Silvia Gregori
Silvia Gregori
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affiliation: San Raffaele Scientific Institute
research area(s): Experimental Medicine, Immunity And Infection
Course: Basic and Applied Immunology
University/Istitution: Università Vita-Salute San Raffaele
Curriculum Vitae
Since 2011: Research Scientist at Fondazione San Raffaele del Monte Tabor and Group Leader at San Raffaele Telethon Institute for Gene Therapy, Milan, Italy.
2006-2010: Research Scientist at Fondazione San Raffaele del Monte Tabor and Project leader at San Raffaele Telethon Institute for Gene Therapy, Milan, Italy.
2003-2006 Project leader at San Raffaele Telethon Institute for Gene Therapy, Milan, Italy.
2001-2002 Senior PostDoctoral Fellow, San Raffaele Telethon Institute for Gene Therapy, Milan, Italy.
1999-2001 PostDoctoral Fellow, Roche Milano Ricerche, Milan, Italy
1999 PhD in Biotechnological Applications, University of Milan, Milan, Italy
1995-1999 Graduate Student, (Prof. L. Adorini's laboratory), Roche Milano Ricerche, Milan, Italy
1994-1995 Fellow, Dept. of Immunologo (Prof. GF Bottazzo's Laboratory), London Hospital, London, England
1994 B.Sc., University of Milan, Milan, Italy
1992-1994 Trainee, Department of Pharmacology, School of Medicine, University of Milan, Italy

Scientific Activities
Published 50 papers in international scientific journals and contributed 6 chapters to books, on the following topics: Cytokines, Regulatory T cells, Immunological Tolerance.
nvited as a speaker to 30 International Meetings or Workshops in the last 5 years.
Co-Inventor in 3 international patents, issued on topics related to tolerogenic compounds and methods to generate regulatory T cells.
Ad hoc reviewer of International Scientific Journals
Member of the Italian Society of Immunology, Editor of the immunology section of the Journal of Biomedicine and Biotechnology, Review Editor of Frontiers in Primary Immuno-deficiencies
Tolerogenic dendritic cells: biology and translational clinical application. Dendritic cells (DC) represent pacemakers of the immune system since they play a major role as antigen presenting cells in inducing adaptive immune responses on the one hand and are critically involved in promoting and maintaining immunological tolerance on the other. The latter function is mediated by specialized subsets of DC, named tolerogenic DC, as well as by DC activated or differentiated in the presence of specific biological or chemical agents in vitro. Suppression by tolerogenic DC is primarily mediated via the induction of regulatory T (Tr) cells. Tr cells are recognized fundamental for the induction and maintenance of immune tolerance. Both naturally occurring Tr (thymic derived; FOXP3+) and adaptive type 1 Tr (Tr1) cells (induced in the periphery; IL-10++IL-4-) have been demonstrated to be effective in controlling immune responses against a variety of antigens in vitro and in vivo. We focus our research in identifying the role of tolerogenic DC in promoting Tr1 cells. We identified and characterized a new subset of tolerogenic DC, termed DC-10 for its outstanding ability to produce IL-10. DC-10 are present in vivo in peripheral blood and secondary lymphoid organs and can be generated in vitro from peripheral monocytes in the presence of exogenous IL-10. DC-10 are characterized by the expression of high levels of membrane-bound HLA-G1 and of other tolerogenic molecules including ILT2, ILT3, and ILT4. In vitro data demonstrate the role of DC-10 in efficiently promoting Tr1 cells, and convert effector allergen-specific effector T cells into Tr1 cells. The expression of high levels of membrane-bound HLA-G, ILT4, and IL-10 by DC-10 is critical to the generation of Tr1 cells.
