Proliferative Assays for T Cell Function

Ada M. Kruisbeek1, Ethan Shevach2, Angela M. Thornton3

1 Netherlands Cancer Institute, Amsterdam, 2 National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, 3 National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 3.12
DOI:  10.1002/0471142735.im0312s60
Online Posting Date:  May, 2004
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Abstract

This addition to UNIT will describe the assays needed to evaluate CD4·CD25·T cell non‐responsiveness and function. Unlike the conventional T cells described in , CD4·CD25· cells do not proliferate to TCR stimuli alone. The conditions required to induce proliferation are described. This addition will also describe the assay in which CD4·CD25·T cells are co‐cultured with conventional T cells in order to assess their suppressive function. Finally, this addition will describe the culture conditions for the activation and expansion of CD4·CD25· cells.

Keywords: Mouse; T cells; Suppressor T cells; T regulatory cells; Proliferation; IL‐2

     
 
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Table of Contents

  • Basic Protocol 1: Activation of Unprimed T Cells
  • Alternate Protocol 1: Activation of Unprimed T Cells with Plate‐Bound Antibodies
  • Alternate Protocol 2: T Cell Proliferation in Mixed Lymphocyte Cultures
  • Support Protocol 1: Depletion of T Cells from Antigen‐Presenting/Stimulator Cell Suspensions
  • Support Protocol 2: Blocking Cellular Division of Accessory/Stimulator Cells
  • Basic Protocol 2: Activation of Primed T Cells
  • Basic Protocol 3: Activation of CD4+CD25+ T Cells and Analysis of Their Suppressive Function
  • Alternate Protocol 3: The Two‐Step Supression Assay: Short‐Term Activation and Expansion of CD4+CD25+ T Cells and Analysis of Their Supressive Function
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Activation of Unprimed T Cells

  Materials
  • Complete RPMI‐5 and RPMI‐10 media ( appendix 2A)
  • Responder cells: lymphocytes from nonimmunized mouse thymus, spleen, or lymph nodes (unit 3.1)
  • Activating agent(s) (Table 3.12.1)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Accessory cells: unfractionated mouse spleen cell suspension, irradiated or treated with mitomycin C (see protocol 5) or T cell–depleted (see protocol 4)
  • [3H]thymidine ( appendix 3D)
  • 15‐ and 4‐ml disposable, polystyrene conical tubes with screw caps
  • Low‐speed centrifuge with Sorvall H‐1000B rotor (or equivalent)
  • 1‐, 5‐, and 10‐ml disposable polystyrene pipets
  • 96‐well flat‐ or round‐bottom microtiter plates with lids (Costar)
  • 25‐ to 100‐µl single‐ and multichannel pipettors with disposable tips
  • Additional reagents and equipment for removing organs (unit 1.9), preparing single‐cell suspensions (unit 3.1), and counting, labeling, and harvesting cells (appendix 3)

Alternate Protocol 1: Activation of Unprimed T Cells with Plate‐Bound Antibodies

  Additional Materials
  • PBS ( appendix 2A), room temperature and 4°C
  • 1 mg/ml purified anti‐CD3 or anti‐TCR MAb in PBS (for nonspecific activation of T cells) or 1 mg/ml purified anti‐Vβ or anti‐TCR‐γδ MAb in PBS (for activation of T cells with specific receptors; see Table 3.12.1)

Alternate Protocol 2: T Cell Proliferation in Mixed Lymphocyte Cultures

  • Responder cells: lymphocytes from nonimmunized mouse thymus, spleen, or lymph nodes (units 1.9 & 3.1) or purified T cells or T cell subpopulations (units 3.1 3.6)
  • Stimulator cells: allogeneic mouse spleen cells that differ from the responder cells at H‐2 or Mls loci, irradiated or treated with mitomycin C (see protocol 5) or T cell–depleted (see protocol 4)

Support Protocol 1: Depletion of T Cells from Antigen‐Presenting/Stimulator Cell Suspensions

  Additional Materials
  • Spleen cells from nonimmunized mice
  • Hanks' balanced salt solution (HBSS; appendix 2A)
  • Low‐Tox rabbit complement (Cedarlane), reconstituted with ice‐cold distilled water and filter‐sterilized
  • Anti‐Thy‐1.2 ascites (HO‐13‐4; ATCC no. TIB 99) or anti‐Thy‐1.1 ascites (HO‐22‐1; ATCC no. TIB 100; alternatively, see Table 97.80.4711 for other anti‐Thy‐1 MAb and unit 2.6 for production of ascites)
  • 70% Percoll solution (see recipe; unit 3.8)

Support Protocol 2: Blocking Cellular Division of Accessory/Stimulator Cells

  • Mitomycin C (Sigma; store in dark)

Basic Protocol 2: Activation of Primed T Cells

  Materials
  • Complete RPMI‐10 medium ( appendix 2A)
  • Responder cells: Purified T cells isolated from lymph nodes (units 3.1 3.6) of in vivo primed mice
  • Antigen: 1 mg/ml sterile protein antigen(s) (unit 3.13), in PBS or suspension of irradiated or mitomycin C–treated stimulator cells expressing alloantigens at 8 × 106 cells/ml (unit 3.11, Support Protocol) in complete RPMI‐10 medium ( appendix 2A)
  • Accessory cells: suspension of irradiated or mitomycin C–treated (or T cell–depleted) spleen cells syngeneic to the responding T cells at 5 × 106 cells/ml in complete RPMI‐10 medium
  • 4‐ml conical tubes
  • 96‐well flat‐bottom microtiter plates with lids

