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• W1977224177 abstract "Type 1 regulatory T (Tr1) cells are an inducible subset of CD4+ Tr cells characterized by high levels of interleukin (IL)-10 production and regulatory properties. Several protocols to generate human Tr1 cells have been developed in vitro. However, the resulting population includes a significant fraction of contaminating non-Tr1 cells, representing a major bottleneck for clinical application of Tr1 cell therapy. We generated an homogeneous IL-10–producing Tr1 cell population by transducing human CD4+ T cells with a bidirectional lentiviral vector (LV) encoding for human IL-10 and the marker gene, green fluorescent protein (GFP), which are independently coexpressed. The resulting GFP+ LV-IL-10–transduced human CD4+ T (CD4LV-IL-10) cells expressed, upon T-cell receptor (TCR) activation, high levels of IL-10 and concomitant low levels of IL-4, and markers associated with IL-10. Moreover, CD4LV-IL-10 T cells displayed typical Tr1 features: the anergic phenotype, the IL-10, and transforming growth factor (TGF)-β dependent suppression of allogeneic T-cell responses, and the ability to suppress in a cell-to-cell contact independent manner in vitro. CD4LV-IL-10 T cells were able to control xeno graft-versus-host disease (GvHD), demonstrating their suppressive function in vivo. These results show that constitutive over-expression of IL-10 in human CD4+ T cells leads to a stable cell population that recapitulates the phenotype and function of Tr1 cells. Type 1 regulatory T (Tr1) cells are an inducible subset of CD4+ Tr cells characterized by high levels of interleukin (IL)-10 production and regulatory properties. Several protocols to generate human Tr1 cells have been developed in vitro. However, the resulting population includes a significant fraction of contaminating non-Tr1 cells, representing a major bottleneck for clinical application of Tr1 cell therapy. We generated an homogeneous IL-10–producing Tr1 cell population by transducing human CD4+ T cells with a bidirectional lentiviral vector (LV) encoding for human IL-10 and the marker gene, green fluorescent protein (GFP), which are independently coexpressed. The resulting GFP+ LV-IL-10–transduced human CD4+ T (CD4LV-IL-10) cells expressed, upon T-cell receptor (TCR) activation, high levels of IL-10 and concomitant low levels of IL-4, and markers associated with IL-10. Moreover, CD4LV-IL-10 T cells displayed typical Tr1 features: the anergic phenotype, the IL-10, and transforming growth factor (TGF)-β dependent suppression of allogeneic T-cell responses, and the ability to suppress in a cell-to-cell contact independent manner in vitro. CD4LV-IL-10 T cells were able to control xeno graft-versus-host disease (GvHD), demonstrating their suppressive function in vivo. These results show that constitutive over-expression of IL-10 in human CD4+ T cells leads to a stable cell population that recapitulates the phenotype and function of Tr1 cells. Interleukin-10 (IL-10) is a pleiotropic cytokine characterized by a broad spectrum of anti-inflammatory activities. Produced by several cells including T, B, and natural killer (NK) cells, dendritic cells (DC), macrophages, mast cells, neutrophils, and eosinophils, when bound to its receptor, IL-10 activates the STAT3-mediated signaling which results in the inhibition of different target genes.1Saraiva M O'Garra A The regulation of IL-10 production by immune cells.Nat Rev Immunol. 