Models for Lymphoma

Arya Biragyn1, Larry W. Kwak1

1 National Cancer Institute, Frederick, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 20.6
DOI:  10.1002/0471142735.im2006s46
Online Posting Date:  February, 2002
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Abstract

This unit describes experimental procedures for development of therapeutic vaccines, particularly “second‐generation” recombinant vaccines. Specifically, a general procedure for handling and culturing lymphoma cell lines in vitro and their subsequent challenge into syngeneic mice is described. Several protocols describe the production of various idiotype (Id)‐based or cellular lymphoma vaccine formulations. In particular, the novel approach of rendering nonimmunogenic lymphoma‐derived scFv or Id immunogenic by fusing it with a chemokine moiety is described. As an alternative, a protocol for expression and purification of these chemokine‐fusion proteins from E. coli is included. A general procedure is included for cloning of cytokine genes, for example, murine GM‐CSF, in A20 lymphoma cells. Support protocols are presented for chemically conjugating intact Ig protein with KLH to produce a prototype protein vaccine and using the Helios Gene Gun System to immunize mice with recombinant DNA tumor vaccines.

     
 
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Table of Contents

  • Basic Protocol 1: Mouse Models of Intraperitoneal or Subcutaneous Lymphomas
  • Basic Protocol 2: Cloning of Lymphoma‐Derived Ig Variable Region Genes (VH and VL) and Construction of Single‐Chain Fv Fusions for Use as Naked DNA Vaccines
  • Basic Protocol 3: Production of scFv Fusion Proteins from Bacteria
  • Basic Protocol 4: Tumor Protection Using Genetically Modified Whole Tumor Cell Vaccines (Granulocyte‐Macrophage Colony‐Stimulating Factor‐Transduced A20 Lymphoma)
  • Basic Protocol 5: In Vivo Tumor Protection and Immunotherapy Experiments
  • Support Protocol 1: ELISA to Determine Proper Folding of scFv Protein
  • Support Protocol 2: ELISA to Measure Anti‐Idiotypic Antibody Production in Immunized Mice
  • Support Protocol 3: Chemical Cross‐Linking of KLH with Lymphoma‐Derived Ig
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Mouse Models of Intraperitoneal or Subcutaneous Lymphomas

  Materials
  • A20 B cell lymphoma (ATCC #TIB‐208; store cell stocks in 1‐ to 2‐ml aliquots in liquid N 2)
  • 38C‐13 B cell lymphoma (Bergman and Haimovich, ; available from authors; store 1–5 × 106 cell/ml stock in 1‐ to 2‐ml aliquots in liquid N 2)
  • RPMI 1640 medium (e.g., Life Technologies)
  • Complete RPMI medium ( appendix 2A) with and without 10% heat‐inactivated FBS
  • Dulbecco's phosphate‐buffered saline (DPBS, without Ca2+ and Mg2+; Life Technologies)
  • 6‐ to 12‐week‐old female C3H/HeN and BALB/c mice
  • 50‐ml conical centrifuge tubes
  • Centrifuge with Sorvall H‐1000B swinging‐bucket rotor, or equivalent
  • 1‐ml disposable syringes and 27.5‐G needles
  • Additional reagents and equipment for counting cells in a hematocytometer and determining cell viability by trypan blue exclusion ( appendix 3B), ear tagging (unit 1.5), and subcutaneous and intraperitoneal injection of mice (unit 1.6)

Basic Protocol 2: Cloning of Lymphoma‐Derived Ig Variable Region Genes (VH and VL) and Construction of Single‐Chain Fv Fusions for Use as Naked DNA Vaccines

