POPULATION OF TREG CELLS FUNCTIONALLY COMMITTED TO EXERT A REGULATORY ACTIVITY AND THEIR USE FOR ADOPTIVE THERAPY
20230323299 · 2023-10-12
Inventors
Cpc classification
C12N2501/04
CHEMISTRY; METALLURGY
C12N5/0637
CHEMISTRY; METALLURGY
C12N2501/01
CHEMISTRY; METALLURGY
C12N2501/02
CHEMISTRY; METALLURGY
International classification
Abstract
Natural Treg (nTreg) can potentially suppress cell immune response. Consequently, these CD4+ CD25+ CD127low Foxp3+ T cells are used in adoptive therapy against autoimmune and GVH disease. One difficulty is the varying functional properties depending on the microenvironment that may cause the loss of their suppressive activity and promote TH17- induced inflammatory effects. By ex vivo transdetermination of CD31 TH0 cells, the inventors established and expanded a Foxp3 regulatory T cell population (CD31d-Treg cells) functionally committed to exert a permanent Ag-specific regulatory activity whichever the microenvironmental conditions are. By contrast to nTreg cells, CD31d-Treg cells do not express the IL1 Receptor whose activation is required for IL-17 production. Accordingly, the present invention relates to a population of CD31d-Treg cells functionally committed to exert a regulatory activity and their use for Treg-based adoptive therapy.
Claims
1. A population of CD31d-Treg cells having the following phenotype: CD4.sup.+CD25.sup.+ CD121a.sup.-CD127.sup.-Foxp3.sup.+.
2. The population of CD3 1d-Treg cells of claim 1 that is a population of CART cells.
3. A method of generating the population of CD31d-Treg cells of claim 1 comprising stimulating naïve CD31+ T cells with antigen-pulsed tolerogenic dendritic cells (tolDC) in the presence of the Treg polarizing medium comprising IL-2, a cAMP activator, a TGFβ pathway activator, and an mTOR inhibitor.
4. The method of claim 3 wherein the tolerogenic DCs express on their surface the major histocompatibility (MHC) class Ia and/or MHC class Ib.
5. The method of claim 3 wherein the tolerogenic DCs are pulsed in the presence of at least one self-peptide antigen, modified self-peptide antigen, over-expressed self-peptide antigen or foreign antigen.
6. The method of claim 3 wherein the cAMP activator is selected from the group consisting of prostaglandin E2 (PGE2), an EP2 or EP4 agonist, a membrane adenine cyclase activator and a metabotropic glutamate receptors agonist.
7. The method of claim 3 wherein the TGFβ pathway activator is selected from the group consisting of TGFβ, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), anti-müllerian hormone (AMH), activin, and nodal.
8. The method of claim 3 wherein the mTOR inhibitor is selected from the group consisting of rapamycin and its analogs ; wortmannin; theophylline; caffeine; epigallocatechin gallate (EGCG); curcumin; resveratrol; genistein; 3, 3-diindolylmethane (DIM); LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one); PP242; PP30; Torin1; Ku-0063794; WAY-600; WYE-687; WYE-354; and mTOR and PI3K dual-specificity inhibitors.
9. The method of claim 3 wherein the naïve CD31+ T cells are cultured for at least 5 days, at least 6 days, at least 7 days, at least 8 days.
10. The method of claim 3 which further comprises a step of expanding the population of CD31d-Treg cells in the presence of the Treg polarizing medium and a hypomethylating agent.
11. The method of claim 3 which further comprises a step of expanding the population of CD31d-Treg cells in the presence of the Treg polarizing medium and a TCRαβ cell activator.
12. The method of claim 11 wherein the TCR αβ activator is an anti-TCR αβ antibody.
13. The method of claim 10 wherein the population of CD3 1d-Treg cells is expanded in culture for at least 5 days, at least 6 days, at least 7 days, or at least 8 days.
14. A method of treating an autoimmune inflammatory disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the population of CD31d-Treg cells of claim 1.
15. A pharmaceutical composition comprising the population of CD31d-Treg cells of claim 1 and at least one pharmaceutically acceptable excipient.
16. (canceled)
17. A method of treating an autoimmune inflammatory disease in a patient in need thereof comprising administering to the patient a therapeutically effective amount of the population of Treg cells of claim 1, wherein the Treg cells are engineered to repress the expression of IL-1R.
