Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

T cells use two directionally distinct pathways for cytokine secretion

Nature Immunology volume 7, pages 247–255 (2006)Cite this article

Abstract

Activated T helper cells produce many cytokines, some of which are secreted through the immunological synapse toward the antigen-presenting cell. Here we have used immunocytochemistry, live-cell imaging and a surface-mediated secretion assay to show that there are two cytokine export pathways in T helper cells. Some cytokines, including interleukin 2 and interferon-γ, were secreted into the synapse, whereas others, including tumor necrosis factor and the chemokine CCL3 (MIP-1α), were released multidirectionally. Each secretion pathway was associated with different trafficking proteins, indicating that they are molecularly distinct processes. These data suggest that T helper cells release some cytokines into the immunological synapse to impart specific communication and others multidirectionally to promote inflammation and to establish chemokine gradients.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Intracellular staining patterns of IL-2 and TNF in representative T cell-APC conjugates.
Figure 2: Synaptic localization of IFN-γ but not TNF.
Figure 3: Live imaging of TNF secretion by TH cells in the presence of TAPI-0.
Figure 4: IL-2 and TNF localize with distinct pools of transport proteins.
Figure 5: Use of the synaptic and multidirectional secretion pathways in both TH1 and TH2 cells.

Similar content being viewed by others

Accession codes

Accessions

Gene Expression Omnibus

References

  1. Bromley, S.K. et al. The immunological synapse. Annu. Rev. Immunol. 19, 375–396 (2001).

    Article  CAS  PubMed  Google Scholar 

  2. Monks, C.R., Freiberg, B.A., Kupfer, H., Sciaky, N. & Kupfer, A. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395, 82–86 (1998).

    Article  CAS  PubMed  Google Scholar 

  3. Grakoui, A. et al. The immunological synapse: a molecular machine controlling T cell activation. Science 285, 221–227 (1999).

    Article  CAS  PubMed  Google Scholar 

  4. Sancho, D. et al. Regulation of microtubule-organizing center orientation and actomyosin cytoskeleton rearrangement during immune interactions. Immunol. Rev. 189, 84–97 (2002).

    Article  CAS  PubMed  Google Scholar 

  5. Kupfer, A. & Singer, S.J. Cell biology of cytotoxic and helper T cell functions: immunofluorescence microscopic studies of single cells and cell couples. Annu. Rev. Immunol. 7, 309–337 (1989).

    Article  CAS  PubMed  Google Scholar 

  6. Davis, S.J. & van der Merwe, P.A. The immunological synapse: required for T cell receptor signalling or directing T cell effector function? Curr. Biol. 11, R289–R291 (2001).

    Article  CAS  PubMed  Google Scholar 

  7. Stinchcombe, J.C., Bossi, G., Booth, S. & Griffiths, G.M. The immunological synapse of CTL contains a secretory domain and membrane bridges. Immunity 15, 751–761 (2001).

    Article  CAS  PubMed  Google Scholar 

  8. Kupfer, A., Mosmann, T.R. & Kupfer, H. Polarized expression of cytokines in cell conjugates of helper T cells and splenic B cells. Proc. Natl. Acad. Sci. USA 88, 775–779 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Kupfer, H., Monks, C.R. & Kupfer, A. Small splenic B cells that bind to antigen-specific T helper (Th) cells and face the site of cytokine production in the Th cells selectively proliferate: immunofluorescence microscopic studies of Th-B antigen-presenting cell interactions. J. Exp. Med. 179, 1507–1515 (1994).

    Article  CAS  PubMed  Google Scholar 

  10. Reichert, P., Reinhardt, R.L., Ingulli, E. & Jenkins, M.K. Cutting edge: in vivo identification of TCR redistribution and polarized IL-2 production by naive CD4 T cells. J. Immunol. 166, 4278–4281 (2001).

    Article  CAS  PubMed  Google Scholar 

  11. Poo, W.J., Conrad, L. & Janeway, C.A., Jr. Receptor-directed focusing of lymphokine release by helper T cells. Nature 332, 378–380 (1988).

