Gut Cytotoxic CD4 T cells in HIV-1 Pathogenesis

摘要 HIV-1相关炎症和慢性免疫激活持续存在，尽管抑制性抗逆转录病毒 治疗，并与并发症的发展和死亡率增加密切相关。的 胃肠道（GI）可能是慢性炎症的主要来源，因为早期HIV-1复制和CD 4 T 肠道中的细胞耗竭导致粘膜炎症和屏障功能障碍，导致 肠道微生物/微生物产物进入固有层和体循环。来深入了解 驱动肠道聚焦慢性免疫激活的分子过程，我们分析了肠道转录组， CD 4 + CD 8 α- T细胞暴露于革兰氏阴性肠道细菌，然后体外感染HIV-1。多 颗粒酶基因在微生物暴露后上调，HIV-1感染进一步增强， 提示协同诱导CD 4细胞毒性T淋巴细胞（CTL）活性。在初步离体研究中， 人肠道CD 4 CTL表型与颗粒酶（GZ）B表达、TCR信号传导、Th 1/17相关 多功能性、对大肠杆菌的反应性和表达GZA、穿孔素和/或CD 107 a的亚群。的 CD 4 CTL表型在来自肠道的CD 4 T细胞中表达的程度比来自外周血的大得多。 血液和淋巴组织。重要的是，HIV-1在离体肠道CD 4 CTL中复制的程度更大。这里我们 假设肠道CD 4 CTL是唯一启动以响应肠道微生物， 对HIV-1介导的杀伤敏感，并通过以下途径在粘膜HIV-1发病机制中发挥关键作用： 细胞溶解和促炎机制。深入了解肠道CD 4 CTL在HIV-1中的作用 发病机制，我们提出了3个目标。在目标1中，我们将确定微生物组物种和配体是否诱导肠道 CD 4 CTL在MHC-II依赖性方式，获得洞察肠道CD 4 CTL的起源和功能，通过单细胞 转录组学，并研究这些细胞离体的稳定性和命运。在目标2中，我们将确定如何 HIV-1感染进一步增加肠道CD 4 T细胞中GZB的产生，并使用我们的离体固有层 聚集体培养（LPAC）模型，确定肠道CD 4 CTL是否会加剧直接和旁观者HIV-1介导的 通过细胞溶解机制的CD 4 T细胞死亡。在目标3中，我们将评估GZ分泌的触发因素， 由微生物暴露的肠道CD 4 T细胞离体分泌的GZ的非细胞溶解、促炎特性。 重要的是，我们将探讨在体内CD 4 CTL频率和粘膜和淋巴结标志物之间的关系。 来自未治疗和未治疗的充分表征的队列的存档血浆和肠活检中的全身性炎症 以及接受治疗的慢性HIV-1感染者。总之，这些研究应该提供关键的 关于这种引人注目的肠道免疫细胞亚群及其对粘膜免疫的贡献的信息 炎症和疾病。

ABSTRACT HIV-1-associated inflammation and chronic immune activation persist despite suppressive antiretroviral therapy and are strongly linked to the development of comorbidities and increased mortality. The gastrointestinal (GI) tract is likely a major source of chronic inflammation, as early HIV-1 replication and CD4 T cell depletion in the gut results in mucosal inflammation and barrier dysfunction, leading to the translocation of enteric microbes/microbial products into the lamina propria and the systemic circulation. To gain insights into the molecular processes that drive gut-focused chronic immune activation, we profiled the transcriptome of gut CD4+CD8α- T cells exposed to gram-negative enteric bacteria and then infected with HIV-1 ex vivo. Multiple granzyme genes were upregulated following microbe exposure that was further enhanced with HIV-1 infection, suggesting the synergistic induction of CD4 cytotoxic T lymphocyte (CTL) activity. In pilot ex vivo studies, the human gut CD4 CTL phenotype was associated with granzyme (GZ) B expression, TCR signaling, Th1/17 polyfunctionality, reactivity to commensal bacteria and subsets expressing GZA, perforin and/or CD107a. The CD4 CTL phenotype was expressed in CD4 T cells from the gut to a much greater extent than from peripheral blood and lymphoid tissue. Importantly, HIV-1 replicated to a greater extent in gut CD4 CTLs ex vivo. Here, we hypothesize that gut CD4 CTLs are uniquely primed to respond to enteric microbes, are highly susceptible to HIV-1 mediated killing, and play critical roles in mucosal HIV-1 pathogenesis via cytolytic and pro-inflammatory mechanisms. To gain insights on the role of gut CD4 CTLs in HIV-1 pathogenesis, we propose 3 aims. In Aim 1, we will determine if microbiome species and ligands induce gut CD4 CTLs in an MHC-II-dependent manner, obtain insights into gut CD4 CTL origin and function by single-cell transcriptomics, and investigate the stability and fate of these cells ex vivo. In Aim 2, we will determine how HIV-1 infection further augments GZB production in gut CD4 T cells and using our ex vivo lamina propria aggregate culture (LPAC) model, determine if gut CD4 CTLs exacerbate direct and bystander HIV-1-mediated CD4 T cell death via cytolytic mechanisms. In Aim 3, we will evaluate the triggers for GZ secretion and dissect the non-cytolytic, pro-inflammatory properties of GZs secreted by microbe-exposed gut CD4 T cells ex vivo. Importantly, we will explore the in vivo relationship between CD4 CTL frequencies and markers of mucosal and systemic inflammation in archived plasma and gut biopsies from well-characterized cohorts of both untreated and treated persons with chronic HIV-1 infection. Altogether, these studies should provide critical information on this striking gut immune cell subpopulation and its contribution to mucosal inflammation and disease.