18. A method of treating an autoimmune inflammatory disease in a subject in need thereof comprising administering to the patient a therapeutically effective amount of an antibody capable of depleting the population of Treg cells that express the IL-1 receptor.
19. A population of Treg cells engineered to repress the expression of the IL-1 receptor.
20. The population of Treg cells of claim 19 that are also engineered to express a chimeric antigen receptor (CAR).
Description
[0106] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.
FIGURES
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[0112]
EXAMPLE 1
Material & Methods
[0113] 1) Human blood samples: Peripheral Blood samples were obtained from healthy donors through Etablissement Français du Sang (EFS, Paris, France). Umbilical cord blood (UCB) samples were obtained from normal term deliveries, after maternal informed consent and stored in the cord blood bank according to approved institutional guidelines (Cellular therapy unit, Saint Louis hospital, Paris, Assistance Publique-Hôpitaux de Paris, France). Blood cells were collected using standard procedures. The study was performed according to the Helsinki declaration, and the study protocol was reviewed and approved by the local Ethics Committee. All samples were de-identified prior to use in this study.
[0114] 2) Cell purification. Peripheral blood mononuclear cells (PBMCs) and UCB were isolated by density gradient centrifugation on Ficoll-Hypaque (Pharmacia, St Quentin en Yvelines, France). PBMCs were used either as fresh cells or stored frozen in liquid nitrogen. CD4+ T cell subsets and T cell-depleted accessory cells (ΔCD3 cells) were isolated from either fresh or frozen PBMCs or UCB. All CD4+ T cells were positively selected with a CD4+ T cell isolation kit (Miltenyi Biotec, Bergisch-Gladbach, Germany), yielding CD4+ T cell populations at a purity of 96-99%. Subsequently, selected CD4+ T cells were labelled with anti-CD25 (B1.49.9)-PC5.5 (Beckman Coulter), anti-CD127 (HIL-7R-M21)-BV421 (BD Biosciences), anti-CD45RA (REA562)-FITC (Miltenyi) and anti-CD31 (WM59)-PE (Biolegend) before being sorted into naive nTreg (CD4+CD127-/lowCD25highCD45RA+) and naive CD31+ or CD31- Tconv (CD4+CD127+CD25neg/ dimCD45RA+CD31+/-) subpopulations using a FACSARIAIII Cell Sorter (Becton Dickinson, Le Pont Claix, France). Postsort analysis confirmed that the purity for each cell type was routinely greater than 90% and that more than 90 % of sorted nTreg expressed FOXP3. T cell-depleted accessory cells (ΔCD3 cells) were isolated by negative selection from PBMCs by incubation with anti-CD3-coated Dynabeads (Dynal Biotech, Oslo, Norway) and were irradiated at 5000 rad (referred to as ΔCD3-feeder).
[0115] 3) Culture. Immature DCs (iDCs) were generated from MACS-isolated CD14+ human monocytes by 6-day cultivation with 100 ng/mL of GM-CSF and 10 ng/mL of IL-4. Their maturation (mDC) was induced by stimulation with LPS (100 ng/mL, Sigma-Aldrich, St. Louis, MO, USA) for an additional 48 h.
[0116] Purified naive nTreg and Tconv cell subsets were cultured separately in IMDM medium containing 2 mM L-glutamine, 100 U/mL penicillin-streptomycin, 1 mM sodium pyruvate and 10% human AB serum, (referred to as complete medium) (Invitrogen, Cergy-Pontoise, France) in 96-well U-bottom plates (Falcon/Becton Dickinson). All cultures were incubated at 37° C. with 5% CO2 and 95% air.
[0117] For polyclonal iTreg generation, cells were stimulated with plate-bound anti-human CD3 (OKT3) mAb (eBioscience, San Diego, CA) at the concentration of 1 .Math.g/mL, soluble anti-human CD28 (CD28.2) mAb (Becton Dickinson, 2 .Math.g/mL), recombinant human IL-2 (Proleukine, Chiron, Amsterdam, 100U/mL), and a tolerogenic cocktail: TGFβ (5 ng/mL), PGE2 (500 nM) and rapamcyin (10 nM), in the presence of ΔCD3-feeder. For plate-bound CD3 stimulation, 100 .Math.L of the anti-CD3 mAb diluted into PBS (Invitrogen) were added to each culture well, placed at 4° C. for 16 h, and then washed twice with PBS. Cells were restimulated every week and harvested for analysis after 21 days of culture.
