Analysis of shark NCR3 family genes reveals primordial features of vertebrate NKp30.

Natural killer (NK) cells play major roles in innate immunity against viruses and cancer. Natural killer receptors (NKR) expressed by NK cells recognize foreign- or self-ligands on infected and transformed cells as well as healthy cells. NKR genes are the most rapidly evolving loci in vertebrates, and it is generally difficult to detect orthologues in different taxa. The unique exception is NKp30, an activating NKR in mammals that binds to the self-ligand B7H6. The NKp30-encoding gene, NCR3, has been found in most vertebrates including sharks, the oldest vertebrates with human-type adaptive immunity. NCR3 has a special, non-rearranging VJ-type immunoglobulin superfamily (IgSF) domain that predates the emergence of the rearranging antigen receptors. Herein we show that NCR3 loci are linked to the shark major histocompatibility complex (MHC), proving NCR3's primordial association with the MHC. We identified eight subtypes of differentially expressed highly divergent shark NCR3 family genes. Using in situ hybridization, we detected one subtype, NS344823, to be expressed by predominantly single cells outside of splenic B cell zones. The expression by non-B cells was also confirmed by PCR in peripheral blood lymphocytes. Surprisingly, high expression of NS344823 was detected in the thymic cortex, demonstrating NS344823 expression in developing T cells. Finally, we show for the first time that shark T cells are found as single cells or in small clusters in the splenic red pulp, also unassociated with the large B cell follicles we previously identified.