HLA-G is a non-classical HLA class I molecule that differs from other HLA class I molecules for (i) low polymorphism, (ii) restricted tissue distribution, (iii) slow turnover, (iv) limited peptide diversity, and (v) its immunosuppressive properties. Despite the reduced variability in the coding region of the HLA-G locus, several polymorphisms have been identified in the 5" up-stream regulatory region (URR) and in the 3" untranslated region (UTR) of the gene which influence HLA-G expression. At least two variants in the 3"UTR of HLA-G are associated with regulation of HLA-G expression, i) the 14 base pair insertion/deletion (14bp ins/del) leads to a shorter and more stable mRNA and to an increased HLA-G expression, and ii) the presence of guanine (G) at position +3142 increases the affinity of microRNA (miRNA) and directly repress HLA-G expression. The 14bp ins/del has been shown to be associated with tolerance in different clinical conditions including autoimmunity, pathological pregnancy, recurrent spontaneous abortions, and pre-eclampsia. So far the biological mechanisms underlying tolerance associate to 3" UTR polymorphisms of HLA-G has been poorly elucidate. The role of membrane-bound HLA-G1 in promoting Tr1 cells via DC-10, prompt us to hypothesize that DC-10 are critical players in tolerance associated with 3" UTR polymorphisms of HLA-G.
The overall objectives of this research project are:
1. To further define DC-10 biology focusing on the identification of specific markers and in vivo localization in healthy and pathological conditions as well as on better defining their mode of tolerance induction in vivo. Translational DC-10 therapy approaches to restore/induce tolerance in T-cell mediated diseases.
2. To elucidate the biological mechanisms of tolerance associated with HLA-G polymorphism assuming the central role played by DC-10.
Role of regulatory T cells in transplantation tolerance. This area of research is part of the core research at HSR-TIGET, aimed at characterizing different subsets of Tr cells and identifying specific molecules involved in their function, and includes the following projects:
1. Cellular and molecular characterization of immunological mechanisms, focusing on tolerogenic DC and Tr cells, in patients affected by genetic diseases who underwent allogeneic HSC transplantations. Thalassemic patients with post-transplant persistent mixed chimerism are included in these studies;
2. To establish protocols to generate homogeneous population of antigen-specific Tr cells by means of gene transfer approach to be use a cell therapy to restore/induce tolerance in T-cell mediated diseases;
3. Immunogenetic studies aimed at identifying genetic factors predisposing to transplant rejection, graft-versus-host disease or engraftment.
1. Roncarolo MG., Gregori S., Lucarelli B., Ciceri F., and Bacchetta R. Clinical Tolerance in Allogeneic Hematopoietic Stem Cell Transplantation. Immunol Rev. 2011, 241(1):145-63.
2. Magnani C.F., Alberigo G., Bacchetta R., Serafini G., Andreani M., Roncarolo M.G., and Gregori S. Type 1 Regulatory T Cells Lyse Myeloid Antigen Presenting Cells via a Granzyme B- and HLA Class I- Dependent Mechanism. Eur J Immunol. 2011
3. Passerini L., Di Nunzio S., Gregori S., Gambineri E., Cecconi M., Seidel M.G., Cazzola G., Perroni L., Tommasini A., Vignola S., Guidi L., Roncarolo M.G., and Bacchetta R. Functional type 1 regulatory T cells develop regardless of FOXP3 mutations in patients with IPEX syndrome. Eur J Immunol. 2011 Apr;41(4):1120-31.
4. Gregori S. Dendritic cells in networks of immunological tolerance. Tissue Antigens. 2011 Feb;77:89-99.
5. Carosella ED., Gregori S., Rouas-Freiss N., Lemaoult J., Menier C., Favier B. The role of HLA-G in immunity and hematopoiesis. Cell Mol Life Sci. 2011 Feb;68(3):353-68.
6. Rossetti M., Gregori S., Hauben E., Brown BD., Sergi LS., Naldini L., Roncarolo MG. HIV-1-Derived Lentiviral Vectors Directly Activate Plasmacytoid Dendritic Cells, Which in Turn Induce the Maturation of Myeloid Dendritic Cells. Hum Gene Ther. 2011 Jan 14.
