On the evolutionary origins of CD1d and semi-invariant natural killer T cells
Semi-invariant natural killer T (NKT) cells recognize both self and foreign lipid antigens when presented by the antigen-presenting molecule CD1d. NKT cells recognize the lipid antigen α-galactosylceramide (α-GalCer). Both mouse and human CD1d molecules present α-GalCer to NKT cells of either species; hence, CD1d and NKT cells are highly conserved between both species. Upon lipid antigen recognition, NKT cells get activated and secrete pro-inflammatory and regulatory cytokines. This response controls cellular and humoral immune responses to microbes to prevent infectious diseases. This project tested the hypothesis that NKT cells are a eutherian (of placental mammals) innovation, meaning that they are not found in other mammals or vertebrates. To test this hypothesis, the soluble form of nine-banded armadillo CD1d and anole lizard CD1 (to serve as a control) molecules were engineered to contain, at the carboxyl terminus, a biotin substrate peptide tag for site-specific biotinylation and a polyhistidine-tag for metal-affinity chromatography. PCR was used to construct the two cDNAs, which were cloned into pLenti vector. The cDNA constructs were transfected into Phoenix line for expression and secretion. Biotinylation was performed using biotin peptide ligase to generate tetramers with fluorescent streptavidin. In future studies, α-GalCer-loaded tetramers will be used to stain mouse and human NKT cells. If positive, these tetramer reagents will be used to track NKT cells in armadillos. Thus, in addition to establishing the evolutionary origins of CD1d and NKT cells, the reagents created in this project will be useful to study immune responses to leprosy, as armadillos are the only model for this dreaded infectious disease.