Ethics approval

All animal experiments were performed in accordance with relevant guidelines and regulations, either in an approved animal facility at Sangamo Therapeutics France (accredited by the French Ministry of Research, Arrêté n° 4566) according to the APAFIS (“Autorisation de Projet utilisant des Animaux à des Fins Scientifiques”)approved protocol APAFIS#23467-2019101811351116v8 and APAFIS#13779-201802156437559v3 and in compliance with the Guide for the Care and Use of Laboratory Animals.

Human cell preparation

The blood of healthy donors was collected by the Etablissement Français du Sang (EFS). Peripheral blood mononuclear cells (PBMC) were isolated from buffy coat by density centrifugation using Ficoll-Paque (GE Healthcare).

Human Treg isolation, transduction, and expansion

Human Tregs and CD4+CD25− responder T cells (Tconv) were isolated from PBMC using EasySep Human CD4+CD127loCD25+ Regulatory T Cell Isolation Kit (STEMCELL Technologies). Naïve CD4+CD127loCD25+CD45RA+ Tregs were further purified by cell sorting and cultured in X-VIVO 15 (Lonza) or OpTmizer media (ThermoFisher Scientific) supplemented with 1000 U/mL recombinant human IL-2 (Proleukin) and anti-CD3/CD28 Dynabeads (ThermoFisher Scientific). Media containing IL-2 were replenished every 2–3 days. Tregs were transduced with lentiviral vectors at day 2 and re-stimulated with anti-CD3/CD28 beads at day 7 and harvested at day 11–13. Briefly, transduction was carried out by loading between 2 and 5 × 106 Transducing Unit (TU) per ml to each well. After 6 hours at 37 °C, viral particles were removed by washout. The plates were then incubated at 37 °C with 5% CO2. The transduction efficiency was measured by flow cytometric analysis of the percentage of GFP positive cells.

Mouse Treg isolation, transduction, and expansion

Mouse Tregs from splenocytes of C57Bl/6 or SJL mice were enriched using EasySep mouse CD4+CD25+ Regulatory T Cell Isolation Kit II (STEMCELL Technologies). In some experiment, naïve Tregs (CD44loCD62hi) were further sorted using a SH800 cell sorter (Sony) and cultured in RPMI 1640, GlutaMAX, HEPES (ThermoFisher Scientific) supplemented with 10% FBS (Sigma-Aldrich), 1 mM Sodium Pyruvate (ThermoFisher Scientific), 0.1 mM non-essential amino acids (ThermoFisher Scientific), 1% Penicillin-streptomycin (ThermoFisher Scientific), 5 µM 2-Mercaptoethanol (ThermoFisher Scientific), 1000 U/mL recombinant IL-2 (Proleukin), 50 nM Rapamycin (Sigma-Aldrich), and anti-CD3/CD28 Dynabeads (ThermoFisher Scientific). Media containing IL-2 was replenished every 2–3 days. Tregs were transduced with lentiviral vectors containing or not luciferase at day 2 and expanded for 5 to 7 days. For FACS sorted Tregs, cells were re-stimulated with anti-CD3/CD28 beads at day 7 until day 11 to 13. Briefly transduction was carried out by loading 2 × 107 Transducing Unit (TU) per ml of CAR vectors to each well plus 10 µg/ml vectofusin-1. Vectofusin-1 (Miltenyi, France) and vectors are mixed for 5 min at 37 °C before being added to the Tregs. A spinoculation was performed at 32 °C, 1000 g for 90 min. After 4 h at 37 °C, viral particles and vectofusin-1 were removed by washout and fresh media containing IL-2 (1000 U/ml) was added. The plates were then incubated at 37 °C with 5% CO2. 4 to 5 days after transduction, the transduction efficiency was measured by flow cytometric analysis of the percentage of NGFR positive cells.