    Article  CAS  PubMed  Google Scholar 

  12. Pagan, J.K. et al. The t-SNARE syntaxin 4 is regulated during macrophage activation to function in membrane traffic and cytokine secretion. Curr. Biol. 13, 156–160 (2003).

    Article  CAS  PubMed  Google Scholar 

  13. Bock, J.B., Matern, H.T., Peden, A.A. & Scheller, R.H. A genomic perspective on membrane compartment organization. Nature 409, 839–841 (2001).

    Article  CAS  PubMed  Google Scholar 

  14. Segev, N. Ypt and Rab GTPases: insight into functions through novel interactions. Curr. Opin. Cell Biol. 13, 500–511 (2001).

    Article  CAS  PubMed  Google Scholar 

  15. Zerial, M. & McBride, H. Rab proteins as membrane organizers. Nat. Rev. Mol. Cell Biol. 2, 107–117 (2001).

    Article  CAS  PubMed  Google Scholar 

  16. Jahn, R., Lang, T. & Sudhof, T.C. Membrane fusion. Cell 112, 519–533 (2003).

    Article  CAS  PubMed  Google Scholar 

  17. Parlati, F. et al. Distinct SNARE complexes mediating membrane fusion in Golgi transport based on combinatorial specificity. Proc. Natl. Acad. Sci. USA 99, 5424–5429 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Allenspach, E.J. et al. ERM-dependent movement of CD43 defines a novel protein complex distal to the immunological synapse. Immunity 15, 739–750 (2001).

    Article  CAS  PubMed  Google Scholar 

  19. Morales-Tirado, V. et al. Cutting edge: selective requirement for the Wiskott-Aldrich syndrome protein in cytokine, but not chemokine, secretion by CD4+ T cells. J. Immunol. 173, 726–730 (2004).

    Article  CAS  PubMed  Google Scholar 

  20. Chen, D.S. et al. Marked differences in human melanoma antigen-specific T cell responsiveness following peptide vaccination using a functional microarray. PloS Med. 2, e265 (2005) published online 20 September 2005 (10.1371/journal.pmed.0020265).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Catalfamo, M. et al. Human CD8+ T cells store RANTES in a unique secretory compartment and release it rapidly after TcR stimulation. Immunity 20, 219–230 (2004).

    Article  CAS  PubMed  Google Scholar 

  22. Darchen, F. & Goud, B. Multiple aspects of Rab protein action in the secretory pathway: focus on Rab3 and Rab6. Biochimie 82, 375–384 (2000).

    Article  CAS  PubMed  Google Scholar 

  23. Riedel, D. et al. Rab3D is not required for exocrine exocytosis but for maintenance of normally sized secretory granules. Mol. Cell. Biol. 22, 6487–6497 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Wendler, F. & Tooze, S. Syntaxin 6: the promiscuous behaviour of a SNARE protein. Traffic 2, 606–611 (2001).

    Article  CAS  PubMed  Google Scholar 

  25. Masuda, E.S. et al. Rab37 is a novel mast cell specific GTPase localized to secretory granules. FEBS Lett. 470, 61–64 (2000).

    Article  CAS  PubMed  Google Scholar 

  26. Antonin, W. et al. A SNARE complex mediating fusion of late endosomes defines conserved properties of SNARE structure and function. EMBO J. 19, 6453–6464 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Hirst, J., Miller, S.E., Taylor, M.J., von Mollard, G.F. & Robinson, M.S. EpsinR is an adaptor for the SNARE protein Vti1b. Mol. Biol. Cell 15, 5593–5602 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Chidambaram, S., Mullers, N., Wiederhold, K., Haucke, V. & von Mollard, G.F. Specific interaction between SNAREs and epsin N-terminal homology (ENTH) domains of epsin-related proteins in trans-Golgi network to endosome transport. J. Biol. Chem. 279, 4175–4179 (2004).

