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. 2014 Dec;9(12):2755-70.
doi: 10.1038/nprot.2014.188. Epub 2014 Nov 6.

A Primary CD4(+) T Cell Model of HIV-1 Latency Established After Activation Through the T Cell Receptor and Subsequent Return to Quiescence

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Free PMC article

A Primary CD4(+) T Cell Model of HIV-1 Latency Established After Activation Through the T Cell Receptor and Subsequent Return to Quiescence

Michelle Kim et al. Nat Protoc. .
Free PMC article

Abstract

A mechanistic understanding of HIV-1 latency depends on a model system that recapitulates the in vivo condition of latently infected, resting CD4(+) T lymphocytes. Latency seems to be established after activated CD4(+) T cells, the principal targets of HIV-1 infection, become productively infected and survive long enough to return to a resting memory state in which viral expression is inhibited by changes in the cellular environment. This protocol describes an ex vivo primary cell system that is generated under conditions that reflect the in vivo establishment of latency. Creation of these latency model cells takes 12 weeks and, once established, the cells can be maintained and used for several months. The resulting cell population contains both uninfected and latently infected cells. This primary cell model can be used to perform drug screens, to study cytolytic T lymphocyte (CTL) responses to HIV-1, to compare viral alleles or to expand the ex vivo life span of cells from HIV-1-infected individuals for extended study.

Figures

Figure 1
Figure 1. Representative results of intracellular Bcl-2 expression levels
Freshly isolated CD4s (blue) and Bcl-2 model cells (purple) were stained with a FITC-conjugated antibody. Cells were also stained with a FITC-conjugated isotype control antibody (gray). 20,000–40,000 cells were measured for each sample.
Figure 2
Figure 2. Structure of the CM6 HIV-1/GFP reporter virus
The second round HIV-1 reporter virus described in this protocol expresses only Tat, Rev, and GFP.
Figure 3
Figure 3. Part I. Schematic of virus production
Two types of viruses are produced in HEK293T cells using a three-plasmid packaging system. After a few days of incubation, supernatant is ultracentrifuged over a sucrose cushion to concentrate virions.
Figure 4
Figure 4. Part II. Schematic of Bcl-2 transduction and expansion
CD4s are isolated by column separation and activated by TCR engagement prior to infection with the Bcl-2 transducing virus produced in Part I. The cells are cultured for a few weeks to allow the untransduced cells to expire. The surviving cells, i.e., the Bcl-2 transduced cells, are isolated by Ficoll density centrifugation. The Bcl-2 cells are then activated by TCR engagement and expanded by incubation with IL-2.
Figure 5
Figure 5. Part III. Schematic of HIV-1/GFP reporter virus infection and isolation of Bcl-2 model cells
Expanded Bcl-2 cells are activated by TCR engagement prior to infection with the HIV-1/GFP reporter virus produced in Part I. Cells are then cultured for several weeks without exogenous cytokines to allow a return to a resting state. By the end of this incubation period, there will be a heterogeneous population: cells that were never infected by the reporter virus (A), cells that have died (B), cells that are actively infected (C), and cells that are latently infected (D). Isolation of GFP- cells by flow cytometry yields a population containing uninfected cells and latently infected cells.
Figure 6
Figure 6. Representative FACS measurement of HIV-1/GFP reporter virus infection efficiency
As described in Step 79, FACS measurement of the infection efficiency of Bcl-2 cells spinoculated with the HIV-1/GFP reporter virus in Steps 70–79. 60,000 cells were measured.
Figure 7
Figure 7. Representative FACS measurement of frequency of latent infection in Bcl-2 model cells
As described in Step 89, FACS measurement of the frequency of latently infected Bcl-2 model cells in the final GFP- population sorted by flow cytometry in Steps 83–84. Two aliquots of Bcl-2 model cells are untreated (left panels) or treated with PMA/Ionomycin (right panels) for 24 hours and then assessed by flow cytometry. Live cells are gated based on forward and side scatter (top panels) and GFP positivity is measured using the FITC channel (lower panels). 50,000 cells were measured for each sample.
Figure 8
Figure 8. TCGF preparation
As described in Box 1. (A) In Step I, slowly tilt the flasks containing PBMCs from the leukopaks to the upright position before removing the culture supernatant by aspiration. (B) When adding fresh WM (Step J) or RPMI-HS (Step L) to the flasks, the liquid should be slowly added along the surface opposite the cells. (C) After adding WM to the flasks, wash the cells by gently and slowly tilting the flask 360° and then returning the flask to the flat position.

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