2010; 10: 170-181Crossref PubMed Scopus (2023) Google Scholar IL-10 acts primarily on antigen-presenting cells inhibiting the upregulation of major histocompatibility complex class II and costimulatory molecules and the release of proinflammatory cytokines and chemokines, overall limiting antigen-presenting cell function.2de Waal Malefyt R Abrams J Bennett B Figdor CG de Vries JE Interleukin 10(IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes.J Exp Med. 1991; 174: 1209-1220Crossref PubMed Scopus (3383) Google Scholar,3Fiorentino DF Zlotnik A Mosmann TR Howard M O'Garra A IL-10 inhibits cytokine production by activated macrophages.J Immunol. 1991; 147: 3815-3822Crossref PubMed Google Scholar IL-10 can also directly inhibit T-cell function and cytokine production,4Taga K Mostowski H Tosato G Human interleukin-10 can directly inhibit T-cell growth.Blood. 1993; 81: 2964-2971Crossref PubMed Google Scholar,5de Waal Malefyt R Yssel H de Vries JE Direct effects of IL-10 on subsets of human CD4+ T cell clones and resting T cells. Specific inhibition of IL-2 production and proliferation.J Immunol. 1993; 150: 4754-4765Crossref PubMed Google Scholar chemotaxis,6Jinquan T Quan S Jacobi HH Madsen HO Glue C Skov PS et al.CXC chemokine receptor 4 expression and stromal cell-derived factor-1alpha-induced chemotaxis in CD4+ T lymphocytes are regulated by interleukin-4 and interleukin-10.Immunology. 2000; 99: 402-410Crossref PubMed Scopus (48) Google Scholar and proliferation.7Groux H Bigler M de Vries JE Roncarolo MG Interleukin-10 induces a long-term antigen-specific anergic state in human CD4+ T cells.J Exp Med. 1996; 184: 19-29Crossref PubMed Scopus (693) Google Scholar IL-10 is involved in the induction and maintenance of peripheral tolerance; specifically, it is implicated in the generation and suppressive function of a subset of adaptive regulatory T cells, namely Tr1.8Groux H O'Garra A Bigler M Rouleau M Antonenko S de Vries JE et al.A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis.Nature. 1997; 389: 737-742Crossref PubMed Scopus (3150) Google Scholar,9Roncarolo MG Gregori S Battaglia M Bacchetta R Fleischhauer K Levings MK Interleukin-10-secreting type 1 regulatory T cells in rodents and humans.Immunol Rev. 2006; 212: 28-50Crossref PubMed Scopus (976) Google Scholar Tr1 cells were initially described in severe combined immunodeficient patients that developed long-term tolerance to stem cell allografts10Roncarolo MG Yssel H Touraine JL Bacchetta R Gebuhrer L De Vries JE et al.Antigen recognition by MHC-incompatible cells of a human mismatched chimera.J Exp Med. 1988; 168: 2139-2152Crossref PubMed Scopus (68) Google Scholar,11Bacchetta R Bigler M Touraine JL Parkman R Tovo PA Abrams J et al.High levels of interleukin 10 production in vivo are associated with tolerance in SCID patients transplanted with HLA mismatched hematopoietic stem cells.J Exp Med. 1994; 179: 493-502Crossref PubMed Scopus (364) Google Scholar and they were subsequently associated with disease prevention and cure in a variety of murine and human pathological conditions.9Roncarolo MG Gregori S Battaglia M Bacchetta R Fleischhauer K Levings MK Interleukin-10-secreting type 1 regulatory T cells in rodents and humans.Immunol Rev. 2006; 212: 28-50Crossref PubMed Scopus (976) Google Scholar For instance, Tr1 cells were detected in the peripheral blood of β-thalassemic patients who developed persistent mixed chimerism associated with tolerance after allogeneic hematopoietic stem cell transplantation.12Serafini G Andreani M Testi M Battarra M Bontadini A Biral E et al.Type 1 regulatory T cells are associated with persistent split erythroid/lymphoid chimerism after allogeneic hematopoietic stem cell transplantation for thalassemia.Haematologica. 2009; 94: 1415-1426Crossref PubMed Scopus (48) Google Scholar Tr1 cells are induced upon chronic exposure to antigen in the presence of IL-10 and are characterized by a distinct cytokine production profile, namely IL-10++TGF-β+IFN-γ+IL-5+IL-4−IL-2low/neg.8Groux H O'Garra A Bigler M Rouleau M Antonenko S de Vries JE et al.A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis.Nature. 