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

CD 4 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，以及它们用于控制结核分枝杆菌（Mtb）的机制 还没有完全定义。在对结核病的适应性免疫高峰期，数百万结核特异性CD 4 T细胞 运输到肺部，但很少有人重新遇到其同源抗原而被激活。的能力 清楚地区分和分离这些少数活化的细胞与更大数量的失活CD 4 T细胞， 能够发现重要的新标志物、效应子功能和克隆扩增的T细胞受体（TCR） 与保护性免疫密切相关的序列。为了研究来自结核分枝杆菌感染肺的活化CD 4 T细胞， 我们使用了一种新的方法，结合：（i）一个报告小鼠，以确定积极接受TCR刺激的细胞 体内（Nur 77-GFP）;（ii）含BSL 3的荧光细胞分选，以将它们从组织中分离出来;和（iii）单个 使用CITE-Seq和TCR免疫分析进行细胞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Nur 77-GFPH 1 CD 4 T细胞表达一系列的 活化标记物和共刺激受体，包括OX 40。该群体富集了T调节基因。 细胞和效应T细胞，TCR克隆型定位于肺实质。通过收养转移，我们发现 Nur 77-GFPHI细胞比它们的Nur 77-GFPLO对应物更具保护性。治疗性地利用 在体内活化的CD 4 T细胞中，我们用激动OX 40的单克隆抗体处理Mtb感染的小鼠， 发现这种治疗减少了肺部细菌负荷，延长了感染小鼠的存活时间>100天 并且没有引起毒性。特异性靶向活化的CD 4 T细胞的免疫疗法是治疗结核病的新方法 这是一种尚未被彻底调查的治疗方法。在本提案的目标1中，我们将定义 使用scRNA-Seq和CITE-Seq在Mtb感染的小鼠的肺中活化的CD 4 T细胞。通过收养转移 和scTCR-Seq，我们将揭示抗原特异性如何塑造不同CD 4 T细胞TCR的功能和命运。 慢性感染中的克隆型。我们将使用我们产生的TCR序列来开发TCR逆转录基因， 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 在目标2中，我们将确定活化标记免疫治疗的潜在机制 介导的TB的CD 4 T细胞控制。我们将检验OX 40激动剂治疗提供保护作用的假设。 通过选择性调节活化的常规T细胞和调节性T细胞的功能。最后我们将 确定OX 40激动如何通过慢性感染影响活化的CD 4 T细胞存活。研究 本文提出的方法首次对活化的CD 4 T细胞小群体进行了高清晰度表征 在Mtb感染部位，第一项探索OX 40活化标志物靶向的潜力的研究 免疫疗法在结核病小鼠模型中的应用。该项目将阐明新的机制， 开发更有效的结核病疫苗，并导致新的治疗方法，以改善结核病的治疗。

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.