T cell receptor diversity vs. efficacy In order to recognize and combat a diverse array of invading pathogens, T cells express a large repertoire of clonotypic ab dimeric T cell receptors (TCR), with few cells on average expressing TCR specific for any given antigen but an enormous number of specificities at the population level within an individual. Accurately estimating total TCR diversity is problematic, as this is a function of both the CDR3 diversity within each TCR chain due to recombination between V, D, and J gene segments, and P- and N-nucleotide addition or deletion at gene segment joints, as well as diversity due to pairing of different TCRa and TCRb chains. Current methods for obtaining both TCRa and TCRb CDR3 sequences from individual cells in large populations are unfeasible; single cell sorting is too expensive and molecular strategies have not been adequately developed. We have developed DNA origami nanostructures that can be transfected into T cells to capture and protect both TCR mRNA, as well as molecular techniques for linking these CDR3 into a single cDNA for paired-end high-throughput next-generation sequencing. We have begun to apply the DNA origami approach for quantitating cellular diversity to cancer in order to refine clinical therapy approaches.
T cell response pathology vs. protection CD8 T cell responses are important for the control of a number of human and animal pathogens at the portal(s) of entry, as well as providing systemic immunity after infection. We are currently evaluating a number of different strategies for selectively targeting vaccine-induced T cell responses to mucosal sites as a means of improving containment at the portal of entry. However, narrowly focused CD8 T cell responses are insufficient to either provide coverage for the large number of circulating strains of some viruses or to prevent selection of mutant viruses after infection that are able to evade CTL recognition. We seek to understand what limits the generation of T cell responses to sub-dominant epitopes, and conversely what facilitates responses to immunodominant epitopes by systematically analyzing the efficiency of generation and stability of epitopes from protein substrates and how these parameters change in different antigen-presenting cell types (including direct and cross-presentation. The data from these studies can then be used to predict which epitopes from an immunogen will be immunodominant after vaccination, to design immunogens to increase the levels of subdominant epitopes on the cell surface as a means of improving responses to these epitopes, and to selectively target public T cell precursors that are present in the na�ve repertoire such that a majority of individuals will be able to generate broader responses.
Linking immunologic and epidemiologic measures of vaccine efficacy Vaccination remains one of the most potent strategies for preventing human diseases caused by pathogens. The empirical approach to vaccine development, employing attenuated versions of pathogens or subunit vaccines has resulted in protective efficacy against a number of viral and bacterial pathogens. However, relatively modest subsequent progress has been made in the improvement of empirically defined vaccines or in the development of new, efficacious vaccines. This is in part due to an incomplete quantitative understanding of the immunological mechanisms that are required to reduce susceptibility to infection, disease pathogenesis, or subsequent transmission. We are using animal models of vaccination and viral infection to explicitly link vaccine-induced immunological responses with epidemiological measures of vaccine efficacy including reduction in susceptibility, disease, and subsequent transmission. This will allow us to develop a quantitative framework for understanding how immunological responses induced by certain types of vaccines might provide protection against infection and/or disease during subsequent pathogen exposure.
Immunotherapy of Persistent Viral Infection and Metastatic Cancer During prolonged antigen stimulation, as occurs during persistent viral infection or prolonged tumor burden, T cell responses become silenced as a means of preventing pathology to the host. However, in some viral infection and cancer settings, it is desirable to reactivate these T cells in order to provide additional protection. We are using persistent viral infection and cancer models to understand the molecular interactions that program T cell exhaustion in order to develop strategies for improving therapeutic approaches. Using both immune checkpoint blockade and metabolic approaches, we have developed new avenues for therapy of refractory tumors and persistent viral infection.