Molecular interactions in T cell development and activation

Fluorescence resonance energy transfer (FRET) imaging of TCR and coreceptor molecular interactions in T cell activation.

Expression of T cell surface proteins as fusions with variants of the green fluorescent protein has enabled us to investigate molecular movement and intermolecular interactions during T cell activation, using live cell fluorescence deconvolution microscopy and FRET microscopy. FRET (e.g. between cyan and yellow fluorescent proteins: CFP, YFP) is effective at <10nm and is therefore ideal to investigate interactions between proteins in living cells. The coreceptor CD8 and the T cell receptor (TCR) signal transducing protein CD3ζ are recruited to the "immunological synapse" where they interact, as shown by FRET, when antigen is presented to the T cell. No FRET occurs when weaker (e.g. TCR antagonist) ligands are used. TCR antagonist ligands inhibit the FRET between CD3ζ-CFP and CD8-YFP induced by agonist ligands, whereas non-stimulatory ligands do not. The antagonists are thought to inhibit both FRET and T cell activation by forming short-lived complexes with MHC-peptide that are kinetically unable to engage the coreceptor. We have compared immunological synapse formation and the TCR-coreceptor interaction in a system where the affinity of the TCR-MHCp interaction is known. The strength of agonists is more closely related to the speed at which they recruit TCR to the synapse and start to induce FRET than to the affinity of the TCR-MHCp interaction. The coreceptor CD4 has been shown by crystallography to exist as a dimer, yet biochemical or biological evidence for this has been weak. We have therefore made CD4 chimeras with CFP and YFP to probe dimerization on the cell surface. We have demonstrated a weak constitutive association, greatly increased at the immunological synapse during antigen-recognition.

Two-photon microscopy of TCR and coreceptor movement in living tissue.

Two-photon microscopy allows visualization of cells deep in tissue, so that T cells interacting with antigen presenting cells during an immune response, or thymocytes interacting with thymic stromal cells during development, can be viewed. We have produced transgenic mice expressing the fluorescent chimeric CD8 and CD3ζ molecules, as well as transgenic TCR, and are collaborating with Dr. Michael Cahalan (UCI) to use two photon microscopy to investigate movement and interaction of TCR and coreceptors. The cell surface molecules can be seen to form synapses within the tissue and we will now investigate how the synapses form during the initiation of an immune response, during responses to a solid tumor and during thymocyte development.

The role of PKCη in the immunological synapse.

We found that the protein kinase Cη isoform is upregulated after TCR ligation in developing thymocytes, and likewise in natural positive selection. Of the PKC isoforms, only PKCθ has been shown to have a special role in T cells, where it is recruited to the immunological synapse during antigen-recognition. Because PKCθ-deficient mice have normal thymic selection, this suggested that PKCη could be replacing PKCθ in the developing thymocytes. We have now 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 may function in place of PKCθ.

Gene expression in early T cell differentiation.

We have identified a novel protein with strongly regulated expression during thymocyte differentiation. It is expressed during the stages of TCR gene rearrangement and interacts with the cell-cycle and DNA damage-repair enzyme ATM, as well as with PLCγ1, important in T cell signaling. We are now using siRNA, gene ablation and transgenic techniques to investigate its possible role 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 two α-chain proteins. However, expression of two 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 is post-translationally regulated. We find that the positively selected αβ combination remains on the surface when stimulated, whereas the other αβ combination is endocytosed. This is controlled by TCR signaling involving the kinase Lck and the ubiquitin ligase Cbl which controls degradation of endocytosed TCRs. We are starting to use FRET imaging to analyze ubiquitination of TCR after endocytosis, using FRET between ubiquitin monomers and TCR subunits labeled with fluorescent proteins.