Visualizing the Mechanisms of Integrin-Mediated Lymphocyte Costimulation

Stephen C. Bunnell, Ph.D.

Tufts University

Funded in June, 2006: $150000 for 3 years
LAY SUMMARY . ABSTRACT . HYPOTHESIS . SELECTED PUBLICATIONS .

LAY SUMMARY

back to top

Identifying Immune System Interactions That are Involved in Autoimmune Diseases

Researchers will use cellular imaging to identify interactions of immune system proteins that are implicated in the development of autoimmune and inflammatory diseases.  The findings could lead to the development of drugs that selectively block these detrimental interactions.

Autoimmune diseases occur when immune cells, usually T cells, mistake the body’s own cells as foreign and attack them.   Implicated in this malfunction of T cells are certain proteins, called “integrins.”  “VLA-4” is an integrin that plays a particularly important role in the immune system.  It helps circulating immune T cells learn the location of inflamed tissues that have been attacked by invaders. In autoimmune diseases, however, the T cells attack the body’s own tissues and produce inflammation.  The VLA-4 integrins then signal more T cells to come to the inflamed site, perpetuating the autoimmune attack.  While strategies that block VLA-4 integrins from signaling T cells might prevent this situation, those strategies also would preclude effective immune responses by T cells to true invaders. 

Instead, the researchers will identify the specific interactions between clusters of VLA-4 integrins and T cell receptors that enable the integrins to influence these signaling clusters and promote T cell activation. These particular proteins may be targets for selectively inhibiting inappropriate T cell activation in otherwise healthy tissues.  The investigators will see if they can identify these specific proteins using high-resolution fluorescence imaging. Once these proteins are identified, drugs might be developed that selectively inhibit these proteins from inappropriately activating T cells to attack healthy tissues.  

Significance:  This research may lead to new types of therapies for preventing autoimmune attacks by selectively blocking signaling that inappropriately stimulates T cell responses against healthy tissues.

ABSTRACT

back to top

Visualizing the Mechanisms of Integrin-Mediated Lymphocyte Costimulation

Integrins influence leukocyte trafficking and sensitize lymphocytes to antigen. Integrin-blocking strategies profoundly inhibit immune responses, but the mechanisms of this inhibition are not well understood. We hypothesize that integrins potentiate T cell activation by immobilizing and stabilizing the signaling clusters induced by the T cell antigen receptor (TCR). To test this hypothesis we have developed dynamic, multicolor, and high-resolution fluorescence imaging techniques that allow us to directly visualize macromolecular assemblies of signaling molecules. Using these imaging techniques in conjunction with assays for T cell function, we have shown that small '\"signaling clusters" containing the crucial adaptor protein, SLP 76, play a decisive role in T cell activation. These clusters arise in association with TCR "microclusters," and persist for several minutes as they move towards the center of the stimulatory contact surface. By applying our imaging tools to T cells costimulated with ligands of the integrin VLA-4, we have shown that integrin-dependent signals dramatically alter the behavior of these signaling clusters.

In this proposal, we will establish how integrin-mediated costimulation affects TCR-induced signaling clusters, and will determine whether these effects are responsible for T cell costimulation. Our working hypothesis is that TCR and VLA-4-associated signaling proteins interact directly to integrate the signals from these receptors. First, we will identify the molecules that link VLA-4 to the TCR, by testing fluorescent chimeras for co localization with these receptors. Direct, nanometer-scale interactions will be confirmed by assessing fluorescence resonance energy transfer (FRET) within our signaling clusters and within immune synapses. Second, we will establish which of these co-localized signaling molecules are essential for VLA-4 mediated costimulation, using dominant-negative and RNAi-mediated knock-down strategies. Third, we will perform mutational analyses to determine how these essential signaling proteins exert their costimulatory functions downstream of VLA-4.

By identifying the proteins that enable VLA-4 to influence TCR-induced signaling clusters and to promote T cell activation, our studies will allow us to understand the costimulatory mechanisms that contribute to autoimmune and inflammatory diseases, such as multiple sclerosis. This knowledge may lead to the development of drugs that selectively block specific aspects of VLA-4 function, and therefore pose reduced risks in clinical settings.

