The immune system is a complex constellation of cells and molecules that is principally responsible for preventing us from certain death from a myriad of pathogenic microorganisms. It performs this enormous task by creating layers of defensive mechanisms whose functions are to identify, isolate, and kill invading organisms. It also has the capacity to neutralize and/or eliminate cancerous cells. We study the immune system of mice and human beings in two distinct ways. First, we try to understand in molecular detail how T lymphocytes, one of the key cell types in the immune system, distinguish foreign entities from self and mount a response. This is a critical event in the immune system and also arguably the best understood model for cell-cell recognition. Second, we are investigating ways to understand the human immune system, both for what it can tell us about immunology in general and because inbred mice have not, in most cases, been a reliable guide for developing treatments for immunological diseases. Given the diverse mechanisms of immunity in humans, we also believe that this is an ideal testing ground for understanding how immune functions work as a system.
Integrating Molecular and Cellular Aspects of T Cell Recognition
To pursue our first goal, we have focused on developing advanced imaging techniques to understand the molecular interactions that underlie the process by which a particular T cell recognizes fragments of an antigen (peptide) that are bound to molecules of the major histocompatibility complex (MHC) and displayed on the surfaces of cells. These peptide-MHC complexes are then recognized by the T cell antigen receptor (TCR), an antibody-like molecule present on most T cells. The TCR is closely associated with the CD3 polypeptides, which mediate intracellular signaling through a kinase cascade. Our previous work and that of others characterized the biochemistry and genetics of TCRs, but in recent years we have focused on how these molecules operate in the larger context of T cell recognition, a context that includes other molecules on the T cell surface that work with the TCR, and on how ligand binding triggers T cell activation. Video microscopy and other advanced imaging techniques, such as super-resolution fluorescence microscopy and three-dimensional electron microscope tomography, have helped us determine many of the key events of this process. In the first few minutes after a T cell recognizes another cell, a dramatic rearrangement of surface molecules and cytoarchitecture takes place to form what is called an immunological synapse. We have helped define the molecular structure and kinetics of synapse formation. Much of the rearrangement is cytoskeletal and depends on engagement of costimulatory receptors such as CD28 and LFA-1, in addition to the TCR. We also showed that synapses may last for many hours but require continuous TCR engagement.
We also developed single peptide-MHC labeling methods that allow us to determine exactly how many ligands are present in an initial T cell synapse and what the subsequent response is. This enables us to correlate ligand number with the consequences for individual cells. We used this method 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 activation—in terms of full calcium elevation and stable synapse formation only occur with 8 to 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 necessary for a cell to embark 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.
We also found that TCRs and other molecules on the T cell surface are clustered in what we term "protein islands" and that these structures segregate specific molecules in the TCR signaling cascade. Specifically, we found that TCRs and the downstream adapter molecule LAT occur on separate islands, but which concatenate upon T cell activation. These islands may be a general organizing principal for cell surface proteins, and the segregation and reorganization that we see with TCR and LAT molecules may be a way of controlling the cellular activation process.
While we have learned a great deal about basic immunology from the laboratory mouse model, it has significant limitations in terms of genetics, environmental exposure, and evolutionary distance from human beings. Thus we have used both peptide-MHC tetramers and systems biology approaches to study the human immune system. With respect to the most T cells, we find that healthy adults have an abundance of self-specific T cells that are capable of being activated and that the effect of negative selection is a reduction in the number of these cells, not their elimination. We suggest that the main objective of the repertoire is to counter pathogens and any of their variants. We also find that in adults, but not in newborns, many memory-phenotype T cells are directed against pathogen epitopes to which the individual has not been exposed. We showed that at least some this effect is likely a result of cross-reactivity of TCRs to other microbes. This may explain why infants and young children are so susceptible to infectious diseases and why a number of vaccines have been shown to decrease significantly mortality from diseases other than the one targeted by the vaccine.
In general, we are finding that studying the human immune system directly in healthy people is highly complementary to mouse studies and, in some cases, provides a broader view. We are also applying systems biology approaches to human immunology, as these hold promise for understanding the role of different immune system components in vaccine responses and disease. These studies involve the analysis of immune biomarkers in twins to assess the role of genetics versus the environment. To determine the effects of the environment and aging, we also compare these biomarkers between cohorts in the developing world and the U.S. and among different age groups.
Grants from the National Institutes of Health, the Bill and Melinda Gates Foundation, and the Ellison Medical Foundation provided partial support for these projects.
As of July 5, 2013