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Calcium oscillations regulate thymocyte motility during positive selection in the three-dimensional thymic environment

Nature Immunology volume 6, pages 143–151 (2005)Cite this article

Abstract

The three-dimensional thymic microenvironment and calcium signaling pathways are essential for driving positive selection of developing T cells. However, the nature of calcium signals and the diversity of their effects in the thymus are unknown. We describe here a thymic slice preparation for visualizing thymocyte motility and signaling in real time with two-photon microscopy. Naive thymocytes were highly motile at low intracellular calcium concentrations, but during positive selection cells became immobile and showed sustained calcium concentration oscillations. Increased intracellular calcium was necessary and sufficient to arrest thymocyte motility. The calcium dependence of motility acts to prolong thymocyte interactions with antigen-bearing stromal cells, promoting sustained signaling that may enhance the expression of genes underlying positive selection.

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Figure 1: Characterization of the acute thymic slice preparation.
Figure 2: Thymocyte motility and [Ca2+]i in nonselecting conditions.
Figure 3: Patterns of thymocyte motility and Ca2+ signaling in conditions of positive selection.
Figure 4: Increased [Ca2+]i is sufficient to inhibit thymocyte motility.
Figure 5: Ca2+ oscillations prolong productive interactions between thymocytes and stromal cells during positive selection.

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References

  1. Huesmann, M., Scott, B., Kisielow, P. & von Boehmer, H. Kinetics and efficacy of positive selection in the thymus of normal and T cell receptor transgenic mice. Cell 66, 533–540 (1991).

    Article  CAS  Google Scholar 

  2. Shortman, K., Vremec, D. & Egerton, M. The kinetics of T cell antigen receptor expression by subgroups of CD4+8+ thymocytes: delineation of CD4+8+32+ thymocytes as post-selection intermediates leading to mature T cells. J. Exp. Med. 173, 323–332 (1991).

    Article  CAS  Google Scholar 

  3. Petrie, H.T. Role of thymic organ structure and stromal composition in steady-state postnatal T-cell production. Immunol. Rev. 189, 8–19 (2002).

    Article  CAS  Google Scholar 

  4. Starr, T.K., Jameson, S.C. & Hogquist, K.A. Positive and negative selection of T cells. Annu. Rev. Immunol. 21, 139–176 (2003).

    Article  CAS  Google Scholar 

  5. Lewis, R.S. Calcium signaling mechanisms in T lymphocytes. Annu. Rev. Immunol. 19, 497–521 (2001).

    Article  CAS  Google Scholar 

  6. 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 330, 179–181 (1987).

    Article  CAS  Google Scholar 

  7. Neilson, J.R., Winslow, M.M., Hur, E.M. & Crabtree, G.R. Calcineurin B1 is essential for positive but not negative selection during thymocyte development. Immunity 20, 255–266 (2004).

    Article  CAS  Google Scholar 

  8. Hayden-Martinez, K., Kane, L.P. & Hedrick, S.M. Effects of a constitutively active form of calcineurin on T cell activation and thymic selection. J. Immunol. 165, 3713–3721 (2000).

    Article  CAS  Google Scholar 

  9. Raman, V. et al. Requirement for Ca2+/calmodulin-dependent kinase type IV/Gr in setting the thymocyte selection threshold. J. Immunol. 167, 6270–6278 (2001).

    Article  CAS  Google Scholar 

  10. Dolmetsch, R.E., Lewis, R.S., Goodnow, C.C. & Healy, J.I. Differential activation of transcription factors induced by Ca2+ response amplitude and duration. Nature 386, 855–858 (1997).

    Article  CAS  Google Scholar 

  11. Dolmetsch, R.E., Xu, K. & Lewis, R.S. Calcium oscillations increase the efficiency and specificity of gene expression. Nature 392, 933–936 (1998).

    Article  CAS  Google Scholar 

  12. Anderson, G., Moore, N.C., Owen, J.J. & Jenkinson, E.J. Cellular interactions in thymocyte development. Annu. Rev. Immunol. 14, 73–99 (1996).