1997; 389: 737-742Crossref PubMed Scopus (3150) Google Scholar,13Bacchetta R Sartirana C Levings MK Bordignon C Narula S Roncarolo MG Growth and expansion of human T regulatory type 1 cells are independent from TCR activation but require exogenous cytokines.Eur J Immunol. 2002; 32: 2237-2245Crossref PubMed Scopus (159) Google Scholar Tr1 cells suppress effector T cells via IL-10- and TGF-β-dependent mechanisms8Groux H O'Garra A Bigler M Rouleau M Antonenko S de Vries JE et al.A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis.Nature. 1997; 389: 737-742Crossref PubMed Scopus (3150) Google Scholar and kill myeloid antigen-presenting cell via granzyme (GZ)-B and perforin production.14Magnani CF Alberigo G Bacchetta R Serafini G Andreani M Roncarolo MG et al.Killing of myeloid APCs via HLA class I, CD2 and CD226 defines a novel mechanism of suppression by human Tr1 cells.Eur J Immunol. 2011; 41: 1652-1662Crossref PubMed Scopus (103) Google Scholar The interest in regulatory T cell-based therapy for treating several immune-mediated pathologies has recently increased. The use of freshly isolated natural CD25+ Treg, expanded CD25+ Treg, and Tr1 cells has been already tested in proof-of-concept clinical trials.15Di Ianni M Falzetti F Carotti A Terenzi A Castellino F Bonifacio E et al.Tregs prevent GVHD and promote immune reconstitution in HLA-haploidentical transplantation.Blood. 2011; 117: 3921-3928Crossref PubMed Scopus (812) Google Scholar,16Brunstein CG Miller JS Cao Q McKenna DH Hippen KL Curtsinger J et al.Infusion of ex vivo expanded T regulatory cells in adults transplanted with umbilical cord blood: safety profile and detection kinetics.Blood. 2011; 117: 1061-1070Crossref PubMed Scopus (815) Google Scholar,17Bacchetta R Sartirana C Lucarelli B Miqueu P Lupo Stanghellini MT Greco R et al.Interleukin-10 anergized donor T cell infusion improves immune reconstitution without severe graft-versus-host disease after haploidentical hematopoietic stem cell transplantation.Blood. 2009; 114 (ASH Annual Meeting Abstract): 45Google Scholar A number of protocols for in vitro induction of human Tr1 cells have been described, where cells are activated in the presence of either recombinant IL-10 and interferon (IFN)-α,18Levings MK Sangregorio R Galbiati F Squadrone S de Waal Malefyt R Roncarolo MG IFN-alpha and IL-10 induce the differentiation of human type 1 T regulatory cells.J Immunol. 2001; 166: 5530-5539Crossref PubMed Scopus (522) Google Scholar or a combination of immunosuppressive drugs (vitamin D3 and dexamethasone),19Barrat FJ Cua DJ Boonstra A Richards DF Crain C Savelkoul HF et al.In vitro generation of interleukin 10-producing regulatory CD4(+) T cells is induced by immunosuppressive drugs and inhibited by T helper type 1 (Th1)- and Th2-inducing cytokines.J Exp Med. 2002; 195: 603-616Crossref PubMed Scopus (980) Google Scholar or anti-CD3 and anti-CD46 monoclonal antibodies,20Kemper C Chan AC Green JM Brett KA Murphy KM Atkinson JP Activation of human CD4+ cells with CD3 and CD46 induces a T-regulatory cell 1 phenotype.Nature. 2003; 421: 388-392Crossref PubMed Scopus (490) Google Scholar or recombinant transforming growth factor (TGF)-β and IL-27.21Pot C Jin H Awasthi A Liu SM Lai CY Madan R et al.Cutting edge: IL-27 induces the transcription factor c-Maf, cytokine IL-21, and the costimulatory receptor ICOS that coordinately act together to promote differentiation of IL-10-producing Tr1 cells.J Immunol. 