Nonclassical MHC-dependent Innate T Cell Ontogeny and Function in the Amphibian Xenopus

University Of Rochester, Rochester NY

Investigators

Abstract

T cells are well known for their role in generating the capacity of the adaptive immune system to identify and respond to diverse non-self antigens. The selectivity of a T cell arises from the T cell receptor variant it displays at its surface, with an individual?s T cell population containing a massive repertoire of distinct T cell receptor variants. However, a subgroup of T cells displays a limited or invariant set of T cell receptors. These so called innate T cells (iT cells) are thought to be rapid responders to infections, recognizing via their T cell receptors conserved molecular determinants in pathogens and providing defense in advance (or absence) of an adaptive immune response. However little is known about how iT cells differentiate or the role they play in immune responses. With preliminary data demonstrating that iT cells are well represented in tadpoles of the frog Xenopus, this project addresses the general hypothesis that the external development of tadpoles requires a capacity to mount rapid immune responses to viruses and bacteria, that the responses be mediated by a small population of immune competent cells, and that these requirements are met by iT cells. This research will extend our understanding of the evolution, biology and importance of iT cells in host/pathogen interactions. It also has the potential to suggest immunologically informed strategies for controlling pathogens and their role in amphibian decline. Undergraduate, graduate and post-graduate students will be extensively involved in all aspects of the research, which includes opportunities to study fundamental immunology, development, microbiology and evolution, as well as to learn a range of modern molecular, cellular and reverse genetic techniques. The PI will recruit one or two undergraduate students during the academic year, and in collaboration with the NSF BIO REU program and the Summer Undergraduate Research Program, at least one additional minority undergraduate each summer. The PI also directs the Xenopus laevis Research Resource for Immunobiology, which provides reagents, animals, information, assistance, training and outreach to scientists and the public. Two specific hypotheses guide this project: (i) ectothermic vertebrates with rapid external development critically rely on iT cells for an innate-like T cell repertoire; and (ii) that class Ib restricted iT cells are pivotal in orchestrating immune responses against viral and bacterial pathogens. This hypothesis will be addressed in vivo in X. laevis tadpoles and adults with innovative reverse genetic loss-of-function approaches that combine transgenesis with RNAi or CRISPr/Cas genome editing technologies to determine the roles of class Ib molecules in the development and function of iT cells in X. laevis. The research will also use and further develop class Ib tetramers and antibodies to identify distinct iT cell subsets. The specific aims are: (1) To identify and characterize class Ib genes regulating the development and function of distinct iT cell lineages using qPCR, immunohistology and in situ hybridization as well as class Ib-deficient transgenic lines; (2) To characterize distinct class Ib-restricted iT cell lineages in tadpoles and adults using a combination of class Ib tetramers and monoclonal antibodies with flow cytometry, cell sorting and fluorescence microscopy; (3) To elucidate the roles of distinct class Ib-restricted iT cell lineages of tadpoles and adults in vivo during immune response against ecologically relevant pathogens including the ranavirus Frog virus 3, Mycobacterium marinum, and Aeromonas hydrophila. The successful application of tools (class Ib tetramers and antibodies) and innovative loss-of-function by transgenesis technologies will further empower the Xenopus research model.