In depth characterization of Mtb specific HLA-E restricted human CD8+ T-cells

1对结核分枝杆菌(结核分枝杆菌)的免疫一直是一系列详细调查的主题 20年。大多数研究都集中在经典受限的(MHC Ia或II类)T细胞上，但最近，非 3经典受限的T细胞已经成为人们越来越感兴趣的对象。非经典递呈分子 4(包括人类CD1a、b、c、d；MR1；人类白细胞抗原-E和其他人类白细胞抗原Ib类分子)已被证明 5向人T细胞递送Mtb抗原。人类白细胞抗原E是一种特别有趣的分子，原因有很多： 6首先，只描述了两个编码变体，它们只有一个氨基酸不同，位于 7肽结合沟，使人类白细胞抗原-E本质上是一种保守的、几乎单态的呈递方式 8分子；其次，相对于人类白细胞抗原Ia类分子，人类白细胞抗原-E相对抵抗HIV介导者 9下调，以便在艾滋病毒感染期间保持抗原呈递；第三，人类白细胞抗原-E是 10存在于含有胞内室的结核分枝杆菌中。最后，我们最近发现人类CD8+T- 结核分枝杆菌感染者血液中存在11个识别人类白细胞抗原E提呈的结核分枝杆菌多肽的细胞。 这些细胞具有非常规的表型和功能：它们具有细胞溶解特性，能够 13控制结核分枝杆菌的细胞内生长，但意外地主要产生包括IL-4、IL-5在内的Th2细胞因子 14和IL-13，并通过IL-4有效地帮助B细胞。 15在本项目中，我们提出了对这些人类白细胞抗原-E限制性T细胞的深入表征，包括 16我们已生成的可用T细胞克隆的详细特征以及通过计数和 17临床相关队列(血液和BAL)中人类白细胞抗原-E限制性T细胞的特征。第一部分 18该项目将表征结核分枝杆菌特异的、人类白细胞抗原E限制的T细胞克隆，首先关注它们的T细胞受体 19(TCR)，包括对TCR的肽识别和分子结构的要求。此外， 将使用20个克隆来破译细胞内结核分枝杆菌生长抑制的机制，因为这将是 21对于理解对结核分枝杆菌的保护性免疫很重要。在项目的第二部分，我们将 22计数结核分枝杆菌感染者和接种结核分枝杆菌疫苗的人群中人类白细胞抗原-E限制性T细胞 23卡介苗，包括静脉注射和雾化吸入。这些人体研究将与平行的卡介苗接种衔接起来。 非人灵长类动物的24项研究，以验证疫苗接种后人类白细胞抗原-E限制性T细胞反应 25以及跟随结核病挑战。对于所有这些研究，将对银行样本进行独特的收集 26由顶尖合作伙伴(来自英国、意大利和SA)提供。最后，对四聚体(TM)阳性细胞进行分选 27以允许RNA测序，以全面表征人类白细胞抗原-E限制的CD8+T细胞反应。 28所有这些研究预计将在功能特征、临床特征方面产生重要的新见解 29人类结核分枝杆菌特异性、人类白细胞抗原E限制性CD8+T细胞的相关性和疫苗动力学。这将提供新颖的 30对它们作为结核病生物标记物和未来结核病疫苗靶标的效力的见解。

1 Immunity to Mycobacterium tuberculosis (Mtb) has been the subject of detailed investigations for a number of 2 decades. Most studies have focused on classically restricted (MHC class Ia or II) T-cells, but recently, non- 3 classically restricted T-cells have become subject of increasing interest. Non-classical presentation molecules 4 (which include human CD1a,b,c,d; MR1; HLA-E and other HLA class Ib molecules) have been shown to 5 present Mtb antigens to human T-cells. HLA-E is a particularly interesting molecule, for a number of reasons: 6 first, there are only 2 coding variants described, which differ in only a single amino acid, located outside the 7 peptide binding groove, such that HLA-E essentially is a conserved, virtually monomorphic presentation 8 molecule; secondly, in contrast to HLA class Ia molecules, HLA-E is relatively resistant to HIV mediated 9 downregulation, such that antigen presentation should be maintained during HIV infection; thirdly, HLA-E is 10 present in Mtb containing intracellular compartments. Finally, we have recently shown that human CD8+ T- 11 cells recognizing Mtb peptides presented by HLA-E are present in the blood of Mtb infected individuals. 12 These cells have an unconventional phenotype and function: they had cytolytic properties, were able to 13 control intracellular outgrowth of Mtb, but unexpectedly produced mainly Th2 cytokines, including IL-4, IL-5 14 and IL-13, and efficiently helped B-cells through IL-4. 15 In the present project we propose an in depth characterization of these HLA-E restricted T-cells, both by 16 detailed characterization of available T-cell clones we have generated as well as by enumeration and 17 characterization of HLA-E restricted T-cells in (blood and BAL of) clinically relevant cohorts. The first part of 18 the project will characterize Mtb specific, HLA-E restricted T-cell clones, focusing first on their T-cell receptor 19 (TCR), including the requirements for peptide recognition and molecular structure of the TCR. In addition, the 20 clones will be employed to decipher the mechanism of intracellular Mtb growth inhibition, as this will be 21 important for understanding protective immunity towards Mtb. In the second part of the project, we will 22 enumerate HLA-E restricted T-cells in cohorts of Mtb infected individuals and individuals vaccinated with 23 BCG, both parenteral and aerosolized. These human studies will be bridged to parallel BCG vaccination 24 studies in non-human-primates, to allow validation of HLA-E restricted T-cell responses following vaccination 25 as well as following TB challenge. For all these studies unique collections of banked samples will be made 26 available by top edge collaborators (from UK, Italy and SA). Finally, tetramer (TM) positive cells will be sorted 27 to allow for RNA sequencing for full characterization of HLA-E restricted CD8+ T-cell responses. 28 All together these studies are expected to yield important new insights in the functional characteristics, clinical 29 relevance and vaccine kinetics of human Mtb specific, HLA-E restricted CD8+ T-cells. This will provide novel 30 insights into their potency as TB biomarkers and future TB vaccine targets.