October 2015
Volume 56, Issue 11
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Immunology and Microbiology  |   October 2015
Inhibition of Noninfectious Uveitis Using Intravenous Administration of Collagen II–Specific Type 1 Regulatory T Cells
Author Affiliations & Notes
  • Hélène Asnagli
    TxCell Valbonne Sophia-Antipolis, France
  • Marie Jacquin
    TxCell Valbonne Sophia-Antipolis, France
  • Nathalie Belmonte
    TxCell Valbonne Sophia-Antipolis, France
  • Julie Gertner-Dardenne
    TxCell Valbonne Sophia-Antipolis, France
  • Marie-Françoise Hubert
    COLA, Jozerand, France
  • André Sales
    VETOPATH, Antibes, France
  • Papa Babacar Fall
    TxCell Valbonne Sophia-Antipolis, France
  • Clémence Ginet
    TxCell Valbonne Sophia-Antipolis, France
  • Irène Marchetti
    TxCell Valbonne Sophia-Antipolis, France
  • Frédérique Ménard
    VETOPATH, Antibes, France
  • Grégory Lara
    TxCell Valbonne Sophia-Antipolis, France
  • Nicole Bobak
    TxCell Valbonne Sophia-Antipolis, France
  • Arnaud Foussat
    TxCell Valbonne Sophia-Antipolis, France
  • Correspondence: Hélène Asnagli, TxCell, Allée de la Nertière, 06560, Valbonne Sophia-Antipolis, France; helene.asnagli@txcell.com
  • Footnotes
     HA and MJ contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Investigative Ophthalmology & Visual Science October 2015, Vol.56, 6456-6466. doi:10.1167/iovs.15-16883
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      Hélène Asnagli, Marie Jacquin, Nathalie Belmonte, Julie Gertner-Dardenne, Marie-Françoise Hubert, André Sales, Papa Babacar Fall, Clémence Ginet, Irène Marchetti, Frédérique Ménard, Grégory Lara, Nicole Bobak, Arnaud Foussat; Inhibition of Noninfectious Uveitis Using Intravenous Administration of Collagen II–Specific Type 1 Regulatory T Cells. Invest. Ophthalmol. Vis. Sci. 2015;56(11):6456-6466. doi: 10.1167/iovs.15-16883.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

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-10high, IL-4low, IFN-γint). 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.

