Nathalie Belmonte

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.

Inhibition of Noninfectious Uveitis Using Intravenous Administration of Collagen II–Specific Type 1 Regulatory T Cells

PURPOSE To evaluate the therapeutic potential of Col-Treg, a collagen II-specific type 1 regulatory T-cell immunotherapy for the treatment of noninfectious uveitis (NIU). METHODS Col-Treg cells were produced from collagen II-specific T cell receptor (TCR) transgenic mice or peripheral blood of healthy donors. Phenotypic characterization was performed by flow cytometry, and cytokine secretion was evaluated with Flowcytomix or ELISA. In vitro functional characterization included ATP hydrolysis, cytotoxicity, and contact-independent T-cell suppression and plasticity assays. Col-Treg migration was assessed by quantitative PCR specific to Col-Treg TCR. Col-Treg cells were administered intravenously in mice displaying experimental autoimmune uveitis (EAU) induced by interphotoreceptor retinoid-binding protein (IRBP) immunizations. Efficacy of Col-Treg was assessed by ophthalmology, histology, and immunohistochemistry. RESULTS Mice Col-Treg cells displayed identity features of type 1 Treg cells with expression of CD25, FoxP3, low surface expression of CD127, and cytokine secretion profile (IL-10(high), IL-4(low), IFN-γ(int)). In vitro functional assays demonstrated Col-Treg suppressive capacity via soluble factor-dependent immunosuppression, cytotoxicity, and ATP hydrolysis. Col-Treg cells expressed granzyme B, CD39, and glucocorticoid-induced TNF-related protein (GITR). Administration of Col-Treg in EAU mice inhibited clinical and morphologic signs of uveitis and decreased ocular leukocyte infiltration. Col-Treg cells homed in the ocular tissues 24 hours after intravenous injection. Human Col-Treg cells were comparable to mice Col-Treg cells in identity and function and did not show the capacity to differentiate into Th17 cells in vitro. CONCLUSIONS These results demonstrate the therapeutic potential of Col-Treg cells as a targeted approach for the treatment of NIU and the feasibility of translating this approach to the human clinical setting.

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.

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.

Comparative analysis of the therapeutic potential of two inducible Treg cell populations in experimental model of arthritis

Backgroundand objectives Adoptive cell transfer of T regulatory (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 this study, the authors compared the therapeutic potential of two IL10-secreting Tregs: Tr1 and CD49b-induced Tregs. The authors previously demonstrated that a single injection of iDC-induced CD49b T cells reversed clinical symptoms of arthritis. This inducible CD49b Treg population shares several phenotypic markers as well as immunosuppressive properties compared with the in vitro-expanded IL-10-secreting Tr1 cells. More recently, the authors provide evidence that injections of Tr1 cells, after the onset of the disease, could protect mice from severe arthritis. In the present study the authors perform adoptive cell transfer experiments of these two inducible Treg cells in order to compare their impact on the immune response. Materials and methods CD49b Treg cells were generated in naive mice following repetitive injections of iDC. The purification was based on the negative selection of CD4 T cells isolated from the spleen and liver of the iDC-vaccinated mice. Cell sorting experiments were realised to obtain 98% pure CD49b T cells. Collagen type II (bCII) specific Tr1 clones were obtained from TCR transgenic mice and expanded in vitro. Selected clones showed in vitro antigen specificity, Tr1 cytokine profile and IL10- and tumour necrosis factor β-dependent suppressive activity. Results Several doses of CD49b or Tr1 cells were injected intravenously at day 28 in established collagen-induced arthritis. 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. The authors defined for both Treg cell populations the dose effect in curative settings experiments. One single dose of 3×106 or 1×106 of Tr1 cell administration could reduce the incidence and severity of CIA. Interestingly, higher dose of 10M of Tr1 cells did not improve the disease. In the same manner, the dose of 105 CD4CD49b+ cells reverse clinical symptom with a lack of efficacy of higher doses. The homing ability and plasticity of Tr1 cells was also investigated. Conclusions These results suggest that even if the Treg cells present some similarities, one should precisely define the dose and type of Treg that will be efficient in each experimental setting.