The role of Cathepsin L in shaping a functional T cell repertoire

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The survival and developmental progression of immature T cells during thymic selection requires productive interactions of their T cell receptor (TCR) with ‘self-peptide’ (p)MHC ligands presented by cortical thymic epithelial cells (cTECs). This process is referred to as positive selection. The traditional view that positive selection supports effective anti-foreign responses by establishing the self-MHC-restriction of the repertoire has recently been challenged. Instead, it was suggested that positive selection mediates the seeding of the periphery with T cells of optimal ‘fitness’. Whether and how the functional competence of mature T cells is lastingly ‘imprinted’ by the pMHC ligands that are encountered during positive selection remains largely enigmatic.The crucial role of cTECs during positive selection is at least in part specified by unique proteolytic machineries that these cells use to generate peptides loaded onto MHC I or II. These proteolytic pathways are likely to endow cTECs with a ‘private’ pMHC ligandome that is different from that on other antigen presenting cells (APCs). For instance, cTECs process substrates of the MHCII loading pathway through Cathepsin L (Ctsl), whereas in other APCs, this occurs mostly through Cathepsin S. Knockout of Ctsl strongly decreases the number of CD4 T cells in the thymus.In preliminary work, we found that the CD4 T cell repertoire of CtslKO mice, irrespective of its reduced cellularity, harbors a ‘normal’ number of cells that recognize a prototypical foreign antigen, Listeriolysin O (LLO). However, these cells failed to undergo proliferative expansion in response to immunization. Similarly, bulk CD4 T cells from Ctsl-deficient mice displayed an impaired response to polyclonal stimulation in vitro and, upon adoptive transfer into lymphopenic hosts, underwent only limited ‘homeostatic’ expansion in vivo. These findings suggest that Ctsl-dependent pMHCII ligands on cTECs not only determine the cellularity, but also the functionality of the CD4 T cell repertoire.In the proposed project, we want to test the hypothesis that there is a substantial overlap in the TCR composition of CD4 T cells in WT or CtslKO mice, but that cells expressing such ‘shared’ TCRs are dysfunctional when selected in the absence of Ctsl. We will employ single-cell TCR sequencing to generate TCR inventories of LLO-specific CD4 T cells from WT and Ctsl-deficient mice to identify TCRs that are found in both genotypes. Representative ‘shared’ TCRs will be re-expressed as TCR transgenes in WT and Ctsl-deficient mice. This will enable us to systematically address the in vivo and in vitro functionality of cells expressing the very same TCR, yet positively selected in either the presence or absence of Ctsl. Furthermore, we will address the biochemical and transcriptional basis of how the functional fitness of CD4 T cells is imprinted through positive selection by Ctsl-dependent pMHC ligands.