COMPOSITIONS AND METHODS FOR ANTIGEN-SPECIFIC TOLERANCE

Abstract

The invention is in the field of immunotherapy. More particularly, the invention provides a composition comprising a Heme Oxygenase-1 (HO-1) and antigens. Also provided herein are methods of administering the compositions of the invention by subcutaneous, intradermal or topical administration in a patient for inducing antigen-specific tolerance.

Claims

1. A method for inducing immune tolerance in a patient suffering from or at risk of a condition related to immune tolerance, comprising administering to the patient a therapeutically effective amount of a composition comprising (i) a Heme Oxygenase-1 (HO-1) inducer and (ii) at least one pathogenic antigen involved in the condition, wherein the composition is administrated topically or intradermally to a patient's skin of the patient.

2. The method of claim 1, wherein the pathogenic antigen is not insulin.

3. The method of claim 1 wherein the HO-1 inducer is not rapamycin.

4. The method according to claim 2, wherein the HO-1 inducer is Cobalt protoporphyrin (CoPP), protoporphyrin IX containing a ferric iron ion (Heme B) with a chloride ligand (Hemin), hematin, iron protoporphyrin or heme degradation products.

5. The method according to claim 2, wherein said pathogenic antigen is an autoantigen, an alloantigen or an allergen.

6. The method according to claim 5, wherein said autoantigen is selected from the group consisting of myelin-related antigen, myelin oligodendrocyte glycoprotein (MOG) and proteolipid protein (PLP).

7. The method according to claim 5, wherein said autoantigen is selected from the group consisting of glutamic acid decarboxylase 65 (GAD65), glial fibrillary acidic protein (GFAP), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), insulinoma-associated antigen-2 (IA-2) and zinc transporter 8 (ZnT8).

8. The method according to claim 5, wherein said autoantigen is type II collagen (CTII).

9. The method according to claim 5, wherein the alloantigen is selected from the group consisting of antigens expressed by the allograft, proteins expressed in the course of gene therapy and therapeutic proteins.

10. The method according to claim 2, wherein said composition is formulated for subcutaneous, intradermal or topical administration.

11. (canceled)

12. The method of claim 1, wherein the immune tolerance is antigen-specific tolerance.

13. A method for preventing or reducing transplant rejection in a patient in need thereof, comprising a step of administering to the patient a therapeutically effective amount of a composition comprising (i) a Heme Oxygenase-1 (HO-1) inducer and (ii) at least one pathogenic antigen relevant to the transplant, wherein the composition is administrated topically or intradermally to skin of the patient, and wherein the pathogenic antigen is not insulin.

14. A method for preventing or treating an autoimmune disease, an unwanted immune response against proteins expressed in the course of gene therapy or therapeutic proteins, and/or an allergy in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a composition comprising (i) a Heme Oxygenase-1 (HO-1) inducer and (ii) at least one pathogenic antigen involved in the autoimmune disease, the unwanted immune response or the allergy, wherein the composition is administrated topically or intradermally to skin of the patient, and wherein the pathogenic antigen is not insulin.

15. The method according to claim 14, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, juvenile oligoarthritis, collagen-induced arthritis, adjuvant-induced arthritis, Sjogren's syndrome, multiple sclerosis, experimental autoimmune encephalomyelitis, inflammatory bowel disease (e.g. Crohn's disease and ulcerative colitis), autoimmune gastric atrophy, pemphigus vulgaris, psoriasis, vitiligo, type 1 diabetes (T1D), non-obese diabetes, myasthenia gravis, Grave's disease, Hashimoto's thyroiditis, sclerosing cholangitis, sclerosing sialadenitis, systemic lupus erythematosis, autoimmune thrombocytopenia purpura, Goodpasture's syndrome, Addison's disease, systemic sclerosis, polymyositis, dermatomyositis, acquired haemophilia and thrombotic thrombocytopenic purpura (TTP).

16. (canceled)

17. An in vitro or ex vivo method for generating a population of tolerogenic antigen-presenting cells (APCs) specific for an antigen of interest, comprising a step of culturing a population of APCs with a culture medium comprising a heme oxygenase-1 (HO-1) inducer and said antigen of interest.

18. A population of antigen-specific tolerogenic APCs generated by the method of claim 17.

19. The method of claim 6, wherein the myelin-related antigen is myelin basic protein (MBP) or MBP83-102 peptide.

20. The method of claim 6, wherein the MOG is a MOG35-55 peptide.

21. The method of claim 6, wherein the PLP is a PLP139-151 peptide.

Description

FIGURES

[0128] FIG. 1A-D: Intradermal injection of CoPP and autoantigen induces HO-1 in MHC-II.sup.+ cells in the draining LN, and tolerizes CTLs. (A, B) Twenty-four hours after intradermal injection of ovalbumin alone or with HO-1 inducer (CoPP), the proportion of HO-1.sup.+ cells among MHC-II.sup.+ cells was determined in splenocytes and draining LNs by FACS. Data are shown as a representative density plot (A), and as mean frequenciess.e.m. of three independent experiments (n>3 mice/group) (B). (C, D) The day after intradermal treatment with the indicated conditions, Rip-OVA.sup.high mice were transferred with 0.510.sup.5 autoreactive CTLs. Mice were monitored for diabetes development. HSA: human serum albumin. Data are shown from the indicated numbers of mice pooled from at least two independent experiments (C). At 6-8 days after treatment, histological analysis was used to determine the extent of insulitis in two different experiments (n=5-6 mice/group), with >150 islets analyzed for both groups (D). *P<0.05; **P<0.01; ***P<0.001.