    Article  CAS  Google Scholar 

  13. Nakayama, T. et al. In vivo calcium elevations in thymocytes with T cell receptors that are specific for self ligands. Science 257, 96–99 (1992).

    Article  CAS  Google Scholar 

  14. Mariathasan, S., Bachmann, M.F., Bouchard, D., Ohteki, T. & Ohashi, P.S. Degree of TCR internalization and Ca2+ flux correlates with thymocyte selection. J. Immunol. 161, 6030–6037 (1998).

    CAS  PubMed  Google Scholar 

  15. Freedman, B.D., Liu, Q.H., Somersan, S., Kotlikoff, M.I. & Punt, J.A. Receptor avidity and costimulation specify the intracellular Ca2+ signaling pattern in CD4+CD8+ thymocytes. J. Exp. Med. 190, 943–952 (1999).

    Article  CAS  Google Scholar 

  16. Haston, W.S., Shields, J.M. & Wilkinson, P.C. Lymphocyte locomotion and attachment on two-dimensional surfaces and in three-dimensional matrices. J. Cell Biol. 92, 747–752 (1982).

    Article  CAS  Google Scholar 

  17. Bousso, P., Bhakta, N.R., Lewis, R.S. & Robey, E. Dynamics of thymocyte-stromal cell interactions visualized by two-photon microscopy. Science 296, 1876–1880 (2002).

    Article  CAS  Google Scholar 

  18. Miller, M.J., Wei, S.H., Cahalan, M.D. & Parker, I. Autonomous T cell trafficking examined in vivo with intravital two-photon microscopy. Proc. Natl. Acad. Sci. USA 100, 2604–2609 (2003).

    Article  CAS  Google Scholar 

  19. Mempel, T.R., Henrickson, S.E. & Von Andrian, U.H. T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases. Nature 427, 154–159 (2004).

    Article  CAS  Google Scholar 

  20. Jenkinson, E.J., Van Ewijk, W. & Owen, J.J. Major histocompatibility complex antigen expression on the epithelium of the developing thymus in normal and nude mice. J. Exp. Med. 153, 280–292 (1981).

    Article  CAS  Google Scholar 

  21. Seder, R.A., Paul, W.E., Davis, M.M. & Fazekas de St Groth, B. The presence of interleukin 4 during in vitro priming determines the lymphokine-producing potential of CD4+ T cells from T cell receptor transgenic mice. J. Exp. Med. 176, 1091–1098 (1992).

    Article  CAS  Google Scholar 

  22. Hare, K.J., Jenkinson, E.J. & Anderson, G. CD69 expression discriminates MHC-dependent and -independent stages of thymocyte positive selection. J. Immunol. 162, 3978–3983 (1999).

    CAS  Google Scholar 

  23. Merkenschlager, M. et al. How many thymocytes audition for selection? J. Exp. Med. 186, 1149–1158 (1997).

    Article  CAS  Google Scholar 

  24. Yamashita, I., Nagata, T., Tada, T. & Nakayama, T. CD69 cell surface expression identifies developing thymocytes which audition for T cell antigen receptor-mediated positive selection. Int. Immunol. 5, 1139–1150 (1993).

    Article  CAS  Google Scholar 

  25. Revy, P., Sospedra, M., Barbour, B. & Trautmann, A. Functional antigen-independent synapses formed between T cells and dendritic cells. Nat. Immunol. 2, 925–931 (2001).

    Article  CAS  Google Scholar 

  26. Negulescu, P.A., Krasieva, T.B., Khan, A., Kerschbaum, H.H. & Cahalan, M.D. Polarity of T cell shape, motility, and sensitivity to antigen. Immunity 4, 421–430 (1996).

    Article  CAS  Google Scholar 

  27. Dustin, M.L., Bromley, S.K., Kan, Z., Peterson, D.A. & Unanue, E.R. Antigen receptor engagement delivers a stop signal to migrating T lymphocytes. Proc. Natl. Acad. Sci. USA 94, 3909–3913 (1997).

