HLA-E restricted T-cell immunity in Mycobacterium tuberculosis infection

1结核分枝杆菌(Mtb)是一种非常成功的致病菌，具有很高的发病率和死亡率。那里 2广泛的共识是，新的疫苗和疫苗接种战略对于控制这种猖獗的疾病至关重要。这样的一个 策略是诱导T细胞对结核分枝杆菌抗原的免疫，这些抗原是通过保守的、非经典的抗原提呈呈现的。 4人类白细胞抗原-E、CD1、MR1等分子。人类白细胞抗原-E在结核病方面特别令人感兴趣：(1)有2个 5个编码变异体，在肽结合沟外只有一个氨基酸不同；(2)人类白细胞抗原-E相对抵抗 6 HIV合并感染介导的下调；(3)人类白细胞抗原-E在结核分枝杆菌噬菌体中富含，有利于其与结核分枝杆菌的装载 在小鼠中，人类白细胞抗原-E(Qa-1b)限制的CD8+T细胞与保护作用有关。我们已经证明了 8多个MTB表位可由人类白细胞抗原E呈递，并可被MTB特异性CD8+T细胞识别，具有独特的 9功能特性，包括抑制细胞内结核分枝杆菌生长的能力。我们工作的长期目标是 10了解结核病中多肽配体、人类白细胞抗原E和T细胞受体(TCR)分子之间的分子相互作用，以及 11将这一知识用于改进的结核病疫苗接种战略。 12我们建议的目标是详细定义人类白细胞抗原-E、多肽和TCR分子之间的分子相互作用 13，并研究感染和疾病过程中人类白细胞抗原-E限制性T细胞对这些多肽的反应。为此， 14我们将定义控制HLA-E、Mtb多肽之间相互作用的确切分子和结构决定因素 15和TCR分子(目标1)。在这个目标中，我们将研究与两者结合的多肽的分子要求。 16个人类白细胞抗原E变异体分子，并利用这些信息识别结核分枝杆菌基因组中具有改进的人类白细胞抗原-E的新表位。 17E绑定和演示属性。其次，将利用现有的TCR序列来生成被转换的TCR 18个细胞株，以评估TCR激活的关键多肽残基。这一目标的预期结果是在 19人类白细胞抗原-E、多肽和TCR分子之间的分子相互作用，导致TCR的有效激活。 20其次，我们将确定Mtb肽/人类白细胞抗原-E/TCR相互作用在非人类中的功能和生物学意义 21只灵长类动物(NHP)和人类结核分枝杆菌感染、疫苗接种和控制人类感染(CHI，使用雾化卡介苗) 22(目标2)。我们将构建和使用Mtb多肽/人类白细胞抗原-E四聚体库来确定Mtb多肽的频率和功能 23来自卡介苗接种者、潜伏期或活动性结核分枝杆菌感染的人样本中的特定CD8+T细胞，以及来自 24小时，将分析气雾剂-卡介苗诱导的粘膜免疫应答的人类白细胞抗原-E限制性T细胞频率 25和功能。为了将人类白细胞抗原-E限制性T细胞与保护性免疫联系起来，我们将四聚体样本 26来自银行的NHP研究，其中包括已知结果的MTB挑战。这一目标的预期结果是 27关于人类和NHP中人类白细胞抗原-E限制性T细胞与感染、疾病和疫苗接种相关性的见解。 28这些结果将影响并显著加深我们对这些非经典T细胞的作用的理解 29在结核分枝杆菌感染、结核病、疫苗接种和控制人类卡介苗感染方面，并为治理铺平道路 30为未来的结核病疫苗设计提供这方面的知识。

1 Mycobacterium tuberculosis (Mtb) is a remarkably successful pathogen that causes high morbidity and mortality. There 2 is wide consensus that novel vaccines and vaccination strategies are essential to control this rampant disease. One such 3 strategy is inducing T-cell immunity to Mtb antigens that are presented by conserved, non-classical antigen presentation 4 molecules such as human HLA-E, CD1 and MR1. HLA-E is particularly interesting in the context of TB: (1) there are 2 5 coding variants that differ in only one amino acid outside the peptide binding groove; (2) HLA-E is relatively resistant to 6 HIV co-infection mediated downregulation; (3) HLA-E is enriched in Mtb phagosomes, facilitating its loading with Mtb 7 peptides; and (4) in mice, HLA-E (Qa-1b) restricted CD8+ T-cells are associated with protection. We have shown that 8 multiple Mtb epitopes can be presented by HLA-E and are potently recognized by Mtb specific CD8+ T-cells with unique 9 functional properties, including the ability to inhibit intracellular Mtb growth. The long-term goal of our work is to 10 understand the molecular interactions between peptide ligands, HLA-E and T-cell receptor (TCR) molecules in TB, and 11 to harness this knowledge into improved TB vaccination strategies. 12 The objective of our proposal is to define in detail the molecular interactions between HLA-E, peptide and TCR molecules 13 and to characterize HLA-E restricted T-cell responses against these peptides during infection and disease. To this end 14 we will define the exact molecular and structural determinants governing interactions between HLA-E, Mtb peptides 15 and TCR molecules (aim 1). In this aim we will investigate the molecular requirements governing peptide binding to both 16 HLA-E variant molecules and utilize that information to identify novel epitopes in the Mtb genome with improved HLA- 17 E binding and presentation properties. Secondly, available TCR sequences will be employed to generate TCR transduced 18 cell lines to assess critical peptide residues for TCR activation. The expected outcome of this aim is detailed insights in 19 the molecular interactions between HLA-E, peptide and TCR molecules that lead to productive TCR activation. 20 Secondly, we will define the functional and biological significance of Mtb peptide/HLA-E/TCR interactions in non-human 21 primates (NHP) and in human Mtb infection, vaccination and controlled human infection (CHI, with aerosolized BCG) 22 (aim 2). We will construct and use Mtb peptide/HLA-E tetramer pools to determine the frequency and functionality of 23 specific CD8+ T-cells in human samples from BCG vaccinees, latent or active Mtb infection, as well as BAL samples from 24 CHI, in which aerosol-BCG induced mucosal immune responses will be analysed for HLA-E restricted T-cell frequencies 25 and functionalities. To correlate HLA-E restricted T-cells with protective immunity, we will tetramer-profile samples 26 from banked NHP studies that included Mtb challenges with known outcome. The expected outcome of this aim is 27 insights in correlations of HLA-E restricted T-cells with infection, disease and vaccination in humans and NHP. 28 Together these results will impact and significantly deepen our understanding of the role of these non-classical T-cells 29 in Mtb infection, TB disease, vaccination and controlled human BCG infection, and pave the way towards harnessing 30 this knowledge for future TB vaccine design.