[0129] FIG. 2: Intradermal injection of CoPP and autoantigen induces HO-1.sup.+ blood-MoDCs. In C57/B6 mice, the cell populations in draining LN were analyzed 24 hours after intradermal injection of ovalbumin (OVA) alone or with HO-1 inducer (CoPP). The number of MHC-II.sup.+F4/80.sup.+CD11b.sup.+Ly6C.sup.+ cells in the draining LN (n=4 mice/group) is reported.

[0130] FIG. 3A-B: HO-1.sup.+ moDCs tolerize autoreactive CTLs. Rip-OVA.sup.high mice were transferred with 0.510.sup.5 autoreactive CTLs, and were co-cultured for 4 hours with MHC-II.sup.+ or CD11b.sup.+CD11c.sup.lowLy6C.sup.highF4/80.sup.+ cells isolated from the draining LN of mice intradermally immunized with OVA or CoPP-OVA (A). Mice were monitored for diabetes development.

[0131] Data shown are from the indicated numbers of mice pooled from three independent experiments. (B) Twenty-four hours after intradermal treatment with the indicated conditions, F4/80-sorted cells from the draining LNs were stimulated with LPS for 24 h. Supernatants were analyzed for IL-12 and IL-10 contents by ELISA. Data show means.e.m. of three experiments.

[0132] FIG. 4A-D: Intradermal injection of CoPP plus autoantigen reduces CTL velocity.

Rip-OVA.sup.high mice were intradermally immunized with OVA or CoPP-OVA. Twenty-four hours after, mice were either transferred with 0.510.sup.5 autoreactive CD45.1.sup.+ CTLs (A-B) or MHC-II.sup.+ cells were isolated from draining LN for in vitro experiments (C, D). (A) On day 2 or 6, in vivo CTL assay was performed and specific lysis was determined 12 hours after target cell transfer; data show means.e.m. of three experiments (n6 mice/group). (B) Two days after CD45.1.sup.+ CTLs transfer, absolute numbers of CD8.sup.+CD45.1.sup.+ cells were determined in spleen, draining LNs, and PLNs; data show means.e.m. of three experiments (n=9 mice/group). For velocity measurement (C) and transwell migration assay (D), data show means.e.m. of three independent experiments. ***P<0.001.

[0133] FIG. 5A-C: Phenotype of human monocyte following HO-1 inducer treatment. (A) Human PBMC were analyzed for HO-1 expression 4 hours after in vitro hemin treatment.

[0134] Data show mean frequenciess.e.m. of five donors. Human monocytic THP-1 cells were analyzed for HO-1 expression 16 hours after in vitro CoPP treatment (B) or clinically relevant hemin treatment (C). Data shown as means.e.m. of three independent experiments (A, B, C). *P<0.05.

[0135] FIG. 6A-G. Intradermal injection of CoPP and autoantigen protect against EAE by inhibiting pathogenic T-cells migration to the CNS. (a) Mice were induced for EAE and treated or not either starting at the time of EAE induction (treatment before onset) or starting after the appearance of the first clinical signs (treatment after onset) three times three days apart. Clinical scores were measured every day and are shown as meanSEM. (b, c) Representative hematoxylin and eosin spinal cord staining are shown (b). The quantification of the infiltrate was calculated by manual measurement of areas after clipping. Using a matrix each infiltrating cell is detectable like a one black pixel (c). One representative experiment out of two is presented (n=4 mice/group). Treatment before and after onset is a pool of three and seven experiments respectively. (d,e) The impact of CoPP treatment on T cell proliferation (day 0 and 3) was evaluated in C57/B6 CD45.1/1 mice immunized with MOG.sub.35-55 peptide that received 610.sup.6 CFSE-loaded 2D2 anti-MOG CD45.2/2 CD4.sup.+ cells. Data are presented as representative dot plots (d), and as the percentage of proliferating cells.e.m. (n=7-8 mice/group) (e). (f, g) CD4.sup.+ T-cells from CNS or DLN were harvested and stained with MOG.sub.38-49-APC tetramer. Data are presented as representative dot plots (f), and as number of cells per organs.e.m. (n=7-8 mice/group) (g).

[0136] FIG. 7A-F. Treatment with clinical hemin in non-human primates induces HO-1 in MHC-II.sup.+ cells in the draining LN and extinguish DTH reaction. (a-d) HO-1 induction with Normosang was evaluated either in vivo after intradermal immunization in Baboons. Twenty-four hours after intradermal injection of clinical grade hemin (Normosang), the proportion of HO-1.sup.+ (a) or CD14.sup.+CD11c.sup.+ (b) in MHC-II.sup.+ cells was determined by intracellular staining in the DLN and in the non-DLN (nDLN) of treated or non-treated baboons. Data show representative FACS profile (a,b) and frequencies (c) with one baboon/condition. (d) The expression of HO-1 was evaluated in MHC-II.sup.+CD11c.sup.+CD14.sup.+ or CD14.sup. cells. (e) Four baboons were immunized with BCG and challenged with tuberculin for DTH assessment before and after Normosang (hemin) treatment. (f) Erythema was measured every days following intradermal injection of tuberculin in all animals.

[0137] FIG. 8. Intravenous injections of CoPP and autoantigen do not protect against induced diabetes. The day after intravenous CoPP and autoantigen (CoPP OVA IV) or autoantigen alone (OVA IV), Rip-OVA.sup.high mice were transferred with 0.510.sup.5 autoreactive CTLs. Mice were monitored for diabetes development. Results are pooled from two independent experiments. Curves are not statistically different.

