Brian J Snow, Nida K Keles, Michael W Grunst, Sanath Kumar Janaka, Ryan T Behrens, David T Evans
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引用次数: 0
摘要
抗体依赖性细胞吞噬作用(ADCP)与保护人体免受 HIV-1 感染有关。然而,目前测量 ADCP 的方法依赖于 gp120 或 gp41 包被珠子的吞噬作用,而这些珠子并不能反映病毒包膜糖蛋白的生理相关构象或病毒感染细胞的大小。因此,我们开发了一种新方法来测量表达 Env 天然构象的 HIV 感染细胞的 ADCP。在有抗体存在的情况下,将单核细胞系(THP-1 细胞)或原代人类单核细胞与表达 eGFP 的 CD4+ T 细胞系孵育,并通过流式细胞术测量 ADCP,即 eGFP+ 物质的积累。通过图像捕获流式细胞术直观地确认了单核细胞内化 HIV 感染细胞的情况。共聚焦显微镜还观察到细胞骨架重塑、假栓形成和吞噬作用。我们发现,强效的广谱中和抗体(bnAbs)而非非中和抗体(nnAbs)能有效地吞噬感染了原代或实验室适配 HIV-1 的细胞。一种针对 gp120 的 CD4 诱导表位(A32)的 nnAb 无法对感染 HIV 的细胞进行 ADCP,但却能介导对 gp120 包被珠的有效吞噬。相反,一种对完整 Env 三聚体具有特异性的 bnAb(PGT145)能介导 HIV 感染细胞的强效 ADCP,但不能促进包被 gp120 的微珠的吸收。这些结果强调了测量表达生理相关构象Env的HIV感染细胞的ADCP的重要性,并表明能够与病毒上的Env三聚体结合以中和病毒传染性的大多数抗体也能够与病毒感染细胞表面的Env结合以介导ADCP。
Antibody-dependent cellular phagocytosis (ADCP) has been implicated in protection against HIV-1. However, methods for measuring ADCP currently rely on the phagocytosis of gp120- or gp41-coated beads that do not reflect physiologically relevant conformations of the viral envelope glycoprotein or the size of a virus-infected cell. We therefore developed a novel approach for measuring ADCP of HIV-infected cells expressing natural conformations of Env. A monocytic cell line (THP-1 cells) or primary human monocytes were incubated with a CD4+ T cell line that expresses eGFP upon HIV-1 infection in the presence of antibodies and ADCP was measured as the accumulation of eGFP+ material by flow cytometry. The internalization of HIV-infected cells by monocytes was confirmed visually by image-capture flow cytometry. Cytoskeletal remodeling, pseudopod formation and phagocytosis were also observed by confocal microscopy. We found that potent broadly neutralizing antibodies (bnAbs), but not non-neutralizing antibodies (nnAbs), mediate efficient phagocytosis of cells infected with either primary or lab-adapted HIV-1. A nnAb to a CD4-inducible epitope of gp120 (A32) failed to enable ADCP of HIV-infected cells but mediated efficient phagocytosis of gp120-coated beads. Conversely, a bnAb specific to intact Env trimers (PGT145) mediated potent ADCP of HIV-infected cells but did not facilitate the uptake of gp120-coated beads. These results underscore the importance of measuring ADCP of HIV-infected cells expressing physiologically relevant conformations of Env and show that most antibodies that are capable of binding to Env trimers on virions to neutralize virus infectivity are also capable of binding to Env on the surface of virus-infected cells to mediate ADCP.
期刊介绍:
Bacteria, fungi, parasites, prions and viruses cause a plethora of diseases that have important medical, agricultural, and economic consequences. Moreover, the study of microbes continues to provide novel insights into such fundamental processes as the molecular basis of cellular and organismal function.