[0118] For specific iTreg generation, cells were stimulated in the tolerogenic medium with autologous immature dendritic cells pre-incubated with OVA or synovial fluid for 24 h. Recombinant human IL-2 was added after 3 days of culture. Cells were restimulated every week with pre-incubated autologous immature dendritic cells. After three stimulations, antigen specific memory T cells (CD45RO+ CD25+) were sorted and either analyzed or either expanded with polyclonal stimulation in the tolerogenic medium, as described previously.
4) Flow Cytometry Analysis:
[0119] a) mAb labelling. A multicolor immunophenotyping approach was used for the identification and analysis of different lymphocyte subpopulations. Immunophenotypic studies were performed on fresh or frozen samples, using 11 to 18-colour flow cytometry. Four common membrane markers (CD4-FITC (13B8.2) Beckman Coulter, CD45RA-BV650 (HI100) BD Biosciences, CD45RO-APC-eF780 (UCHL1) Invitrogen, CD25-BV785 (M-A251) BD Biosciences, IL-1RI-PE R&D), an intracellular marker (FOXP3-PECF594 (236A/E7), BD Biosciences) and a viability dye were constantly present in all aliquots. Cells were stained for membrane markers (at 4° C. in the dark for 30 min) using cocktails of Ab diluted in PBS containing BSA/NaN3 (0.5% BSA, 0.01% NaN3) (FACS buffer). FOXP3 intracellular staining was performed according to the manufacturer’s instructions. Appropriate isotype control Abs were used for each staining combination. Samples were acquired on BD LSR-Fortessa flow cytometer using FACSDiva software (Beckton Dickinson). Flow data were analysed using FlowJo software (FlowJo, LLC).
[0120] b) CFSE staining. nTreg cells or Tconvs were stained with 1 .Math.M CFSE (Cell Trace cell proliferation kit; Molecular Probes/Invitrogen) in PBS for 8 min at 37° C. at a concentration of 1 × 10.sup.6 cells/mL. The labelling was stopped by washing the cells twice with RPMI-1640 culture medium containing 10% FBS. The cells were then re-suspended at the desired concentration and subsequently used for proliferation assays.
5) Functional Assays.
[0121] a) Polyclonal nTreg cell-contact mediated suppression. CFSE-labelled Tconvs (4 × 10.sup.4/well), used as responder cells, were cultured with ΔCD3-feeder (4 × 10.sup.4/well) in the presence or absence of defined amounts of nTreg cells or iTreg (0.4 × 10.sup.4 to 4 × 10.sup.4 cells/well) for 4-5 days. Cultures were performed in round bottom wells coated with 0.2 .Math.g/mL anti-CD3 mAb in 200 .Math.L of complete medium. Varying concentrations of soluble anti-CD28 mAb were added when indicated. Results are expressed either as the percentage of proliferating CFSE low T cells or as a percentage of suppression calculated as follows: (100 × [(percentage of Tconv CFSE low cells -- percentage of Tconv CFSE low in coculture with nTreg cells)/percentage of Tconv CSFE low cells]).
[0122] b) Ag-specific HLA-DR-restricted nTreg suppressive assay. Pre-activated CFSE-labelled Tconv cells (4 × 10.sup.4/well) with soluble anti-CD3 (4 .Math.g/mL) and anti-CD28 (4 .Math.g/mL) were stimulated with autologous iDC (10.sup.5 cells/well of each cell type) in the presence or absence of defined amounts of autologous nTreg or iTreg (0.4 × 10.sup.4 to 4 × 10.sup.4 cells/well) for 4-5 days.
[0123] 6) Cytokines quantification. IL-17 levels in cell culture supernatants (SN) were determined by luminex technology.