    Article  CAS  PubMed  Google Scholar 

  29. Murray, R.Z., Wylie, F.G., Khromykh, T., Hume, D.A. & Stow, J.L. Syntaxin 6 and Vti1b form a novel SNARE complex, which is up-regulated in activated macrophages to facilitate exocytosis of tumor necrosis factor-α. J. Biol. Chem. 280, 10478–10483 (2005).

    Article  CAS  PubMed  Google Scholar 

  30. Huber, L.A., Dupree, P. & Dotti, C.G. A deficiency of the small GTPase rab8 inhibits membrane traffic in developing neurons. Mol. Cell. Biol. 15, 918–924 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Ang, A.L., Folsch, H., Koivisto, U.M., Pypaert, M. & Mellman, I. The Rab8 GTPase selectively regulates AP-1B-dependent basolateral transport in polarized Madin-Darby canine kidney cells. J. Cell Biol. 163, 339–350 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Stinchcombe, J.C. et al. Rab27a is required for regulated secretion in cytotoxic T lymphocytes. J. Cell Biol. 152, 825–834 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Butz, E.A. & Bevan, M.J. Massive expansion of antigen-specific CD8+ T cells during an acute virus infection. Immunity 8, 167–175 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Murali-Krishna, K. et al. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8, 177–187 (1998).

    Article  CAS  PubMed  Google Scholar 

  35. Boehm, U., Klamp, T., Groot, M. & Howard, J.C. Cellular responses to interferon-γ. Annu. Rev. Immunol. 15, 749–795 (1997).

    Article  CAS  PubMed  Google Scholar 

  36. Pestka, S. et al. Interleukin-10 and related cytokines and receptors. Annu. Rev. Immunol. 22, 929–979 (2004).

    Article  CAS  PubMed  Google Scholar 

  37. Grouard, G. et al. The enigmatic plasmacytoid T cells develop into dendritic cells with interleukin (IL)-3 and CD40-ligand. J. Exp. Med. 185, 1101–1111 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Locksley, R.M., Killeen, N. & Lenardo, M.J. The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell 104, 487–501 (2001).

    Article  CAS  PubMed  Google Scholar 

  39. Sallusto, F., Mackay, C.R. & Lanzavecchia, A. The role of chemokine receptors in primary, effector, and memory immune responses. Annu. Rev. Immunol. 18, 593–620 (2000).

    Article  CAS  PubMed  Google Scholar 

  40. Schmitz, J. et al. Induction of interleukin 4 (IL-4) expression in T helper (Th) cells is not dependent on IL-4 from non-Th cells. J. Exp. Med. 179, 1349–1353 (1994).

    Article  CAS  PubMed  Google Scholar 

  41. Glimcher, L.H. & Murphy, K.M. Lineage commitment in the immune system: the T helper lymphocyte grows up. Genes Dev. 14, 1693–1711 (2000).

    CAS  PubMed  Google Scholar 

  42. Okada, T. et al. Antigen-engaged B cells undergo chemotaxis toward the T zone and form motile conjugates with helper T cells. PLoS Bio 3, e150 (2005) published online 3 May 2005 (10.1371/journal.pbio.0030150).

    Article  CAS  Google Scholar 

  43. Croft, M. & Swain, S.L. B cell response to fresh and effector T helper cells. Role of cognate T-B interaction and the cytokines IL-2, IL-4, and IL-6. J. Immunol. 146, 4055–4064 (1991).

    CAS  PubMed  Google Scholar 

  44. Calderhead, D.M., Kosaka, Y., Manning, E.M. & Noelle, R.J. CD40–CD154 interactions in B-cell signaling. Curr. Top. Microbiol. Immunol. 245, 73–99 (2000).

    CAS  PubMed  Google Scholar 

  45. Boisvert, J., Edmondson, S. & Krummel, M.F. Immunological synapse formation licenses CD40–CD40L accumulations at T-APC contact sites. J. Immunol. 173, 3647–3652 (2004).