    Article  CAS  Google Scholar 

  28. Stewart, M.P., Cabanas, C. & Hogg, N. T cell adhesion to intercellular adhesion molecule-1 (ICAM-1) is controlled by cell spreading and the activation of integrin LFA-1. J. Immunol. 156, 1810–1817 (1996).

    CAS  Google Scholar 

  29. Petrie, H.T. Cell migration and the control of post-natal T-cell lymphopoiesis in the thymus. Nat. Rev. Immunol. 3, 859–866 (2003).

    Article  CAS  Google Scholar 

  30. Campbell, J.J., Pan, J. & Butcher, E.C. Cutting edge: developmental switches in chemokine responses during T cell maturation. J. Immunol. 163, 2353–2357 (1999).

    CAS  PubMed  Google Scholar 

  31. Uehara, S., Song, K., Farber, J.M. & Love, P.E. Characterization of CCR9 expression and CCL25/thymus-expressed chemokine responsiveness during T cell development: CD3highCD69+ thymocytes and γδTCR+ thymocytes preferentially respond to CCL25. J. Immunol. 168, 134–142 (2002).

    Article  CAS  Google Scholar 

  32. Ueno, T. et al. CCR7 signals are essential for cortex-medulla migration of developing thymocytes. J. Exp. Med. 200, 493–505 (2004).

    Article  CAS  Google Scholar 

  33. Adachi, S., Amasaki, Y., Miyatake, S., Arai, N. & Iwata, M. Successive expression and activation of NFAT family members during thymocyte differentiation. J. Biol. Chem. 275, 14708–14716 (2000).

    Article  CAS  Google Scholar 

  34. Amasaki, Y., Masuda, E.S., Imamura, R., Arai, K. & Arai, N. Distinct NFAT family proteins are involved in the nuclear NFAT-DNA binding complexes from human thymocyte subsets. J. Immunol. 160, 2324–2333 (1998).

    CAS  PubMed  Google Scholar 

  35. Oukka, M. et al. The transcription factor NFAT4 is involved in the generation and survival of T cells. Immunity 9, 295–304 (1998).

    Article  CAS  Google Scholar 

  36. Amasaki, Y. et al. A constitutively nuclear form of NFATx shows efficient transactivation activity and induces differentiation of CD4+CD8+ T cells. J. Biol. Chem. 277, 25640–25648 (2002).

    Article  CAS  Google Scholar 

  37. Tomida, T., Hirose, K., Takizawa, A., Shibasaki, F. & Iino, M. NFAT functions as a working memory of Ca2+ signals in decoding Ca2+ oscillation. EMBO J. 22, 3825–3832 (2003).

    Article  CAS  Google Scholar 

  38. Donnadieu, E., Bismuth, G. & Trautmann, A. Antigen recognition by helper T cells elicits a sequence of distinct changes of their shape and intracellular calcium. Curr. Biol. 4, 584–595 (1994).

    Article  CAS  Google Scholar 

  39. Dustin, M.L. Stop and go traffic to tune T cell responses. Immunity 21, 305–314 (2004).

    Article  CAS  Google Scholar 

  40. Jacobelli, J., Chmura, S.A., Buxton, D.B., Davis, M.M. & Krummel, M.F. A single class II myosin modulates T cell motility and stopping, but not synapse formation. Nat. Immunol. 5, 531–538 (2004).

    Article  CAS  Google Scholar 

  41. Buxton, D.B. & Adelstein, R.S. Calcium-dependent threonine phosphorylation of nonmuscle myosin in stimulated RBL-2H3 mast cells. J. Biol. Chem. 275, 34772–34779 (2000).

    Article  CAS  Google Scholar 

  42. Stewart, M.P., McDowall, A. & Hogg, N. LFA-1-mediated adhesion is regulated by cytoskeletal restraint and by a Ca2+-dependent protease, calpain. J. Cell Biol. 140, 699–707 (1998).

    Article  CAS  Google Scholar 

  43. Merkenschlager, M., Benoist, C. & Mathis, D. Evidence for a single-niche model of positive selection. Proc. Natl. Acad. Sci. USA 91, 11694–11698 (1994).