7. Bacchetta R.*, Gregori S.*, Serafini G., Sartirana C., Schulz U., Zino E., Tomiuk S., Jansen U., Ponzoni M., Paties CT., Fleischhauer K., Roncarolo MG. Molecular and functional characterization of allogantigen-specific anergic T cells suitable for cell therapy. *Equal contributors. Haematologica. 2010 Dec;95(12):2134-43.
8. Rossetti M., Gregori S., Roncarolo MG. Granulocyte-colony stimulating factor drives the in vitro differentiation of human dendritic cells that induce anergy in naïve T cells. Eur J Immunol. 2010 Nov;40(11):3097-106. Epub 2010 Oct 19.
9. Gregori S., Tomasoni D., Pacciani V., Scirpoli M., Battaglia M., Magnani CF, Hauben E, Roncarolo MG. Differentiation of type 1 T regulatory cells (Tr1) by tolerogenic DC-10 requires the IL-10-dependent ILT4/HLA-G pathway. Blood. 2010 Aug 12;116(6):935-44.
10. Pacciani V.*, Gregori S.*, Chini L., Corrente S., Chianca M., Moschese V., Rossi P., Roncarolo MG, and Angelini F. Modulation of Allergen-specific T-cell Responses via Tolerogenic Dendritic Cells in House Dust Mites-Allergic Children. * Equal contribution J Allergy Clin Immunol. 2010
11. Gregori S., Magnani C.F., and Roncarolo MG., Role of HLA-G in the induction of adaptive Type 1 regulatory T cells. Human Immunology, 2009 70 (12):966-969.
12. Allan SE., Broady R., Gregori S., Himmel ME., Locke N., Roncarolo MG., Bacchetta R., Levings MK. CD4+ T-regulatory cells: toward therapy for human diseases. Immunol Rev. 2008 Jun;223:391-421.
13. Gregori S., Bacchetta R., Passerini L., Levings MK, and Roncarolo MG. Isolation, expansion and characterization of human natural and adaptive regulatory T cells. Methods Mol Biol. 2007;380:83-106.
14. Roncarolo MG., Gregori S., Battaglia M., Bacchetta R., Fleischhauer K., Levings MK. IL-10 secreting type 1 regulatory T cells in rodents and humans. Immunol Rev. 2006 Aug;212:28-50.
15. Gregori S., Mangia P., Bacchetta R., Tresoldi E., Bottazzoli F., Aversa G., Traversari K., Korthäuer U., and Roncarolo MG. An anti-CD45RO/RB monoclonal antibody modulates T-cell responses via induction of apoptosis and generation of regulatory T cells. JEM 2005 April 18;201(8):1293-1305.
16. Levings MK.*, Gregori S.*, Tresoldi E., and Roncarolo MG. Differentiation of Tr1 cells by immature dendritic cells requires IL-10 but not CD25+CD4+ Treg cells. Blood 2005 Feb 1;105(3):1162-9. Epub 2004 Oct 12. * Equal contribution.
17. Gregori S., Bacchetta R., Hauben H., Battaglia M., and Roncarolo MG. Regulatory T cells: prospective for clinical application in hematopoietic stem cells transplantation. Current Opinion in Haematology. 2005 Nov 12(6): 451-6.
18. Gregori S., Giarratana N., and Adorini L. Dynamics of pathogenic and suppressor T cells in the development of autoimmune diabetes. J Immunol. 2003 Oct 15;171(8):4040-7
19. Adorini L., Gregori S., Harrison L.C.. Understanding autoimmune diabetes: insights from mouse models. Trends Mol Med. 2002 Jan;8(1):31-8.
20. Gregori S., Giarratana N., Casorati M, and Adorini L. Vitamin D3 analogue administration enhanced regulatory T cells and protects NOD mice from autoimmune diabetes. Diabetes 2002, May;51(5):1367-74.
