Analysis of the Targeting Properties of Fc Chimeric Proteins and Antibodies in Mice In Vivo

Luisa Martinez‐Pomares1

1 Faculty of Medicine and Health Sciences, University of Nottingham, Nottingham, United Kingdom
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 18.18
DOI:  10.1002/0471142735.im1818s97
Online Posting Date:  April, 2012
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Abstract

This unit includes protocols for the analysis of the targeting properties of Fc chimeric proteins and antibodies in mice in vivo. We present procedures to prepare the reagents to be tested under endotoxin‐free conditions, suitable injection sites, methodology for tissue collection, and processing, and labeling procedures to detect the injected material in situ. Curr. Protoc. Immunol. 97:18.18.1‐18.18.12. © 2012 by John Wiley & Sons, Inc.

Keywords: macrophages; antibodies; chimeric Fc proteins

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Tissue Samples for Histological Analysis
  • Basic Protocol 2: Detection of Targeted Chimeric Fc Proteins and Rat Antibodies In Situ
  • Basic Protocol 3: Preparation of Single‐Cell Suspensions from Mouse Lymph Nodes
  • Basic Protocol 4: Labeling of Single‐Cell Suspensions with Fc Chimeric Protein
  • Support Protocol 1: Expression of Fc Chimeric Proteins
  • Support Protocol 2: Purification of Fc Chimeric Proteins and Rat IgG Under Endotoxin‐Free Conditions
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Tissue Samples for Histological Analysis

  Materials
  • Purified Fc chimeric proteins ( protocol 6)
  • Rat IgG (purified monoclonal antibody specific for the receptor of interest or isotype‐matched control)
  • Immune stimulus [e.g., purified endotoxin (LPS, ligand for toll‐like receptor 4, TLR4), flagellin (ligand for TLR5), polyinosine‐polycytidylic acid (polyI:C, ligand for TLR3), Pam3CSK‐Ser‐(lys)4 (ligand for TLR2); all are available from InvivoGen (doses need to be titrated for each application)]
  • Cross‐linking reagent [antibody specific against the Fc portion of the Fc‐chimeric protein; for Fc‐chimeric proteins containing a human IgG Fc portion, use mouse F(ab)2 anti‐human Fc (Jackson ImmunoResearch)]; optimal Fc‐chimeric protein:cross‐linking reagent ratio needs to be calculated for each application.
  • Dulbecco's phosphate‐buffered saline (D‐PBS; Sigma, cat. no. D8537)
  • Animals (e.g., BALB/c and C57BL/6 strains; both wild‐type or knockout animals)
  • Set up to inhaled anesthesia (Isoflurane)
  • OCT Tissue‐Tek compound (Bayer Diagnostics)
  • Isopentane (VWR)
  • Dry Ice
  • Syringe and needles (e.g., BD Mirco‐Fine insulin needles)
  • Dissecting tools
  • Dissecting board
  • Plastic molds (Raymond Lamb)
  • 500‐ml evaporating flat‐bottom cylindrical basin (no spout; Pyrex)
  • Ice container
  • Cryostat
  • Gelatin‐coated or charged slides

Basic Protocol 2: Detection of Targeted Chimeric Fc Proteins and Rat Antibodies In Situ

  Materials
  • Tissue on slides (see protocol 1)
  • Fixative solution (see recipe)
  • Dulbecco's phosphate‐buffered saline (D‐PBS; Sigma, cat. no. D8537)
  • Permeabilizing solution: 0.1% Triton X‐100 in PBS.
  • Blocking solution: 5% normal serum in PBS sources of normal sera [e.g., donkey (Sigma), goat (Vector Laboratories), mouse (Sigma), rabbit (Vector Laboratories), or rat (Sigma)]
  • Avidin/Biotin Blocking kit (Vector Laboratories), optional
  • Biotinylated goat anti‐human IgG (Vector Laboratories; see Table 18.18.1)
  • Alexa 488‐conjugated Goat anti‐human IgG (Molecular Probes; see Table 18.18.1)
  • Alexa 488‐conjugated Goat anti‐rat IgG (Molecular Probes; see Table 18.18.1)
  • Alexa 647‐conjugated Goat anti‐rat IgG (Molecular Probes; see Table 18.18.1)
  • Cy5‐conjugated Streptavidin (Jackson Immunoresearch; see Table 18.18.1)
  • Alexa 488‐conjugated Streptavidin (Molecular Probes; see Table 18.18.1)
  • Coplin jars
  • Shandon Sequenza Slide Rack (Thermo Scientific)
  • Shandon Coverplate (Thermo Scientific)
    Table 8.8.1   MaterialsExamples of Labeling Procedures for the Analysis of the Targeting Properties of Fc Chimeric Proteins and Rat IgG in Mouse Tissues

