NK Tolerance and Hematopoietic Cell Transplantation

描述（由申请人提供）：自然杀伤（NK）细胞是先天免疫系统的重要组成部分，能够杀死病毒感染细胞和恶性细胞。自身免疫NK活性可通过抑制性受体（如杀手igg样受体（KIR））来避免，这些受体可识别存在于所有自体组织细胞上的人类白细胞抗原（HLA） I类分子。然而，NK细胞经常表达个体缺乏适当HLA I类配体的KIRs，然而这些NK细胞不表现出自身反应行为。这导致了“许可模型”，即只有NK细胞对自身hla配体表达KIR才具有功能能力和效应活性。表达KIR的NK细胞如何实现自身HLA的功能能力，而表达KIR的NK细胞如何实现对非自身HLA的耐受是我们拟研究的重点。使用6色流式细胞术对NK反应进行单细胞分析，研究了功能性KIR库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，自我hla类I的抑制性KIR表达赋予NK细胞功能能力，细胞内IFN-？生产达到I类负指标。此外，效应函数的增加与：1)自身HLA特异性KIR的高定性数量和2)HLA的高NK表达之间存在相关性。我们假设功能能力部分源于细胞固有的HLA和KIR分子，它们的相互作用是效应功能所必需的。在Specific Aim 1中，我们将使用慢病毒构建体引入I类shRNA或KIR/HLA cDNA，研究KIR或HLA在原代NK细胞和NK细胞系中的表达改变如何扰乱NK细胞的功能能力。同种异体造血细胞移植（HCT）后，我们发现表达KIR的NK细胞最初被赋予非自身HLA的功能能力，但在第100天变得低反应和对自身的耐受。这一观察结果支持了一个模型，即初始NK细胞在与自体细胞相互作用循环一段时间后实现自我耐受，从所有抑制性KIR受体都能够识别缺乏配体（“缺失配体”）的状态过渡到成熟状态，其中适当的抑制性KIR识别缺乏自我配体（“缺失自我”）。同种异体HCT提供了一种独特的体内环境，在这种环境中，移植后早期的造血功能为检查NK细胞的发育提供了一个窗口。我们假设功能能力可能通过与表达HLA的自体细胞的相互作用来维持，而潜在的表达KIR的非自体HLA的自体反应性细胞由于缺乏I类参与而变得无能。特异性目标2试图证明，如果在表达异体朗格汉斯细胞或基质细胞转染物的非自体I类配体的环境中培养，本来会采取低反应命运的细胞可以保持功能能力。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，而且对NK细胞作为恶性肿瘤的过继细胞治疗的明智应用也有影响。

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.