Effector Mechanisms in Graft Tolerance

描述（由申请人提供）：免疫耐受被认为是通过接受同种异体皮肤移植来衡量的“冷漠或无反应性”。虽然被认为是由于缺乏免疫反应，但我们现在知道，除了克隆能量外，主动抑制对移植物的持久性至关重要。这一建议的重点是积极的，有效的机制，基础上的同种异体移植物在耐受受体的寿命。移植物耐受的细胞复杂性可以通过使用定义明确的异体抗原特异性的效应T细胞（Teff）和调节性T细胞（Treg）的TCR Tg群体来降低。我们提出了一个TCR Tg系统，可用于研究移植排斥和体内耐受的细胞动力学，并有助于确定Treg如何控制Teff体内功能的潜在分子机制。本研究的具体目的是：1)了解Treg如何控制初始和启动宿主体内同种异体CD4+和CD8+ T细胞的初始扩增和效应功能，从而控制移植物排斥反应。天然Treg （Treg）和自适应Treg （Tr1）对一个单一的，定义的类限制性，同种异体表位在体外和体内阻止Teff反应。Treg到单一定义表位如何在完整的H-2差异中阻止同种异体反应将是本目标的重点。探讨抗原特异性Treg对CD4+和CD8+ Teff生长、分化、效应因子功能和基因表达的影响，以及Treg自身的调控条件。最后，数据表明Treg也可能具有控制记忆T细胞反应的能力，因此将确定Treg是否可以减轻移植物在致敏宿主中的排斥反应。2)描述允许移植物长时间存活的细胞机制。虽然Treg最初建立耐受状态，但有证据表明Treg具有催化和“传染性”。研究建议描述抑制如何随着时间的推移而传播。通过创建含有Foxp3gfp敲入等位基因（Foxp3表达GFP）的同种异体反应性TCR Tg T细胞（Treg和Teff），可以确定Teff在体内向抗原特异性适应性Treg的分化。不同Treg亚群对移植物存活的功能重要性将通过使用遗传策略来确定转移后删除定义的Treg亚群。我们假设随着时间的推移，与移植物存活相关的Treg亚群将发生变化。3. 定义控制同种异体T细胞反应并允许移植物长时间存活的接触依赖因子和可溶性因子。我们专注于已知的（TGFb和IL-10）和新的介质，旨在解决这些分子如何控制耐受性。本文描述了确定TGFb和IL-10对耐受性“感染性”方面的贡献的深入研究。其次，数据显示Treg产生的颗粒酶B （granzyme B， GZB）参与体内移植物耐受。研究提出了确定GZB的细胞靶点及其在维持移植物存活中的作用模式。新的证据强烈暗示IL-9和肥大细胞可能是外周耐受性的重要因素，并且研究表明它们在功能上的参与。

DESCRIPTION (provided by applicant): Immunologic tolerance was conceived as an "indifference or non-reactivity" measured by the acceptance of allogeneic skin grafts. While thought to be due to the lack of immune responsiveness, we now know that, in addition to clonal anergy, active suppression is essential for graft persistence. This proposal focuses on the active, effector mechanisms that underlie the longevity of allografts in the tolerant recipient. The cellular complexity of graft tolerance can be reduced by using well-defined, alloantigen-specific, TCR Tg populations of effector T cells (Teff) and regulatory T cells (Treg). We present a TCR Tg system can be used to study the cellular dynamics of graft rejection and tolerance in vivo, and can help to define the underlying molecular mechanisms of how Treg control Teff function in vivo. The specific aims of this proposal are: 1) To understand how Treg control the initial expansion and effector function of alloreactive CD4+ and CD8+ T cells in the naive and primed host to control graft rejection. Natural Treg (Treg) and adaptive Treg (Tr1) to a single, defined class ll-restricted, allogeneic epitope arrest Teff responses in vitro and in vivo. How Treg to a single defined epitope can arrest allogeneic responses across complete H-2 disparities will be the focus of this aim. The impact of antigen-specific Treg on CD4+ and CD8+ Teff growth, differentiation, effector function and gene expression will be explored, as well as the conditions that control Treg themselves. Finally, data suggests that Treg may also have the capacity to control memory T cell responses and thus it will be determined if Treg can mitigate graft rejection in the sensitized host. 2) To delineate the cellular mechanisms that allow for long-lived graft survival. While Treg initially establish a state of tolerance, evidence suggest that Treg are catalytic and "infectious". Studies are proposed to delineate how suppression spreads" over time. By creating alloreactive TCR Tg T cells (Treg and Teff) containing the Foxp3gfp knock- in allele (that expresses GFP upon Foxp3 expression), the differentiation of Teff to antigen-specific adaptive Treg in vivo can be determined. The functional importance of distinct Treg subsets to graft survival will be determined by using genetic strategies to delete defined Treg subsets following transfer. We hypothesize that over time, the relevant Treg subsets to graft survival will change. 3. To define the contact-dependent and soluble factors that control alloreactive T cell responses and allow long-lived graft survival. We focus on known (TGFb and IL-10) and new mediators with the intent of solving how these molecules control tolerance. Incisive studies to determine the contribution of TGFb and IL-10 to "infectious" aspects of tolerance are described. Second, data shows that granzyme B (GZB) production by Treg is involved in graft tolerance in vivo. Studies are presented to identify the cellular targets of GZB and its mode of action in sustaining graft survival. New evidence strongly implicates IL-9 and mast cells as elements that may be important to peripheral tolerance, and studies are presented to functionally implicate their involvement.