2009; 183: 797-801Crossref PubMed Scopus (375) Google Scholar Tr1 cells can also be differentiated by repetitive stimulation of naive T cells with allogeneic immature monocyte-derived DC22Levings MK Gregori S Tresoldi E Cazzaniga S Bonini C Roncarolo MG Differentiation of Tr1 cells by immature dendritic cells requires IL-10 but not CD25+CD4+ Tr cells.Blood. 2005; 105: 1162-1169Crossref PubMed Scopus (398) Google Scholar or by a single stimulation with IL-10-treated tolerogenic DC, named DC-10.23Bacchetta R Gregori S Serafini G Sartirana C Schulz U Zino E et al.Molecular and functional characterization of allogantigen-specific anergic T cells suitable for cell therapy.Haematologica. 2010; 95: 2134-2143Crossref PubMed Scopus (54) Google Scholar,24Gregori S Tomasoni D Pacciani V Scirpoli M Battaglia M Magnani CF et al.Differentiation of type 1 T regulatory cells (Tr1) by tolerogenic DC-10 requires the IL-10-dependent ILT4/HLA-G pathway.Blood. 2010; 116: 935-944Crossref PubMed Scopus (411) Google Scholar In addition, virally activated plasmacytoid DC can induce Tr1 cells in vitro.25Ito T Yang M Wang YH Lande R Gregorio J Perng OA et al.Plasmacytoid dendritic cells prime IL-10-producing T regulatory cells by inducible costimulator ligand.J Exp Med. 2007; 204: 105-115Crossref PubMed Scopus (516) Google Scholar In most protocols, however, only a small fraction of the cells produces IL-10, suggesting that few Tr1 cells can be obtained. The presence of contaminating effector T cells in the resulting population represents a major barrier toward the clinical application of adoptive Tr1 cell therapy in humans. One potential strategy to overcome this limitation is to generate a homogeneous IL-10–producing T cell population through direct IL-10 gene transfer. Previous murine studies showed that after retroviral gene transfer of either murine or viral IL-10, CD4+ T cells acquire the ability to suppress T cell-mediated responses in vitro26Brandt C Yang J Schmitt-Knosalla I Siepert A Hammer MH Vogt K et al.Allo-specific T-cells encoding for viral IL-10 exert strong immunomodulatory effects in vitro but fail to prevent graft rejection.Am J Transplant. 2005; 5: 268-281Crossref PubMed Scopus (6) Google Scholar and in vivo in various T cell-mediated diseases, i.e., rheumatoid arthritis,27Setoguchi K Misaki Y Araki Y Fujio K Kawahata K Kitamura T et al.Antigen-specific T cells transduced with IL-10 ameliorate experimentally induced arthritis without impairing the systemic immune response to the antigen.J Immunol. 2000; 165: 5980-5986Crossref PubMed Scopus (38) Google Scholar,28Guichelaar T ten Brink CB van Kooten PJ Berlo SE Broeren CP van Eden W et al.Autoantigen-specific IL-10-transduced T cells suppress chronic arthritis by promoting the endogenous regulatory IL-10 response.J Immunol. 2008; 180: 1373-1381Crossref PubMed Scopus (22) Google Scholar colitis,29Van Montfrans C Rodriguez Pena MS Pronk I Ten Kate FJ Te Velde AA Van Deventer SJ Prevention of colitis by interleukin 10-transduced T lymphocytes in the SCID mice transfer model.Gastroenterology. 2002; 123: 1865-1876Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar and allergen-induced Th2-dependent airway hyperreactivity.30Oh JW Seroogy CM Meyer EH Akbari O Berry G Fathman CG et al.CD4 T-helper cells engineered to produce IL-10 prevent allergen-induced airway hyperreactivity and inflammation.J Allergy Clin Immunol. 