    Article  CAS  Google Scholar 

  44. Merkenschlager, M. Tracing interactions of thymocytes with individual stromal cell partners. Eur. J. Immunol. 26, 892–896 (1996).

    Article  CAS  Google Scholar 

  45. Yasutomo, K., Lucas, B. & Germain, R.N. TCR signaling for initiation and completion of thymocyte positive selection has distinct requirements for ligand quality and presenting cell type. J. Immunol. 165, 3015–3022 (2000).

    Article  CAS  Google Scholar 

  46. Miller, M.J., Hejazi, A.S., Wei, S.H., Cahalan, M.D. & Parker, I. T cell repertoire scanning is promoted by dynamic dendritic cell behavior and random T cell motility in the lymph node. Proc. Natl. Acad. Sci. USA 101, 998–1003 (2004).

    Article  CAS  Google Scholar 

  47. Adachi, S. & Iwata, M. Duration of calcineurin and Erk signals regulates CD4/CD8 lineage commitment of thymocytes. Cell. Immunol. 215, 45–53 (2002).

    Article  CAS  Google Scholar 

  48. Yasutomo, K., Doyle, C., Miele, L., Fuchs, C. & Germain, R.N. The duration of antigen receptor signalling determines CD4+ versus CD8+ T-cell lineage fate. Nature 404, 506–510 (2000).

    Article  CAS  Google Scholar 

  49. Grynkiewicz, G., Poenie, M. & Tsien, R.Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3450 (1985).

    CAS  Google Scholar 

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Acknowledgements

We thank M. Davis and M. Prakriya for comments on the manuscript; D. Madison for use of the Vibratome and advice on culturing slices; S. Smith and N. O'Rourke for help in designing and constructing the two-photon microscope; V. Li for assistance with immunohistochemistry; and V. Sohal and members of the Lewis lab for stimulating discussion. Supported by the Medical Scientist Training Program at Stanford (N.R.B. and D.Y.O.) and by US National Institutes of Health (R01 GM45374) and the Mathers Foundation (R.S.L.).

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Authors and Affiliations

  1. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, 94305, California, USA

    Nirav R Bhakta, David Y Oh & Richard S Lewis

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Correspondence to Richard S Lewis.

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Supplementary information

Supplementary Fig. 1

Surface phenotype of 5C.C7 thymocytes from B6 mice (PDF 82 kb)

Supplementary Fig. 2

Reticular structure of stromal cells in thymic slices. (PDF 233 kb)

Supplementary Fig. 3

Additional examples of Ca2+ signals representative of cells in B10.BR slices (positive selection conditions). (PDF 89 kb)

Supplementary Fig. 4

Characterization of Ca2+ oscillations and motility in thymocytes under positive selection conditions. (PDF 85 kb)

Supplementary Fig. 5

Long-term tracking of a single cell under positive selection conditions. (PDF 126 kb)

Supplementary Fig. 6

Cells undergoing Ca2+ oscillations during positive selection undergo significant displacement only after [Ca2+]i falls. (PDF 26 kb)

Supplementary Video 1

FITC-stained slice showing the packing density and motility of endogenous cells. This C57BL/6 slice was stained with 400 μg/ml FITC in PBS plus 5% FBS for 6 min and subsequently put on the microscope stage for perfusion and imaging. An excitation wavelength of 870 nm was used and emission at 535 nm was collected from a plane 40 μm in from the cut surface. Frames were collected every 10 s for a total of 15 min. Note the movement of brightly stained stromal cell processes as thymocytes migrate through the tissue. Frame rate: 10 fps (100x time compression). Scale bar = 10 μm. (MOV 3863 kb)

Supplementary Video 2

Z series through an FM4-64 labeled slice (red) reseeded with calcein-AM-loaded thymocytes (green). Thymocytes were labeled with 1 μM calcein-AM (Molecular Probes) for 30 min at room temperature and allowed to migrate into slices for 3 hr. Slices were then stained with 10 μM FM4-64 (Molecular Probes) for 20 min at room temperature. 870 nm excitation was used, and emission was collected through 535 bandpass and 580 LP filters. Sections are 2 μm apart starting at the cut surface and ending 70 μm into the slice. Scale bar = 10 μm. (MOV 2211 kb)