EXAMPLES

Example 1: Tolerization of Ongoing CTL Response in Type 1 Diabetes (T1D) by Monocyte-Derived Dendritic Cells Induced by Intradermal Injection of Heme-Oxygenase-1 Inducer

[0138] Material & Methods

[0139] Cells:

[0140] Autoreactive CTL generation: Autoreactive CTLs were generated as described previously (34). Briefly, CD8.sup.+ cells were isolated by magnetic selection (Miltenyi Biotech) from OVA-specific class I-restricted T cells (OT-I) mice (35) spleen and lymph node single-cell suspensions. 110.sup.6 purified OT-I CD8.sup.+ T cells were stimulated with 510.sup.6 mitomycin-treated and ovalbumin (257-264) peptide-loaded syngeneic spleen cells in 2 ml complete DMEM high glucose with stable glutamine (PAA) supplemented with 10% FCS (Eurobio) containing, 5 ng/ml IL-2 (Roche Applied Science), and 20 ng/ml IL-12 (R&D Systems). On day 3, the cultures were split into four aliquots and fed with fresh medium containing IL-2. On day 6, cells were collected and washed with culture medium at three times.

[0141] Isolation of murine APCs and co-culture with autoreactive CTLs: For MHC-II.sup.+ and F4/80.sup.+ cells isolation, skin-draining LNs were removed 24 hours after intradermal injection in the back or in the ear. Single-cell suspensions were prepared by enzymatic lymph node disaggregation with collagenase D (Sigma-Aldricht). Cells were stained with anti-MHC-II-FITC (clone AF6-120.1, BD pharmingen) or anti-F4/80-PE (clone BM8, eBioscience) monoclonal antibodies as primary labeling reagent and respectively with anti-FITC or anti-PE microbeads (Miltenyi) as secondary reagent. After magnetic separation, we checked that purity was >82% for CD11b.sup.+CD11c.sup.lowLy6C.sup.highF4/80.sup.+ cells in CoPP-OVA immunized mice. These cells were co-cultured with autoreactive CTLs (respectively 1:1 and 1:5 APC to CTL ratio) overnight for velocity measurement and transwell migration assay.

[0142] Human THP-1 monocytic cell line and co-culture with CD8.sup.+ T cells clones: THP-I cells were cultured in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (Eurobio), 1% glutamine (Gibco), 50 IU/mL penicillin 50 IU/mL streptomycin (Gibco) and 200 nM PMA (Sigma-Aldrich) under standard conditions as previously described (36).

[0143] For co-culture experiments with CD8.sup.+ T cells clones, THP-1 were treated with hemin (25 M or 50 M) or CoPP (12.5 M or 25 M) overnight, pulsed 30 min at 37 C. with 5 M of MUCI (950-958) or NYESO-1 (156-165) in culture medium, thoroughly washed and cocultured respectively with HLA-A*0201/MUC1(950-958)-specific T-cell clone (N5.14) (37) or HLA-A*0201/NYESO-1 156-165)-specific CD8.sup.+ T-cell clone (M117.167) (38) at ratio 1:1 overnight for velocity measurement.

[0144] Human and non-human primate PBMC: Human PBMCs were obtained at the Etablissement Franais du Sang in Nantes from blood of healthy donors. After Ficoll-Paque density gradient centrifugation (GE Healthcare), PBMC were cultured in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (Eurobio), 1% glutamine (Gibco), 50 IU/mL penicillin 50 IU/mL streptomycin (Gibco) in presence or not of CoPP or hemin during 4 hours.

[0145] Animals:

[0146] Non-Human primates: Baboons (Papio anubis, from the CNRS Primatology Center, Rousset, France) were negative for all quarantine tests. Animals were housed at the large animal facility of our laboratory following the recommendations of the Institutional Ethical Guidelines of the Institut National de la Sant Et de la Recherche Mdicale, France. All experiments were performed under general anaesthesia with Zoletil (Virbac, Carron, France). Three Baboons were injected intradermally in the inguinal fold with respectively 6.25 mg (500 L), 12.5 mg (500 L) or 25 mg (1 mL) of clinical hemin (Normosang). A non-treated baboon has served as control. Inguinal lymph nodes were surgically removed 24 hours after intradermal injection. Single-cell suspensions for flow cytometry analysis were prepared by enzymatic lymph node disaggregation with Collagenase D (Sigma-Aldricht).

[0147] Generation of pIi-TTA-TetO-HO-1 transgenic NOD mice: pIi-TTA mice were a kind gift from Christophe Benoist (21). For generation of the TetO-HO-1 mice, the human HO-1 cDNA, the human -globin intron located upstream of the cDNA sequence and the bovine growth hormone polyA located downstream of the cDNA was cloned at the Not-I/Xho-I sites into pBluKSM-tet-O-CMV vector containing the Tet-responsive-element (TRE) downstream the minimal CMV promoter followed by the human -globin intron and the bovine growth hormone polyA. Transgenic mice were generated by pronuclear microinjection of CBA/C57BL6 eggs with the XhoI-NotI fragment of the vector described. Seventeen different founders were carrying the transgene as tested by PCR and southern blot. Of the 17 lines, 3 founders contained high copies of the hHO-1 cDNA. One of these was further analyzed by crossing with actin-rtTA mice. hHO-1 expression was confirmed by western blotting. Finally, both strains pIi-TTA and TetO-HO-1 were backcrossed to NOD/LtJ mice (Charles River, France) for at least twelve generations. Only females where used in experiments.

[0148] Other mice: The Rip-OVAhigh transgenic mice (39) express OVA in pancreatic islets and the OT-I CD45.1.sup.+ transgenic mice express a TCR-specific for the H2Kb restricted epitope of OVA and the CD45.1 congenic marker. Rip-OVAhigh and OT-I CD45.1.sup.+ and C57/BL6 mice were obtained respectively through Jackson Laboratory, Charles River and Janvier. All animal breeding and experiments were performed under conditions in accordance with the Inserm and European Union Guidelines.

[0149] Doxycycline treatment: Doxycycline hyclate powder (Sigma-Aldricht) was diluted in drinking water at different concentration (200 g/mL up to 800 g/mL) and protected from light. HO-1 transgenic mice have been treated during 3 days for HO-1 expression analysis and up to 200 days for diabetes incidence experiments.

