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fbtwitterlinkedinvimeoflicker grey 14rssslideshare1
Pentier, Johanne
Languages: English
Types: Doctoral thesis
Subjects: QH426, R1
Human T-cells play a major role in controlling and clearing Mycobacterial infections. The adaptive immune system deploys a complex network of specialised T-cell subsets in order to tailor an optimum immune response. Two categories of T-cells have been described that are characterised by the ligands they recognise: “conventional” T-cells (polymorphic, HLA-restricted, peptide-specific) and “unconventional” T-cells (non-polymorphic, restricted by HLA-like molecules, non-peptide-specific). Both T-cell categories were shown to be important for the elimination of cells infected with Mycobacterium tuberculosis (M. tuberculosis) and their role, specificities and functionalities are under active investigation in order to develop optimum vaccination strategies. A large interest in unconventional T-cells, such as MR1-restricted MAITs or CD1-specific T-cells, and their role in mycobacterial infections has recently arisen. I initiated my studies by dissecting T-cell responses generated during direct ex vivo boosting of PBMCs with antigen presenting cells that had phagocytosed Mycobacterium smegmatis (M. smegmatis). M. smegmatis is a non-pathogenic bacterium and is mainly eliminated by the innate immune system. However, T-cells might respond to M. smegmatis antigens and therefore play a role in clearing the pathogen. Using polychromatic flow cytometry, I successfully identified major CD3+ conventional and unconventional M. smegmatis-specific T-cell populations and evaluated their respective frequencies and distribution. The identification of a significant frequency of M. smegmatis-specific unconventional MAITs pushed me to further analyse the specificity of this interesting T-cell subset. At the time of my studies, the ligand(s) presented by MR1 to MAITs were still undiscovered. However, structural models of MR1 groove moiety provided evidences that MR1 could potentially present peptides to MAITs. Therefore, I attempted to identify the molecular and cellular mechanisms by which an M. tuberculosis-specific MAIT clone recognises peptide loaded on MR1 and to refold this MHC-like protein. Vaccination strategies have been mainly focusing on targeting CD8 T-cells, known to be essential for the host defence against mycobacterial infections. Therefore a huge effort is made to discover new immunodominant mycobacterial epitopes. Collaborators isolated the HLA-A*0201-restricted D454 T-cell clone specific to the LLDAHIPQL epitope derived from the highly immunogenic Esx-G protein. The LLDAHIPQL sequence is conserved across mycobacterial species thus offering potential for pan-mycobacterial vaccination. I aimed at proving that D454 TCR binds to HLA-A*0201-LLDAHIPQL. I successfully obtained an HLA-A*0201-LLDAHIPQL crystal structure, the first bacterially-derived HLA-peptide complex, and identified the key mechanisms involved in the molecular recognition of HLA-A*0201-LLDAHIPQL by a conventional TCR.
  • The results below are discovered through our pilot algorithms. Let us know how we are doing!

    • Introduction ..................................................................................................51 T-cell responses to bacteria ............................................................................51 Mycobacterium smegmatis as a model to study T-cell responses to bacteria 52 Aims of the study............................................................................................52 Results............................................................................................................53 Experimental Approach..................................................................................53 Monitoring growth of M. smegmatis in culture..............................................54 Using DC to present M. smegmatis antigens..................................................56 A lung epithelial cell line phagocytoses M. smegmatis..................................58 M. smegmatis-loaded A549 cells induce specific T-cell responses ...............61 Dissection of M. smegmatis-specific T-cell responses in multiple donors M.
    • D454 TCR size exclusion chromatogram of pooled anion exchange fractions. ...........125 Figure 5.11. Second purification of D454 TCR by size exclusion chromatography. A.
    • D454 TCR size exclusion chromatogram of pooled anion exchange fractions. ...........126 Figure 5.12. Estimation of the D454 binding affinity to HLA-A*0201-LLDAHIPQL and HLA-A*0201-MIDAHIPQV. ................................................................................127 Figure 5.13. Comparison of the conformation of LLDAHIPQL and MIDAHIPQV peptides inside the A2 groove. ......................................................................................130 Figure 5.14. Comparison of LLDAHIPQL and MIDAHIPQV residues at position P1, P2 and P9. .....................................................................................................................132 Figure 6.1 MAIT TCR recognition of MR1-vitamin antigens......................................138 monocytes and macrophages (Yu and Gaffen, 2008, Liang et al., 2006). In addition, Fifteen year follow up of trial of BCG vaccines in south India for tuberculosis prevention. 1999. Indian J Med Res, 137, 14 p following p571.
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