Generation and Maintenance of Cloned Human T Cell Lines

Hans Yssel1, Hergen Spits2

1 INSERM U454, Montpellier, France, 2 The Netherlands Cancer Institute, Amsterdam, Netherlands
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.19
DOI:  10.1002/0471142735.im0719s47
Online Posting Date:  May, 2002
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Abstract

This unit describes protocols for the generation of human (allo‐) antigen‐specific T cell lines, and T lymphocyte subpopulations with distinct cytokine production profiles from purified peripheral or cord blood CD4+ T cells, respectively. Methods for the cloning and maintenance of these cell lines are given, as well as a protocol for freeze/thaw procedures.

     
 
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Table of Contents

  • Basic Protocol 1: Generation of (Allo‐)Antigen‐Specific T Cell Lines
  • Basic Protocol 2: Generation of CD4+ T Cell Lines with a TH1 or TH2 Cytokine Production Profile
  • Support Protocol 1: Establishment of Clones from T Cell Lines
  • Support Protocol 2: Maintenance of Cloned T Cell Lines
  • Support Protocol 3: Cryopreservation of T Cell Clones
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Generation of (Allo‐)Antigen‐Specific T Cell Lines

  Materials
  • For soluble antigen‐specific T cell clones:
  •  Unfractionated peripheral blood mononuclear cells (PBMC) from an antigen sensitized donor (unit 7.1)
  •  Antigen of interest (native antigen or peptide)
  •  Unfractionated PBMC from a donor with appropriate HLA type
  •  Irradiated, autologous or HLA‐matched unfractionated PBMC
  •  Autologous Epstein‐Barr virus transformed (EBV‐) B cells (unit 7.22 to be used for T cell lines or clones with known peptide specificity)
  • For allo‐antigen‐specific T cell clones:
  •  Allogeneic EBV‐B cells of appropriate HLA type
  • recipe Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% human AB+ serum (culture medium; see recipe)
  • Phosphate buffered saline (PBS) without Ca2+/Mg2+ ( appendix 2A)
  • 24‐well culture plates (Linbro, Hampton Research)
  • 37°C, 5% CO 2 incubator
  • 96‐well round‐bottom culture plates (Becton Dickinson Labware)
  • 15‐ml and 50‐ml centrifuge tubes (Becton Dickinson Labware)
  • Additional reagents and equipment for Ficoll‐Hypaque density centrifugation (unit 7.1), T cell proliferation and cytokine production assay (unit 7.10), counting cells using a hemacytometer ( appendix 3A), trypan blue test for cell viability ( appendix 3B)

Basic Protocol 2: Generation of CD4+ T Cell Lines with a TH1 or TH2 Cytokine Production Profile

  • Human umbilical cord blood
  • Purified CD4+ umbilical cord blood or CD4+, CD45RA+ peripheral blood T cells (optional)
  • Anti‐CD45RO+ mAb (unit 7.4, optional)
  • Mouse fibroblasts (L cells), co‐transfected with cDNA encoding CD32 (F cRγII), CD58 and CD80 molecules (unit 10.14 for transfection; unit 10.13 for L cell maintenance)
  • IMDM, supplemented with 10% (w/v) fetal bovine serum (FBS), filtered through a 0.1‐µm filter
  • Anti‐CD3 and anti‐CD28 mAbs (OKT‐3, UCHT‐1, 9.3, L293, or equivalent)
  • rIL‐2, rIL‐4, rIL‐10, rIL‐12 (PeproTech, R&D Systems), neutralizing anti‐IL‐4, anti‐IL‐12, and anti‐IFN‐γ mAbs (R&D Systems, Pharmingen)
  • 50‐ml heparin‐containing blood collection tubes
  • Magnetic beads coated with CD3 and anti‐CD28 mAbs (Dynal; optional)
  • Magnetic particle separator (Dynal or equivalent; optional)
  • Additional reagents and equipment for Ficoll‐Hypaque density centrifugation (unit 7.1)

Support Protocol 1: Establishment of Clones from T Cell Lines

  • Unfractionated peripheral blood mononuclear cells (PBMC) from a healthy donor, preferentially from a buffycoat
  • EBV‐B cell line
  • Phytohemagglutinin (PHA, Murex)
  • rIL‐2, specific activity = ∼107 U/ml (PeproTech of R&D Systems)

Support Protocol 2: Maintenance of Cloned T Cell Lines

  Materials
  • T cell clones
  • DMSO (Sigma)
  • RPMI‐1640 or any medium without HEPES
  • Human or bovine serum
  • PBS ( appendix 2A), cold
  • recipeCulture medium (see recipe)
  • 15‐ml centrifuge tubes
  • Platform shaker
  • Cryovials (Corning or Nunc)
  • 1°C cryocontainer (Nalgene)
  • 37°C water bath
  • 2‐ml pipets
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Figures

Videos

Literature Cited

Literature Cited
   Abbas, A.K., Murphy, K.M., and Sher, A. 1996. Functional diversity of helper T lymphocytes. Nature 383:787‐793.
   Gillis, S. and Smith, K.A. 1977. In vitro generation of tumor‐specific cytotoxic T lympocytes. Secondary allogeneic mixed tumor lymphocyte culture of noral murine spleen cells. J. Exp. Med. 146:486‐482.
   Glasebrook, A.L. and Fitch, F.W. 1979.. T‐cell lines which cooperate in generation of specific cytolytic activity. Nature 278:171‐173.
   Inouye, H., Hank, J.A., Chardonnens, X., Segall, M., Alter, B.J., and Bach, F.H. 1980. Cloned primed‐lymphocyte‐test reagents in the dissection of HLA‐D. J. Exp. Med. 152:143‐155.
   Kornbluth, J. and Dupont, B. 1980. Cloning and functional characterization of primary alloreactive human T lymphocytes. J. Exp. Med. 152:164‐181.
   Morgan, D.A., Ruscetti, F.W., and Gallo, R. 1976. Selective in vitro growth of T lymphocytes from normal human bone marrows. Science 193:1007‐1008.
   Nowell, P. 1960. Phytohaemagglutinin: An initiator of mitosis in cultures of normal human leukocytes. Cancer Res. 20:462‐466.
   Spits, H., Yssel, H., Terhorst, C., and de Vries, J.E. 1982. Establishment of human T lymphocyte clones highly cytotoxic for an EBV‐transformed B cell line in serum‐free medium: Isolation of clones that differ in phenotype and specificity. J. Immunol. 128:95‐99.
   Yssel, H., de Vries, J.E., Koken, M., Van Blitterswijk, W., and Spits, H. 1984. Serum‐free medium for the generation and propagation of functional human cytotoxic and helper T cell clones. J. Immunol. Methods 72:219‐227.
Key References
   Morgan et al., 1976. See above
  The seminal reference discussing the use of TCGF for the in vitro growth of T cells.
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