INTRODUCTION: Crohn's disease (CD) is a disabling chronic enteropathy sustained by a harmful T-cell response toward antigens of the gut microbiota in genetically susceptible subjects. Growing evidence highlights the safety and possible efficacy of mesenchymal stem cells (MSCs) as a new therapeutic tool for this condition. Therefore, we aimed to investigate the effects of bone marrow-derived MSCs on pathogenic T cells with a view to clinical application. METHODS: T-cell lines from both inflamed and non-inflamed colonic mucosal specimens of CD patients and from healthy mucosa of control subjects were grown with the antigen muramyl-dipeptide in the absence or presence of donors' MSCs. The MSC effects were evaluated in terms of T-cell viability, apoptotic rate, proliferative response, immunophenotype, and cytokine profile. The role of the indoleamine 2,3-dioxygenase (IDO) was established by adding a specific inhibitor, the 1-methyl-DL-tryptophan, and by using MSCs transfected with the small interfering RNA (siRNA) targeting IDO. The relevance of cell-cell contact was evaluated by applying transwell membranes. RESULTS: A significant reduction in both cell viability and proliferative response to muramyl-dipeptide, with simultaneous increase in the apoptotic rate, was found in T cells from both inflamed and non-inflamed CD mucosa when co-cultured with MSCs and was reverted by inhibiting IDO activity and expression. A reduction of the activated CD4(+)CD25(+) subset and increase of the CD3(+)CD69(+) population were also observed when T-cell lines from CD mucosa were co-cultured with MSCs. In parallel, an inhibitory effect was evident on the expression of the pro-inflammatory cytokines tumor necrosis factor-α, interferon-γ, interleukin-17A and -21, whereas that of the transforming growth factor-β and interleukin-6 were increased, and production of the tolerogenic molecule soluble HLA-G was high. These latter effects were almost completely eliminated by blocking the IDO, whose activity was upregulated in MSCs co-cultured with CD T cells. The use of a semipermeable membrane partially inhibited the MSC immunosuppressive effects. Finally, hardly any effects of MSCs were observed when T cells obtained from control subjects were used. CONCLUSION: MSCs exert potent immunomodulant effects on antigen-specific T cells in CD through a complex paracrine and cell-cell contact-mediated action, which may be exploited for widespread therapeutic use.

Centro di Ricerca di Medicina Rigenerativa Fondazione IRCCS Policlinico San Matteo Piazzale Golgi 19 Pavia 27100 Italy

Chirurgia Generale 1 Fondazione IRCCS Policlinico San Matteo Università di Pavia Piazzale Golgi 19 Pavia 27100 Italy

Clinica Medica 1 Dipartimento di Medicina Interna Fondazione IRCCS Policlinico San Matteo Università di Pavia Piazzale Golgi 19 Pavia 27100 Italy

Clinica Medica 1 Dipartimento di Medicina Interna Fondazione IRCCS Policlinico San Matteo Università di Pavia Piazzale Golgi 19 Pavia 27100 Italy Centre for the Study and Cure of Inflammatory Bowel Disease Clinica Medica 1 IRCCS San Matteo Hospital Foundation University of Pavia Piazzale Golgi 19 Pavia 27100 Italy

Clinica Medica 3 Dipartimento di Medicina Interna Fondazione IRCCS Policlinico San Matteo Università di Pavia Piazzale Golgi 19 Pavia 27100 Italy

Dipartimento di Medicina Occupazionale Ergonomia e Disabilità Laboratorio di Nanotecnologia Fondazione IRCCS Salvatore Maugeri Università di Pavia Via Maugeri 8 10 Pavia 27100 Italy Dipartimento di Medicina Molecolare Centro di Ingegneria Tissutale INSTM UdR Pavia Università di Pavia Pavia 27100 Italy

Dipartimento di Onco Ematologia Pediatrica e Medicina Trasfusionale Ospedale Pediatrico Bambino Gesù Via Sant'Onofrio 4 Rome 00165 Italy

International Clinical Research Center St Anne's University Hospital and Masaryk University Pekarska 53 Brno 656 91 Czech Republic

Laboratori di Oncoematologia Pediatrica Fondazione IRCCS Policlinico San Matteo Piazzale Golgi 19 Pavia 27100 Italy

Laboratorio di Ematologia Fondazione IRCCS Policlinico San Matteo Piazzale Golgi 19 Pavia 27100 Italy

Servizio di Immunogenetica Immunoematologia e Medicina Trasfusionale Fondazione IRCCS Policlinico San Matteo Università di Pavia Piazzale Golgi 19 Pavia 27100 Italy

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