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Direct access to CD4+ T cells specific for defined antigens according to CD154 expression

Nature Medicine volume 11, pages 1118–1124 (2005)Cite this article

Abstract

The direct assessment of T helper (TH)-cell responses specific for antigens is essential to evaluate pathogenic and protective immunity. Presently, analysis and isolation of antigen-specific TH cells is restricted to cells that produce cytokines, or can be performed only with a rare selection of specific peptide major histocompatibility complex class II (MHC II) multimers. Here we report a new method that enables the assessment and isolation of TH cells specific for a defined antigen according to CD154 expression induced after stimulation in vitro. We show that antigen-induced CD154 expression is highly sensitive and specific for human and mouse antigen-specific TH cells. Moreover, the isolation of antigen-specific CD154+ TH cells necessitates only surface staining with antibodies, thereby enabling the fast generation of antigen-specific TH cell lines. Our approach allows assessment of TH cells with a defined specificity for the combined quantitative and qualitative analysis of TH-cell immunity as well as for the isolation of specific TH cells for targeted cellular immunotherapies.

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Figure 1: Cytometric assessment of antigen-specific TH cells according to intracellular antigen-induced CD154 expression.
Figure 2: Combined qualitative and quantitative assessment of antigen-specific TH cells.
Figure 3: Cytometric assessment of antigen-induced CD154 surface expression.
Figure 4: Isolation of live CMV pp65–specific TH cells from PBMCs.

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References

  1. Altman, J.D. et al. Phenotypic analysis of antigen-specific T lymphocytes. Science 274, 94–96 (1996).

    Article  CAS  Google Scholar 

  2. Casares, S., Bona, C.A. & Brumeanu, T.D. Enzymatically mediated engineering of multivalent MHC class II-peptide chimeras. Protein Eng. 14, 195–200 (2001).

    Article  CAS  Google Scholar 

  3. Casares, S. et al. Down-regulation of diabetogenic CD4+ T cells by a soluble dimeric peptide-MHC class II chimera. Nat. Immunol. 3, 383–391 (2002).

    Article  CAS  Google Scholar 

  4. Hackett, C.J. & Sharma, O.K. Frontiers in peptide-MHC class II multimer technology. Nat. Immunol. 3, 887–889 (2002).

    Article  CAS  Google Scholar 

  5. Manz, R., Assenmacher, M., Pfluger, E., Miltenyi, S. & Radbruch, A. Analysis and sorting of live cells according to secreted molecules, relocated to a cell-surface affinity matrix. Proc. Natl. Acad. Sci. USA 92, 1921–1925 (1995).

    Article  CAS  Google Scholar 

  6. Waldrop, S.L., Pitcher, C.J., Peterson, D.M., Maino, V.C. & Picker, L.J. Determination of antigen-specific memory/effector CD4+ T cell frequencies by flow cytometry: evidence for a novel, antigen-specific homeostatic mechanism in HIV-associated immunodeficiency. J. Clin. Invest. 99, 1739–1750 (1997).

    Article  CAS  Google Scholar 

  7. Suni, M.A., Picker, L.J. & Maino, V.C. Detection of antigen-specific T cell cytokine expression in whole blood by flow cytometry. J. Immunol. Methods 212, 89–98 (1998).

    Article  CAS  Google Scholar 

  8. Brosterhus, H. et al. Enrichment and detection of live antigen-specific CD4(+) and CD8(+) T cells based on cytokine secretion. Eur. J. Immunol. 29, 4053–4059 (1999).

    Article  CAS  Google Scholar 

  9. Maino, V.C., Suni, M.A. & Ruitenberg, J.J. Rapid flow cytometric method for measuring lymphocyte subset activation. Cytometry 20, 127–133 (1995).

    Article  CAS  Google Scholar 

  10. Kahi, S. et al. A rapid flow cytometric method to explore cellular immunity against Toxoplasma gondii in humans. Clin. Diagn. Lab. Immunol. 5, 745–748 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Michalek, J. et al. Definitive separation of graft-versus-leukemia- and graft-versus-host-specific CD4+ T cells by virtue of their receptor beta loci sequences. Proc. Natl. Acad. Sci. USA 100, 1180–1184 (2003).

    Article  CAS  Google Scholar 

  12. Thiel, A., Scheffold, A. & Radbruch, A. Antigen-specific cytometry–new tools arrived!. Clin. Immunol. 111, 155–161 (2004).

    Article  CAS  Google Scholar 

  13. Armitage, R.J. et al. Molecular and biological characterization of a murine ligand for CD40. Nature 357, 80–82 (1992).

