Investigation of in-vivo antigen-specific T cell responses
Autor: Univ. Prof. Dr. med. Dirk H. Busch
The major focus of the Busch laboratory is to visualize and track antigen-specific T cell populations during in vivo antigen challenge. These studies are designed to increase our understanding of how T cell responses are regulated in vivo and how protective and long lasting immunity is established. This knowledge is of special interest for adoptive immunotherapy, diagnostic monitoring of T cell mediated immunity, and the development of new vaccination strategies.
The T cell receptor (TCR) of T cells recognizes antigen in the context of MHC (major histocompatibility complex) molecules. MHC class I molecules present small peptides (epitopes) processed from intracellular antigens, such as viruses and intracellular bacteria, to cytotoxic CD8+ T cells. MHC class II molecules receive their peptides mainly from extracellular and soluble antigens and present them to CD4+ T helper cells. Antigen-specific T cell responses are of major importance in the control of infection and the development of protective immunity. T cells can also mediate anti-tumor effects and, in the case of autoimmune syndromes, can contribute to the development and pathology of disease.
Over the last few decades, numerous epitopes recognized by antigen-specific T cells have been identified, and general features of T cell responses have been revealed. Due to the difficulty of identifying antigen-specific T cells directly ex vivo, however, many basic questions regarding the in vivo regulation of antigen-specific T cell responses and the generation of protective immunity are still unsolved. With the recent development of new immunological methods, especially MHC tetramer technology, we are now able to directly identify and isolate T cells depending on their antigen specificity.
The goal of this lab is to further develop these new advances in immunological techniques, to investigate antigen-specific T cell responses in an animal model (infection of mice with the facultative intracellular bacterium Listeria monocytogenes), and to test direct clinical applications of the technology.
1. MHC Tetramers
The specificity of T cells is determined by the TCR. T cell populations activated and expanded during most in vivo antigen challenges are highly complex, with diverse TCR repertoires, complicating the detection of these cells. The ideal reagent to identify complex epitope-specific T cell populations would be the natural ligand of the TCR, the MHC/epitope complex. However, the affinity of the TCR-MHC/epitope interaction is very low; the association is characterized by a particularly high dissociation rate. To increase the overall avidity of this interaction, MHC/epitope complexes are multimerized into e.g. tetramers. MHC tetramer reagents conjugated with a fluorescent dye can be used in flow cytometry, allowing highly specific detection and isolation of (complex) epitope specific T cell populations directly ex vivo.
The generation of MHC class I multimer reagents has become well established over the past few years. Beta-2-microglobulin and the heavy chains (HC) of MHC molecules are expressed as recombinant proteins in bacteria and subsequently refolded in vitro in the presence of high concentrations of peptide (epitope). A specific biotinylation site at the C-terminus of the HC allows enzymatic addition of one biotin per MHC molecule. Tetrameric MHC/peptide complexes are formed in the presence of streptavidin, which has 4 biotin binding sites. We are in the process of generating a large ‘bank’ of expression vectors for human an, d murine MHC molecules. MHC class I tetramer reagents with epitopes derived from a variety of different pathogens (e.g. Listeria monocytogenes, HIV, HCMV, EBV, HCV) and tumor antigens are being generated for experimental and clinical studies.
Conventional MHC multimer reagents are powerful tools for phenotypical ex vivo analysis of antigen-specific T cell populations. However, functional analysis and in vivo transfer of MHC multimer-stained cells is hampered by the persistence of T cell receptor (TCR)-MHC interactions and subsequently induced signaling events. Since MHC monomers do not stably bind to TCRs, we recently postulated that targeted disassembly of multimers into MHC monomers would result in dissociation of surface-bound TCR ligands. We generated a new type of MHC multimers (Streptamers), which can be monomerized in the presence of a competitor, resulting in rapid loss of the staining reagent even at low temperatures (preferably at 4°C). Following staining and dissociation at low temperatures, the T cells are phenotypically and functionally indistinguishable from untreated cells. We are currently testing this “reversible” T cell staining procedure, which maintains the specificity and sensitivity of MHC multimer staining while preserving the functional status of T lymphocytes, for ex vivo investigation of T cell functions and clinical applications.
The generation of MHC class II multimers for the detection of CD4+ T helper cells is more difficult than the generation of class I reagents. We are currently testing different methods for the effective generation of MHC class II reagents. Special focus will be on the developemt of a reversible staining procedure for epitop-specific T helper cells.
2. Ex vivo analysis of pathogen-specific T cell responses
Sublethal infection of mice with the Gram-positive bacterium Listeria monocytogenes results in the development of very effective and long-lasting protective immunity, mediated primarily by CD8+ and CD4+ Listeria-specific T cells. Using MHC multimer reagents, we want to characterize the determinants of effective protective immunity in this experimental model: how, when and where are Listeria-specific memory T cell populations generated in vivo? What is the contribution of different Listeria-specific subpopulations (defined by their lineage, epitope-specificity, phenotype, functional status) to protective immunity? Is there functional interaction/synergism between different Listeria-specific T cell subpopulations which is important for the quality of protective immunity? Different experimental strategies are used to address these questions (e.g. gene knockout mice, transgenic mice, ENU mutagenesis). These studies are designed to shed light on the basic requirements for effective protective immunity; a better understanding of immunological memory will facilitate the development of new, more effective vaccine strategies. An additional clinical application of MHC tetramers is the diagnostic monitoring of antigen-specific T cell immunity.
3. Adoptive transfer of protective immunity
MHC multimer technology allows both identification and isolation of epitope-specific T cells. Especially with the help of the recently developed reversible MHC-multimer staining (Streptamers) it may be possible to purify and adoptively transfer epitope-specific T cell populations into recipients, a technique that would have broad clinical applications. We are pursuing the development of methods to efficiently purify tetramer-positive T cells and prepare them for adoptive transfer. Adoptive transfer of epitope-specific T cells will initially be tested in the murine Listeriosis model in order to address some basic experimental questions: Is it possible to transfer pathogen-specific protective immunity into naïve individuals by adoptive transfer of purified tetramer-positive, epitope-specific T cells? What are the minimal requirements (absolute number, epitope specificity, lineage, functional status) for effective adoptive transfer of protective immunity? Similar studies will be performed in other experimental models (e.g. chronic infections).
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