Skip to main content
NCBI home page
Immunology logoLink to Immunology
. 1995 Feb;84(2):183–192.

Developmental regulation of TCR-CD3-dependent [Ca2+]i responses of individual normal and pp59fyn-deficient T lymphocytes.

K E Hedin 1, M W Appleby 1, D E Clapham 1
PMCID: PMC1415114  PMID: 7750993

Abstract

The aim of this study was to ascertain whether different types of T-cell receptor (TCR)-mediated [Ca2+]i signals could begin to explain the different cellular responses of mature and immature T cells to ligation of the TCR-CD3 complex. Using a digital fluorescence imaging system, we measured and compared [Ca2+]i of individual cells from immature and mature murine T-cell populations following application of CD3-epsilon monoclonal antibody (mAb). Our approach revealed distinctions among developmental subsets which were not seen by previous measurements of [Ca2+]i in bulk cell populations. The CD3-mediated [Ca2+]i responses of individual thymocytes were very complex. Latencies to peak [Ca2+]i varied greatly among thymocytes, but the responses of splenic T cells were synchronized, novel evidence that the timing of [Ca2+]i responses may be an important informative parameter for TCR-CD3 signalling. In addition, among cells responding to CD3 mAb, higher peak [Ca2+]i responses correlated with maturity (CD4+ CD8+ thymocytes < single-positive thymocytes < splenic T cells). Examination of cells from pp59fyn-deficient mice showed that pp59fyn deficiency affects the amplitude and probability, but not the latency or synchrony, of CD3-mediated [Ca2+]i responses of CD4+ CD8+ and CD4+ CD8- thymocytes. All subsets showed equivalent receptor-independent mobilization of [Ca2+]i. These developmentally distinct [Ca2+]i features most probably reflect meaningful developmental changes in how the TCR-CD3 complex couples to intracellular signalling machinery including pp59fyn. By clearly showing how [Ca2+]i responses change during development, these results support the hypothesis that distinctive types of [Ca2+]i responses drive thymocyte differentiation.

