Understanding the T Cell Receptor Signaling Complex
T cells are integral to the immune system, and their sensitivity to activation is critical to a healthy immune response. If T cells are too sensitive, allergies and more severe autoimmune diseases, such as multiple sclerosis and type 1 diabetes, can occur. If T cells are not sensitive enough, the immune system malfunctions and severe infection can result. Bunnell has created a technological tool kit to correlate cell physiology with real-time observations of proteins in living T cells.
T cell activation begins upon contact with an antigen-presenting cell, which presents a fragment of a foreign protein, an antigen, to the receptor protein on the T cell. The antigen-presenting cell also presents the T cell with surface proteins — co-stimulatory molecules that tell the T cell the circumstances under which the antigen was contacted. “It might say something like ‘I ate this during an inflammatory response,’ which means ‘Red flag! Look at me!’” says Bunnell. In that case, a strong immune response is needed. Or it might say something like “I was walking around the body the other day and I came across this foreign object, but there was no inflammation, so it’s probably not a big deal.” In that case, either no response or a weak response is appropriate. A T cell has to understand the antigen-presenting cell’s message to be able to initiate the appropriate immune response. In this way, cell surface interactions in effect “tune” the immune response.
T cells also have cell surface determinants. One group, the integrins, bind to their counterparts, the integrin ligands, on antigen-presenting cells, sticking the two cells together so they can communicate more easily. Integrins also transmit signals into the T cell. Bunnell is investigating how these integrins influence the activation process. “In a T cell, having the integrin around changes the sensitivity of the interaction between antigen and cell by two orders of magnitude,” says Bunnell. “We want to know how that works.” Bunnell has data suggesting that integrin binding is an important factor in the creation of a signaling complex that triggers T cell activation. This complex is not present in quiescent T cells but can be formed by signaling and cytoskeletal proteins in response to antigen recognition. If the signaling complex must be assembled to activate a T cell, anything that influences the formation of that complex is going to affect the ability of T cells to become activated. The signaling complex, and the mechanisms that allow integrins to regulate its formation, may be prime candidates for drug intervention both for immunodeficiencies and autoimmune diseases.
Bunnell uses advanced microscopy to see how intermediates of T cell signaling move during activation. A specialized confocal microscope in his laboratory takes movies of T cells in action, tracking differentially labeled fluorescent tags for proteins of interest. Bunnell explains: "We trigger the T cell to become activated, then we ask: 'What changes occur inside that cell? What changes fail to occur if the cells are missing a specific protein or lack the capacity to generate an immune response?'" The research group is using Jurkat cells (T cell lines derived from a human leukemia) to take advantage of genetic models, and primary human cells to measure properties that have direct clinical relevance.
Signaling intermediates that Bunnell investigates include ZAP-70, a T cell receptor–associated tyrosine kinase, and the signaling adapters LAT, Grb2, Gads, and SLP-76. The key molecules combine in specific stoichiometries (ratios) and specific spatial relationships to activate T cells. “If you want to make the cell move forward, you need something that distinguishes frontward from backward,” says Bunnell. “If you want the cell to respond by secreting some effector molecule in a specific direction, it needs to be able to differentiate place. The molecules that integrate the signals interact as scaffolds and as functional molecules with enzymatic activities that are facilitated by their proximity to one another. So essentially, by bringing everything together, you make the transmission of information more efficient.”
In addition to expanding his research, Bunnell is offering expertise in imaging techniques as a member of the Study Center for the Immunogenetics of Infectious Disease (SCIID), a new organization at Tufts that focuses on the interplay among immunological and microbiological aspects of infectious disease. Bunnell plans to collaborate with Mercio Perrin on research into how trypanosomes, the parasites that cause Chagas disease, get into neurons. “The process seems to be analogous to T cell signaling,” says Bunnell. “We are developing a collaboration where we will use fluorescent methods to look at parasites getting into neurons and watch how they move inside neurons.”
In related research Joan Mecsas, an investigator in the Department of Molecular Biology and Microbiology, studies how the pathogenic bacterium Yersinia pseudotuberculosis evades the host immune response. “What this clever little bug does is it essentially builds a little biological syringe and it stuffs proteins into the opposing cells of the immune system to perturb their function,” says Bunnell. The injected proteins, known as Yops, target some components of the T cell signaling complex that Bunnell studies. The Mecsas group uses model systems that permit them to manipulate the pathogen or the host and get at both sides of the interaction. Says Bunnell, “we can take infected cells and look at them in our imaging system and ask ‘What did those proteins the bugs injected do to the triggering structure?’ We know biochemically that Yops should have some impact, but we don’t know what, when, or how. So whatever the pathogen hits to turn off the immune response is something you might want a drug to turn off, to suppress an excessive immune response.”
To see movies of T cells in action, go to http://www.tufts.edu/sackler/immunology/bunnell/research.html.