Lentiviral vector production and titration

CAR-expressing lentiviral vectors (LVs) were produced using the classical 4-plasmid lentiviral system. Briefly, HEK293T cells (Lenti-X, Ozyme) were transfected with the CAR-expressing transfer vector, the plasmid expressing HIV-1 Gag/pol (pMDLg/pRRE), HIV-1 Rev (pRSV.Rev) and the VSV-G glycoprotein (pMD2.G) (Didier Trono, EPFL, Switzerland). 24-hours post-transfection, viral supernatants were harvested, concentrated by centrifugation, aliquoted and frozen at -80 °C for long term storage. The infectious titers expressed in transducing units per milliliter (TU/ml) were obtained after transduction of the Jurkat T cell line with a serial dilution of viral supernatants and transduction efficiency evaluated after 4 days by monitoring GFP expression.

Flow cytometry

Cells from in vitro experiments were washed with phosphatebuffered saline (PBS)/4% bovine serum albumin and stained for cell surface markers. All antibodies were purchased from Miltenyi Biotec unless otherwise stated. The anti-human antibody clones used included: CD4-VioBlue (VIT4), CD45RA-FITC (REA1047), CD127-APC (MB15-18C9), CD25-PE (STEMCELL Technologies, 2A3), CD4-VioGreen (REA623), CD69-APC (REA824), GARP-PE (REA166), HELIOS-eF450 (eBioscience, 22F6), CD25-PE (REA570), CTLA-4-PE-Cy7 (BioLegend, BNI3), FOXP3-AlexaFluor647 (BD Biosciences, 259D/C7), CD127-APC-Vio770 (REA614), CD45-FITC (REA747), CD3-PerCP-Vio700 (REA613), CD127-AlexaFluor700 (BioLegend, A019D5), CD86-APC (REA968), HLA-DR-VioBlue (REA805), CD80-PE (REA661), CD40-APC-Vio770 (REA733), CD209-PE-Vio770 (REA617).

The anti-mouse antibodies were purchased from BD Biosciences otherwise stated. Antibody clones used included: CD45.1-BV721 (NDS58), CD45.2-APC-cy7 or BV510 (104), CD4 -BV510, V450 or APC-cy7 (RM4-5), NGFR-BV515 (REA844, Miltenyi), FOXP3-APC (REA788, Miltenyi), CD69-PE (H12F3), CTLA-4-PE-CF594 (UC10-4F10), IFNγ-FITC (XMG1.2), IL-17 A-APC (TC-11-18H10.1). FcR blocking reagent was used prior to surface marker staining. Dead cells were excluded using DAPI, PI, 7-AAD, or fixable viability dye.

Following in vivo experiments, spleen and brain samples were passed through a 70 μm cell strainer to obtain a single cell suspension. Brain cells were then incubated in RPMI/5% fetal calf serum (FCS) with 2.5 mg/ml collagenase D (#11088858001, SigmaAldrich, France) at 37 °C under agitation, washed in RPMI/5% FCS, and centrifuged. The cell pellet was resuspended in 70% (v/v) Percoll solution (#GE17-0891-01, SigmaAldrich, France), centrifuged at 500 g over 37% (v/v) Percoll, and cells at the interface of the Percoll 70/37 gradient were recovered. Red blood cells were lysed with Red Blood Cell Lysing Buffer (#R7757, SigmaAldrich, France) or ammonium chloride. Cells were washed with PBS/2% FCS and incubated with mouse Fc block (#553142, BD Biosciences, France), then washed with PBS/4% bovine serum albumin and stained. Fixable viability dyes eFluor780 or eFluor506 (#65086514 and #65086614, ThermoFisher, France) were used. Cells were stained with cell surface markers including anti mouse CD45.2, CD45.1, CD4, NGFR, and then fixed and permeabilized with the forkhead box P3 (FOXP3) staining buffer set (#00-5523-00, ThermoFisher, France), and stained with anti-mouse FOXP3 (PE or Alexa Fluor 647, BD Biosciences, France).

