WO2018024893A1 - Lymphocytes t régulateurs foxp3+ invariants et leurs utilisations thérapeutiques - Google Patents

Lymphocytes t régulateurs foxp3+ invariants et leurs utilisations thérapeutiques Download PDF

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WO2018024893A1
WO2018024893A1 PCT/EP2017/069822 EP2017069822W WO2018024893A1 WO 2018024893 A1 WO2018024893 A1 WO 2018024893A1 EP 2017069822 W EP2017069822 W EP 2017069822W WO 2018024893 A1 WO2018024893 A1 WO 2018024893A1
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foxp3
cells
invariant
regulatory
cell
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PCT/EP2017/069822
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Hélène LE BUANEC
Daniel Zagury
Sophie Duchez
Valérie SCHIAVON
Armand Bensussan
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Université Paris Diderot - Paris 7
Centre National De La Recherche Scientifique (Cnrs)
Medecine Et Innovation
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Publication of WO2018024893A1 publication Critical patent/WO2018024893A1/fr

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Definitions

  • the present invention relates to a novel population of invariant Foxp3 + regulatory T cells, an ex vivo method generating and expanding them and therapeutic uses thereof.
  • Tregs The history of Tregs begins in 1970 (under the name of "suppressor T cells"). Unfortunately, due to lack of a molecular marker, research on suppressor T cells was stopped, until many years later, when Sakaguchi et al. (1995) identified CD25 as a phenotypic marker for suppressive CD4+ T cells in mice, which upon adoptive transfers could prevent development of autoimmune disease. These suppressive T cells were named nTregs and were later also found in humans within the CD4+CD25 hlgh T cell population.
  • Tregs T regulatory cells
  • Tregs T regulatory cells
  • Tregs can develop both in the thymus (tTregs, thymic-derived Tregs formerly natural or nTreg) and in the periphery (pTregs, peripheral-induced Tregs, formerly induced, or iTreg) and silence autoreactive cells by cell-cell contact.
  • Tregs are routinely identified by the use of a combination of phenotypic markers: CD4, CD25, CD127, CD45RA and the canonical transcription factor FOXP3. Tregs may also exhibit others functional markers (CTLA-4, CD28, ICOS, GITR, CD39, TIGIT, FCLR3, TIM-3, Helios), maturation markers (HLA-DR, CD95) and homing molecules (CXCR3, CCR7, CXCR3, CCR5, CCR4, CLA) whose frequency and level depend on their maturation stages. Trl T cells originate from most immune-stimulated CD4+ helper T cell subtypes and down-modulate immune response by releasing IL-10.
  • the inventors isolated a new population of regulatory T cells, which are invariant Foxp3 + regulatory T cells.
  • the present invention thus provides the characteristics of this new population of invariant Foxp3 + regulatory T cells, a method for ex vivo generating and expanding said invariant Foxp3 + regulatory T cells and therapeutic uses of said invariant Foxp3 + regulatory T cells.
  • the present invention relates to an isolated population of invariant Foxp3 + regulatory T cells having the following phenotype: CD3 + Va24 + Foxp3 + .
  • the isolated population of invariant Foxp3 + regulatory T cells of the invention further expressing CD4 and CD25, thereby having the following phenotype: CD3 + CD4 + Va24 + CD25 + Foxp3 + .
  • the present invention also relates to a method for generating ex vivo invariant Foxp3 + regulatory T cells having the following phenotype: CD3 + Va24 + Foxp3 + , comprising - culturing CD3 + Va24 + T cells in the presence of an invariant T cell activator and the following agents: i) an cAMP (Cyclic adenosine monophosphate) activator, ii) a TGF (Transforming growth factor beta) pathway activator, iii) a mTOR inhibitor, optionally iv) at least one cytokine selected in the group of IL-2, IL-7, IL-15 and TSLP, and optionally v) at least one TET enzymes activator (preferably selected from vitamin C and a NaHS hydrogen sulfide releasing agent) and/or at least one DNMT inhibitor (such as, for example, RG108, DAC or 5AC), for at least 5 days.
  • an cAMP Cyclic adenosine
  • the invariant T cell activator is a polyclonal invariant T cell activator, preferably a Va24 activator.
  • the invariant T cell activator is an antigen-specific invariant T cell activator, preferably tolerogenic dendritic cells (DCs) expressing CDl and pulsed with at least one non peptide lipid antigen.
  • the cAMP activator is selected from the group comprising prostaglandin E2 (PGE2), an EP2 or EP4 agonist, a membrane adenine cyclase activator and metabotropic glutamate receptors agonists.
  • the TGF pathway activator is selected from the group comprising TGF , bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), anti-mullerian hormone (AMH), activin and nodal .
  • BMPs bone morphogenetic proteins
  • GDFs growth and differentiation factors
  • AH anti-mullerian hormone
  • the mTOR inhibitor is selected from the group comprising rapamycin, rapamycin 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, Torinl , Ku-0063794, WAY-600, WYE-687, WYE-354, GNE477, NVP-BEZ235, PI-103, XL765 and WJD008.
  • the method of the invention further comprises an expansion step, wherein the invariant Foxp3 + regulatory T cells obtained by the generation method as described above are cultured in the presence of an invariant T cell activator and the following agents: i) an cAMP (Cyclic adenosine monophosphate) activator, ii) a TGF (Transforming growth factor beta) pathway activator, iii) a mTOR inhibitor, optionally iv) at least one cytokine selected in the group of IL-2, IL-7, IL-15 and TSLP, and optionally v) at least one TET enzymes activator (preferably selected from vitamin C and a NaHS hydrogen sulfide releasing agent) and/or at least one DNMT inhibitor (such as, for example, RG108, DAC or 5 AC), for at least 5 days.
  • an cAMP Cyclic adenosine monophosphate
  • TGF Transforming growth factor beta pathway activator
  • iii) a mTOR inhibitor
  • the present invention also relates to an ex vivo generated invariant Foxp3 + regulatory T cell population obtainable by the method according to the invention, wherein said Foxp3 + regulatory T cells have the following phenotype: CD3 + Va24 + Foxp3 + .
  • Another object of the present invention is an ex vivo generated and expanded invariant Foxp3 + regulatory T cell population obtainable by the method according to the invention, wherein said Foxp3 + regulatory T cells have the following phenotype: CD3 + Va24 + Foxp3 + .
  • a further object of the present invention is an immunogenic product comprising inactivated invariant Foxp3 + regulatory T cells having the following phenotype: CD3 + Va24 + Foxp3 + or blebs of invariant Foxp3 + regulatory T cells having the following phenotype: CD3 + Va24 + Foxp3 + or immunogenic dendritic cells loaded with blebs of invariant Foxp3 + regulatory T cells having the following phenotype: CD3 + Va24 + Foxp3 + .
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising inactivated invariant Foxp3 + regulatory T cells having the following phenotype: CD3 + Va24 + Foxp3 + or blebs of invariant Foxp3 + regulatory T cells having the following phenotype: CD3 + Va24 + Foxp3 + or immunogenic dendritic cells loaded with blebs of invariant Foxp3 + regulatory T cells having the following phenotype: CD3 + Va24 + Foxp3 + and at least one pharmaceutically acceptable excipient.
  • the present invention further relates to a vaccine composition
  • a vaccine composition comprising inactivated invariant Foxp3 + regulatory T cells having the following phenotype: CD3 + Va24 + Foxp3 + or blebs of invariant Foxp3 + regulatory T cells having the following phenotype: CD3 + Va24 + Foxp3 + or immunogenic dendritic cells loaded with blebs of invariant Foxp3 + regulatory T cells having the following phenotype: CD3 + Va24 + Foxp3 + and at least one adjuvant.
  • Another object of the present invention is the immunogenic product, pharmaceutical composition or vaccine composition according to the present invention for use in treating cancer.
  • a further object of the present invention is a pharmaceutical composition comprising invariant Foxp3 + regulatory T cells having the following phenotype: CD3 + Va24 + Foxp3 + and at least one pharmaceutically acceptable excipient.
  • the present invention also relates to a pharmaceutical composition according to the invention for use in cell therapy.
  • the present invention further relates to a pharmaceutical composition according to the invention for use in treating inflammatory or autoimmune diseases or for preventing transplant rejection or graft versus host disease (GVHD).
  • GVHD transplant rejection or graft versus host disease
  • regulatory T cells refers to cells functionally committed, i.e. capable of suppressive activity (i.e. inhibiting proliferation of conventional T cells), either by cell-cell contact or by MLR suppression (Mixed Lymphocytes Reaction). These cells include different subpopulations including but not limited to, MHCII restricted CD4 + Foxp3 + regulatory T cells, ⁇ Foxp3 + regulatory T cells and invariant Foxp3 + regulatory T cells.
  • invariant Foxp3 + regulatory T cells refers to cells having the following phenotype: CD3 + Va24 + Foxp3 + . These cells recognize non peptide lipid antigens under CD1 restriction.
  • ⁇ Foxp3 + regulatory T cells refers to cells having the following phenotype: y5TCR + Foxp3 + . These cells recognize non peptide phospho antigens with no MHC (major histocompatibility complex) restriction.
  • MCHII restricted CD4 + Foxp3 + regulatory T cells refers to cells having the following phenotype: CD4 + CD25 + Foxp3 + . These cells are thymic derived or peripherally induced. These cells can be identified by their a TCR (T cell receptor) and recognize peptides (including foreign or self peptides) presented by restricted MHC class II (major histocompatibility complex class II) molecules.
  • TCR T cell receptor
  • MHC class II major histocompatibility complex class II
  • treatment refers to therapeutic treatment and prophylactic and preventive measures, wherein the object is to prevent or slow down (lessen, diminish) the targeted pathological disorder or condition.
  • Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.
  • a subject or mammal is successfully "treated” for a disease if, after receiving a therapeutic amount of invariant Foxp3 + regulatory T cells or a therapeutically amount of inactivated invariant Foxp3 + regulatory T cells according to the present invention, the patient shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of pathogenic cells; reduction in the percent of total cells that are pathogenic; and/or relief to some extent, of one or more of the symptoms associated with the specific disease or condition; reduced morbidity and mortality, and improvement in quality of life issues.
  • the above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.
  • therapeutically effective amount refers to the number of invariant Foxp3 + regulatory T cells or of inactivated invariant Foxp3 + regulatory T cells that is aimed at inducing a therapeutic response, without causing significant negative or adverse side effects to the target.
  • a therapeutically effective amount may be administered prior to the onset of the disease to be treated, for a prophylactic or preventive action. Alternatively or additionally, the therapeutically effective amount may be administered after initiation of the disease to be treated, for a therapeutic action.
  • therapeutic response refers to a therapeutic benefit induced by the invariant Foxp3 + regulatory T cell therapy or the invariant Foxp3 + regulatory T cell vaccination in a subject.
  • a therapeutic response may include the fact of (1) delaying or preventing the onset of the disease to be treated; (2) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of the disease to be treated; (3) bringing about ameliorations of the symptoms of the disease to be treated; (4) reducing the severity or incidence of the disease to be treated; or (5) curing the disease to be treated.
  • subject or patient refers to a mammal, preferably a human.
  • the terms subject and patient may be used with the same meaning.
  • non-human mammal include a pet such as a dog, a cat, a domesticated pig, a rabbit, a ferret, a hamster, a mouse, a rat and the like; a primate such as a chimp, a monkey, and the like; an economically important animal such as cattle, a pig, a rabbit, a horse, a sheep, a goat.
  • the subject is awaiting the receipt of, or is receiving medical care or was/is/will be the object of a medical procedure, or is monitored for the development of a disease.
  • the subject is an adult (for example a subject above the age of 18).
  • the subject is a child (for example a subject below the age of 18).
  • the subject is a male.
  • the subject is a female.
  • allogeneic cells refers to cells isolated from one subject (the donor) and infused in another (the recipient or host).
  • autologous cells refers to cells that are isolated and infused back into the same subject (recipient or host).
  • the present invention relates to an isolated population of Foxp3 + regulatory T cells having the following phenotype: CD3 + Va24 + Foxp3 + .
  • the present invention relates to an isolated population of invariant Foxp3 + regulatory T cells having the following phenotype: CD3 + Va24 + Foxp3 + .
  • invariant as used herein includes the term “semi-invariant", where the semi-invariant T cells are T cells not expressing ⁇ ⁇ .
  • the isolated population of the invention is a population of semi- invariant Foxp3 + T cells having the following phenotype: CD3 + Va24 + Foxp3 + Vpi .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + .
  • the invariant Foxp3 + regulatory T cells CD3 + Va24 + Foxp3 + CD4 + express the TCR ⁇ chain.
  • the invariant Foxp3 + regulatory T cells CD3 + Va24 + Foxp3 + CD4 + express the TCR ⁇ 2 chain.
  • the invariant Foxp3 + regulatory T cells CD3 + Va24 + Foxp3 + CD4 + express the TCR ⁇ 4 chain.
  • the invariant Foxp3 + regulatory T cells CD3 + Va24 + Foxp3 + CD4 + express the TCR ⁇ 8 chain.
  • the invariant Foxp3 + regulatory T cells CD3 + Va24 + Foxp3 + CD4 + express the TCR ⁇ 5.1 chain.