The unmet medical need of noninfectious uveitis (NIU) treatment remains particularly relevant considering that this condition represents one of the leading causes of blindness in the Western world. Currently, virtually all approved treatments remain steroid based, even though immunosuppressors and biologics are also used. While steroid therapy normally provides fast initial relief of symptoms in uveitis, the effect is limited and associated with significant local and systemic side effects. Moreover, steroid treatment is clearly unsatisfactory, with incomplete responses and refractoriness to treatment in a significant fraction of the patients.1 Immunosuppressors and biologics, of which some are in development for the treatment of NIU, are used off-label and appear to provide some additional help in the management of the most severe cases. Nevertheless, a large proportion of patients are also refractory to these treatments and experience reduction of vision.24 A new well-tolerated alternative is thus needed that would represent a significant benefit for the patients. 
CD4+ regulatory T lymphocytes (Tregs) are involved in the control of unwanted immune responses.5,6 Among Treg subpopulations, type 1 Treg cells, also known as Tr1 cells, were firstly described in 19977 and are characterized by a specific cytokine profile with high production of IL-10, low or no production of IL-4, and intermediate production of IFN-γ.7,8 We have previously demonstrated the therapeutic efficacy of antigen-specific type 1 Treg cells (Ag-Tregs) in animal models of inflammatory colitis, dermatitis, and arthritis.79 Moreover, a clinical trial with Ag-Treg cells for the treatment of refractory Crohn's disease (CD) has shown signs of efficacy together with good tolerability.10 In this study, the type 1 Treg cells administered to CD patients are specific for the dietary antigen ovalbumin, which although not linked with the pathology under treatment, is distributed in the inflamed gut, and thus allows local triggering of the Treg anti-inflammatory and immunoregulatory activities.10 
The role of Tregs in NIU has been mainly studied in the model of experimental autoimmune uveitis (EAU) in mice. This model has been described as a self-remitting ocular pathology where Treg cells are involved in the natural remission of inflammation.11 Indeed, transfer of Treg cells harvested from EAU mice during the active period of inflammation is able to inhibit EAU development in newly disease-prone recipients.12 Several modes of in vivo induction of Treg cells have also been described that allow inhibition of EAU.1316 Moreover, de novo Treg induction has been observed in NIU patients treated with anti–TNF-α monoclonal antibodies.17 Also, patients with active uveitis show a deregulated Treg blood compartment with decreased frequency of circulating Tregs among T cells.18 Interestingly, molecules such as IL-10 and IL-13, two anti-inflammatory cytokines highly produced by Ag-Tregs, are able to decrease ocular inflammation in EAU models.19,20 Antigen specificity of Treg cells has been described as a key Treg attribute to allow local inhibition of inflammation. Indeed, when administered intravenously, Treg cells specific for an ocular antigen, but not polyclonal Treg cells, can inhibit uveitis.21 Altogether, these observations suggest that restoration of a functional Treg compartment or induction of a Treg cell suppressive activity in NIU patients could be an effective therapeutic strategy. 
Here, we describe the nonclinical development of Col-Treg, a collagen II–specific type 1 Treg cellular immunotherapy for the treatment of NIU. Collagen II expression has been described, mainly in cartilaginous tissues but also in the retina, and is constitutively present in the human vitreous.22,23 In the present study, we identified its ocular expression in uveitis in mice, in particular in vitreous, ciliary bodies, and retina (shown in Supplementary Fig. S1). These findings strongly suggest that type II collagen could represent a local activation triggering for Col-Treg cells in the context of ocular inflammation, allowing inhibition of NIU. 
Materials and Methods
Mice
DBA-1 mice (Janvier, Le Genest Saint Isle, France) and transgenic mice carrying the rearranged Vα11.1 and Vβ8.2 TCR chain genes isolated from a collagen type II (Col-II)–specific T-cell hybridoma (kindly provided by R Toes, LUMC, Leiden, The Netherlands) with approval from W Ladiges,24 were used. Experiments were performed in accordance with national guidelines and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and approved by the National Ethics Committee. 
Generation of Mouse Col-Treg Cells, Cytokine Profile, Phenotypic Characterization, and Col-Treg Cell In Vivo Tracking Studies
These methods have been previously described.9 Briefly, at day 0, splenocytes were stimulated in presence of bovine type II collagen (0.5 μg/mL; MD Biosciences, Zurich, Switzerland), recombinant human (rhu) IL-10 (50 ng/mL; R&D Systems, Abingdon, UK), and anti–IL-4 antibodies (10 μg/mL, clone 11B11; eBiosciences, Paris, France). Recombinant murine IL-10 (5 ng/mL; R&D Systems) was added at day 2 and cells were cloned by limiting dilution in the presence of coated anti-CD3 monoclonal antibodies (mAbs; 5 μg/mL, clone 145.2C11; BD Biosciences, Le Pont de Claix, France) and in the presence of irradiated syngeneic splenocytes. T-cell clones were then expanded in the presence of rhu-IL-2 (Proleukin; Norvartis, Basel, Suisse) with coated anti-CD3 (5 μg/mL) and soluble anti-CD28 (1 μg/mL) mAbs. T cells were further expanded in the presence of anti-CD3/CD28 mAbs paramagnetic coated beads (Invitrogen, Villebon sur Yvette, France) with addition of IL-2 at day 2 and at each cell division. 
Antigen Specificity Assay
Mouse Col-Treg cells were stimulated with irradiated syngeneic splenocytes for 5 days in the presence of bovine Coll-II (5–20 μg/mL). For the last 6 hours, proliferating cells were labeled with bromodeoxyuridine (BrdU) and proliferation was detected by specific anti-BrdU ELISA (Roche, Meylan, France). As positive control for polyclonal stimulation, Col-Treg cells were stimulated in the presence of coated anti-CD3 (1 μg/mL) mAb and soluble anti-CD28 Ab (0.1 μg/mL). Human Col-Treg cells were stimulated with 2 μg/mL human collagen type II (Millipore, Saint Quentin en Yvelines, France) for 48 hours. Interleukin 10 secretion was measured by Flowcytomix (eBiosciences). 
In Vitro Suppression Assays
Immunosuppressive function of murine Col-Treg clones was evaluated by cell contact–independent in vitro assay.25 Briefly, mouse Col-Treg cells were activated by polyclonal stimulation in transwell assay on top of anti-CD3 (0.05 μg/mL)–activated DBA-1 splenocytes. Splenocyte proliferation was assessed by carboxyfluorescein diacetate succinimidyl ester (CFSE) staining after 72 hours. Levels of secreted IFN-γ and IL-17A from the suppression assay's culture supernatants were measured by ELISA and Cytometry Beads Array (BD Biosciences). Suppression mediated by soluble factors of human Col-Treg cells was evaluated by the effect of Col-Treg cell culture supernatants on activated Th1- and Th17-like T-cell populations differentiated in the presence of PHA (Sigma-Aldrich Corp., St. Louis, MO, USA) or recombinant TGF-β, IL-23, and IL-21, as previously described.26 After 3 days in the presence of Col-Treg supernatants, Th1-like and Th17 cells were collected and after 2 days of polyclonal stimulation, levels of IL-17A and IFN-γ secretion were measured by Flowcytomix (eBiosciences). 
For suppression mediated by cytolysis, Col-Treg cells were coincubated at different effector to target ratios with myeloid target cells (U937 human cell line or mouse dendritic cells) stained with Dye450 (eBiosciences). After 4 hours, the percentage of dead target cells was assessed with ethidium homodimer staining gated on Dye450+ cells and analyzed by cytometry. 
For suppression mediated by metabolic disruption, inorganic phosphate generated by CD39 (ATPDase) from Col-Treg cells were measured in culture supernatant by the malachite green colorimetric assay according to manufacturer's instructions (R&D Systems). 
Induction and Clinical Evaluation of EAU
At day 0, DBA-1 mice were immunized subcutaneously with a mixture of two human IRBP peptides (150 μg/mL each) emulsified (pIRBP1-20-pIRBP160-181; Biosynthesis, Lewisville, TX, USA) in complete Freund adjuvant (CFA) containing 2 mg/mL Mycobacterium tuberculosis (MD Biosciences). At days 0 and 3, mice received intraperitoneal administration of pertussis toxin (1.5 μg; Sigma-Aldrich Corp.). One week later, mice received subcutaneously the same CFA-IRBP1-20/160-181 emulsion. The following day, mice received either saline or Col-Treg cells (106 cells per animal) via tail vein injection (six to nine animals per group). Ophthalmic examinations were performed by an independent, masked veterinarian, using indirect ophthalmoscopy with interposition of a 60-diopter lens and slit lamp biomicroscopy after topical instillation of a mydriatic solution (0.5% tropicamide; Théa, Clermont-Ferrand, France). Clinical scores were based on the ocular lesions for anterior (0–4), intermediate (0–3), and posterior (0–4) uveitis with an overall clinical score ranging from 0 to 11. Details on the scoring are given in Supplementary Table S1. The parameters included episcleritis, cornea edema, and/or neovascularization, iritis, and/or cyclitis, vitritis, retinitis, or chorioretinitis. 
Histology
Eyes were collected and fixed with Davidson's fixative for histopathologic analysis on 4-μm sections stained with haemalum/eosin/saffron. The EAU severity scoring was performed randomly by an independent histopathologist veterinarian on one to four sections of individual eye, in order to ensure full ocular representation (including lens, optic nerve). The scoring was adapted from Copland et al.,27 with criteria such as cellular infiltration and morphologic changes including retinal folds. Details of histologic score are given in Supplementary Table S2
Immunohistochemistry for Leukocyte Antigens in Ocular Tissues
Eye sections were processed for detection of T cells, B cells, neutrophils, and macrophages with indirect immunoperoxidase staining as described.28 Briefly, one to three sections (including lens, retina, and optic nerve) were stained with antibodies specific for T cells (anti-CD3 mAb), B cells (anti-CD45/B220 mAb), neutrophils (antimyeloperoxidase/MPO mAb), or macrophages (anti–arginase I mAb); stained by specific antibodies coupled with immunoperoxidase; and revealed by 3,3′-diaminobenzidine (DAB) substrate and counterstained with Mayer's hematoxylin. An independent and masked histopathologist performed microscopic examination and counting of ciliary bodies, vitreous body and retina. Cell counting was performed either at microscopic examination only or after digital image analysis (for staining of more than 10 positive cells, ImageJ analysis program [http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA] was used). Data represent mean ± SEM of positive cells counted per eye, with P value < 0.05 (*) or < 0.01 (**) (ANOVA). One representative experiment of three is shown. Immunohistochemical staining and evaluation were performed by a board-certified veterinary pathologist at the Mouse and Animal Pathology Laboratory of Fondazione Filarete (Italy). 
Evaluation of Human Col-Treg Plasticity
Col-Treg or freshly isolated Treg cells from healthy volunteers (control Tregs) were cultured for 7 days under control (IL-2/IL-4) or Th17 conditions as described.26 Interleukin 17-A secretion was measured after 48 hours of polyclonal stimulation by Flowcytomix (eBiosciences). Retinoid-related orphan receptor C (RORC) expression was analyzed by RT-PCR using RNA extraction and cDNA synthesis kits RNAqueous (AMBION-Thermofisher Scientific, Saint Aubin, France) and Verso Kit (AMBION-ThermoFisher Scientific), according to the manufacturer's instructions. Quantitative real-time PCR was performed by using TaqMan universal PCR Master Mix (Applied Biosystems, Foster, CA, USA) and the Pikoreal system (AMBION-ThermoFisher Scientific). For rorc, the Hs01076112_m1 probe was used. RORC expression was calculated as a ratio relative to the β-2 microglobulin as the housekeeping gene. 
Statistical Methods
t-test and analysis of variance were performed by using Graph PAD Instat software (GraphPad, La Jolla, CA, USA). For data with non-Gaussian distribution, Kruskal-Wallis and Mann-Whitney U tests were performed. 
Results
Phenotypic Characterization and In Vitro Function of Col-Treg Cells
The production of Col-Treg cells involved the differentiation of collagen II–specific CD4+ T cells from collagen II–specific TCR transgenic mice into the type 1 Treg cell type, using IL-10 and cloning of the differentiated cell population by limiting dilution. Type 1 Treg selected clones were further expanded in vitro. This selection included (1) collagen II specificity, demonstrated by the expression of the transgenic TCR Vβ chain and the specific in vitro proliferation capacity in response to collagen II (Figs. 1A, 1B); (2) Treg identity demonstrated by the expression of the Treg markers as CD25 and FoxP3 with low surface expression of the IL-7 receptor α chain, CD127 (Fig. 1C), with CD62L being also expressed at low levels; and (3) type 1 Treg differentiation status demonstrated by the cytokine secretion profile after polyclonal activation showing high IL-10 secretion together with low IL-4 secretion and intermediate production of IFN-γ. Interleukin 13 was also produced in relatively high quantities by Col-Treg cells, whereas IL-17 production was absent (Fig. 1D). 
Figure 1
 
