Functional and phenotypical characterization of peptide-specific PLZF+ innate-like T cells

• Applicant: Dr. Hristo Georgiev
• Subject Area: Immunology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 470136623

Peptide-specific PLZF+ innate-like (PIL) T cells are a recently described T cell subset exhibiting innate-like features. Reminiscent of invariant natural killer T (iNKT) cells, PIL T cells are positively selected on double positive (DP) thymocytes and differentiate into three functional effector subsets during their development in the thymus. However, in contrast to iNKT cells, which recognize lipid antigens, PIL T cells recognize peptide antigens.In a recent study, we have generated a novel transgenic mouse line allowing specific expansion of PIL T cells under steady state conditions. This study elucidated the mechanisms governing PIL T cell selection but did not aim to provide in depth analysis of their T cell receptor (TCR) repertoire diversity nor of their essential function(s) under steady state or disease conditions.In this project, we will perform high-throughput single cell TCR sequencing and transcriptome analysis of sorted PIL T cells, which will allow us to identify PIL T cell developmental trajectories, compare PIL T cells to iNKT cells at transcriptome level and explore the diversity within the PIL T cell TCR repertoire. These data will be crucial to determine characteristic TCR clones present in the PIL T cell pool. Further, we will examine if a defined TCR specificity can commit developing thymocytes to the innate-like T cell developmental pathway by generating retrogenic mice harboring monoclonal T cells expressing TCR sequences selected from the PIL T cell TCR repertoire. In addition, we will establish a set of NFAT-GFP reporter cell lines expressing defined PIL T cell TCR sequences. We will exploit this system to screen antigen presenting cells from various tissues for antigen recognition in in vitro assays. Therefore, these experiments will help us to elucidate the systemic distribution and abundance of putative peptide antigens involved in PIL T cell selection and activation. Finally, we will investigate PIL T cell distribution and localization in peripheral organs, and inquire for anti-tumor function in a melanoma tumor model. By monitoring the composition and the activation status of infiltrating lymphocytes, we will gain insights on whether PIL T cells can infiltrate solid tumor tissues and if they can modulate local anti-tumor immune responses.

• DFG Programme: Research Grants