NK Tolerance and Hematopoietic Cell Transplantation

描述(申请人提供)：自然杀伤(NK)细胞是先天免疫系统的重要组成部分，能够杀死病毒感染的细胞和恶性细胞。自身免疫NK活性是通过抑制受体来避免的，例如识别存在于所有自体组织细胞上的自体人类白细胞抗原(HLAI)类分子的杀伤性Ig样受体(KIR)。然而，NK细胞经常表达个体缺乏适当的HLAI类配体的KIR，但这些NK细胞并不表现出自身反应行为。这导致了“许可模式”，在这种模式下，只有表达KIR的NK细胞才具有功能活性和效应器活性。表达KIR的NK细胞如何实现自身的功能，而表达非自身的KIR的NK细胞如何实现对自身的耐受是我们研究的重点。用6色流式细胞术对NK细胞的反应进行单细胞分析，以研究其功能KIR谱几乎完全由抑制性受体组成的个体，我们证明在稳态个体中，以细胞内干扰素？产量降至I类负面目标。此外，效应器功能的增强与：1)自身人类白细胞抗原特异性KIR定性数增加，2)人类白细胞抗原NK表达增加有关。我们假设，功能功能部分源于细胞固有的人类白细胞抗原和KIR分子，它们的相互作用是效应器功能所必需的。在特定目的1中，我们将利用慢病毒载体导入I类shRNA或KIR/HLAc DNA，研究KIR或HLAs在原代NK细胞和NK细胞系中的表达变化如何扰乱NK细胞的功能。异基因造血细胞移植(HCT)后，我们发现表达非自身人类白细胞抗原KIR的NK细胞最初被赋予功能，但到第100天变得对自身低反应和耐受。这一观察结果支持这样的模型，即在与自体细胞相互作用的循环中经过一段时间后，天然NK细胞实现了自我耐受，从所有抑制性KIR受体能够识别缺乏配体的状态过渡到成熟状态，在成熟状态中，适当的抑制性KIR识别缺乏自我配体(“缺少自我”)。同种异体血细胞移植提供了一个独特的体内环境，移植后早期的造血为检查NK细胞的发育提供了一个窗口。我们假设功能能力可能是通过与表达HLA的自体细胞的反式相互作用来维持的，而表达非自身HL A的KIR的潜在自身反应细胞由于缺乏I类参与而变得无能。特定目的2试图证明，如果在发育过程中培养的细胞在异体朗格汉斯细胞或基质细胞转染剂呈现的表达非自我I类配体的环境中培养，原本将采用低反应性命运的细胞可以保持功能能力。阐明NK细胞是如何获得功能的，不仅对移植环境中的NK同种异体反应性有意义，而且对于将NK细胞作为恶性肿瘤过继细胞治疗的明智应用也具有重要意义。

DESCRIPTION (provided by applicant): 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.