A new cell line for assessing HIV-1 antibody dependent cellular cytotoxicity against a broad range of variants

Abstract

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, CD16⁺KHYG-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.

Keywords: Antibody dependent cellular cytotoxicity; Envelope; Human immunodeficiency virus type 1.

Figure 1.. CEM.NKr-CCR5-Luc fail to produce adequate luciferase levels after exposure to strains of interest.

Representative examples of relative luminescence units (y-axis) after CEM.NKr-CCR5-Luc cells were exposed to virus using different protocols (A), varying multiplicity of infection (MOI) (B), and sampled at different days post infection (PI) (C). Each bar represents the mean and standard error mean from three replicates for NL4–3 (black bar), REJO (blue bar), swarm with maternal envelopes 1431 (red bar), and uninfected cells (white bar).

Figure 2.. MT4-CCR5-Luc, but not PM1-CCR5-Luc, cells are resistant to CD16⁺KHYG-1 cell killing in the absence of antibodies.

Relative light units (y-axis) over time (x-axis) in NL43-infected PM1-CCR5-Luc (A) and MT4-CCR5-Luc (B) cells without antibody or NK cells (red circle), with NK cells but no antibody (blue triangle), and with NK cells and 500 μg/mL of HIVIG (black square). ADCC is determined as the percent difference as graphed. Uninfected cells (green diamond) represents background RLU reading. Data from 2 independent experiments with 3 replicates each. Bars shows standard error mean (SEM).

Figure 3.. PM1-CCR5-Luc, MT4-CCR5-Luc, and CEM.NKr-CCR5-Luc cells yield similar ADCC estimates.

Percent ADCC (y-axis) observed against exclusively CXCR4-using virus NL4–3 infected MT4-CCR5-Luc (red), PM1-CCR5-Luc (blue), and CEM.NKr-CCR5-Luc (green) cells with serially diluted HIVIG (x-axis). Bars show SEM from 5 independent experiments with each condition tested in triplicate.

Figure 4.. PM1-CCR5-Luc and MT4-CCR5-Luc as compared to CEM.NKr-CCR5-Luc support replication of diverse HIV-1 variants.

Relative light unit (RLU) fold induction 4 days post infection in MT4-CCR5-Luc (red), PM1-CCR5-Luc (blue), and CEM.NKr-CCR5-Luc cells (green) infected with various viruses compared to uninfected cells. Viruses and co-receptor usage are indicated at the bottom. Bars represent standard error of the mean (SEM) from minimum of 2 independent experiments.

Figure 5.. Proposed ADCC assay has been validated via flow cytometry.

MT4-CCR5-Luc cells were stained with eFluor670 prior to use in assay. (A) Gating strategy is shown for the condition with uninfected MT4-CCR5-Luc cells and NK cells, which represents background signal. The final panel shows live GFP+ target cells (Q1). (B) NL43-infected MT4-CCR5-Luc cells and NK cells (no antibody added). (C) NL43-infected MT4-CCR5-Luc cells, NK cells, and HIVIG. (D) Percent ADCC calculated using luciferase readout and percent ADCC calculated as the change in percentage of live GFP+ target cells (Q1) in the flow cytometry analysis (p = 0.25, Wilcoxon matched-pair test). (E) NL43-ΔEnv-Luc-VSVG-infected MT4-CCR5-Luc cells and NK cells (no antibody added). (F) NL43-ΔEnv-Luc-VSVG-infected MT4-CCR5-Luc cells, NK cells, and HIV Ig. (G) Nonspecific cytotoxicity of NL43-ΔEnv-Luc-VSVG-infected MT4-CCR5-Luc cells observed using luciferase and flow cytometry readout.

Figure 6.. Plasma contents, besides IgG, do not impact ADCC estimates.

Percent ADCC (y-axis) observed against NL4–3 infected MT4-CCR5-Luc cells with plasma from an antiretroviral naïve individual versus IgG isolated from that plasma (top dilution 1:50) (A) and 500 μg/mL HIVIG versus 500 μg/mL HIVIG spiked into heat inactivated HIV-1 seronegative plasma (B). The bars show SEM and values are from 2–3 independent experiments with each condition done in triplicate. Dashed line at y = 25 denotes threshold for nonspecific cytotoxicity.

Figure 7.. MT4-CCR5-Luc cells can be used to estimate ADCC against viruses with primary Envs of interest.

(A) MT4-CCR5-Luc cells were infected with viruses representing the circulating strains present in an infected mother. Percent ADCC (y-axis) was determined after incubating infected cells with NK cells and a serial dilution (x-axis) of either maternal plasma (MpL) or infant plasma (IpL). Top dilution was 1:50 of plasma. Bars show SEM from three independent experiments with each condition tested in triplicate. (B) Heterologous ADCC responses of maternal plasma against BJOX2000-infected cells (y-axis) were plotted against neutralization responses (x-axis) from the same samples against the same virus. Dashed line at y = 25 denotes threshold for nonspecific cytotoxicity.