2002; 110: 460-468Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar Notably, in these studies, retroviral vectors were used to promote IL-10 expression in murine CD4+ TCR transgenic cells and in one report in CD4+ polyclonal cells, but no extensive characterization of the resulting murine IL-10–transduced T cells was performed. Lentiviral vectors (LVs) encoding for IL-10 were directly injected intragraft in preclinical experimental models of heart transplantation31Zhao J Pettigrew GJ Bolton EM Murfitt CR Carmichael A Bradley JA et al.Lentivirus-mediated gene transfer of viral interleukin-10 delays but does not prevent cardiac allograft rejection.Gene Ther. 2005; 12: 1509-1516Crossref PubMed Scopus (21) Google Scholar and intracorneal to prevent uveitis.32Trittibach P Barker SE Broderick CA Natkunarajah M Duran Y Robbie SJ et al.Lentiviral-vector-mediated expression of murine IL-1 receptor antagonist or IL-10 reduces the severity of endotoxin-induced uveitis.Gene Ther. 2008; 15: 1478-1488Crossref PubMed Scopus (36) Google Scholar In these models specific downregulation of the immune response without general immunosuppression was observed. LVs have been shown to guarantee higher transduction efficiency in T cells with preservation of immune competence in vitro.33Cavalieri S Cazzaniga S Geuna M Magnani Z Bordignon C Naldini L et al.Human T lymphocytes transduced by lentiviral vectors in the absence of TCR activation maintain an intact immune competence.Blood. 2003; 102: 497-505Crossref PubMed Scopus (128) Google Scholar,34Marktel S Magnani Z Ciceri F Cazzaniga S Riddell SR Traversari C et al.Immunologic potential of donor lymphocytes expressing a suicide gene for early immune reconstitution after hematopoietic T-cell-depleted stem cell transplantation.Blood. 2003; 101: 1290-1298Crossref PubMed Scopus (110) Google Scholar To generate a homogeneous population of Tr1 cells in vitro, which can be administered in vivo, we transduced human CD4+ T cells with LV encoding human IL-10. To this aim, we developed a bidirectional LV, similar to the one previously described,35Amendola M Venneri MA Biffi A Vigna E Naldini L Coordinate dual-gene transgenesis by lentiviral vectors carrying synthetic bidirectional promoters.Nat Biotechnol. 2005; 23: 108-116Crossref PubMed Scopus (253) Google Scholar where expression of IL-10 and that of a marker gene, in this case green fluorescent protein (GFP), are driven by different promoters (LV-IL-10/GFP). We found that the resulting CD4LV-IL-10 T cells produced high levels of IL-10, upregulated the expression of IL-10R and other Tr1-related tolerogenic markers (i.e., human leukocyte antigen-G (HLA-G), inducible costimulator (ICOS), inducible costimulator-ligand (ICOS-L)), were anergic and acquired lytic and suppressive activities in vitro and in vivo in NOD.scid mice in which xeno graft-versus-host disease (xeno-GvHD) was induced. These results demonstrate that constitutive over-expression of hIL-10 confers Tr1 cell features to human CD4+ T cells. To generate LVs co-encoding for IL-10 and GFP, as marker gene, (LV-IL-10/GFP), we cloned the complementary DNA of human IL-10 into a bidirectional LV35Amendola M Venneri MA Biffi A Vigna E Naldini L Coordinate dual-gene transgenesis by lentiviral vectors carrying synthetic bidirectional promoters.Nat Biotechnol. 2005; 23: 108-116Crossref PubMed Scopus (253) Google Scholar under the control of the PGK promoter whereas the mCMV promoter controlled GFP expression, as shown in Figure 1a. A bidirectional LV co-encoding for GFP (LV-GFP) and ΔNGFR (in IL-10 position) was used as control (Figure 1a). To obtain a high transduction rate, human CD4+ T cells were preactivated for 48 hours with low concentrations of soluble anti-CD3 (30 ng/ml) and anti-CD28 (1 µg/ml) monoclonal antibodies (mAbs) in the presence of rhIL-2 (50 U/ml). Preactivated cells were then infected with LVs overnight (multiplicity of infection: 20). Forty to eighty percent of CD4+ T cells were transduced, as confirmed by GFP expression (Figure 1b). Due to the presence of the bidirectional promoter, hIL-10/ΔNGFR and GFP genes are translated into separate proteins, but their expression is coordinated and the levels of expression are comparable (Figure 1c), as previously demonstrated.35Amendola M Venneri MA Biffi A Vigna E Naldini L Coordinate dual-gene transgenesis by lentiviral vectors carrying synthetic bidirectional promoters.Nat Biotechnol. 