[0124] 7) qRT-PCR. Total RNA was isolated from nTreg or iTreg conserved in RLT buffer, using miRNeasy Micro Kit (Quiagen, Courtaboeuf, France). cDNA was synthesized from total RNA using a reverse transcription kit PrimerScriptTM 1.sup.st strand cDNA Synthesis (Takara Bio Europe S.A.S., Saint-Germain en Laye, France). RNA quantitation was performed with a Nanodrop instrument (Thermo Fisher Scientific, Courtaboeur, France). The expression levels of IL-1RI was tested by real-time quantitative PCR. Real-time quantitative PCR was carried out using the kit Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, California, USA). A LightCycler 480 II thermocycler (Roche Applied Science, Meylan, France) was used. PCR conditions were 95° C. for 5 min, followed by 45 cycles of 95° C. for 15 s and 60° C. for 1 min. At the end of the amplification reaction, a melting curve analysis was performed to confirm the specificity as well as the integrity of the PCR product by the presence of a single peak. Absence of cross-contamination and primer dimers was verified on a blank water control. The geometric means of three reference genes (YWHAZ, TOP1 and ATP5B) was used for normalization. The relative expression levels of mRNA were determined using the ΔΔCt formula; fold changes were calculated as 2-ΔΔCt. Only means of duplicates with a CV of <15% were analysed.
[0125] 8) Bisulfite pyrosequencing. Primer Design: All primers used in this study are listed and were purchased from Eurofin Genomics. Reverse PCR primers were biotinylated for downstream pyrosequencing experiments. For FOXP3 upstream enhancer 1 and proximal promoter regions, the pyrosequencing primers were designed using the SNP Primer design software (Qiagen). Bisulfite conversion: Bisulfite conversion of DNA was performed on DNA from 5,000-100,000 fixed cell using the EZ DNA Methylation -DirectTM Kit (Zymo Research) according to the manufacturer’s instruction in an elution volume of 12-20 .Math.L.
[0126] PCR amplification: For each region, PCR reactions were performed using 1 .Math.L of bisulfite treated DNA as template in a 20 .Math.L PCR mix including 200 nM of each primer, 1x HotStar Taq DNA polymerase Buffer, 1.6 mM of additional MgCl2, 200 .Math.M of each dNTPs, and 2 U of HotStar Taq DNA polymerase. The reaction was performed in a Mastercyler Pro S (Eppendorf) and the cycling conditions included an initial denaturation step performed for 10 min at 95° C., followed by 50 cycles of 30 sec denaturation at 95° C., 30 sec annealing at Ta and 30 sec elongation at 72° C. The final step included 5 min elongation at 72° C. The optimal primer annealing temperatures were determined for each assay using the same PCR and cycling conditions except for the annealing step performed using a gradient temperature program ranging from 50° C. to 70° C., followed by the analysis of 5 .Math.l of the PCR reaction by electrophoresis on a 2% agarose gel.
[0127] DNA methylation analysis by pyrosequencing: 10 .Math.L of PCR product were supplemented with 2 .Math.L of Sepharose beads, 40 .Math.L of binding buffer (10 mM Tris-HCl, 2 M NaCl, 1 mM EDTA, 0.1% Tween 20; pH 7.6) and 28 .Math.L of water and incubated under constant mixing (1400 rpm) for 10 min at room temperature. PCR products were then purified and rendered single-stranded using the PyroMark Q96 Vacuum Workstation (Qiagen) after three successive baths of 70 % ethanol, 0.2 M NaOH denaturing solution and 1x washing buffer (10 mM Tris-acetate; pH 7.6). Final elution was performed in a pyrosequencing plate (PyroMark Q96 Plate Low, Qiagen) including 4 pmol of the pyrosequencing primer and 12 .Math.L of annealing buffer (20 mM Tris-acetate, 2 mM Mg-acetate; pH 7.6). DNA methylation analysis was performed using PyroMark Gold SQA Q96 Kit (Qiagen) on a PyroMark Q96 MD (Qiagen) and analyzed with PyroMark CpG software (Qiagen).
[0128] 9) Immunofluorescence. CD4+ CD25high live cells were sorted from CD31d Foxp3 Treg or naive nTreg after 21 days of culture in tolerogenic medium and 50.000 were cytospinned on slides. Cells were stained using the Image IT Fixation/Permeabilization kit (Molecular Probes, Eugene, Oregon, USA). The following antibodies were used for staining: biotinylated anti-Foxp3 (1:100) and anti-biotin AF594-labeled antibody as a secondary antibody (1:100). Cells were incubated in a medium with 4′6-diamidino-2-phenylindole (DAPI, Vector Laboratories) to trace cell nuclei. Slides were evaluated in a LSM 780 confocal microscope with a 63X NA 1,4 oil immersion objective.
