Novel Mycobacterium tuberculosis specific HLA-A*0201 restricted T cell epitopes in Tb patients

Marc Jacobsen¹, Helmy Rachman¹, Andrea Gutschmidt¹, Jörg Hakenberg², Hans J. Mollenkopf¹, Albert Neher³, Knut Feldmann⁴, Stefan H.E. Kaufmann¹

¹ Max-Planck-Insitute for Infection Biology, Dept. of Immunology, 10117 Berlin, Germany;
² Humboldt-University, Institute for Computer Science, Dept. Knowledgemanagement in Bioinformatics, 12489 Berlin, Germany;
³ Institute of Laboratory Medicine, Asklepios Clinics, Gauting, Germany ;
⁴ Zentralkrankenhaus Gauting, Germany;
^* Corresponding author. Present adress: Max-Planck-Institute for Infection Biology, 10117 Berlin, Germany.

Abstract

Previous studies suggested an important role for CD8⁺ T cells in the host defense against Mycobacterium tuberculosis (M.tb), the etiologic agent for human tuberculosis. To further characterize this host response we focused on M.tb specific proteins determined by genome comparison of twelve different mycobacterial species performing microarray analysis. Genome comparison confirmed by PCR analysis revealed seven genes which were identified to be exclusive in M.tb. We employed the MAPPP (MHC-class I Antigenic Peptide Processing Prediction) program to predict immunogenic peptides from these proteins. Restrictive criteria for proteasome cleavage and HLA-A*0201 binding revealed 92 peptides probably recognized by CD8⁺ T cells. These 9-10mer peptides and seven described immunodominant HLA-A*02 restricted M.tb epitopes were commercially synthesized and tested in short term immune cell assays for their capability to induce IFN-γ in peripheral blood mononuclear cells (PBMCs) from tuberculosis patients, PPD⁺ and PPD^- control donors. Intracellular IFN-γ straining and ELISPOT analysis revealed two peptides RLRRDNAEL (Rv795) and VLYDECDTL (Rv2655c) which stimulated CD8⁺ memory t cells only from tuberculosis patients. About 30% of the HLA-matched patients hah peptide specific T cells and the amount was approximately 0.1% of CD8⁺ T cells. Nearly identical results were observed for the described immunogenic peptides VLTDGNPPEV (19-kDa protein), GLPVEYLQV (Antigen 85A), and KLAGGVAVI (Mhsp65). PBMCs from control donors showed no reactivity with any peptide.
Our investigations revealed two novel M.tb specific epitopes which induce IFN-γ expression in CD8⁺ T cells from tuberculosis patients predicted by MAPPP. Further studies will determine the frequency of these immune cells during disease course and the role of the mycobacterial proteins coded by the genes Rv795 and Rv2655c in the host response against M.tb infection

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MAPPP - MHC-class I Antigenic Peptide Processing Prediction

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