  Materials
  • DEPC‐treated (RNase‐free) H 2O (unit 10.11)
  • ANA‐1 murine monocyte cell line (Blasi et al., ; gift of Luigi Varesio, Instituto G. Gaslini, Genoa, Italy) or LPS‐induced murine monocytes
  • recipeSolution D (see recipe; also see unit 10.11 for general discussion of guanidinium extraction of RNA)
  • 2 M sodium acetate, pH 4.0 (unit 10.11)
  • Phenol, water‐saturated (unit 10.11)
  • 49:1 (v/v) chloroform/isoamyl alcohol
  • Isopropanol
  • 70% and 100% (absolute) ethanol
  • 25 mM MgCl 2
  • 10× PCR buffer II (Perkin‐Elmer)
  • 2.5 mM 4dNTP mix: 2.5 mM each dNTP in water; store at −20 °C
  • RNAsin (human placental ribonuclease inhibitor; Promega)
  • Random hexamer primer mixture (e.g., Pharmacia Biotech)
  • 200 U/µl AMV reverse transcriptase
  • 30 µM forward and reverse PCR primers: to clone V H and V L from 38C‐13 and A20 murine B cell lymphomas (Table 20.6.2) and IP‐10 and MCP‐3 chemokines (Table 20.6.3)
  • AmpliTaq Gold DNA polymerase (Perkin‐Elmer Applied Biosystems)
  • Klenow fragment of DNA polymerase
  • 10× loading buffer (unit 10.4)
  • Low‐melting‐temperature agarose (Type XI, Sigma; also see unit 10.4)
  • Ethidium bromide solution (unit 10.4)
  • TAE electrophoresis buffer (unit 10.4)
  • DNA size ladder: e.g., λ phage DNA cut with HindIII
  • 5 M NaCl ( appendix 2A)
  • Phenol, buffer‐saturated (unit 2.NaN)
  • 25:24:1 phenol/chloroform/isoamyl alcohol (unit 10.1)
  • TE buffer, pH 7.5( appendix 2A)
  • 10 U/µl T4 polynucleotide kinase and 10× polynucleotide kinase buffer
  • 10 mM ATP
  • T4 DNA ligase and 10× DNA ligase buffer
  • Restriction endonucleases: NcoI, EcoRI, HindIII, SmaI or XmaI, DraI, XhoI, SalI,BamHI, and BglII
  • 3 M sodium acetate ( appendix 2A)
  • myc‐his‐pcDNA3.1 plasmid: pcDNA3.1(+) (Invitrogen) modified by inserting c‐myc and His‐tags (Fig. ; for plasmid modification, see Chapter 10 of this manual and Ausubel et al. )
  • Competent E. coli cells ( appendix 3N) or XL1 Blue competent cells (Stratagene)
  • LB medium or other bacterial complete medium without antibiotics
  • LB medium (unit 10.3) and LB plates (unit 10.19) with 100 µg/ml ampicillin (Amp)
  • recipeGTE solution (optional; unit 10.3)
  • 0.2 N NaOH/1% (w/v) SDS (prepare just before use)
  • 3 M potassium acetate, pH 4.8 (unit 10.3)
  • AmpliTaq DNA polymerase (Perkin‐Elmer)
  • Agarose (for routine use, Sigma; not LMP)
  • Qiagen P‐2500 column plasmid purification kit
  • Gold particles (0.8 to 1 µm; Degussa; also available at higher price from Bio‐Rad)
  • 0.1 M spermidine (store frozen in single‐use aliquots)
  • 2.5 M CaCl 2
  • Ultrapure nitrogen gas
  • Heating block
  • Thermal cycler (see unit 10.20)
  • Water bath sonicator
  • Tefzel tubing (0.104‐in. i.d. × 0.192‐in. o.d.;Bio‐Rad)
  • Tubing Prep Station (Bio‐Rad; provided with Helios Gene Gun System)
  • Additional reagents and equipment for RNA isolation by the guanidinium thiocyanate method (see units 2.12 & 10.11), PCR amplification of DNA (unit 10.20), agarose gel electrophoresis (unit 10.4), alkaline lysis to isolate DNA from bacteria (unit 10.3), and transient transfection (units 10.14 & 10.15) and immunoblotting (unit 8.10)
NOTE: Use endotoxin‐free ultra‐pure distilled water, tissue culture grade (Advanced Biotechnologies) at all points where water is called for.

