Tansiri Yada, Rowland-Jones Sarah L, Ananworanich Jintanat, Hansasuta Pokrath
Division of Virology, Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
Nuffield Department of Medicine, NDM Research Building, University of Oxford, Old Road Campus, Headington, OX3 7FZ, Oxford, United Kingdom.
PLoS One. 2015 Mar 12;10(3):e0118871. doi: 10.1371/journal.pone.0118871. eCollection 2015.
In this cross-sectional study we evaluated T-cell responses using several assays to determine immune correlates of HIV control that distinguish untreated viraemic controllers (VC) from noncontrollers (NC) with similar CD4 counts. Samples were taken from 65 ART-naïve chronically HIV-infected VC and NC from Thailand with matching CD4 counts in the normal range (>450 cells/μl). We determined HIVp24-specific T-cell responses using standard Interferon-gamma (IFNγ) ELISpot assays, and compared the functional quality of HIVp24-specific CD8+ T-cell responses using polychromatic flow cytometry. Finally, in vitro HIV suppression assays were performed to evaluate directly the activity of CD8+ T cells in HIV control. Autologous CD4+ T cells were infected with primary patient-derived HIV isolates and the HIV suppressive activity of CD8+ T cells was determined after co-culture, measuring production of HIVp24 Ag by ELISA. The HIVp24-specific T-cell responses of VC and NC could not completely be differentiated through measurement of IFNγ-producing cells using ELISpot assays, nor by the absolute cell numbers of polyfunctional HIVp24-specific CD8+ T cells. However, in vitro HIV suppression assays showed clear differences between VC and NC. HIV suppressive activity, mediated by either ex vivo unstimulated CD8+ T cells or HIVp24-specific T-cell lines, was significantly greater using cells from VC than NC cells. Additionally, we were able to demonstrate a significant correlation between the level of HIV suppressive activity mediated by ex vivo unstimulated CD8+ T cells and plasma viral load (pVL) (Spearman r = -0.7345, p = 0.0003). This study provides evidence that in vitro HIV suppression assays are the most informative in the functional evaluation of CD8+ T-cell responses and can distinguish between VC and NC.
在这项横断面研究中，我们使用多种检测方法评估T细胞反应，以确定区分未经治疗的病毒血症控制者（VC）和具有相似CD4计数的非控制者（NC）的HIV控制免疫相关因素。样本取自65名来自泰国的初治慢性HIV感染者，这些VC和NC的CD4计数在正常范围内（>450个细胞/μl）。我们使用标准的干扰素-γ（IFNγ）酶联免疫斑点分析（ELISpot）来确定HIV p24特异性T细胞反应，并使用多色流式细胞术比较HIV p24特异性CD8+ T细胞反应的功能质量。最后，进行体外HIV抑制试验以直接评估CD8+ T细胞在HIV控制中的活性。用原发性患者来源的HIV分离株感染自体CD4+ T细胞，并在共培养后通过ELISA测量HIV p24抗原的产生来确定CD8+ T细胞的HIV抑制活性。通过ELISpot分析测量产生IFNγ的细胞，或通过多功能HIV p24特异性CD8+ T细胞的绝对细胞数，无法完全区分VC和NC的HIV p24特异性T细胞反应。然而，体外HIV抑制试验显示VC和NC之间存在明显差异。使用来自VC的细胞比NC细胞，由体外未刺激的CD8+ T细胞或HIV p24特异性T细胞系介导的HIV抑制活性显著更高。此外，我们能够证明体外未刺激的CD8+ T细胞介导的HIV抑制活性水平与血浆病毒载量（pVL）之间存在显著相关性（Spearman相关系数r = -0.7345，p = 0.0003）。这项研究提供了证据，表明体外HIV抑制试验在CD8+ T细胞反应的功能评估中最具信息价值，并且可以区分VC和NC。
