Julie Quentin

Adoptive transfer of IL-10-secreting CD4+CD49b+ regulatory T cells suppresses ongoing arthritis

We have previously demonstrated, in the collagen-induced arthritis model (CIA), that repetitive injections of immature bone-marrow-derived dendritic cells (iDCs) induce the expansion of a population of CD4CD49b-expressing cells, and that their adoptive transfer results in protection against CIA in a prophylactic setting. However, the in vivo mechanism responsible for their expansion, as well as their therapeutic potential in established disease remains to be defined. In the present study, we show that expression of the MHC class II molecules on iDCs is required for their expansion thus identifying these cells as MHC class II-restricted T cells. Using adoptive transfer of Thy1.1 positive cells, it is shown that iDC-induced CD4(+)CD49b(+) T cells home to the lymph nodes draining the inflamed tissue. The high immunomodulatory potential of these cells was underscored following their adoptive transfer in a model of contact hypersensitivity. Finally, we assessed and compared the therapeutic potential of iDC-inducible CD4(+)CD49b(+) T cells with that of iDCs in established CIA. Repetitive injections of iDCs in arthritic mice failed to decrease the severity of established disease. In contrast however, a single injection of iDC-induced CD4(+)CD49b(+) T cells reversed clinical symptoms of arthritis and provided long-lasting protection. Together, our data indicate that iDC-induced CD4(+)CD49b(+) T cells are bona fide T regulatory cells with strong immunomodulatory properties that are not only able to prevent disease onset, but also to interfere with an ongoing inflammatory immune response.

Type 1 regulatory T cells specific for collagen type II as an efficient cell-based therapy in arthritis

IntroductionRegulatory T (Treg) cells play a crucial role in preventing autoimmune diseases and are an ideal target for the development of therapies designed to suppress inflammation in an antigen-specific manner. Type 1 regulatory T (Tr1) cells are defined by their capacity to produce high levels of interleukin 10 (IL-10), which contributes to their ability to suppress pathological immune responses in several settings. The aim of this study was to evaluate the therapeutic potential of collagen type II–specific Tr1 (Col-Treg) cells in two models of rheumatoid arthritis (RA) in mice.MethodsCol-Treg clones were isolated and expanded from collagen-specific TCR transgenic mice. Their cytokine secretion profile and phenotype characterization were studied. The therapeutic potential of Col-Treg cells was evaluated after adoptive transfer in collagen-antibody– and collagen-induced arthritis models. The in vivo suppressive mechanism of Col-Treg clones on effector T-cell proliferation was also investigated.ResultsCol-Treg clones are characterized by their specific cytokine profile (IL-10highIL-4negIFN-γint) and mediate contact-independent immune suppression. They also share with natural Tregs high expression of GITR, CD39 and granzyme B. A single infusion of Col-Treg cells reduced the incidence and clinical symptoms of arthritis in both preventive and curative settings, with a significant impact on collagen type II antibodies. Importantly, injection of antigen-specific Tr1 cells decreased the proliferation of antigen-specific effector T cells in vivo significantly.ConclusionsOur results demonstrate the therapeutic potential of Col-Treg cells in two models of RA, providing evidence that Col-Treg could be an efficient cell-based therapy for RA patients whose disease is refractory to current treatments.

Development of tripartite polyion micelles for efficient peptide delivery into dendritic cells without altering their plasticity

For many years, a great deal of interest has been focusing on the optimization of peptide presentation by dendritic cells (DCs) using peptide-encapsulated particles, in order to enhance the immune response. Nowadays, DCs are also known to be involved in peripheral tolerance, inducing anergy or regulatory T lymphocytes. To preserve the plasticity of DCs, we formulated non-cytotoxic pH-sensitive polyion complex micelles based on an original tripartite association of polymethacrylic acid-b-polyethylene oxide, poly-L-lysine and fluorescent-peptide: OVAFITC peptide, as a model drug. We demonstrated that the OVAFITC peptide was successfully entrapped into the micelles, released into DC endosomes thanks to the pH-sensitivity property of the micelles, and efficiently loaded onto MHC class II molecules. The phenotype as well as the cytokinic secretion profile of the mature and immature DCs loaded with peptide-encapsulated micelles was unaltered by the tripartite polyion micelles. The efficient loading of the peptide by immature and mature DCs was shown by the in vitro proliferation of OVA-specific transgenic T cells. Therefore, the present results show that the tripartite polyion complex micelles can be used as efficient peptide vectors immunogically inert for ex vivo DCs engineering without modifying their intrinsic immune plasticity.

