Measurement of CD40 Ligand (CD154) Expression on Resting and In Vitro–Activated T Cells

Maurice R.G. O'Gorman1

1 Northwestern University Medical School, Children's Memorial Hospital, Chicago, Illinois
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.7
DOI:  10.1002/0471142956.cy0607s12
Online Posting Date:  May, 2001
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Measurement of the ability of in vitro activated T lymphocytes to express CD40 ligand (CD154) is a valuable tool for diagnosis of the X‐linked hyper‐IgM syndrome (XHIM) and for investigating abnormalities in the costimulatory functions of T lymphocytes. Resting peripheral blood lymphocytes do not normally express any appreciable levels of CD154, necessitating in vitro activation. This unit details the in vitro activation procedure, the three‐color monoclonal antibody panels used to label the appropriate lymphocyte subsets, the controls required to interpret the assay, the setup and acquisition of listmode data files, and the gating and analysis protocols used to interpret the data.

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:

  • 1 mg/ml PMA solution (see recipe)
  • Complete RPMI ( appendix 2A) without serum
  • 1 mg/ml calcium ionophore solution (see recipe)
  • Sample:
  •  Venous blood drawn into sodiul heparin Vacutainer tube, or
  •  Peripheral blood mononuclear cells (PBMC): separated from whole blood by ficoll‐hypaque density‐gradient centrifugation (unit 5.1), washed twice in complete RPMI, and brought to 107 cells/ml
  • Ca2+‐ and Mg2+‐free PBS ( appendix 2A)
  • Monoclonal antibodies:
  •  Phycoerythrin‐labeled anti‐CD40 ligand (CD40L‐PE), clone TRAP1 (Pharmingen)
  •  PE‐labeled mouse IgG1 (MsIgG1‐PE;Pharmingen) isotype control
  •  Fluorescein isothiocyanate–labeled anti‐CD8 (CD8‐FITC; Pharmingen)
  •  FITC‐labeled anti‐CD3 (CD3‐FITC; Pharmingen)
  •  PE‐labeled anti‐CD69 (CD69‐PE: Becton Dickinson)
  •  PE‐cyanine‐5‐labeled anti‐CD3 (CD3‐PE‐Cy5; Pharmingen)
  • 1× FACS lysing solution: 1/10 aqueous dilution of 10× stock (Becton Dickenson Immunocytometry), stable 1 month at room temperature
  • Flow cytometry wash solution (see recipe)
  • 1% (w/v) paraformaldehyde fixative (see recipe)
  • 12 × 75–mm polystyrene tubes (Falcon, Becton Dickinson Labware)
  • Flow cytometer with a 488‐nm line, able to detect simultaneously at least three different emission wavelengths plus forward and right‐angle light‐scatter signals, and with the ability to store listmode data
  • Flow cytometric analysis software (e.g., Cellquest, Becton Dickinson; Winlist, Verity Software House)
CAUTION: Phorbol 12‐myristate 13‐acetate is toxic and a potential carcinogen. Handle, store, and dispose of appropriately.
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Literature Cited

