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

Alina Suzann Fichtner, Mohindar Murugesh Karunakaran, Lisa Starick, Richard W. Truman, Thomas Herrmann

1-5% of human blood T cells are Vγ9Vδ2 T cells whose T cell receptor (TCR) contain aTRGV9/TRGJPrearrangement and aTRDV2comprising 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 thatTRGV9, TRDV2, andBTN3genes coevolved and emerged together with placental mammals. Furthermore, we identified alpaca (Vicugna pacos) 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 (Tursiops truncatus) and the nine-banded armadillo (Dasypus novemcinctus) with genomic sequences encoding open reading frames forTRGV9, TRDV2, and the extracellular part ofBTN3. Dolphins have been shown to express Vγ9- and Vδ2-like TCR chains and possess a predictedBTN3-like gene homologous to humanBTN3A3. The other candidate, the armadillo, is of medical interest since it serves as a natural reservoir forMycobacterium leprae. In this study, we analyzed the armadillo genome and found evidence for multiple non-functionalBTN3genes including genomic context which closely resembles the organization of the human, alpaca, and dolphinBTN3A3loci. However, noBTN3transcript could be detected in armadillo cDNA. Additionally, attempts to identify a functionalTRGV9/TRGJPrearrangementviaPCR failed. In contrast, completeTRDV2gene segments preferentially rearranged with aTRDJ4homolog 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-RT1Blcells and HMBPP. So far, the lack of expression ofTRGV9rearrangements andBTN3renders 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.