Supplementary Video 3

Motility of thymocytes in a non-selecting environment. 5C.C7 thymocytes from a B6 mouse were loaded with indo-PE3 and allowed to migrate into a B10 slice. 4 images at 5-μm intervals spanning a distance of 15-30 μm into the slice were collected every 35 s for a total of 42 min. For each time point of the movie, each image plane (675SP emission image) was encoded with a different color (red, green, blue, gray from superficial to deep), and superimposed. The capsule is seen near the top. Frame rate: 10 fps (350x time compression). Scale bar = 10 μm. (MOV 2665 kb)

Supplementary Video 4

[Ca2+]i and motility of 5C.C7/B6 thymocytes in a B6 slice (non-selecting conditions). Frames were collected every 10 s for 19 min. For Videos 4-7, the 675SP/390 indo-PE3 emission ratio is displayed using a rainbow spectrum lookup table ranging from blue (low [Ca2+]i) to red (high [Ca2+]i). In this video, blue corresponds to [Ca2+]i ≅ 75, yellow to 700, and red to 3000 nM. Frame rate: 8 fps (80x time compression). Scale bar = 10 μm. (MOV 728 kb)

Supplementary Video 5

[Ca2+]i and motility of 5C.C7/B6 thymocytes in a B10.BR slice (positively selecting conditions). Blue corresponds to [Ca2+]i ≅ 75, yellow to 700, and red to 3000 nM. Frames were collected every 10 s for 62 min. Frame rate: 8 fps (80x time compression). Scale bar = 10 μm. (MOV 2970 kb)

Supplementary Video 6

Elevation of [Ca2+]i reversibly immobilizes thymocytes in the thymic slice. The movie shows the indo-PE3 emission ratio of 5C.C7/B6 thymocytes in a B10.BR slice pretreated with thapsigargin (see Methods). The bath solution was changed from 1.25 mM Ca2+ to 0 Ca2+ and back to 1.25 mM Ca2+ as indicated. Blue corresponds to [Ca2+]i ≅ 250 and yellow to 2000 nM. Frames were collected every 10 s for 53 min. Frame rate: 30 fps (300x time compression). Scale bar = 10 μm. (MOV 796 kb)

Supplementary Video 7

High extracellular [K+] reduces [Ca2+]i and relieves motility arrest. This movie shows B6 thymocytes in a B6 slice pretreated with thapsigargin as in Movie 8. The bath solution is changed from 5 to 132 mM K+ in the constant presence of 0.4 mM Ca2+. Blue corresponds to [Ca2+]i ≅ 250, yellow to 1500 nM. Frames were collected every 20 s for 29 min. Frame rate: 15 fps (300x time compression). Scale bar = 10 μm. (MOV 293 kb)

Supplementary Video 8

Effect of terminating Ca2+ oscillations on cell motility. 5C.C7/B6 thymocytes loaded with indo-PE3 were imaged in a B10.BR (positively selecting) slice. 4 images at 7-μm intervals spanning a distance of 13-34 μm into the slice were collected every 38 s for a total of 41 min. Cell depth is displayed as described for Supplementary Video 3. A stationary cell undergoing Ca2+ oscillations is marked by a white square at its center (cell shown in Fig. 5 in the main text). A 10-μm-radius white circle is centered on the cell's starting position. After the bath solution is changed from 1.25 mM Ca2+ to 0 Ca2+, the cell becomes motile and leaves the circle. Frame rate: 8 fps (304x time compression). Scale bar = 10 μm. (MOV 1050 kb)

Supplementary Note (PDF 32 kb)

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Bhakta, N., Oh, D. & Lewis, R. Calcium oscillations regulate thymocyte motility during positive selection in the three-dimensional thymic environment. Nat Immunol 6, 143–151 (2005). https://doi.org/10.1038/ni1161

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