CD8 T 细胞病毒抑制实验方法

Yinyan  Xu; Ann Marie Weideman; Maria  Abad-Fernandez; Katie R. Mollan; Sallay  Kallon; Shahryar  Samir; Joanna A. Warren; Genevieve  Clutton; Nadia  Roan; Adaora A. Adimora; Nancie  Archin; JoAnn  Kuruc; Cindy  Gay; Michael G. Hudgens; Nilu Goonetilleke

doi:10.21769/BioProtoc.4354

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Peer-reviewed

CD8 T Cell Virus Inhibition Assay Protocol

CD8 T 细胞病毒抑制实验方法

YX Yinyan Xu ^*

AW Ann Marie Weideman ^*

MA Maria Abad-Fernandez

MH Michael G. Hudgens

NG Nilu Goonetilleke email

(*contributed equally to this work) 发布: 2022年03月20日第12卷第6期 DOI: 10.21769/BioProtoc.4354 浏览次数: 2479

评审: Alessandro DidonnaDhiman Sankar PalAnonymous reviewer(s)

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Abstract

The human immunodeficiency virus (HIV)-1 viral inhibition assay (VIA) measures CD8⁺ T cell-mediated inhibition of HIV replication in CD4⁺ T cells and is increasingly used for clinical testing of HIV vaccines and immunotherapies. Different VIAs that differ in length of CD8:CD4 T cell culture periods (6–13 days), purity of CD4 cultures [isolated CD4⁺ T cells or CD8⁺ depleted peripheral blood mononuclear cells (PBMCs)], HIV strains (laboratory strains, isolates, reporter viruses) and read-outs of virus inhibition (p24 ELISA, intracellular measurement of p24, luciferase reporter expression, and viral gag RNA) have been reported.

Here, we describe multiple modifications to a 7-day VIA protocol, the most impactful being the introduction of independent replicate cultures for both HIV infected-CD4 (HIV-CD4) and HIV-CD4:CD8 T cell cultures. Virus inhibition was quantified using a ratio of weighted averages of p24⁺ cells in replicate cultures and the corresponding 95% confidence intervals. We identify methodological and analysis changes that could be incorporated into other protocols to improve assay reproducibility. We found that in people living with HIV (PLWH) on antiretroviral therapy (ART), CD8 T cell virus inhibition was largely stable over time, supporting the use of this assay and/or analysis methods to examine therapeutic interventions.

Graphic abstract:

Keywords: CD4 (CD4)

Virus inhibition (病毒抑制)

Background

A CD8⁺ T cell virus inhibition assay (VIA) measures the in vitro ability of CD8⁺ T cells to inhibit human immunodeficiency virus (HIV)-1 replication in autologous CD4⁺ T cells. This assay captures the full range of CD8⁺ T cell anti-viral activity in response to in vitro antigen presentation of virus-derived peptides by infected CD4 T cells. VIAs are increasingly used in clinical studies but mostly as an exploratory assay, because the complex co-culture of the VIA has substantial assay variability and a limited dynamic range. Different VIAs have been reported (Yang, O. O. et al., 1997; Sáez-Cirión et al., 2009, 2010; Spentzou et al., 2010; Julg et al., 2010; Freel et al., 2011; Yang, H. et al., 2012, 2013; Naarding et al., 2014; Slichter et al., 2014; Hancock et al., 2015) that differ in length of CD8:CD4 T cell culture periods (6–13 days), purity of CD4 cultures (isolated CD4⁺ T cells or CD8⁺ depleted PBMCs), HIV strains (laboratory strains, isolates, reporter viruses), and read-outs of virus inhibition (p24 ELISA, intracellular measurement of p24, luciferase reporter expression, and viral gag RNA). Here, we describe multiple modifications to a 7-day VIA protocol, the most impactful being the introduction of independent replicate cultures for both HIV infected-CD4 (HIV-CD4) and HIV-CD4:CD8 T cell cultures. Virus inhibition was quantified using a ratio of weighted averages of p24⁺ cells in replicate cultures and the corresponding 95% confidence intervals (CI). We found that in people living with HIV (PLWH) on antiretroviral therapy (ART), CD8⁺ T cell virus inhibition was largely stable over time, supporting the use of this assay and/or analysis methods to examine therapeutic interventions.