For IFN-γ and IL-17A intracellular cytokine staining of pathogenic cells recovered from the CNS, cells were incubated with or without 10 µg/ml MOG 35-55 peptide in RPMI-10%FCS for 2 h prior addition of brefeldin A 0.5 mg/ml. Cells were incubated at 37 °C 5% CO2 for further 16 h prior intracellular cytokine staining using standard protocol.

Cells were analysed on an Attune™ NxT flow cytometer and Attune™ NxT software (ThermoFisher, France) and data analysis were performed with FlowJo V10.

Human and mouse Treg activation assay

For human Tregs, activation assay was performed at day 9 of the culture. Briefly, 0.05 × 106 Treg were seeded in 96 U-bottom plate alone or in presence of anti-CD3/CD28 beads (in a 1 to 1 Treg to beads ratio, ThermoFisher), or with MOG coated beads (in a 1 to 1 Treg to beads ratio) in a 200 µl final volume.

For mouse Tregs, activation assay was performed at day 7 of the culture. Briefly, 0.05 × 106 Treg were seeded in 96 U-bottom plate alone or in presence of anti-CD3/CD28 beads (in a 1 to 1 Treg to beads ratio; ThermoFisher Scientific, France), or with MOG coated beads (in a 1 to 1 Treg to beads ratio) in a 200 µl final volume.

For activation assay using mouse spinal cord, the spinal cord of C57BL/6 mice was harvested by flushing and co-cultured with human or mouse Tregs in RPMI-10%FCS for 24 h.

After 24 h at 37 °C, 5% CO, cells were stained for CD4 and CD69 and then analyzed by flow cytometry. The monitoring of the CD69 spontaneous expression in CAR Treg cells, compared to control Treg cells, allowed to determine the tonic signaling intensity.

Suppression assay of human Tconv proliferation

The suppressive assays were performed at day 9 of the culture. Briefly, Treg were harvested, counted and activated either through the TCR using anti-CD3/CD28 beads (in a 1 to 1 Treg to beads ratio, ThermoFisher), or through the CAR using MOG coated beads (in a 1 to 1 Treg to beads ratio) or kept unstimulated to evaluate their spontaneous suppressive activity. In parallel, allogeneic Tconv were thawed, stained with Cell Trace Violet (CTV, ThermoFisher) and activated with anti-CD3/CD28 beads (in a 3 to 1 Tconv to beads ratio). The day after, beads were removed from Tconv before their coculture with un-activated or activated Treg (untransduced or transduced). At day 3, cells were harvested, and proliferation of Tconv was assessed by flow cytometry through the determination of CTV dilution. The percentage of inhibition of Tconv proliferation was calculated as follows:

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Human monocyte-derived DC generation and DC suppression assay

Monocytes were isolated from PBMCs using Classical Monocyte Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Monocytes were differentiated for 6 days in X-VIVO 15 media (Lonza) supplemented with 100 ng/mL GM-CSF and 50 ng/mL IL-4 (Miltenyi Biotec) to generate immature monocyte-derived dendritic cells (imDC), followed by 24 h treatment with 100 ng/mL LPS (Sigma) to generate mature DC (mDC).

MOG-CAR Tregs were treated with or without anti-CD3/CD28 beads or MOG coated-plates prior to co-culture with imDC. After 3-day co-culture, Tregs were labelled with anti-CD2 and CD3 microbeads and depleted using magnetic columns (Miltenyi Biotec). DC phenotype was analysed by their surface expression of DC-SIGN(CD209), costimulatory molecules CD80, CD86, CD40 and antigen presentation molecule HLA-DR using flow cytometry.

Mouse EAE models

C57BL/6 mice and BALB/c mice were purchased from Charles River (Lyon, France) and SJL mice were purchased from Janvier Labs (Le Genest-saint-Isle, France). Mice were housed in an opportunistic pathogen free facility, in individually, positively ventilated polysulfone cages with HEPA filtered air, controlled 12 h light/dark cycle, temperature of 20–26 °C, and relative humidity of 30–70%. Filtered tap water and standard rodent chow were provided ad libitum. For experiments, mice were age matched but distributed randomly to treatment groups.

