Evaluation of B Cell Function in Patients with HIV

Clarisa M. Buckner1, Lela Kardava1, Susan Moir1

1 National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 12.13
DOI:  10.1002/0471142735.im1213s100
Online Posting Date:  February, 2013
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Abstract

HIV disease is associated with abnormalities in all major lymphocyte populations, including B cells. B cell dysfunction in HIV infection is largely driven by alterations in the subsets of B cells that circulate in the blood or reside in tissues. Meaningful functional assays are thus dependent on the identification and isolation of B cell subsets present in the starting material. This unit describes several assays designed to phenotype, fractionate, and assess functional properties of B cells that circulate in the blood of HIV‐infected individuals. The four protocols, which have been adapted from standard techniques, are tailored to evaluate B cells in peripheral blood mononuclear cells (PBMCs) of individuals infected with HIV, but can also be applied to other disease settings and PBMCs isolated from healthy individuals. Curr. Protoc. Immunol. 100:12.13.1‐12.13.18. © 2013 by John Wiley & Sons, Inc.

Keywords: B cell; subset isolation; immunophenotyping; HIV; ELISPOT; proliferation; CFSE

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Multiparameter Flow Cytometric Analysis of B Cell Subsets in Peripheral Blood of HIV‐Infected Individuals
  • Basic Protocol 2: Isolation of B Cells Followed by Two‐Step Fractionation to Generate B Cell Subsets
  • Alternate Protocol 1: One‐Step Double Fractionation for Isolation of Tissue‐Like Memory (CD21−/CD27−) B Cells
  • Basic Protocol 3: Measurement of B Cell Proliferation
  • Basic Protocol 4: Measurement Total Ig(G/M/A) and HIV‐Specific Ig(G/M/A) Response in Memory B Cells
  • Alternate Protocol 2: Measurement Total Ig(G/M/A) and HIV‐Specific Ig(G/M/A) Response in Plasmablasts
  • Alternate Protocol 3: Measurement of Responses Using Biotinylated Antigen
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Multiparameter Flow Cytometric Analysis of B Cell Subsets in Peripheral Blood of HIV‐Infected Individuals

  Materials
  • Whole blood or leukapheresis product or PBMCs (unit 7.1)
  • Wash buffer (WB): Phosphate‐buffered saline (PBS) without Ca2+ and Mg2+ containing 2% fetal bovine serum (FBS)
  • Monoclonal antibodies (MAbs) against cell surface markers: CD19, CD20, CD21, CD27, CD10
  • Optional MAbs: CD3, CD95 (see Fig. for details)
  • Fc blocking mouse IgG at 2 µg/ml (suggested source: Jackson ImmunoResearch, cat. no. 015‐000‐003)
  • Staining buffer: Phosphate‐buffered saline (PBS) without Ca2+ and Mg2+ and containing 0.03% sodium azide and 3% fetal bovine serum (FBS)
  • FACS lysing solution that contains fixative (BD Biosciences)
  • 96‐well plate round‐bottom microtiter plate
  • Centrifuge
  • FACS tubes
  • Flow cytometer

Basic Protocol 2: Isolation of B Cells Followed by Two‐Step Fractionation to Generate B Cell Subsets

  Materials
  • PBMCs (unit 7.1)
  • Custom Human B Cell Enrichment Cocktail (StemCell Technologies; includes MAbs against CD2, CD3, CD14, CD16, CD56, glycophorin A, CD66b, CD36, CD123, CD42b)
  • Tetrameric anti‐CD10 (StemCell Technologies)
  • EasySep Magnetic Microparticles (StemCell Technologies)
  • Wash buffer (WB): PBS without Ca2+ and Mg2+ containing 2% FBS
  • Biotinylated anti‐CD27 MAb at 0.5 mg/ml (eBioscience)
  • Anti‐biotin magnetic microbeads (Miltenyi Biotec)
  • FITC‐conjugated anti‐CD21 MAb (Beckman Coulter)
  • Anti‐FITC magnetic microbeads (Miltenyi Biotec)
  • Centrifuge
  • EasySep magnet or RoboSep (StemCell Technologies)
  • 5‐ml polypropylene snap‐cap tubes (Falcon)
  • MS columns (Miltenyi Biotec)
  • Magnet and stand (Miltenyi Biotec)
  • 15‐ml conical tubes
  • Plunger
NOTES: Unless otherwise stated, perform all steps on ice or at 4°C using cold buffers and reagents. The fractionation procedure assumes that the number of magnetically labeled cells does not exceed 107, the maximum capacity of MS columns. When labeling more than 107 cells, either scale up with either an LS column or multiple MS columns. Working volumes should be adjusted accordingly.

