Characterization of lymphoid development in lamprey
Lampreys and hagfishes exhibit an alternative adaptive immune system, provding an unprecedented opportunity to examine common features of vertebrate adaptive immunity. Our identification and characterization of thymus-like lympho-epithelial structures – termed thymoids – in the gill basket of lamprey larvae established that the similarities underlying the dual nature of the adaptive immune systems in the two sister groups of vertebrates extend to primary lymphoid organs. We have now turned our attention to genetic studies of the development of lymphoid lineages in lampreys. The opportunity to raise in vitro fertilized animals in the laboratory has allowed us to determine the ontogenetic sequence of lamprey immune system development. For instance we found that functionally assembled VLRB antigen receptor genes first appear 10 weeks after fertilization, with VLRA and VLRC assemblies being detectable about 2 weeks later. These studies not only established the baseline for our systematic investigations of mutant lampreys, they also showed that the development of the B cell system precedes that of the T cell arm of lamprey immunity. From a large-scale analysis of cytidine deaminases of metazoans in general, we concluded that CDA1 and CDA2 represent “institutionalized” versions of evolutionarily ancient anti-viral effector molecules, which were coopted by vertebrates to achieve somatic diversification of VLR (lampreys, and presumably also hagfish), and Ig genes (jawed vertebrates), in addition to affinity maturation and, in some species, class switch of antibodies (jawed vertebrates). Based on the expression profiles of the CDA2 gene of lampreys, it was hypothesized that it is involved in the somatic assembly of the VLRB antibody genes. Indeed, targeting the CDA2 gene abolishes assembly of VLRB antigen receptor genes. Surprisingly, mutant animals appear to be able to survive, suggesting the presence of redundancy among adaptive facilities and/or strong activities of innate immune components.
It is known that, in addition to VLRA, the genes encoding the VLRC-type of VLRs are assembled in the thymoid of lamprey larvae. Structures of intermediates of the assembly process indicated that sequence elements encoding the internal LRR-repeats are appended to the incomplete germ-line encoded VLRC gene in a step-wise fashion proceeding from both ends; interestingly, VLRA and VLRC loci, encoding the two different T-like antigen receptors of lamprey, are situated in close proximity in the genome of L. planeri causing some of the internal LRR modules to be shared between the two receptors, reminiscent of the situation in TCRδ/α locus in jawed vertebrates. Moreover, we found that the sequences of mature VLRCs isolated from the thymoid and blood differ at specific sites, possibly indicating selection for particular structural characteristics; in support of this, we could demonstrate that the sites exhibiting signs of selection all locate to one surface of the VLRC molecule.
In order to examine the possibility that repertoire selection is a consequence of interaction with polymorphic antigen-presenting cells (akin to pMHC/TCR interactions in jawed vertebrates), we have now embarked on an extensive search for sequence polymorphisms of genes expressed in presumptive antigen-presenting cells in the blood and the thymoid of lamprey. This analysis is being done with a view to identifying candidates for potential restriction elements (MHC equivalents).