Induction of Polyclonal Immunoglobulin Synthesis

Stephen P. James1

1 National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.11
DOI:  10.1002/0471142735.im0711s00
Online Posting Date:  May, 2001
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This unit is designed to examine the effects of T cells or lymphokines on B cell differentiation in situations where the antigen specificity of the B cells is not of interest. In these cases, antibody production induced by polyclonal stimuli (e.g., mitogens, antibodies, Epstein‐Barr virus (EBV), or lymphokines) can be measured instead of antigen‐specific immunoglobulin production. Because B cells in the circulation and tissues are pleomorphic (containing subpopulations of cells that may be resting, cells that may have had prior antigenic exposure, and cells that may have undergone prior isotype commitment), the antibody responses of these subpopulations to various growth and differentiation factors differ. Therefore, the choice of which lymphocyte subpopulation to culture and which activation signal to use is determined by the particular experimental question.

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Table of Contents

  • Basic Protocol 1: Polyclonal Mitogenic Stimulation of PBMC
  • Reagents and Solutions
  • Commentary
  • Tables
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Basic Protocol 1: Polyclonal Mitogenic Stimulation of PBMC

  • Peripheral blood mononuclear cell suspension (PBMC; unit 7.1)
  • Complete RPMI‐5 and ‐10 media ( appendix 2A)
  • recipePokeweed mitogen (PWM) solution
  • 96‐well flat‐bottomed microtiter plates (Costar #3596)
  • Additional reagents and equipment for counting cells ( appendix 3A), ELISPOT (unit 7.14), reverse plaque assay (unit 7.13) or ELISA (unit 7.12)
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Literature Cited

Literature Cited
   Bertolini, J.N. and Benson, E.M. 1990. The role of human interleukin‐6 in B cell isotype regulation and differentiation. Cell Immunol 125:197‐209.
   Bich‐Thuy, I. and Fauci, A.S. 1986. Recombinant interleukin 2 and gamma interferon act synergistically on distinct steps of in vitro terminal human B cell maturation. J. Clin. Invest 77:1173‐1179.
   Del Prete, G., Maggi, E., Parronchi, P., Chretien, I., Macchia, D., Ricci, M., Banchereau, J., DeVries, J., and Romagnani, S. 1988. IL‐4 is an essential factor for the IgE synthesis induced in vitro by human T cell clones and their supernatants. J. Immunol 140:4193‐4198.
   Hirohata, S. and Lipsky, P.E. 1989. T cell regulation of human B cell proliferation and differentiation. Regulatory influences of CD45R+ and CD45R− T4 cell subsets. J. Immunol 152:2597‐2607.
   Jelinek, D.F. and Lipsky, P.E. 1988. Inhibitory influence of IL‐4 on human B cell responsiveness. J. Immunol 141:164‐173.
   Lipsky, P.E. 1989. The control of antibody production by immunomodulatory molecules. Arthritis Rheum 32:1345‐1355.
   Nakagawa, T., Hirano, R., Nakagawa, N., Yoshizaki, K., and Kishimoto, T. 1985. Effect of recombinant IL‐2 and γ‐IFN on proliferation and differentiation of human B cells. J. Immunol 134:959‐966.
   Sherr, E.H., Saxon, A., and Wells, J.R. 1988. Functional and phenotypic characterization of human B cell lymphocyte subsets isolated by unit gravity sedimentation. Int. Arch. Allergy Appl. Immunol 85:1540‐1560.
   Tosato, G., Blaese, R.M., and Yarchoan, R. 1985. Relationship between immunoglobulin production and immortalization by Epstein Barr virus. J. Immunol 135:959‐964.
   Yokota, T., Coffman, R.L., Hagiwara, H., Rennick, D.M., Takebe, Y., Yokota, K., Gemmell, L., Shrader, B., Yang, G., Meyerson, P., Luh, J., Hoy, P., Pene, J., Briere, F., Spits, H., Banchereau, J., DeVries, J., Lee, F.D., Arai, N., and Arai, I.‐I. 1987. Isolation and characterization of lymphokine cDNA clones encoding mouse and human IgA enhancing factor and eosinophil colony stimulating factor activities: Relationship to interleukin 5. Proc. Natl. Acad. Sci. U.S.A 84:7388‐7392.
Key References
   Hirohata and Lipsky, 1989. See above.
  Describes in detail the regulatory effects of T cell subpopulations on B cell differentiation using PWM‐ or anti‐CD3‐stimulated cultures. Emphasizes the complexities of the enhancing or suppressing effects of T cells and cytokines on B cell differentiation.
   Lipsky, 1989. See above.
  Reviews the positive and negative effects of different cytokines on human B cell differentiation.
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