Scientific Report 2005

Immunology

Molecular Interactions in T-Cell Development and Activation

N.R.J. Gascoigne, J. Ampudia, G. Fu, K. Holmberg, H.-C. Hung, H.-O. Kim, C. Lotz, A. Munshi, N. Niederberger, G. Sternik, S. Vallee, P. Yachi, M.A. Zal, T. Zal, M. Gronski,* P. Ohashi,* M.Y. Lin,** S.M. Hedrick,** N. Bosco,***
R. Ceredig,*** M. Cahalan****

* Ontario Cancer Institute, Toronto, Ontario
** University of California, San Diego, California
*** U548 INSERM, Grenoble, France
**** University of California, Irvine, California

Imaging Molecular Interactions in Living Cells in T-Cell Activation

We used live-cell fluorescence deconvolution microscopy and fluorescence resonance energy transfer (FRET) microscopy to investigate molecular movement and intermolecular interactions during T-cell activation. FRET between cyan and yellow fluorescent proteins is effective at ranges of less than 10 nm and is therefore ideal to investigate interactions between proteins in living cells.

Using FRET, we showed that the coreceptor CD8 and the T-cell receptor (TCR) signal-transducing protein CD3ζ are recruited to the “immunologic synapse,” where they interact when antigen is presented to the T cell. No FRET occurs when weaker (e.g., TCR antagonist) ligands are used. We compared formation of the immunologic synapse and the TCR-coreceptor interaction in a system in which the affinity of the interaction between TCRs and MHC-peptides is known. We found that the strength of weak agonists is more closely related to the speed at which they recruit TCRs to the synapse and start to induce FRET than it is to the affinity of the interactions between TCRs and MHC-peptides.

The induction of FRET appears to explain why some agonists are stronger or weaker than would be predicted on the basis of their affinities. By controlling the level of antigenic MHC-peptide complexes presented to T cells, in the presence or absence of natural endogenous nonstimulatory MHC-peptides, we found that the endogenous complexes aided in the recognition of the antigenic complexes. The interaction between CD8 and the endogenous MHC-peptide improves the TCR recognition of the antigenic MHC-peptide, including the ability to associate with CD8. This surprising finding suggests how T cells can respond to small amounts of antigens in a “sea” of nonstimulatory MHC-peptides.

In a series of experiments in collaboration with P. Ohashi, Ontario Cancer Institute, Toronto, Ontario, we found that the strength of TCR interaction with MHC peptide ligands alters the ability of an autoantigen to induce diabetes, so that a reduced affinity results in a reduced incidence of disease. We also used fluorescent tetrameric class I MHC molecules to measure binding of TCR-MHC-peptide complexes on T cells to compare binding of negative- and positive-selecting ligands.

Although crystallographic studies indicate that the coreceptor CD4 exists as a dimer, biochemical or biological evidence for this finding has been weak. We made CD4 chimeras with cyan and yellow fluorescent proteins to probe dimerization on the cell surface. We found a weak constitutive association, which was greatly increased at the immunologic synapse during antigen recognition.

Two-Photon Microscopy of TCR and Coreceptor Movement in Living Tissues

Two-photon microscopy allows visualization of cells deep in tissues and thus allows observation of T cells interacting with antigen-presenting cells during an immune response or of thymocytes interacting with thymic stromal cells during development. We produced transgenic mice that express the fluorescent chimeric CD8 and CD3ζ molecules, as well as transgenic TCRs, and are collaborating with M. Cahalan, University of California, Irvine, to use 2-photon microscopy to investigate movement and interaction of TCRs and coreceptors. We found that the cell-surface molecules form synapses within tissues, and we are investigating how the synapses form during the initiation of an immune response, during responses to a solid tumor, and during thymocyte development.

Role of the Protein Kinase η Isoform in the Immunologic Synapse

We found that the η isoform of protein kinase C (PKCη) is upregulated after TCR ligation in developing thymocytes and in natural positive selection. Of the PKC isoforms, only PKCθ is known to have a special role in T cells, where it is recruited to the immunologic synapse during antigen recognition. The finding that mice deficient in PKCθ have normal thymic selection suggested that PKCη could be replacing PKCθ in the developing thymocytes. We found that PKCη is also naturally recruited to the synapse in mature thymocytes and T cells. In the absence of PKCθ, PKCη is expressed at an earlier stage of thymocyte development, where it functions in place of PKCθ.

Gene Expression in Early T-Cell Differentiation

We identified a novel protein with strongly regulated expression during thymocyte differentiation. The protein is expressed during the stages of TCR gene rearrangement. It interacts with the cell-cycle and DNA damage–repair enzyme ATM and with phospholipase Cγ1, which is important in T-cell signaling. We are now using small interfering RNA, gene ablation, and transgenic techniques to investigate the possible role of the protein in development and T-cell signaling.

TCR Endocytosis, Recycling, and Ubiquitination

Because allelic exclusion of the TCR α-chain is poor, many mature T cells express 2 α-chain proteins. However, expression of 2 α-chains on the cell surface is quite rare. We previously showed that functional allelic exclusion is attained in the thymus at the start of positive selection and that this exclusion is posttranslationally regulated. We found that the positively selected αβ combination remains on the surface when the TCR is stimulated, whereas the other αβ combination is endocytosed. Endocytosis and allelic exclusion are controlled by TCR signaling involving the kinase Lck and the ubiquitin ligase Cbl, which controls degradation of endocytosed TCRs. To analyze ubiquitination of TCRs after endocytosis, we are using FRET between ubiquitin monomers and TCR subunits labeled with fluorescent proteins. In collaboration with R. Ceredig, INSERM, Grenoble, France, we examined the TCR α-chain repertoire of specialized CD25⁺CD4⁺ regulatory T cells. We found that the repertoire is as diverse as that of mainstream CD4⁺ T cells.

Publications

Bosco, N., Hung, H.-C., Pasqual, N., Jouvin-Marche, E., Marche, P.N., Gascoigne, N.R.J., Ceredig, R. Role of the T cell receptor α-chain in the development and phenotype of naturally arising CD25⁺CD4⁺ T cells. Mol. Immunol., in press.

Gronski, M.A., Boulter, J.M., Moskophidis, D., Nguyen, L.T., Holmberg, K., Elford, A.R., Deenick, E.K., Kim, H.O., Penninger, J.M., Odermatt, B., Gallimore, A., Gascoigne, N.R.J., Ohashi, P.S. TCR affinity and negative regulation limit autoimmunity. Nat. Med. 10:1234, 2004.

Lin, M.Y., Zal, T., Ch’en, I.L., Gascoigne, N.R.J., Hedrick, S.M. A pivotal role for the multifunctional calcium/calmodulin-dependent protein kinase II in T cells: from activation to unresponsiveness. J. Immunol. 174:5583, 2005.

Niederberger, N., Buehler, L.K., Ampudia, J., Gascoigne, N.R.J. Thymocyte stimulation by anti-TCRβ, but not by anti-TCRα, leads to induction of developmental transcription program. J. Leukoc. Biol. 77:830, 2005.

Yachi, P.P., Ampudia, J., Gascoigne, N.R.J., Zal, T. Nonstimulatory peptides contribute to antigen-induced CD8-T cell receptor interaction at the immunological synapse. Nat. Immunol. 6:785, 2005.