21. Trembleau S., Gregori S., Penna G., Gorny I., and Adorini L. IL-12 administration reveals diabetogenic T cells in genetically resistant I-Ealpha-transgenic nonobese diabetic mice: resistance to autoimmune diabetes is associated with binding of Ealpha-derived peptides to the I-A(g7) molecule. J Immunol. 2001, Oct 1;167(7):4104-14.
22. Gregori S., Casorati M., Amuchastegui S., Smiroldo S., Davalli A.M., and Adorini L. Regulatory T cells induced by MMF/1,25(OH)2D3 treatment mediate transferable transplantation tolerance. J. Immunol. 2001, Vol. 167: 1945-53.
Project Title:
Antigen-specific IL-10 engineered T cells: generation in vitro and preclinical validation in vivo
CD4+ type 1 regulatory T (Tr1) cells are important players in modulating immune responses. Tr1 cells produce high levels of IL-10, but no IL-4, and suppress T-cell responses via cytokine-mediated mechanisms and selective killing of target cells. Tr1 cells have been demonstrated to be effective in controlling immune responses against a variety of antigens(Ags) in vitro and in vivo. We established a method to generate Tr1 cells in vitro after priming of T cells in the presence of allogeneic antigen presenting cells and exogenous IL-10 or using tolerogenic dendritic cells (DC-10). Infusion of allo-specific Tr1 cells in patient with high risk of hematological malignancies after hematopoietic stem cell transplantation (HSCT) demonstrated the safety and feasibility of this approach. In line with these promising results, we will define new in vitro methods to obtain higher number and more potent/stable allo-specific Tr1 cells that will be used as cell therapy in vivo. To this aim, we constructed a lentiviral vector encoding human IL-10 and carrying a marker gene(LV-hIL-10). LV-hIL-10-transduced human CD4+ T cells acquired a Tr1-like phenotype and function.
The goal of this project is to generate Ag-specific Tr1 cells starting from naïve CD4+ cells or pathogenic effector T cells by means of IL-10 gene transfer with LV-hIL-10. Biological and functional in vitro characterization of the resulting T cells will be carried out. Using animal models we will perform in vivo safety studies and explore the efficacy of murine IL-10 engineered T cells. Studies in gene reporter mice we define the mode of tolerance induction mediated by these cells. Finally, humanized pre-clinical models of xenoGvHD and GvL will be used to determine the efficacy of human IL-10 engineered T cells in vivo. Results will lead to develop of an innovative therapeutic approach of immunotherapy for preventing GvHD after HSCT in patients affected by hematological malignancies.


References:
1. Pacciani V.*,Gregori S.*, Chini L., Corrente S., Chianca M., Moschese V., Rossi P., Roncarolo MG, and Angelini F. Modulation of Allergen-specific T-cell Responses via Tolerogenic Dendritic Cells in House Dust Mites-Allergic Children. * Equal contribution J Allergy Clin Immunol. 2010 Feb 10. [Epub ahead of print].
2. Gregori S., Tomasoni D., Pacciani V., Scirpoli M., Battaglia M., Magnani CF, Hauben E, Roncarolo MG. Differentiation of type 1 T regulatory cells (Tr1) by tolerogenic DC-10 requires the IL-10-dependent ILT4/HLA-G pathway. Blood. 2010 Aug 12;116(6):935-44. Epub 2010 May 6 IF: 10.555; Time cited 4, Source Scopus
3. Bacchetta R.*, Gregori S.*, Serafini G., Sartirana C., Schulz U., Zino E., Tomiuk S., Jansen U., Ponzoni M., Paties CT., Fleischhauer K., Roncarolo MG. Molecular and functional characterization of allogantigen-specific anergic T cells suitable for cell therapy. *Equal contributors. Haematologica. 2010 Dec;95(12):2134-43. Epub 2010 Aug 16.