2005; 23: 108-116Crossref PubMed Scopus (253) Google Scholar Quantitative real-time reverse transcription-PCR for IL-10 and GFP performed on CD4+ T cells transduced with LV-IL-10/GFP (CD4LV-IL-10) confirmed these findings (data not shown). CD4LV-IL-10 T cells acquired the ability to release significantly higher levels of IL-10 compared to CD4+ T cells transduced with LV-GFP (CD4LV-GFP) (on average 18.1 ng/ml versus 0.1 ng/ml in culture supernatant 6 days after transduction, respectively, n = 8, P = 0.0009, data not shown).Figure 1Bidirectional LV-IL-10/GFP and LV-GFP efficiently transduce human CD4+ T cells. (a) Scheme of the lentiviral vectors. hIL-10 cDNA replaced the ΔNGFR coding sequence in the LV-GFP vector to obtain LV-IL-10/GFP. The presence of the bidirectional promoter (mCMV/PGK) allows the coregulated expression GFP and IL-10/ΔNGFR genes. Ψ, encapsidation signal including the 5′ portion of GAG gene (GA); cPPT, central poly-purine tract; CTE, constitutive transport element; polyA, poly-adenylation site from the simian Virus 40; RRE, Rev-responsive element; WPRE, woodchuck hepatitis virus post-transcription regulatory element. (b) Human CD4+ or CD4+CD45RO− T cells were activated with soluble anti-CD3, soluble anti-CD28 mAbs, and rhIL-2 for 48 hours before transduction with LV-GFP or LV-IL-10/GFP at MOI of 20. After 6 days, transduced cells were analyzed by FACS. The frequency of GFP+ cells are indicated on top of each quadrant. One representative donor of at least eight analyzed is presented. (c) Coordinated expression of ΔNGFR/IL-10 and GFP. FACS-sorted CD4+GFP+ cells were expanded in feeder mixture as described in Materials and Methods. At the end of the feeder mixture, cells were analyzed for the expression of the ΔNGFR and GFP by flow cytometry. cDNA, complementary DNA; CMV, cytomegalovirus; FACS, fluorescence-activated cell sorting; GFP, green fluorescent protein; IL, interleukin; LV, lentiviral vector; mAb, monoclonal antibody; MOI, multiplicity of infection.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The LV-IL-10/GFP-transduced CD4+ T (CD4LV-IL-10) cells were fluorescence-activated cell sorted according to CD4 and GFP expression and expanded in vitro using feeder mixture, as described in Materials and Methods. In all experiments cells transduced with LV-GFP (CD4LV-GFP) and untransduced cells were included as controls. Notably, the expansion of CD4LV-GFP T cells was significantly higher than that of CD4LV-IL-10 T cells (fold increase of 10.4 ± 4.6 versus 3.6 ± 2.2, respectively, n = 5, p = 0.0313), suggesting that in the latter expansion was impaired by IL-10 secretion (data not shown). The vector copy number was on average 1.6 ± 0.3 for sorted CD4LV-GFP and 2.5 ± 0.3 for sorted CD4LV-IL-10 T cells (data not shown). In vitro expanded CD4LV-IL-10 and CD4LV-GFP T cells displayed a memory phenotype (CD45RO+, CD45RA−) but the CD4LV-IL-10 T cells showed predominantly a central memory profile (CD62Lhi) (Figure 2a and Supplementary Figure S1). No significant differences in the expression of CD40L, CD40, CD25, FOXP3, CD161, and CD137 were observed between CD4LV-IL-10 and CD4LV-GFP T cells (Figure 2a and Supplementary Figure S2a). Interestingly, expression of markers associated with IL-10 (Figure 2b) such as PD-1,36Akdis M Verhagen J Taylor A Karamloo F Karagiannidis C Crameri R et al.Immune responses in healthy and allergic individuals are characterized by a fine balance between allergen-specific T regulatory 1 and T helper 2 cells.J Exp Med. 2004; 199: 1567-1575Crossref PubMed Scopus (896) Google Scholar