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 细胞对于预防结核病 (TB) 至关重要。但目前的结核病治疗方法没有利用它们的潜在益处以及它们用来控制结核分枝杆菌 (Mtb) 的机制尚未完全定义。在结核病适应性免疫的高峰期，数以百万计的结核分枝杆菌特异性 CD4 T 细胞运输到肺部，但很少有它们重新遇到同源抗原而被激活。有能力清楚地区分这些少数活化细胞并将其与大量非活性 CD4 T 细胞区分开来能够发现重要的新标记物、效应子功能和克隆扩展的 T 细胞受体 (TCR) 与保护性免疫密切相关的序列。为了研究 Mtb 感染肺部的活化 CD4 T 细胞，我们使用了一种新方法，结合了：(i) 报告小鼠来识别积极接受 TCR 刺激的细胞体内（Nur77-GFP）； (ii) 包含 BSL3 的荧光细胞分选，将它们从组织中分离出来； (iii) 单身使用 CITE-Seq 和 TCR 免疫分析进行细胞 RNA 测序 (scRNA-Seq)，以探究其功能并抗原特异性。在我们的前期工作中，我们发现 Nur77-GFPHI CD4 T 细胞表达一系列激活标记物和共刺激受体，包括 OX40。这个群体的 T 调节能力得到了丰富具有定位于肺实质的 TCR 克隆型的细胞和效应 T 细胞。通过采用收养转移，我们发现 Nur77-GFPHI 细胞比 Nur77-GFPLO 细胞更具保护性。治疗性利用为了在体内激活 CD4 T 细胞，我们用一种可激动 OX40 的单克隆抗体治疗 Mtb 感染的小鼠，发现这种治疗方法减少了肺部细菌负担，将受感染小鼠的存活时间延长了 100 天以上并且没有引起毒性。专门针对活化的 CD4 T 细胞的免疫疗法是治疗结核病的一种新方法尚未彻底研究的治疗方法。在本提案的目标 1 中，我们将定义以下功能：使用 scRNA-Seq 和 CITE-Seq 激活 Mtb 感染小鼠肺部的 CD4 T 细胞。通过收养转移和 scTCR-Seq，我们将揭示抗原特异性如何影响不同 CD4 T 细胞 TCR 的功能和命运整个慢性感染的克隆型。我们将使用我们生成的TCR序列来开发TCR逆转录能够识别保护性 Mtb 抗原并测试抗原特异性塑造 T 细胞的假设的小鼠结核病表型在目标 2 中，我们将确定激活标记免疫疗法的潜在机制介导的 CD4 T 细胞对结核病的控制。我们将检验 OX40 激动剂治疗提供保护的假设通过选择性调节激活的常规 T 细胞和调节性 T 细胞的功能。最后，我们将确定 OX40 激动剂如何影响慢性感染过程中激活的 CD4 T 细胞的存活。研究这里提出的代表了小群激活的 CD4 T 细胞的第一个高清特征在 Mtb 感染部位，第一项研究探索了 OX40 激活标记物的潜力结核病小鼠模型的免疫治疗。该项目将阐明新的机制，可以为开发更有效的结核病疫苗并产生新的治疗方法来改善结核病治疗。