4. Magnani C.F., Alberigo G., Bacchetta R., Serafini G., Andreani M., Roncarolo M.G., and Gregori S. Type 1 Regulatory T Cells Lyse Myeloid Antigen Presenting Cells via a Granzyme B- and HLA Class I- Dependent Mechanism. Eur J Immunol. 2011 Apr 6. doi: 10.1002/eji.201041120. [Epub ahead of print]
5. Roncarolo MG., Gregori S., Lucarelli B., Ciceri F., and Bacchetta R. Clinical Tolerance in Allogeneic Hematopoietic Stem Cell Transplantation. Immunol Rev.


Project Title:
Characterization of molecular signature and of the in vivo mode of action of HLA-G-expressing tolerogenic dendritic cells (DC-10)
Dendritic cells (DC) are initiators of the immune response. Increasing evidence support a key role for DC in limiting immunity and in promoting and maintaining peripheral tolerance. We recently identified a new subset of DC, termed DC-10 for its outstanding ability to produce IL-10. In vitro data demonstrate the role of DC-10 in efficiently promoting adaptive type 1 regulatory T (Tr1) cells, and convert effector allergen-specific effector T cells into Tr1 cells. In addition to their ability to secrete high levels of IL-10, DC-10 highly express the tolerogenic molecules ILT4 and HLA-G that are critically involved in the differentiation of Tr1 cells in vitro. DC-10 are present in vivo in peripheral blood and secondary lymphoid organs and can be generated in vitro. We are currently defining the molecular signature of DC-10. We performed compared microarray-based transcriptional profiling of in vitro generated DC-10 and immunogenic mature DC. We are validating the expression of the identified molecules using both in vitro differentiated and circulating DC-10. These analyses will allow us to define selective markers of DC-10 for their in vivo identification, not only in peripheral blood, but also in tissues. In addition to the specific markers, we expect to identify the expression of molecules that can be associated with homing of DC-10, which will allow to define their role in promoting tolerance in vivo.
The final goal of the project is to further characterize the role of DC-10 in promoting tolerance by defining their in vivo localization in tissues, and the tolerogenic properties of DC-10. Using pre-clinical mouse models as well as biological human samples we will investigate the tolerogenic properties of DC-10, focusing primarily on their ability to promote Tr1 cell differentiation. Results will highlight the tolerogenic properties of DC-10 and will open new perspectives in the use of these cells as therapeutics.

References:
1. Pacciani, V.*, Gregori, S.*, Chini, L., Corrente, S., Chianca, M., Moschese, V., Rossi, P., Roncarolo, M.G., and Angelini, F. (2010). Induction of anergic allergen-specific suppressor T cells using tolerogenic dendritic cells derived from children with allergies to house dust mites. J Allergy Clin Immunol 125, 727-736.* Equal contributors
2. Gregori, S., Tomasoni, D., Pacciani, V., Scirpoli, M., Battaglia, M., Magnani, C.F., Hauben, E., and Roncarolo, M.G. (2010). Differentiation of type 1 T regulatory cells (Tr1) by tolerogenic DC-10 requires the IL-10-dependent ILT4/HLA-G pathway. Blood 116, 935-944.
3. Gregori S. Dendritic cells in networks of immunological tolerance. Tissue Antigens. 2011 Feb;77:89-99.
4. Bacchetta R.*, Gregori S.*, Serafini G., Sartirana C., Schulz U., Zino E., Tomiuk S., Jansen U., Ponzoni M., Paties CT., Fleischhauer K., Roncarolo MG. Molecular and functional characterization of allogantigen-specific anergic T cells suitable for cell therapy. *Equal contributors. Haematologica. 2010 Dec;95(12):2134-43. Epub 2010 Aug 16.
5. Carosella ED., Gregori S., Rouas-Freiss N., Lemaoult J., Menier C., Favier B. The role of HLA-G in immunity and hematopoiesis. Cell Mol Life Sci. 2011 Feb;68(3):353-68. Epub 2010 Nov 30.