Materials and Reagents

Falcon^TM Round-Bottom Polystyrene Test Tubes (Falcon^TM, catalog number: 352054)
Falcon 50 mL Conical Centrifuge Tubes (Corning, catalog number: 352070)
Falcon 15 mL Conical Centrifuge Tubes (Falcon^TM, catalog number: 352097)
Phytohaemagglutinin (PHA) (Sigma-Aldrich, catalog number: L8902-5MG)
Benzonase^® (Sigma-Aldrich, catalog number: NucleaseE1014)
CD4⁺ T Cell Isolation Kit (MACS, Miltenyi-Biotec, catalog number: 130-096-533)
HIV-1 JR-CSF Infectious Molecular Clone (pYK-JRCSF) NIH reagent program
IL-2 (Prometheus Proleukin, Aldesleukin, catalog number: 407682M)
IL-7 (PeproTech, catalog number: 200-07-10UG)
Polybrene (Santa Cruz Biotechnology, catalog number: NC9840454)
RPMI (Corning^®,catalog number: 10-040-CV)
FBS (VWR Life Science, catalog number: 97068-091)
L-Glutamine (Corning^TM, catalog number: 25005CI)
Sodium Pyruvate (Corning^TM, catalog number: 25-000-CIR)
Penicillin-streptomycin (Gibco^TM, catalog number: 15070063)
HEPES (Corning^TM, catalog number: 25-060-CI)
PBS (Corning^TM, catalog number: 21-030-CM)
BSA (Sigma, catalog number: A8412)
EDTA (Corning^TM, catalog number: 46-034-CI)
Lysolecithin (Sigma, catalog number: L4129-25MG)
Paraformaldehyde (PFA) (Santa Cruz Biotechnology, catalog number: sc-281692)
Methanol (Sigma, catalog number: 34860-1L-R)
Nonidet P-40 (Biotang Inc, catalog number: BTBB914)
Zombie NIR Fixable Viability Kit (Biolegend, catalog number: 423106)
p24-FITCKC57-FITC (catalog number: 6604665, Beckman)
CD3-BV421 (BD Biosciences, catalog number: BDB562426)
CD4-AF488 (Biolegend, catalog number: 317434)
CD8-BV510 (Biolegend, catalog number: 344732)
Anti-mouse Ig, κ compensation beads (BD^TM, catalog number: 552843)
PBMCs: Isolated from PLWH receiving antiretroviral therapy (HIVART) and seronegative individuals (healthy donor (HD))
R-10+ (see Recipes)
R-20+ (see Recipes)
Cell isolation buffer (see Recipes)
50% Methanol/PBS (see Recipes)
0.1% NP-40/PBS (see Recipes)

Equipment

Centrifuges (Thermo Scientific, ST40R TX-1000, catalog number: 50144036)
Muse Cell Analyzer (EMD Millipore Corporation, catalog number: 0500-3115)
Incubator (Thermo Scientific, catalog number: 190408340396)
MACS magnet (Miltenyi Biotech, OctoMACS Separator, catalog number: 130-042-108)
Flow Cytometer (BD LSRFortessa)
FINNPIPETTE F1 GLP (Thermo Scientific, catalog number: 4700850N)

Software

Excel (Microsoft)
Flow Jo (Flow Jo, LLC/ FlowJo_v10.8.0, https://www.flowjo.com/)

Procedure

English

中文翻译

文章信息

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如何引用

Xu, Y., Weideman, A. M., Abad-Fernandez, M., Mollan, K. R., Kallon, S., Samir, S., Warren, J. A., Clutton, G., Roan, N., Adimora, A. A., Archin, N., Kuruc, J., Gay, C., Hudgens, M. G. and Goonetilleke, N. (2022). CD8 T Cell Virus Inhibition Assay Protocol. Bio-protocol 12(6): e4354. DOI: 10.21769/BioProtoc.4354.