Functional genetics of human innate immunity in the bimodal gamma delta T cell response to Epstein-Barr Virus and in education of NK cells and their re-education to respond to autologous cells

这个项目调查了人类基因变异如何使对感染的先天免疫反应多样化 癌症。目的1涉及对爱泼斯坦-巴尔病毒(EBV)的先天免疫，EBV是导致癌症的一种原因。我们发现了人类 根据他们对EB病毒的反应形成两个同等规模的小组。第一组制造强大的自然杀伤(NK)细胞 和Vγ9Vδ2T细胞反应，而组2结合了强大的NK细胞反应和较差的Vγ9Vδ2T 细胞反应。这种差异与性别、人类白细胞抗原类型、既往接触EBV或CMV、 两组V-γ9V-δ-2T细胞的丰度或其功能。我们建议这种均衡的平衡 应答者和非应答者的出现是因为使Vγ9Vδ2 T细胞应答的成本和收益 EB病毒。我们之前工作中的遗传先例是两种杀伤细胞在功能上的独特单倍型 免疫球蛋白样受体(KIR)基因家族和人类白细胞抗原I类基因家族。我们将确定基因 以及区别第一组和第二组捐赠者的功能差异，有三种方法。首先，我们将定义 γδT细胞受体用于第1组和第2组供者的静息和EB病毒激活的γδT细胞。第二，我们 将确定第1组和第2组中EB病毒应答Vγ9Vδ2T细胞和NK细胞的表型和功能 个人。第三，用高通量核糖核酸比较第一组和第二组个体的γδT细胞 测序。目的2检测NK细胞和γδT细胞对EB病毒的反应.我们的初步数据显示 在EB病毒的背景下，γδT细胞对NK细胞的功能有抑制作用，并且这种作用是明显的 第1组VS第2组供者。我们建议对NK细胞和γδT细胞的串扰进行检查 NK细胞教育状态和二分性γδT细胞对EB病毒的反应。AIM 3检查交互作用 KIR与控制NK细胞发育和对感染或恶性反应的HLA-A、B和C之间的关系 低表达HLAI类的细胞。这些相互作用涉及四个HLAI类表位和四个 抑制KIR(IKIR)。KIR和人类白细胞抗原的多态性及其独立分离和随机KIR 表达，意味着大多数人表达没有受体的配体，反之亦然。在开发过程中， 表达一个HLA表位及其同源的iKIR的NK细胞被培养成杀死缺乏该表位的细胞。 因此，受过教育的NK细胞可以激发白血病患者的移植物抗肿瘤(GVT)反应 在异基因造血细胞移植后。GVT反应清除残留的肿瘤细胞并改善 病人存活率。初步实验表明，NK细胞可以在体外进行再培养以杀死恶性肿瘤 细胞。我们将确定代表所有12种常见基因的捐赠者队列中再教育的可能性 4个人类白细胞抗原表位的组合。这将为NK细胞的培养提供新的视角和基础 用于再培养自体NK细胞作为白血病的免疫疗法。总而言之，这项工作将定义 三种现象的遗传和机制参数：Vγ9Vδ2T细胞对EB病毒的二分反应， Vγ9Vδ2T细胞与NK细胞的串扰，并在体外对NK细胞进行再培养。

This project investigates how human genetic variation diversifies innate immune responses to infection and cancer. Aim 1 concerns innate immunity to Epstein-Barr Virus (EBV), a cause of cancer. We found humans form two equally sized groups based on their response to EBV. Group 1 make strong Natural Killer (NK) cell and Vγ9Vδ2 T cell responses, whereas Group 2 combine a vigorous NK cell response with a poor Vγ9Vδ2 T cell response. This difference does not correlate with sex, HLA type, prior exposure to EBV or CMV, abundance of Vγ9Vδ2 T cells, or their functionality in the two groups. We propose that this even balance of responders and non-responders arises because of costs and benefits in making a Vγ9Vδ2 T cell response to EBV. Genetic precedents from our previous work are functionally distinctive haplotypes of both the killer cell immunoglobulin-like receptor (KIR) gene family, and the HLA class I gene family. We will determine genetic and functional differences that distinguish Group 1 and 2 donors, with three approaches. First, we will define the γδ T cell receptor repertoires for resting and EBV-activated γδ T cells of Group 1 and 2 donors. Second, we will define the phenotype and function of EBV-responding Vγ9Vδ2 T cells and NK cells in Group 1 and 2 individuals. Third, the γδ T cells of Group 1 and 2 individuals will be compared by high throughput RNA sequencing. Aim 2 examines NK cell and γδ T cell crosstalk in response to EBV. Our preliminary data shows that γδ T cells have an inhibitory effect on NK cell function in the context of EBV, and that this effect is distinct in Group 1 vs Group 2 donors. We propose an examination of NK cell and γδ T cell crosstalk that incorporates both NK cell educational status, and the dichotomous γδ T cell responses to EBV. Aim 3 examines interactions between KIR and HLA-A, B and C that control NK cell development and responses to infected or malignant cells that have low expression of HLA class I. These interactions involve four HLA class I epitopes and four inhibitory KIR (iKIR). The polymorphism of KIR and HLA, their independent segregation and stochastic KIR expression, mean that most humans express ligands without receptors and vice versa. During development, NK cells expressing an HLA epitope and its cognate iKIR, become educated to kill cells that lack the epitope. Consequently, educated NK cells can instigate a graft-versus-tumor (GVT) response in leukemia patients following allogeneic hematopoietic cell transplant. The GVT response clears residual tumor cells and improves patient survival. Preliminary experiments demonstrate that NK cells can be re-educated in vitro to kill malignant cells. We will determine the potential for re-education in a donor cohort representing all 12 common genetic combinations of the 4 HLA epitopes. This will give new insight on NK cell education and provide the foundation for re-educating autologous NK cells as immunotherapy for leukemia. In summary, this work will define the genetic and mechanistic parameters of three phenomena: the dichotomous Vγ9Vδ2 T cell response to EBV, Vγ9Vδ2T cell crosstalk with NK cells, and in vitro NK cell re-education.