Full text

PDF
183

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Appleby M. W., Gross J. A., Cooke M. P., Levin S. D., Qian X., Perlmutter R. M. Defective T cell receptor signaling in mice lacking the thymic isoform of p59fyn. Cell. 1992 Sep 4;70(5):751–763. doi: 10.1016/0092-8674(92)90309-z. [DOI] [PubMed] [Google Scholar]
  2. Blackman M., Kappler J., Marrack P. The role of the T cell receptor in positive and negative selection of developing T cells. Science. 1990 Jun 15;248(4961):1335–1341. doi: 10.1126/science.1972592. [DOI] [PubMed] [Google Scholar]
  3. Chaffin K. E., Beals C. R., Wilkie T. M., Forbush K. A., Simon M. I., Perlmutter R. M. Dissection of thymocyte signaling pathways by in vivo expression of pertussis toxin ADP-ribosyltransferase. EMBO J. 1990 Dec;9(12):3821–3829. doi: 10.1002/j.1460-2075.1990.tb07600.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chan A. C., Iwashima M., Turck C. W., Weiss A. ZAP-70: a 70 kd protein-tyrosine kinase that associates with the TCR zeta chain. Cell. 1992 Nov 13;71(4):649–662. doi: 10.1016/0092-8674(92)90598-7. [DOI] [PubMed] [Google Scholar]
  5. Cooke M. P., Abraham K. M., Forbush K. A., Perlmutter R. M. Regulation of T cell receptor signaling by a src family protein-tyrosine kinase (p59fyn). Cell. 1991 Apr 19;65(2):281–291. doi: 10.1016/0092-8674(91)90162-r. [DOI] [PubMed] [Google Scholar]
  6. Crabtree G. R. Contingent genetic regulatory events in T lymphocyte activation. Science. 1989 Jan 20;243(4889):355–361. doi: 10.1126/science.2783497. [DOI] [PubMed] [Google Scholar]
  7. Davidson D., Chow L. M., Fournel M., Veillette A. Differential regulation of T cell antigen responsiveness by isoforms of the src-related tyrosine protein kinase p59fyn. J Exp Med. 1992 Jun 1;175(6):1483–1492. doi: 10.1084/jem.175.6.1483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Donnadieu E., Bismuth G., Trautmann A. Calcium fluxes in T lymphocytes. J Biol Chem. 1992 Dec 25;267(36):25864–25872. [PubMed] [Google Scholar]
  9. Donnadieu E., Cefai D., Tan Y. P., Paresys G., Bismuth G., Trautmann A. Imaging early steps of human T cell activation by antigen-presenting cells. J Immunol. 1992 May 1;148(9):2643–2653. [PubMed] [Google Scholar]
  10. Dustin M. L., Springer T. A. Role of lymphocyte adhesion receptors in transient interactions and cell locomotion. Annu Rev Immunol. 1991;9:27–66. doi: 10.1146/annurev.iy.09.040191.000331. [DOI] [PubMed] [Google Scholar]
  11. Finkel T. H., Cambier J. C., Kubo R. T., Born W. K., Marrack P., Kappler J. W. The thymus has two functionally distinct populations of immature alpha beta + T cells: one population is deleted by ligation of alpha beta TCR. Cell. 1989 Sep 22;58(6):1047–1054. doi: 10.1016/0092-8674(89)90503-5. [DOI] [PubMed] [Google Scholar]
  12. Finkel T. H., McDuffie M., Kappler J. W., Marrack P., Cambier J. C. Both immature and mature T cells mobilize Ca2+ in response to antigen receptor crosslinking. Nature. 1987 Nov 12;330(6144):179–181. doi: 10.1038/330179a0. [DOI] [PubMed] [Google Scholar]
  13. Gardner P., Alcover A., Kuno M., Moingeon P., Weyand C. M., Goronzy J., Reinherz E. L. Triggering of T-lymphocytes via either T3-Ti or T11 surface structures opens a voltage-insensitive plasma membrane calcium-permeable channel: requirement for interleukin-2 gene function. J Biol Chem. 1989 Jan 15;264(2):1068–1076. [PubMed] [Google Scholar]
  14. Gelfand E. W., Cheung R. K., Grinstein S., Mills G. B. Characterization of the role for calcium influx in mitogen-induced triggering of human T cells. Identification of calcium-dependent and calcium-independent signals. Eur J Immunol. 1986 Aug;16(8):907–912. doi: 10.1002/eji.1830160806. [DOI] [PubMed] [Google Scholar]
  15. Gelfand E. W., Cheung R. K., Grinstein S. Role of membrane potential in the regulation of lectin-induced calcium uptake. J Cell Physiol. 1984 Dec;121(3):533–539. doi: 10.1002/jcp.1041210312. [DOI] [PubMed] [Google Scholar]
  16. Golstein P., Ojcius D. M., Young J. D. Cell death mechanisms and the immune system. Immunol Rev. 1991 Jun;121:29–65. doi: 10.1111/j.1600-065x.1991.tb00822.x. [DOI] [PubMed] [Google Scholar]