    Injected reagent Fc chimeric protein (Fc region of human IgG1) Rat IgG
    Reagents to detect injected reagent 1‐ Anti‐human Fc (gamma chain‐specific) biotinylated; 2‐ Alexa‐488 conjugated streptavidin Donkey anti‐rat IgG conjugated to Alexa 488; Goat anti‐rat IgG conjugated to Cy5
    Additional blocking step Blocking with purified rat IgG (100 µg/ml)
    Primary reagent to identify targeted cells Rat IgG against mouse cell marker Biotinylated rat IgG against mouse cell marker; Rabbit IgG (adsorbed to rat IgG) against mouse cell marker
    Secondary reagent to identify targeted cells Donkey anti‐rat conjugated to Alexa‐647 Streptavidin conjugated to Cy5; Donkey anti‐rabbit conjugated to FITC
    Reagent to detect cell nucleus 4′,6‐Diamidino‐2‐phenylindole,dilactate (DAPI; Sigma) DAPI
    Mounting Fluorescent mounting medium Fluorescent mounting medium
    Observation Fluorescent microscope Fluorescent microscope
    Analysis Adobe Photoshop Adobe Photoshop

Basic Protocol 3: Preparation of Single‐Cell Suspensions from Mouse Lymph Nodes

  Materials
  • RPMI medium
  • Collagenase D (Roche)
  • DNase I (Roche)
  • EDTA
  • Bovine serum albumin (BSA)
  • Trypan blue solution (Sigma)
  • 6‐well plates
  • 25‐G needles
  • 2‐ml syringe barrels
  • Sterile blades
  • Shaker at 37°C
  • Pasteur pipets
  • 70‐µm cell strainer (BD Biosciences)
  • Additional reagents and equipment for removal of lymphoid organs (see unit 1.9) and assessing viability of cells using trypan blue exclusion ( appendix 3B)

Basic Protocol 4: Labeling of Single‐Cell Suspensions with Fc Chimeric Protein

  Materials
  • Cells
  • FACS blocking buffer (see recipe)
  • FcγRII/III blocking mAb, clone 2.4G2 (Mouse BD Fc Block; BD Pharmingen)
  • Purified Fc‐chimeric protein
  • FACS washing buffer: PBS, 0.5% w/v BSA, 5 mM EDTA, 2 mM NaN 3
  • FACS fixing solution: PBS supplemented with 1% (v/v) formaldehyde (Sigma)
  • Alexa 488‐conjugated goat anti‐human IgG (Molecular Probes)
  • Biotinylated goat anti‐human IgG (Vector Laboratories)
  • Alexa 488‐conjugated streptavidin (Molecular Probes)
  • Refrigerated centrifuge

Support Protocol 1: Expression of Fc Chimeric Proteins

  Materials
  • 293T cells
  • Growth medium: Dulbecco's minimal essential medium (DMEM) containing glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% of fetal bovine serum (DMEM‐10)
  • Serum free medium: Opti‐MEM (Invitrogen)
  • GeneJuice (Novagen)
  • Expression plasmid purified using an endotoxin‐free method (18 µg/175‐cm2 flask)
  • 175‐cm2 flasks
  • 37°C, 5% CO 2 incubator
  • 14‐ml polypropylene tubes
  • Centrifuge

Support Protocol 2: Purification of Fc Chimeric Proteins and Rat IgG Under Endotoxin‐Free Conditions