Basic Protocol 3: Activation of CD4+CD25+ T Cells and Analysis of Their Suppressive Function

  Materials
  • CD4+CD25 and CD4+CD25+ T cells (unit 3.5)
  • Complete RPMI‐10 medium ( appendix 2A)
  • Accessory cells: T‐cell depleted mouse spleen cell suspension, irradiated (unit 3.5 or protocol 4)
  • Purified anti‐CD3 (BD PharMingen)
  • Mouse or human IL‐2 (PeproTech or Roche)
  • Purified anti‐CD28 (BD PharMingen)
  • [3H] thymidine ( appendix 3D)
  • 96‐well flat bottom microtiter plates with lids
  • 37°C, 5% to 7% CO 2 humidified incubator
  • Semiautomated sample harvester ( appendix 3A)
  • β scintillation counter
  • Additional reagents and equipment for removing organs (unit 1.9), preparing single‐cell suspensions (unit 3.1), and counting, labeling, and harvesting cells ( appendix 3A)

Alternate Protocol 3: The Two‐Step Supression Assay: Short‐Term Activation and Expansion of CD4+CD25+ T Cells and Analysis of Their Supressive Function

  • PBS ( appendix 2A)
  • Purified CD4+ T cells from a TCR transgenic mouse
  • Peptide corresponding to the TCR transgenic
  • 24‐well flat bottom plates with lids
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Figures

Videos

Literature Cited

   Ashwell, J.D., DeFranco, A.L., Paul, W.E., and Schwartz, R.H. 1984. Antigen presentation by resting B cells: Radiosensitivity of the antigen‐presentation function and two distinct pathways of T cell activation J. Exp. Med. 159:861‐869.
   Baecher‐Allen, C., Brown, J.A., Freeman, G.J., and Hafler, D.A. 2001. CD4+CD25high regulatory cells in human peripheral blood. J. Immunol. 167:1245‐1253.
   Cohen, J.L., Trenado, A., Vasey, D., Klatzmann, D., and Salomon, B.L. 2002. CD4+CD25+ immunoregulatory T cells: New therapeutics for graft‐versus‐host disease. J. Exp. Med. 196:401‐406.
   Gunter, K.C., Malek, T.R., and Shevach, E.M. 1984. T cell activating properties of an anti‐Thy‐1 monoclonal antibody: Possible analogy to OKT3/Leu‐4. J. Exp. Med. 159:716‐730.
   Hathcock, K.S., Segal, D.M., and Hoder, R.J. 1989. Activation of Lyt2+ (CD8+) and Lyt2−(CD4+) T cell subsets by anti‐receptor antibody. J. Immunol. 142:2181‐2186.
   Jenkins, M.K., Chen, C., Jung, G., Mueller, D.L., and Schwartz, R.H. 1990. Inhibition of antigen‐specific proliferation of type 1 murine T cell clones after stimulation with immobilized anti‐CD3 monoclonal antibody. J. Immunol. 144:16‐22.
   Marrack, P. and Kappler, J. 1989. The staphylococcal enterotoxins and their relatives. Science 248:705‐711.
   Piccirillo, C.A. and Shevach, E.M. 2001. Cutting edge: Control of CD8+ T cell activation by CD4+CD25+ immunoregulatory cells. J. Immunol. 167:1137‐1140.
   Shevach, E.M. 2003. CD4+CD25+ suppressor T cell: More questions than answers. Nat. Rev. Immunol. 2:389‐400.
   Staerz, U.D. and Bevan, M.J. 1986. Activation of resting T lymphocytes by a monoclonal antibody directed against an allotypic determinant on the T cell receptor. Eur. J. Immunol. 16:263‐268.
   Thornton, A.M. and Shevach, E.M. 1998. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cells activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 188:287‐296.
   Thornton, A.M. and Shevach, E.M. 2000. Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific. J. Immunol. 164:183‐190.
Key References
   Corradin, G., Etlinger, H.M., and Chiller, M. 1977. Lymphocyte specificity to protein antigens. I. Characterization of the antigen‐induced in vitro T cell dependent proliferative response with lymph node cells from primed mice. J. Immunol. 119:1048‐1055.
  Both of the above describe how specific proliferation of antigen‐primed T cells can be measured.
   Rosenwasser, L.J. and Rosenthal, A.S. 1978. Adherent cell function in murine T lymphocyte antigen recognition. I. A macrophage‐dependent T cell proliferation assay in the mouse. J. Immunol. 120:1991‐1998.
  Details the MLC proliferation assay.
   Strong, D.M., Ahmed, A.A., Thurman, G.B., and Sell, K.W. 1973. In vitro stimulation of murine spleen cells using a microculture system and a multiple automated sample harvester. J. Immunol. Methods 2:279‐287.
  Describes the non‐responsiveness of CD4+CD25+ T cells and the assay to measure their suppressive function.
   Thornton, A.M. and Shevach, E.M. 1998. See above.
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