| Nonclassical MHC class Ib (class Ib) molecules are increasingly implicated in tumorigenesis and tumor immunity, however, their function is not well understood. To investigate the fundamental role of class Ib genes in anti-tumor immune responses we capitalized on a well characterized comparative immuno-cancer model in Xenopus LG isogenetic clones. Previous work demonstrated the ability of the heat shock proteins gp96 and HSP70 to generate potent CD8 T cell responses against a transplantable thymic lymphoid tumor (15/0) that expresses several class Ib genes, but no classical MHC class Ia (class Ia) molecules. In the absence of class Ia, we hypothesized that HSP70 proteins can prime class Ib anti-tumor unconventional T cells in an antigen-dependent manner. To test this, we produced recombinant Xenopus inducible hsp72 and constitutive hsc73 from stable 15/0 tumor transfectants. Using a cross-presentation priming assay we showed that hsp72-Ag complexes, but neither hsc73-Ag nor Ag-negative HSP70s, elicit class Ib-mediated T cells responses resulting in protection from 15/0 tumor challenge.;To further elucidate the role of class Ib molecules in anti-tumor immunity, we developed a powerful reverse genetic loss of function approach by combining transgenesis and RNAi technology. We found that the I-SceI meganuclease was more effective than two alternative transgenesis techniques, the ΦC31 integrase and the Sleeping Beauty transposase, for generating transgenic clones; it resulted in stable nonmosaic germline transmission (100%). To impair class Ia and Ib function in vivo, we generated F0 and F1 transgenic LG clones with stable knockdown expression of β2-microglobulin (β2-m). Transgenic F1 frogs with β2-m knockdown have significant downregulation of class Ia molecules on the surface of red blood cells and lymphocytes, and exhibit delayed MHC-mismatched skin graft rejection. Furthermore, β2-m silencing in transgenic larvae increased susceptibility to viral infection. Therefore, we have built the foundation for determining the effects of β2-m knockdown on tumor immunity, as well as silencing specific class Ib genes.;Overall, this study provides evidence of the fundamental role of class Ib molecules in HSP70-mediated anti-tumor immunity. Furthermore, the loss of function strategy we developed has a unique potential to unveil the importance of class Ib molecules in immune function. |
