By Emily Burke
Luc Teyton, associate professor at The Scripps Research Institute (TSRI), describes himself as an "old-fashioned biologist"and loves to discuss the cutting-edge technologies he uses in his work. But the phrase "old-fashioned" is not out of place: his motivation is common to most scientiststhe desire to answer the deceptively simple question, "How does it work?"
In Teyton's case, he uses the modern techniques of structural biology to piece together an understanding of basic immune function and dysfunction.
Ready for Battle
The human immune system is our defense force, evolved over the millennia to combat onslaughts of invading microorganisms. This sophisticated army is composed of two basic units: innate immunity, a non-specific defense mounted at the first sign of a foreign invader; and adaptive immunity, a defense mounted against a specific entity.
Immune cells that make up the core of the adaptive immune response are referred to as lymphocytes, and come in two flavors: B cells and T cells. Lymphocytes bear variable receptors on their cell surfaces to capture their target, an "antigen"broadly defined as any substance capable of being recognized by the adaptive immune system. The variability of these receptors arises from genetic recombination events that occur during lymphocyte development. The result is the ability to recognize a diverse set of antigens. Upon antigen recognition, the lymphocyte is activated and proliferates, creating many progeny cells specific for the activating antigen.
One key difference between B cells and T cells is the context in which each recognizes antigen. Once activated, B cells produce antibody molecules that target pathogens outside of the cell. The recognition of pathogens inside the cell is the domain of T-cells.
Stowaway pathogens usually unwittingly betray their presence inside a host cell. This occurs when protein components of an invading microorganism are degraded into peptide fragments in the cytoplasm of an infected cell. Specialized host proteins known as major histocompatibility complex (MHC) molecules bind peptide fragments and transport them to the cell surface.
At the cell surface, the antigen remains bound to the MHC molecule, and this foreign peptide-MHC duo activates T cells. An activated T cell will then proliferate, and its progeny will hunt down other host cells displaying the same foreign peptide, resulting in either cell death (by killer T cells) or further activation of B cells and macrophages (via helper T cells) that also seek to destroy the initiating antigen.
The Enemy Within
As a former clinician, Teyton has first-hand knowledge of the ravages of the immune system gone awry. He has treated patients with autoimmune diseases, in which the immune system targets its own antigen. For example, in Type 1 diabetes the immune system attacks insulin-producing cells in the pancreas. Without insulin, the body is unable to convert blood sugar to energy and patients suffer from weakness, hunger, weight-loss, excessive thirst, frequent urination, and sudden irritability. Left untreated, diabetes can be fatal. Type 1 diabetes typically strikes children and young adults. About 151,000 people less than 20 years of age have diabetes. Approximately one in every 400 to 500 children and adolescents has Type 1 diabetes, and needs several insulin injections a day or an insulin pump to survive.
While working as a rheumatologist in Paris, Teyton began to feel frustrated because "we really didn't understand what was going on with the patients." This frustration led him to pursue a career in research, where he was able to follow his desire to understand the basic mechanisms of autoimmunity.
From Bedside to Bench
Teyton first sought to tackle the question of autoimmunity by developing a better understanding of MHC class II function. MHC-II molecules present peptides derived from pathogens present in intracellular compartments, while MHC-I present peptides generated in the cytosol. Teyton was particularly interested in the role of the MHC-II-associated invariant chaina protein that had been ignored.
Like many cell-surface proteins, MHC molecules are actually glycoproteins, which means that they contain various sugar moieties that are added to the protein in a specialized sub-cellular compartment known as the endoplasmic reticulum (ER). Proper folding of the protein subunits also occurs in the ER.
By expressing recombinant MHC-II and invariant chain molecules and monitoring their movement through the ER, Teyton demonstrated that a role of the invariant chain was to associate with the MHC-II molecule as it is being processed. This association prevents binding of cellular peptides that are also present in the ER. The final role of the invariant chain is to direct the MHC-II molecule from the ER to acidified intracellular vesicles that contain peptides derived from pathogens. Once inside these acidic vesicles, the invariant chain is cleaved by various proteases, leaving the MHC-II molecule free to bind pathogen-derived peptides.
