Visualizing the Mechanisms of Integrin-Mediated Lymphocyte Costimulation

Lay Summary

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

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.