HYPOTHESIS

back to top

Hypothesis:
Integrins are cellular adhesion molecules that play important roles in lymphocyte migration and activation. Strikingly, the co-engagement of integrins with the T cell antigen receptor (TCR) enables T cells to respond to small amounts of antigen. However, the mechanisms by which integrins sensitize T lymphocytes to antigens are not well understood. Previously, we have shown that the engagement of the TCR elicits the formation of macromolecular signaling clusters. The persistence of these membrane-associated structures correlates strongly with effective T cell activation. We propose that integrins increase the stability of these antigen-induced signaling clusters, and thereby lower the threshold for T cell activation. In this manner, integrins may contribute to the recognition of self-antigens in autoimmune diseases such as multiple sclerosis.

Goals:
The goal of these studies is to determine how integrins facilitate T cell activation. We have proposed that integrins promote T cell activation by directly influencing TCR-proximal signaling clusters. Our preliminary data indicate that costimulatory signals delivered through integrins stabilize these clusters.

1. Employing dynamic imaging techniques and fluorescence resonance energy transfer (FRET), we will identify integrin-dependent signaling proteins that interact with TCR-induced signaling clusters, and may therefore influence their stability and efficacy.

2. Using RNA interference, we will identify integrin-dependent signaling proteins that are required for cluster stabilization and enhanced T cell activation.

3. By reconstituting cells with mutant proteins, we will identify specific protein-protein interactions that contribute to integrin-induced cluster stabilization and T cell costimulation.

These studies will identify specific protein-protein interactions that facilitate the recognition of antigen following integrin ligation. Compounds perturbing these protein-protein interactions may prove useful in the treatment of autoimmune disorders.

Methods:

  • Assays for T cell activation.
  • Fluorescent labeling of lymphocyte signaling proteins.
  • Multicolor dynamic cellular imaging.
  • FRET assays to establish molecular proximity.
  • Automated particle tracking and movement analysis.
  • Dynamic visualization of the immune synapses formed by living T cells.
  • RNA interference.

SELECTED PUBLICATIONS

back to top

Nguyen K., Sylvain N., and Bunnell S.C. T cell costimulation via the integrin VLA-4 inhibits the actin-dependent centralization of signaling microclusters containing the adaptor SLP-76.  Immunity. 2008 Jun;28(6):810-21.

Bunnell S.C., Singer A.L., Hong D.I., Jacque B.H., Jordan M.S., Seminario M.C., Barr V.A., Koretzky G.A., and Samelson L.E.  The persistence of cooperatively stabilized signaling clusters drives T cell activation.  Mol Cell Biol. 2006 Oct;26(19):7155-66.

Zipfel P.A., Bunnell S.C., Witherow D.S., Gu J.J., Chislock E.M., Ring C., and Pendergast A.M.  Role for the Abi/wave protein complex in T cell receptor-mediated proliferation and cytoskeletal remodeling. Curr Biol. 2006 Jan 10;16(1):35-46.

Barda-Saad M., Braiman A., Titerence R., Bunnell S.C., Barr V.A., and Samelson L.E.  Dynamic molecular interactions linking the T cell antigen receptor to the actin cytoskeleton. Nat Immunol. 2005 Jan;6(1):80-9.

Singer A.L., Bunnell S.C., Obstfeld A.E., Jordan M.S., Wu J.N., Myung P.S., Samelson L.E., and Koretzky G.A.  Roles of the proline-rich domain in SLP-76 subcellular localization and T cell function.  J Biol Chem. 2004 Apr 9;279(15):15481-90.

Bunnell S.C., Barr V.A., Fuller C.L., and Samelson L.E.  High-resolution multicolor imaging of dynamic signaling complexes in T cells stimulated by planar substrates.  Sci STKE. 2003 Apr 8;2003(177):PL8.

Bunnell S.C., Hong D.I., Kardon J.R., Yamazaki T., McGlade C.J., Barr V.A., and Samelson L.E.  T cell receptor ligation induces the formation of dynamically regulated signaling assemblies.  J Cell Biol. 2002 Sep 30;158(7):1263-75.