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

1对结核分枝杆菌（Mtb）的免疫力已经成为许多研究的主题。 二十年了大多数研究都集中在经典限制（MHC Ia或II类）T细胞，但最近，非- 3经典限制性T细胞已成为越来越感兴趣的主题。非经典呈递分子 4（包括人CD 1a、B、c、d; MR 1; HLA-E和其它HLA Ib类分子）已被证明 5将Mtb抗原呈递给人T细胞。HLA-E是一种特别有趣的分子，原因如下： 首先，仅描述了2种编码变体，其仅在单个氨基酸上不同，位于编码变体之外。 7肽结合沟，使得HLA-E基本上是保守的，实际上是单态性呈递 第二，与HLA Ia类分子相比，HLA-E对HIV介导的免疫应答具有相对抗性。 9下调，使得在HIV感染期间抗原呈递应得以维持;第三，HLA-E下调， 10存在于含有Mtb的细胞内区室中。最后，我们最近表明，人CD 8 + T- 识别HLA-E呈递的Mtb肽的细胞存在于Mtb感染个体的血液中。 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Mtb的细胞内生长，但出乎意料地主要产生Th 2细胞因子，包括IL-4、IL-5 14和IL-13，并通过IL-4有效地帮助B细胞。 在本项目中，我们提出了这些HLA-E限制性T细胞的深入表征， 16我们已经生成的可用T细胞克隆的详细表征以及通过计数和 图17临床相关群组（的血液和BAL）中HLA-E限制性T细胞的表征。的第一部分 该项目将表征Mtb特异性HLA-E限制性T细胞克隆，首先关注其T细胞受体 19（TCR）中所描述的，包括对TCR的肽识别和分子结构的要求。此外该 将采用20个克隆来破译细胞内Mtb生长抑制的机制，因为这将是 21对于理解对Mtb的保护性免疫很重要。在项目的第二部分，我们将 22计数Mtb感染的个体和接种Mtb疫苗的个体的群组中的HLA-E限制性T细胞。 23卡介苗，注射和雾化。这些人体研究将与平行的卡介苗接种相衔接 24项非人灵长类动物研究，以验证接种疫苗后HLA-E限制性T细胞应答 25、结核病的治疗。对于所有这些研究，将收集独特的库存样本 26个由顶级合作者提供（来自英国、意大利和南非）。最后，将分选四聚体（TM）阳性细胞 27以允许RNA测序用于HLA-E限制性CD 8 + T细胞应答的完全表征。 28所有这些研究预计将在功能特征、临床 人Mtb特异性、HLA-E限制性CD 8 + T细胞的相关性和疫苗动力学。这将提供新的 30个关于它们作为结核病生物标志物和未来结核病疫苗靶点的潜力的见解。

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.