[0150] Intradermal immunization of mice and diabetes induction: Eight to ten week-old Rip-OVAhigh mice received two intradermal injections in the back with 70 g of CoPP (Livchem) prepared as described and 20 g of endofree ovalbumin (Hyglos) in 10 l. One hundred and forty micrograms of MnPP (Livchem) prepared as described (18) was added to the preparation for HO-1-inhibition experiments. Forty micrograms of Alexa Fluor 488 ovalbumin (Molecular Probes) has been used for phagocytosis assay. For diabetes induction, mice were injected i.v. the following day with 0.510.sup.5 autoreactive cytotoxic OT-I CD8.sup.+ T cells (purity >95%) previously co-cultured or not with MHC-II.sup.+ or F4/80.sup.+ cells during 4 hours.

[0151] Diabetes follow-up: Diabetes monitoring has been done by urine glycosuria analysis and confirmed in positive mice (5.5 mmol/L) by blood glycemia measurement. Mice were considered diabetic when blood glycemia was superior to 180 mg/dL during two consecutive weeks for HO-1 transgenic NOD models or two consecutive days for Rip-OVAhigh model.

[0152] Flow Cytometry: Single-cells suspensions of spleen and lymph nodes were stained with anti-CD8 (clone 53-6.7), anti-CD45.1 (clone A20), anti-CD49b/2 (clone DX5), anti-CD49d/4 (clone RI-2), anti-ITGAE/E (clone M290), anti-CD29/1-integrin (clone Ha2/5), anti-CD18/2-integrin (clone C71/16), anti-CD61/3-integrin (clone 2C9.G2), anti-CD62E (clone 10E9.6), anti-CD62L (clone Mel14), anti-PSGL1 (clone 2PH1), anti-ICAM-1 (clone 3E2), anti-VCAM-1 (clone 429), anti-CD183/CXCR3 (clone CXCR3-173), anti-CD195/CCR5 (clone C34-3448), anti-GITR (clone DTA-1), anti-PD-1 (clone J43), anti-CD28 (clone 37.51), anti-CD38 (clone 90), anti-CD69 (clone H1.2F3), anti-CD44 (clone IM7), anti-CD107b (clone ABL-93) antibodies. All antibodies are from BD Biosciences. Intracellular staining of HO-1 using BD cytofix/cytoperm kit following manufacturers' recommendations were performed with anti-HO-1 antibody (clone HO-1-2, abcam) as primary labeling reagent and with anti-anti-IgG1 (clone MOPC-21) monoclonal antibody conjugated to a fluorochrome as secondary labeling reagent.

[0153] Isotype antibodies (Immunotech) were used as a negative control. Staining was assessed using a FACScanto flow cytometer and Diva 6.1 software (Becton Dickinson).

[0154] In vivo cytotoxicity assay: Spleen cells from C57/B16 mice were pulsed or not with 5 M of H2-Kb Ovalbumin (257-264) peptide for 30 min at 37 C. and incubated 5 min at RT in PBS containing 5 and 0.5 M CFSE respectively. For in vivo cytotoxic test, 310.sup.6 of OVA (257-264) peptide and unpulsed splenocytes were co-injected i.v. into recipient mice. Mice were killed 12 h later, and spleen cells were analyzed by flow cytometry. Cytolytic activity was determined by calculating the percentage of specific lysis using the following formula: 100-([(% Ovalbumin (257-264)-pulsed splenocytes/% unpulsed splenocytes) with autoreactive CTLs/(% Ovalbumin (257-264)-pulsed splenocytes/% unpulsed splenocytes) without autoreactive CTLs]100).

[0155] Histological analysis: For evaluation of insulitis, pancreata were snap-frozen and cryosections (8 m thick) were acetone-fixed. Sections were stained with H&E (Thermo Electron Corp.), and the degree of insulitis was evaluated microscopically.

[0156] Light-sheet-based fluorescent microscopy analysis: Animals were perfused with PFA 4% and washed with PBS. Excised pancreases were clarified using the 3Disco method (40, 41). Briefly, they were dehydrated at room temperature in successive bathes from 50% TetraHydroFuran, anhydrous (THF, Sigma, Saint Quentin Falavier, France) (vol/vol) overnight, 80% THF (vol/vol) for 2 hours to 100% THF two times 1 hour. Then the organs are incubated in a solution of 100% Di-ChloroMethan (DCM, Sigma, Saint Quentin Falavier, France) for 45 minutes and transferred overnight into in the clearing medium 100% DiBenzylEther 98% (DBE, Sigma, Saint Quentin Falavier, France). To image whole adult mice pancreas, we use a home-made light-sheet ultramicroscope (40). The specimen was placed into a cubic cuvette filed with DBE placed on the Z-stage of the bench. It was illuminated with planar sheets of light, formed by cylinder lenses. The light coming from a multi-wavelength (405, 488, 561, 635 nm) laser bench (LBX-4C, Oxxius, Lannion, France) was coupled via two single mode optical fibers into the setup, allowing illumination from one or two sides. Illumination intensity using the 488 nm laser excitation was 10 to 40 mW per light sheet. We used two-sided illumination. The specimen was imaged from above with a MVX10 macroscope, through a PlanApo 1X/0.25 NA or 2X/0.5 NA objective (Olympus, Rungis, France), which was oriented perpendicular to the 488nm light sheet. For GFP fluorescence imaging, we used a 525/50 band pass filter on the turret. Images were captured using a CCD Camera (ORCA AG, Hamamatsu, France) synchronized with the z-stage moving the sample through the light sheet. The home-made ultramicroscope is managed by Micro-manager software and z-stacks of images were taken every 20, 10 or 5 microns depending on the sample and magnification. The images stacks are analyzed using ImageJ software (Rasband, W. S., ImageJ, U. S. National Institutes of Health, Bethesda, Md., USA, http://imagej.nih.gov/ij/, 1997-2012).