  17. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  18. Hall C. G., Sancho J., Terhorst C. Reconstitution of T cell receptor zeta-mediated calcium mobilization in nonlymphoid cells. Science. 1993 Aug 13;261(5123):915–918. doi: 10.1126/science.8346442. [DOI] [PubMed] [Google Scholar]
  19. Havran W. L., Poenie M., Kimura J., Tsien R., Weiss A., Allison J. P. Expression and function of the CD3-antigen receptor on murine CD4+8+ thymocytes. Nature. 1987 Nov 12;330(6144):170–173. doi: 10.1038/330170a0. [DOI] [PubMed] [Google Scholar]
  20. Hess S. D., Oortgiesen M., Cahalan M. D. Calcium oscillations in human T and natural killer cells depend upon membrane potential and calcium influx. J Immunol. 1993 Apr 1;150(7):2620–2633. [PubMed] [Google Scholar]
  21. Hoth M., Penner R. Depletion of intracellular calcium stores activates a calcium current in mast cells. Nature. 1992 Jan 23;355(6358):353–356. doi: 10.1038/355353a0. [DOI] [PubMed] [Google Scholar]
  22. Imboden J. B., Stobo J. D. Transmembrane signalling by the T cell antigen receptor. Perturbation of the T3-antigen receptor complex generates inositol phosphates and releases calcium ions from intracellular stores. J Exp Med. 1985 Mar 1;161(3):446–456. doi: 10.1084/jem.161.3.446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Janeway C. A., Jr, Bottomly K. Signals and signs for lymphocyte responses. Cell. 1994 Jan 28;76(2):275–285. doi: 10.1016/0092-8674(94)90335-2. [DOI] [PubMed] [Google Scholar]
  24. Janeway C. A., Jr The T cell receptor as a multicomponent signalling machine: CD4/CD8 coreceptors and CD45 in T cell activation. Annu Rev Immunol. 1992;10:645–674. doi: 10.1146/annurev.iy.10.040192.003241. [DOI] [PubMed] [Google Scholar]
  25. June C. H., Fletcher M. C., Ledbetter J. A., Schieven G. L., Siegel J. N., Phillips A. F., Samelson L. E. Inhibition of tyrosine phosphorylation prevents T-cell receptor-mediated signal transduction. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7722–7726. doi: 10.1073/pnas.87.19.7722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Koyasu S., D'Adamio L., Clayton L. K., Reinherz E. L. T-cell receptor isoforms and signal transduction. Curr Opin Immunol. 1991 Feb;3(1):32–39. doi: 10.1016/0952-7915(91)90073-a. [DOI] [PubMed] [Google Scholar]
  27. Lechleiter J., Girard S., Clapham D., Peralta E. Subcellular patterns of calcium release determined by G protein-specific residues of muscarinic receptors. Nature. 1991 Apr 11;350(6318):505–508. doi: 10.1038/350505a0. [DOI] [PubMed] [Google Scholar]
  28. Lewis R. S., Cahalan M. D. Mitogen-induced oscillations of cytosolic Ca2+ and transmembrane Ca2+ current in human leukemic T cells. Cell Regul. 1989 Nov;1(1):99–112. doi: 10.1091/mbc.1.1.99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lytton J., Westlin M., Hanley M. R. Thapsigargin inhibits the sarcoplasmic or endoplasmic reticulum Ca-ATPase family of calcium pumps. J Biol Chem. 1991 Sep 15;266(26):17067–17071. [PubMed] [Google Scholar]
  30. Malissen B., Schmitt-Verhulst A. M. Transmembrane signalling through the T-cell-receptor-CD3 complex. Curr Opin Immunol. 1993 Jun;5(3):324–333. doi: 10.1016/0952-7915(93)90049-x. [DOI] [PubMed] [Google Scholar]
  31. Nakayama T., June C. H., Munitz T. I., Sheard M., McCarthy S. A., Sharrow S. O., Samelson L. E., Singer A. Inhibition of T cell receptor expression and function in immature CD4+CD8+ cells by CD4. Science. 1990 Sep 28;249(4976):1558–1561. doi: 10.1126/science.2120773. [DOI] [PubMed] [Google Scholar]
  32. Nakayama T., Samelson L. E., Nakayama Y., Munitz T. I., Sheard M., June C. H., Singer A. Ligand-stimulated signaling events in immature CD4+CD8+ thymocytes expressing competent T-cell receptor complexes. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):9949–9953. doi: 10.1073/pnas.88.22.9949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nakayama T., Ueda Y., Yamada H., Shores E. W., Singer A., June C. H. In vivo calcium elevations in thymocytes with T cell receptors that are specific for self ligands. Science. 1992 Jul 3;257(5066):96–99. doi: 10.1126/science.1621102. [DOI] [PubMed] [Google Scholar]
  34. Paul W. E., Seder R. A. Lymphocyte responses and cytokines. Cell. 1994 Jan 28;76(2):241–251. doi: 10.1016/0092-8674(94)90332-8. [DOI] [PubMed] [Google Scholar]