In vitro characterization of mouse Col-Treg cells. Mouse Col-Treg cells were analyzed for the expression of TCR Vβ8.1/8.2 on CD3+ gated cells by cytometry (A). Eleven days after polyclonal expansion, Col-Treg cells were stimulated in vitro for 5 days (B), in the presence of the indicated doses of bovine collagen type II or in presence of coated anti-CD3 and soluble anti-CD28 monoclonal antibodies. Proliferation based on BrdU incorporation was revealed by anti-BrdU ELISA. Mouse Col-Treg cells were analyzed for the expression of markers associated with Treg identity: CD25, FoxP3, CD127, and CD62L (C). The data represent the percentage of positive cells gated on CD4+ cells and are representative of the mean ± SEM of two Col-Treg products. Interleukin 10, IL-4, IFN-γ, IL-13, and IL17 secretion after polyclonal stimulation with CD3/CD28 monoclonal antibodies after 48 hours (D). Results represent the mean ± SEM. Data are pooled results, n = 3 to 5 mouse Col-Treg products.
Figure 1
 
In vitro characterization of mouse Col-Treg cells. Mouse Col-Treg cells were analyzed for the expression of TCR Vβ8.1/8.2 on CD3+ gated cells by cytometry (A). Eleven days after polyclonal expansion, Col-Treg cells were stimulated in vitro for 5 days (B), in the presence of the indicated doses of bovine collagen type II or in presence of coated anti-CD3 and soluble anti-CD28 monoclonal antibodies. Proliferation based on BrdU incorporation was revealed by anti-BrdU ELISA. Mouse Col-Treg cells were analyzed for the expression of markers associated with Treg identity: CD25, FoxP3, CD127, and CD62L (C). The data represent the percentage of positive cells gated on CD4+ cells and are representative of the mean ± SEM of two Col-Treg products. Interleukin 10, IL-4, IFN-γ, IL-13, and IL17 secretion after polyclonal stimulation with CD3/CD28 monoclonal antibodies after 48 hours (D). Results represent the mean ± SEM. Data are pooled results, n = 3 to 5 mouse Col-Treg products.
Col-Treg cells were further characterized in terms of immunoregulatory properties. Firstly, as Col-Treg cells expressed several anti-inflammatory soluble factors, the suppressive capacity of activated Col-Treg cell culture supernatants was evaluated on proinflammatory T-cell activation. Soluble factors expressed by Col-Treg cells were able to inhibit the proliferation of activated CD4+ T cells in a transwell suppression assay (Fig. 2A). Col-Treg cell culture supernatant also inhibited the secretion of IL-17A by proinflammatory Th17 cells as well as the production of IFN-γ by proinflammatory Th1 cells (Figs. 2B, 2C). In addition to the production of anti-inflammatory soluble factors, Col-Treg cells expressed surface and intracellular markers generally associated with Treg functions. Col-Treg cells expressed GITR, high levels of intracellular granzyme B, as well as the ectonuclease CD39, but expressed low levels of CTLA-4 (Fig. 2D). The observed high level of granzyme B expression was consistent with the identification of a strong in vitro contact-dependent capacity of Col-Treg to lyze dendritic cells, and the levels of CD39 expression was associated with a capacity to hydrolyze extracellular ATP (Figs. 2E, 2F). 
Figure 2
 
In vitro functions of mouse Col-Treg cells. Col-Treg cells ([AC]; n = 4) were activated by polyclonal stimulation in transwell assay on top of anti-CD3–activated DBA-1 splenocytes. Splenocyte proliferation was assessed by CFSE staining (A), and secretion of mouse IFN-γ (B) and IL-17A (C) was measured 72 hours after stimulation. Expression of marker for Treg functions (D) was analyzed on n = 3 to 4 Col-Treg products. Cytolytic activity was assessed on freshly isolated dendritic cell targets by flow cytometry (n = 3) (E). Adenosine triphosphatase hydrolysis was tested on Col-Treg by colorimetric assay (n = 3) (F). Data are expressed as mean ± SEM. (*) and (**) indicate P values below 0.05 and 0.01, respectively, with t-test statistical analysis.
Figure 2
 
In vitro functions of mouse Col-Treg cells. Col-Treg cells ([AC]; n = 4) were activated by polyclonal stimulation in transwell assay on top of anti-CD3–activated DBA-1 splenocytes. Splenocyte proliferation was assessed by CFSE staining (A), and secretion of mouse IFN-γ (B) and IL-17A (C) was measured 72 hours after stimulation. Expression of marker for Treg functions (D) was analyzed on n = 3 to 4 Col-Treg products. Cytolytic activity was assessed on freshly isolated dendritic cell targets by flow cytometry (n = 3) (E). Adenosine triphosphatase hydrolysis was tested on Col-Treg by colorimetric assay (n = 3) (F). Data are expressed as mean ± SEM. (*) and (**) indicate P values below 0.05 and 0.01, respectively, with t-test statistical analysis.
Altogether, these data demonstrated the specific and multimodal immunoregulatory capacities of Col-Treg cells in vitro, underlying the relevance of testing the therapeutic activity of Col-Treg cells in vivo in models of inflammatory diseases, such as uveitis or arthritis, where collagen II is present in the inflamed tissues, as previously shown.9 
Experimental Autoimmune Uveitis Characterization in DBA-1 Mice
Antigen-specific T-cell adoptive transfer experiments require major histocompatibility complex (MHC) matching between the administered cells and the recipients, allowing efficient antigen presentation and T-cell activation. Col-Treg cells were derived from mice with a DBA-1 background and DBA-1 mice are described to be weakly sensitive to the classical EAU induction with a single IRBP immunization,29 with a disease that does not always lead to retinal damage and strong morphologic changes.30,31 Here, the EAU protocol was optimized by addition of an IRBP-boost immunization 7 days after the first one and the use of two IRBP peptides (see Materials and Methods). DBA-1 immunized mice developed a mild uveoretinitis with onset of clinical signs from day 12 and a peak of disease between day 14 and 15 followed by self-remission (Fig. 3A). Using biomicroscopy and indirect ophthalmoscopy, various clinical signs of anterior, intermediate, and posterior uveitis were observed. In the anterior area of the uveal tract, findings consisted of dilatation of the perilimbal vessels (episcleritis), hypopyon and/or lenticular precipitates of inflammatory exudates and cells (cyclitis), as well as posterior synechiae of the iris (iritis). In some mice, severe episcleritis was associated with edema and/or neovascularization of the cornea. Vitritis was evidenced by turbidity of the vitreous gel. Retinitis with retinal vessel changes (vasculitis and perivasculitis) was present alone or associated with discrete retinal folds and/or papilledema (chorioretinitis). From day 12, because of severe anterior uveitis, vitreous and fundus were not observable in some eyes. Despite a second immunization, immune response of DBA-1 mice against IRBP antigens did not lead to severe uveitis with marked retinal damages. However, a clear significant infiltration of anterior, intermediate, as well as posterior areas, mainly found within the ciliary bodies, vitreous, and retina, demonstrated significant signs of uveitis, allowing the evaluation of Col-Treg effect on EAU (Figs. 3B, 3C). 
Figure 3
 
