Detection and Isolation of Cytokine Secreting Cells Using the Cytometric Cytokine Secretion Assay

Mario Assenmacher1, Max Löhning2, Andreas Radbruch2

1 Miltenyi Biotec, Bergisch‐Gladbach, Germany, 2 Deutsches Rheumaforschungszentrum Berlin, Berlin, Germany
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 6.27
DOI:  10.1002/0471142735.im0627s46
Online Posting Date:  February, 2002
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The cell‐surface affinity matrix technology, otherwise called “secretion assay” or “capture assay,” represents a new, innovative method for the analysis and enrichment of viable cells according to secreted molecules such as antibodies or cytokineof T cells to induce cytokine secretion is included. Alternate protocols provide very rapid and simple procedures for the direct detection of antigen‐specific T cells from small numbers of PBMCs or whole blood without magnetic enrichment.

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Table of Contents

  • Basic Protocol 1: Cytokine Secretion Assay
  • Alternate Protocol 1: Rapid Cytokine Secretion Assay Using PBMCs
  • Alternate Protocol 2: Rapid Cytokine Secretion Assay Using Whole Blood Detection
  • Reagents and Solutions
  • Commentary
  • Figures
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Basic Protocol 1: Cytokine Secretion Assay

  • Culture medium, ice cold and warm (37°): e.g., RPMI‐1640 ( appendix 2A) containing 10% (v/v) human plasma type AB or autologous serum for human cells, or mouse serum for murine cells
  • Antigen (e.g., 1 to 10 µg/ml peptide, 1 to 100 µg/ml protein)
  • Control antigen (optional)
  • 1 µg/ml staphylococcal enterotoxin B (Sigma; optional)
  • Phosphate‐buffered saline (PBS), pH 7.2 ( appendix 2A), containing 0.5% (w/v) BSA and 2 mM EDTA ( appendix 2A), ice cold
  • Cytokine catch reagent: anti‐cytokine monoclonal antibody conjugated to cell surface (i.e., CD45)–specific monoclonal antibody (Miltenyi Biotec)
  • Recombinant cytokine (optional)
  • Cytokine detection antibody (Miltenyi Biotec): anti‐cytokine monoclonal antibody conjugated to phycoerythrin (PE), allophycocyanin (APC), or fluorescein isothiocyanate (FITC)
  • Fluorochrome‐conjugated staining reagents (optional)—e.g., human or mouse (as appropriate) anti‐CD4‐FITC, human anti‐CD14‐peridinin chlorophyll protein (PerCP; Becton Dickinson), mouse anti‐CD45R/B220‐PerCP
  • Anti‐PE MicroBeads: colloidal super‐paramagnetic MicroBeads conjugated to monoclonal mouse anti‐PE antibody (Miltenyi Biotec; optional)
  • 96‐ or 24‐well culture plates or 6‐cm petri dishes
  • Cell scraper or equivalent
  • 1.5‐ to 15‐ml V‐bottom tubes or deep‐well culture plates.
  • Shaker or rotator (optional)
  • Additional reagents and equipment for preparing human peripheral blood mononuclear cells (PBMC; unit 7.1) or mouse spleen/lymph node (LN) cells (unit 3.1), counting cells ( appendix 3A), determining cell viability ( appendix 3B), and performing flow cytometry (Chapter 5)

Alternate Protocol 1: Rapid Cytokine Secretion Assay Using PBMCs

  • Sodium‐heparinized whole blood
  • recipe1× lysing solution, fresh (see recipe)
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Literature Cited

Literature Cited
   Assenmacher, M., Löhning, M., Scheffold, A., Manz, R. A., Schmitz, J., and Radbruch, A. 1998. Sequential production of IL‐2,IFN‐γ and IL‐10 by individual staphylococcal enterotoxin B‐activated T helper lymphocytes. Eur. J. Immunol. 28:1534‐1543.
   Brosterhus, H., Brings, S., Leyendeckers, H., Manz, R.A., Miltenyi, S., Radbruch, A., Assenmacher, M., and Schmitz, J. 1999. Enrichment and detection of live antigen‐specific CD4+ and CD8+ T cells based on cytokine secretion. Eur. J. Immunol. 29:4053‐4059.
   Farrar, J.D., Ouyang, W., Löhning, M., Assenmacher, M., Radbruch, A., Kanagawa, O., and Murphy, K.M. 2001. An instructive component in T helper cell type 2 (Th2) development mediated by GATA‐3. J. Exp. Med. 193:643‐648.
   Hu‐Li, J., Pannetier, C., Guo, L., Löhning, M., Gu, H., Watson, C., Assenmacher, M., Radbruch, A., and Paul, W.E. 2001. Regulation of expression of IL‐4 alleles: Analysis using a chimeric GFP/IL‐4 gene. Immunity 14:1‐11.
   Manz, R., Assenmacher, M., Pfluger, E., Miltenyi, S., and Radbruch, A. 1995. Analysis and sorting of live cells according to secreted molecules relocated to a cell‐surface affinity matrix. Proc. Natl. Acad. Sci. U.S.A. 92:1921‐1925
   Oelke, M., Kurokawa, T., Hentrich, I., Behringer, D., Cerundolo, V., Lindemann, A., and Mackensen, A. 2000a. Functional characterization of CD8+ antigen‐specific cytotoxic T lymphocytes after enrichment based on cytokine secretion: Comparison with the MHC‐tetramer technology. Scand. J. Immunol. 52:544‐549.
   Oelke, M., Moehrle, U., Chen, J.L., Behringer, D., Cerundolo, V., Lindemann, A., and Mackensen, A. 2000b. Generation and purification of CD8+ melan‐A‐specific cytotoxic T lymphocytes for adoptive transfer in tumor immunotherapy. Clin. Cancer Res. 6:1997‐2005.
   Ouyang, W., Löhning, M., Gao, Z., Assenmacher, M., Ranganath, S., Radbruch, A., and Murphy, K.M. 2000. Stat6‐independent GATA‐3 autoactivation directs IL‐4‐independent Th2 development and commitment. Immunity 12:27‐37.
   Pittet, M.J., Zippelius, A., Speiser, D.E., Assenmacher, M., Guillaume, P., Valmori, D., Lienard, D., Lejeune, F., Cerottini, J.C., and Romero, P. 2001. Ex vivo IFN‐gamma secretion by circulating CD8 T lymphocytes. Implications of a novel approach for T cell monitoring in infectious and malignant diseases. J. Immunol. 166:7634‐7640.
   Smits, H.H., van Rietschoten, J.G., Hilkens, C.M., Sayilir, R., Stiekema, F., Kapsenberg, M.L., and Wierenga, E.A. 2001. IL‐12‐induced reversal of human Th2 cells is accompanied by full restoration of IL‐12 responsiveness and loss of GATA‐3 expression. Eur. J. Immunol. 31:1056‐1065.
Key References
   Manz et al., 1995. See above.
  The original description of the cell‐surface affinity matrix technology.
   Brosterhus et al., 1999. See above.
  Description of an actual cytokine secretion assay and its use for the analysis and isolation of antigen‐specific T cells.
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