Mechanisms of antigen-recognition by gamma/delta T lymphocytes
Project management at the University of Würzburg:
Vg9/d2 T lymphocytes represent a T cell subpopulation which is suggested to play a role as a first line of defense in the immune system and to have a strong immunmodulatory effect. To understand the function of these still enigmatic cells, it is important to elucidate their mechanism of antigen recognition, which substantially differs from that of MHC restricted ab T cells.Initially we focused on isolating and characterizing bacterial Vg9/d2 T cell ligands. We identified 3-formyl-1-butyl pyrophosphate and its +14 amu longer chain homologue as the Vg9/d2 T cell stimulating phosphoantigens in E. coli . Furthermore, we demonstrated that the 1-deoxy-D-xylulose 5-phosphate pathway of isoprenoid biosynthesis is essential for the Vg9/d2 T cell stimulatory activity of bacteria. Therefore this part of our work was successfully brought to an end.In addition, we discovered the aminobisphosphonates as a new class of Vg9/d2 T cell stimulating antigens. The structural requirements for recognition of these compounds by the Vg9/d2 T cell receptor (TCR) will be studied in the next period. This will be done by synthesizing different aminobisphosphonates (variation of the carbon chain, introduction of diverse substituents) and testing them in our bioassays. Furthermore, we will address the question how these aminobisphosphonates are recognized by Vg9/d2 T cells. By using fluorescence- or radioactive labeled pamidronate we will investigate if these compounds are bound to extra cellular structures or are active by intracellular mechanisms. A possible accumulation of phosphoantigens caused by aminobisphosphonates will be analyzed by means of direct detection of these compounds.In order to perform a molecular analysis of the interaction of Vg9/d2 TCR with its ligands, cell lines have been established, which, after transduction with genes of the TCR and costimulatory receptors, can be activated by phosphoantigens. With the help of these lines, sites of TCR-ligand interaction shall be mapped in detail by mutagenesis of the TCR. Different types of ligands (Pyrophosphate containing ligands, aminobisphosphonates, SEA) will be compared for their stimulation of TCR mutants. Also, the newly established heterologous system (stimulation of Vg9/d2 TCR expressing mouse cell lines by phospholigands presented by human cells) shall be used as device for the generation of monoclonal antibodies interfering with the recognition of phosphoantigens. Finally, the experiments will be continued on the development of soluble, oligomeric TCR which than will be used as a new tool for the identification of cell bound ligands of Vg9/d2 TCR.
Projekt period: from 05.1999 to 04.2004
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