T cell receptors, Igs, and MHC genes are present in jawed cartilaginous fish (Chondrichthyes: sharks, skates, rays, and chimaeras), but not in more ancestral vertebrates (e.g., lamprey and hagfish) (1
). Thus, sharks represent the oldest living vertebrates with the basic components of the adaptive immune system in mammals. Their study provides a window into the natural history of the genes critical to the system as well as the most fundamental aspects of its physiology.
Ig genes of cartilaginous fish are not arranged in a single large translocon organization common to other vertebrates. Instead, sharks employ many clusters or mini-loci to generate a diverse Ab repertoire (2
). Such a system in an ancient vertebrate has confounding implications for the origins of allelic and isotypic exclusion, problems that are only beginning to be addressed (3
). In contrast to Ig, the organization of shark and skate TCR loci was suggested to parallel that of other vertebrates, with evidence for a single translocon locus encoding each chain (5
). The many Ig loci in the shark could increase the possibility of Ig-TCR trans-rearrangement between juxtaposed or even distant Ag receptor loci normally thought to provide receptors on distinct B and T cells. The translocon TCR organization yet multiple cluster Ig arrangement was consistent with two distinct trends emerging among vertebrate Ag receptors: a plasticity of isotypes, primary lymphoid organs, and diversification mechanisms for the BCR, whereas TCRs showed much more evolutionary conservation (perhaps a result of MHC restriction, at least for the TCR α- and β-chains) (8
). However, interesting variants also have been found in vertebrate TCRs, including wide variation in number of gene segments (9
), bizarre CDR1 and -2 lengths in pathogen-specific Vs (10
), and both allelic polymorphism (11
) and multiple, distinct C region loci (13
). Sandbar shark was shown to use (B cell-like) somatic hypermutation at the TCRγ locus (7
). In the nurse shark, we previously reported the first doubly rearranging Ag receptor, the new Ag receptor (NAR)-TCR δ-chain (15
). This longer (three-domain) form of TCRδ uses an N-terminal V domain generated by VDJ rearrangement that is very similar to IgNAR, a shark Ig H chain isotype that does not associate with L chains (16
). Subsequently, a fifth TCR chain (μ) expressed in a marsupial was identified that also was predicted to contain two V domains that are IgVH-like. TCRμ is related to TCRδ, but it is not orthologous to NAR-TCR despite the fact that both Ag receptors have three domains (17
). These recent findings demonstrate that like Ig, the TCR, especially γδ TCR, is more evolutionarily plastic than previously appreciated. TCR employing IgH domains in disparate vertebrates via convergent evolution prompted a comprehensive study of the TCR gene products and their tissue localization in the shark.
Like the molecular hardware of adaptive immunity, the required primary (thymus) and secondary lymphoid tissues (spleen) are also found in jawed vertebrates but not in agnathans. Cartilaginous fish possess a thymus derived from the first pharyngeal pouches that is composed of lobes with a distinct cortex and medulla (18
) and has high expression of TCR genes (19
). In mammals, T cell progenitors enter the thymus through the corticomedullary blood vessels and accumulate in the subcapsular region of the cortex. These cells proliferate and migrate deeper into the cortex as they mature into αβ or γδ cells. In the cortex, the αβ thymocytes that do not bind self-MHC with high enough affinity fail positive selection and die by apoptosis. As the thymocytes pass from the cortex into the medulla, to generate self-tolerance, they scan a complex set of organ-specific Ags under the control of the AIRE gene expressed by medullary epithelial cells. The thymus has been characterized anatomically and cytochemically in several elasmobranchs (20
), yet gene expression and development of T lymphocytes in the most ancient organisms with a thymus has not been studied in detail. Analysis of nurse shark secondary lymphoid tissue (spleen) has been performed for B cells and APCs, but in fact, T cells have never been identified by TCR expression in cartilaginous fish (21
In this work, we have studied the repertoire of nurse shark TCR chains and complemented the molecular work with TCR expression analysis by in situ hybridization. We have discovered unexpected trans-rearrangements between Ig and TCR loci. Whether these transcripts are by-products of the multiple Ig clusters in shark or if the Ig V segments are functional on T cells is not addressed, but the adaptive repertoires of sharks (7
), noneutherian mammals (22
), and now higher poikilothermic vertebrates (Z.E. Parra, Y. Ohta, M.F. Criscitiello, M.F. Flajnik, and R.D. Miller, submitted for publication) have blurred the boundary between B and T cell Ag receptors. Thus, these findings of genetic and mechanistic plasticity may be redefining the fundamental boundaries of Ag receptor repertoires.