Infection of CD4+ Primary T Cells and Cell Lines, Generation of Chronically Infected Cell Lines, and Induction of HIV Expression

Elisa Vicenzi1, Guido Poli1

1 San Raffaele Scientific Institute, Milan
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 12.3
DOI:  10.1002/0471142735.im1203s69
Online Posting Date:  November, 2005
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Abstract

Acute infection of most primary cells and cell lines with HIV depends upon the sequential engagement of CD4 (primary receptor) and a chemokine coreceptor (usually CCR5 or CXCR4) by gp120 Env. Chronically infected cell lines and clones are currently used as sources of virus for infecting other cell types, as “factories” for large‐scale production of virions or viral components, and as model systems for studies of regulation of virus expression. This unit provides detailed protocols for acute in vitro HIV infection of primary T cell blasts, interleukin‐2‐stimulated PBMC, and resting PBMC. The unit also contains information on how to determine the chemokine coreceptor usage of the virus for experimental infections. The use of cell lines as targets of acute infection is also described. Finally, protocols for generating and studying chronically HIV‐infected cell lines are provided.

     
 
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Table of Contents

  • Acute in Vitro Infection of CD4+ Primary T Cells With HIV
  • Basic Protocol 1: Acute Infection of T Cell Blasts
  • Alternate Protocol 1: Acute Infection of IL‐2‐Stimulated PBMC
  • Alternate Protocol 2: Acute HIV Infection of “Resting” PBMC
  • Basic Protocol 2: Determination of HIV Chemokine Coreceptor Usage in U87MG.CD4‐Derived Cell Lines
  • Basic Protocol 3: Acute in Vitro Infection of CD4+ Cell Lines with HIV
  • Basic Protocol 4: Generation of Chronically Infected Cell Lines
  • Basic Protocol 5: HIV Induction in Chronically Infected Cell Lines
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Acute Infection of T Cell Blasts

  Materials
  • PBMC from healthy HIV‐seronegative donors, isolated by Ficoll‐Hypaque density gradient (unit 7.1)
  • Complete RPMI‐10 medium (see recipe)
  • Phytohemagglutinin P (PHA‐P, purified; e.g., Difco; reconstitute lyophilized material according to manufacturer's instructions)
  • Recombinant human interleukin 2 (rhIL‐2)
  • HIV stock (unit 12.1)
  • 50‐ml conical polypropylene centrifuge tube
  • Tabletop centrifuge (e.g., IEC Clinical)
  • Tissue culture plates or flasks
  • Additional reagents and equipment for culture of HIV‐infected cells (unit 12.2) and assays for p24 Gag production or RT activity (unit 12.5)

Alternate Protocol 1: Acute Infection of IL‐2‐Stimulated PBMC

  Materials
  • PBMC from healthy HIV‐seronegative donors, isolated by Ficoll‐Hypaque density gradient (unit 7.1)
  • Complete DMEM‐15 medium (see recipe)
  • Recombinant human interleukin 2 (rhIL‐2)
  • HIV stock (unit 12.1)
  • 75‐cm2 tissue culture flasks
  • Centrifuge tubes
  • Cell scrapers (for isolation of moncytes‐macrophages)
  • Tabletop centrifuge (e.g., IEC Clinical)
  • 24‐ or 48‐well tissue culture plates
  • Additional reagents and equipment for culture of HIV‐infected cells (unit 12.2) and assays for p24 Gag production and RT activity (unit 12.5)

Alternate Protocol 2: Acute HIV Infection of “Resting” PBMC

  Materials
  • PBMC from healthy HIV‐seronegative donors, isolated by Ficoll‐Hypaque density gradient (unit 7.1)
  • Complete RPMI‐10 medium (see recipe; use endotoxin‐free FBS)
  • HIV stock (unit 12.1)
  • Phytohemagglutinin P (PHA‐P, purified; e.g., Difco; reconstitute lyophilized material according to manufacturer's instructions) or anti‐CD3 plus anti‐CD28 MAb‐coated beads (∼150 fg/bead for each MAb; Dynal; Levine et al., )
  • Recombinant human interleukin 2 (rhIL‐2)
  • 24‐ or 48‐well tissue culture plates
  • Additional reagents and equipment for culture of HIV‐infected cells (unit 12.2) and assays for p24 Gag production or RT activity (unit 12.5)

Basic Protocol 2: Determination of HIV Chemokine Coreceptor Usage in U87MG.CD4‐Derived Cell Lines

  Materials
  • U87MG‐derived cell lines expressing human CD4 and individual chemokine receptors (CCR5 and CXCR4 are fundamental; a complete characterization should include at least CCR1, CCR2b, and CCR3)
  • Complete DMEM‐15 medium (see recipe) with and without the selection antibiotics G‐418 (300 µg/ml) and puromycin (1 µg/ml)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Trypsin‐EDTA (e.g., Invitrogen)
  • HIV stock (unit 12.1)
  • 25‐ or 75‐cm2 tissue culture flasks
  • 24‐well tissue culture plates
  • Additional reagents and equipment for culture of HIV‐infected cells (unit 12.2) and assays for p24 Gag production or RT activity (unit 12.5)

Basic Protocol 3: Acute in Vitro Infection of CD4+ Cell Lines with HIV

  Materials
  • Target CD4+ cell line (Table 12.3.1)
  • Complete medium appropriate for the cell line (e.g., complete RPMI; appendix 2A)
  • Tabletop centrifuge
  • HIV stock (unit 12.1)
  • Additional reagents and equipment for testing cell viability by trypan blue dye exclusion ( appendix 3A) and assays for p24 Gag production and RT activity (unit 12.5)

Basic Protocol 4: Generation of Chronically Infected Cell Lines

  Materials
  • CD4+ cell line (Table 12.3.1)
  • Complete medium appropriate for the cell line (e.g., complete RPMI‐10; appendix 2A)
  • HIV stock (unit 12.1)
  • 96‐well round‐bottom microtiter plates
  • Inverted microscope
  • 48‐ or 24‐well flat‐bottom tissue culture plates
  • Additional reagents and equipment for determination of HIV activity by RT activity or p24 Gag antigen production assays (unit 12.5).

Basic Protocol 5: HIV Induction in Chronically Infected Cell Lines

  Materials
  • Chronically infected cell line
  • Complete medium appropriate for the cell line (e.g., complete RPMI‐10; appendix 2A)
  • Stimuli of interest dissolved in complete medium
  • 96‐well flat‐bottom microtiter plates or tissue culture flasks
  • Additional reagents and equipment for determining cell viability by trypan blue dye exclusion ( appendix 3A) and determination of HIV activity by RT activity or p24 Gag antigen production assays (unit 12.5)
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Figures

Videos

Literature Cited

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