Tripartite siRNA micelles as controlled delivery systems for primary dendritic cells

Dendritic cells (DCs) are key cells in immunology that are able to stimulate or inhibit the immune response. RNA interference has appeared of great interest to modulate the expression of immunogenic or tolerogenic molecules. In our study, pH-sensitive polyion complex micelles based on a double-hydrophilic block copolymer and poly-l-lysine were formulated to entrap a small interfering RNA (siRNA). We show that siRNA-loaded micelles were cytotolerant and efficiently endocytosed by DCs. siRNA targeting eGFP, used as model siRNA, was released into the cytosol following endocytosis of the micelles and the silencing of eGFP expression was observed in DC isolated from transgenic mice. Our results underscore the potential of pH-sensitive polyion complex micelles to formulate therapeutic siRNA for DC engineering in order to maintain the homeostasis of the immune response.

Versatile polyion complex micelles for peptide and siRNA vectorization to engineer tolerogenic dendritic cells

Dendritic cells (DCs) are professional antigen-presenting cells that play a critical role in maintaining the balance between immunity and tolerance and, as such are a promising immunotherapy tool to induce immunity or to restore tolerance. The main challenge to harness the tolerogenic properties of DCs is to preserve their immature phenotype. We recently developed polyion complex micelles, formulated with double hydrophilic block copolymers of poly(methacrylic acid) and poly(ethylene oxide) blocks and able to entrap therapeutic molecules, which did not induce DC maturation. In the current study, the intrinsic destabilizing membrane properties of the polymers were used to optimize endosomal escape property of the micelles in order to propose various strategies to restore tolerance. On the first hand, we showed that high molecular weight (Mw) copolymer-based micelles were efficient to favor the release of the micelle-entrapped peptide into the endosomes, and thus to improve peptide presentation by immature (i) DCs. On the second hand, we put in evidence that low Mw copolymer-based micelles were able to favor the cytosolic release of micelle-entrapped small interfering RNAs, dampening the DCs immunogenicity. Therefore, we demonstrate the versatile use of polyionic complex micelles to preserve tolerogenic properties of DCs. Altogether, our results underscored the potential of such micelle-loaded iDCs as a therapeutic tool to restore tolerance in autoimmune diseases.

A8.26 Inducible IL-10 secreting CD49b +Treg cells as cell based-therapy for rheumatoid arthritis

Background Adoptive transfer of regulatory T cells (Tregs) is a promising approach to restore tolerance in autoimmune diseases. However based on the heterogeneity of the Tregs, we need to precisely establish which Tregs will be able to dampen efficiently the immune response in the various settings. We previously showed the potential of CD49b+ Treg cells to protect and prevent an experimental model of arthritis. Nevertheless the optimal injection dose, the phenotype and the in vivo-suppressive mechanism of these Treg cells remain unknown. In our study, we investigated and compared the therapeutic potential of CD25+FoxP3+ and induced IL10-secreting CD49b+ Treg cells in an experimental model of arthritis, the collagen-induced arthritis (CIA). Materials and Methods IL-10 secreting CD49b+ Treg cells were generated in naïve mice following repetitive injections of immature DCs (iDCs). Treg purification was based on the negative selection of CD4 T cells isolated from spleen and liver of the iDC-vaccinated mice. Cell sorting was performed to obtain 98% pure CD49b+ or CD25+ Treg cells. Several doses of CD49b+ were intravenously (i.v.) injected at day 28 in established CIA. Clinical signs of arthritis were scored, as well as biological parameters such as the level of anti-bCII antibodies in sera and the cytokine profile of bCII specific T cells. Phenotypes of both Treg cells were compared as well as their suppressive activity in vitro and in vivo. Results Several doses of CD49+ Treg cells were tested in curative settings experiments. The dose of 105 CD49b+ or CD25+ cells reverse clinical symptoms of arthritis while interestingly, a lack of efficacy was observed after higher doses. In vitro suppressive experiments confirmed the similar efficiency of both populations and phenotype analyses of CD49b+ Treg cells showed expression profile of several Treg specific markers (LAP+, LAG+, CTLA-4high). Moreover, in an OVA-specific model of inflammation, we demonstrated the high impact of the CD49b Treg cells on the proliferation of effector cells in vivo. Conclusions Altogether, our results confirm the therapeutic potential of IL-10 secreting T cells in experimental model of arthritis in curative settings and unravel their mechanism of suppression.