For bioluminescence experiment, SJL mice were immunized with subcutaneous injection of proteolipid protein PLP 139−151 peptide in CFA (Hooke laboratories emulsion EK-0120, USA) under anesthesia with isofluorane in air 4% (induction) and 1–2% (maintenance). MOG-CAR Tregs were injected on day 7 post-immunization. For bioluminescence analysis, mice were injected I.P with luciferin 100 mg/kg and anesthetized with Isofluorane /air 4% (induction) and 1–2% (maintenance) for imaging at Day 9, Day11, Day14 and Day18 in an IVIS Lumina S5 bioimager (Perkin Elmer, USA).

For passive EAE induction, CD45.1 C57Bl/6 mice were immunized with subcutaneous injection of MOG35 − 55 peptide in CFA (216μg MOG35 − 55 (Biosynthesis, USA), and 350μg mycobacterium tuberculosis H37 RA (ThermoFisher scientific, France) in 100 µl). 2 weeks later, pathogenic cells were harvested from the spleen and inguinal lymph nodes and restimulated with MOG35 − 55 peptide 20 µg/ml (Hooke Labs, USA) for 3 days in vitro in RPMI1640-Glutamax (Thermo Life Tech, France) supplemented with 10% FCS (Sigma, France), 1mM sodium Pyruvate, 0.1mM non-essential amino acids, 1% Penicillin-Streptavidin and 5 µM 2-βetamercapto-ethanol (all from ThermoLife Tech, France) and with a polarizing cytokine cocktail comprising IL-1β 20ng/ml, IL-23 20ng/ml, IL-6 20ng/ml (all from Biolegend, USA) and anti-TGFβ 10ng/ml (Euromedex, France). After 3 days, 25 to 10 million cells were injected intraperitoneally (I.P). EAE onset typically started between day 8 and 10. 1 million MOG-CAR or Ctrl-CAR Tregs were injected I.V. 24 h after the injection of pathogenic cells. Mice were sacrificed at day 11 or 12. Disease severity was scored daily on a 5-point scale: 0, no neurological signs; 1, tail atony; 2, hind limb weakness; 3, hind limb paralysis; 4, forelimb paralysis and 5, moribund.

Spinal cord histopathology and immunostaining

Histopathology and immunostaining were performed by Atlantic Bones using proprietary SOPs. Spinal cord from untreated, MOG-CAR Treg treated or MOG-CAR control Treg treated mice with passive EAE were provided fixed in 4% paraformaldehyde and trimmed by Atlantic bones according to the RITA guidelines30 and embedded into paraffin blocks. The blocks were then sectionned with a microtome (Leica) at room temperature and placed in a water bath at 48 °C. The sections of 3–4 μm were placed onto Superfrost Plus microscope slides (ThermoFisher) then dried at 60 °C before deparaffinization using Xylene, Ethanol and distilled water baths. Sections were then stained with Luxol Fast blue using Atlantic Bones validated procedure. For immunostaining of Iba-1, after antigen retrieval procedure endogenous peroxidases were inhibited and sections were incubated for 1 h at room temperature with primary antibody against Iba-1 (Wakochemicals, USA) after blocking step. Then sections were stained with anti-Rabbit HRP for 30 min at room temperature and staining was revealed using DAB. Slides were counterstained with hematoxylin and mounted on slides. Microscopic slides were then scanned with a Nanozoomer S60 slide scanner (Hamamatsu, France) using the 20X magnification (transmitted light). Slides were analyzed by a pathologist. For Iba-1 staining, images were analyzed with Image J software.

Multiple sclerosis patient samples

PBMCs from RR-MS patients were collected by Sanguine bio and provided as frozen vials. Age range between 18 and 85 years old and patients with criteria for active disease, defined as fitting one of the following were selected: 1 or more relapse in the last year or 2 MS relapses in the last 2 years, presence of a new/enlarge T2-hyperintense, T1 gadolinium-enhancing (GdE) lesion on brain MRI in the previous year. Subject were undergoing Ocrevus or Kesimpta treatment.