Quantitation of Functional T Cells by Limiting Dilution

Richard A. Miller1

1 University of Michigan School of Medicine, Ann Arbor, Michigan
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 3.15
DOI:  10.1002/0471142735.im0315s35
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Culturing by limiting dilution (LD) can be used to estimate the proportion of T cells in a cell preparation which can respond to an activating stimulus to produce interleukin 2 or to generate cytotoxic T lymphocyte (CTL) activity. In the first basic protocol, IL‐2‐producing murine T cells are measured following stimulation by the mitogen Con A. This protocol can be modified as described in the first alternate protocol to quantitate responses to alloantigens, anti‐CD3 antibody, protein antigens such as keyhole limpet hemocyanin (KLH), and viruses. The second alternate protocol provides a modification for using human responder cells. The second basic protocol is used for estimating the proportion of cells that can generate a clone of cytotoxic effector cells when stimulated by Con A with the addition of IL‐2.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: LD Analysis of Mitogen‐Stimulated IL‐2‐Producing T Cells
  • Alternate Protocol 1: Alternate Stimuli for Activation of IL‐2‐Producing T Cells
  • Alternate Protocol 2: Activation of Human Responder T Cells
  • Support Protocol 1: Preparation of Peritoneal Feeder Cells
  • Support Protocol 2: High‐Sensitivity Colorimetric Assay for IL‐2
  • Basic Protocol 2: LD Analysis of Mitogen‐Stimulated Cytotoxic T Cells
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: LD Analysis of Mitogen‐Stimulated IL‐2‐Producing T Cells

  Materials
  • recipeComplete RPMI‐10 medium, supplemented (see )
  • T cell–depleted feeder peritoneal cells, preferably from syngeneic mice, suspended in recipesupplemented RPMI‐10 at 2 × 105 cells/ml (first protocol 4support protocol)
  • Responder cells: T cell–enriched suspension from mouse spleen or lymph node (units 3.1 3.4)
  • recipeWash medium
  • 8 µg/ml Con A prepared in recipesupplemented RPMI‐10
  • 8‐ or 12‐channel pipettor
  • 96‐well cone‐ and flat‐bottom microtiter plates (tissue culture grade)
  • Sorvall H‐1000B rotor (or equivalent)
  • 5‐ to 10‐ml round‐bottom plastic tubes

Alternate Protocol 1: Alternate Stimuli for Activation of IL‐2‐Producing T Cells

  Additional Materials
  • Irradiated (5000 rad; unit 3.12) JY feeder cells
  • 80 µg/ml phytohemagglutinin‐P (PHA‐P; Difco) or 12 µg/ml Con A stock solutions

Alternate Protocol 2: Activation of Human Responder T Cells

  Additional Materials
  • Donor mice
  • 70% ethanol
  • recipeWash medium, ice‐cold
  • recipeAnti‐Thy‐1 MAb ascites fluid, ice‐cold (see )
  • recipeRabbit complement, optimally diluted (see )
  • Forceps
  • 10‐ml syringe equipped with 21‐G, 1‐in. needle
  • 50‐ml conical centrifuge tubes, ice‐cold

Support Protocol 1: Preparation of Peritoneal Feeder Cells

  Additional Materials
  • IL‐2‐dependent indicator cell line (e.g., CTLL‐2 or HT‐2; see unit 6.3)
  • recipeRat spleen cell–conditioned medium
  • MTT solution (5 mg/ml MTT in PBS; e.g., Sigma #2128)
  • 0.04 N HCl in isopropyl alcohol