[0129] 10) Statistical analysis. Difference between groups was assessed using Student’s T-Test. Error bars on graphs represent either s.e.m. or interquartile range. Statistical analysis was performed using GraphPad Prism. P values under or equal to 0.05 were considered as statistically significant. In the figures, P values are displayed according to the following representation: * P< 0.05, ** P<0.005, *** P<0.001.
Results
Ex Vivo Generation of CD31d-Foxp3 Treg Cells by Functional Reprogrammation of Activated TH0 Cells
[0130] In the prospect of using autologous cells for nTreg adoptive transfer, we investigated whether naïve CD4+ T cells isolated from PBMCs could be functionally reprogrammed (transdetermined) to Foxp3 regulatory T cells under a tolerogenic microenvironment, as can be naïve CD4+ T cells isolated from cord blood (Schiavon et all). We purified naïve human CD4+ CD25- CD127+ CD45RA+ T cells by FACS from PBMCs and CBMCs. These cells will be referred to as PBMCS and CBMCs naïve CD4+ T cells, respectively and ex vivo naïve nTreg cells isolated from PBMCs were used as positive control.
[0131] Furthermore we show that these ex vivo generated CD31d-Foxp3 Treg cells display a similar suppressive activity as fresh nTreg when using the standard polyclonal cell-cell contact Treg suppression assay (
[0132] Exploring which phenotypic or epigenetic characteristics could explain this different behaviour, we found that transdetermined CD31d Foxp3+ T cells exhibit lower levels of IL-1R1 mRNA (
Antigen Specific CD31d-Foxp3 Treg Cells Generation and Expansion.
[0133] Clinical transfer of polyclonal Treg cells has been shown to be safe in patients as treatment for GVHD and T1D (10,12,13). However, due to their TCR polyclonality, a large number of T cells (in the 10.sup.9-10.sup.10 range) need to be administered, thus representing a limitation to nTreg based adoptive therapy. In order to overcome this drawback, we set up a method to generate and expand Ag-specific Treg exhibiting highly potent suppressive activity whichever culture condition of stimulation. This protocol consists of generating antigen-specific CD31d-Treg cells by stimulating naïve CD31+ T cells with antigen-pulsed tolerogenic DC (tolDC) in presence of the Treg polarizing medium comprising the combination of IL-2, TGFβ, PGE2 and rapamycin. Briefly, after 3 stimulations under the same conditions, CD4+ T cells that still retained the naive phenotype CD45RA+RO- were removed from the culture and the antigen specific memory T-cells were further expanded with periodic stimulation with anti-CD3 Ab and anti-CD28 Ab in the presence of the same tolerogenic medium as described in Mat and Methods.
Conclusion
[0134] nTreg based adoptive therapy represents a promising medication to autoimmune pathologies, such as type I diabetes, and in prevention of GVHD, or of allogeneic transplant rejection in solid organ transplantation. However, nTreg cell preparations currently administered in patients exhibit intrinsic characteristics, which per se represent a source of clinical complications and limitations. Indeed, they are most often neither autologous nor Ag specific. Furthermore, they may exert a TH-17 like activity under a pro-inflammatory tissue stromal context (Le Buanec /VS PNAS). The present study focuses on the CD31d Foxp3 Treg cells. The structural and functional characteristics of these Treg cells are distinct from those of the currently administered Treg cells including fresh or expanded nTreg cells of thymic origin, ex vivo induced nTreg (ref) and car Treg cells (ref). They are optimal for the use of CD31d Foxp3 Treg cells in Treg-based adoptive therapies, considering that 1) these cells can be generated from autologous cells, 2) they can be expanded under appropriate conditioned medium for over 21 days remaining Ag specific, 3) following Ag specific stimulation their number could be administered at much lower doses than ex vivo-expanded polyclonally stimulated Treg cells. Given that the number of antigen specific CD31d Foxp3 Treg cells generated from only 1 million blood TH0 could exceed 8-10 million cells, the number of these cells required for adoptive therapy should not represent a limitation, as compared to the polyclonally expanded cells administered in current trials to treat both auto and allo-immune disease range from 3 to 300 million cells (Table 1). In this line it is noteworthy to consider that following Ag specific stimulation with auto-immune tissue extracts, as described in this study with synovial liquid originated from rheumatoid arthritis patients, CD31d Foxp3 Treg cells could control in auto-immune pathologies the different pathogenic autoreactive clones induced by the distinct auto-antigens even when unidentified. 4) Most importantly, given that these cells do not released IL-17, their adoptive transfer for therapeutic purpose does not entail any risk of inflammatory complication.
