Targeted cell-depleting immunotherapy for TB and HIV

项目总结 在结核肉芽肿中，Tregs和MDSCs已成为抑制效应免疫的关键抑制细胞。 此外，Tregs还与HIV潜伏期的维持有关。我们已经产生了两个选择性的 利用白喉毒素(DT)融合蛋白有效去除Treg和MDSCs的生物制剂 技术：DT-IL2靶向IL2-R阳性(CD25)细胞(Tregs)，DT-IL4靶向IL4-R阳性(CD124) 细胞(MDSCs)。我们的初步数据表明：(I)DT-IL2和DT-IL4降低了Treg和 (Ii)DT-IL2和DT-IL4可降低结核分枝杆菌感染小鼠的能力。 在急性小鼠结核病感染模型中增殖，(Iii)具有强大的抗MDSC活性的药物他喹莫特， 降低了结核分枝杆菌在急性结核病感染模型中的增殖能力，并伴随着MDSCs的减少， 以及(Iv)将DT-IL2注射到来自受控HIV患者的CD4T细胞，可重新激活病毒复制。 这些数据支持以下假设：(1)Tregs和MDSCs在结核分枝杆菌中发挥重要的抑制作用 遏制，以及(2)Treg细胞在HIV潜伏期维持中发挥作用。这是一个核心的科学前提 应用是进一步定义Tregs和MDSCs在结核病和艾滋病毒感染中的免疫抑制作用 将揭示重要的免疫致病机制，可能被利用来开发改进的 针对这两种疾病的宿主导向疗法(HDTS)。在这项提案中，我们寻求通过以下方式解决这一前提 目标有以下几点。目的1：通过靶向CD25细胞去除来确定Treg细胞在结核肉芽肿中的作用 使用DT-IL2。我们将(A)评估细胞(C57BL/6，WT，均为sst1R)和 应用单细胞RNA-Seq技术检测有无CD25细胞缺失的坏死性(C57BL/6 sst1S)小鼠肉芽肿 与传统方法一样，(B)评估靶向CD25细胞去除治疗慢性粒细胞白血病的疗效。 接受和不接受抗结核药物治疗的小鼠结核病，以及(C)评估Treg细胞在该过程中的贡献 使用一种新的兔空洞性结核病模型进行空化(肉芽肿破裂)的研究。目标2：确定 用DT-IL4靶向CD124细胞去除结核肉芽肿中的MDSCs。我们将(A)评估细胞 应用SSRS技术检测CD124细胞缺失和不缺失的小鼠细胞肉芽肿和坏死性肉芽肿的成分 和传统方法一样，(B)评估靶向CD124细胞去除对慢性小鼠结核病的疗效 在接受和不接受抗结核药物治疗的情况下，(C)评估CD25和CD124双重细胞耗竭对结核病的影响 使用SSRS和传统方法对B6sst1R和sst1S小鼠进行肉芽肿治疗，以及(D)评估 双重CD25和CD124细胞去除在抗结核药物治疗前后对慢性小鼠结核病的疗效。 目标3：通过(I)卡介苗接种和(Ii)HIV进行CD25细胞耗竭的翻译应用 重新激活。我们将(A)评估靶向CD25细胞去除作为疫苗佐剂的有效性 卡介苗接种和(B)评估靶向CD25细胞去除对外周血单核细胞中HIV重新激活的影响 来自艾滋病毒感染得到控制的患者。

PROJECT SUMMARY Tregs and MDSCs have emerged as key suppressor cells that inhibit effector immunity in the TB granuloma. In addition, Tregs have been implicated in the maintenance of HIV latency. We have generated two selective biologic agents for potent depletion of Tregs and and MDSCs using diphtheria toxin (DT) fusion protein technology: DT-IL2 targets IL2-R-positive (CD25+) cells (Tregs), and DT-IL4 targets IL4-R positive (CD124+) cells (MDSCs). Our preliminary data demonstrate that: (i) DT-IL2 and DT-IL4 reduce the abundance of Treg and MDSC cells, respectively, in mouse lungs and spleens, (ii) DT-IL2 and DT-IL4 reduces the ability of M. tb to proliferate in an acute murine TB infection model, (iii) the drug, tasquinimod which has potent anti-MDSC activity, reduced the ability of M. tb to proliferate in an acute TB infection model with a concomitant reduction in MDSCs, and (iv) administration of DT-IL2 to CD4+ T cells from patients with controlled HIV reactivates viral replication. These data support the hypotheses that (1) Tregs and MDSCs play important inhibitory roles in M. tb containment, and (2) that Treg cells play a role in HIV latency maintenance. A central scientific premise of this application is that further definition of the immunosuppressive roles of Tregs and MDSCs in TB and HIV infection will reveal important immunopathogenesis mechanisms that may be exploited towards developing improved host-directed therapies (HDTs) for both diseases. In this proposal we seek to address this premise by pursuing the following aims. Aim 1: define the role of Treg cells in the TB granuloma using targeted CD25+ cell depletion with DT-IL2. We will (a) evaluate the cellular composition of both cellular (C57BL/6, WT which are sst1R) and necrotic (C57BL/6 sst1S) murine granulomas with and without CD25+ cell depletion using single-cell RNA-Seq (SSRS) as well as traditional methods, (b) evaluate the efficacy of targeted CD25+ cell depletion in chronic murine TB with and without anti-TB drug therapy, and (c) evaluate the contribution of Treg cells to the process of cavitation (granuloma breakdown) using a novel model of rabbit cavitary TB. Aim 2: define the role of MDSCs in the TB granuloma using targeted CD124+ cell depletion with DT-IL4. We will (a) evaluate the cellular composition of both cellular and necrotic murine granulomas with and without CD124+ cell depletion using SSRS as well as traditional methods, (b) evaluate the efficacy of targeted CD124+ cell depletion in chronic murine TB with and without anti-TB drug therapy, (c) assess the impact of dual CD25+ and CD124+ cell depletion in the TB granuloma with B6 sst1R and sst1S mice using SSRS as well as traditional methods, and (d) evaluate the efficacy of dual CD25+ and CD124+ cell depletion in chronic murine TB with and without anti-TB drug therapy. Aim 3: conduct translational applications of CD25+ cell depletion with (i) BCG vaccination and (ii) HIV reactivation. We will (a) evaluate the efficacy of targeted CD25+ cell depletion as a vaccine adjuvant prior to BCG vaccination and (b) evaluate the impact of targeted CD25+ cell depletion on HIV reactivation in PBMCs from patients with controlled HIV infection.