    Article  CAS  Google Scholar 

  14. Graf, D., Korthauer, U., Mages, H.W., Senger, G. & Kroczek, R.A. Cloning of TRAP, a ligand for CD40 on human T cells. Eur. J. Immunol. 22, 3191–3194 (1992).

    Article  CAS  Google Scholar 

  15. Yellin, M.J. et al. CD40 molecules induce down-modulation and endocytosis of T cell surface T cell-B cell activating molecule/CD40-L. Potential role in regulating helper effector function. J. Immunol. 152, 598–608 (1994).

    CAS  PubMed  Google Scholar 

  16. Misumi, Y., Miki, K., Takatsuki, A., Tamura, G. & Ikehara, Y. Novel blockade by brefeldin A of intracellular transport of secretory proteins in cultured rat hepatocytes. J. Biol. Chem. 261, 11398–11403 (1986).

    CAS  PubMed  Google Scholar 

  17. Choi, Y.W. et al. Interaction of Staphylococcus aureus toxin “superantigens” with human T cells. Proc. Natl. Acad. Sci. USA 86, 8941–8945 (1989).

    Article  CAS  Google Scholar 

  18. Hong, S.C., Waterbury, G. & Janeway, C.A., Jr. Different superantigens interact with distinct sites in the Vbeta domain of a single T cell receptor. J. Exp. Med. 183, 1437–1446 (1996).

    Article  CAS  Google Scholar 

  19. Murphy, K.M., Heimberger, A.B. & Loh, D.Y. Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo. Science 250, 1720–1723 (1990).

    Article  CAS  Google Scholar 

  20. Lindgren, H., Axcrona, K. & Leanderson, T. Regulation of transcriptional activity of the murine CD40 ligand promoter in response to signals through TCR and the costimulatory molecules CD28 and CD2. J. Immunol. 166, 4578–4585 (2001).

    Article  CAS  Google Scholar 

  21. Stinski, M.F. Sequence of protein synthesis in cells infected by human cytomegalovirus: early and late virus-induced polypeptides. J. Virol. 26, 686–701 (1978).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Wills, M.R. et al. The human cytotoxic T-lymphocyte (CTL) response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity, and T-cell receptor usage of pp65-specific CTL. J. Virol. 70, 7569–7579 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Beninga, J., Kropff, B. & Mach, M. Comparative analysis of fourteen individual human cytomegalovirus proteins for helper T cell response. J. Gen. Virol. 76, 153–160 (1995).

    Article  CAS  Google Scholar 

  24. Czerkinsky, C.C., Nilsson, L.A., Nygren, H., Ouchterlony, O. & Tarkowski, A. A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J. Immunol. Methods 65, 109–121 (1983).

    Article  CAS  Google Scholar 

  25. Sallusto, F., Lenig, D., Forster, R., Lipp, M. & Lanzavecchia, A. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401, 708–712 (1999).

    Article  CAS  Google Scholar 

  26. Ludewig, B., Henn, V., Schroder, J.M., Graf, D. & Kroczek, R.A. Induction, regulation, and function of soluble TRAP (CD40 ligand) during interaction of primary CD4+ CD45RA+ T cells with dendritic cells. Eur. J. Immunol. 26, 3137–3143 (1996).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank A. Radbruch for critical discussion and U. Klein for critical reading of the manuscript. We thank Q. Phan, F. Kellndorfer and D. Huscher for technical help. This work was supported by the European Sixth Framework Program, grants LSHP-CT-2003-503240, LSHG-CT-2005-005203 and LSHB-CT-2004-512074, grants PTJ 0311597 and PTJ 0312102 (all grants were to A.T. and A.S.).

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Authors and Affiliations

  1. Clinical Immunology Group, Deutsches Rheuma-Forschungszentrum, Berlin, Germany

    Marco Frentsch, Olga Arbach, Beate Moewes, Martin Rothe & Andreas Thiel

  2. Immunomodulation Group, Deutsches Rheuma-Forschungszentrum, Schumannstr. 21/22, Berlin, 10117, Germany

    Dennis Kirchhoff & Alexander Scheffold

  3. Clinic for Dermatology, Venerology and Allergology, Charité Campus Mitte, Schumannstr. 20/21, Berlin, 10117, Germany

    Margitta Worm & Martin Rothe

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  1. Marco Frentsch
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Correspondence to Andreas Thiel.

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Supplementary information

Supplementary Fig. 1

Combined qualitative and quantitative assessment of Th cells specific for different antigens. (PDF 872 kb)

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Frentsch, M., Arbach, O., Kirchhoff, D. et al. Direct access to CD4+ T cells specific for defined antigens according to CD154 expression. Nat Med 11, 1118–1124 (2005). https://doi.org/10.1038/nm1292

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