[0157] Velocity assay: Autoreactive CTLs were cocultured overnight with MHC-II.sup.+ cells isolated from DLNs of OVA or CoPP-OVA immunized mice. After magnetic depletion of MHC-II.sup.+cells, re-isolated CTL were settled on a confluent SVEC cells monolayer seeded on ibidi slide. Live cell imaging was performed at 37 C. using DMI 6000 B microscope (Leica Microsystems, Nanterre, France) under 20 magnification. Images were acquired every 15 s for 30 min with a Cool Snap HQ2 camera (Roper, Tucson, Ariz.) and analyzed with Metafluor 7.1 imaging software (Universal Imaging, Downington, Pa.). The images were imported into the ImageJ software program, and manual cell tracking was performed using the MTrackJ plug-in (developed by Erik Meijering at the University Medical Center Rotterdam, Rotterdam, The Netherlands;http://www.imagescience.org/meijering/software/mtrackj/). The average velocity was calculated for each cell by dividing the total distance traveled by the cell during the last ten minutes.

[0158] Transwell migration assay: Autoreactive CTLs were cocultured with MHC-II.sup.+ cells isolated from DLNs of OVA or CoPP-OVA immunized mice. After magnetic depletion of MHC-II+ cells, re-isolated CTL were deposited on the upper chamber containing a polycarbonate transwell membrane filter (5-m pore size; Corning). The lower chamber contained 100 ng/ml CXCL12, 100 ng/ml CCL19, or 80 ng/ml CCL17 in DMEM complete medium. The recovered cells after 2 hours at 37 C. were analyzed by flow cytometry.

[0159] Cytokine measurement: MHC-II.sup.+ mice cells or PMA-activated THP-1 cells were treated with CoPP or hemin as described for mouse and cultured for 24 hours with LPS at 1 g/mL. Supernatants were serially diluted, and cytokine concentration assessed in duplicate by enzyme-linked immunosorbent assay (ELISA). Mouse ELISA kits for IL-12 and IL-10, as well as human IL-1 ELISA kits (BD) were used.

[0160] Statistics: For diabetes incidence, significance was calculated using the log-rank test. For all other parameters, significance was calculated by t-paired test, Mann-Whitney nonparametric t-test or one-way anova using Prism software: *p<0.05, **p<0.01, and ***p<0.001.

[0161] Results

[0162] Genetic and pharmacological manipulation of HO-1 expression prevents T1D: Since DCs play a critical role in T1D onset (19), and because HO-1 exerts its immunosuppressive effect mainly via APCs (17, 18), here we investigated whether HO-1 expression was impaired in DCs from NOD mice. Indeed, we found that compared to their non-diabetic counterparts (NOR mice) (20), female NOD mice exhibited a lower percentage of HO-1-expressing cells among CD11c.sup.+ cells in the spleen.

[0163] To further determine whether HO-1 expression in APCs could impact diabetes, we used pIi-TTA mice, a strain in which the Tet ON system is under the control of the MHC-II invariant chain (E-Ii) promoter (21). pIi-TTA mice were crossed with TetO-HO-1 transgenic NOD mice, in which the HO gene is under the control of the hybrid CMV-Tet operator. In the resultant pIi-TTA/TetO-HO-1 double-transgenic mice, doxycycline administration induced dose-dependent increases in HO-1 expression in both bone-marrow derived dendritic cells (BMDCs) and splenic DCs. In female NOD mice, doxycycline-induced HO-1 expression was mainly observed in DCs, and restored HO-1 levels to those of NOR mice.

[0164] When doxycycline was provided in drinking water from four weeks of age, the doxycycline-treated pIi-TTA/Tet-O-HO-1 female mice exhibited lower T1D incidence compared to treated single transgenic or untreated littermates. Among doxycycline-treated animals, pIi-TTA/Tet-O-HO-1 mice exhibited reduced leukocyte infiltration compared to Tet-O-HO-1 mice. This phenomenon was dose-dependent. Altogether, our data demonstrated that HO-1 overexpression in MHC-II.sup.+ APCs reduced T1D incidence in female NOD mice.

[0165] HO-1 inducers inhibit autoreactive CTL-mediated damage: We next investigated whether T1D could be prevented by HO-1 inducers, such as cobalt protoporphyrin (CoPP). For these experiments, we used RIP-OVA.sup.high transgenic mice in which OVA is selectively expressed in pancreatic B cells (22). Intradermal injection of CoPP increased HO-1 expression (5.8 fold1.8) in MHC-II.sup.+cells in draining lymph nodes (LNs) (FIG. 1A and B). This induction was restricted to the draining LNs, was observed as early as 24 hours after CoPP injection, and lasted for at least 72 hours.

[0166] When adoptively transferred with activated OVA-specific CTLs, RIP-OVA.sup.high transgenic mice rapidly developed T1D, as previously reported (22). However, co-injecting RIP-OVA.sup.high transgenic mice with OVA and CoPP one day before CTL transfer reduced both diabetes (FIG. 1C) and insulitis as analyzed by immunohistology at day 6-8 (FIG. 1D) and by light-sheet-based fluorescent microscopy at 18 h. Such protection was not observed in mice injected with either OVA or CoPP alone or with CoPP and human albumin (FIG. 1C), suggesting that CoPP mediated this protection by interfering with antigen-specific immune responses. Administration of the HO-1 enzymatic inhibitor MnPP also abolished protection, further implying that this protection relied on increased HO-1 enzymatic activity (FIG. 1C). Interestingly, protection was not achieved when CoPP and OVA were injected i.v. (FIG. 8). Altogether, these results demonstrated that activated antigen-specific CTLs could be tolerized in vivo upon intradermal injection of their cognate antigen together with CoPP.