Literature Cited
   Allen, R.C., Armitage, R.J., Conley, M.E., Rosenblatt, J., Jenkins, N.A., Copeland, N.G., Bedell, M.A., Edelhoff, S., Disteche, C.M., Simoneaux, D.K. et al. 1993. CD40 ligand gene defects responsible for X‐linked hyper IgM syndrome. Science. 259:990‐993.
   Armitage, R.J., Fanslow, W.C., Strockbine, L., Sato, T.A., Clifford, K.N., Macduff, B.M., Anderson, D.M., Gimpel, S.D., Davis‐Smith, T., Maliszewski, C.R. et al. 1992. Molecular and biological characterization of a murine ligand for CD40. Nature. 357:80‐82.
   Armitage, R.A., Macduff, B.A., Spriggs, M.K., and Fasnslow, W.C. 1993. Human B cell proliferation and Ig secretion induced by recombinant CD40 ligand are modulated by soluble cytokines. J. Immunol. 150:3671‐3680.
   Arrufo, A., Farrington, M., Hollembaugh, D., Li, X., Milatovich, A., Nonoyama, S., Bajorath, J., Grosmaire, L.S., Stenkamp, R., Neubauer, M. et al. 1993. The CD40 ligand, gp39, is defective in activated T cells from patients with X‐linked hyper‐IgM syndrome. Cell. 72:291‐300.
   Conley, M.E. 1992. Molecular approaches to the analysis of X‐linked immunodeficiencies. Annu. Rev. Immunol. 10:215‐238.
   DiSanto, J.P., Bonnefoy, J.Y., Gauchat, J.F., Fischer, A., and De Saint Basile, F. 1993. CD40 ligand mutations in X‐linked immunodeficiency with hyper IgM. Nature. 361:539‐543.
   DuChateau, B.K., Yogev, R., and O'Gorman, M.R.G. 1998. Evaluation of CD154 expression on the surface of CD4+ lymphocytes obtained from pediatric HIV‐1 infected patients. Cytometry Suppl. 9:101.
   Farrington, M., Grosmaire, L.S., Nonoyama, S., Fischer, S.H., Hollenbaugh, D., Ledbetter, J.A., Noelle, R.J., Aruffo, A., and Ochs, H.D. 1994. CD40 ligand expression is defective in a subset of patients with common variable immunodeficiency. Proc. Natl. Acad. Sci. U.S.A. 91:1099‐1103.
   Fuleihan, R., Ramesh, N., Loh, R., Jabara, H.F., Rosen, S., Chatila, T., Fu, S.M., Stamenkovic, I., and Geha, R.S. 1993. Defective expression of the CD40 ligand in X chromosome linked immunoglobulin deficiency with normal or elevated IgM. Proc. Natl. Acad. Sci. U.S.A. 90:2170‐2173.
   Grewal, I.S., Foellmer, H.G., Grewal, K.D., Xu, J., Hardardottir, F., Baron, J.L., Janeway, C.A., and Flavell, R.A. 1996. Requirement for CD40 ligand in costimulation induction, T cell activation, and experimental allergic encephalomyelitis. Science 272:1864‐1867.
   Hollenbaugh, D., Grosmaire, L.S., Kullas, C.D., Chalupny, N.J., Braesch‐Andersen, S., Noelle, R.J., Stamenkovic, I., Ledbetter, J.A., and Aruffo, A. 1992. The human T cell antigen gp39, a member of the TNF gene family, is a ligand for the CD40 receptor: Expression of a soluble form of p39 with B cell co‐stimulatory activity. EMBO J. 11:4313‐4321.
   Korthäuer, U., Graf, D., Mages, H.W., Briere, F., Padayachee, M., Malcolm, S., Ugazio, A.G., Notarangelo, L.D., Levinsky, R.J., and Kroczek, R.A. 1993. Defective expression of T‐cell CD40 ligand causes X‐linked immunodeficiency with hyper‐IgM. Nature 361:539‐541.
   Liu, Y.‐D., Joshua, D.E., Williams, G.T., Smith, C.A., Gordon, J., and MacLennan, I.C.M. 1989. Mechanisms of antigen‐driven selection in germinal centers. Nature 342:929‐931.
   Notarangelo, L.D., Duse, M., and Ugazio, A.G. 1992. Immunodeficiency with hyper IgM (HIM). Immunodef. Rev. 3:101‐122.
   O'Gorman, M.R.G., Zaas, D., Paniagua, M., Corrochano, V., Scholl, P.R., and Pachman, L.M. 1997. Development of a rapid whole blood flow cytometry procedure for the diagnosis of X‐linked hyper‐IgM syndrome patients and carriers. Clin. Immunol. Immunopathol. 85:172‐181.
   Rousset, F., Garcia, E., and Banchereau, J. 1991. Cytokine‐induced proliferation and immunoglobulin production of human B lymphocytes triggered through their CD40 antigens. J. Exp. Med. 173:705‐710.
   Yang, Y. and Wilson, J.M. 1996. CD40 ligand‐dependent T cell activation: Requirement of B7‐CD28 signaling through CD40. Science. 273:1862‐1864.
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