Evolution and Immune Functions of Nonclassical MHC Class Ib Genes in the Clawed Frogs Xenous laevis and Silurana tropicalis

Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Microbiology and Immunology, 2010.Major histocompatibility complex (MHC) class Ia (class Ia) are evolutionary conserved genes in jawed vertebrates encoding highly polymorphic and ubiquitously expressed surface molecules essential for the development and activation of CD8 T cells. By contrast, nonclassical MHC class Ib (class Ib) are a group of heterogeneous genes encoding molecules structurally similar to class Ia but with a more limited tissue distribution and polymorphism. In mammals, class Ibs have diverse, and often not well characterized, functions that include stress responses, detection of malignancy, immune regulation and the differentiation of CD8 T cells. Although class Ib genes have been identified in all taxa of jawed vertebrates, information about their function is even more limited and their phylogeny is uncertain. This is usually attributed to their fast rate of evolution. The subfamily Xenopodinae, including species of the clawed frog genera Xenopus and Silurana, which diverged from a common ancestor 80 million years ago (MYA), provides a powerful non-mammalian model to further investigate the evolution and immune functions of class Ib genes. Xenopus laevis is one of the best characterized and most widely used non-mammalian animal models to study immunity. We used this species to investigate the role of class Ib in early T cell ontogeny and in tumor immunity. The genome of the diploid Silurana tropicalis has been fully sequenced and annotated. We used this species to evaluate the genomic and molecular evolution of class Ib genes. Genomic, molecular and phylogenetic studies reveal an unexpected degree of conservation (9 out of 13 class Ib subfamilies are homologous between X. laevis and S. tropicalis) of class Ib genes in Xenopodinae throughout a period of time (80 MY) similar to that which separates primates and rodents from a common ancestor. This study also unveils the ancient origin and uniqueness of one X. laevis class Ib gene, XNC10, which has an unequivocal ortholog in S. tropicalis, SNC10. XNC10 is preferentially expressed by CD8 thymocytes and by a subset of peripheral effector CD8 T cells in adults. In larvae, XNC10 is expressed from the onset of thymus organogenesis during which no class Ia protein expression is detected. These data strongly suggest that XNC10 is involved in T cell ontogeny. To investigate how conserved the involvement of class Ib gene products are in tumor immunity, we took advantage of a X. laevis transplantable lymphoid tumor cell line (15/0) that is class Ia deficient, and whose killing involves unconventional CD8 cytotoxic T cells that are antigen-specific and class Ia-unrestricted (CCU-CTL). We generated, for the first time in ectothermic vertebrates, stable tumor transfectants expressing shRNA effectively silencing either β2-microglobulin, to prevent class Ib surface expression, or class Ib directly. Both types of 15/0 tumor transfectants are more resistant to CCU-CTL killing and more tumorigenic, while more susceptible to NK-like cell killing. These data are consistent with a conserved role of class Ib molecules in regulating anti-tumor NK and CD8 T cell responses. Overall, this study highlights the unusual evolutionary conservation of class Ib genes in Xenopodinae, and their involvement in tumor immunity and in the development of CD8 T cells. Finally, this work gives a foundation for Xenopodinae to serve as a useful comparative non-mammalian model system to decipher the evolution and function of class Ib genes in immunity