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD25 + .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 CTLA4 + .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 CD45RO + .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 CD127 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 CD161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD25 + .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CTLA4 + .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD45RO + .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD 127 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD25 + CTLA4 + .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD25 + CD45RO + .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD25 + CD 127 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD25 + CD 161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD25 + CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD45RO + CD 127 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD45RO + CD 161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD45RO + CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD 127 " CD161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD127 " CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD161 CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD25 + CTLA4 + .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD25 + CD45RO + .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD25 + CD127 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD25 + CD 161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 CD25 + CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CTLA4 + CD45RO + .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CTLA4 + CD 127 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CTLA4 + CD161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CTLA4 + CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD45RO + CD127 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD45RO + CD 161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD45RO + CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD127 " CD 161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD127 " CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD161 CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD25 + CTLA4 + CD45RO + .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD25 + CTLA4 + CD 127 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD25 + CTLA4 + CD 161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD25 + CTLA4 + CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD25 + CD45RO + CD 127 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD25 + CD45RO + CD161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD25 + CD45RO + CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD25 + CD 127 " CD 161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD25 + CD127 " CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD25 + CD161 CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CTLA4 + CD45RO + CD 127 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CTLA4 + CD45RO + CD 161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CTLA4 + CD45RO + CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CTLA4 + CD127 " CD 161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CTLA4 + CD127 " CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CTLA4 + CD161 " CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD45RO + CD 127 " CD 161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD45RO + CD127 " CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD45RO + CD161 CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD127 CD161 CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD25 + CTLA4 + CD45RO + .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD25 + CTLA4 + CD 127 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD25 + CTLA4 + CD161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 CD25 + CTLA4 + CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD25 + CD45RO + CD 127 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 CD25 + CD45RO + CD 161 .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD25 + CD45RO + CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD25 + CD 127 " CD 161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 CD25 + CD127 " CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 CD25 + CD161 " CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CTLA4 + CD45RO + CD 127 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CTLA4 + CD45RO + CD161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CTLA4 + CD45RO + CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CTLA4 + CD 127 " CD 161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CTLA4 + CD127 CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CTLA4 + CD161 CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD45RO + CD 127 " CD 161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD45RO + CD127 " CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD45RO + CD161 CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD127 CD161 " CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD25 + CTLA4 + CD45RO + CD 127 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD25 + CTLA4 + CD45RO + CD161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD25 + CTLA4 + CD45RO + CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD25 + CTLA4 + CD127 CD161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD25 + CTLA4 + CD127 " CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD25 + CD127 CD161 " CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD25 + CD45RO + CD 127 " CD161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD25 + CD45RO + CD127 " CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD25 + CD45RO + CD161 " CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD25 + CD127 CD161 " CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CTLA4 + CD45RO + CD 127 " CD161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD25 + CD45RO + CD127 CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CTLA4 + CD45RO + CD161 " CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CTLA4 + CD127 CD161 " CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD45RO + CD127 CD161 " CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD25 + CTLA4 + CD45RO + CD 127 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD25 + CTLA4 + CD45RO + CD161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 CD25 + CTLA4 + CD45RO + CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD25 + CTLA4 + CD 127 " CD 161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD25 + CTLA4 + CD127 " CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 CD25 + CTLA4 + CD161 CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD25 + CD45RO + CD 127 " CD161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CD25 + CD45RO + CD127 " CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 CD25 + CD45RO + CD161 CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 CD25 + CD127 " CD161 CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CTLA4 + CD45RO + CD127 CD161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CTLA4 + CD45RO + CD127 CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CTLA4 + CD45RO + CD161 CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 + CD4 + CTLA4 + CD127 " CD161 CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 CD4 + CD45RO + CD12T CD161 CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 CD25 + CTLA4 + CD45RO + CD 127 " CD 161 " .
  • the invariant Foxp3 regulatory T cells are CD3 + Va24 + Foxp3 + CD25 + CTLA4 + CD45RO + CD127 " CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 CD25 + CTLA4 + CD45RO + CD161 CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 CD25 + CTLA4 + CD127 " CD161 CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 CTLA4 + CD45RO + CD127 " CD161 CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 CD25 + CTLA4 + CD45RO + CD127 " CD161 CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 CD4 + CTLA4 + CD45RO + CD127 " CD161 CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 CD4 + CD25 + CD45RO + CD127 " CD161 CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 CD4 + CD25 + CTLA4 + CD127 " CD161 CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 CD4 + CD25 + CTLA4 + CD45RO + CD161 CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 CD4 + CD25 + CTLA4 + CD45RO + CD127 " CD56 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 CD4 + CD25 + CTLA4 + CD45RO + CD 127 " CD161 " .
  • the invariant Foxp3 + regulatory T cells are CD3 + Va24 + Foxp3 CD4 + CD25 + CTLA4 + CD45RO + CD127 " CD161 CD56 " .
  • the invariant Foxp3 + regulatory T cells are Va24 + Jal8 + . In one embodiment, the invariant Foxp3 + regulatory T cells of the invention are human cells.
  • the invariant Foxp3 + regulatory T cells express Foxp3 with a median fluorescence intensity (MFI) at least equivalent to the Foxp3 MFI measured in naive regulatory T cells.
  • MFI median fluorescence intensity
  • naive regulatory T cells refer to T cells having for phenotype Foxp3 + CD45RA + CD4 + CD25 + CD127 ⁇
  • the invariant Foxp3 + regulatory T cells express Foxp3 with a median fluorescence intensity (MFI) of at least 2000.
  • MFI median fluorescence intensity
  • the invariant Foxp3 + regulatory T cells express Foxp3 with a median fluorescence intensity (MFI) of at least 2 or 3 fold the Foxp3 MFI measured in naive regulatory T cells.
  • MFI median fluorescence intensity
  • the invariant Foxp3 + regulatory T cells express Foxp3 with a median fluorescence intensity (MFI) of at least 2000, 3000, 4000, 5000, 10000, 20000, 30000, 40000, 50000, 60000, 70000.
  • MFI median fluorescence intensity
  • the invariant Foxp3 + regulatory T cells population comprises at least about 65% of the CD3 + Va24 + cells expressing Foxp3.
  • the expression "at least about 65%” includes, without limitation, about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 752%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%.
  • the invariant Foxp3 + regulatory T cell population is isolated.
  • isolated population refers to a cell population that is removed from its natural environment (such as the peripheral blood) and that is isolated, purified or separated, and is at least about 75% free, 80%> free, 85% free and preferably about 90%>, 95%, 96%, 97%, 98%, 99% free, from other cells with which it is naturally present, but which lack the cell surface markers based on which the cells were isolated.
  • the Foxp3 + regulatory T cell or the Foxp3 + regulatory T cell population is frozen.
  • the term "expression" may refer alternatively to the transcription of a molecule (i.e. expression of the mRNA) or to the translation (i.e. expression of the protein) of a molecule.
  • detecting the expression may correspond to an intracellular detection.
  • detecting the expression may correspond to a surface detection, i.e. to the detection of molecule expressed at the cell surface.
  • detecting the expression may correspond to an extracellular detection, i.e. to the detection of secretion.
  • detecting the expression may correspond to intracellular, surface and/or extracellular detections.
  • Methods for determining the expression level include, without limitation, determining the transcriptome (in an embodiment wherein expression relates to transcription of a molecule) or proteome (in an embodiment wherein expression relates to translation of a cytotoxic molecule) of cells.
  • the expression of the molecules is assessed at the mRNA level.
  • Methods for assessing the transcription level of a molecule are well known in the prior art. Examples of such methods include, but are not limited to, RT-PCR, RT- qPCR, Northern Blot, hybridization techniques such as, for example, use of microarrays, and combination thereof including but not limited to, hybridization of amplicons obtained by RT-PCR, sequencing such as, for example, next-generation DNA sequencing (NGS) or RNA-seq (also known as "Whole Transcriptome Shotgun Sequencing") and the like.
  • NGS next-generation DNA sequencing
  • RNA-seq also known as "Whole Transcriptome Shotgun Sequencing”
  • the expression of the molecules is assessed at the protein level. Methods for determining a protein level in a sample are well-known in the art.
  • Such methods include, but are not limited to, immunohistochemistry, Multiplex methods (Luminex), western blot, enzyme-linked immunosorbent assay (ELISA), sandwich ELISA, fluorescent-linked immunosorbent assay (FLISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), flow cytometry (FACS) and the like.
  • determining the expression level of at least one molecule corresponds to detecting and/or quantifying binding of a ligand to a molecule.
  • said ligand is an antibody specific of said molecule
  • the method of the invention comprises detecting and/or quantifying a complex formed between said antibody and said molecule.
  • the complex can be detected if the ligand has been for example, but not limited to, covalently coupled with a detectable molecule such as an antibody constant fragment (Fc) or a fluorescent compound (e.g. Cyanine dye, Alexa dye, Quantum dye, etc).
  • the complex can also be detected if the ligand has been tagged with different means well known to the person skilled in the art.
  • a tag used with the invention can be a tag selected from the group comprising or consisting of Hemaglutinin Tag, Poly Arginine Tag, Poly Histidine Tag, Myc Tag, Strep Tag, S-Tag, HAT Tag, 3x Flag Tag, Calmodulin-binding peptide Tag, SBP Tag, Chitin binding domain Tag, GST Tag, Maltose-Binding protein Tag, Fluorescent Protein Tag, T7 Tag, V5 Tag and Xpress Tag.
  • the use of the ligand therefore allows on the one hand the identification and detection of the molecule depending on the ligand used, and on the other hand the quantification of the complex formed.
  • determining the expression level of molecules is conducted by flow cytometry, immunofluorescence or image analysis, for example high content analysis.
  • the determination of the expression level of molecules is conducted by flow cytometry.
  • cells are fixed and permeabilized, thereby allowing detecting intracellular proteins.
  • determining the expression level of a molecule in a cell population comprises determining the percentage of cells of the cell population expressing the molecule (i.e. cells "+" for the molecule). Preferably, said percentage of cells expressing the molecule is measured by FACS.
  • expressing (or +) and “not expressing (or -)” are well known in the art and refer to the expression level of the cell marker of interest, in that the expression level of the cell marker corresponding to "+” is high or intermediate, also referred as "+/-".
  • the cell marker corresponding to "-” is a null expression level of the cell marker or also refers to less than 10 % of a cell population expressing the said cell marker.
  • the expression level of the cell marker of interest is determined by comparing the Median Fluorescence Intensity (MFI) of the cells from the cell population stained with fluorescently labeled antibody specific for this marker to the fluorescence intensity (FI) of the cells from the same cell population stained with fluorescently labeled antibody with an irrelevant specificity but with the same isotype, the same fluorescent probe and originated from the same specie (referred as Isotype control).
  • MFI Median Fluorescence Intensity
  • FI fluorescence intensity
  • the invariant Foxp3 + regulatory T cells are a different population than the MHCII restricted CD4 + Foxp3 + regulatory T cells as shown in Figure 1, wherein MHCII restricted CD4 + Foxp3 + regulatory T cells have the following phenotype CD3 + CD4 + TCRa + CD25 + Foxp3 + CD 127 " CTLA-4 + whereas invariant Foxp3 + regulatory T cells have the following phenotype CD3 + CD4 + Va24 + Foxp3 + CTLA4 + CD25 + CD127 " . As shown in Figure 1, invariant Foxp3 + regulatory T cells have a Foxp3 MFI superior to the Foxp3 MFI of MHCII restricted CD4 + Foxp3 + regulatory T cells.
  • the invariant Foxp3 + regulatory T cells are a different population than the invariant NK T cells as shown in Figure 2, wherein the invariant NK T cells have the following phenotype Va24 + CD56 +/" CD161 + whereas the invariant Foxp3 + regulatory T cells have the following phenotype Va24 + CD56 " CD161 " .
  • the invariant NK T cells have the following phenotype CD127 + CD25 " CTLA4 " whereas the invariant Foxp3 + regulatory T cells have the following phenotype CD 127 " CD25 + CTLA4 + .
  • the invariant Foxp3 + regulatory T cells of the invention do not present a regulatory T cells specific demethylated region (TSDR) of the gene Foxp3. In one embodiment, the invariant Foxp3 + regulatory T cells of the invention present a regulatory T cells specific demethylated region (TSDR) of the gene Foxp3. In one embodiment, the invariant Foxp3 + regulatory T cells present a percentage of demethylation of the TSDR of the gene FOXP3 superior to at least 30%, 40%>, 50%>, whereas the invariant NK T cells present a percentage of Foxp3 promoter demethylation less than 10%. A protocol for measuring promoter demethylation percentage is shown in the Material and Method part of the Examples.
  • the invariant Foxp3 + regulatory T cells present a percentage of enrichment of acetylated histone in Foxp3 promoter region superior to at least 10%, 20%, 30%, 40% or 50%.
  • a protocol for measuring enrichment of acetylated histones in percentage is shown in the Material and Method part of the Examples.
  • the invariant Foxp3 + regulatory T cells of the invention present a percentage of enrichment of acetylated histone in Foxp3 promoter region superior to at least 10%>, 20%>, 30%>, 40%> or 50%>.
  • a protocol for measuring enrichment of acetylated histones in percentage is shown in the Material and Method part of the Examples.
