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

CD4T细胞对预防结核病(TB)至关重要。但目前的结核病治疗方法已经 没有利用他们的潜在利益，以及他们用来控制结核分枝杆菌(Mtb)的机制 还没有被完全定义。在结核病获得性免疫的高峰期，数百万结核分枝杆菌特异性CD4T细胞 交通进入肺部，但很少有人再次遇到他们的同源抗原而被激活。有能力 从大量非活动的CD4T细胞中清楚地区分和分离这些少数激活的细胞可以 能够发现重要的新标记物、效应器功能和克隆扩增的T细胞受体(TCR) 与保护性免疫密切相关的序列。为了研究结核分枝杆菌感染肺中活化的CD4T细胞， 我们使用了一种新的方法，它结合了：(I)报告鼠来识别主动接受TCR刺激的细胞 体内(Nur77-GFP)；(Ii)含有BSL3的荧光细胞分选，以将它们从组织中分离出来；以及(Iii)单个 用CITE-Seq和TCR免疫图谱进行细胞RNA测序(scRNA-Seq)以询问它们的功能和 抗原特异性。在我们的初步工作中，我们发现Nur77-GFPHI CD4T细胞表达一系列 激活标记和共刺激受体，包括OX40。这群人是为了T调节而丰富的 TCR克隆型的细胞和效应T细胞定位于肺实质。使用收养转移，我们发现 Nur77-GFPHI细胞比其对应的Nur77-GFPLO细胞具有更强的保护作用。以治疗性的方式驾驭 体内活化的CD4T细胞，我们用一种能刺激OX40和OX40的单抗治疗结核分枝杆菌感染的小鼠 发现这种治疗方法减少了肺部细菌负荷，延长了感染小鼠100天的存活时间 并且没有造成毒性。针对活化的CD4T细胞的免疫治疗是治疗结核病的一种新方法 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 用scRNA-Seq和Cite-Seq激活结核分枝杆菌感染小鼠肺中的CD4T细胞。通过领养转移 和scTCR-Seq，我们将揭示抗原特异性如何塑造不同的CD4T细胞TCR的功能和命运 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 能够识别保护性结核分枝杆菌抗原并检验抗原特异性塑造T细胞的假设的小鼠 在Aim 2中，我们将确定激活标记免疫治疗的潜在机制 介导的CD4T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 通过选择性地调节激活的T常规细胞和T调节细胞的功能。最后，我们会 确定OX40激动剂如何通过慢性感染影响活化的CD4T细胞存活。这些研究 这里提出的第一个高清晰度表征激活的CD4T细胞的小群体 结核分枝杆菌感染部位及OX40激活标志物靶向潜能的初步研究 免疫疗法在结核病小鼠模型中的应用。这个项目将阐明新的机制，可能会告知 开发更有效的结核病疫苗，并导致改进结核病治疗的新治疗方法。

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.