[0167] MoDCs tolerize CTL in mice treated with HO-1 inducers: Since co-injection of CoPP and OVA dramatically increased the number of HO-1.sup.+ MHC-II.sup.+ cells in draining LNs (FIG. 1A and B), we further characterized these cells for surface molecule expression and for their ability to tolerize preactivated CTLs. Through this, we aimed to elucidate the mechanisms by which CTLs were tolerized in CoPP/OVA-treated mice. Twenty-four hours after intradermal injection of CoPP and OVA, the vast majority of MHC-II.sup.+HO-1.sup.+ cells recruited in the draining LNs exhibited a CD11b.sup.+CD11c.sup.lowF4/80.sup.+CD64.sup.+Ly6C.sup.+FcRI.sup.+ surface phenotype, which is a distinctive feature of Monocyte-derived DCs (MoDCs) (23). Following CoPP/OVA immunization, these cells were massively recruited in the draining LNs in contrast to OVA immunization (FIG. 2). Previous studies have demonstrated that MoDC cannot be recruited in CCR2 deficient mice and, accordingly, the number of HO-1.sup.+ MoDCs in the LNs of CoPP/OVA immunized mice was dramatically reduced in CCR2-deficient mice (1.510.sup.3 cells/LN, n=3) compared to in WT animals (2.1510.sup.5 cells/LN, n=3), confirming the monocytic origin of the HO-1.sup.+ APCs in CoPP/OVA immunized mice.

[0168] MoDC can be recruited in the draining LN from circulating monocytes present either in the blood or in the dermis (24). Administration of the D.sub.2 prostaglandin receptor agonist BW245c as a dermal migration inhibitor did not affect MoDC recruitment, suggesting that

[0169] MoDCs were recruited directly from the blood. This result was confirmed by the extent of MoDC recruitment after excision of the CoPP-injected site one hour after CoPP injection, which was incompatible with a skin origin of the MoDCs.

[0170] To further investigate whether HO-1.sup.+ MoDCS were responsible for T-cell tolerization in CoPP-injected mice, MHC-II.sup.+ cells were purified from the draining LN of mice immunized with CoPP and OVA, and incubated for four hours with pre-activated OVA-specific CTLs. Next, these cells were adoptively transferred into RIP-OVA.sup.high mice, which were then monitored for T1D. CTLs that were incubated with MHC-II.sup.+ cells from CoPP/OVA-immunized animals exhibited a decreased ability to induce T1D (FIG. 3A).

[0171] Similar results were obtained with CTLs incubated with CD11b.sup.+CD11c.sup.lowLy6C.sup.highF4/80.sup.+ cells isolated from the draining LN of CoPP/OVA treated mice, further suggesting that HO-1.sup.+ MoDCs were responsible for the tolerization of OVA-specific CTLs observed in mice co-injected with CoPP and OVA. Supporting this hypothesis, we also found that F4/80.sup.+ cells from the draining LN of mice co-injected with CoPP and OVA secreted lower IL-12 levels and higher IL-10 levels in response to LPS (FIG. 3B), and expressed lower surface levels of co-stimulatory molecules compared to cells purified from mice injected with OVA alone. Moreover, HO-1.sup.+ MoDCs efficiently endocytosed OVA in accordance with the antigen-specific tolerization properties assigned to these cells.

[0172] HO-1.sup.+ MoDCs impair CTL velocity and response to chemokines: We next investigated the mechanism of tolerance of CTL by MoD, to this aim we compared the cytotoxic activities and distributions of OVA-specific CTLs in mice immunized with CoPP/OVA or OVA alone. CTL activities were measured using an in vivo cytolytic assay, and were similar in both groups (FIG. 4A). Furthermore, both groups exhibited similar numbers of OVA-specific CTLs in the spleen, and in draining and pancreatic LNs at two days (FIG. 4B) and six days after injection. In striking contrast, imaging experiments performed 18 hours after T-cell injection showed that OVA-specific CTLs in pancreatic islets were less abundant in CoPP/OVA-immunized mice compared to in OVA-immunized control animals thus suggesting that HO-1.sup.+ MoDCs altered the ability of CTL to migrate to non-lymphoid tissues.

[0173] To further investigate this issue, we performed in vitro experiments measuring these cells' velocity and ability to respond to a chemokine gradient in a transwell migration assay. Compared to non-tolerized control cells, tolerized CTLs exhibited a reduced cell velocity (FIG. 4C) and a decreased ability to migrate along a chemokine gradient (FIG. 4D).

[0174] Monitoring the expressions of 20 surface markers involved in CD8.sup.+ T-cell migration to inflamed tissues, including chemokine receptors and adhesion molecules, revealed that none was differentially expressed in CoPP/OVA- and OVA-immunized mice.

[0175] HO-1 inducers selectively recruit MoDCs across species: We next investigated whether a similar phenomenon occurred in humans. With this aim, we incubated PBMCs from healthy human volunteers with hemin. Four hours later, these cells were analyzed for HO-1 expression. As observed in baboons, hemin induced highest HO-1 expression in human MHC-II.sup.+CD11c.sup.lowCD14.sup.+ cells (FIG. 5A). Accordingly, cells from the human monocytic cell line THP-1 dose-dependently expressed HO-1 upon incubation with CoPP (FIG. 5B) or hemin (FIG. 5C). Since THP-1 cells do not express IL-10 and IL-12 upon stimulation we could not compare their expression. However compared to untreated THP-1 cells, HO-1.sup.+ THP-1 cells secreted reduced levels of the inflammatory cytokine IL-1 and expressed lower surface levels of the co-stimulation markers CD40 and CD86. Furthermore, HO-1.sup.+ THP-1 cells reduced human CTL velocity. Altogether, these results showed that clinically approved HO-1 inducers selectively target MoDCs across species, and suggested that these cells could tolerize CTLs in both mice and humans.