    Article  CAS  PubMed  Google Scholar 

  46. Maldonado, R.A., Irvine, D.J., Schreiber, R. & Glimcher, L.H. A role for the immunological synapse in lineage commitment of CD4 lymphocytes. Nature 431, 527–532 (2004).

    Article  CAS  PubMed  Google Scholar 

  47. Taylor, P.C., Williams, R.O. & Feldmann, M. Tumour necrosis factor α as a therapeutic target for immune-mediated inflammatory diseases. Curr. Opin. Biotechnol. 15, 557–563 (2004).

    Article  CAS  PubMed  Google Scholar 

  48. Kontoyiannis, D., Pasparakis, M., Pizarro, T.T., Cominelli, F. & Kollias, G. Impaired on/off regulation of TNF biosynthesis in mice lacking TNF AU-rich elements: implications for joint and gut-associated immunopathologies. Immunity 10, 387–398 (1999).

    Article  CAS  PubMed  Google Scholar 

  49. Grivennikov, S.I. et al. Distinct and nonredundant in vivo functions of TNF produced by T cells and macrophages/neutrophils: protective and deleterious effects. Immunity 22, 93–104 (2005).

    CAS  PubMed  Google Scholar 

  50. Steinman, L. Immune therapy for autoimmune diseases. Science 305, 212–216 (2004).

    Article  CAS  PubMed  Google Scholar 

  51. Spiliotis, E.T. & Nelson, W.J. Spatial control of exocytosis. Curr. Opin. Cell Biol. 15, 430–437 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Boniface, J.J., Reich, Z., Lyons, D.S. & Davis, M.M. Thermodynamics of T cell receptor binding to peptide-MHC: evidence for a general mechanism of molecular scanning. Proc. Natl. Acad. Sci. USA 96, 11446–11451 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Purbhoo, M.A., Irvine, D.J., Huppa, J.B. & Davis, M.M. T cell killing does not require the formation of a stable mature immunological synapse. Nat. Immunol. 5, 524–530 (2004).

    Article  CAS  PubMed  Google Scholar 

  54. Irvine, D.J., Purbhoo, M.A., Krogsgaard, M. & Davis, M.M. Direct observation of ligand recognition by T cells. Nature 419, 845–849 (2002).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank L. Steinman, H. McDevitt, W.J. Nelson and K. Pham for discussions and for critical reading of this manuscript; S. Pfeffer for comments; E. Gallo for assistance with the Affymetrix data; L. Klein for advice with colocalization; J. Mulholland and K. Lee for assistance with confocal microscopy; N. Prado and J. Fabian for technical assistance; and other members of the Davis lab for discussions and support. Supported by the Helen Hay Whitney Foundation (M.H.), Giannini Family Foundation (M.H.), Human Frontiers Science Program (B.F.L.), Cancer Research Institute (M.S.K.), National Institutes of Health (M.M.D.) and Howard Hughes Medical Institute (M.M.D.).

Author information

Authors and Affiliations

  1. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, 94305, California, USA

    Morgan Huse, Björn F Lillemeier, Michael S Kuhns, Daniel S Chen & Mark M Davis

  2. The Howard Hughes Medical Institute, Stanford, 94305, California, USA

    Daniel S Chen & Mark M Davis

Authors
  1. Morgan Huse
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Björn F Lillemeier
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michael S Kuhns
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Daniel S Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mark M Davis
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mark M Davis.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Gradual appearance of non-synapse associated TNF compartments. (PDF 43 kb)

Supplementary Fig. 2

TAPI-0-mediated blockade of TNF release. (PDF 72 kb)

Supplementary Fig. 3

Stability of TNF puncta over time. (PDF 282 kb)

Supplementary Fig. 4

Intracellular IL-4 has Golgi-associated and Golgi-independent pools. (PDF 109 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Huse, M., Lillemeier, B., Kuhns, M. et al. T cells use two directionally distinct pathways for cytokine secretion. Nat Immunol 7, 247–255 (2006). https://doi.org/10.1038/ni1304

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni1304

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing