@phdthesis{Fichtner2020,
  author    = {Fichtner, Alina Suzann},
  title     = {Alpaca, armadillo and cotton rat as new animal models for nonconventional T cells: Identification of cell populations and analysis of antigen receptors and ligands},
  doi       = {10.25972/OPUS-16910},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-169108},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {In this thesis, three species were investigated for the conservation of two non-conventional T cell systems, the CD1d/ iNKT cell system and the BTN3/ Vγ9Vδ2 T cell system. Non-conventional T cells are αβ or γδ T cells that do not fit into the classical mode of antigen recognition and adaptive responses. These T cells recognize antigens different from classical peptide antigens and are not restricted to the polymorphic MHC molecules but rather to non-polymorphic antigen-presenting molecules. The iNKT cell subset is restricted by the lipid antigen-presenting molecule CD1d and carries out immunomodulatory functions by rapid cytokine secretion. The molecular basis of this system, the semi-invariant iNKT TCR chains and CD1d were proven to be expressed and compared to homologs in human and rodents. Cotton rats possess multiple members of the AV14 and BV8 family and only one isoform of CD1d which is comparable to findings in the rat. Moreover, the reactivity of primary cells to glycolipid antigens could be shown, and an iNKT cell-like population was detected in primary cells using newly developed cotton rat CD1d oligomers. These were also applied to test the capacity of CD1d to present typical glycolipid antigens to iNKT TCR transductants. In addition, expression of cotton rat iNKT TCR α and β chains in TCR-negative cell lines was used to show successful pairing and detection of glycolipids in the context of CD1d. In summary, the conservation of a functional CD1d/iNKT cell system in the cotton rat could be shown, and tools were developed to study this cell subset in the course of infectious diseases. The Vγ9Vδ2 T cell subset is the major γδ T cell subset in human peripheral blood and has the unique ability to contribute to immune surveillance by detecting pyrophosphorylated metabolites of isoprenoid synthesis that indicate cell stress, transformation or infection. Up to this date, phosphoantigen-reactive γδ T cells have only been shown in primate species. However, evidence for the existence and functional conservation of the genes implied in the BTN3/Vγ9Vδ2 T cell system was found in several placental mammal species, and two candidate species were chosen for further investigation. The nine-banded armadillo, a valuable model for leprosy research, was shown to possess homologous genes to TRGV9, TRDV2 and BTN3. In this study, the expression of productive rearrangements of TRDV2 gene segments could be shown in peripheral blood samples, but no evidence was found for the expression of a functional TRGV9 rearrangement or BTN3 molecules. Moreover, determinants of phosphoantigen-reactive Vγ9Vδ2 T cells and functional BTN3 molecules were found to still be prevalent in armadillo genes. This makes the armadillo an interesting model to study the structural determinants that allow phosphoantigen recognition by a functional Vγ9Vδ2 T cell subset although this species is merely a witness for a functional system in a placental mammal ancestor. In contrast, alpacas were shown to express functional Vγ9Vδ2 T cells which conserved many features of the human counterpart. Expression of Vγ9Vδ2 pairings could be shown by single-cell PCR and functional phosphoantigenreactive pairings were observed. This phosphoantigen reactivity was also shown in PBMC cultures with a newly developed antibody specific for alpaca Vδ2Jδ4 chains. Moreover, a more detailed study of the alpaca TCR repertoire showed similarities to "γδ high" species like camelids and cattle which possess an extended family of TRDV genes. The γ and δ loci of alpaca TCR genes were drafted based on genomic information and cDNA studies and provide an overview for more detailed studies. Conservation of phosphoantigen recognition by the single BTN3 molecule of alpacas was shown in 293T knock out cell lines, and BTN3 detection on PBMCs was investigated with a newly developed alpaca BTN3-specific antibody. These findings prove the existence of a functional BTN3-dependent phosphoantigen-reactive Vγ9Vδ2 T cell subset and provide a basis for the future study of this cell system in a non-primate species. Moreover, as the first non-primate candidate species with the BTN3/Vγ9Vδ2 T cell system the alpaca is an important outgroup for research in this field. The use of a single BTN3 variant in contrast to three human isoforms that work together renders the alpaca a unique and to this date indispensable model for Vγ9Vδ2 T cells. In conclusion, this study provides an overview of the applicability of new animal models in the study of the non-conventional T cell subsets iNKT cells and Vγ9Vδ2 T cells and leads the way for a better understanding of structural and functional relationships.},