This intimate association with the regulation of peptide binding suggested that the invariant chain might play a role in autoimmune diseaseand prompted several questions: What does the invariant chain look like? How does its structure relate to this critical function - and is it a potential therapeutic target? Attempts to determine the invariant chain's structure were not successfulbut they whetted Teyton's appetite for the powerful techniques of structural biology as a means of answering basic immunological questions.
There is a great deal of genetic diversity among individuals in the type of MHC-encoding gene expressed. At the time, it was known that people carrying the MHC-II gene known as "HLA-DQ" were much more likely to have Type 1 diabetes than those who did not. This suggested a new question to Teyton: Do these genetic differences translate into differences in protein structure that might ultimately affect function, thereby leading to the disease state?
Obtaining the structure of MHC molecules had already proven to be an elusive goal for many labs, due to the difficulty in producing large amounts of soluble, properly paired MHC-II molecules. Teyton solved this problem by engineering soluble mouse MHC-II molecules whose pairing was forced by the addition of leucine zipper peptide dimers at their COOH-terminus. Subsequent proteolytic removal of the leucine zipper left stable MHC-II dimers that bound peptide in the same manner as the native molecule.
A Picture is Worth a Thousand Words
This technical innovation made it possible to obtain large amounts of soluble protein molecules, enabling Teyton to grow crystals and to determine the crystal structure of mouse MHC-II. Teyton obtained structural data for two different mouse MHC molecules: the mouse MHC-II I-Ag7, which is expressed in the mouse model of Type 1 diabetes; and the mouse MHC-II I-Ad, which is not linked to diabetes.
"We had hypothesized that there were unusual structural features of I-Ag7 that accounted for its role in autoimmunity," Teyton said. "However, after solving the structure, we saw that the I-Ag7 is a normal class II MHC molecule in its structural features and in the way that it binds peptides."
Thus, many questions remain to be answered in the quest to understand the mechanisms of diabetic autoimmunity, such as: Is diabetes caused by a peptide-specific mechanism? The specific antigen recognized by auto-reactive T-cells has yet to be identified. Teyton's lab is part of a multi-center National Institutes of Health (NIH) project that is working on this question.
A second possibility is that the MHC-II molecule itself somehow leads to abnormal T-cell selection. Although there are no obvious structural differences between the MHC-II molecules that are linked to Type 1 diabetes and those that are not, there are potentially significant sequence differences between them. This sequence difference results in a net negative charge on the peptide-binding portion of the MHC-II not linked to autoimmune diabetes, while a neutral charge in this portion of the molecule is associated with disease susceptibility. This difference in charge could confer selectivity for peptides that cause diabetes, or could play a role in abnormal T-cell selection or activation.
To further explore this possibility, Teyton is conducting structural studies of the interaction of the diabetes-associated MHC-II and the T-cell receptor, through the use of interference microscopy, a new technique that allows the visualization of single molecules at the cell surface. This enables Teyton to examine the interactions between reconstituted T-cell receptors and MHC-II moleculesand to detect any differences in this interaction that are dependent on the type of MHC-II expressed. Ultimately, this will lead to a better understanding of the mechanisms of T-cell activation and the abnormalities associated with autoimmune responses.
Says Teyton, "If we can detect the structural basis for a problem, we will have a basis for specific clinical intervention. The fundamental question remains: How are T-cells activated, and can we specifically prevent the activation of particular subsets?"
Teyton states his goals with intensity, recalling the frustration of being a clinician who could not explain why his patients were suffering. But Teyton's commitment to figuring out how things work, combined with the advanced technological resources at his disposal, may someday help to alleviate that frustration for future physicians and patients.