促炎细胞因子IL-17的亚群。但是，NKT17细胞是如何获得这种能力的，以及是什么选择性地触发了它们的激活，目前还不完全清楚。由于我们之前发现细胞因子受体CD122 （IL-15受体的一个关键成分）是NKT1细胞分化的一个非冗余因子，我们质疑细胞因子受体是否也可能参与NKT17细胞的分化。通过筛选一系列gc和非gc家族细胞因子的表达，我们最近发现TNF超家族细胞因子受体死亡受体3 （DR3）在胸腺NKT17细胞上特异性表达，而在其他胸腺iNKT亚群上大多缺失（Luo S et al., 2023, Cell Mol Life Sci）。重要的是，通过使用激动性抗dr抗体连接DR3分子，以及通过其天然配体TL-1A刺激，我们发现DR3刺激促进了胸腺NKT17细胞的体内活化，并对激动性α - galcer刺激提供了共刺激作用。因此，这些结果确定了胸腺NKT17细胞的特异性表面标记物，可以触发它们的激活并增强它们在体内和体外的效应功能。我们认为这些发现很重要，因为它们扩大了iNKT细胞生成和分化过程中细胞因子受体信号传导的范围和相关性。这些结果进一步为解读小鼠NKT17细胞的作用和功能以及了解iNKT细胞的发育和激活机制提供了新的见解（Liman N and Park JH, 2023, Exp. Mol Med）。其他细胞因子受体，特别是gc家族的细胞因子受体，是否有助于亚群分化仍然是一个问题，我们目前正在用其他基因工程小鼠模型解决这个问题。然而，我们的初步结果表明，细胞因子的作用和需求在很大程度上取决于特定的T细胞类型以及组织环境，并且在评估给定细胞因子的个体作用时需要考虑这些因素。在这方面，我们最近记录了胸腺和外周iNKT细胞的生存需求有很大不同，因此胸腺中的iNKT细胞严重依赖IL-15，而外周的iNKT细胞需要IL-7才能存活（Park JY., 2022, Cell Rep）。这些结果表明，组织特异性细胞因子需求可能与个体细胞因子受体的选择性或优先表达有关，但这些假设需要进一步研究。因此，我们将继续研究IL-7和gc受体表达的调控机制，从而根据组织起源和细胞类型解决细胞因子表达的转录和表观遗传方面的问题。这些研究的主要部分是解决染色质重塑锌指转录因子Ikaros （Ikfz1）的作用，我们提出了作为控制IL-7Ra基因位点可及性的新调节剂。我们的数据表明，Ikfz1在胸腺早期T细胞发育过程中控制细胞表面IL-7Ra的表达，并影响周围T细胞生存/稳态中多种细胞因子受体的表达。为了了解Ikaros如何促进细胞因子受体的调节，特别是IL-7Ra的表达，我们目前正在分析一系列ikfz1条件敲除小鼠，其中ikfz1修饰的小鼠与CD2-Cre重组酶转基因小鼠或CD4-Cre转基因小鼠等杂交。在这些研究的同时，我们还生成了Ikfz1转基因小鼠，以测试IL-7R信号背景下Ikfz1过表达对T细胞生成和稳态的影响。在IL-7R和其他细胞因子受体表达的背景下分析这些小鼠的T细胞发育和分化目前正在研究中。

In this project, our main goal has been to address the role of cytokine receptor expression in the development and differentiation of T cells, specifically focusing on the role of gc family cytokine receptors. In the thymus, gc expression is critical for thymopoiesis and for CD8 lineage commitment, whereby IL-7 signaling provides pro-survival and pro-metabolic cues but also induces the expression of Runx3d that specifies CD8 lineage differentiation. In addition to these effects, we recently identified a new role for gc expression in the generation of innate-like CD8 T cells in the thymus. Innate-like CD8 T cells correspond to a small population of terminally differentiated cytotoxic effector T cells that phenotypically appear as antigen-experienced memory cells, expressing large amounts of CD44, and that functionally resemble proinflammatory CD8 T cells, expressing copious amounts of IFNg. However, innate-like CD8 T cells are distinct from conventional effector-memory CD8 T cells as they acquire phenotypic and functional maturity during their generation in the thymus, and prior to antigen exposure. Moreover, innate-like CD8 T cells are thymus-resident while conventional effector memory cells are found in peripheral tissues. Understanding the molecular mechanisms that drive the thymic development and differentiation of innate-like CD8 T cells is an intensely studied subject in T cell immunity. However, the selecting ligand and the environmental cues that promote their generation remain poorly understood. In our recent studies, we identified the cytokine receptor gc as a critical mediator of innate CD8 T cell generation that, surprisingly, promotes their positive selection of innate CD8 T cells even in the absence of the classical MHC-I molecules, H-2 KbDb. Classical MHC-I molecules are critical for the generation of conventional CD8 T cells in the thymus so that KbDb-deficiency results in severe paucity of naive CD8 T cells. Surprisingly, however, we found that the generation of innate CD8 T cells was independent of classical MHC-I molecules. Specifically, we found that overexpression of gc resulted in a dramatic increase of innate CD8 T cells, not only in WT mice, but also in KbDb-deficient mice (Won HY et al., 2023, J. Autoimmunity). These results indicated that the TCR-mediated selection of innate CD8 T cells is independent of classical MHC-I molecules, at least, when driven by the increased abundance of gc protein in thymocytes. We further assessed and mapped its underlying mechanism to the expansion of IL-4-producing invariant NKT (iNKT) cells, so that it is the increased availability of intrathymic IL-4 that augments the selection of innate CD8 T cells. Collectively, these results unravel the selection of innate CD8 T cells being mediated by non-classical MHC-I molecules and being modulated by the abundance of the gc cytokine, IL-4. Because we found that gc expression can substantially alter the subset composition of thymic iNKT cells, we expanded our studies into assessing the effect of other cytokine