The EAU model in DBA-1 mice. The structures of the eyes from EAU or naïve mice were examined with indirect ophthalmoscopy and slit lamp biomicroscopy after topical instillation of a mydriatic solution. Clinical observation (A) started before immunization (day 0, naïve) and then from day 8 to day 30 post immunization. Eyes with nonobservable posterior segment, due to severe lesions of anterior and/or intermediate segment, were excluded as indicated on the top of each histogram (n = number of eyes with fundus and vitreous observable/total number of eyes examined). Representative images (B) of eye from EAU mice at disease peak with criteria assessed for the histology scoring. Histology scores (C) comparing control naïve mice (n = 12) and EAU mice (n = 30). Data are expressed as mean ± SEM of individual fully observable eyes' clinical scores and individual eyes' animal histology scores. (*) and (***) indicate P values below 0.05 and 0.001, respectively, with Kruskal-Wallis analysis of variance.
Figure 3
 
The EAU model in DBA-1 mice. The structures of the eyes from EAU or naïve mice were examined with indirect ophthalmoscopy and slit lamp biomicroscopy after topical instillation of a mydriatic solution. Clinical observation (A) started before immunization (day 0, naïve) and then from day 8 to day 30 post immunization. Eyes with nonobservable posterior segment, due to severe lesions of anterior and/or intermediate segment, were excluded as indicated on the top of each histogram (n = number of eyes with fundus and vitreous observable/total number of eyes examined). Representative images (B) of eye from EAU mice at disease peak with criteria assessed for the histology scoring. Histology scores (C) comparing control naïve mice (n = 12) and EAU mice (n = 30). Data are expressed as mean ± SEM of individual fully observable eyes' clinical scores and individual eyes' animal histology scores. (*) and (***) indicate P values below 0.05 and 0.001, respectively, with Kruskal-Wallis analysis of variance.
Immunohistochemistry experiments on eyes from EAU mice revealed that the cellular infiltrate was constituted by a mix of T cells, B cells, neutrophils, and macrophages, as described within susceptible strain and classical EAU model (Fig. 4). Cellular infiltrates were analyzed in ciliary bodies, vitreous, and chorioretinal region. All populations were found in the anterior, intermediate, and posterior areas with particular incidence in the vitreous, confirming the clinical findings throughout the uveal tract. In addition, cytokine measurements at the ocular level demonstrated the presence of IL-6, TNF-α, IFN-γ, and IL-1β, with IL-6 levels already detectable at day 8, after the first IRBP immunization (data not shown). 
Figure 4
 
Intravenous administration of Col-Treg reduces ocular cellular infiltrates. Sections of eyes from EAU mice (n = 8) treated with saline or 106 Col-Treg cells were stained with antibodies specific for for T cells, neutrophils and macrophages (A) and B lymphocytes (B); secondary antibodies coupled with immunoperoxidase; and revealed by DAB substrate and counterstained with Mayer's hematoxylin. Data represent mean ± SEM of positive cells counted per eye, within ciliary bodies, vitreous, and retina (P < 0.05 [*] or < 0.01 [**] with ANOVA, saline-treated mice versus Col-Treg–treated mice). One representative experiment of three is shown.
Figure 4
 
Intravenous administration of Col-Treg reduces ocular cellular infiltrates. Sections of eyes from EAU mice (n = 8) treated with saline or 106 Col-Treg cells were stained with antibodies specific for for T cells, neutrophils and macrophages (A) and B lymphocytes (B); secondary antibodies coupled with immunoperoxidase; and revealed by DAB substrate and counterstained with Mayer's hematoxylin. Data represent mean ± SEM of positive cells counted per eye, within ciliary bodies, vitreous, and retina (P < 0.05 [*] or < 0.01 [**] with ANOVA, saline-treated mice versus Col-Treg–treated mice). One representative experiment of three is shown.
To ensure that collagen II expression was detected in the eyes of EAU DBA-1 mice, immunohistochemistry studies were performed with a specific antibody for collagen II. The data confirmed a strong expression in cartilaginous tracheal tissues (Supplementary Fig. S1B) as well as the vitreous and ciliary bodies (Supplementary Figs. S1C, S1E, S1F) and at a weaker level in the cornea (Fig. 1D). 
Col-Treg Cells Reduce EAU
106 Col-Treg cells or saline were administrated intravenously to EAU mice, 1 day after the boost of immunization (day 8 post immunization). The EAU severity was assessed by ophthalmoscopy/biomicroscopy at day 15 and by histology at day 17. Clinical evaluation revealed a statistically significant inhibition of anterior signs of uveitis together with a reduction, although not reaching statistical significance, of intermediate/posterior uveitis after Col-Treg cell administration (Figs. 5A–C). The absence of statistical significance for areas of intermediate and posterior uveitis is likely linked to the fact that clinical scores were measured on fully observable eyes only (i.e., from anterior to posterior areas) since, as previously mentioned, from day 12 vitreous and fundus were not observable in some eyes (three in the saline group and one in the Col-Treg–treated group). 
Figure 5
 
Intravenous administration of Col-Treg reduces EAU. At day 8 post EAU induction, DBA-1 mice received either saline (n = 7) or 106 (n = 7) Col-Treg cells per mouse intravenously. Clinical ophthalmology analysis was assessed at peak of disease (day 15) post EAU induction by an independent, masked veterinary. Clinical scores (A) for anterior, intermediate, and posterior uveitis are represented for saline- and Col-Treg–treated groups. Data are expressed as mean ± SEM of individual clinical ophthalmology scores of fully observable eyes (n = number of eyes with fundus and vitreous observable/total number of eyes examined). Statistical analysis on the different (anterior, intermediate, and posterior) eye segments of Col-Treg–treated mice versus saline-treated mice with P value < 0.05 (*). Eyes were collected after clinical ophthalmology analysis at day 16 post EAU induction and scored randomly by an independent histopathologist. Grading is based on parameters described in Materials and Methods. Total histology scores of uveitis (B), including infiltration scores within anterior, intermediate, and posterior areas and morphologic scores. Data represent mean of individual mean values ± SEM collected per eye. (*) and (**) indicate P values below 0.05 and 0.01, respectively, with Mann-Whitney statistical analysis. Histology images (C) from naïve mice (left), EAU mice from saline-treated group (middle), Col-Treg–treated group (right) from anterior (top panels) and intermediate/posterior areas (bottom panels).
Figure 5
 