  Materials
  • PBS
  • Starting material: supernatants from transfected 293T cells or hybridoma prepared under serum‐free conditions
  • Protein A‐Sepharose or Protein G‐Sepharose (Pharmacia)
  • 0.1 M Glycine, pH 2.9 (endotoxin free)
  • 1 M Tris⋅Cl, pH 8 (endotoxin free)
  • Protein quantification reagent (BCA, Perbio)
  • Endotoxin detection kit (e.g., Invitrogen), optional
  • Columns (e.g., Econocolumns and accessories are available from Bio‐Rad)
  • Chromatography equipment (if in doubt, treat with 0.5 M NaOH and rinse extensively with PBS to reduce possible endotoxin contamination)
  • 1.5‐ml tubes
  • Dialysis cassettes (Perbio)
NOTE: All steps should be performed at 4°C.
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Figures

  •   FigureFigure 18.18.1 Targeting of the Fc chimeric proteins CR‐Fcmut and CRW117A‐Fcmut in vivo. Recombinant proteins CR‐Fcmut and CRW117A‐Fcmut optimally cross‐linked using mouse anti‐human Fc F(ab')2 were injected i.v. into BALB/c mice (20 µg of recombinant protein per mouse), and spleens were removed 15 min later. The recombinant proteins (green) were detected with a biotinylated anti‐human Fc and streptavidin‐conjugated Alexa 488, and Sialoadhesin+ met macrophages were identified with 3D6 and goat anti‐rat IgG‐conjugated Alexa 647 (red). CR‐Fcmut predominantly localized to the Siaoloadhesin+met macrophages, with some protein detectable in the outer marginal zone. The protein CRW117A‐Fcmut showed enhanced localization in the outer marginal zone but no trapping on the met macrophages. This is consistent with its inability to recognize sulfated carbohydrates in vitro due to the substitution of tryptophan at position 117 with alanine. The outer marginal zone staining corresponds to marginal zone macrophages.
  •   FigureFigure 18.18.2 Targeting of an anti‐MR antibody to medullary macrophages in draining lymph nodes. Medullary macrophages (Lyve), but not lymphatic endothelia (Lyve+) can be efficiently targeted in vivo using specific anti‐MR antibodies. BALB/c animals were injected s.c. in the forelimb with rat anti‐mouse MR6C3 or isotype control mAbs. Secondary lymphoid tissues were collected 24 hr later and processed for immunofluorescence. Injected mAbs were detected in tissue sections using a goat anti‐rat IgG reagent (green) and lyve‐1‐expressing cells using a rabbit anti‐mouse Lyve‐1 and Cy5‐conjugated anti‐rabbit (red).

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Literature Cited

Literature Cited
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   Martinez‐Pomares, L., Crocker, P.R., Da Silva, R., Holmes, N., Colominas, C., Rudd, P., Dwek, R., and Gordon, S. 1999. Cell‐specific glycoforms of sialoadhesin and CD45 are counter‐receptors for the cysteine‐rich domain of the mannose receptor. J. Biol. Chem. 274:35211‐35218.
   McKenzie, E.J., Taylor, P.R., Stillion, R.J., Lucas, A.D., Harris, J., Gordon, S., and Martinez‐Pomares, L. 2007. Mannose receptor expression and function define a new population of murine dendritic cells. J. Immunol. 178:4975‐4983.
   Phan, T.G., Green, J.A., Gray, E.E., Xu, Y., and Cyster, J.G. 2009. Immune complex relay by subcapsular sinus macrophages and noncognate B cells drives antibody affinity maturation. Nat. Immunol. 10:786‐793.
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   Taylor, P.R., Zamze, S., Stillion, R.J., Wong, S.Y., Gordon, S., and Martinez‐Pomares, L. 2004. Development of a specific system for targeting protein to metallophilic macrophages. Proc. Natl. Acad. Sci. U.S.A. 101:1963‐1968.
   Yu, P., Wang, Y., Chin, R.K., Martinez‐Pomares, L., Gordon, S., Kosco‐Vibois, M.H., Cyster, J., and Fu, Y.X. 2002. B cells control the migration of a subset of dendritic cells into B cell follicles via CXC chemokine ligand 13 in a lymphotoxin‐dependent fashion. J. Immunol. 168:5117‐5123.
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