Injection of antigen-specific regulatory Tr1 lymphocytes protects mice from severe collagen-induced arthritis

Introduction Tr1 cells have been characterised as induced T regulatory lymphocytes (Treg) inhibiting inflammation in various chronic inflammatory models. Based on these data, a phase I/II clinical trial is currently under investigation in Crohns disease (TxCell). However, the therapeutic potential of these cells has not yet been evaluated in rheumatoid arthritis. In this study, the authors investigated the therapeutic potential of bovine type II collagen (bCII) specific Tr1 cells, isolated from TBC mice, in the experimental model of collagen-induced arthritis (CIA). Methods Collagen type II specific Tr1 clones were obtained from T cell receptor transgenic mice and expanded in vitro. Selected clones showed in vitro antigen specificity, Tr1 cytokine profile (IL-10high/IL-4neg) and IL-10- and transforming growth factor β-dependent suppressive activity. Male DBA/one mice were immunised with bCII and 10×106, 3×106, 1×106, 0.3×106 of Tr1 cells were injected (intravenously) 28 days postimmunisation. Hind paws swelling and clinical signs of arthritis were scored, as well as biological parameters such as the level of anti-bCII antibodies in the sera of treated mice and the cytokine profile of bCII specific T cells. Results One single injection of 3×106 or 1×106 of Tr1 cells at day 28, in ongoing arthritis, significantly inhibits the development of arthritic disease, shown by reduction of disease severity and incidence. In contrast the injection of 0.3×106 and 10 M of Tr1 cells did not improve the clinical signs of arthritis. The analysis of the bCII specific T cell responses following euthanasia of the mice injected with 3×106 and 1×106 of Tr1 cells revealed a decrease of anti-bCII specific antibodies in the sera, a decrease of proliferation of bCII specific T cells and a slight increase of IL-10 secreted by activated splenocytes. Importantly, these preliminary data indicate that a single injection of Tr1 cells at disease onset could reduce disease severity and incidence in experimental arthritis. Conclusions Single dose 3×106, 1×106 of Tr1 cell administration showed a reduction of disease incidence and severity in CIA demonstrating the therapeutic potential of Tr1 cells in arthritis and confirming the clinical potential of these induced Treg.

A6.5 Versatile polyion complex micelles for peptide and sirna vectorization to engineer tolerogenic dendritic cells