receptors in iNKT cell differentiation. iNKT cells correspond to a population of thymus-generated T cells with innate-like characteristics and effector functions. Depending on their effector cytokine production, three major subsets of iNKT cells have been identified, namely IFNg-producing NKT1, IL-4-producing NKT2, and IL-17-producing NKT17 cells. Importantly, NKT17 is the only subset that produces the proinflammatory cytokine IL-17. But, how NKT17 cells acquire this ability and what would selectively trigger their activation remain incompletely understood. Because we previously identified the cytokine receptor CD122, a critical component of the IL-15 receptor, as a non-redundant factor in NKT1 cell differentiation, we questioned whether cytokine receptors could be also involved in the differentiation of NKT17 cells. Be screening the expression of a series of gc and non-gc family cytokines, we recently identified the TNF superfamily cytokine receptor Death Receptor 3 (DR3) being specifically expressed on thymic NKT17 cells and mostly absent on other thymic iNKT subsets (Luo S et al., 2023, Cell Mol Life Sci). Importantly, ligation of DR3 molecules using agonistic anti-DR antibodies, but also by stimulation with its natural ligand TL-1A, we showed that DR3 stimulation promoted the in vivo activation of thymic NKT17 cells and provided costimulatory effects upon agonistic alpha-GalCer stimulation. Thus, these results identified a specific surface marker for thymic NKT17 cells that can trigger their activation and augments their effector functions both in vivo and in vitro. We consider these findings important as they expand the scope and relevance of cytokine receptor signaling in iNKT cell generation and differentiation. Thes result further provide new insights for deciphering the role and function of murine NKT17 cells and for understanding the development and activation mechanisms of iNKT cells in general (Liman N and Park JH, 2023, Exp. Mol Med). Whether other cytokine receptors, specifically of the gc family, contribute to the subset differentiation remains a question that we are currently addressing with other genetically engineered mouse models. However, our preliminary results indicate that the role and requirement for cytokines substantially differ depending on the specific T cell type as well as on the tissue environment, and that these factors need to be considered when assessing the individual role of a given cytokine. In this regard, we have recently documented that the survival requirement for thymic and peripheral iNKT cells dramatically differ, so that iNKT cells in the thymus critically depend on IL-15 but iNKT cells in the periphery require IL-7 for their survival (Park JY., 2022, Cell Rep). These results suggested that the tissue-specific cytokine requirement could have been linked to selective or preferential expression of individual cytokine receptors, but these postulations need to be further investigated. As such, we are continuing our efforts to interrogate the regulatory mechanisms of IL-7 and gc receptor expression whereby we address both the transcriptional and epigenetic aspects of cytokine expression, based on tissue origin and cell type. A major part of these studies is addressing the role of the chromatin remodeling zinc finger transcription factor Ikaros (Ikfz1), which we propose as a novel regulator of controlling the accessibility of IL-7Ra gene locus. Our data showed that Ikfz1 controls the cell surface IL-7Ra expression during the early T cell development in the thymus, and affected multiple cytokine receptor expression in the survival/homeostasis of peripheral T cells. To understand how Ikaros contributes to cytokine receptor regulation in general, and specifically to IL-7Ra expression, we are currently analyzing a series of Ikfz1-conditional knock out mice, whereby Ikfz1-floxed mice were crossed with CD2-Cre recombinase transgenic mice or CD4-Cre transgenic mice, among others. In parallel to these studies, we also generated Ikfz1 transgenic mice to test the effect of Ikfz1 overexpression on T cell generation and homeostasis in the context of IL-7R signaling. Analyzing the T cell development and differentiation of these mice in context of IL-7R and other cytokine receptor expression is currently under investigation.