[0176] Discussion

[0177] HO-1 induced by chemicals (CoPP) and heme degradation products (e.g., CO) have been shown to inhibit T1D in NOD mice when administered systemically before disease onset (14, 16). However, the mechanisms of action for these treatments have not yet been completely elucidated. Furthermore, these systemic treatments can have secondary effects. In the present paper, we have shown that selective induction of HO-1 in MHC class II-positive cells in 4-week-old mice was sufficient to inhibit T1D in NOD mice. This finding provides a mechanistic explanation for the efficacy of HO-1-based treatment. It is also noteworthy that spleen CD11c.sup.+ cells from NOD mice expressed lower basal levels of HO-1 than those from their non-diabetic counterpart, NOR mice. This suggests that HO-1, or more likely one of its upstream regulators, could be one of the many genes involved in T1D development in NOD mice. It is possible that inhibition of IL-12 cytokine secretion and increased or maintained IL-10 secretion by HO-1-overexpressing dendritic cells (17, 18) or macrophages (25) may account for these results (26, 27). The explanation may also involve the ability of dendritic cells to tolerize naive anti- islet CD8.sup.+ T cells through decreased integrin expression upon exposure to carbon monoxide produced by HO-1 (28).

[0178] We reasoned that localized and not systemic HO-1 induction could be used as a tolerogenic strategy for T1D treatment. Parenteral or intranasal vaccination, and oral route administration of self-antigens have been used to induce specific tolerance to -cell antigens (29). These treatments have occasionally been reported to induce a delay in disease onset (30) or in the decline of C-peptide levels (31), but overall the results have been disappointing.

[0179] Notably, the results of such studies have suggested that the antigen and adjuvant selection may be paramount for ensuring the success of this strategy. HO-1 induction confers tolerogenic properties to classical DCs, which inhibit the priming of pathogenic T-cells, as previously demonstrated by our group (18) and others (14).

[0180] One of the most striking results in the present study was that MoDCs induced to express HO-1 could inhibit pre-activated CTLs in vitro and in vivo. MoDCs are also called inflammatory DCs because they are recruited in parallel to inflammatory macrophages (23) but contrary to macrophages MoDCs have been shown to cross present efficiently antigens to CD8.sup.+ cells both in mice and humans. This report provides the first evidence that they can present tolerogenic properties towards activated CD8.sup.+ T cells. The molecular mechanisms responsible for this phenomenon remain unknown, but several clues are provided in the present results. We found that HO-1.sup.+ MoDCs secreted high IL-10 levels, and expressed lower levels of costimulatory molecules. This phenotype of HO-1.sup.+ MoDCs is compatible with a tolerogenic function. Indeed, in the natural process of apoptotic erythroid cells engulfment (a process called hemophagocytic) by MoDC lead to both increase HO-1 expression (32) and IL-10 secretion that moderate anti-viral CTL activity (33). Regarding the mechanism of CTLs tolerisation, we found that their proliferation or lytic activity was not impaired following exposure to HO-1.sup.+ MoDCs in vivo. In striking contrast, tolerized CTLs were impaired in their ability to migrate to non-lymphoid tissues, as demonstrated by their absence in the pancreatic islets of RIP-OVA.sup.high mice. This defect was also associated with both a decreased velocity and lower ability to respond to a chemokine gradient in vitro. This finding provides a probable mechanistic explanation for the phenotype of tolerized CTLs.

[0181] Representing an improvement over the results of previous strategies using HO-1 or its derivatives as tolerogenic agents to cure autoimmune diseases (14, 16), the tolerance observed following intradermal injection of HO-1 inducer was clinically relevant and antigen-specific. As observed in mice, we found that the intradermal injection of HO-1 inducers in non-human primates resulted in the appearance of HO-1.sup.+ MoDCs in draining LNs. Most importantly, cells from a human monocyte cell line that were induced to express HO-1 upon incubation with HO-1 inducers show reduced velocity of two different human CTL clones, further suggesting that the same tolerizing mechanisms occur across species. We believe that this is an important finding because HO-1 inducers such as Normosang and Panhematin have been already approved for the treatment of acute porphyria in humans (12), therefore paving the way for the use of this molecule to prevent the development of T1D in humans.

Example 2: Tolerization of Ongoing CTL Response in Experimental Autoimmune Encephalomyetis (EAE)

[0182] Material & Methods

[0183] Animals: C57BL/6 mice were maintained under safety condition approved by the Inserm and European Union Guidelines. Mice were used between 6 and 10 weeks of age.

[0184] Experimental Autoimmune Encephalomyelitis (EAE) induction: Briefly, C57BL/6 were immunized by a subcutaneous injection of emulsified complete freund adjuvant (CFA, sigma aldrich) complemented with mycobacterium tuberculosis (400 g, BD) and MOG35-55 peptide (200 g, genecust). Pertussis toxin is injected (200 ng i.v, VWR) at the time of immunization and two days later. Clinical signs of EAE were evaluated daily and scored as follows: 0, normal; 1, limp tail; 2, partial paralysis of the hind limbs; 3, complete paralysis of the hind limbs; 4, hind-limb paralysis and forelimb weakness; 5, moribund or deceased

[0185] Treatments: For prophylactic treatment, mice were treated at the time of immunization with one injection in each ear of: CoPP alone (70 g) or CoPP (70 g) and MOG peptide (20 g) or MOG peptide alone (20 g), or CoPP (70 g) with irrelevant class II peptide OVA (20 g). Same injections were repeated twice at three days interval. For curative treatment, mice were treated with one injection in each ear at the EAE onset (i.e., mean clinical score, 0.720.1) with the same quantity than in prophylactic treatment. Same injections were repeated twice at three days interval after EAE onset.