Background Dendritic cells (DCs) are professional antigen-presenting cells that play a critical role in maintaining the balance between immunity and tolerance and, as such are a promising immunotherapy tool to induce immunity or to restore tolerance. The main challenge to harness the tolerogenic properties of DCs is to preserve their immature phenotype. We recently developed polyion complex micelles, formulated with double hydrophilic block copolymers of poly (methacrylic acid) and poly (ethylene oxide) blocks and able to entrap therapeutic molecules, that did not induce DC maturation. In the current study, the intrinsic destabilising membrane properties of the polymers were used to optimise endosomal escape property of the micelles in order to propose various strategies to restore tolerance. Material and method PMAA- b-PEO copolymers with low (PMAA2100- b-PEO5000, PMAA3500- b-PEO5000) and high Mw (PMAA2500- b-PEO12000, PMAA16200- b-PEO30000) were used to formulate either OVA peptide-entrapped or siRNA-entrapped micelles. siRNA- and peptide-entrapment efficiency was monitored using fluorescent molecules. Peptide presentation was monitored using transgenic T cells and si-RNA silencing of target gene was followed by FACS. Results On the first hand, we showed that high molecular weight (Mw) copolymer-based micelles were efficient to favour the release of the micelle-entrapped peptide into the endosomes, and thus to improve peptide presentation by immature (i) DCs. On the second hand, we put in evidence that low Mw copolymer-based micelles were able to favour the cytosolic release of micelle-entrapped small interferent RNAs, dampening the DCs immunogenicity. Conclusion Therefore, we demonstrate the versatile use of polyionic complex micelles to preserve tolerogenic properties of DCs. Altogether, our results underscored the potential of such micelle-loaded iDCs as a therapeutic tool to restore tolerance in autoimmune diseases.

AB0053 Increased Frequency of Plasmacytoid Dentritic Cells in Rheumatoid Arthritis Patients in Response to IL-6R Blockade

Background Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that can induce either immunity or tolerance. Myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) represent the 2 major subsets of DCs, with human pDCs defined as CD4+CD11c-CD123hiCD1c-CD303+HLADR+ cells, and mDCs defined as CD4+CD1c+CD123loCD303-CD45RO-HLADR+ cells. Some studies reported that patients with therapy-induced remission of rheumatoid arthritis (RA) exhibited higher numbers of circulating pDCs. Objectives Indeed, in vivo effects of IL-6 inhibition on DCs are partially unknown. Tocilizumab (TCZ) is the first therapeutic agent targeting IL-6 to be effective in RA. Our aim was to monitor the changes in circulating mDCs and pDCs during TCZ therapy in patients with RA. Methods DC subsets were characterized by flow cytometry in the peripheral blood of patients with RA (n=15) and in healthy volunteers (n=10). In patients with RA, these levels were measured before and during TCZ therapy (8 mg/kg every 4 weeks). Response to TCZ therapy was evaluated at 12 weeks. All the patients fulfilled the 1987 ACR criteria for RA. At baseline, the mean DAS28 score was 5.2 (±1.3). At 3 months, 9 patients were classified as responders (DAS28 1.8±0.8) and 6 patients were classified as non-responders (DAS28 4.0±0.4). Statistical analyses were performed by Mann-Whitney U tests or Wilcoxon signed-rank tests. Results At baseline, patients with active RA were characterized by a significantly decrease frequency of circulating pDCs compared to healthy donors (40.2% vs 60.9%; p<0.01). After 3 months of TCZ therapy, pDC frequency has not changed (48.5% vs 40.2%) while mDC frequency decrease significantly (31.1% vs 22.3%; p<0.05). Moreover, TCZ-treated patients who reached low disease activity at Week 12 had significant increase frequency of pDC (52.0% vs 38.7%; p<0.05) as compared with non-responders patients (43.2% vs 42.5%). Conclusions Our study reveals an increased frequency of circulating pDC in responder patients to TCZ therapy. We previously demonstrated that the clinical response was also associated with increased frequencies of CD39+ Treg cells. Our results suggest a crosstalk between DC and Tregs to restore/induce tolerance. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4336

Inducible Treg cell populations as cell based-therapy for rheumatoid arthritis

Background Adoptive cell transfer of Treg cells is a promising approach to restore tolerance in autoimmune disease. However the various type of Tregs, their doses of injection and their in vivo-suppressive mechanism need to be precisely define to clearly establish which Tregs will be able to dampen efficiently the immune response in the various settings. In our study, we compared the therapeutic potential of induced CD25FoxP3 and two IL10-secreting Tregs: Tr1 and CD49b-induced Tregs.