This project combines structural and functional studies to decipher TSSP function. Because TSSP is a critical regulator of autoimmune diabetes, these results could to new strategies to control TSSP function and consequently the development of autoimmune diabetes.
TSSP is a new protease that controls by yet unknown mechanisms the development of self-reactive T cells directly involved in the development of autoimmune diabetes. The main objective of this project are to elucidate TSSP function and finds potential substrates. Beside fundamental knowledge in the understanding of the mechanisms of central tolerance, this project may lead to the identification of modulators of TSSP function with clinical interest in the field of autoimmune diabetes.
This project combine crystallographic studies, a critical step in the understanding of the enzymatic properties of a protease (type of substrates and design of inhibitors) as well as enzymatic and biological studies in vitro and in vivo that collectively will permit an integrated view of TSSP function.
We successfully produced recombinant protein, a key step for the analysis of a 3-D structure and enzymatic activity. We also identified several potential substrates that will help develop enzymatic assays. Our preliminary studies points to an expected and original function of TSSP in the regulation of the antigen presentation pathway that will be further characterized in this project.
Deciphering TSSP 3D-structure and defining enzymatic assays are critical step to evaluate TSSP function in Human and to design new strategies to control autoimmune diabetes. Our recent observations on TSSP function open new perspective on the mechanisms that may control the maturation of antigen presenting cells that, if confirmed, will be study in future projects.
This project contributed to the publication of 3 scientific articles and 1 revew.
The functional T cell repertoire is shaped in the thymus by positive and negative selection through interactions with MHC/peptide complexes expressed by thymic epithelial cells and bone marrow-derived antigen (Ag) presenting cells. The size of the CD4 T cell compartment and its diversity are determined by the complexity of the self-peptide repertoire presented by MHC class II molecules in the thymus. Several cysteine and aspartic proteases have been involved in the generation of MHC class II/ peptide complexes, but indirect evidence suggest that serine and metalloproteases may also participate to Ag processing.
We identified a novel serine protease named Thymus Specific Serine Protease (TSSP) that is highly expressed in the endosomal compartment of thymic cortical epithelial cells suggesting that TSSP may have a role in the selection of the CD4 T cell repertoire. TSSP is however also expressed moderately by activated B cells and bone marrow-derived dendritic cells (DC). TSSP was initially described as a gene linked to a diabetes susceptibility locus in humans.
We showed that TSSP-deficient mice (TSSP-/-) express normal MHC class II level, have normal number of all immune cells, including CD4 T cells and show no gross immune dysfunction. While TSSP-deficiency has no quantitative impact on the development of CD4 T cells expressing polyclonal TCR, we found that TSSP is necessary for the intra-thymic development of some CD4 T cell specificities. Hence, positive selection of I-Ab restricted OTII and Marylin CD4 T cells are impaired in B6-TSSP-/- mice. Since TSSP was linked to a diabetes susceptibility locus in humans we analyzed whether TSSP-deficiency may affect the development of the functional polyclonal CD4 T cell repertoire in NOD-TSSP-/- mice. Concerning foreign Ags we found that while the response of these mice to five different protein were unaffected, the CD4 T cell response to HEL was severely impaired. Comparative analysis of the CDR3 sequences of HEL-specific CD4 T cells showed that TSSP is necessary for the diversification of the HEL-specific CD4 T cell repertoire and development of high affinity HEL-reactive CD4 T cells. In a similar analysis of the response of NOD-TSSP-/- mice to immunodominant peptide of islet Ags showed that these mice are tolerant to one epitope of Phogin (IA2ß755-777) out of seven Ags tested. Tolerance in this case resulted from negative selection by TSSP-deficient DC of the specific CD4 T cells in the thymus. These analyses suggest that the development of roughly 15% (2 out of 13 Ags) of the functional CD4 T cell repertoire requires TSSP function in the thymus. The limited and specific impact of TSSP-deficiency on CD4 T cell development points to a role of TSSP in the class II presentation pathway. In contrast to Cathepsin L, TSSP has no generic role in this process but would edit the peptide repertoire presented by class II molecules in the thymus and consequently shape the functional CD4 T cell repertoire through either positive or negative selection.
Such editing function of TSSP has important physiological consequence. Indeed we showed that NOD-TSSP-/- mice are completely protected from diabetes and severe insulitis. Diabetes resistance resulted from impaired intra-thymic development of diabetogenic T cells.
TSSP is therefore the first example of a protease that may through peptide repertoire editing in the thymus shape the functional CD4 T cell repertoire and consequently immune responsiveness.
In this project we propose to decipher the function of TSSP and its impact on the development of the functional CD4 T cell repertoire. By combining structural and functional studies we also aim at characterizing TSSP substrates, some of which may be islet-Ags to which TSSP-deficient NOD mice are tolerant. Thus, while providing fundamental knowledge on this novel protease, this project may also permit to identify novel islet Ags that may be essential for diabetes development.
Madame Sylvie GUERDER (INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE - Délégation régionale Midi-Pyrénées Limousin) – firstname.lastname@example.org
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
INSERM U 624 INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE - DELEGATION PACA
Université Aix Marseille 2 - AFMB UNIVERSITE AIX-MARSEILLE II [DE LA MEDITERRANEE]
INSERM-CPTP INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE - Délégation régionale Midi-Pyrénées Limousin
Help of the ANR 570,000 euros
Beginning and duration of the scientific project: - 36 Months