Evolutionarily Conserved TCR Binding Sites, Identification of T Cells in Primary Lymphoid Tissues, and Surprising Trans-Rearrangements in Nurse Shark

Abstract

Cartilaginous fish are the oldest animals that generate RAG-based Ag receptor diversity. We have analyzed the genes and expressed transcripts of the four TCR chains for the first time in a cartilaginous fish, the nurse shark (Ginglymostoma cirratum). Northern blotting found TCR mRNA expression predominantly in lymphoid and mucosal tissues. Southern blotting suggested translocon-type loci encoding all four chains. Based on diversity of V and J segments, the expressed combinatorial diversity for γ is similar to that of human, α and β may be slightly lower, and δ diversity is the highest of any organism studied to date. Nurse shark TCRδ have long CDR3 loops compared with the other three chains, creating binding site topologies comparable to those of mammalian TCR in basic paratope structure; additionally, nurse shark TCRδ CDR3 are more similar to IgH CDR3 in length and heterogeneity than to other TCR chains. Most interestingly, several cDNAs were isolated that contained IgM or IgW V segments rearranged to other gene segments of TCRδ and α. Finally, in situ hybridization experiments demonstrate a conservation of both α/β and γ/δ T cell localization in the thymus across 450 million years of vertebrate evolution, with γ/δ TCR expression especially high in the subcapsular region. Collectively, these data make the first cellular identification of TCR-expressing lymphocytes in a cartilaginous fish.