ICOS-L, ICOS,37Häringer B Lozza L Steckel B Geginat J Identification and characterization of IL-10/IFN-gamma-producing effector-like T cells with regulatory function in human blood.J Exp Med. 2009; 206: 1009-1017Crossref PubMed Scopus (131) Google Scholar and IL-10R, was significantly higher in CD4LV-IL-10 compared to CD4LV-GFP T cells, although a variability among T-cell lines generated from different donors was observed, (on average PD-1+ cells 16.2 ± 3.8 versus 7.1 ± 1.6%, n = 6, P = 0.053, ICOS-L+ cells 11.9 ± 2.4 versus 4.5 ± 0.66%, n = 14, P = 0.0074, ICOS+ cells 6.6 ± 1.6 versus 2.9 ± 0.82%, n = 14, P = 0.0047, and IL-10R+ cells 8.6 ± 1.5 versus 3.5 ± 0.75%, n = 11, P = 0.0058, Figure 2b). The relative low expression of IL-10R on CD4LV-IL-10 T cells might be dependent on the binding of IL-10, which increases the receptor's occupancy limiting the labeling by anti-IL-10R antibody. To overcome this limitation, we measured the IL-10R expression levels by real-time reverse transcription-PCR (see Supplementary Materials and Methods). Results confirmed that CD4LV-IL-10 T cells express significantly higher levels of IL-10R messenger RNA as compared to control CD4LV-GFP T cells (on average a fold increase expression of 38 ± 14 versus 11.9 ± 6.4, n = 3, in CD4LV-IL-10 and CD4LV-GFP T cells, respectively, Supplementary Figure S3). The expression of the non-classical HLA class I molecule HLA-G on CD4LV-IL-10 T cells was also investigated, because we recently demonstrated that IL-10 derived from tolerogenic DC promotes the upregulation of HLA-G on T cells.24Gregori S Tomasoni D Pacciani V Scirpoli M Battaglia M Magnani CF et al.Differentiation of type 1 T regulatory cells (Tr1) by tolerogenic DC-10 requires the IL-10-dependent ILT4/HLA-G pathway.Blood. 2010; 116: 935-944Crossref PubMed Scopus (411) Google Scholar CD4LV-IL-10 T cells expressed significantly higher levels of HLA-G than CD4LV-GFP T cells (on average 26.6 ± 4.8 versus 8.7 ± 2.4%, n = 7, P = 0.0063, Figure 2b). The effect of IL-10 was specific for HLA-G, because the expression of classical HLA class I molecules was comparable between CD4LV-GFP and CD4LV-IL-10 T cells (Figure 2a). Upregulation of above IL-10–related genes and in particular of HLA-G increased after long-term in vitro culture (more than three rounds of feeders), suggesting that the Tr1 phenotype was maintained by the autocrine action of IL-10. Indeed, IL-10 levels were significantly elevated in CD4LV-IL-10 long-term cultures ranging from 5 to 40 ng/ml (data not shown). Altogether, our results show that stable enforced expression of IL-10 in human CD4+ T cells by means of LV transduction induces a Tr1 cell phenotype, with high expression of PD-1, ICOS-L, ICOS, IL-10R, and HLA-G. We next determined the cytokine production profile of GFP+ sorted and in vitro expanded CD4LV-IL-10 in comparison to CD4LV-GFP T cells unstimulated or upon polyclonal stimulation with anti-CD3/anti-CD28 mAbs in the presence or absence of rhIL-2 or after stimulation with LAK (leukocyte activation cocktail: TPAphorbol 12-myristate 13-acetate (PMA)/ionomycin). As shown in Figure 3a, upon activation, LV-IL-10–transduced cells but not controls (CD4LV-GFP) concomitantly upregulated IL-10 and GFP expression. The increased production of IL-10 was more evident when the mean fluorescence intensity (MFI) for IL-10-PE was plotted (Figure 3b,c). Results obtained with intracellular staining were further confirmed by measurement of IL-10 in culture supernatants. CD4LV-IL-10 T cells secrete spontaneously significantly higher levels of IL-10 as compared to CD4LV-GFP T cells (on average 0.51 ± 0.44 versus 0.007 ± 0.009 ng/ml respectively, n = 10, P = 0.0012), which increased upon TCR stimulation (on average 4.7 ± 1.5 versus 