Conference Highlight

Immunology and Cell Biology (1999) 77, 186–187; doi:10.1046/j.1440-1711.1999.00813.x

Visualizing lymphocyte recognition

Christoph Wülfing1, Yueh-Hsiu Chien1 and Mark M Davis1

1Howard Hughes Medical Institute and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA

Correspondence: Dr M Davis, Howard Hughes Medical Institute, Department of Microbiology and Immunology, Stanford University School of Medicine, 279 Campus Drive, Stanford, CA 94305-5323, USA. Email: mdavis@cmgm.stanford.edu

Received 12 January 1999; Accepted 12 January 1999.

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Abstract

Studies of T cell recognition have entered new territory now that some of the basic issues of genetics, biochemistry and structure have been addressed, at least in outline form. In the present work, the focus is on a new aspect of T cell recognition that goes beyond classical biochemistry to ask, 'how do TCR and other cell surface molecules cooperate to initiate and control recognition?'

Keywords:

CDR3, costimulation, recognition, T cell receptors, video microscopy

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Introduction

There is now extensive information on the biochemistry of T cell receptor binding to peptide/MHC complexes and this can be used to predict, albeit imperfectly, the likely biological consequences (activation, antagonism etc.).1 Similar information has been developed on other molecules and their ligands that are involved in T cell recognition (LFA-1/ ICAM-1, CD2/CD48, etc.), but what is lacking is the technical means to see what sort of 'molecular choreography' these proteins follow in real life; that is, what is happening on the cell surface while a particular peptide/MHC on another cell is being recognized. In order to address this need, we have made fusions of various cell surface proteins with green fluorescent protein (GFP) and expressed them in B cells and Chinese hamster ovary (CHO) cells. We then perform video microscopy in which we simultaneously monitor T cell calcium levels and the movements of GFP-labelled molecules (ICAM-1, CD48, I-Ek). This has given us the means to investigate the molecular biology of T cell recognition in situ, and a number of interesting observations have emerged concerning signal amplification and the nature of costimulation. In addition, the elegant work of Monks et al. has recently shown that the organization of cell surface molecules at the T cell:APC interface is not random, but has a distinct order in cell couples that is likely to result in successful activation.2

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Results and discussion

Green fluorescent protein fusions with B cell surface molecules were made by adding a linker of 15 amino acids followed by a GFP mutant to the C-termini of ICAM-1, I-Ek, and CD48 as described.3 They were then transfected into the B cell lymphoma CH27 or into CHO cells. Stable cell clones that expressed moderate amounts of GFP were selected and used for further analysis (as illustrated with ICAM-1 by Wülfing et al.3).

Interestingly, we find that all three of these molecules on B cells rapidly accumulate at the interface with the T cell, beginning ~ 20 s after the first rise in intracellular calcium. This occurs with B cells as APC, not with CHO cells, and thus seems to be a property of 'professional' APC. At least with respect to ICAM-1, this increased density acts to stabilize the rise in intracellular calcium levels, thus contributing to the B cell's effectiveness as an APC.3

This clustering phenomenon appears to be driven by the T cell, because blocking actin polymerization with cytocholasin D in the T cell, but not the B cell, disrupts this effect. In order to assay cortical cytoskeletal movements on the T cell more directly, we have attached large (2.8–4.5 mum) beads to the T cells prior to engagement with APC. We have done this in three different ways: either by coupling beads with antibodies to ICAM-1; or using strepavidin beads with surface biotinylated, or biotin-lipid-incorporated, T cells. In all cases, the beads move from the front to the back of the T cell when it is migrating, as seen with other mobile cells, consistent with the movement of the cortical actin cytoskeleton from the front of a moving cell to the back. Interestingly, when T cells with these beads attached recognize a B cell APC (or dendritic cells), ~ 4 min after the first calcium flux the beads begin moving towards the interface. This indicates that there is a major movement of cell-surface and other cortical cytoskeleton-linked molecules towards the interface, and it would explain why ligands for these molecules on B cells would be clustering there as well. This novel cytoskeletal movement is closely linked to costimulatory signals because it does not occur when CHO cells are the APC, and can be blocked on B cells with antibodies to either B7 or ICAM-1.4 That costimulatory signals (together with TCR engagement) are sufficient for this effect can be seen from the fact that CHO cells transfected with ICAM-1 or B7 partially reconstitute bead movement, and also that CHO transfectants expressing ICAM-1 together with anti-CD28 antibodies added to the culture, restore this effect to B cell levels.4

Thus we have identified a novel cytoskeletal movement that is dependent on costimulatory signals. While many efforts to delineate the mechanism of costimulation have focused on finding a nuclear factor or factors that would enhance the changes in gene expression initiated by TCR engagement, these efforts have thus far not been successful (as reviewed by Shaw and Dustin5). The data summarized here and presented in more detail by Wülfing and Davis suggest an alternative mechanism, which is that costimulatory signals trigger a particular cytoskeletal movement, probably dependent on myosin motors, that results in a continuous delivery of cytoskeleton-linked membrane and associated molecules to the T cell–APC interface.4 This would, at the least, replace molecules that have been internalized and would also probably elevate the concentration of these molecules in this location in the 1 h or so that the T cell needs to finalize its commitment to activation. It is clear from the work of Cornall et al. and theoretical models that even modest differences in concentration (two-fold) can have profound effects on such decisions.6, 7, 8

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Future work

Currently we are working to extend the findings summarized in the previous section using GFP-labelled molecules expressed on T cells and a new video instrument that can provide 3-D fluorescent images. Ultimately we hope to provide a complete description of the dynamics and causal relationships between the major surface molecules and how this relates to previously described signalling cascades.

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References

  1. Davis MM, Boniface JJ, Reich Z et al. Ligand recognition by alphabeta T cell receptors. Ann. Rev. Immunol. 1998; 16: 523–44. | Article | ChemPort |
  2. Monks CR, Freiberg BA, Kupfer H, Sciaky N, Kupfer A. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 1998; 395: 82–6. | Article | PubMed | ISI | ChemPort |
  3. Wülfing C, Sjaastad MD, Davis MM. Visualizing the dynamics of T cell activation: ICAM-1 migrates rapidly to the T cell:B cell interface and acts to sustain calcium levels. Proc. Natl Acad. Sci. USA 1998; 95: 6302–7. | Article | PubMed | ISI | ChemPort |
  4. Wülfing C, Davis MM. A receptor/cytoskeletal movement triggered by co-stimulation during T cell activation. Science 1998; 282: 2266–9. | Article | PubMed | ISI | ChemPort |
  5. Shaw AS, Dustin M. Making the T cell receptor go the distance: A topological view of T cell activation. Immunity 1997; 6: 361–9. | Article | PubMed | ISI | ChemPort |
  6. Cornall RJ, Cyster JG, Hibbs ML et al. Polygenic autoimmune traits: Lyn, CD22, and SHP-1 are limiting elements of a biochemical pathway regulating BCR signaling and selection. Immunity 1998; 8: 497–508. | Article | PubMed | ISI | ChemPort |
  7. McKeithan TW. Kinetic proofreading in T-cell receptor signal transduction. Proc. Natl Acad. Sci. USA 1995; 92: 5042–6. | Article | PubMed | ChemPort |
  8. Rabinowitz JD, Beeson C, Lyons DS, Davis MM, McConnell HM. Kinetic discrimination in T cell activation. Proc. Natl Acad. Sci. USA 1996; 93: 1401–5. | Article | PubMed | ChemPort |
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Acknowledgements

We thank the Howard Hughes Medical Institute and the NIH for grant support.