Antigen-specific mdscs induce immunological tolerance in an experimental model of multiple sclerosis. generation of human mdscs from hematopoietic progenitors as a therapeutic tooll

En estudis previs realitzats en el nostre laboratori vam demostrar que la infusio de cel·lules de medul·la ossia (MO) transduides amb un autoantigen (MOG40-55), dirigit a la via de presentacio d'antigens per MHC de classe II, induien tolerancia immunologica en un model experimental d'esclerosi multiple, la encefalomielitis autoimmune experimental (EAE), tant en un abordatge preventiu com terapeutic (Eixarch et al. 2009). Per altra banda, l'absencia d'empelt de les cel·lules que expressaven la MOG va permetre eliminar la mieloablacio i ens va conduir a la hipotesis de que l'efecte terapeutic observat no estava mediat per cel·lules amb capacitat d'empeltar sino mes aviat per unes cel·lules mes madures que presentaven l'autoantigen de forma tolerogenica. Estudis posteriors van revelar que la majoria de les cel·lules que es generaven en els cultius de transduccio amb retrovirus de cel·lules de MO eren d'origen mieloide i que, de fet, eren cel·lules mieloide supressores (MDSCs, sigles en angles) (Gomez et al.2014). Per tant, la primera part d'aquesta tesi es va iniciar amb el proposit de caracteritzar millor aquestes MDSCs i de determinar si eren elles les responsables de la induccio de tolerancia immunologica observada en la EAE a mes d'estudiar els possibles mecanismes d'accio implicats. Amb aquesta finalitat es van transduir cel·lules de MO amb un vector retroviral que codificava per l'autoantigen (MOG40-55) o amb un vector control. Es van aillar les MDSCs i es van administrar als ratolins set dies abans (brac preventiu) o 13-14 dies despres (brac terapeutic) de la induccio de la EAE. Els resultats mostren que una unica administracio de MDSCs antigen-especifiques va induir tolerancia immunologica in vivo i que va prevenir i millorar els signes clinics de la EAE de manera antigen-especifica. A mes a mes, els animals toleritzats presentaven una neuropatologia reduida, proporcions de limfocits activats reduides i proporcions de limfocits B amb un fenotip regulador augmentades. D'altra banda, aquestes MDSCs generades ex vivo expressaven la molecula inhibitoria programmed death ligand 1 (PD-L1) i produien especies reactives d'oxigen, mecanismes associats amb la inhibicio dels limfocits T autoreactius i amb la induccio de tolerancia immunologica. Despres d'obtenir resultats prometedors amb les MDSCs antigen-especifiques de ratoli, vam decidir donar un pas mes i la segona part d'aquesta tesi esta enfocada a desenvolupar metodes eficients per generar MDSCs humanes a partir de progenitors hemopoetics per la seva potencial aplicacio clinica. Es van cultivar progenitors CD34+ obtinguts de productes d'aferesis de donants sans durant 9, 14 i 20 dies en presencia de diferents combinacions de citoquines basicament formades per SCF, TPO, FLT3-L, IL-3, GM-CSF i IL-6. Els resultats mostren que les MDSCs es poden generar de manera eficient a partir de progenitors hemopoetics i que les seves proporcions augmentaven amb els dies de cultiu. A mes, aquestes cel·lules expressaven la molecula immunosupressora PD-L1, eren molt poc al·loreactives, suprimien la proliferacio de cel·lules T induida per un estimul policlonal i reduien els nivells de les citoquines proinflamatories mentre que augmentaven el nivell de la citoquina immunomoduladora IL-10. Per aquests motius creiem que les MDSCs generades in vitro son una eina potencial pel tractament de malalties autoimmunes i per evitar la malaltia de l'empelt contra l'hoste despres del trasplantament. In previous studies conducted at our laboratory we demonstrated that infusion of bone marrow (BM) cells transduced with a self-antigen (MOG40-55), targeted to the MHC class II antigen presentation pathway, induced immunological tolerance in an experimental model of multiple sclerosis, the experimental autoimmune encephalomyelitis (EAE), both preventively and therapeutically (Eixarch et al. 2009). Moreover, the absence of engraftment of the MOG-expressing cells allowed to eliminate myeloablation and hypothesize that the therapeutic effect observed was not mediated by cells with engrafting potential but rather by a more mature cell type that expressed the self-antigen in a tolerogenic manner. Subsequent studies revealed that the majority of cells generated in standard retroviral transduction cultures of BM cells were of myeloid origin and that, indeed, were myeloid-derived suppressor cells (MDSCs) (Gomez et al. 2014). Therefore, the first part of this thesis was initiated with the purpose of better characterizing these MDSCs generated in BM retroviral transduction cultures and determining whether these cells were responsible for the induction of the immunological tolerance observed in the EAE model as well as studying the potential mechanisms of action involved. To this end, BM cells were transduced either with a retroviral vector encoding for the self-antigen or with a control vector. Both BM cells and isolated MDSCs were infused to the animals seven days before (preventive arm) or 13-14 days after (therapeutic arm) EAE induction. Results showed that a single infusion of antigen-specific MDSCs induced immunological tolerance in vivo and was able to prevent and ameliorate established EAE in an antigen-specific manner. In addition, tolerized animals presented a decreased neuropathology, reduced proportions of activated T cells and increased proportions of B cells with a regulatory phenotype. Moreover, the ex vivo generated MDSCs expressed the inhibitory molecule programmed death ligand 1 (PD-L1) and produced reactive oxygen species, mechanisms associated with the inhibition of autoreactive T cells and with the induction of immunological tolerance. After obtaining these promising results with the murine antigen-specific MDSCs, we decided to move one step further and, therefore, the second part of this thesis was aimed at developing efficient methods to generate human MDSCs from hematopoietic progenitor cells for its potential clinical application. To this end, CD34+ progenitor cells from apheresis products of healthy donors were cultured for 9, 14 and 20 days in the presence of different cytokine combinations mainly consisting of SCF, TPO, FLT3-L, IL-3, GM-CSF and IL-6. Results showed that MDSCs can be efficiently generated from hematopoietic progenitor cells and that their proportions significantly increased along with the days of the culture. Moreover, these cells expressed the immunosuppressive molecule PD-L1, had very little alloreactivity, suppressed polyclonal-induced T-cell proliferation and decreased the levels of proinflammatory cytokines while increasing the levels of the immunomodulatory cytokine IL-10. For these reasons, we believe that in vitro generated MDSCs constitute a potential tool for the treatment of autoimmune diseases and to prevent graft-versus-host disease (GVHD) in transplantation settings.