[0186] Results

[0187] Given that HO-1 induction have shown some anti-inflammatory properties for CD4+ T-cells in the EAE model (14), we hypothesized that HO-1-mediated protection may exert an antigen-specific action if both HO-1 expression and antigens presentation occurs at the same time in the same APC. When using a low dose of CoPP (0.2 mg/mouse), we found that i.d. injection of CoPP alone is not sufficient to tolerize against EAE in preventive (administration CoPP treatment on day 0, 3 and 6 after MOG immunization) or curative (CoPP administration 3 times, 3 days apart, after the first clinical signs of EAE appeared) settings (FIG. 6A). However when a low dose of CoPP is co-administered intradermally with cognate antigen (MOG.sub.35-55 class II peptide) tolerization of myelin-specific CD4.sup.+ T-cells occurs preventively or curatively (FIG. 6A). Such protection was not observed in mice injected with MOG.sub.35-55 alone or with CoPP and irrelevant peptide, demonstrating that CoPP-induced protection is antigen-specific (FIG. 6A). Likewise CoPP-induced CTLs tolerisation, MOG-specific CD4.sup.+ T-cell have the same proliferation capacity (FIG. 6B,C) in CoPP injected mice and untreated mice. This protection was associated with an inhibition of infiltration in the CNS of CoPP MOG treated mice (FIG. 6D,E). We observed a significant decrease of the absolute number of MOG-specific CD4.sup.+ T-cells in the CNS in CoPP MOG-treated mice compared to control (FIG. 6F,G) for preventive settings, and for curative settings. Interestingly, in peripheral compartment (spleen and lymph node) the number of MOG specific T cell is unchanged suggesting an impairment of MOG-specific CD4.sup.+ T-cells migration in the CNS.

[0188] Altogether, these results demonstrate that ongoing autoreactive CD4.sup.+ T cells response is tolerized in vivo upon i.d. injection of their cognate autoantigen and CoPP.

Example 3: Tolerization of Ongoing Th1 Response in Delayed-Type Hypersensitivity (DHG) in Non-Human Primates

[0189] Material & Methods

[0190] Animals: Non-Human primates: Baboons (Papio anubis, from the CNRS Primatology Center, Rousset, France) were negative for all quarantine tests. Animals were housed at the large animal facility of our laboratory following the recommendations of the Institutional Ethical Guidelines of the Institut National de la Sant Et de la Recherche Mdicale, France.

[0191] Intradermal immunization with Normosang: Three Baboons were injected intradermally in the inguinal fold with respectively 6.25 mg (500 L), 12.5 mg (500 L) or 25 mg (1 mL) of clinical hemin (Normosang). A non-treated baboon has served as control.

[0192] Inguinal lymph nodes were surgically removed 24 hours after intradermal injection. Single-cell suspensions for flow cytometry analysis were prepared by enzymatic lymph node disaggregation with Collagenase D (Sigma-Aldricht). All experiments were performed under general anaesthesia with Zoletil (Virbac, Carron, France).

[0193] For DTH assays, four baboons were injected intradermally in the two inguinal folds with 25 mg plus 2000 UI of tuberculin-purified protein derivative (PPD; Symbiotics Corporation, San Diego, Calif., USA) in 1.2 mL

[0194] BCG vaccination and DTH assay: Baboons were immunized intradermally (i.d.) twice with a bacillus Calmette-Gurin (BCG) vaccine (0.1 nil; 2-810 5 UFS; Sanofi Pasteur MSD, Lyon, France) in the upper region of the leg, 4 and 2 weeks before the DTH skin test. Intradermal reactions (IDR) were performed in the back with duplicate intradermal injections of two doses (2000 UI or 40 UI) of tuberculin-purified protein derivative (PPD; Symbiotics Corporation, San Diego, Calif., USA) in 0.1 ml in the skin on the right back of the animals. Saline (0.1 ml) was used as a negative control. Dermal responses at the injection sites were measured using a caliper square. The diameter of each indurated erythema was measured by two observers from days 3-8, and were considered positive when >4 mm in diameter. The mean of the reading was recorded. Other IDRs were performed 4 days, one, two and three month after animals received one i.d. injection of hemin (Normosang) and 2000 UI of tuberculin-purified protein derivative.

[0195] Results

[0196] To investigate whether HO-1 inducers could induce HO-1.sup.+ MoDCs in primates, we injected baboons intradermally with clinical-grade hemin (Normosang), an HO-1-inducer that has been approved for the treatment of acute porphyrias in humans.sup.10. Hemin injection dose-dependently increased the frequency of HO-1.sup.+ cells in draining LN but not in the contralateral LNs (FIG. 7A,C). These HO-1.sup.+ cells expressed MHC-II, CD11c, and CD14 (FIG. 7B,C) by contrast to MHC-II.sup.+, CD11c.sup.+, and CD14 negative cells (FIG. 7D), further suggesting that they were MoDCs.

[0197] As a first step to investigate whether HO-1 inducers could be used to induce antigen-specific tolerance in primates, we used a delayed type hypersensitivity (DTH) model in baboons. Animals were immunized with BCG vaccine and challenged three consecutive times over five months period with tuberculin intradermal reaction (IDR) (FIG. 7E). These animals showed a measurable and reproducible erythema (minimal size of 4 mm) lasting from 3 to 7 days after injection of tuberculin purified protein derivative (PPD) (FIG. 7F). Six month after the last IDR, animals were tolerized by i.d. injection of Normosang and tuberculin. Four days, one, two and three month after tolerization, IDR was assessed. A strong reduction of IDR was observed following tolerization protocol with a progressive reappearance at three month (FIG. 7F).

[0198] As conclusion, i.d. administration of clinical hemin and tuberculin in BCG vaccinated baboons resulted in suppression of T cell memory response against tuberculin for at least two months.

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