Harnessing activated CD4 T cells to define new mechanisms of protection in tuberculosis

利用活化的 CD4 T 细胞定义结核病的新保护机制

基本信息

批准号：
10735439
负责人：
Tyler Dallas Bold
金额：
$ 59.02万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-01 至 2028-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10735439
关键词：
Address Adoptive Transfer Agonist Antibiotic Therapy Antibodies Antigens Blood Vessels CD4 Positive T Lymphocytes Cause of Death Cell Communication Cell Count Cell Separation Cell Survival Cell physiology Cells Cellular Indexing of Transcriptomes and Epitopes by Sequencing Characteristics Chronic Clonal Expansion Data Development Differentiation Antigens Disease Failure Fluorescence Genetic Transcription Immunity Immunodominant Epitopes Immunologic Receptors Immunotherapeutic agent Immunotherapy Infection Knowledge Lung Mediating Mediator Membrane Proteins Monoclonal Antibodies Mus Mycobacterium tuberculosis Mycobacterium tuberculosis antigens OX40 Persons Phenotype Population Positioning Attribute RNA Regulatory T-Lymphocyte Reporter Resolution Shapes Signal Transduction Site Specificity Structure of parenchyma of lung T cell receptor repertoire sequencing T-Cell Activation T-Cell Receptor T-Lymphocyte Testing Therapeutic Time Tissues Toxic effect Tuberculosis Tuberculosis Vaccines Vaccines Work adaptive immunity chronic infection effector T cell fighting improved in vivo innovation mortality mouse model novel novel marker novel strategies novel therapeutic intervention prevent programs protein expression pulmonary function receptor single-cell RNA sequencing therapeutic target trafficking tuberculosis immunity tuberculosis treatment

项目摘要

CD4 T cells are essential for protection against tuberculosis (TB). But current approaches to TB therapy have not harnessed their potential benefit, and the mechanisms they use to control Mycobacterium tuberculosis (Mtb) have not been completely defined. At the peak of adaptive immunity to TB, millions of Mtb-specific CD4 T cells traffic to the lungs, but very few of them re-encounter their cognate antigen to become activated. The ability to clearly discriminate and isolate these few activated cells from the greater population of inactive CD4 T cells could enable the discovery of important new markers, effector functions, and clonally expanded T cell receptor (TCR) sequences closely associated with protective immunity. To study activated CD4 T cells from Mtb-infected lungs, we used a new approach that combines: (i) a reporter mouse to identify cells actively receiving TCR stimulation in vivo (Nur77-GFP); (ii) BSL3-contained fluorescence cell sorting to isolate them live from tissues; and (iii) single cell RNA sequencing (scRNA-Seq) with CITE-Seq and TCR immune-profiling to interrogate their function and antigen-specificity. In our preliminary work, we discovered that Nur77-GFPHI CD4 T cells express an array of activation markers and costimulatory receptors including OX40. This population was enriched for T regulatory cells and effector T cells with TCR clonotypes localizing to lung parenchyma. Using adoptive transfer, we found that Nur77-GFPHI cells are more protective than their Nur77-GFPLO counterparts. To therapeutically harness activated CD4 T cells in vivo, we treated Mtb-infected mice with a monoclonal antibody that agonizes OX40 and found that this treatment reduced the lung bacterial burden, prolonged survival of infected mice by >100 days and did not cause toxicity. Immunotherapy specifically targeting activated CD4 T cells is a novel approach to TB treatment that has not been thoroughly investigated. In Aim 1 of this proposal, we will define the function of activated CD4 T cells in the lungs of Mtb-infected mice using scRNA-Seq and CITE-Seq. With adoptive transfer and scTCR-Seq, we will uncover how antigen specificity shapes the function and fate of different CD4 T cell TCR clonotypes throughout chronic infection. We will use TCR sequences we generate to develop TCR retrogenic mice that can identify protective Mtb antigens and test the hypothesis that antigen specificity shapes T cell phenotype in TB In Aim 2, we will determine the mechanisms underlying activation marker immunotherapy mediated CD4 T cell control of TB. We will test the hypothesis that OX40 agonist treatment provides protection by selectively modulating the function of both activated T conventional and T regulatory cells. Finally, we will determine how OX40 agonism impacts activated CD4 T cell survival through chronic infection. The studies proposed here represent the first high-definition characterization of the small population of activated CD4 T cells at the site of Mtb infection and the first study exploring the potential of OX40 activation marker targeted immunotherapy in the mouse model of TB. This project will elucidate new mechanisms that may inform the development of more effective TB vaccines and lead to new therapeutic approaches to improve TB treatment.

CD4 T细胞对于防止结核病（TB）至关重要。但是当前的结核病治疗方法已有没有利用其潜在的好处，也没有用于控制结核分枝杆菌的机制（MTB）尚未完全定义。在自适应免疫对结核病的峰值上，数百万MTB特异性CD4 T细胞肺部流量，但很少有人会重新遇到其同源抗原以被激活。能力显然会区分和分离这几个活化的细胞与较大的无活性CD4 T细胞的群体可以启用重要的新标记，效应器功能和克隆扩展的T细胞受体（TCR）序列与保护性免疫密切相关。为了研究来自MTB感染肺的激活CD4 T细胞，我们使用了一种结合结合的新方法：（i）记者鼠标来识别主动接收TCR刺激的细胞体内（NUR77-GFP）; （ii）含有BSL3的荧光细胞分类，使它们从组织中分离出来；（iii）单带有Cite-Seq和TCR免疫促进的细胞RNA测序（SCRNA-SEQ），以询问其功能抗原特异性。在我们的初步工作中，我们发现NUR77-GFPHI CD4 T细胞表达了活化标记和包括OX40在内的共刺激受体。该人群丰富了调节性细胞和效应T细胞，具有TCR clonotypes定位于肺实质。使用收养转移，我们发现该NUR77-GFPHI细胞比其NUR77-GFPLO对应物更具保护性。在治疗上利用激活的CD4 T细胞在体内，我们用一种使OX40和发现这种治疗减轻了肺细菌负担，感染小鼠的长期存活率> 100天并且没有引起毒性。特异性靶向活化CD4 T细胞的免疫疗法是TB的一种新方法尚未彻底研究的治疗。在本提案的目标1中，我们将定义使用SCRNA-SEQ和CITE-SEQ活化的MTB感染小鼠肺中的CD4 T细胞。与收养转移和sctcr-seq，我们将发现抗原特异性如何塑造不同CD4 T细胞TCR的功能和命运整个慢性感染中的克隆型。我们将使用我们生成的TCR序列来发展TCR后源性可以识别保护性MTB抗原并检验抗原特异性形状T细胞的假设的小鼠 AIM 2中TB中的表型，我们将确定激活标记物免疫疗法的机制介导的TB的CD4 T细胞控制。我们将测试OX40激动剂治疗的假设通过选择性调节激活的T传统和T调节细胞的功能。最后，我们会的确定OX40激动剂如何通过慢性感染影响激活的CD4 T细胞存活。研究此处提出的代表了少量活化的CD4 T细胞的第一个高清表征在MTB感染部位和第一项研究，探索了OX40激活标记的潜力 TB小鼠模型中的免疫疗法。该项目将阐明可能告知该机制开发更有效的结核病疫苗并导致新的治疗方法改善结核病治疗。