The Armadillo (): A Witness but Not a Functional Example for the Emergence of the Butyrophilin 3/Vγ9Vδ2 System in Placental Mammals.

1-5% of human blood T cells are Vγ9Vδ2 T cells whose T cell receptor (TCR) contain a rearrangement and a comprising Vδ2-chain. They respond to phosphoantigens (PAgs) like isopentenyl pyrophosphate or (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMBPP) in a butyrophilin 3 (BTN3)-dependent manner and may contribute to the control of mycobacterial infections. These cells were thought to be restricted to primates, but we demonstrated by analysis of genomic databases that , and genes coevolved and emerged together with placental mammals. Furthermore, we identified alpaca () as species with typical Vγ9Vδ2 TCR rearrangements and currently aim to directly identify Vγ9Vδ2 T cells and BTN3. Other candidates to study this coevolution are the bottlenose dolphin () and the nine-banded armadillo () with genomic sequences encoding open reading frames for , and the extracellular part of . Dolphins have been shown to express Vγ9- and Vδ2-like TCR chains and possess a predicted -like gene homologous to human . The other candidate, the armadillo, is of medical interest since it serves as a natural reservoir for . In this study, we analyzed the armadillo genome and found evidence for multiple non-functional genes including genomic context which closely resembles the organization of the human, alpaca, and dolphin loci. However, no transcript could be detected in armadillo cDNA. Additionally, attempts to identify a functional rearrangement PCR failed. In contrast, complete gene segments preferentially rearranged with a homolog were cloned and co-expressed with a human Vγ9-chain in murine hybridoma cells. These cells could be stimulated by immobilized anti-mouse CD3 antibody but not with human RAJI-RT1B cells and HMBPP. So far, the lack of expression of rearrangements and renders the armadillo an unlikely candidate species for PAg-reactive Vγ9Vδ2 T cells. This is in line with the postulated coevolution of the three genes, where occurrence of Vγ9Vδ2 TCRs coincides with a functional BTN3 molecule.