Production of Mouse T Cell Hybridomas

Ada M. Kruisbeek1

1 Netherlands Cancer Institute, Amsterdam, The Netherlands
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 3.14
DOI:  10.1002/0471142735.im0314s24
Online Posting Date:  May, 2001
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Abstract

T cell hybridomas can be obtained by fusing activated T cells with tumor cells. A heterogeneous population of hybridomas can be cloned by limiting dilution to obtain hybridomas that express specificity to one T cell receptor (TCR). This unit describes cell fusion and selection of T cell hybridomas. A protocol is provided for screening of T cell hybridomas for expression of the CD3‐TCR complex by flow cytometry analysis. Those hybridomas expressing a CD3‐TCR complex are then further tested for expression of antigen specificity by screening for specific antigen‐induced lymphokine production. The procedures for establishment of stable hybridoma lines and cloning of stable lines by limiting dilution are identical to those described for B cell hybridomas and can be found in UNIT 2.5. Protocols for freezing and thawing T cell hybridomas are identical to those described for B cell hybridomas and other cell lines.

     
 
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Table of Contents

  • Basic Protocol 1: Cell Fusion and Selection of T Cell Hybridomas
  • Support Protocol 1: Screening Primary Hybridomas for CD3‐TCR Expression
  • Support Protocol 2: Screening Hybridomas for Antigen Specificity
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Cell Fusion and Selection of T Cell Hybridomas

  Materials
  • Mice—for in vivo priming of T cells against soluble protein or peptide antigen
  • Complete Freunds adjuvant
  • Responder cells (unprimed lymph node T cells) and stimulator cells (spleen cells expressing desired alloantigens)—for activating T cells in secondary MLC
  • BW5147 lymphoma cell line (ATCC #TIB 48: full name is BW5147.G.1.4; drug marked, dies in HAT, resistant to 6‐thioguanine)
  • Complete DMEM‐20 and ‐10 media ( appendix 2A)
  • Soluble protein antigen and antigen‐presenting cells (unit 3.12)
  • recipe2× HAT plating medium (see recipe)
  • Wash medium: DMEM without FBS or HEPES (i.e., unsupplemented DMEM)
  • recipe50% (w/v) PEG in wash medium
  • Mouse thymocytes (of same strain as that used for responder T cells; unit 3.1)
  • recipe1× HT plating medium (see recipe)
  • 25‐, 75‐, and 175‐cm2 tissue culture flasks
  • 400‐ and 600‐ml beakers
  • Stopwatch
  • Laminar flow hood with vacuum connection and vacuum flask
  • 4‐ and 50‐ml conical polypropylene centrifuge tubes
  • Low‐speed centrifuge with Sorvall H‐1000B rotor (or equivalent) at room temperature
  • 1‐, 2‐, 10‐, and 25‐ml pipets
  • 96‐ and 24‐well flat‐bottom microtiter plates
  • Additional reagents and equipment for mouse euthanasia (unit 1.80), priming mice (units 2.5 3.12 & 3.13), lymph node removal (unit 3.1), preparing activated T cells in secondary MLC (for alloreactive T cells; units 3.12 & 3.13) or in an in vitro culture with primed responder lymph node T cells (for T cells reactive with protein antigens; units 3.12 & 3.13), preparing thymocyte suspension (unit 3.1),removing dead cells by Ficoll‐Hypaque centrifugation (unit 3.1), and counting cells and assessing viability ( 3.NaN)

Support Protocol 1: Screening Primary Hybridomas for CD3‐TCR Expression

  • Growing hybridomas (see protocol 1, step )
  • recipeWash buffer (see recipe)
  • Fluorescein isothyocyanate (FITC)‐conjugated anti‐mouse‐CD3‐ɛ MAb (Pharmingen HM‐CD3; see Table 97.80.4711)
  • FITC‐conjugated control MAb (i.e., MAb recognizing any protein that is not present on T cells)
  • 4‐ml nonsterile round‐bottom centrifuge tubes (Falcon or equivalent)
  • Additional materials for FITC conjugation (unit 5.3) and flow cytometry analysis (unit 5.4)