Intravenous administration of Col-Treg reduces EAU. At day 8 post EAU induction, DBA-1 mice received either saline (n = 7) or 106 (n = 7) Col-Treg cells per mouse intravenously. Clinical ophthalmology analysis was assessed at peak of disease (day 15) post EAU induction by an independent, masked veterinary. Clinical scores (A) for anterior, intermediate, and posterior uveitis are represented for saline- and Col-Treg–treated groups. Data are expressed as mean ± SEM of individual clinical ophthalmology scores of fully observable eyes (n = number of eyes with fundus and vitreous observable/total number of eyes examined). Statistical analysis on the different (anterior, intermediate, and posterior) eye segments of Col-Treg–treated mice versus saline-treated mice with P value < 0.05 (*). Eyes were collected after clinical ophthalmology analysis at day 16 post EAU induction and scored randomly by an independent histopathologist. Grading is based on parameters described in Materials and Methods. Total histology scores of uveitis (B), including infiltration scores within anterior, intermediate, and posterior areas and morphologic scores. Data represent mean of individual mean values ± SEM collected per eye. (*) and (**) indicate P values below 0.05 and 0.01, respectively, with Mann-Whitney statistical analysis. Histology images (C) from naïve mice (left), EAU mice from saline-treated group (middle), Col-Treg–treated group (right) from anterior (top panels) and intermediate/posterior areas (bottom panels).
Histologic examination (Fig. 5) revealed that cellular infiltration of the anterior, vitreous, as well as posterior areas was significantly impacted by Col-Treg treatment with a statistically significant difference between saline-treated and Col-Treg–treated mice (Fig. 5B). Morphologic changes were also reduced after Col-Treg treatment with differences between saline- and Col-Treg–treated mice close to statistical significance. The absence of statistical significance is likely linked to the very mild severity of these signs in the DBA-1 mice, as previously mentioned. Nevertheless, immunohistochemistry experiments revealed that infiltrating leukocytes in the ciliary bodies, vitreous, and retina, including T cells, B cells, neutrophils, and macrophages, were significantly reduced after treatment with Col-Treg cells (Fig. 4). These data suggest that Col-Treg administration impacts overall severity of the uveitis mostly by blocking the level of infiltrating cells. 
Migratory Properties of Col-Treg Cells in Noninfectious Uveitis
The migratory properties of Col-Treg cells were analyzed by tracking injected cells in mouse tissues by using quantitative PCR specific for the TCR Vβ chain of Col-Treg cells. Col-Treg cells were administrated intravenously at the peak of disease at a time when eyes show inflammatory signs. After 24 hours, tissues were collected for analysis. Col-Treg cells migrated into the inflamed eyes as well as in highly vascularized tissues such as lung, liver, and spleen as well as in lymph nodes (Fig. 6A). The detection of cellular-based products in highly vascularized tissues, such as lung, liver, and spleen, post intravenous administration, is not particular to Col-Treg cells but has been largely described for other cellular-based therapies in nonclinical and clinical settings (T lymphocytes and other cell types).3234 When injected in healthy animals, Col-Treg cells migrated to the same extent in highly vascularized tissues and lymph nodes but were not detected in the eyes (Fig. 6B). This suggests that inflammation drives the homing of Col-Treg cells in the eyes of EAU mice. Interestingly, the number of infiltrated Col-Treg cells in the eyes correlated with the severity of the disease (Fig. 6C). One week to 1 month after intravenous administration, Col-Treg cells were no longer detected in the tissues of EAU mice, indicating a lack of persistence together with absence of uncontrolled proliferation even in the constitutive presence of the specific antigen collagen II in mice tissues (data not shown). In addition, Col-Treg cells expressed high levels of LFA-1 and PSGL-1, two adhesion molecules involved in the recognition of inflamed vascular endothelium (Fig. 6D). 
Figure 6
 
Biodistribution of Col-Treg after intravenous administration. The EAU (n = 7) (A) or naïve (n = 5) (B) mice received either 107 or 3 × 106 Col-Treg cells intravenously. Twenty-four hours post administration, organs were collected and gDNA was extracted for quantification of Col-Treg cells by qPCR. Results are expressed as number of cells per milligram or per organ (*). Ophthalmologic scores were performed and correlated to the number of Col-Treg cells (C) detected per eye from EAU mice (closed circles) or naïve mice (open triangles, n = 5). Expression of LFA-1, PSGL-1, and VLA-4 (composed of CD29 and CD49d) integrins was assessed on Col-Treg cells (n = 4) by flow cytometry (D). Data are expressed as mean ± SEM. gDNA, genomic deoxyribonucleic acid; qPCR, quantitative polymerase chain reaction.
Figure 6
 
Biodistribution of Col-Treg after intravenous administration. The EAU (n = 7) (A) or naïve (n = 5) (B) mice received either 107 or 3 × 106 Col-Treg cells intravenously. Twenty-four hours post administration, organs were collected and gDNA was extracted for quantification of Col-Treg cells by qPCR. Results are expressed as number of cells per milligram or per organ (*). Ophthalmologic scores were performed and correlated to the number of Col-Treg cells (C) detected per eye from EAU mice (closed circles) or naïve mice (open triangles, n = 5). Expression of LFA-1, PSGL-1, and VLA-4 (composed of CD29 and CD49d) integrins was assessed on Col-Treg cells (n = 4) by flow cytometry (D). Data are expressed as mean ± SEM. gDNA, genomic deoxyribonucleic acid; qPCR, quantitative polymerase chain reaction.
Human Col-Treg Production and Characterization
The data presented above highlight the relevance of Col-Treg use for NIU treatment. To assess the feasibility of the approach in humans, we produced human Col-Treg cells and assessed their phenotypic profile as well as their in vitro function. Col-Treg cells were produced from the peripheral blood of healthy donors, activated with native collagen II. The Col-II–activated cells were cultured at low density, and cell suspensions showing a collagen II specificity, based on IL-10–induced secretion upon collagen II stimulation (data not shown) and a type 1 Treg cytokine secretion profile after polyclonal stimulation (Fig. 7A), were further expanded and characterized. Human Col-Treg cells were comparable to their rodent counterparts in terms of marker expression and in vitro suppressive function (Figs. 7B, 7C, 7D). Indeed, human Col-Treg cells exerted soluble factor–mediated suppression of proinflammatory Th17 or Th1-like populations, indicated by reduction of IL-17 or IFN-γ secretion (Figs. 7E, 7F) as well as contact-dependent myeloid cell cytotoxicity (Fig. 7G) and ATP hydrolysis (Fig. 7H). Expression of adhesion molecules by Col-Treg from both humans and mice was also comparable with the exception of VLA-4, composed of a heterodimer of CD29 and CD49d (Fig. 7D), highly expressed by human Col-Treg cells. 
Figure 7
 
In vitro characterization of human Col-Treg. Human Col-Treg cells were selected by their cytokine secretion profile (A). The expression of markers associated with Treg identity (B), Treg functions (C), and adhesion molecules (D) were analyzed by flow cytometry. The data represent the percentage of positive cells gated on CD4+ cells and are expressed as mean ± SEM (n = 5). Suppressive activity of Col-Treg supernatants (n = 5), obtained after polyclonal activation, was assessed on IL-17A (E) and IFN-γ (F) production by either Th17- or Th1-like cells, respectively. Cytolytic activity of Col-Treg (n = 4) was assessed on myeloid cell line (U937) by flow cytometry (G), and ATP hydrolysis (H) was tested on Col-Treg (n = 5) by colorimetric assay.
Figure 7
 