EXAMPLE 2
Material and Method
[0135] 1) Human blood samples. Peripheral Blood samples were obtained from healthy donors through Etablissement Français du Sang (EFS, Paris, France) and from treated or untreated patients with Systemic Sclerosis through Discovery Life Science, Alabama, United States or with Systemic Lupus Erythematosus through Hôpital Saint Louis, Paris.
[0136] 2) Cell purification. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation on Ficoll-Hypaque (Pharmacia, St Quentin en Yvelines, France). PBMCs were stored frozen in liquid nitrogen. CD4+ T cell subsets and T cell-depleted accessory cells (ΔCD3 cells) were isolated from frozen PBMCs. All CD4+ T cells were positively selected with a CD4+ T cell isolation kit (Miltenyi Biotec, Bergisch-Gladbach, Germany), yielding CD4+ T cell populations at a purity of 96-99%. Subsequently, selected CD4+ T cells were labelled with anti-CD25 (B1.49.9)-PC5.5 (Beckman Coulter), anti-CD45RA (REA562)-FITC (Miltenyi) and anti-CD31 (WM59)-PE (Biolegend) before being sorted into naive CD31+ naïve Tcells (CD4+ CD25neg/ dim CD45RA+CD31+/-) subpopulations using a FACSARIAIII Cell Sorter (Becton Dickinson, Le Pont Claix, France) (
[0137] 3) Culture. For polyclonal iTreg generation, cells were stimulated with plate-bound anti-human CD3 (OKT3) mAb (eBioscience, San Diego, CA) at the concentration of 1 .Math.g/mL, soluble anti-human CD28 (CD28.2) mAb (Becton Dickinson, 2 .Math.g/mL), recombinant human IL-2 (Proleukine, Chiron, Amsterdam, 100U/mL), and a tolerogenic cocktail: TGFβ (5 ng/mL), PGE2 (500 nM) and rapamcyin (10 nM), in the presence of ΔCD3-feeder. For plate-bound CD3 stimulation, 100 .Math.L of the anti-CD3 mAb diluted into PBS (Invitrogen) were added to each culture well, placed at 4° C. for 16 h, and then washed twice with PBS. Cells were restimulated at Day 6 and harvested for analysis at Day 12 of culture.
4) Flow Cytometry
[0138] a) Phenotypic analysis of unstimulated PBMCs. PBMCs were stained with 2 different antibody panels (Table 3) to evaluate CD31 expression in naïve CD4+ T cells gated either as CD3+ CD4+ CD45RA+ CD25-(Table 3 Panel 1) or as CD3+ CD4+ CD45RA+ CCR7+ (Table 3 Panel 2).
[0139] b) Phenotypic analysis of naïve CD4+ T cells stimulated under tolerogenic conditions. CD4+ T cells were stained with the antibody panel described in Table 3 Panel 3.
Results
Patients With Systemic Sclerosis and Systemic Lupus Erythematosus Exhibit Naïve CD4+ T Cells Expressing CD31
[0140] We wanted to see if patients with autoimmune diseases could present naive CD4+ T cells expressing CD31, in order to generate and expand in vitro stable regulatory T cells regardless of their microenvironment. As shown in Table 4A and Table 4B, patients with Systemic Lupus Erythematosus or Systemic Sclerosis expressed similar level of CD31 in naïve CD4+ T cells as Healthy Donors.
CD4+ Expressing CD25 and FOXP3 Can Be Generated in Vitro From Naive CD4+ CD31+ T Cells Isolated From Patients with Systemic Sclerosis
[0141] As a result of the tolerogenic medium used to transduce the expression of CD25 and Foxp3 in the population of interest (CD4+ CD25neg/ dimCD45RA+CD31+) during 12 days, we analyzed the expression of Foxp3 and CD25 which are marker of regulatory T-cells (Treg) (Table 5). There was no significant change between the Healthy Donor and the 2 Systemic Sclerosis patients with a population between 82.4% (Patient A) and 69.1 % (Patient B) expressing Foxp3 compared to control HD Stimulated (91.2%) with high level of CD25. In contrary unstimulated Healthy Donor HD do express only very few Foxp3 with 4.7 % (
[0142] Those experiments show the effect of i) cell sorting of the CD4 naïve T-cell CD31+ subpopulation and ii) the tolerogenic medium to transduce the expression of CD25/Foxp3 which characterizes regulatory T-cells (Treg).
