NK Tolerance and Hematopoietic Cell Transplantation

摘要 自然杀伤(NK)细胞是先天免疫系统的重要组成部分，能够 杀死受病毒感染的细胞和恶性细胞。通过抑制来避免自身免疫的NK活性 识别自身人类白细胞抗原的受体，如杀伤Ig样受体(KIR) (HL A)类分子存在于所有自体组织细胞上。然而，NK细胞经常 个体缺乏适当的HLAI类配体的快速KIR，但这些NK 细胞不会表现出自身反应行为。这导致了只有自然杀伤细胞(NK细胞)的“许可模式” 表达KIR的自身-人类白细胞抗原配体具有功能活性和效应性。 表达自身人类白细胞抗原KIR的NK细胞是如何实现功能的 表达非我人类白细胞抗原KIR实现对自身的耐受是我们提出的重点 学习。应用六色流式细胞术分析个体NK细胞应答 其功能的KIR谱系几乎完全由抑制性受体组成，我们 证明在稳定期个体中，自身HLAI类抑制性KIR的表达 通过细胞内干扰素的产生来评估NK细胞的功能 我否定了目标。此外，增加的效应器功能与：1) 自身人类白细胞抗原KIR定性数高，2)人类白细胞抗原NK高表达。我们 假设功能能力部分源于人类白细胞抗原和KIR分子所固有的 细胞，其相互作用是效应器功能所必需的。在特定目标1中，使用慢病毒 构建引入I类shRNA或KIR/HLAc DNA，我们将研究如何改变 原代NK细胞和NK细胞株中的KIR或HLA干扰了NK细胞的功能。 异基因造血细胞移植(HCT)后，我们发现NK细胞 表达KIR的非自身人类白细胞抗原最初被赋予功能能力，但后来 到第100天，对自己反应迟钝和宽容。这一观察结果支持了这样一种模型，即 NK细胞在与自体细胞相互作用的循环中经过一段时间后达到自我耐受， 从所有抑制性KIR受体能够识别缺乏配体的状态过渡 (“缺失配体”)到成熟状态，其中适当的抑制性KIR识别缺乏自我 配基(“失落的自我”)。同种异体红细胞移植提供了一个独特的体内环境，在这个环境中， 在移植后早期为研究NK细胞的发育提供了一个窗口。我们 假设功能能力可能通过与人类白细胞抗原-1的反式相互作用来维持 表达自体细胞，以及表达非自身人类白细胞抗原KIR的潜在自身反应细胞 由于缺乏第一类参与而变得麻木。具体目标2试图证明 如果培养，原本会采取低反应性命运的细胞可以保持功能能力 在表达非自我I类配体的环境中的开发过程中，如 同种异体朗格汉斯细胞或基质细胞转染体。阐明NK细胞如何实现 功能能力不仅对移植环境中的NK同种异体反应性有影响，而且 也用于明智地应用NK细胞作为恶性肿瘤的过继细胞治疗。

Abstract Natural killer (NK) cells are an important component of the innate immune system and are capable of killing virally infected cells and malignant cells. Autoimmune NK activity is avoided by inhibitory receptors such as the killer Ig-like receptors (KIR) that recognize self-human leukocyte antigens (HLA) class I molecules present on all autologous tissue cells. NK cells, however, frequently express KIRs for which the individual lacks the appropriate HLA class I ligand, and yet these NK cells do not exhibit autoreactive behavior. This has led to the "licensing model" where only NK cells expressing KIR for self-HLA ligands are functionally competent and capable of effector activity. How NK cells expressing KIR for self-HLA achieve functional competence while NK cells expressing KIR for non-self HLA achieve tolerance to self is the focus of our proposed studies. Using 6-color flow cytometry for single cell analysis of NK response to study individuals whose functional KIR repertoire is comprised nearly completely by inhibitory receptors, we demonstrate that in the steady state individual, expression of inhibitory KIR for self-HLA class I confers functional competence to the NK cell as assessed by intracellular IFN-¿ production to class I negative targets. Furthermore, there is a correlation between increased effector function and: 1) higher qualitative numbers of KIR specific for self-HLA, and 2) higher NK expression of HLA. We hypothesize that functional competence in part results from HLA and KIR molecules inherent to the cell, whose interaction is necessary for effector function. In Specific Aim 1, using lentiviral constructs to introduce class I shRNA or KIR/HLA cDNA, we will study how altered expression of KIR or HLA in primary NK cells and NK cell lines perturbs the functional capacity of NK cells. Following allogeneic hematopoietic cell transplantation (HCT), we have found that NK cells expressing KIR for non-self HLA are initially endowed with functional capacity, but become hyporesponsive and tolerant to self by day 100. This observation supports the model that the na¿ve NK cell achieves self-tolerance after a period in the circulation of interaction with autologous cells, transitioning from a state where all inhibitory KIR receptors are capable of recognizing lack of ligand ("missing ligand") to a mature state where the appropriate inhibitory KIR recognize lack of self- ligand ("missing self"). Allogeneic HCT offers a unique in vivo environment in which hematopoiesis in the early post-transplant period provides a window to examine NK cell development. We hypothesize that functional competence may be sustained through trans-interaction with HLA- expressing autologous cells, and potentially autoreactive cells expressing KIR for non-self HLA are rendered anergic through lack of class I engagement. Specific Aim 2 seeks to demonstrate that cells that would otherwise adopt a hyporesponsive fate can retain functional competence if cultured during development in an environment expressing non-self class I ligands as presented by allogeneic Langerhans cells or stromal cell transfectants. Elucidating how NK cells achieve functional competence not only has implications for NK alloreactivity in the transplant setting, but also for judicious application of NK cells as adoptive cellular therapy for malignancies.