  35. Perlmutter R. M., Levin S. D., Appleby M. W., Anderson S. J., Alberola-Ila J. Regulation of lymphocyte function by protein phosphorylation. Annu Rev Immunol. 1993;11:451–499. doi: 10.1146/annurev.iy.11.040193.002315. [DOI] [PubMed] [Google Scholar]
  36. Rabinovitch P. S., June C. H., Grossmann A., Ledbetter J. A. Heterogeneity among T cells in intracellular free calcium responses after mitogen stimulation with PHA or anti-CD3. Simultaneous use of indo-1 and immunofluorescence with flow cytometry. J Immunol. 1986 Aug 1;137(3):952–961. [PubMed] [Google Scholar]
  37. Samelson L. E., Phillips A. F., Luong E. T., Klausner R. D. Association of the fyn protein-tyrosine kinase with the T-cell antigen receptor. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4358–4362. doi: 10.1073/pnas.87.11.4358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Secrist J. P., Burns L. A., Karnitz L., Koretzky G. A., Abraham R. T. Stimulatory effects of the protein tyrosine phosphatase inhibitor, pervanadate, on T-cell activation events. J Biol Chem. 1993 Mar 15;268(8):5886–5893. [PubMed] [Google Scholar]
  39. Secrist J. P., Karnitz L., Abraham R. T. T-cell antigen receptor ligation induces tyrosine phosphorylation of phospholipase C-gamma 1. J Biol Chem. 1991 Jul 5;266(19):12135–12139. [PubMed] [Google Scholar]
  40. Smith C. A., Williams G. T., Kingston R., Jenkinson E. J., Owen J. J. Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T cells in thymic cultures. Nature. 1989 Jan 12;337(6203):181–184. doi: 10.1038/337181a0. [DOI] [PubMed] [Google Scholar]
  41. Stein P. L., Lee H. M., Rich S., Soriano P. pp59fyn mutant mice display differential signaling in thymocytes and peripheral T cells. Cell. 1992 Sep 4;70(5):741–750. doi: 10.1016/0092-8674(92)90308-y. [DOI] [PubMed] [Google Scholar]
  42. Tsien R. Y., Pozzan T., Rink T. J. T-cell mitogens cause early changes in cytoplasmic free Ca2+ and membrane potential in lymphocytes. Nature. 1982 Jan 7;295(5844):68–71. doi: 10.1038/295068a0. [DOI] [PubMed] [Google Scholar]
  43. Tsunoda Y. Oscillatory Ca2+ signaling and its cellular function. New Biol. 1991 Jan;3(1):3–17. [PubMed] [Google Scholar]
  44. Tsygankov A. Y., Bröker B. M., Fargnoli J., Ledbetter J. A., Bolen J. B. Activation of tyrosine kinase p60fyn following T cell antigen receptor cross-linking. J Biol Chem. 1992 Sep 15;267(26):18259–18262. [PubMed] [Google Scholar]
  45. Volarević S., Niklinska B. B., Burns C. M., Yamada H., June C. H., Dumont F. J., Ashwell J. D. The CD45 tyrosine phosphatase regulates phosphotyrosine homeostasis and its loss reveals a novel pattern of late T cell receptor-induced Ca2+ oscillations. J Exp Med. 1992 Sep 1;176(3):835–844. doi: 10.1084/jem.176.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wallace V. A., Fung-Leung W. P., Timms E., Gray D., Kishihara K., Loh D. Y., Penninger J., Mak T. W. CD45RA and CD45RBhigh expression induced by thymic selection events. J Exp Med. 1992 Dec 1;176(6):1657–1663. doi: 10.1084/jem.176.6.1657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Weiss A., Imboden J., Hardy K., Manger B., Terhorst C., Stobo J. The role of the T3/antigen receptor complex in T-cell activation. Annu Rev Immunol. 1986;4:593–619. doi: 10.1146/annurev.iy.04.040186.003113. [DOI] [PubMed] [Google Scholar]
  48. Weiss A., Koretzky G., Schatzman R. C., Kadlecek T. Functional activation of the T-cell antigen receptor induces tyrosine phosphorylation of phospholipase C-gamma 1. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5484–5488. doi: 10.1073/pnas.88.13.5484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Weiss A., Littman D. R. Signal transduction by lymphocyte antigen receptors. Cell. 1994 Jan 28;76(2):263–274. doi: 10.1016/0092-8674(94)90334-4. [DOI] [PubMed] [Google Scholar]
  50. Zweifach A., Lewis R. S. Mitogen-regulated Ca2+ current of T lymphocytes is activated by depletion of intracellular Ca2+ stores. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6295–6299. doi: 10.1073/pnas.90.13.6295. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Immunology are provided here courtesy of British Society for Immunology

RESOURCES