Alternate Protocol 1: One‐Step Double Fractionation for Isolation of Tissue‐Like Memory (CD21−/CD27−) B Cells

  • Biotinylated anti‐CD21 MAb at 1 mg/ml (Ancell)
NOTE: Unless otherwise stated, perform all steps on ice or at 4°C using cold buffers and reagents. The fractionation procedure assumes that the number of magnetically labeled cells does not exceed 107, the maximum capacity of MS columns. When labeling more than 107 cells, either scale up with either an LS column or multiple MS columns. Working volumes should be adjusted accordingly.

Basic Protocol 3: Measurement of B Cell Proliferation

  Materials
  • B cells or B cell subsets ( protocol 2)
  • Complete RPMI medium ( appendix 2A)
  • CpG oligodeoxynucleotide type B (CpG‐B) at 2.5 µg/ml
  • CD40 ligand at 500 ng/ml or anti‐CD40 MAb at 100 ng/ml
  • Anti‐IgM at 10 µg/ml (suggested source: Goat F(ab') 2 anti‐IgM from Jackson ImmunoResearch, cat. no. 309‐006‐043)
  • 48‐ or 96‐well flat‐bottom plates
  • 37°C incubator

Basic Protocol 4: Measurement Total Ig(G/M/A) and HIV‐Specific Ig(G/M/A) Response in Memory B Cells

  Materials
  • Cells
  • Staphylococcus aureus Cowan (SAC) at 1/10,000
  • CpG oligodeoxynucleotide type B (CpG‐B) at 2.5 µg/ml
  • MAbs to cell surface markers: CD19, CD27
  • Optional MAb to surface markers: CD20
  • Staining buffer: Phosphate‐buffered saline (PBS) without Ca2+ and Mg2+ and containing 0.03% sodium azide and 3% fetal bovine serum (FBS)
  • FACS lysing solution that contains fixative (BD Biosciences)
  • Permeabilization solution (BD Biosciences)
  • Biotinylated reagents for detection: anti‐IgG at 1/20,000; anti‐IgA at 1/10,000; and anti‐IgM at 1/10,000
  • MAbs to intracellular markers: IgD, IgG, IgA, IgM
  • 35% ethanol
  • Phosphate‐buffered saline (PBS; Lonza)
  • Coating antigens, each at 5 µg/ml: Goat anti‐kappa and anti‐lambda light chain antibodies (Rockland), HIV gp120, and KLH
  • 5%, 10% FBS in RPMI medium, freshly prepared
  • Complete RPMI medium
  • PBS‐T: 0.25% (w/v) Tween 20 in PBS
  • Primary antibodies
  • 0.25% PBS‐Tween containing 1% FBS
  • BSA/PBS: 1% BSA (w/v) in PBS
  • ELISPOT Blue Color Module (Streptavidin‐AP and BCIP‐NBT; R&D Systems, cat. no. SEL002)
  • 24‐well plates
  • Centrifuge
  • FACS tubes
  • Flow cytometer
  • 96‐well Multi‐Screen‐IP plate (Millipore)
  • 15‐ml conical tubes
  • Automated cell counter
  • 37°C incubator
  • Clean gauze or paper towels
  • Microplate reader

Alternate Protocol 2: Measurement Total Ig(G/M/A) and HIV‐Specific Ig(G/M/A) Response in Plasmablasts

  • ELISPOT: coat plate with antigen (see protocol 5)

Alternate Protocol 3: Measurement of Responses Using Biotinylated Antigen

  • Biotinylated reagents for detection: HIV gp120 and KLH
  • ELISPOT: coat plate with goat anti‐kappa and anti‐lambda light chain antibodies (see protocol 5)
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Figures

Videos

Literature Cited

Literature Cited
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