  • the invariant Foxp3 + regulatory T cells are capable of suppressive activity similar to the suppressive activity of naive CD4 + CD25 + CD45RA + CD127 " regulatory T cells. Determination of the suppressive activity of a cell population is well known in the art and can be performed by conventional assays such as the standard polyclonal cell-cell contact Treg suppression assay or the autologous MLR suppression assay as described in the Examples.
  • the present invention also relates to a method for generating ex vivo invariant Foxp3 + regulatory T cells as defined here above.
  • the method for generating ex vivo invariant Foxp3 + regulatory T cells comprises:
  • CD3 + Va24 + T cells preferably CD3 + Va24 + CD45RA+ T cells, in the presence of an invariant T cell activator and the following agents: i) an cAMP (Cyclic adenosine monophosphate) activator, ii) a TGF (Transforming growth factor beta) pathway activator, iii) a mTOR inhibitor, optionally iv) at least one cytokine selected from the group of IL-2, IL-7, IL-15 and TSLP (Thymic stromal lymphopoietin), and optionally v) at least one TET enzymes activator and/or at least one DNMT inhibitor, for at least 5 days,
  • the CD3 + Va24 + T cells are obtained by any technic well known in the art from a blood sample.
  • the CD3 + Va24 + T cells preferably CD3 + Va24 + CD45RA + T cells, are isolated from PBMCs (peripheral blood mononuclear cells) by flow cytometry.
  • the CD3 + Va24 + T cells preferably CD3 + Va24 + CD45RA + T cells, may be isolated from frozen PBMCs.
  • the obtainment of isolated CD3 + Va24 + T cells may be improved by an optional first to a purification step.
  • the CD3 + Va24 + T cells preferably CD3 + Va24 + CD45RA + T cells, are stimulated with antigen pulsed tolerogenic DCs (for example ovalbumin pulsed tolerogenic DCs) in the presence of soluble anti-CD28 and anti-CD40 antibodies.
  • the time of stimulation ranges between 1 hour and 24 hours, preferably between 10 hours and 20 hours, more preferably during about 16 hours. After stimulation, cells are washed, for example with PBS, and stained with anti-CD 154 and anti-CD4 antibodies for sorting.
  • the purified CD3 + Va24 + CD154 + T cells are enriched and may be used for the following activation step.
  • the CD3 + Va24 + T cells are activated in the presence of an invariant T cell activator.
  • Said invariant T cell activator can be a polyclonal invariant T cell activator or an antigen-specific invariant T cell activator.
  • the polyclonal invariant T cell activator is a Va24 activator.
  • Va24 activator include, but are not limited to, anti-Va24 antibody such as 6B11 antibody (Montoya CJ et al. Immunology.
  • CD1 ligands including CD la ligands, CD lb ligands, CDlc ligands and CD Id ligands, preferably CD Id ligands such as a-galactosylceramide (a-GalCer) and analogs thereof, such as for example HS44 (a synthetic amino cyclitolic ceramide analogue in which the sugar head group is a carba cyclitol ring that mimics glucose instead of galactose, and which has the O-glycosidic linkage replaced with an amide group), a-GalCer analogs of the table 1 herein below: Table 1 : Structure of a-GalCer analogs
  • R is C0 2 Me, C0 2 H or CH 2 OH
  • a-glucuronyl- and a-galacturonyl-ceramides and analogs thereof iGb3 (Isoglobotriosylceramide); N-glycolyl (NGc) gangliosides such as for example NGcGM3; glycosphingo lipids or phosphoglycero lipids such as phosphatidylinositol, phosphatidylethanolamine, and phosphatidylglyerol presented preferably by CD Id.
  • the polyclonal invariant T cell activator is an anti-Va24 antibody, preferably a m6Bl 1 antibody.
  • the polyclonal invariant T cell activator is soluble in the culture medium. In another embodiment, the polyclonal invariant T cell activator is coated to the culture plate.
  • the polyclonal invariant T cell activator is used in the presence of feeder cells, preferably autologous feeder cells.
  • Feeder cells include, but are not limited to, ACD3 cells (T cell-depleted accessory cells), irradiated PBMCs, irradiated DCs, artificial APCs (antigen presenting cells), Sf9 cells, insect cells, a pool of PBMCs or a pool of B cells from different subjects, KCD40L cells EBV-transformed B cell lines and EBV-transformed lymphoblastoid cells (LCL).
  • the feeder cells used in the invention are ACD3 cells that are isolated by negative selection from PBMCs by incubation with anti-CD3 coated beads and then irradiated at 3000 rad.
  • the ratio T cells / feeder cells ranges from 1:100 to 1: 10000, preferably from 1:1000 to 1:5000.
  • the expression "from 1 : 100 to 1 : 10000” includes, without limitation, 1 : 100, 1 :200, 1 :300, 1 :400, 1 :500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:1250, 1:1500, 1: 1750, 1:2000, 1:2250, 1:2500, 1: 2750, 1:3000, 1:3250, 1:3500, 1: 3750, 1:4000, 1:4250, 1:4500, 1: 4750, 1:5000, 1:5250, 1:5500, 1: 5750, 1:6000, 1:6250, 1:6500, 1: 6750, 1:7000, 1:7250, 1:7500, 1: 7750, 1:8000, 1:8250, 1:8500, 1: 8750, 1:9000, 1:9250, 1
  • the antigen-specific invariant T cell activator is tolerogenic dendritic cells (DCs) expressing CD1, i.e. CD la, CD lb, CDlc and/or CD Id, and pulsed with at least one non peptide lipid antigen.
  • DCs dendritic cells
  • the tolerogenic DCs express CD Id.
  • tolerogenic DCs express on their surface the major histocompatibility (MHC) class la and/or MHC class lb.
  • MHC class la presentation refers to the "classical” presentation through HLA-A, HLA-B and/or HLA-C molecules whereas the MHC class lb presentation refers to the "non-classical" antigen presentation through HLA-E, HLA-F, HLA-G and/or HLA-H molecules.
  • tolerogenic DCs express 50% of MHC class la molecules and 50%> of MHC class lb molecules on their surface. In one embodiment, tolerogenic DCs express 45% of MHC class la molecules and 55% of MHC class lb molecules on their surface. In one embodiment, tolerogenic DCs express 40%> of MHC class la molecules and 60%> of MHC class lb molecules on their surface. In one embodiment, tolerogenic DCs express 35% of MHC class la molecules and 65% of MHC class lb molecules on their surface. In one embodiment, tolerogenic DCs express 30% of MHC class la molecules and 70%> of MHC class lb molecules on their surface.
  • tolerogenic DCs express 25% of MHC class la molecules and 75% of MHC class lb molecules on their surface. In one embodiment, tolerogenic DCs express 20%> of MHC class la molecules and 80%> of MHC class lb molecules on their surface. In one embodiment, tolerogenic DCs express 15% of MHC class la molecules and 85% of MHC class lb molecules on their surface. In one embodiment, tolerogenic DCs express 10%> of MHC class la molecules and 90%> of MHC class lb molecules on their surface. In one embodiment, tolerogenic DCs express 5% of MHC class la molecules and 95% of MHC class lb molecules on their surface. In one embodiment, tolerogenic DCs express only MHC class lb molecules on their surface.
  • tolerogenic DCs express 50% of HLA-A, HLA-B and/or HLA-C molecules and 50% of HLA-E molecules on their surface. In one embodiment, tolerogenic DCs express 45% of HLA-A, HLA-B and/or HLA-C molecules and 55% of HLA-E molecules on their surface. In one embodiment, tolerogenic DCs express 40% of HLA-A, HLA-B and/or HLA-C molecules and 60%> of HLA-E molecules on their surface. In one embodiment, tolerogenic DCs express 35% of HLA-A, HLA-B and/or HLA-C molecules and 65% of HLA-E molecules on their surface.
  • tolerogenic DCs express 30% of HLA-A, HLA-B and/or HLA-C molecules and 70% of HLA-E molecules on their surface. In one embodiment, tolerogenic DCs express 25% of HLA-A, HLA-B and/or HLA-C molecules and 75% of HLA-E molecules on their surface. In one embodiment, tolerogenic DCs express 20% of HLA-A, HLA-B and/or HLA-C molecules and 80% of HLA-E molecules on their surface. In one embodiment, tolerogenic DCs express 15% of HLA-A, HLA-B and/or HLA-C molecules and 85% of HLA-E molecules on their surface.
  • tolerogenic DCs express 10% of HLA-A, HLA-B and/or HLA-C molecules and 90% of HLA-E molecules on their surface. In one embodiment, tolerogenic DCs express 5% of HLA-A, HLA-B and/or HLA-C molecules and 95% of HLA-E molecules on their surface. In one embodiment, tolerogenic DCs express only HLA-E molecules on their surface.
  • the non peptide lipid antigen is a recombinant antigen a- galactosylceramide and analogs such as for example HS44 (a synthetic amino cyclitolic ceramide analogue in which the sugar head group is a carba cyclitol ring that mimics glucose instead of galactose, and which has the O-glycosidic linkage replaced with an amide group), a-GalCer analogs of the table 1 herein above, and homodimeric a- galactosylceramide analogs including the following:
  • R is C0 2 Me, C0 2 H or CH 2 OH
  • a-glucuronyl- and a-galacturonyl-ceramides and analogs iGb3 (Isoglobotriosylceramide); N-glycolyl (NGc) gangliosides such as for example NGcGM3; glycosphingo lipids or phosphoglycero lipids such as phosphatidylinositol, phosphatidylethanolamine, and phosphatidylglyerol presented preferably by CD 1 d.
  • the non peptide lipid antigen is derived from immunogenic apoptotic bodies from cancer cells or immunogenic blebs from cancer cells or derived from tissue lysate.
  • Cancer cells may derive from tumor biopsy or from expansion of circulatory cancer cells.
  • Immunogenic apoptotic bodies from cancer cells may be obtained for example with anthracyclines including, without limitation, doxorubicin, daunorubicin, idarubicin and mitoxanthrone; oxaliplatin, UVC or ⁇ -radiation treated cancer cells releasing apoptotic bodies or can be directly isolated from anthracyclines including doxorubicin, daunorubicin, idarubicin and mitoxanthrone; oxaliplatin; UVC or ⁇ -radiation treated cancer.
  • Blebs constitute an important immunogenic particle. They are heterogenous vesicle formed at the surface of apoptotic cells. In one embodiment, the size of blebs ranges from 0.05 - 5 um, preferably from 0.1 to 1 ⁇ .
  • Various Immunogenic cell death (ICD) inducers can induce the release of blebs from apoptotic or autophagic cells, such as, for example, irradiation at 5000 rad and several antineoplastic agents, including doxorubicin, oxaliplatin and cisplatin.
  • Immunogenic cancer cell blebs may, in particular, be obtained from apoptotic cancer cells or from cancer cell autophagy following treatment by chemical or physical inducers.
  • tissue lysate examples include, but are not limited to, synovial liquid or inflammatory tissue lysate.
  • tolerogenic DCs refers to DCs capable to induce tolerance.
  • tolerogenic DCs are capable of secreting more suppressive cytokines such as IL-10 and TGF than proinflammatory cytokines such as IL-12, IL-23 or TNFa.
  • DCs are defined as tolerogenic when they secrete IL-10 and IL-12 in a ratio IL-10: IL-12 > 1.
  • An exemplary method is the generation of tolerogenic DCs from CD14 + monocytes.
  • CD14 + monocytes are cultured in the presence of GM-CSF and IL-4, or in the presence of GM- CSF and IFNa, for the generation of immature DCs.
  • TAP transporter transporter associated with antigen processing
  • MHC class la molecules thereby promoting HLA-E molecules expression on the surface of tolerogenic DCs.
  • exemplary methods to inhibit the TAP transporter in the endoplasmic reticulum include, but are not limited to, CRISPR-CAS-9 technology, silencing RNA, transfected DCs with the UL-10 viral protein from the CMV (cytomegalovirus) or the use of viral proteins.
  • viral proteins able to inhibit the TAP transporter include, but are not limited to, HSV-1 ICP47 protein, varicella- virus UL49.5 protein, cytomegalovirus US6 protein or gammaherpesvirus EBV BNLF2a protein.
  • Another method is the use of a chemical product to inhibit the expression of MHC class la molecules without changing HLA-E expression on the surface of tolerogenic DCs.
  • chemical products include, but are not limited to, 5'- methyl-5 '- thioadenosine or leptomycin B.
  • CD la i.e. CD la, CD lb, CDlc and/or CD Id on the surface of the tolerogenic DCs
  • sulfatide can be used for the expression of CD la; rifampin and a number of its derivative (e.g., rifabutin) for the expression of CD lb; cholesteryl esters and acylated steryl glycosides for the expression of CDlc and rosiglitazone; retinoic acid; RARa agonist such as AM580, CD437, AM80, BMS961, NRX195183, All-trans-retinoic acid, 9-cis-Retinoic acid, Ch55, TTNPB (4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)- l-propenyl]benzoic acid), tamibaro
  • rosiglitazone can be used at a concentration ranging from 0.1 ⁇ to 10 ⁇ .
  • AM580 can be used at a concentration ranging from 1 nM to 10 ⁇ .
  • pioglitazone can be used at a concentration ranging from 1 nM to 10 ⁇ .
  • BMS961 can be used at a concentration ranging from 1 nM to 100 nM.
  • TTNPB can be used at a concentration ranging from 1 nM to 1 mM.
  • the cAMP activator added in the culture allows the activation of the cAMP pathway.
  • cAMP activators include, but are not limited to PGE2 (prostaglandin E2), an EP2 or EP4 agonist, a membrane adenine cyclase activator such as forskolin, or metabotropic glutamate receptors agonists.
  • PGE2 examples include, but are not limited to, PGE2 of ref P5640 or P0409 (Sigma- Aldrich), PGE2 of ref 2296 (R&D Systems), PGE2 of ref 2268 (Bio Vision), PGE2 of ref 72192 (Stemcell), PGE2 of ref abl44539 (Abeam), and PGE2 of ref 14010 (Cayman Chemical).
  • the cAMP activator preferably PGE2 is used at a concentration ranging from 0.01 ⁇ to 10 ⁇ .
  • the expression "from 0.01 ⁇ to 10 ⁇ " includes, without limitation, 0.02 ⁇ , 0.03 ⁇ , 0.04 ⁇ , 0.05 ⁇ , 0.06 ⁇ , 0.07 ⁇ , 0.08 ⁇ , 0.09 ⁇ , 0.1 ⁇ , 0.2 ⁇ , 0.3 ⁇ , 0.4 ⁇ , 0.5 ⁇ , 0.6 ⁇ , 0.7 ⁇ , 0.8 ⁇ , 0.9 ⁇ , 1 ⁇ , 1.5 ⁇ , 2 ⁇ , 2.5 ⁇ , 3 ⁇ , 3.5 ⁇ , 4 ⁇ , 4.5 ⁇ , 5 ⁇ , 6 ⁇ , 7 ⁇ , 8 ⁇ , 9 ⁇ .
  • PGE2 is at a concentration ranging from 0.03 ⁇ to 1.5 ⁇ .
  • the TGF pathway activator added in the culture allows the activation of the TGF pathway.
  • TGF pathway activators include, but are not limited to, TGF family (TGF i, TGF 2, TGF 3), bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), anti-mullerian hormone (AMH), activin, and nodal.
  • TGF examples include, but are not limited to, TGF 1 of ref T7039 (Sigma- Aldrich), TGF 2 of ref T2815 (Sigma- Aldrich), TGF 3 of ref T5425 (Sigma- Aldrich), human TGF l of ref P01137 (R&D system), human TGF l of ref 580702 (Biolegend), TGF l of ref HZ-1011 (HumanZyme), human TGF l of ref 14-8348-62 (Affymetrix eBioscience).
  • the pathway activator is used at a concentration ranging from 1 ng/ml to 20 ng/ml.
  • the expression "from 1 ng/ml to 20 ng/ml” includes, without limitation, 2 ng/ml, 2.5 ng/ml, 3 ng/ml, 3.5 ng/ml, 4 ng/ml, 4.5 ng/ml, 5 ng/ml, 5.5 ng/ml, 6 ng/ml, 6.5 ng/ml, 7 ng/ml, 7.5 ng/ml, 8 ng/ml, 8.5 ng/ml, 9 ng/ml, 9.5 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml, 15 ng/ml, 16 ng/ml, 17 ng/ml, 18 ng/ml, 19 ng/ml.
  • TGFP is at a concentration ranging from 2.5 ng/ml to 7.5 ng/ml.
  • the mTOR inhibitor added in the culture allows the inhibition of the mTOR pathway.
  • mTOR inhibitor include, but are not limited to, rapamycin (also named sirolimus) and its analogs (termed rapalogs); wortmannin; theophylline; caffeine; epigallocatechin gallate (EGCG); curcumin; resveratrol; genistein; 3, 3- diindolylmethane (DIM); LY294002 (2-(4-morpholinyl)-8-phenyl-4H-l-benzopyran-4- one); PP242; PP30; Torinl; Ku-0063794; WAY-600; WYE-687; WYE-354; and mTOR and PI3K dual-specificity inhibitors such as GNE477, NVP-BEZ235, PI- 103, X
  • rapamycin examples include, but are not limited to, rapamycin of ref R0395 (Sigma-Aldrich), rapamycin of ref S1039 (Selleckchem), rapamycin of ref 1292 (Tocris), rapamycin of ref R-5000 (LC Laboratories), rapamycin of ref tlrl-rap (InvivoGen), rapamycin of ref abl20224 (Abeam), rapamycin of ref R0395 (Sigma-Aldrich).
  • Examples of compounds of the same chemical class than rapamycin used clinically include, but are not limited to, Everolimus (code name RADOOl), Temsirolimus (code name CCI-779, NSC 683864), Zotarolimus (code name ABT-578).
  • the mTOR inhibitor preferably rapamycin
  • the expression "from 0.1 nM to 50 nM” includes, without limitation, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 21 nM, 22 nM, 23 nM, 24 nM, 25 nM, 26 nM, 27 nM, 28 nM, 29 nM
  • At least one cytokine selected from IL-2, IL-7, IL-15 and TSLP can be added in the culture.
  • IL-2 is used at a concentration ranging from 10 IU/ml to 1000 IU/ml.
  • the expression "from 10 IU/ml to 1000 IU/ml” includes, without limitation, 15 IU/ml, 20 IU/ml, 25 IU/ml, 30 IU/ml, 35 IU/ml, 40 IU/ml, 45 IU/ml, 50 IU/ml, 55 IU/ml, 60 IU/ml, 65 IU/ml, 70 IU/ml, 75 IU/ml, 80 IU/ml, 85 IU/ml, 90 IU/ml, 95 IU/ml, 100 IU/ml, 150 IU/ml, 200 IU/ml, 250 IU/ml, 300 IU/ml, 350 IU/ml, 400 IU/ml, 450 IU/ml, 500 IU/ml, 550 IU/ml, 600 IU/ml, 650 IU/ml, 700 IU/ml, 750 IU/ml, 800 IU/
  • IL-7 is used at a concentration ranging from 1 ng/ml to 100 ng/ml.
  • the expression "from 1 ng/ml to 100 ng/ml” includes, without limitation, 1 ng/ml, 5 ng/ml, 10 ng/ml, 15 ng/ml, 20 ng/ml, 25 ng/ml, 30 ng/ml, 35 ng/ml, 40 ng/ml, 45 ng/ml, 50 ng/ml, 55 ng/ml, 60 ng/ml, 65 ng/ml, 70 ng/ml, 75 ng/ml, 80 ng/ml, 85 ng/ml, 90 ng/ml, 95 ng/ml, 100 ng/ml.
  • IL-15 is used at a concentration ranging from 1 ng/ml to 50 ng/ml.
  • the expression "from 1 ng/ml to 50 ng/ml” includes, without limitation, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 15 ng/ml, 20 ng/ml, 25 ng/ml, 30 ng/ml, 35 ng/ml, 40 ng/ml, 45 ng/ml.
  • IL-15 is used at a concentration ranging from 10 ng/ml to 30 ng/ml.
  • TSLP is used at a concentration ranging from 1 ng/ml to 100 ng/ml.
  • the expression "from 1 ng/ml to 100 ng/ml” includes, without limitation, 1 ng/ml, 5 ng/ml, 10 ng/ml, 15 ng/ml, 20 ng/ml, 25 ng/ml, 30 ng/ml, 35 ng/ml, 40 ng/ml, 45 ng/ml, 50 ng/ml, 55 ng/ml, 60 ng/ml, 65 ng/ml, 70 ng/ml, 75 ng/ml, 80 ng/ml, 85 ng/ml, 90 ng/ml, 95 ng/ml, 100 ng/ml.
  • TET activators include but are not limited to vitamin C and NaHS hydrogen sulfide releasing agents (also known as H2S donors).
  • vitamin C is used at a concentration ranging from 1 to 100 ⁇ g/ml.
  • NaHS hydrogen sulfide releasing agent is used at a concentration ranging from 0.25 to 8 mM.
  • DNMT inhibitors include but are not limited to 2-(l,3-dioxo-l,2-dihydro- 2H-isoindol-2-yl)-3-(lH-indol- 3-yl) propanoic acid (RG108), 5-aza-2 , -deoxycytidine (decitabine or DAC) and 5-azacytidine (5 AC).
  • RG108 is used at a concentration ranging from 20 to 500 ⁇ . In one embodiment, DAC is used at a concentration ranging from 0.1 to 2 ⁇ .
  • 5 AC is used at a concentration ranging from 0.1 to 10 ⁇ .
  • neutralizing antibodies can be added to the culture to prevent the generation of other populations of regulatory T cells.
  • neutralizing antibodies include, but are not limited to, anti-IFNy, anti-IL-4, and/or anti-IL12 antibodies.
  • anti-IFNy antibodies examples include, but are not limited to, Affymetrix eBioscience (Ref 14-7318), R&D systems (Ref MAB285), Novus Biologicals (Ref AF-485-NA).
  • anti-IL-4 antibodies include, but are not limited to, R&D Systems (Ref MAB304, MAB204, or MAB204), Affymetrix eBioscience (Ref 14-7048), GeneTex (Ref GTX10755).
  • anti-IL-12 antibodies include, but are not limited to, Affymetrix eBioscience (Ref 16-7129 or 16-8126), Biolegend (Ref 508803), R&D systems (Ref MAB219, AF- 219, or AB-219).
  • the culture medium used in the culture of the invention comprises (i) one or more pH buffering system(s); (ii) inorganic salt(s); (iii) trace element(s); (vi) hormone(s); (vii) carbon/energy source(s).
  • inorganic salts include, but are not limited to, calcium bromide, calcium chloride, calcium phosphate, calcium nitrate, calcium nitrite, calcium sulphate, magnesium bromide, magnesium chloride, magnesium sulphate, potassium bicarbonate, potassium bromide, potassium chloride, potassium dihydrogen phosphate, potassium disulphate, di- potassium hydrogen phosphate, potassium nitrate, potassium nitrite, potassium sulphite, potassium sulphate, sodium bicarbonate, sodium bromide, sodium chloride, sodium disulphate, sodium hydrogen carbonate, sodium dihydrogen phosphate, di-sodium hydrogen phosphate, sodium sulphate and a mix thereof.
  • Trace elements include, but are not limited to cobalt (Co), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), molybdenum (Mo), nickel (Ni), selenium (Se), zinc (Zn) and the salts thereof.
  • free amino acids include, but are not limited to, L-alanine, L-arginine, L- asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, taurine, L-threonine, L-tryptophan, L-tyrosine, L-valine and a mix thereof.
  • vitamins include, but are not limited to, biotin (vitamin H); D-calcium- pantothenate; choline chloride; folic acid (vitamin B9); myo-inositol; nicotinamide; pyridoxal (vitamin B6); riboflavin (vitamin B2); thiamine (vitamin Bl); cobalamin (vitamin B 12); acid ascorbic; a-tocopherol (vitamin E) and a mix thereof.
  • biotin vitamin H
  • D-calcium- pantothenate choline chloride
  • folic acid vitamin B9
  • myo-inositol nicotinamide
  • pyridoxal vitamin B6
  • riboflavin vitamin B2
  • thiamine vitamin Bl
  • cobalamin vitamin B 12
  • acid ascorbic a-tocopherol (vitamin E) and a mix thereof.
  • carbon/energy sources include, but are not limited to D-glucose; pyruvate; lactate; ATP; creatine; creatine phosphate; and a mix thereof.
  • the culture medium is a commercially available cell culture medium, in particular selected in a group comprising the IMDM (Iscove's Modified Dulbecco's Medium) from GIBCO® or the RPMI 1640 medium from GIBCO®.
  • IMDM Iscove's Modified Dulbecco's Medium
  • the culture medium is a serum-free culture medium such as the AIM-V medium from GIBCO®, the X-VIVO 10, 15 and 20 media from LONZA.
  • the culture medium can be further supplemented with additional compound(s), in particular selected in a group comprising foetal bovine serum, pooled human AB serum, cytokines and growth factors; antibiotic(s), in particular selected in a group comprising penicillin, streptomycin and a mix thereof.
  • additional compound(s) in particular selected in a group comprising foetal bovine serum, pooled human AB serum, cytokines and growth factors; antibiotic(s), in particular selected in a group comprising penicillin, streptomycin and a mix thereof.
  • the culture medium is IMDM.
  • the culture medium comprises IMDM cell culture medium; from 1 % (w/w) to 5% (w/w) of foetal bovine serum; from 10 IU/ml to 200 IU/ml of penicillin; from 10 IU/ml to 200 IU/ml of streptomycin; from 0.1 mM to 10 mM of a mixture of non-essential amino acids, in particular amino acids selected in a group comprising alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, and tyrosine; from 0.5 mM to 10 mM of glutamine from 10 mM to 25 mM of HEPES pH 7.6-7.8.
  • the medium is a nTreg polarizing medium.
  • the inventors define a "nTreg polarizing medium" as a medium such as RPMI medium comprising at least one cAMP activator as described hereabove, at least one TGF pathway activator as described here above and at least one mTor inhibitor as described hereabove.
  • the "nTreg polarizing medium” refers to a RPMI medium comprising TGF , rapamycin and PGE2.
  • the medium is an inflammatory medium.
  • the inventors define an "inflammatory medium" as a medium such as IMDM comprising inflammatory cytokines such as for example IL- ⁇ (10 ng/ml), IL-6 (30 ng/ml), IL-21 (50 ng/ml), IL- 23 (30 ng/ml), IL-2 (100 Ul/ml).
  • the culture for generating the invariant Foxp3 + regulatory T cells of the invention is performed during at least 5 days, at least 6 days, at least 7 days, at least 8 days.
  • the expression "at least 5 days” includes, without limitation, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days.
  • a portion of the culture medium is discarded once, twice, three times, four times or five times during the time course of the generation culture and replaced with the same volume of fresh culture medium.
  • portion is intended to mean at least 20% (v/v), at least 25% (v/v), at least 30%> (v/v), at least 35% (v/v), at least 40% (v/v), at least 45% (v/v), at least 50% (v/v), at least 55% (v/v), at least 60% (v/v), at least 65% (v/v), at least 70% (v/v), at least 75% (v/v) of the volume of the culture medium.
  • fresh culture medium refers to a culture medium that has not been in contact with any CD3+ T cells.
  • the method for generating ex vivo invariant Foxp3 + regulatory T cells comprises: - culturing CD3 + Va24 + T cells, preferably CD3 + Va24 + CD45RA+ T cells, in the presence of ACD3 feeder cells and coated 6B11 mAb and the following agents: i) PGE2, ii) TGFp, iii) rapamycin, optionally iv) IL-2 and/or IL-15, and optionally v) vitamin C for at least 5 days,
  • invariant Foxp3 + regulatory T cells ex vivo generated, preferably from invariant naive (CD45RA + ) T cells.
  • the method for generating ex vivo invariant Foxp3 + regulatory T cells comprises:
  • CD3 + Va24 + T cells preferably CD3 + Va24 + CD45RA+ T cells
  • tolerogenic DCs expressing CD Id incubation with AM580 during about 24h
  • aGalactosyleramide in the presence of ACD3 feeder cells and the following agents: i) PGE2, ii) TGFp, iii) rapamycin, optionally iv) IL-2 and/or IL-15, and optionally v) vitamin C, for at least 5 days, thereby obtaining a population of invariant Foxp3 + regulatory T cells ex vivo generated, preferably from invariant naive (CD45RA + ) T cells.
  • the present invention also relates to an ex vivo method of generation and expansion of invariant Foxp3 + regulatory T cells, comprising:
  • invariant Foxp3 + regulatory T cells as described here above, expanding the invariant Foxp3 + regulatory T cells generated by contacting them with an invariant T cell activator (preferably either ACD3 feeder cells and coated
  • 6B11 mAb or tolerogenic DCs expressing CD Id (incubation with AM580 during about 24h) and pulsed with aGalactosyleramide in the presence of ACD3 feeder cells) in the presence of the following agents: i) an cAMP (Cyclic adenosine monophosphate) activator (preferably PGE2), ii) a TGFP (Transforming growth factor beta) pathway activator (preferably TGFP), iii) a mTOR inhibitor
  • cytokine selected in the group of IL-2, IL-7, IL-15 and TSLP (preferably IL-2 and/or IL-15), and optionally v) at least one TET enzymes activator (preferably selected from vitamin C and a NaHS hydrogen sulfide releasing agent) and/or at least one DNMT inhibitor (such as, for example, RG108, DAC or 5AC), for at least 5 days, thereby obtaining an expanded population of invariant Foxp3 + regulatory T cells.
  • TET enzymes activator preferably selected from vitamin C and a NaHS hydrogen sulfide releasing agent
  • DNMT inhibitor such as, for example, RG108, DAC or 5AC
  • the invariant Foxp3 + regulatory T cell population generated ex vivo is isolated by flow cytometry based on the following phenotype: CD3 + Va24 + CD45RO + Foxp3 + .
  • the isolated invariant Foxp3 + regulatory T cell population thus obtained is then expanded ex vivo by culturing these cells in the presence of a polyclonal T cell activator.
  • polyclonal T cell activator examples include, but are not limited to, mitogen such as PMA/ionomycin, super- antigen, anti-CD3 antibody...
  • the anti-CD3 monoclonal antibody is coated.
  • the polyclonal T cell activator can be used in the presence of feeder cells as described here above.
  • the isolated invariant Foxp3 + regulatory T cell population thus obtained is then expanded ex vivo by culturing these cells in the presence of antigen- specific invariant T cell activator as described here above.
  • the antigen-specific T cell activator can be used in the presence of feeder cells as described here above.
  • the culture for expanding the ex vivo generated invariant Foxp3 + regulatory T cells of the invention is performed during at least 5 days, at least 6 days, at least 7 days, at least 8 days.
  • the expression "at least 5 days” includes, without limitation, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days.
  • a portion of the culture medium is discarded once, twice, three times, four times or five times during the time course of the generation culture and replaced with the same volume of fresh culture medium.
  • portion is intended to mean at least 20% (v/v), at least 25% (v/v), at least 30%> (v/v), at least 35% (v/v), at least 40% (v/v), at least 45% (v/v), at least 50% (v/v), at least 55% (v/v), at least 60% (v/v), at least 65% (v/v), at least 70% (v/v), at least 75% (v/v) of the volume of the culture medium.
  • fresh culture medium refers to a culture medium that has not been in contact with any CD3+ T cells.
  • invariant Foxp3 + regulatory T cells are generated ex vivo by culturing CD3 + Va24 + CD45RA + T cells obtained from PBMCs by flow cytometry (5.10 3 cells/ml) in the presence of autologous ACD3 feeder cells (125 10 5 cells/ml) and coated 6B11 mAb (2 ⁇ g/ml) in the presence of PGE2 (1 ⁇ ), TGF (5 ng/ml), Rapamycin (10 nM) and IL- 2 (100 Ul/ml) in IMDM-5. On day 1, IL-2 (100 Ul/ml) and IL-15 (10 ng/ml) are added to the culture.
  • invariant Foxp3 + regulatory T cells are generated ex vivo by culturing CD3 + Va24 + CD45RA + T cells (5.10 3 cells/ml) obtained from PBMCs by flow cytometry (5.10 3 cells/ml) in the presence of tolerogenic DCs expressing CD Id (incubation with AM580 during about 24h) and pulsed with aGalactosyleramide and in the presence of ACD3 feeder cells (125 10 5 cells/ml), PGE2 (1 ⁇ ), TGF (5 ng/ml), Rapamycin (10 nM) and IL-2 (100 Ul/ml) in IMDM-5.
  • CD3 + Va24 + CD45RA + T cells 5.10 3 cells/ml obtained from PBMCs by flow cytometry (5.10 3 cells/ml) in the presence of tolerogenic DCs expressing CD Id (incubation with AM580 during about 24h) and pulsed with aGalactosyleramide and in the presence of ACD3
  • IL-2 100 Ul/ml
  • IL- 15 10 ng/ml
  • TGF 5 ng/ml
  • PGE2 50 nM
  • TGF 5 ng/ml
  • Rapamycin 1 nM
  • IL-2 100 Ul/ml
  • IL-15 10 ng/ml
  • cells Once cells begin to expand, they can be split every 2 or 3 days and restimulated every 9 days with tolerogenic DCs pulsed with aGalactosylceramide in the presence of ACD3 feeder cells and PGE2 (1 ⁇ ), TGF (5 ng/ml), Rapamycin (10 nM) and IL-2 (100 Ul/ml).
  • tolerogenic DCs were obtained by culturing CD14 + monocytes isolated from PBMCs in the presence of AIMV supplemented with GMCSF (100 ng/ml), IL-4 (10 ng/ml) and AM580 (100 nM). At day 3 and 6, the medium is discarded and replaced by fresh medium comprising GM-CSF and IL-4. On day 6, the tolerogenic DCs are pulsed for 24 hours in the presence of aGalactosylceramide (100 ng/ml) and AM580 (100 nM).
  • the present invention also relates to invariant Foxp3 + regulatory T cells obtainable by the ex vivo generation method as described here above.
  • the present invention also relates to invariant Foxp3 + regulatory T cells obtainable by the ex vivo generation and expansion method as described here above.
  • the population of invariant Foxp3 + regulatory T cells obtained by the generation and expansion method of the invention comprises at least 10 6 , 10 7 , 10 8 , 10 9 , 10 10 cells.
  • the population of invariant Foxp3 + regulatory T cells obtained by the generation and expansion method of the invention has a phenotype similar to the phenotype of isolated invariant Foxp3 + regulatory T cells as described here above.
  • the population of invariant Foxp3 + regulatory T cells obtained by the generation and expansion method of the invention has the property to remain functionally stable when placed in inflammatory condition.
  • IL-17 IL-17
  • IL-17 IL-17
  • inflammatory condition refers to a medium enriched in aromatic acid, preferably in tryptophan, such as for example IMDM, comprising inflammatory cytokines such as for example IL- ⁇ (10 ng/ml), IL-6 (30 ng/ml), IL-21 (50 ng/ml), IL-23 (30 ng/ml), IL-2 (100 Ul/ml).
  • a method for determining if a population of regulatory T cells remains functionally stable in inflammatory condition comprises culturing the regulatory T cells in the inflammatory condition medium as described here above in the presence of anti-CD3 (4 ⁇ g/ml), preferably coated, and anti-CD28 (4 ⁇ g/ml), preferably in a soluble form.
  • IL-17 in the culture supernatant is measured.
  • the recognition of IL-17 in the culture supernatant may be carried out by conventional methods known in the art such as, for example, a sandwich ELISA anti-IL- 17. Briefly, after coated the plate with a capture anti-IL-17 antibody, the culture supernatant is added to each well with a dilution series. After incubation, a detection anti- IL-17 antibody is added to each well.
  • the ELISA is developed by any colorimetric means known in the art such as, for example, using detection antibody labelled with biotin, a poly-streptavidin HRP amplification system and an o-phenylenediamine dihydrochloride substrate solution.
  • An IL-17 level inferior to 200 ng/ml, 100 ng/ml, 50 ng/ml corresponds to no secretion or low secretion of IL-17.
  • the inventors state that the stroma of malignant tumor cells comprises TILs (Tumor-infiltrating lymphocytes) that are highly enriched in regulatory T cells and that exert an immune suppressive activity, in particular on NK cells, which likely accounts for the local cancer immune escape.
  • TILs Tumor-infiltrating lymphocytes
  • the inactivated invariant Foxp3 + regulatory T cells may represent an antigenic target to induce an immune response directed against the invariant Foxp3 + regulatory T cells present in the TILs, thereby preventing their immune suppressive activity and allowing the cytotoxic activity of effector cells such as NK cells against the tumor cells.
  • the inventors thus suggest using a vaccine composition comprising inactivated invariant Foxp3 + regulatory T cells as active principle.
  • One object of the invention is an immunogenic product comprising inactivated invariant Foxp3 + regulatory T cells as described here above.
  • the immunogenic product comprises, consists essentially of or consists of inactivated invariant Foxp3 + regulatory T cells having the following phenotype CD3 + Va24 + Foxp3 + , as described hereinabove.
  • the term "consisting essentially of, with reference to a pharmaceutical composition, vaccine, immunogenic product or medicament, means that the at least one invariant Foxp3 + regulatory T cell population or antibody of the invention is the only one therapeutic agent or agent with a biologic activity within said pharmaceutical composition, vaccine, immunogenic product or medicament.
  • the immunogenic product comprises, consists essentially of or consists of inactivated invariant Foxp3 + regulatory T cells having the following phenotype CD3 + Va24 + Foxp3 + generated and optionally expanded ex vivo by the method as described here above.
  • One object of the invention is an immunogenic product comprising blebs from invariant Foxp3 + regulatory T cells as described here above.
  • the immunogenic product comprises, consists essentially of or consists of blebs of invariant Foxp3 + regulatory T cells having the following phenotype CD3 + Va24 + Foxp3 + , as described hereinabove.
  • One object of the invention is an immunogenic product comprising immunogenic dendritic cells (immunogenic DC) loaded with blebs from invariant Foxp3 + regulatory T cells as described here above.
  • immunogenic dendritic cells immunogenic DC
  • the immunogenic product comprises, consists essentially of or consists of immunogenic dendritic cells (immunogenic DC) loaded with blebs of invariant Foxp3 + regulatory T cells having the following phenotype CD3 + Va24 + Foxp3 + , as described hereinabove.
  • immunogenic dendritic cells immunogenic DC
  • blebs of invariant Foxp3 + regulatory T cells having the following phenotype CD3 + Va24 + Foxp3 + , as described hereinabove.
  • immunogenic DCs refers to DCs capable to induce an immunogenic response.
  • immunogenic DCs have the following phenotype: CD80 hi g h CD83 high CD86 high HLA class jhigh and HLA class jjhigh and secrete IL _ 10 and
  • IL-12 with a ratio IL-12/IL-10 >1.
  • An exemplary method the generation of immunogenic DCs from CD14 + monocytes.
  • CD14 + monocytes are cultured in the presence of GM-CSF (about 20 ng/mL) and IFNa (about 10 ng/mL) for obtaining MoDC.
  • Maturation of MoDC may be induced by a cytokine cocktail consisting of IL- ⁇ (about 10 ng/ml), IL-6 (about 10 ng/ml) TNFa (about 200 U/ml), and PGE2 (about 10 ng/ml).
  • Another object of the invention is a pharmaceutical composition comprising, consisting essentially of or consisting of the immunogenic product as described here above and at least one pharmaceutically acceptable excipient.
  • Another object of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising, consisting essentially of or consisting of inactivated invariant Foxp3 + regulatory T cells having the following phenotype CD3 + Va24 + Foxp3 + and at least one pharmaceutically acceptable excipient.
  • Another object of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising, consisting essentially of or consisting of inactivated invariant Foxp3 + regulatory T cells having the following phenotype CD3 + Va24 + Foxp3 + generated and expanded ex vivo by the method as described here above and at least one pharmaceutically acceptable excipient.
  • Another object of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising, consisting essentially of or consisting of blebs of invariant Foxp3 + regulatory T cells having the following phenotype CD3 + Va24 + Foxp3 + as described here above and at least one pharmaceutically acceptable excipient.
  • Another object of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising, consisting essentially of or consisting of immunogenic DC loaded with blebs of invariant Foxp3 + regulatory T cells having the following phenotype CD3 + Va24 + Foxp3 + as described here above and at least one pharmaceutically acceptable excipient.
  • excipient refers to any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by regulatory offices, such as, for example, FDA Office or EMA.
  • pharmaceutically acceptable is meant that the ingredients of a pharmaceutical composition are compatible with each other and not deleterious to the subject to which it is administered.
  • compositions comprising, consisting essentially of or consisting of the immunogenic product as described here above.
  • Another object of the invention is a vaccine composition comprising, consisting essentially of or consisting of inactivated invariant Foxp3 + regulatory T cells having the following phenotype CD3 + Va24 + Foxp3 + .
  • Another object of the invention is a vaccine composition comprising, consisting essentially of or consisting of inactivated invariant Foxp3 + regulatory T cells having the following phenotype CD3 + Va24 + Foxp3 + generated and expanded ex vivo by the method as described here above.
  • Another object of the invention is a vaccine composition
  • a vaccine composition comprising, consisting essentially of or consisting of blebs of invariant Foxp3 + regulatory T cells having the following phenotype CD3 + Va24 + Foxp3 + as described here above.
  • Another object of the invention is a vaccine composition
  • a vaccine composition comprising, consisting essentially of or consisting of immunogenic DC loaded with blebs of invariant Foxp3 + regulatory T cells having the following phenotype CD3 + Va24 + Foxp3 + as described here above.
  • inactivated T cells refers to T cells that are viable but has reduced or no effector function, i.e. have lost any pathogenic potential.
  • cell surface markers of inactivated T cells include, but are not limited to, 7-Aminoactinomycin D (7- AAD), calreticulin and heat shock protein 90 (HSP-90). Therefore, inactivated T cells express 7-AAD and/or calreticulin and/or HSP-90.
  • the inactivated invariant Foxp3 + regulatory T cells of the invention have lost their suppressive activity but are still immunogenic.
  • An example of T cell effector function assay is, but not limited to, T-cell proliferation assay. T-cell proliferation may be assessed on fixed T cells versus non- fixed T cells.
  • the T-cell proliferation assay aims at determining the percentage of living proliferating cells in fixed versus non- fixed T cells by flow cytometry. After staining the T cells with CFSE, anti-CD3 antibody and 7-AAD, the living proliferating cells are defined as the CFSE low fraction in gated CD3 + 7-AAD " cells.
  • the invariant Foxp3 + regulatory T cells are inactivated by any method well-known in the art.
  • methods for inactivating cells include, but are not limited to, irradiation, preferably with about 2500 to 3000 rads and/or chemical inactivation such as exposure to cisplatin, carboplatin, oxaliplatin, mitomycine C or antracycline.
  • the vaccine composition of the invention further comprises at least one adjuvant.
  • adjuvant that can be used in the vaccine composition include, but are not limited to, ISA51; emulsions such as CFA, MF59, montanide, AS03 and AF03; mineral salts such as alum, calcium phosphate, iron salt, zirconium salt, and AS04; TLR ligands such as TLR2 ligands (such as outer-surface protein A or OspA), TLR3 ligands (such as poly I:C), TLR4 ligands (such as MPL and GLA), TLR5 ligands, TLR7/8 ligands (such as imiquimod), TLR9 ligands (such as CpG ODN); polysacharrides such as chitin, chitosan, a-glucans, ⁇ -glucans, fructans, mannans, dextrans, lentinans, inulin- based adjuvants (such as TLR
  • the inactivated invariant Foxp3 + regulatory T cells are specific of at least one non-peptide lipid antigens presented by CDl molecules as described here above. In another embodiment, the inactivated invariant Foxp3 + regulatory T cells are specific of at least one non-peptide lipid antigens presented by CDl molecules that were present on apoptotic bodies of cancer cells.
  • the inactivated invariant Foxp3 + regulatory T cells are specific of at least one non-peptide lipid antigens presented by CDl molecules that were present on blebs of cancer cells.
  • the inactivated invariant Foxp3 + regulatory T cells present in the immunogenic product, pharmaceutical composition or vaccine composition of the invention are human invariant Foxp3 + regulatory T cells.
  • the inactivated invariant Foxp3 + regulatory T cells present in the immunogenic product, pharmaceutical composition or vaccine composition of the invention are autologous invariant Foxp3 + regulatory T cells.
  • the inactivated invariant Foxp3 + regulatory T cells present in the immunogenic product, pharmaceutical composition or vaccine composition of the invention are allogenic invariant Foxp3 + regulatory T cells.
  • the immunogenic product, pharmaceutical composition or vaccine composition of the invention may be personalized for a patient.
  • a "personalized" immunogenic product or vaccine composition refers to the use of invariant Foxp3 + regulatory T cells generated and expanded ex vivo with at least one patient specific epitope.
  • the invariant Foxp3 + regulatory T cells to be used as immunogenic product or in the vaccine composition are generated and expanded ex vivo in the presence of apoptotic bodies of cancer cells or of blebs obtained from the patient, thereby providing at least one patient specific epitope.
  • the immunogenic product, pharmaceutical composition or vaccine composition of the invention comprises, consists essentially of or consists of inactivated invariant Foxp3 + regulatory T cells as active principle.
  • the immunogenic product, pharmaceutical composition or vaccine composition of the invention comprises, consists essentially of or consists of at least 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 inactivated invariant Foxp3 + regulatory T cells as active principle.
  • the immunogenic product, pharmaceutical composition or vaccine composition of the invention comprises, consists essentially of or consists of about 10 4 , 5xl0 4 , 10 5 , 5xl0 5 , 10 6 , 5xl0 6 , 10 7 , 5xl0 7 , 10 8 , 5xl0 8 , 10 9 , 5xl0 9 , 10 10 , inactivated invariant Foxp3 + regulatory T cells as active principle.
  • the invariant Foxp3 + regulatory T cells, the inactivated invariant Foxp3 + regulatory T cells, the immunogenic product, the pharmaceutical composition or the vaccine composition of the invention are/is frozen.
  • the immunogenic product, pharmaceutical composition or vaccine composition of the invention may be administrated to the subject by subcutaneaous, intramuscular, intraperitoneal or intravenous injection, or directly into the tumor.
  • the immunogenic product, pharmaceutical composition or vaccine composition of the invention may be administrated to the subject at least once, twice, 3 times, 4 times, 5 times in a year.
  • Example of regime of administration includes, but is not limited to, administration of the immunogenic product or vaccine composition at day 0, 4 weeks after day 0, 8 weeks after day 0, 12 weeks after day 0 and 24 weeks after day 0.
  • Another object of the invention is a method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of inactivated invariant Foxp3 + regulatory T cells or of the immunogenic product, pharmaceutical composition or vaccine composition of the invention as described here above.
  • Another object of the invention is a method for eliciting an immune response against invariant Foxp3 + regulatory T cells present in the TILs of a subject affected with a cancer, comprising administering to the subject a therapeutically effective amount of inactivated invariant Foxp3 + regulatory T cells or of the immunogenic product, pharmaceutical composition or vaccine composition of the invention as described here above.
  • cancers that can be treated with the immunogenic product, pharmaceutical composition or vaccine composition of the invention include, but are not limited to, adrenocortical carcinoma, anal cancer, bladder cancer, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal, pineal tumors, hypothalamic glioma, breast cancer, carcinoid tumor, carcinoma, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, ewings family of tumors (pnet), extracranial germ cell tumor, eye cancer, intraocular melanoma, gallbladder cancer, gastric cancer, germ cell tumor, extragonadal, gestational trophoblastic tumor, head and neck cancer, hypopharyngeal cancer, islet cell carcinoma, laryngeal cancer, leukemia, acute lymphoblastic leukemia, oral cavity cancer, liver cancer, lung cancer, small cell lymphoma, AIDS-related, lympho
  • the cancer that can be treated with the immunogenic product, pharmaceutical composition or vaccine composition of the invention include, but is not limited to, breast cancer, prostate cancer, ovarian cancer and glioblastoma.
  • Another object of the invention is a method for preparing the immunogenic product of the invention, comprising:
  • a biological sample preferably a blood sample, from the subject to be treated and optionally a tumor sample, from the subject to be treated
  • CD3 + Va24 + CD45RA+ T cells isolated from the biological sample
  • the generation and expansion steps are carried out in the presence of tolerogenic dendritic cells (DCs) expressing CD1, i.e. CD la, CD lb, CDlc and/or CD Id, and pulsed with apoptotic tumor bodies or blebs obtained from the tumor sample of the subject.
  • DCs tolerogenic dendritic cells
  • Another object of the invention is a method for treating cancer in a subject in need thereof, comprising administrating to the subject the immunogenic product, pharmaceutical composition or vaccine composition of the invention.
  • Another object of the invention is a method for treating cancer in a subject in need thereof, comprising:
  • the inventors suggest that the invariant Foxp3 + regulatory T cells of the invention, which are committed to exert immune suppressive function, may be capable of inhibiting autoreactive pathogenic immune effector cells including CD4+, CD8+, B cells or innate NK cells, which, in turn, are no longer able to exert their cytotoxic properties towards the self-cells.
  • One object of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising, consisting essentially of or consisting of the invariant Foxp3 + regulatory T cells or invariant Foxp3 + regulatory T cell population as described here above and at least one pharmaceutically acceptable excipient.
  • Another object of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising, consisting essentially of or consisting of invariant Foxp3 + regulatory T cells having the following phenotype CD3 + Va24 + Foxp3 + and at least one pharmaceutically acceptable excipient.
  • Another object of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising, consisting essentially of or consisting of invariant Foxp3 + regulatory T cells having the following phenotype CD3 + Va24 + Foxp3 + generated and expanded ex vivo by the method as described here above and at least one pharmaceutically acceptable excipient.
  • One object of the invention is the invariant Foxp3 + regulatory T cells or the invariant Foxp3 + regulatory T cell population or the pharmaceutical composition as described here above for use in adoptive therapy.
  • Another object of the invention is the invariant Foxp3 + regulatory T cells or the invariant Foxp3 + regulatory T cell population or the pharmaceutical composition as described here above for use in treating inflammatory or autoimmune diseases.
  • inflammatory or autoimmune diseases include, but are not limited to, acute disseminated encephalomyelitis, acute necrotizing haemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, antiphospho lipid syndrome, autoimmune angioedema, autoimmune aplastic anaemia, autoimmune dysautonomia, autoimmune haemo lytic anaemia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease, autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura, autoimmune thyroid disease, autoimmune urticaria, axonal and neuronal neuropathies, Balo disease, Behcet's disease,
  • inflammatory or autoimmune diseases include, but are not limited to, rheumatoid arthritis, type 1 diabetes, and multiple sclerosis.
  • Another object of the invention is the invariant Foxp3 + regulatory T cells or the invariant Foxp3 + regulatory T cell population or the pharmaceutical composition as described here above for use in preventing transplant rejection or graft versus host disease (GVHD).
  • GVHD transplant rejection or graft versus host disease
  • the invariant Foxp3 + regulatory T cells are specific of at least one non-peptide lipid antigens presented by CD1 molecules as described here above.
  • the invariant Foxp3 + regulatory T cells are specific of at least one non-peptide lipid antigens presented by CD1 molecules that were present in tissue lysates.
  • tissue lysate examples include, but are not limited to, synovial liquid, Langerhans islets lysate, beta islets lysate...
  • the pharmaceutical composition of the invention comprises, consists essentially of or consists of at least 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 invariant Foxp3 + regulatory T cells as active principle.
  • the pharmaceutical composition of the invention comprises, consists essentially of or consists of about 10 4 , 5xl0 4 , 10 5 , 5xl0 5 , 10 6 , 5xl0 6 , 10 7 , 5xl0 7 , 10 8 , 5xl0 8 , 10 9 , 5xl0 9 , 10 10 invariant Foxp3 + regulatory T cells as active principle.
  • the invariant Foxp3 + regulatory T cells, the invariant Foxp3 + regulatory T cell population or the pharmaceutical the invention are/is frozen.
  • the invariant Foxp3 + regulatory T cells present in the pharmaceutical composition of the invention are human invariant Foxp3 + regulatory T cells.
  • the invariant Foxp3 + regulatory T cells present in the pharmaceutical composition of the invention are autologous invariant Foxp3 + regulatory T cells. In one embodiment, the invariant Foxp3 + regulatory T cells present in the pharmaceutical composition of the invention are allogenic invariant Foxp3 + regulatory T cells.
  • the pharmaceutical composition of the invention may be administrated to the subject by subcutaneaous, intramuscular, intraperitoneal or intravenous injection.
  • the pharmaceutical composition of the invention may be administrated to the subject at least once, twice, 3 times, 4 times, 5 times per week.
  • the pharmaceutical composition of the invention may be administrated to the subject at least once, twice, 3 times, 4 times, 5 times per month. In another embodiment, the pharmaceutical composition of the invention may be administrated to the subject at least once, twice, 3 times, 4 times, 5 times per 3 months.
  • Another object of the invention is a method for treating inflammatory or autoimmune diseases in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the invariant Foxp3 + regulatory T cells or the invariant Foxp3 + regulatory T cell population or the pharmaceutical composition as described here above.
  • T cell vaccination induces regulatory networks that specifically suppress the immunogenic T cells by activating T cells specific for a clono type-specific determinant (anti- idiotypic response).
  • anti-ergotypic responses directed at activation markers may also partially account for the suppression of the regulatory T cell population targeted.
  • Another object of the invention is an antibody recognizing the TCR (T cell receptor) of the invariant Foxp3 + regulatory T cells of the invention.
  • the antibody recognizing the TCR of the invariant Foxp3 + regulatory T cells of the invention recognizes at least one of the CDR1, CDR2 and CDR3 (complementary determining region 1, 2 and 3) of the TCR.
  • the antibody recognizing the TCR of the invariant Foxp3 + regulatory T cells of the invention recognizes the CDR3 of the TCR.
  • Another object of the invention is a pharmaceutical composition comprising, consisting essentially of or consisting of said antibody and at least one pharmaceutically acceptable excipient.
  • Another object of the invention is the use of said antibody for treating cancer in a subject in need thereof.
  • the antibodies directed against the invariant Foxp3 + regulatory T cells of the invention consist of antibodies produced following immunization of a mammal, including a human, with the immunogenic composition as described here above.
  • the antibodies may also be obtained by cloning the relevant DNA material encoding them, starting for example from B cells obtained from the said mammal, including from the said human.
  • the antibodies may also be obtained by sequencing the amino acid sequences of the antibodies collected from the said mammal, including from the said human, and then synthesize a DNA molecule encoding the antibody or a portion thereof comprising the CDR thereof, for producing relevant recombinant antibodies directed against the invariant Foxp3 + regulatory T cells of the invention.
  • Preparing antibodies directed against the invariant Foxp3 + regulatory T cells of the invention by immunization with the immunogenic composition of the invention may be easily performed by a skilled in the art, using the common technical knowledge from the state in the art.
  • the antibodies directed against the invariant Foxp3 + regulatory T cells of the invention may be obtained after immortalization of the human B lymphocytes producing them; their cDNA can also be cloned and used further for producing them or their derivatives through recombinant DAN technology.
  • antibody herein is used to refer to a molecule having a useful antigen binding specificity. Those skilled in the art will readily appreciate that this term may also cover polypeptides which are fragments of or derivatives of antibodies yet which can show the same or a closely similar functionality. Such antibody fragments or derivatives are intended to be encompassed by the term antibody as used herein.
  • antibody or “antibody molecule” for the purpose of passive immunotherapy, it is intended herein not only whole immunoglobulin molecules but also fragments thereof, such as Fab, F(ab')2, Fv and other fragments thereof that retain the capacity to bind and inactivate the invariant Foxp3 + regulatory T cells.
  • the term antibody includes genetically engineered derivatives of antibodies such as single chain Fv molecules (scFv) and domain antibodies (dAbs).
  • an antibody directed against the invariant Foxp3 + regulatory T cells of the invention consists of a polyclonal antibody.
  • an antibody directed against the invariant Foxp3 + regulatory T cells of the invention consists of a monoclonal antibody.
  • monoclonal antibody is used herein to encompass any isolated Ab's such as conventional monoclonal antibody hybridomas, but also to encompass isolated monospecific antibodies produced by any cell, such as for example a sample of identical human immunoglobulins expressed in a mammalian cell line.
  • variable heavy (VH) and variable light (VL) domains of the antibody are involved in antigen recognition, a fact first recognized by early protease digestion experiments. Further confirmation was found by "humanization" of rodent antibodies. Variable domains of rodent origin may be fused to constant domains of human origin such that the resultant antibody retains the antigenic specificity of the rodent parented antibody (Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81, 6851-6855). That antigenic specificity is conferred by variable domains and is independent of the constant domains is known from experiments involving the bacterial expression of antibody fragments, all containing one or more variable domains.
  • ScFv molecules encompasses molecules wherein the VH and VL partner domains are linked via a flexible oligopeptide.
  • Engineered antibodies, such as ScFv antibodies can be made using the techniques and approaches described in J. Huston et al, (1988) "Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single chain Fv analogue produced in E. coli", Proc. Natl. Acad. Sci. USA, 85, pp. 5879-5883, and in A. Pluckthun, (1991) "Antibody engineering; Advances from use of E. coli expression systems", Bio/technology 9 (6): 545-51, incorporated herein by reference.
  • Suitable monoclonal antibodies which are reactive as described herein may be prepared by known techniques, for example those disclosed in “Monoclonal Antibodies; A manual of techniques", H Zola (CRC Press, 1988) and in “Monoclonal Hybridoma Antibodies: Techniques and Application", S G R Hurrell (CRC Press, 1982).
  • a further embodiment encompasses humanized antibodies where the regions of the murine antibody that contacted the antigen, the Complementarity Determining Regions (CDRs) were transferred to a human antibody framework.
  • CDRs Complementarity Determining Regions
  • Such antibodies are almost completely human and seldom cause any harmful antibody responses when administered to patients.
  • Several chimeric or humanized antibodies have been registered as therapeutic drugs and are now widely used within various indications (Borrebaeck & Carlsson, 2001, Curr. Opin. Pharmacol. 1 : 404-408).
  • the antibody is a humanized antibody.
  • Suitably prepared non-human antibodies can be "humanized” in known ways, for example by inserting the CDR regions of mouse antibodies into the framework of human antibodies. Humanized antibodies can be made using the techniques and approaches described in Verhoeyen et al (1988) Science, 239, 1534-1536, and in Kettleborough et al, (1991) Protein Engineering, 14 (7), 773-783.
  • antibodies also encompass completely human antibodies, which may be produced using recombinant technologies.
  • large libraries comprising billions of different antibodies are used.
  • this technology does not rely on immunization of animals to generate the specific antibody.
  • the recombinant libraries comprise a huge number of pre-made antibody variants wherein it is likely that the library will have at least one antibody specific for any antigen.
  • the frequency of administration may be determined clinically by following the decline of antibody titers in the serum of patients over time, but in any event may be at a frequency of 1 to 52 times per year, and most preferably between 1 and 12 times per year.
  • Quantities of antibody may vary according to the severity of the disease, or half-life of the antibody in the serum, but preferably will be in the range of 1 to 10 mg/kg of patient body weight, and preferably within the range of 1 to 5 mg/kg of patient, and most preferably 1 to 2 mg/kg of patient.
  • Figure 1 Detection of FOXP3 expression in a CD3 + invTCR Va24 + CD 1 -restricted T cell subset within peripheral blood.
  • A-B Representative histograms showing Foxp3 expression (%) and fluorescence intensity (MFI) in (A) CD3 + TCR ⁇ MHCII-restricted T cell subset and in (B) CD3 + invTCR Va24 + CD 1 -restricted T cell subset.
  • C Phenotypic and functional regulatory capacity of both Foxp3 + CD3 + T cell subsets: frequency (%) and expression levels (MFI) ofTreg-associated markers including, CD 127, CD25 and CTLA-4 and cell-cell contact suppressive capacity as evaluated by the standard polyclonal cell-cell contact suppression assay.
  • Figure 2 FOXP3 + CD3 + TCR Va24 + CD 1 -restricted T cell subset do not display invariant NKT cell membrane phenotypic markers, including CD161 and CD56, while expressing Treg cell phenotypic hallmarks.
  • FIG. 3 TCR ⁇ diversity analysis in circulating CD3 + TCRVa24 + CD4 + expressing FOXP3 + .
  • TCR ⁇ repertoire was determined by seven-color flow cytometry antibodies directed against CD3, CD4, CD8, Va24 and Foxp3 + and 24 ⁇ families. Distribution of the 24 ⁇ segments in CD3 + Va24 + CD4 + FOXP3 + cells is shown.
  • Figure 4 Different frequencies and phenotypic characteristics between FOXP3 + and FOXP3 " CD3 + T cell populations, as defined by their variable TCR recognition in human peripheral blood (PBMCs) and in TIL isolated from breast tumor.
  • Figure 5 In vitro induction of tumor- Ag specific CD3 + TCR Va24 + CD 1 -restricted T cells (invTreg) from stimulated naive CD3 + TCR Va24 + T cells with different nTreg polarizing medium.
  • invTreg tumor- Ag specific CD3 + TCR Va24 + CD 1 -restricted T cells
  • Naive CD3 + TCR Va24 + T cells were stimulated for 21 days with tumor - apoptotic breast tumor cell line pulsed autologous tDC as described in Fig 4 in presence of IL-2 (100 IU/ml) and IL-15 (10 ng/ml). Where indicated, TGF , RAPA and PGE2 were added.
  • A Overlay histogram displaying Foxp3 expression profiles of each of the generated invTreg.
  • B Frequency and (C) expression level (evaluated by MFI) of Foxp3 in CD3 + T cell culture. Dashed black line represents in (B) and (C) the frequency and the expression level of FOXP3 in naive Treg phenotypically defined by the expression a high level of CD45RA and CD25 and a low level of CD127.
  • FIG. 6 Combination of TGF , PAPA and PGE2 induce the establishment and the expansion of tumor Antigen specific FOXP3 + CD3 + TCR Va24 + CD 1 -restricted T cell cells committed to exclusively exert regulatory activity, with an autologous MLR assay.
  • CD3 + TCR Va24 + CD45RA + T cells were stimulated with autologous tolerogenic DC pulsed with apoptotic breast tumor cell lines in presence of IL-2, IL-15 and nTreg polarizing medium. After 21 days of in vitro expansion in nTreg polarizing medium, suppressive capacity of ex vivo generated Tumor Ag-specific invariant Foxp3 + Treg was evaluated in the presence of (A) a low or (B) high inflammatory medium. Fresh naive Treg were used as control.
  • Tumor tissue sample originated from patient with Luminal A and Luminal B Breast cancer (Institut Jean Godinot, Reims).
  • PBMCs Peripheral blood mononuclear cells
  • ACD3 cells T cell-depleted accessory cells
  • ACD3 cells T cell-depleted accessory cells
  • CD4 + T cells are negatively selected with a CD4 + T-cell isolation kit (Miltenyi Biotec, yielding CD4 + T-cell populations at a purity of 96-99%. Sub- sequent ly, selected CD4 + T cells are labeled with anti-CD4 (13B8.2)-FITC (Beckman Coulter), anti-CD25(4E3)- APC (Miltenyi Biotec), and anti-CD127(R34.34)-PE (Beckman Coulter) before being sorted into CD4 CD 127 "/lo CD25 high (pTregs) and CD4 CD 127 + CD25 neg/dim [conventional helper CD4 T cells (Tconv)] subpopulations using a FACSAria III Cell Sorter (Becton Dickinson).
  • CD14 + monocytes are isolated from PBMCs by positive selection using a MACS system.
  • CD3 + CD4 + CD127 + CD45RA + CD25 TCRaP + MHCII restricted are isolated from PBMCs after magnetic enrichment (MACS system: CD4 microbeads) and FACs sorting. Before the sorting step, enriched CD3 + CD4 + T cells are stained with anti-CD4 (13B8.2)-FITC (Beckman Coulter), anti-CD25(4E3)-APC (Miltenyi Biotec), and anti-CD 127(R34.34)-PE (Beckman Coulter), anti-TCR ⁇ - ⁇ 421 (IP26) (Biolegend).
  • CD3 + CD45RA + invTCR Va24 + CD 1 -restricted T cells are isolated from PBMCs after magnetic enrichment (MACS system: anti-iNKT microbeads and FACS sorting. Before the sorting step, enriched CD3 + invTCR Va24 + T cells are stained with anti-CD3 (UCHT- 1) V450 anti-invariant TCR Va24-JaQ (6B11)-PE (inv TCR Va24-JaQ (Becton Dickinson) and anti-CD45RA (T6D11)-FITC (Miltenyi Biotec).