Support Protocol 2: High‐Sensitivity Colorimetric Assay for IL‐2

  Materials
  • Donor mice for feeder cell preparation
  • recipeWash medium
  • recipeComplete RPMI‐10 medium, supplemented
  • 12 µg/ml Con A in recipesupplemented RPMI‐10 medium
  • recipe12% rat spleen cell–conditioned medium
  • P815 target cells (unit 3.11)
  • 1 mCi/ml Na 251CrO 4 (51Cr) solution (stored at 4°C)
  • Phytohemagglutinin solution (PHA‐P; Difco), reconstituted to 10 ml and diluted 1:500
  • Paraffin wax
  • 96‐well cone‐bottom microtiter plates (tissue culture grade)
  • Multichannel pipettor
  • Sorvall H‐1000B rotor (or equivalent)
  • Conical vials and racks ( Sarstedt #73.1055 and #95.1046, respectively)
  • Additional reagents and equipment for handling and restraint of mice (unit 1.3), euthanasia (unit 1.8), removal of mouse spleen (unit 1.10), preparation of single‐cell suspension (unit 3.1), counting cells ( appendix 3A) and CTL assay (unit 3.11)
NOTE: Carry out all manipulations at room temperature.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Eichmann, K., Falk, I., and Simon, M.M. 1980. Quantitative studies on T cell diversity. I. Determination of the precursor frequencies for two types of Streptococcus A‐specific helper cells in nonimmune, polyclonally activated splenic T cells. J. Exp. Med. 152:477‐492.
   Fazekas de St. and Groth, S. 1982. The evaluation of limiting dilution assays. J. Immunol. Methods. 49:R11‐R23.
   Kelso, A. and MacDonald, H.R. 1982. Precursor frequency analysis of lymphokine‐secreting alloreactive T lymphocytes. Dissociation of subsets producing interleukin 2, macrophage‐activating factor, and granulocyte‐macrophage colony stimulating factor on the basis of Lyt‐2 phenotype. J. Exp. Med. 156:1366‐1379.
   Lefkovits, I. and Waldmann, H. 1979. Limiting Dilution Analysis of Cells in the Immune System. Cambridge University Press, Cambridge and New York.
   Miller, R.A. and Reiss, C.S. 1984. Limiting dilution cultures reveal latent influenza virus‐specific helper T cells in virus‐primed mice. J. Mol. Cell. Immunol. 1:357‐368.
   Moretta, A., Pantaleo, G., Moretta, L., and Mingari, M.C. 1983. Direct demonstration of the clonogenic potential of every human peripheral blood T cell: Clonal analysis of HLA‐DR expression and cytolytic activity. J. Exp. Med. 157:743‐754.
   Mosmann, T. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods. 65:55‐63.
   Rocha, B.B. 1987. Population kinetics of precursors of IL 2‐producing peripheral T lymphocytes: Evidence for short life expectancy, continuous renewal, and post‐thymic expansion. J. Immunol. 139:365‐372.
   Rozans, M.K., Smith, B.R., Emerson, S., Crimmins, M., Laurent, G., Reichert, T., and Miller, R.A. 1986. Functional assessment of T cell depletion from bone marrow prior to therapeutic transplantation using limiting dilution methods. Transplantation. 42:380‐387.
   Taswell, C. 1981. Limiting dilution assays for the determination of immunocompetent cell frequencies. I. Data analysis. J. Immunol. 126:1614‐1619.
   Vie, H. and Miller, R.A. 1986. Limiting dilution analysis of IL‐2 producing helper T cells: Measurement of IL‐2 produced by single, lymphokine‐secreting effector cells. J. Immunol. 136:3292‐3297.
   Wilson, A., Chen, W.‐.F., and Shortman, K. 1982. Semi‐automated limit‐dilution assay and clonal expansion of all T‐cell precursors of cytotoxic lymphocytes. J. Immunol. Methods. 52:283‐306.
Key References
   Lefkovits, I. and Waldmann, H. 1979. See above.
  Good overview of the principles of LD analysis in the context of T and B lymphocyte function, with examples of results expected under good and bad experimental conditions.
   Miller, R. A. and Stutman, O. 1982. Enumeration of IL 2‐secreting helper T cells by limiting dilution analysis, and demonstration of unexpectedly high levels of IL‐2 production per responding cell. J. Immunol. 128:2258‐2264.
  Contains examples of assays for IL‐2‐producing cells in responses to major and Mls alloantigens by CD8+ and CD8− mouse splenic cells.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library