Support Protocol 2: Screening Hybridomas for Antigen Specificity

  • Growing hybridomas (see protocol 1, step )
  • Syngeneic spleen cell suspension (from same strain of mouse used as donor of activated T cells; for testing specificity against soluble protein antigens) or allogeneic spleen cell suspension from same strain of mouse used as donor of stimulator cells for the MLC; for testing specificity against alloantigens (unit 3.1)
  • Soluble protein or peptide antigen
  • Additional reagents and equipment for IL‐2 and IL‐4 detection (unit 6.3)
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Figures

Videos

Literature Cited

Literature Cited
   Born, W., White, J., O'Brien, R., and Kubo, R. 1988. Development of T cell receptor expression: Studies using T cell hybridomas. Immunol. Res. 7:279‐291.
   Burgert, H.G., White, J., Weltzien, H.U., Marrack, P., and Kappler, J.W. 1989. Reactivity of Vβ17α CD8 T cell hybrids: Analysis using a new CD8 T cell fusion partner. J. Exp. Med. 170:1887‐1904.
   Fathman, C.G., Fitch, F.W., Davis, K.A., and Witte, O.N. 1989. Long‐term culture of immunocompetent cells.. In Fundamental Immunology, 2nd ed. (W.E. Paul, ed.) pp. 803‐815. Raven Press, New York.
   Hedrick, S.M., Matis, L.A., Hecht, T.T., Samelson, L.E., Longo, D.L., Heber‐Katz, E., and Schwartz, R.H. 1982. The fine specificity of antigen and Ia determinant recognition by T cell hybridoma clones specific for pigeon cytochrome C. Cell. 30:141‐152.
   Hyman, R. and Stalling, V. 1974. Complementation patterns of Thy1 variants and evidence that antigen loss variants “pre‐exist” in the parental population. J. Nat. Cancer Inst. 51: 429‐434.
   Kanagawa, O. and Maki, R. 1989. Inhibition of MHC class II–restricted T cell response by Lyt‐2 alloantigen. J. Exp. Med. 170:901‐912.
   Kappler, J.W., Skidmore, B., White, J., and Marrack, P. 1981. Antigen‐inducible H‐2 restricted, interleukin‐2‐producing T cell hybridomas. Lack of independent antigen and H‐2 recognition. J. Exp. Med. 153:1198‐1208.
   Kaufmann, Y., Berke, G., and Eshhar, Z. 1981. Cytotoxic T lymphocyte T cell hybridomas that mediate specific tumor cell lysis in vitro. Proc. Natl. Acad. Sci. U.S.A. 78:2502‐2505.
   Köhler, G. and Milstein, C. 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 256:495‐497.
   Rock, K.L., Rothstein, L., and Gamble, S. 1990. Generation of class I MHC–restricted T‐T hybridomas. J. Immunol. 145:804‐811.
   Schwartz, R.H. 1990. A cell culture model for T lymphocyte clonal energy. Science. 248:1349‐1356.
   Silva, A., MacDonald, H.R., Conzelmann, A., Corthesy, P., and Nabholz, M. 1983. Rat × mouse T cell hybrids with inducible cytolytic activity. Immunol. Rev. 76:105‐122.
   Taniguchi, M. and Miller, J.F.A.P. 1978. Specific suppressive factors produced by hybridomas derived from the fusion of enriched suppressor T cell and a T lymphoma cell line. J. Exp. Med. 148:373‐382.
   White, J., Blackman, M., Bill, J., Kappler, J., Marrack, P., and Born, W. 1989. Two better cell lines for making hybridomas expressing specific T cell receptors. J. Immunol. 143:1822‐1825.
Key References
   Kappler et al., 1981. See above.
   Kühler and Milstein, 1975. See above.
   Taniguchi and Miller, 1978. See above.
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