In vitro characterization of human Col-Treg. Human Col-Treg cells were selected by their cytokine secretion profile (A). The expression of markers associated with Treg identity (B), Treg functions (C), and adhesion molecules (D) were analyzed by flow cytometry. The data represent the percentage of positive cells gated on CD4+ cells and are expressed as mean ± SEM (n = 5). Suppressive activity of Col-Treg supernatants (n = 5), obtained after polyclonal activation, was assessed on IL-17A (E) and IFN-γ (F) production by either Th17- or Th1-like cells, respectively. Cytolytic activity of Col-Treg (n = 4) was assessed on myeloid cell line (U937) by flow cytometry (G), and ATP hydrolysis (H) was tested on Col-Treg (n = 5) by colorimetric assay.
Several studies have highlighted the fact that Treg cells have the capacity to differentiate into Th17 proinflammatory cells in an inflammatory environment, a phenomenon known as plasticity.26 We evaluated in vitro the plasticity potential of Col-Treg, using culture conditions known to drive Th17 differentiation. Results demonstrated that, in contrast to primary blood-derived Treg cells, Col-Treg cells do not express ROR-γt or produce IL-17A, two hallmarks of Th17 proinflammatory cells (Figs. 8A, 8B). 
Figure 8
 
Absence of in vitro plasticity of human Col-Treg cells under Th17 conditions. After 7 days of culture under control or Th17 conditions (see Materials and Methods), Col-Treg or control Treg cells (primary blood-derived Treg) from healthy volunteers were stimulated with anti-CD3 and anti-CD28 antibodies, supernatants were collected after 48 hours for evaluation of IL-17A levels (A) or cells were collected for RNA extraction and evaluation of RORC expression by RT-PCR (B). The data are expressed as mean ± SEM (n = 3).
Figure 8
 
Absence of in vitro plasticity of human Col-Treg cells under Th17 conditions. After 7 days of culture under control or Th17 conditions (see Materials and Methods), Col-Treg or control Treg cells (primary blood-derived Treg) from healthy volunteers were stimulated with anti-CD3 and anti-CD28 antibodies, supernatants were collected after 48 hours for evaluation of IL-17A levels (A) or cells were collected for RNA extraction and evaluation of RORC expression by RT-PCR (B). The data are expressed as mean ± SEM (n = 3).
Discussion
Here we described the use and possible therapeutic benefit of Col-Treg, a regulatory T cell–based immunotherapy for the treatment of NIU in mice and the potential for this immunotherapy to be translated in humans. Firstly, characterization results showed that Col-Treg belongs to the type 1 Treg cell subpopulation. The therapeutic benefit of type 1 Treg adoptive transfer has been previously demonstrated in models of skin inflammation,7 asthma,8 rheumatoid arthritis,9 and type-1 diabetes.35 In all these settings, type 1 Treg homed preferentially to the inflamed tissues, as observed here with a Col-Treg migration in inflamed, but not in healthy eyes. Interestingly, the migration of Col-Treg cells to inflamed eyes correlated to the degree of inflammation at the time of their administration, suggesting that Col-Treg cells might be particularly active in a severe inflammatory context. High expression of LFA-1 and PSGL-1 are likely to be involved in this migration by inducing strong adhesion to the activated vascular endothelium of the inflamed ocular tissues.36 Indeed, P-selectin and ICAM-1, the respective ligands of PSGL-1 and LFA-1, are known to be present in inflamed eye vascular endothelium in EAU. Together with this capacity to home to inflammatory lesions, antigen-specificity is also a key parameter for type 1 Treg cell efficacy in vivo, allowing them to be effective only in a specific location. Indeed, in inflammatory bowel disease (IBD) models, specificity to ovalbumin has been used to trigger the immunoregulatory function of ovalbumin-specific type 1 Treg (Ova-Treg) cells in the inflamed gut.7,8 Absence of ovalbumin negates the therapeutic activity of Ova-Treg despite the presence of cells in the inflamed areas, demonstrating that in situ antigen recognition is a crucial step in the mode of action of antigen-specific type 1 Treg–based immunotherapy. Here, collagen II is a constitutive antigen of the eye, with reported higher protein expression in vitreous and weaker signal in retinal epithelium, cornea, and sclera.23 This pattern of murine collagen type II expression was confirmed in the present study in the EAU model at the peak of disease (Supplementary Fig. S1). It is postulated that as Col-Treg cells migrate to the inflamed eye, antigen recognition occurs at the ocular level, leading to local Col-Treg activation and bystander suppressive effect. Despite the use of DBA-1 mice, in which only mild EAU can be obtained upon immunization with IRBP peptides, the results from clinical and histologic evaluations show Col-Treg inhibition of uveitis-related changes in the ocular tissues. Here, although Col-Treg administration at day 8 after the first IRBP immunization (1 day after the boost of immunization) cannot be seen as a curative treatment because of lack of clinical symptoms, it shows an inhibition of an ongoing systemic and ocular proinflammatory immune response witnessed by elevated proinflammatory cytokine levels at day 8 of the disease. 
One particularity of cell-based therapy, in contrast to biologics, resides in multiple effector activities that can act in synergy to dampen inflammation. As previously shown for type 1 Treg cells,37 Col-Treg cells can kill myeloid cells, inhibit proinflammatory T-cell activity through cell contact and anti-inflammatory soluble factors,7,37,38 and modify the extracellular compartment by ATP hydrolysis.39 These multiple mechanisms of action suggest firstly that cell-based approaches can be more effective than monotarget approaches such as monoclonal antibodies; and secondly, that resistance of patients to autologous cell-based therapies is likely to be less important than resistance to monoclonal antibody–based therapies. 
Translating T-cell immunotherapy from the research setting to the clinic is a challenge taking into account the complexity of any cellular entity. Here, we described the phenotypic and functional comparability between mice and human Col-Treg cells. In view of the efficacy of Col-Treg cells in the EAU model, translating Col-Treg cell treatment to the clinics, especially in refractory patients, would bring a new therapeutic option to physicians and patients. Clinical experience has already been gathered on the use of antigen-specific type 1 Treg cells for treatment of chronic inflammatory diseases in a first human study in which CD patients have been given Ovasave, an autologous Ova-Treg cell–based product.10 Tolerability is good with no adverse events related to the Ova-Treg cells. Indeed, the autologous nature of these types of products renders unlikely immune rejection/reaction against the patient's own cells or the endogenous secreted soluble factors. 
Several potential risks associated with Treg-based cell therapies have been anticipated.40,41 Firstly, uncontrolled proliferation and tumorigenicity could be developed upon chronic stimulation and could lead to T-cell malignancies. We demonstrated that Col-Treg cells are not maintained in a long-term manner in injected animals and have a limited survival capacity upon chronic stimulation in vitro (data not shown), highlighting the absence of tumorigenicity potential of Col-Treg. Secondly, chronic activation could lead to undesired or off-target immunosuppression. Col-Treg acts locally and only in inflamed tissues with concomitant collagen II expression. This mechanism of action reduces the risks of systemic or off-target undesired immunosuppression. A third potential risk is related to the presence of proinflammatory Th17 cellular impurities or to the plasticity described for Treg cell subpopulations.4143 Human Col-Treg cells did not reveal in vitro plasticity potential. In addition, IL-17A secretion was totally absent from human Col-Treg cells, demonstrating the absence of Th17 cells in the injected cells. 
Altogether, these results suggest that the Col-Treg safety profile is in line with its potential translation to human clinical settings. The feasibility of this translation is particularly underlined by the comparability between mouse and human Col-Treg cells, strongly suggesting the potential of Treg-cell–based therapy for inflammatory ocular diseases, especially, NIU. 
Acknowledgments
Special thanks to Eugenio Scanziani (Filarete Foundation, Milano, Italy) for technical help and expertise in histopathology. 
Disclosure: H. Asnagli, TxCell (E); M. Jacquin, TxCell (E); N. Belmonte, TxCell (E); J. Gertner-Dardenne, TxCell (E); M.-F. Hubert, None; A. Sales, None; P.B. Fall, TxCell (E); C. Ginet, TxCell (E); I. Marchetti, TxCell (E); F. Ménard, None; G. Lara, TxCell (E); N. Bobak, TxCell (E); A. Foussat, TxCell (E) 
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Figure 1
 