[0143] We also looked at the expression of CD31 in the “final” population (i.e. after the 12 days of harvesting with the tolerogenic medium): about 37 to 47% of cells do express CD31, which is not the objective of this population of interest. We only want to have Foxp3/CD25 cells indifferently of the expression of CD31, unlike some previous publications.
[0144] It confirms the previous results with diseased PBMC from Systemic Sclerosis which is a major auto-immune disease with unbalanced Treg/Th17 in favor of Th17.
Tables
[0145] TABLE-US-00001 Published results on Treg adoptive transfer in human disease. Publication Diseases Cells Dose Expansion Trzonkowski and al, 2009 GVHD Polyclonal expanded Treg 1 × 10.sup.5 or 3 × 10.sup.6 Treg / kg αCD3 + αCD28 + IL-2 Brunstein and al, 2011 GVHD Polyclonal expanded UCB Treg 0.1-30 × 10.sup.5 Treg /kg αCD3 + αCD28 + IL-2 Di Ianni and al, 2011 GVHD Fresh polyclonal Treg 2-4 × 10.sup.6 Treg / kg No expansion Marek-Trzonkowska and al, 2012 T1DM Polyclonal expanded Treg 10-20 × 10.sup.6 Treg /kg αCD3 + αCD28 + IL-2 Desreumaux and al, 2012 Crohn’s disease OVA-specific Tr1 1 × 10.sup.6 - 1 × 10.sup.9 Treg αCD3 + IL-2 + OVA then selection of OVA-specific IL-10-producing cells Bachetta and al, 2014 GVHD IL-10 DLI 1 × 10.sup.5 - 3 × 10.sup.6 T cells / kg Donor T cells pretreated with IL-10 Martelli and al, 2014 GVHD Fresh polyclonal Treg 2.5 × 10.sup.6 Treg / kg No expansion Bluestone and al, 2015 T1DM Polyclonal expanded Treg 0.05-26 × 10.sup.8 Treg αCD3 + αCD28 + IL-2 Theil and al, 2015 GVHD Polyclonal expanded Treg 2.4 × 10.sup.6 Treg / kg αCD3 + αCD28 + IL-2 + rapamcyin Todo and al, 2016 Liver transplant ation Regulatory lymphocytes 23.30 × 10.sup.6 T cells /kg Allo-reactive regulatory lymphocytes Chandran and al, 2017 Kidney transplant ation Polyclonal expanded Treg 320 × 10.sup.6 Treg αCD3 + αCD28 + IL-2 Mathew and al, 2018 Kidney transplant ation Polyclonal expanded Treg 0.5-5 × 10.sup.9 Treg αCD3 + αCD28 + IL-2 + TGFβ + rapamcyin Kellner and al, 2018 GVHD Fucosylated polyclonal expanded UCB Treg 1 × 10.sup.6 Treg / kg αCD3 + αCD28 + IL-2 Thonhoff and al, 2018 Amyotrop hic lateral sclerosis Autologous polyclonal expanded Treg 1 × 10.sup.6 Treg / kg αCD3 + αCD28 + IL-2 + rapamcyin Abbreviations: GVHD, graft-versus-host disease; T1DM, type I diabetes mellitus
TABLE-US-00002 Sorting iTreg: Percentage CD4 naïve CD31+ Sample % CD45RA+CD31+ HD H61 16.6 Patient A 12.8 Patient B 16.2 Percentage (%) of CD45RA+ CD31+ from Naïve CD4 CD25- cells.
