T-Cell Signalling

(Click on figures to enlarge and view figure legends)

 

In the adaptive immune response, the T-cell antigen receptor (TCR) on the T-cell surface can “interrogate” the major histocompatibility complex (MHC) protein on the surface of the APC. If the TCR binds peptide-bound MHC (pMHC) of the right complementarity, then the interaction results in tyrosine phosphorylation of the TCR (termed “triggering”) and the transmission of intracellular signals that activate the T cell.  We have been interested in understanding the biochemical and biophysical basis of this signaling response.  While we are not a dedicated immunology lab, we have sought to apply our strengths in biochemical and microscopy to this problem.

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A graduate student Adam Douglass (now a new faculty at U. Utah) first began to apply TIRF and confocal microscopy techniques to image signaling molecules in Jurkat T cells, including at the single molecule level (Douglass et al., 2005).  The study was initially motivated to look for lipid rafts although we did not find evidence for them (at our temporal, spatial resolution) in this study.  However, the work did reveal the formation of clusters of signaling proteins in the T cell membrane and that work suggested that these clusters were held together by multi-valent protein-protein interactions.  In addition, single molecule work suggested that these signaling clusters could trap some molecules and acted as diffusion barriers and excluded other molecules.

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The above work was performed with anti-TCR placed on a glass surface.  We then began to look at a more dynamic situation where ligands (ICAM-1, CD58, anti-TCR) were placed on planar lipid bilayers and could interact with Jurkat T cells (Kaisuka et al, 2007; Kaizuka et al, 2009).  This work showed the formation of signaling clusters, but also showed that TCR co-clustered with the co-receptor CD2 but segregated away from the adhesion molecule LFA-1.  These clusters could interact with actin and centripetal actin movement carried these clusters towards the center of the cell, resulting in structure like the immunological synapse described previously for native T cells.

In more recent microscopy studies, we have developed an imaging based FRET sensor for the TCR that can be used to examine its phosphorylation in living cells (Yudushkin and Vale, 2010).  Somewhat surprisingly, this study revealed considerable phosphorylation of the TCR in endosomes in addition to the plasma membrane.  We are continuing with follow up the role of the endosomal TCR in T cell signaling pathways (with Ivan Yudushkin, now in IMBA Vienna).

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More recently, we have turned to reconstitution and biochemical approaches for addressing the problem of TCR signaling. Reconstitution of a biological phenomenon with defined components is a very powerful means to dissect mechanism at the molecular level (as we have done with motility; see sections on molecular motors).  One approach we have used is reconstituting the minimal signaling apparatus in non-immune cells (HEK-293T) (James and Vale, 2012). The genes encoding the TCR and other proteins required for regulating its phosphorylation are either transiently or stably expressed in these cells. We use Western blotting and fluorescence microscopy to determine whether these reconstituted cells can recapitulate all the hallmarks of native TCR triggering when these cells interact with APCs. Since each protein can be introduced separately and is amenable to genetic engineering, this system has allowed us to test models of TCR triggering and the roles of individual proteins in a manner that is difficult to achieve with native T cells. This has revealed a physical mechanism for TCR triggering that differs from dimerization or conformational change models proposed for many cell surface receptors. We found that the binding energy of the TCR-pMHC interaction generates an exclusion force for membranes proteins with large and/or unligated extracellular domains. By having an inhibitory phosphatase activity linked to a transmembrane protein (CD45) that is subject to the exclusion force and an activating kinase linked to the inner leaflet of the membrane (Lck) that is not, the TCR-pMHC mediated exclusion shifts the kinase-phosphatase balance and thus triggers the TCR, as first suggested by the kinetic segregation model. Furthermore, receptor clustering through minimizing unfavorable bending of the plasma membrane seems to provide a simple and effective means to both cluster the ligated receptor and exclude other proteins. This model also appears to extend to other entirely artificial receptors, as well as chimeric antigen receptors that are currently being used in gene therapeutics for cancer treatment.

In a complementary approach, we are also attempting to reconstitute aspects of the TCR triggering pathway in vitro. The kinases, phosphatases and TCR form a complex signaling network that controls the phosphorylation state of the cytoplasmic tails of the TCR. By reconstituting the TCR/kinase/phosphatase signaling network into lipid bilayers, the network can be studied in a reduced, well-defined membrane system, where both concentrations and spatial relationships of these proteins can be readily manipulated. Using data generated from this very tractable system, we hope to build a complete model summarizing the behavior of the TCR/kinase/phosphatase network, and then directly test key hypotheses for TCR triggering.

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Results from a completely in vitro phosphorylation reaction.  Phosphorylation of the TCR zeta chain on liposomes was measured using a FRET assay in response to different concentrations of Lck kinase and CD45 phosphatase (also on liposome membranes). 

 

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See additional movies and images here.

References:

  • (pdf) - James, J.R., and Vale, R.D. (2012) Biophysical mechanism of T-cell receptor triggering in a reconsistuted system. Nature 487: 64-69.
  • (pdf) - Yudushkin, I.A., and Vale, R.D. (2010) Imaging T-cell receptor activation reveals accumulation of tyrosine-phosphorylated CD3{zeta} in the endosomal compartment. Proc Natl Acad Sci USA 107: 22128-22133
  • (pdf) - Yu, C.H., Wu, H.J., Kaizuka, Y., Vale, R.D. & Grove. J.T. (2010) Altered actin centripetal retrograde flow in physically restricted
    immunological synapses. PLoS One 5: e11878.
  • (pdf) - Kaizuka, Y., Douglass, A.D., Vardhana, S., Dustin, M.L. and Vale, R.D. (2009) The coreceptor CD2 uses plasma membrane microdomains to transduce signals in T cells. J Cell Biol 185: 521-534.
  • (pdf) - Douglass, A.D. and Vale, R.D. (2008) Single-molecule imaging of fluorescent proteins. Methods Cell Biol 85: 113-125.
  • (pdf) - Kaizuka, Y., Douglass, A.D., Varma, R., Dustin, M L. and Vale, R.D. (2007) Mechanisms for segregating T cell receptor and adhesion molecules during immunological synapse formation in Jurkat T cells. Proc Natl Acad Sci USA 104: 20296-20301.
  • (pdf) - Douglass, A.D. and Vale, R.D. (2005) Single-molecule microscopy reveals plasma membrane microdomains created by protein-protein networks that exclude or trap signaling molecules in T cells. Cell 121: 937-950.

 

updated 9/13/2012