Basic Protocol 3: Production of scFv Fusion Proteins from Bacteria

  Materials
  • 100 ng/µl pET11 bacterial expression plasmid, containing the T7 promoter element (Stratagene)
  • Competent E. coli cells: XL1 Blue and BL21(DE3) (Stratagene)
  • LB medium (unit 10.3)
  • LB plates (unit 10.19) with 100 µg/ml ampicillin (Amp)
  • recipeGlycerol growth medium (see recipe)
  • 20% (v/v) glycerol
  • recipeInduction medium (see recipe)
  • 20% (w/v) sucrose solution in H 2O, ice cold
  • recipeTE buffer for proteins (see recipe)
  • 50 mg/ml lysozyme (store frozen)
  • recipe5 M NaCl ( appendix 2A)
  • 25% (v/v) Triton X‐100
  • recipe6 M guanidinium solution (see recipe)
  • BSA Protein Assay Kit (Pierce) or equivalent
  • Dithioerythritol (DTE)
  • recipeRefolding solution (see recipe), prechilled to 10°C
  • recipe20 mM Tris⋅Cl, pH 7.5 ( appendix 2A) containing 100 mM ultrapure urea
  • Heparin Sepharose CL‐6B (Pharmacia Biotech)
  • recipe20 mM Tris⋅Cl, pH 7.5 ( appendix 2A)
  • recipeGlycerol column buffers with 50 mM, 300 mM, and 1 M NaCl (see recipe)
  • Triton X‐100
  • recipePhosphate‐buffered saline ( appendix 2A) containing an additional 300 mM NaCl (∼450 mM total) with or without 5% to 10% (v/v) glycerol
  • Heating block
  • 2‐liter Ehrlenmeyer flask
  • 30°C incubator with shaker
  • Beckman Avanti J‐251 centrifuge with JLA‐10.5 and JA‐17 rotors (or equivalent)
  • Tissue‐Tearor Homogenizer (Fisher)
  • 10°C water bath
  • Dialysis membrane (Spectra/Por, MWCO 6000 to 8000, 50‐mm width; Spectrum)
  • 2‐liter plastic container
  • Chromatography column: Poly‐Prep, 0.8 × 4–cm (Bio‐Rad)
  • Additional reagents and equipment for dialysis ( appendix 3H), polyacrylamide gel electrophoresis (Ausubel et al., ), determination of proper folding of scFv ( protocol 6), and in vitro chemotaxis assay (unit 6.12)

Basic Protocol 4: Tumor Protection Using Genetically Modified Whole Tumor Cell Vaccines (Granulocyte‐Macrophage Colony‐Stimulating Factor‐Transduced A20 Lymphoma)

  Materials
  • 1 mg/ml mammalian expression plasmid, encoding murine GM‐CSF, dissolved in TE buffer, pH 8.0 (linearized by single restriction cut, purified by phenol/chloroform extraction and ethanol precipitation; see Ausubel et al., 2001, and Chapter in this manual)
  • A20 cells (ATCC #TIB‐208) growing in exponential phase
  • RPMI 1640 medium
  • Complete RPMI medium with 10% FBS ( appendix 2A)
  • MPRO cells (ATCC # CRL‐11422; Tsai and Collins, ) growing in exponential phase
  • Complete DMEM medium with 10% FBS ( appendix 2A)
  • recipeHanks' balanced salt solution ( appendix 2A) without Ca2+ and Mg2+
  • 6‐ to 12‐week‐old BALB/c female mice
  • Gene Pulser II System for electroporation (Bio‐Rad)
  • Sterile electroporation cuvettes (0.4‐cm electrode gap; Bio‐Rad)
  • 96‐well flat‐bottom microtiter plates (Costar)
  • Sorvall centrifuge with H‐1000B swinging‐bucket rotor (or equivalent)
  • 50‐ml conical polypropylene centrifuge tubes (e.g., Falcon)
  • Additional reagents and equipment for culturing cells cells (see appendix 33), ELISA (unit 2.1), immunoblotting (unit 8.10), labeling cells for determination of [3H]thymidine uptake ( appendix 3D and unit 3.12), and injection of mice (unit 1.6)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper aseptic technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2, 5% O 2 incubator unless otherwise indicated.

