Control of Treg Homeostasis and Function by the Lipid Phosphatase PTEN

描述（由申请人提供）：我们的实验室对移植和自身免疫的免疫耐受有着长期的兴趣，特别关注调节性T细胞（Tregs）。Tregs在人和小鼠中的主要群体是通过表达x连锁转录因子Foxp3来定义的。虽然这些细胞是正常免疫稳态所必需的，但越来越多的数据表明，这种细胞谱系可能是不稳定的（例如，由于IL-2、TCR和CD28刺激不足，或由于暴露于炎症细胞因子），这意味着treg可以恢复/转化为效应T细胞，从而导致对自身或移植同种异体移植物的耐受性丧失。控制淋巴细胞谱系规范和反应性的关键途径之一是磷酸肌苷激酶（PI3K）途径，该途径可以通过多种表面受体激活，包括CD28和IL-2R。虽然这一途径对传统的T细胞反应至关重要，但它在Tregs中的功能可能有限。事实上，过度激活PI3K通路会显著抑制Treg的发育，而同样通过IL-2R激活的STAT5通路则会强烈促进Treg的发育。T细胞中PI3K活性的主要调节因子是脂质磷酸酶PTEN（10号染色体上的磷酸酶和张力素同源物）。本课题的目的是确定Treg中的PTEN如何控制Treg稳态，PI3K和STAT5信号的整合，以及药物靶向PI3K通路是否可以稳定Treg。为了实现这一目标，我们通过培养PTENfl/fl等位基因Foxp3-YFP-Cre敲入的小鼠或BAC转基因动物来产生PTEN-¿Treg小鼠，从而在Treg中特异性地缺失PTEN。令人惊讶的是，尽管这些小鼠的treg数量升高，但这些细胞中CD25-和CD62Llo的比例很高，并且这些动物患上了严重的多克隆淋巴细胞增生性疾病。这使我们提出了一个假设，即PTEN的丢失会破坏Treg的稳态，原因是细胞因子受体表达减少、迁移改变和调节能力明显丧失。本资助的目的是确定Treg中的PTEN如何控制Treg稳态，PI3K和STAT5信号的整合，以及靶向PI3K通路的药物是否可以稳定Treg。为此，我们有两个目标。在Aim #1中，使用Treg特异性PTEN缺失的Treg命运定位小鼠，我们将在稳态条件下和模型自身免疫性疾病中检查PTEN缺失对自然和适应性Treg稳定性和功能的影响。在Aim #2中，我们将剖析CD25、PI3K和STAT5的下游信号，以确定哪些是导致我们观察到的Treg表型和功能变化的原因。我们的研究将对Treg信号通路产生新的见解，并为增强免疫耐受提供潜在的治疗策略。

DESCRIPTION (provided by applicant): Our laboratories have a long-standing interest in immune tolerance in transplantation and autoimmunity, with a particular focus on regulatory T cells (Tregs). The primary population of Tregs in humans and mice is defined by expression of the X-linked transcription factor Foxp3. While these cells are required for normal immune homeostasis increasing data suggests that this lineage of cells may be unstable (e.g., as a result for example of inadequate IL-2, TCR and CD28 stimulation, or due to exposure to inflammatory cytokines), meaning that Tregs can revert/convert to effector T cells and thus contribute to loss of tolerance to self or to transplanted allografts. One of the key pathways controlling lymphocyte lineage specification and responsiveness is the phosphoinositide 3-kinase (PI3K) pathway, which can be activated via multiple surface receptors, including, most prominently, CD28 and the IL-2R. While this pathway is essential for conventional T cell responses, it may have limited, if any, function in Tregs. In fact, over activation of the PI3K pathway dramatically inhibits Treg development, while the STAT5 pathway, which is also activated through the IL-2R, strongly promotes Tregs. The primary regulator of PI3K activity in T cells is the lipid phosphatase PTEN (phosphatase and tensin homolog on chromosome 10). The goal of this proposal is to determine how PTEN in Tregs controls Treg homeostasis, the integration of PI3K and STAT5 signals, and whether drug targeting of the PI3K pathway can stabilize Tregs. To accomplish this, we created mice with PTEN deleted specifically in Tregs by breeding mice with a PTENfl/fl allele with Foxp3-YFP-Cre knock-in or BAC transgenic animals to generate PTEN-¿Treg mice. Surprisingly, although these mice have elevated numbers of Tregs, a high proportion of those cells are CD25- and CD62Llo, and the animals develop a severe polyclonal lymphoproliferative disorder. This has led us to formulate the hypothesis that PTEN loss disrupts Treg homeostasis due to reduced cytokine receptor expression, altered migration and apparent loss of regulatory capacity. The goal of this grant is to determine how PTEN in Tregs controls Treg homeostasis, the integration of PI3K and STAT5 signals, and whether drug targeting of the PI3K pathway can stabilize Tregs. To do so, we have two aims. In Aim #1, employing Treg fate mapping mice with Treg specific deletion of PTEN, we will examine the effects of loss of PTEN on natural and adaptive Treg stability and function, both under homeostatic conditions and in a model autoimmune disease. In Aim #2, we will dissect the signals downstream of CD25, PI3K and STAT5 to determine which are responsible for the Treg phenotypic and functional changes we have observed. Our studies will yield new insights into Treg signaling pathways and provide potential therapeutic strategies to enhance immune tolerance.