In vitro characterization of mouse Col-Treg cells. Mouse Col-Treg cells were analyzed for the expression of TCR Vβ8.1/8.2 on CD3+ gated cells by cytometry (A). Eleven days after polyclonal expansion, Col-Treg cells were stimulated in vitro for 5 days (B), in the presence of the indicated doses of bovine collagen type II or in presence of coated anti-CD3 and soluble anti-CD28 monoclonal antibodies. Proliferation based on BrdU incorporation was revealed by anti-BrdU ELISA. Mouse Col-Treg cells were analyzed for the expression of markers associated with Treg identity: CD25, FoxP3, CD127, and CD62L (C). The data represent the percentage of positive cells gated on CD4+ cells and are representative of the mean ± SEM of two Col-Treg products. Interleukin 10, IL-4, IFN-γ, IL-13, and IL17 secretion after polyclonal stimulation with CD3/CD28 monoclonal antibodies after 48 hours (D). Results represent the mean ± SEM. Data are pooled results, n = 3 to 5 mouse Col-Treg products.
Figure 1
 
In vitro characterization of mouse Col-Treg cells. Mouse Col-Treg cells were analyzed for the expression of TCR Vβ8.1/8.2 on CD3+ gated cells by cytometry (A). Eleven days after polyclonal expansion, Col-Treg cells were stimulated in vitro for 5 days (B), in the presence of the indicated doses of bovine collagen type II or in presence of coated anti-CD3 and soluble anti-CD28 monoclonal antibodies. Proliferation based on BrdU incorporation was revealed by anti-BrdU ELISA. Mouse Col-Treg cells were analyzed for the expression of markers associated with Treg identity: CD25, FoxP3, CD127, and CD62L (C). The data represent the percentage of positive cells gated on CD4+ cells and are representative of the mean ± SEM of two Col-Treg products. Interleukin 10, IL-4, IFN-γ, IL-13, and IL17 secretion after polyclonal stimulation with CD3/CD28 monoclonal antibodies after 48 hours (D). Results represent the mean ± SEM. Data are pooled results, n = 3 to 5 mouse Col-Treg products.
Figure 2
 
In vitro functions of mouse Col-Treg cells. Col-Treg cells ([AC]; n = 4) were activated by polyclonal stimulation in transwell assay on top of anti-CD3–activated DBA-1 splenocytes. Splenocyte proliferation was assessed by CFSE staining (A), and secretion of mouse IFN-γ (B) and IL-17A (C) was measured 72 hours after stimulation. Expression of marker for Treg functions (D) was analyzed on n = 3 to 4 Col-Treg products. Cytolytic activity was assessed on freshly isolated dendritic cell targets by flow cytometry (n = 3) (E). Adenosine triphosphatase hydrolysis was tested on Col-Treg by colorimetric assay (n = 3) (F). Data are expressed as mean ± SEM. (*) and (**) indicate P values below 0.05 and 0.01, respectively, with t-test statistical analysis.
Figure 2
 
In vitro functions of mouse Col-Treg cells. Col-Treg cells ([AC]; n = 4) were activated by polyclonal stimulation in transwell assay on top of anti-CD3–activated DBA-1 splenocytes. Splenocyte proliferation was assessed by CFSE staining (A), and secretion of mouse IFN-γ (B) and IL-17A (C) was measured 72 hours after stimulation. Expression of marker for Treg functions (D) was analyzed on n = 3 to 4 Col-Treg products. Cytolytic activity was assessed on freshly isolated dendritic cell targets by flow cytometry (n = 3) (E). Adenosine triphosphatase hydrolysis was tested on Col-Treg by colorimetric assay (n = 3) (F). Data are expressed as mean ± SEM. (*) and (**) indicate P values below 0.05 and 0.01, respectively, with t-test statistical analysis.
Figure 3
 
The EAU model in DBA-1 mice. The structures of the eyes from EAU or naïve mice were examined with indirect ophthalmoscopy and slit lamp biomicroscopy after topical instillation of a mydriatic solution. Clinical observation (A) started before immunization (day 0, naïve) and then from day 8 to day 30 post immunization. Eyes with nonobservable posterior segment, due to severe lesions of anterior and/or intermediate segment, were excluded as indicated on the top of each histogram (n = number of eyes with fundus and vitreous observable/total number of eyes examined). Representative images (B) of eye from EAU mice at disease peak with criteria assessed for the histology scoring. Histology scores (C) comparing control naïve mice (n = 12) and EAU mice (n = 30). Data are expressed as mean ± SEM of individual fully observable eyes' clinical scores and individual eyes' animal histology scores. (*) and (***) indicate P values below 0.05 and 0.001, respectively, with Kruskal-Wallis analysis of variance.
Figure 3
 
The EAU model in DBA-1 mice. The structures of the eyes from EAU or naïve mice were examined with indirect ophthalmoscopy and slit lamp biomicroscopy after topical instillation of a mydriatic solution. Clinical observation (A) started before immunization (day 0, naïve) and then from day 8 to day 30 post immunization. Eyes with nonobservable posterior segment, due to severe lesions of anterior and/or intermediate segment, were excluded as indicated on the top of each histogram (n = number of eyes with fundus and vitreous observable/total number of eyes examined). Representative images (B) of eye from EAU mice at disease peak with criteria assessed for the histology scoring. Histology scores (C) comparing control naïve mice (n = 12) and EAU mice (n = 30). Data are expressed as mean ± SEM of individual fully observable eyes' clinical scores and individual eyes' animal histology scores. (*) and (***) indicate P values below 0.05 and 0.001, respectively, with Kruskal-Wallis analysis of variance.
Figure 4
 
Intravenous administration of Col-Treg reduces ocular cellular infiltrates. Sections of eyes from EAU mice (n = 8) treated with saline or 106 Col-Treg cells were stained with antibodies specific for for T cells, neutrophils and macrophages (A) and B lymphocytes (B); secondary antibodies coupled with immunoperoxidase; and revealed by DAB substrate and counterstained with Mayer's hematoxylin. Data represent mean ± SEM of positive cells counted per eye, within ciliary bodies, vitreous, and retina (P < 0.05 [*] or < 0.01 [**] with ANOVA, saline-treated mice versus Col-Treg–treated mice). One representative experiment of three is shown.
Figure 4
 
Intravenous administration of Col-Treg reduces ocular cellular infiltrates. Sections of eyes from EAU mice (n = 8) treated with saline or 106 Col-Treg cells were stained with antibodies specific for for T cells, neutrophils and macrophages (A) and B lymphocytes (B); secondary antibodies coupled with immunoperoxidase; and revealed by DAB substrate and counterstained with Mayer's hematoxylin. Data represent mean ± SEM of positive cells counted per eye, within ciliary bodies, vitreous, and retina (P < 0.05 [*] or < 0.01 [**] with ANOVA, saline-treated mice versus Col-Treg–treated mice). One representative experiment of three is shown.
Figure 5
 