Basic Protocol 5: In Vivo Tumor Protection and Immunotherapy Experiments

  Materials
  • 6‐ to 12‐week‐old female C3H/HeN mice (for experiments with 38C13 lymphoma) or BALB/c mice (for experiments with A20 lymphoma)
  • Gene Gun cartridges coated with plasmid DNA vaccines (see protocol 2)
  • Protein vaccines: Ig‐KLH (for positive control; see protocol 8) and chemokine fusion scFv formulations (see protocol 3)
  • Dulbecco's phosphate‐buffered saline (DPBS, without Ca2+ and Mg2+; Life Technologies)
  • Tumor cells (also see protocol 1):
  •  A20 B cell lymphoma cells (ATCC)
  •  38C‐13 B cell lymphoma (Bergman and Haimovich, ; gift of Ronald Levy, Stanford Univ.)
  •  Cytokine (e.g., GM‐CSF) ‐transduced or modified A20 cells
  • Hanks' balanced salt solution without Ca2+ and Mg2+ (HBSS; appendix 2A), ice‐cold
  • Electric clippers
  • Helios Gene Gun (Bio‐Rad)
  • Compressed Ultra Pure Helium gas; maximum pressure, 2600 psi – with high‐pressure helium tank gas regulator
  • Hearing protection (e.g., ear plugs)
  • 1‐ml disposable syringes and 27.5‐G needles
  • γ irradiator (e.g., Nordion Gammacell)
  • Sorvall centrifuge with H1000B swinging‐bucket rotor, or equivalent
  • Additional reagents and equipment for immunization using Gene Gun (unit 2.14), handling and restraint (unit 1.3) and bleeding (unit 1.7) of mice, ELISA to measure anti‐idiotypic antibody production ( protocol 7), injection of mice (unit 1.6), growth of tumor cells and tumor cell challenge (see protocol 1), counting viable cells using trypan blue exclusion ( appendix 3B)
NOTE: The gene gun and tank regulator are supplied as part of the complete Helios Gene Gun System (Bio‐Rad) or can be purchased separately from Bio‐Rad.

Support Protocol 1: ELISA to Determine Proper Folding of scFv Protein

  Materials
  • 10 µg/ml lymphoma‐derived IgM (for 38C‐13) or IgG2a (for A20): available from Kwak Laboratory ( )
  • Polyclonal anti‐idiotypic sera or monoclonal 38C‐13 Id‐specific antibody S1C5
  • Coating buffer, pH 9.6 (unit 7.12)
  • recipeELISA wash buffer (see recipe)
  • 5% (w/v) fat‐free powdered milk in recipePBS (see appendix 2A for PBS)
  • Diluent buffer: 2% (w/v) BSA in recipePBS
  • scFv protein (see protocol 3)
  • Horseradish peroxidase (HRPO)–conjugated goat anti‐mouse IgG or IgG 1 antibody (Caltag)
  • ABTS peroxidase substrate (Kirkegaard & Perry)
  • 96‐well flat‐bottom ELISA plates (e.g., Immunol 4, Dynatech) and plate sealers
  • 96‐well U‐bottom tissue culture plates (Costar)
  • Multiwell scanning spectrophotometer
  • Additional reagents and equipment for ELISA (unit 2.1)

Support Protocol 2: ELISA to Measure Anti‐Idiotypic Antibody Production in Immunized Mice

  • Serum from 38C‐13 or A20‐immunized mice

Support Protocol 3: Chemical Cross‐Linking of KLH with Lymphoma‐Derived Ig

  Materials
  • KLH in saturated ammonium sulfate slurry (Calbiochem‐Novabiochem)
  • IgM derived from 38C‐13 lymphoma (Eshar et al., ; Maloney et al., ) or IgG2a from A20 (hybridoma is available from the Kwak laboratory; )
  • Glutaraldehyde (Sigma)
  • Dulbecco's recipePBS (without Ca2+ and Mg2+; Life Technologies; also see appendix 2A)
  • Dialysis membrane, MWCO 10,000 (Spectra/Por; Spectrum)
  • Additional reagents and equipment for dialysis ( appendix 3H)
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Figures

Videos

Literature Cited

Literature Cited
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