  subject      = {T-Lymphozyt},
  language  = {en}
}
@article{HerrmannFichtnerKarunakaran2020,
  author    = {Herrmann, Thomas and Fichtner, Alina Suzann and Karunakaran, Mohindar Murugesh},
  title     = {An Update on the Molecular Basis of Phosphoantigen Recognition by Vγ9Vδ2 T Cells},
  series = {Cells},
  volume    = {9},
  journal   = {Cells},
  number    = {6},
  issn      = {2073-4409},
  doi       = {10.3390/cells9061433},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-207937},
  year      = {2020},
  abstract  = {About 1-5\% of human blood T cells are Vγ9Vδ2 T cells. Their hallmark is the expression of T cell antigen receptors (TCR) whose γ-chains contain a rearrangement of Vγ9 with JP (TRGV9JP or Vγ2Jγ1.2) and are paired with Vδ2 (TRDV2)-containing δ-chains. These TCRs respond to phosphoantigens (PAg) such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is found in many pathogens, and isopentenyl pyrophosphate (IPP), which accumulates in certain tumors or cells treated with aminobisphosphonates such as zoledronate. Until recently, these cells were believed to be restricted to primates, while no such cells are found in rodents. The identification of three genes pivotal for PAg recognition encoding for Vγ9, Vδ2, and butyrophilin (BTN) 3 in various non-primate species identified candidate species possessing PAg-reactive Vγ9Vδ2 T cells. Here, we review the current knowledge of the molecular basis of PAg recognition. This not only includes human Vγ9Vδ2 T cells and the recent discovery of BTN2A1 as Vγ9-binding protein mandatory for the PAg response but also insights gained from the identification of functional PAg-reactive Vγ9Vδ2 T cells and BTN3 in the alpaca and phylogenetic comparisons. Finally, we discuss models of the molecular basis of PAg recognition and implications for the development of transgenic mouse models for PAg-reactive Vγ9Vδ2 T cells.},
  language  = {en}
}
@article{HerrmannKarunakaranFichtner2020,
  author    = {Herrmann, Thomas and Karunakaran, Mohindar Murugesh and Fichtner, Alina Suzann},
  title     = {A glance over the fence: Using phylogeny and species comparison for a better understanding of antigen recognition by human γδ T-cells},
  series = {Immunological Reviews},
  volume    = {298},
  journal   = {Immunological Reviews},
  number    = {1},
  doi       = {10.1111/imr.12919},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-218373},
  pages     = {218 -- 236},
  year      = {2020},
  abstract  = {Both, jawless and jawed vertebrates possess three lymphocyte lineages defined by highly diverse antigen receptors: Two T-cell- and one B-cell-like lineage. In both phylogenetic groups, the theoretically possible number of individual antigen receptor specificities can even outnumber that of lymphocytes of a whole organism. Despite fundamental differences in structure and genetics of these antigen receptors, convergent evolution led to functional similarities between the lineages. Jawed vertebrates possess αβ and γδ T-cells defined by eponymous αβ and γδ T-cell antigen receptors (TCRs). "Conventional" αβ T-cells recognize complexes of Major Histocompatibility Complex (MHC) class I and II molecules and peptides. Non-conventional T-cells, which can be αβ or γδ T-cells, recognize a large variety of ligands and differ strongly in phenotype and function between species and within an organism. This review describes similarities and differences of non-conventional T-cells of various species and discusses ligands and functions of their TCRs. A special focus is laid on Vγ9Vδ2 T-cells whose TCRs act as sensors for phosphorylated isoprenoid metabolites, so-called phosphoantigens (PAg), associated with microbial infections or altered host metabolism in cancer or after drug treatment. We discuss the role of butyrophilin (BTN)3A and BTN2A1 in PAg-sensing and how species comparison can help in a better understanding of this human Vγ9Vδ2 T-cell subset.},
  language  = {en}
}