Ontogeny of Xenopus NK cells in the absence of MHC class I antigens

Abstract

This paper explores the ontogeny of NK cells in control and early-thymectomized (Tx) Xenopus laevis through phenotypic analysis of cells expressing the NK cell antigen 1F8 and by performing in vitro cytotoxic assays. Dual color flow cytometry reveals that a few 1F8positive splenocytes first emerge in late larval life, at ∼7-weeks post-fertilization. This is about 2-weeks after the time when surface MHC class Ia expression can first be detected. The proportion of splenocytes expressing 1F8 remains very low in 3–4 month-old froglets, but by 1 year there is a sizeable 1F8positive population, which is proportionally elevated in Tx frogs. The ontogeny of NK cell function is monitored by a 5 h DNA fragmentation (JAM) assay. Control and Tx larval splenocytes (from either 5- or 7-week-old tadpoles) fail to kill MHC-deficient thymus-derived tumor cell targets. Such in vitro killing is still relatively poor in 3–4 month froglets, compared with high levels of tumor cell cytotoxicity mediated by splenocytes from older frogs. Immunoprecipitation studies identify that the major ligand for the 1F8 mAb is a 55 kDa polypeptide. Finally, further evidence is provided that 1F8positive lymphocytes are indeed bona fide NK cells, distinct from T cells, since purified 1F8positive splenocytes from Tx Xenopus fail to express fully rearranged TCRβ V region transcripts. We conclude that NK cells fail to develop prior to MHC class I protein expression and, therefore, do not contribute to the larval immune system, whereas they do provide an important backup for T cells in the adult frog by contributing to anti-tumor immunity.