TABLE-US-00003 antibody panels for flow cytometry analysis Markers Fluorochrome CD3 FITC Viab V506 CD4 APC-H7 CD25 BV786 CD45RA BV421 CD31 PE Panel 1: ex vivo phenotypic analysis
TABLE-US-00004 Markers Fluorochrome CD3 FITC Viab V506 CD4 APC-H7 CCR7 BV786 CD45RA BV421 CD31 PE Panel 2: ex vivo phenotypic analysis
TABLE-US-00005 Markers Fluorochrome CD3 BUV737 Viab V506 CD.sub.4 BUV 395 CD25 BV786 CD45RA BV650 CD31 PE Foxp3 PeCF594 Panel 3: phenotypic analysis after in vitro stimulation in presence of tolerogenic condition
TABLE-US-00006 Frequencies of CD31+ T cells within the CD3+ CD4+ naïve compartment from patients with Systemic Lupus Erythematosus (SLE) under treatment. Gating Strategy Healthy Donor 1 Healthy Donor 2 Event Count % of Total % of parent Event Count % of Total % of parent Ungated 953200 588512 Lymphocytes 523749 54.95 54.95 343002 58.28 58.28 CD3+ 366149 38.41 69.91 241035 40.96 70.27 CD3+ CD4+ 224810 23.58 61.4 108586 18.45 45.05 Naive CD3+ CD4+ 118631 12.45 52.77 32315 5.49 29.76 Naive CD3+ CD4+ CD31+ 88427 9.28 74.54 24638 4.19 76.24 Gating Strategy Patient 1 with Systemic Lupus Erythematosus under treatment Patient 2 with Systemic Lupus Erythematosus under treatment Event Count % of Total % of parent Event Count % of Total % of parent Ungated 788256 1204760 Lymphocytes 671068 85.13 85.13 1003262 83.27 83.27 CD3+ 439875 55.8 65.55 860753 71.45 85.8 CD3+ CD4+ 305775 38.79 69.51 591459 49.09 68.71 Naive CD3+ CD4+ 243651 30.91 79.68 303375 25.18 51.29 Naive CD3+ CD4+ CD31+ 181368 23.01 74.44 203841 16.92 67.19 Frequencies of CD31 + T cells within the CD3+ CD4 + naive compartment. (A) PBMCsfrom 2 Healthy Donors and 2 SLE patients under treatment were analyzed by flow cytometry with a panel using antibodies anti CD3, CD4, CD45RA, CCR7, and CD31 to identify naive (CD45RA+, CCR7+) CD4+ expressing CD31 as a marker of recent thymic emigrants.
TABLE-US-00007 Frequencies of CD31 + T cells within the CD3+ CD4 + naive compartment from patients with systemic sclerosis. Healthy Donor 3 Patient 1 with systemic sclerosis under treatment Gating Strategy Event Count % of Total % of parent Event Count % of Total % of parent Ungated 1982394 1146256 Lymphocytes 440014 22.2 22.2 474505 41.4 41.4 CD3+ 240586 12.1 55.3 297568 26 63 CD3+ CD4+ 166152 8.38 69.1 250006 21.8 84 Naive CD3+ CD4+ 76860 3.88 46.3 175216 15.3 70.1 Naive CD3+ CD4+ CD31+ 26923 1.36 35 94770 8.27 54.1 Patient 2 with systemic sclerosis under treatment Patient 3 with systemic sclerosis without treatment Gating Strategy Event Count % of Total % of parent Event Count % of Total % of parent Ungated 3022731 Lymphocytes 1740055 57.6 57.6 1025239 46.8 46.8 CD3+ 788848 26.4 45.9 313365 14.3 31.3 CD3+ CD4+ 441398 14.6 56 240931 11 76.9 Naive CD3+ CD4+ 184512 6.1 41.8 84058 3.84 34.9 Naive CD3+ CD4+ CD31+ 55935 1.85 30.3 39463 1.8 46.9 Healthy Donor 3 anti-CD3 / anti CD28 Ab IL-2 4.7 96.2 11070 anti-CD3 / anti CD28 Ab IL-2 + tolerogenic coktail 91.2 96.3 29236 Patient 1 with systemic sclerosis under treatment anti-CD3 / anti CD28 Ab IL-2 + tolerogenic coktail 82.4 96.3 22502 Patient 2 with systemic sclerosis under treatment anti-CD3 / anti CD28 Ab IL-2 + tolerogenic coktail 69.1 95.6 28546 (A) PBMCsfrom 1 Healthy Donor and from 3 patients with sytemic sclerosis with or without treatment were analyzed by flow cytometry with a panel using antibodies anti CD3, CD4, CD45RA, CD25, and CD31 to identify naive (CD45RA+, CD25-) CD4+ expressing CD31 as a marker of recent thymic emigrants.
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