Production of TH1 and TH2 Cell Lines and Clones

Frank W. Fitch1, Thomas F. Gajewski1, Jane Hu‐Li2

1 University of Chicago, Chicago, Illinois, 2 National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 3.13
DOI:  10.1002/0471142735.im0313s72
Online Posting Date:  May, 2006
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Abstract

This unit describes protocols for the generation of polyclonal TH1 and TH2 cell lines from naive CD4+ T cells as well as for generation of antigen‐specific cell lines from TCR‐transgenic mice and antigen‐specific T cell clones from primed mice. Also described are methods for the preparation and maintenance of alloreactive murine helper T (TH) lymphocyte and cytotoxic T lymphocyte (CTL) clones using the limiting dilution technique, as well as derivation of TH clones reactive with soluble protein antigens, including a method for the selection of either TH1 or TH2 lymphocyte subsets. These two subsets of TH cells exhibit helper function in different ways and can be distinguished by the patterns of cytokines they synthesize. Support protocols describe a micromanipulation method for cloning T cells and a roadmap for using protocols published elsewhere in this series to assess cytokine production by T cell clones and lines.

Keywords: TH1; TH2; T cells; IL‐4; IFNγ

     
 
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Table of Contents

  • Basic Protocol 1: Production of Polyclonal CD4+ TH1 AND TH2 Cell Lines
  • Alternate Protocol 1: Production of TH1 and TH2 Cell Lines and Clones from TCR‐Transgenic Mice
  • Alternate Protocol 2: Derivation and Maintenance of TH Clones Reactive with Soluble Protein Antigens
  • Alternate Protocol 3: Derivation and Maintenance of Alloreactive TH and CTL Clones
  • Support Protocol 1: Cloning by Micromanipulation
  • Support Protocol 2: Assays Used to Measure Cytokine Production by TH1 and TH2 Cell Lines and Clones
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Production of Polyclonal CD4+ TH1 AND TH2 Cell Lines

  Materials
  • Mice of interest as source of CD4+ cells
  • Complete RPMI‐10 or DMEM‐10 (see recipe and appendix 2A)
  • Syngeneic mice as source of antigen‐presenting cells (APC)
  • Human recombinant IL‐2 (PeproTech)
  • Anti‐ IL‐4 monoclonal antibody from clone11B11 (BD Pharmingen)
  • Mouse interleukin 12 (IL‐12; R&D Systems)
  • Mouse interleukin 4 (IL‐4; PeproTech)
  • Anti‐IFN‐γ monoclonal antibody, clone XMG1.2 (BD Pharmingen)
  • Anti‐IL‐12 monoclonal antibody, clone C17.8 (BD Pharmingen)
  • Anti‐CD3 monoclonal antibody, clone 2C11 (BD Pharmingen)
  • Anti‐CD28 monoclonal antibody, clone 37.51 (BD Pharmingen)
  • Hanks' balanced salt solution (HBSS; appendix 2A)
  • 6‐, 12‐, and 24‐well plates (Costar)
  • Low‐speed Sorvall centrifuge RT‐6000 (or equivalent)
  • 15‐ or 50‐ml disposable conical tubes with screw caps
  • Additional reagents and equipment for removing organs (unit 1.9), preparing single‐cell suspensions (unit 3.1), counting viable cells by trypan blue dye exclusion ( appendix 3B), immunomagnetic depletion of T cells (unit 3.5), irradiation of cells (unit 3.12), and cloning by limiting dilution (unit 2.5) or micromanipulation ( protocol 5 in this unit)

Alternate Protocol 1: Production of TH1 and TH2 Cell Lines and Clones from TCR‐Transgenic Mice

  • TCR‐transgenic mice: it is preferable to use mice that are maintained on a RAG‐deficient or SCID background; these mice lack T cells that express endogenous TCRs in addition to the transgenic TCR (also see appendix 1C)
  • Syngeneic mice as source of antigen‐presenting cells (APC)
  • TCR‐specific antigen or peptides of interest
  • Anti‐CD44 and anti‐CD62L monoclonal antibodies (BD Pharmingen; optional, needed only for sorting naive CD4+ cells)

Alternate Protocol 2: Derivation and Maintenance of TH Clones Reactive with Soluble Protein Antigens

  Materials
  • Mice for immunization
  • Protein antigen
  • Dulbecco's phosphate‐buffered saline (e.g., Life Technologies)
  • Complete Freund's adjuvant
  • Complete DMEM‐5 medium, supplemented (see recipe)
  • Irradiated (2000 rad; see unit 3.12) syngeneic spleen cells in complete DMEM‐5 medium
  • Source of growth factors: recombinant cytokines such as human recombinant IL‐2 (hrIL‐2; e.g., PeproTech) and murine recombinant IFN‐γ (mrIFN‐γ; e.g., Genentech)
  • 96‐well flat‐bottom microtiter plates (Costar)
  • 24‐well microtiter plates (preferably Linbro; alternatively Costar)
  • Additional reagents and equipment for parenteral injection of mouse (unit 1.6) and preparation of single‐cell suspensions of lymph node cells (unit 3.1), and purification of T cells (unit 3.5)
NOTE: All reagents and cells are prepared in supplemented DMEM‐5 prior to addition of cultures.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Alternate Protocol 3: Derivation and Maintenance of Alloreactive TH and CTL Clones

  Materials
  • C57BL/6‐responding murine spleen cells (unit 3.1)
  • Complete DMEM‐5 and DMEM‐20 medium, supplemented (see recipe)
  • Irradiated (2000 rad; unit 3.12) allogeneic murine spleen cells
  • Human recombinant IL‐2 (PeproTech)
  • 50‐ml plastic upright tissue culture flasks
  • 50‐ml conical polypropylene centrifuge tubes
  • Tabletop centrifuge
  • 96‐well flat‐bottom microtiter plates (Costar)
  • 24‐well microtiter plates (preferably Linbro; alternatively Costar)
  • Additional reagents and equipment for counting cells ( appendix 3A), flow cytometry (units 5.3& 5.4), and assaying for cytolytic activity (unit 3.11)
NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 1: Cloning by Micromanipulation

  Materials
  • Microcapillary tube (e.g., plain microhematocrit tubes; Becton Dickinson)
  • Tissue culture burner
  • Small‐bore rubber or plastic tubing
  • 60 × 15–mm tissue culture plates (Becton Dickinson Labware)
  • Additional reagents and equipment for measuring cytolytic activity (unit 3.11) secretion of lymphokines (units 6.3& 6.9) or proliferation (unit 3.12)
NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.
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Figures

Videos

Literature Cited

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Key Reference
  Fathman, C.G. and Fitch, F.W. (eds.) 1982. Isolation, Characterization, and Utilization of T Lymphocyte Clones. Academic Press, San Diego.
  Although published several years ago, this volume contains useful general information regarding various aspects of the isolation and utilization of T lymphocyte clones.
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