Department of Microbiology, Boston University School of Medicine, Boston, MA, USA.
Department of Medicine, Boston University School of Medicine, Boston, MA, USA.
J Immunol Methods. 2020 May;480:112766. doi: 10.1016/j.jim.2020.112766. Epub 2020 Mar 2.
Human immunodeficiency virus type 1 (HIV-1) studies suggest that antibody-dependent cellular cytotoxicity (ADCC) influences both virus acquisition and subsequent disease outcome. Technical issues with currently available assays, however, have limited the ability to comprehensively assess the impact of ADCC on transmission and disease progression. Commonly used ADCC assays use a target cell line, CEM.NKr-CCR5-Luc, that often does not support replication of relevant HIV-1 variants. Thus, the extent of ADCC responses against a large panel of HIV-1 strains often cannot be assessed using the currently available methods. We developed two new reporter cell-lines (MT4-CCR5-Luc and PM1-CCR5-Luc) to overcome these issues. MT4-CCR5-Luc cells are resistant, whereas PM1-CCR5-Luc cells are susceptible, to killing by a natural killer cell line, CD16KHYG-1, in the absence of antibody. Polyclonal HIVIG gave similar ADCC estimates against HIV-1 isolate, NL4-3, regardless of which of the three cell lines were used as the targets. In contrast to CEM.NKr-CCR5-Luc and PM1-CCR5-Luc, however, MT4-CCR5-Luc target cells produce significantly higher luciferase after exposure to various HIV-1 strains, including transmitted founder variants and viruses incorporating specific envelopes of interest. This higher luciferase expression does not yield spurious results because ADCC estimates are similar when killing is assessed by both reporter protein expression and flow cytometry. Furthermore, ADCC estimates derived from MT4-CCR5-Luc cells are not skewed by non-antibody contents present in human plasma. In aggregate, the MT4-CCR5-Luc cell line can be used to estimate monoclonal antibody or plasma-induced ADCC responses against a diverse range of HIV-1 envelopes relevant for transmission and disease progression studies.
人类免疫缺陷病毒 1 型（HIV-1）研究表明，抗体依赖性细胞毒性（ADCC）会影响病毒的获得和随后的疾病结果。然而，目前可用的检测方法存在技术问题，限制了全面评估 ADCC 对传播和疾病进展影响的能力。常用的 ADCC 检测方法使用靶细胞系 CEM.NKr-CCR5-Luc，该细胞系通常不支持相关 HIV-1 变体的复制。因此，目前可用的方法通常无法评估针对大量 HIV-1 株的 ADCC 反应程度。我们开发了两种新的报告细胞系（MT4-CCR5-Luc 和 PM1-CCR5-Luc）来克服这些问题。MT4-CCR5-Luc 细胞对自然杀伤细胞系 CD16KHYG-1 的杀伤具有抗性，而 PM1-CCR5-Luc 细胞在没有抗体的情况下易受杀伤。多克隆 HIVIG 对 HIV-1 分离株 NL4-3 的 ADCC 估计值相似，无论使用哪种三种细胞系作为靶细胞。然而，与 CEM.NKr-CCR5-Luc 和 PM1-CCR5-Luc 不同，MT4-CCR5-Luc 靶细胞在暴露于各种 HIV-1 株后会产生明显更高的荧光素酶，包括传播的创始变体和包含感兴趣特定包膜的病毒。这种更高的荧光素酶表达不会产生虚假结果，因为通过报告蛋白表达和流式细胞术评估杀伤时，ADCC 估计值相似。此外，来自 MT4-CCR5-Luc 细胞的 ADCC 估计值不受人血浆中存在的非抗体成分的影响。总之，MT4-CCR5-Luc 细胞系可用于估计针对与传播和疾病进展研究相关的各种 HIV-1 包膜的单克隆抗体或血浆诱导的 ADCC 反应。
