T lymphocytes play a number of critical roles in the immune system. Some subsets of T cells are able to kill virally infected or transformed cells directly; others seem specifically designed to mobilize other cells, particularly B cells, in the course of an immune response. Both activities can be mediated through the same cell surface recognition apparatus, the αβ T cell receptor (TCR) heterodimer, in close association with the CD3 polypeptides. A central feature of T cell recognition by this receptor is that antigens are often (if not always) "seen" as peptide fragments complexed with either class I or class II molecules of the major histocompatibility complex (MHC).
One of our goals is to understand the biochemical and structural characteristics of this form of recognition and whether it is fundamentally distinguishable from antibody-antigen interactions. Another goal is to understand the mechanics of early T cell activation, particularly with respect to how the various cell surface molecules redistribute themselves. A third goal is to investigate how T cell activation and selection are controlled and where critical decision points in this process lie.
The Biochemistry and Thermodynamics of T Cell Receptor Binding
We have found evidence that TCR binding occurs in two distinct stages: the TCR first contacts portions of the MHC molecule that straddle the peptide-binding groove; later it fully engages with the antigenic peptide. This two-step model suggests how TCRs on a T cell could quickly orient themselves to MHC molecules on other cells to "scan" their bound peptides. It also suggests a mechanism for TCR cross-reactivity to different peptides bound to either the same or a related "foreign" MHC (alloreactivity): once binding is initiated with a particular MHC, the TCR could fold onto the peptide in a potentially large number of different conformations. This mechanism could explain how TCRs could be selected in the thymus for weak reactivity to self-peptide-MHC and yet how some emerge later with strong reactivity to peptides derived from foreign entities.
We are also interested in the thermodynamics of TCR binding to ligand. Earlier we and others showed that most TCRs bind peptide-MHCs with an induced-fit mechanism, i.e., a loss of entropy and a gain in enthalpy. In a survey of peptide variants that bind to the same TCR (and form a complex with the same MHC), we found general agreement with this principal but a wide variation in the values for entropy, enthalpy, and heat capacity, despite structural studies (in collaboration with K. Christopher Garcia, HHMI, Stanford University) that showed only slight differences in the peptide-MHC ligand. This suggests that the TCR can have considerable variations in its binding strategy. Wide variations in heat capacity, which is a measure of conformational changes and/or changes in flexibility, were particularly interesting. This binding parameter seems able to substitute for stability as a predictor of T cell activation, suggesting that more subtle forces are at work in the mechanism of T cell activation. We are pursuing the ramifications of this possibility.
Integrating Molecular and Cellular Aspects of T Cell Recognition
We are also interested in the larger context of T cell recognition, including the other molecules on the T cell surface that work with the TCR and how ligand binding triggers activation. Video microscopy has helped us determine some of the key events of this process. In the first few minutes of T cell recognition of another cell, a dramatic rearrangement of surface molecules and cytoarchitecture occurs that has been called an immunological synapse. We have helped to define the molecular structure and kinetics of synapse formation. Much of the rearrangement is cytoskeletal and depends on the engagement of costimulatory receptors such as CD28 and LFA-1, in addition to the TCR. We have also shown that synapses can last for many hours but require continuous TCR engagement.
Recently, we have developed a peptide-labeling method that allows us to determine exactly how many ligands are present in an initial T cell synapse and what the subsequent response is. This gives us the ability to correlate ligand number with the consequences for individual cells. We have used this to show that all T cells surveyed thus far can detect a single molecule of peptide-MHC on the surface of another cell but that full calcium elevation and stable synapse formation only occurs with 8—10 ligands. This shows that T cells can be as sensitive to foreign antigens as any sensory cells in the nervous system. Killing by cytotoxic T cells requires three or more ligands, showing that sensitivity is distinct from the threshold that a cell may have for embarking on an irreversible action. We hypothesize that different kinds of T cells will have different thresholds and that these are also likely to change with the different developmental stages.
Our finding that even a single ligand can initiate T cell activation presented a puzzle, in that our previous results and those of others had indicated that TCR dimerization is a key event in T cell triggering. We have suggested a psuedodimer model in which an otherwise nonstimulatory endogenous peptide-MHC ligand, together with CD4, synergizes with an agonist to trigger helper T cells. Recently, we have determined that this model is substantially correct, in that synthetic heterodimers consisting of agonist ligands plus certain endogenous peptide-MHC ligands joined together can stimulate specific T cells. Experiments with these ligands on cell surfaces (but not covalently linked) corroborate these results. In this novel mechanism, the endogenous ligands are extremely weak TCR binders compared to the agonists. We are interested in knowing more about the biochemical and structural basis of this binding and how it triggers activation.
It is also likely that the same peptides that synergize with agonist peptides in the periphery help to select T cells in the thymus. Previously it was thought that thymic selection for weak self-peptide-MHC reactivity (positive selection) was solely to enrich the TCR repertoire for cells that could work with a given individual's MHC molecules, but the work described above and that of others has shown that that this property may be relevant to the enhancement of T cell sensitivity as well.
Tracking Specific T cell Responses
We are also interested in developing new ways to analyze T cell responsiveness, particularly in humans, where the need is greatest. This interest began with our development of peptide-MHC tetramers as a method to identify and quantitate specific populations of T cells in conjunction with flow cytometry. Most recently, in collaboration with Patrick Brown (HHMI, Stanford University) and his colleagues, we have developed a new, array-based methodology that is able to generate much more information about the specificities of T cells in a population and their functional capabilities. We have shown that this methodology can be used to analyze patient samples from a peptide vaccine trial of melanoma patients (in collaboration with Jeffrey Weber, University of Southern California, Los Angeles) and that these patients show a marked variability in their responses to the different melanoma antigens. This heterogeneity may explain why most cancer vaccine trials elicit such a wide range of patient outcomes. We have also begun characterizing the response to influenza antigens in both younger and older people receiving a standard influenza vaccine. This vaccine is effective in healthy people much of the time but does not work as well in the elderly. The reasons for this, especially in terms of the T cell response, are not well understood. Ultimately we wish to derive metrics for immunological health versus disease that would be both useful clinically and help us learn more about the underlying mechanisms.