1.3 ± 0.42 ng/ml, n = 13, P = 0.04, (Figure 3d). These results confirm the Tr1 “nature” of CD4LV-IL-10 T cells, which were able to produce high amounts of IL-10 in an inducible manner, upon activation. We further characterized the cytokine signature of CD4LV-IL-10 T cells. In addition to secrete significantly higher amounts of IL-10 compared to CD4LV-GFP, CD4LV-IL-10 T cells secrete significantly higher levels of TGF-β1 (on average 1.05 ± 0.45 versus 0.29±0.22 ng/ml, n = 2, P = 0.042) and significantly lower amounts of IL-4 (on average 70 ± 34 versus 275 ± 58 pg/ml, n = 12, P = 0.0063) and IL-5 (on average 8.2 ± 2.6 versus 18.9 ± 2.4 ng/ml, n = 9, P = 0.0131; Figure 4). No statistically significant differences in IL-2, IFN-γ, and tumor necrosis factor-α production were observed (Figure 4). In line with their cytokine production profile, CD4LV-IL-10 T cells expressed similar levels of T-bet, but lower levels of Gata-3, compared to CD4LV-GFP T cells (n = 3, Supplementary Figure S4). These results indicate that human CD4+ T cells stably transduced with IL-10 acquire a cytokine production profile consistent with that of Tr1 cells. A prominent characteristic of Tr1 cells is that they are anergic upon re-stimulation in vitro. The proliferative capacity measured by thymidine incorporation of CD4LV-IL-10 T cells upon anti-CD3/CD28 stimulation in vitro was lower compared to that of CD4LV-GFP T cells (6 ± 2.3 versus 26.1 ± 11.7 × 103 cpm, n = 6), with a mean reduction in proliferation of 67.1 ± 23.7% (P < 0.005) (Figure 5a). The anergic state of CD4LV-IL-10 T cells was reverted by addition of 100 U/ml of rhIL-2 to the culture (Figure 5b). Next, we evaluated the anergic state of CD4LV-IL-10 T cells by measuring their proliferation in the presence of third party allogeneic peripheral blood mononuclear cells (PBMCs). CD4LV-IL-10 T cells were stained with Vybrant or eFluor670 and stimulated with anti-CD3/CD28 mAbs in the absence or presence of unlabeled allogeneic PBMCs at increasing ratio and their proliferation was determined by flow cytometry. Results in Figure 5c show that CD4LV-IL-10 T cells are profoundly anergic also in the presence of allogeneic PBMCs. CD4LV-GFP T cells under the same culture conditions showed increased proliferation, especially at low PBMCs number. The suppressive activity of LV-IL-10–transduced cells in vitro was next examined. Allogeneic Vybrant/eFluor670-stained CD4+ responder T cells were stimulated with anti-CD3/CD28 mAbs alone or in combination with CD4LV-IL-10 or control CD4LV-GFP T cells at 1:1 ratio and their proliferation (Vybrant/eFluor670 dilution) was determined by flow cytometry. Results in Figure 5d,e show that proliferation of CD4+ T cells was suppressed by the addition of allogeneic CD4LV-IL-10 T cells (one representative donor shown in Figure 5d; mean suppression of proliferation of CD4+ responder cells: 39.25 ± 5.17, n = 22, P < 0.0001, Figure 5e). CD4LV-IL-10 T cells suppress proliferation of responder CD4+ T cells independently from their proliferative capacity. Moreover, the proliferation of purified allogeneic eFluor670-stained CD8+ responder cells stimulated with anti-CD3/CD28 mAbs was also significantly suppressed by the addition of CD4LV-IL-10 T cells (one representative donor shown in Figure 5f; mean suppression of proliferation of CD8+ responder cells: 47.22 ± 13.67%, n = 4," @default.
• W1977224177 created "2016-06-24" @default.
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• W1977224177 date "2012-09-01" @default.
• W1977224177 modified "2023-10-04" @default.
• W1977224177 title "Enforced IL-10 Expression Confers Type 1 Regulatory T Cell (Tr1) Phenotype and Function to Human CD4+ T Cells" @default.
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