Intravenous administration of Col-Treg reduces EAU. At day 8 post EAU induction, DBA-1 mice received either saline (n = 7) or 106 (n = 7) Col-Treg cells per mouse intravenously. Clinical ophthalmology analysis was assessed at peak of disease (day 15) post EAU induction by an independent, masked veterinary. Clinical scores (A) for anterior, intermediate, and posterior uveitis are represented for saline- and Col-Treg–treated groups. Data are expressed as mean ± SEM of individual clinical ophthalmology scores of fully observable eyes (n = number of eyes with fundus and vitreous observable/total number of eyes examined). Statistical analysis on the different (anterior, intermediate, and posterior) eye segments of Col-Treg–treated mice versus saline-treated mice with P value < 0.05 (*). Eyes were collected after clinical ophthalmology analysis at day 16 post EAU induction and scored randomly by an independent histopathologist. Grading is based on parameters described in Materials and Methods. Total histology scores of uveitis (B), including infiltration scores within anterior, intermediate, and posterior areas and morphologic scores. Data represent mean of individual mean values ± SEM collected per eye. (*) and (**) indicate P values below 0.05 and 0.01, respectively, with Mann-Whitney statistical analysis. Histology images (C) from naïve mice (left), EAU mice from saline-treated group (middle), Col-Treg–treated group (right) from anterior (top panels) and intermediate/posterior areas (bottom panels).
Figure 5
 
Intravenous administration of Col-Treg reduces EAU. At day 8 post EAU induction, DBA-1 mice received either saline (n = 7) or 106 (n = 7) Col-Treg cells per mouse intravenously. Clinical ophthalmology analysis was assessed at peak of disease (day 15) post EAU induction by an independent, masked veterinary. Clinical scores (A) for anterior, intermediate, and posterior uveitis are represented for saline- and Col-Treg–treated groups. Data are expressed as mean ± SEM of individual clinical ophthalmology scores of fully observable eyes (n = number of eyes with fundus and vitreous observable/total number of eyes examined). Statistical analysis on the different (anterior, intermediate, and posterior) eye segments of Col-Treg–treated mice versus saline-treated mice with P value < 0.05 (*). Eyes were collected after clinical ophthalmology analysis at day 16 post EAU induction and scored randomly by an independent histopathologist. Grading is based on parameters described in Materials and Methods. Total histology scores of uveitis (B), including infiltration scores within anterior, intermediate, and posterior areas and morphologic scores. Data represent mean of individual mean values ± SEM collected per eye. (*) and (**) indicate P values below 0.05 and 0.01, respectively, with Mann-Whitney statistical analysis. Histology images (C) from naïve mice (left), EAU mice from saline-treated group (middle), Col-Treg–treated group (right) from anterior (top panels) and intermediate/posterior areas (bottom panels).
Figure 6
 
Biodistribution of Col-Treg after intravenous administration. The EAU (n = 7) (A) or naïve (n = 5) (B) mice received either 107 or 3 × 106 Col-Treg cells intravenously. Twenty-four hours post administration, organs were collected and gDNA was extracted for quantification of Col-Treg cells by qPCR. Results are expressed as number of cells per milligram or per organ (*). Ophthalmologic scores were performed and correlated to the number of Col-Treg cells (C) detected per eye from EAU mice (closed circles) or naïve mice (open triangles, n = 5). Expression of LFA-1, PSGL-1, and VLA-4 (composed of CD29 and CD49d) integrins was assessed on Col-Treg cells (n = 4) by flow cytometry (D). Data are expressed as mean ± SEM. gDNA, genomic deoxyribonucleic acid; qPCR, quantitative polymerase chain reaction.
Figure 6
 
Biodistribution of Col-Treg after intravenous administration. The EAU (n = 7) (A) or naïve (n = 5) (B) mice received either 107 or 3 × 106 Col-Treg cells intravenously. Twenty-four hours post administration, organs were collected and gDNA was extracted for quantification of Col-Treg cells by qPCR. Results are expressed as number of cells per milligram or per organ (*). Ophthalmologic scores were performed and correlated to the number of Col-Treg cells (C) detected per eye from EAU mice (closed circles) or naïve mice (open triangles, n = 5). Expression of LFA-1, PSGL-1, and VLA-4 (composed of CD29 and CD49d) integrins was assessed on Col-Treg cells (n = 4) by flow cytometry (D). Data are expressed as mean ± SEM. gDNA, genomic deoxyribonucleic acid; qPCR, quantitative polymerase chain reaction.
Figure 7
 
In vitro characterization of human Col-Treg. Human Col-Treg cells were selected by their cytokine secretion profile (A). The expression of markers associated with Treg identity (B), Treg functions (C), and adhesion molecules (D) were analyzed by flow cytometry. The data represent the percentage of positive cells gated on CD4+ cells and are expressed as mean ± SEM (n = 5). Suppressive activity of Col-Treg supernatants (n = 5), obtained after polyclonal activation, was assessed on IL-17A (E) and IFN-γ (F) production by either Th17- or Th1-like cells, respectively. Cytolytic activity of Col-Treg (n = 4) was assessed on myeloid cell line (U937) by flow cytometry (G), and ATP hydrolysis (H) was tested on Col-Treg (n = 5) by colorimetric assay.
Figure 7
 
In vitro characterization of human Col-Treg. Human Col-Treg cells were selected by their cytokine secretion profile (A). The expression of markers associated with Treg identity (B), Treg functions (C), and adhesion molecules (D) were analyzed by flow cytometry. The data represent the percentage of positive cells gated on CD4+ cells and are expressed as mean ± SEM (n = 5). Suppressive activity of Col-Treg supernatants (n = 5), obtained after polyclonal activation, was assessed on IL-17A (E) and IFN-γ (F) production by either Th17- or Th1-like cells, respectively. Cytolytic activity of Col-Treg (n = 4) was assessed on myeloid cell line (U937) by flow cytometry (G), and ATP hydrolysis (H) was tested on Col-Treg (n = 5) by colorimetric assay.
Figure 8
 
Absence of in vitro plasticity of human Col-Treg cells under Th17 conditions. After 7 days of culture under control or Th17 conditions (see Materials and Methods), Col-Treg or control Treg cells (primary blood-derived Treg) from healthy volunteers were stimulated with anti-CD3 and anti-CD28 antibodies, supernatants were collected after 48 hours for evaluation of IL-17A levels (A) or cells were collected for RNA extraction and evaluation of RORC expression by RT-PCR (B). The data are expressed as mean ± SEM (n = 3).
Figure 8
 
Absence of in vitro plasticity of human Col-Treg cells under Th17 conditions. After 7 days of culture under control or Th17 conditions (see Materials and Methods), Col-Treg or control Treg cells (primary blood-derived Treg) from healthy volunteers were stimulated with anti-CD3 and anti-CD28 antibodies, supernatants were collected after 48 hours for evaluation of IL-17A levels (A) or cells were collected for RNA extraction and evaluation of RORC expression by RT-PCR (B). The data are expressed as mean ± SEM (n = 3).
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