  • CD3 + CD45RA + CD27 + TCRy6 + unrestricted T cells are isolated from PBMCs after magnetic enrichment (MACS system: TCRy6 + T cell isolation kit) and FACS sorting. Before the sorting step, enriched CD3 + TCRy6 + T cells are stained with anti-CD3 (UCHT-1) V450, anti- TCR pany6 + PE (IMMU510) (Beckman Coulter), anti-CD27- APC efluor 780 (0323) (ebioscience) and anti-CD45RA (T6D11)-FITC (Miltenyi Biotec).
  • T cell subsets are cultured either in IMDM supplemented with 5% SVF, 100 IU/ml penicillin/streptomycin, 1 mM sodium pyruvate, 1 mM nonessential amino acids, glutamax and 10 mM HEPES (IMDM-5 media) in hypoxia 2%.
  • MCF-7 human breast cancer cell line and culture.
  • the human breast cancer cell line MCF-7 was obtained from the American Type Culture Collection (USA). Cells are maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS). MCF-7 cells are treated with 5 ⁇ g/ml Doxorubicin for 24 h or by ⁇ irradiation (20 Gy). Extent of apoptosis is monitored by flow cytometric analysis (FACS). Cells are extensively washed prior to feeding DCs.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • TIL isolation Tumor tissue was minced with scalpels and enzymatically digested by overnight incubation in collagenase Type IV (2 mg/ mL, Roche Diagnostic GmbH) in DMEM High Glucose medium supplemented with 2 mM glutamine (Gibco), 50 mg/mL gentamycin and 0.25 % Human Serum Albumin, at 37 °C on a rotary shaker.
  • T cells are seeded at 2,5 10 5 /well in 48-well plates and stimulated with plate-bound anti-CD3 mAb (4 ⁇ g/ml) in the presence of ACD3- feeder (1 M).
  • Cells are cultured in IMDM-5 media (IMDM supplemented with 5% SVF, 100 IU/ml penicillin/streptomycin, 1 mM sodium pyruvate, 1 mM nonessential amino acids, glutamax and 10 mM HEPES) with PGE2 1 ⁇ , TGF 5 ng/ml, Rapa 10 nM.
  • IL-2 (lOOIU/ml) are added to the culture. Every three days, half of the supernatant volume is discarded and replaced with fresh IMDM-5 with IL-2 (100 Ul/ml).
  • these CD4 + T-cell lines were further expanded by restimulation with plate-bound anti- CD3 Abs (4 ⁇ g/ml). The restimulations were performed in the presence of ACD3-feeder, PGE2 1 ⁇ , TGFp 5 ng/ml, Rapa 10 nM and IL-2 (100 Ul/ml). Then every three days, half of the supernatant volume is discarded and replaced with fresh IMDM-5 with IL-2 (100 UI/ml).
  • the phenotype of the expanded CD4 + T cells was assessed by flow cytometry. 75% of the stimulated naive conventional T cells that became CD45RO + express FOXP3 + .
  • T cells are seeded at 1 x 10 3 /well in 96-well plates and stimulated with plate-bound anti-inv TCR Va24-JaQ (6B11) mAb (2 ⁇ / ⁇ 1) in the presence of ACD3- feeder (2.5x10 5 ).
  • Cells are cultured in IMDM-5 media with PGE2 1 ⁇ , TGFp 5 ng/ml, Rapa 10 nM, IL-2 (100 UI/ml) and IL-15 (10 ng/ml). Every three days, IL-2 (100 UI/ml) and IL-15 (10 ng/ml) are added to the culture.
  • T cells are further expanded by restimulation with plate-bound anti- anti-inv TCR Va24-JaQ (6B11) mAb (2 ⁇ g/ml) in the presence of ACD3-feeder, PGE2 1 ⁇ , TGFp 5 ng/ml, Rapa 10 nM IL-2 (100 UI/ml) and IL-15 (10 ng/ml). Then every three days, half of the supernatant volume is discarded and replaced with fresh IMDM-5 with IL-2 (100 UI/ml) and IL-15 (10 ng/ml). On day 21, cells are analyzed by flow cytometry. 70% of the stimulated CD3 + invTCR Va24 + RA + T cells that became CD45RO + express Foxp3 + .
  • T cells are seeded at 1 x 10 3 /well in 96-well plates and stimulated with plate-bound anti-TCRy6 mAb (2 in the presence of ACD3-feeder (2.5X10 5 ).
  • IMDM-5 media IMDM supplemented with 5% SVF, 100 IU/ml penicillin/streptomycin, 1 mM sodium pyruvate, 1 mM nonessential amino acids, glutamax and 10 mM HEPES
  • PGE2 1 ⁇ PGE2 1 ⁇
  • TGF 5 ng/ml Rapa 10 nM
  • IL-2 100 UI/ml
  • IL-15 10 ng/ml
  • T cells were further expanded by restimulation with plate-bound anti-pan TCR ⁇ Abs (2 ⁇ g/ml).
  • the restimulations were performed in the presence of ACD3-feeder, PGE2 1 ⁇ , TGFp 5 ng/ml, Rapa 10 nM and IL-2 (100 UI/ml) and IL-15 (10 ng/ml).
  • ACD3-feeder PGE2 1 ⁇
  • TGFp 5 ng/ml TGFp 5 ng/ml
  • Rapa 10 nM and IL-2 100 UI/ml
  • IL-15 10 ng/ml
  • cells are analyzed by flow cytometry. 65% of the stimulated CD3 + CD45RA + CD27 + TCRy6 + T cells that became CD45RO + express Foxp3 + .
  • monocytes are cultured in 48-well flat-bottom plates containing 0,5 ml of AIMV per well supplemented with 100 ng/ml recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and 10 ng/ml human recombinant IL-4 for the generation of immature DC.
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • IL-4 human recombinant IL-4
  • Tol-Mo-DC are 1) removed from the wells, washed twice with IMDM-5 (IMDM supplemented with 5% SVF, 100 IU/ml penicillin/streptomycin, 1 mM sodium pyruvate, 1 mM nonessential amino acids, glutamax and 10 mM HEPES, 2) added to wells of a 48-well plate at a concentration of 3 X 10 5 /ml in IMDM-5 and 3) pulsed in IMDM-5 with specific
  • ovalbumin pulsed tDC are 1) washed twice with IMDM-5 and 2) added to wells of a 48-well plate at a concentration of 3 X 10 5 /ml in IMDM-5 in the presence of 2 X 10 5 irradiated autologous feeders, PGE2 1 ⁇ , and Rapa 10 nM.
  • Purified naive conventional CD4 + T cells are added to the pulsed tDC.
  • T cell cloning medium On day 1, IL-2 (lOOIU/ml) and TGF (5ng/ml) are added to the coculture. Every three days, half of the supernatant volume is discarded and replaced with fresh IMDM-5 with IL-2 (100 Ul/ml (T cell cloning medium). On day 12, these T-cells are further expanded by restimulation with ova-pulsed tDC in the presence of ACD3-feeder, PGE2 1 ⁇ , TGF 5 ng/ml, Rapa 10 nM, IL-2 (100 Ul/ml). Once T cells begin to expand, they can be split every 2 to 3 days with T cell cloning medium and irradiated feeder. On day 21, cells are analyzed by flow cytometry.
  • nTreg polarizing medium comprising the combination of IL-2, TGF , PGE2 and rapamycin
  • TH-17 polarizing medium IMDM medium containing IL-2 IL-1 IL-6, IL-21 IL-23 cytokines
  • the 21-day- expanded-Foxp3 expressing CD3 + CD4 + TCRa,p + MHCII restricted T cells are stimulated with plate-bound anti-CD3 mAb (4 ⁇ g/ml) in the presence of ACD3- feeder (1 M) in 48-well plates and every three days, half of the supernatant volume is discarded and replaced with fresh T cell cloning medium or TH-17 polarizing medium for 21 days.
  • monocytes are cultured in 48-well flat-bottom plates containing 0,5 ml of AIMV per well supplemented with 100 ng/ml recombinant human granulocyte-macrophage colony- stimulating factor (GM-CSF) and 10 ng/ml human recombinant IL-4 and AM580 (100 nM) for the generation of immature DC expressing CDld.
  • GM-CSF granulocyte-macrophage colony- stimulating factor
  • AM580 100 nM
  • tDCs are co-cultured with apoptotic MCF-7 cells at a DC/tumor cell ratio of 1 :2 for 24h in AIMV with GM-CSF (100 ng/niL), IL-4 (10 ng/mL).
  • Another portion of tDC are freezed at 2 x 10 6 /per vial vial- in 90% FBS - 10% DMSO.
  • tumor-antigen pulsed tDC are 1) washed twice with IMDM-5 and 2) added to wells of a 48-well plate at a concentration of 3 X 10 5 /ml in IMDM- 5 in the presence of 2 X 10 5 irradiated autologous feeders, PGE2 1 ⁇ , and Rapa 10 nM.
  • Purified CD3 + CD45RA + invTCR Va24 + CD 1 -restricted T cells are added to the pulsed tDC.
  • IL-2 (lOOIU/ml), IL-15 (10 ng/ml) and TGF (5ng/ml) are added to the coculture. Every three days, half of the supernatant volume is discarded and replaced with fresh IMDM-5 with IL-2 (100 Ul/ml) and IL-15 (10 ng/ml) (T cell cloning medium). On day 12, these T-cells are further expanded by restimulation with tumor Ag-pulsed tDC in the presence of ACD3-feeder, PGE2 1 ⁇ , TGF 5 ng/ml, Rapa 10 nM, IL-2 (100 Ul/ml) and IL-15 (10 ng/ml).
  • T cells Once T cells begin to expand, they can be split every 2 to 3 days with T cell cloning medium and irradiated feeder. On day 21, cells are analyzed by flow cytometry. 75 % of the stimulated CD3 + CD45RA + invTCR Va24 + cells that became CD45RO + express Foxp3 + .
  • Tolerogenic DC from CD14 + monocytes
  • Tol-Mo-DC monocytes are cultured in 48-well flat-bottom plates containing 0,5 ml of AIMV per well supplemented with 100 ng/ml recombinant human granulocyte-macrophage colony- stimulating factor (GM-CSF) and 10 ng/ml human recombinant IL-4 for the generation of immature DC.
  • GM-CSF granulocyte-macrophage colony- stimulating factor
  • IL-4 human recombinant IL-4
  • IMDM-5 IMDM supplemented with 5% SVF, 100 IU/ml penicillin/streptomycin, 1 mM sodium pyruvate, 1 mM nonessential amino acids, glutamax and 10 mM HEPES, freezed or used for the generation and expansion of phospho-antigen specific functionally committed FOXP3 expressing CD3 + TCRy5 + unrestricted T cells.
  • T cell cloning medium IMDM-5 with IL-2 (100 Ul/ml) and IL-15 (10 ng/ml) (T cell cloning medium).
  • these T-cells are further expanded by restimulation with tDC in the presence of ACD3-feeder, PGE2 1 ⁇ , TGF 5 ng/ml, Rapa 10 nM, IL-2 (100 Ul/ml), IL-15 (10 ng/ml) and zoledronic acid (100 nM).
  • T cells can be split every 2 to 3 days with T cell cloning medium and irradiated feeder.
  • cells are analyzed by flow cytometry. 75 % of the stimulated CD3 + CD45RA + TCRy6 + T cells that became CD45RO + express Foxp3 + .
  • monocytes are cultured in 48- well flat-bottom plates containing 0,5 ml of RPMI-5 per well supplemented with 20 ng/ml recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and 20 ng/ml human recombinant IL-4 for the generation of immature DC (iDC).
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • iDC immature DC
  • iDC a portion of iDC are co- cultured with apoptotic MCF-7 cells at a DC/tumor cell ratio of 1 :2 for 24h in RPMI 1640 supplemented with GM-CSF (20 ng/mL), IL-4 (20 ng/mL) and 5 % FBS.
  • Another portion of iDC are freezed at 2 x 10 6 / per vial - in 90% FBS -10% DMSO.
  • pulsed DCs are matured with tumor necrosis factor a (TNF-a; 20 ng/mL final) and PGE2 (1 ⁇ ) for 2 days (mDC).
  • TNF and PGE2 at the same concentrations
  • lipopolysaccharide LPS; 10-1000 ng/mL; Sigma
  • Antigen- loaded DC stimulators are irradiated at 30 Gy.
  • CD3 + T cells anti- CD4(SK3)-PerCP-eFluor 710, anti-TCRa (IP26)-APC (ebio science), anti-CD25 (B1.49.9)-PeCy55, anti-CD127(R34.34)-APC-AF700 (Beckman Coulter), anti- CD3(UCHT1)- BB515 anti-invariant TCR Va24-JaQ (6B11)-PE, anti-Foxp3 (259D/C7)-PE-CF594 and anti-CD152 (BNI3)-BV421, anti-CD 161 (DX12) BV605 and anti-CD56(NCAM 16.2) BU395 (Becton Dickinson), anti-TCR ⁇ - ⁇ 421 (IP26) (Bio legend), anti- TCR pany5 + PE (IMMU510) (Beckman Coulter) and anti
  • CFSE staining Tconv are stained with 1 ⁇ carboxy- fluorescein succinimidyl ester (CFSE) (CellTrace cell proliferation kit; Molecular Probes/Invitrogen) in PBS for 8 min at 37 °C at a concentration of 1 x 10 7 cells/mL. The labeling is stopped by washing the cell twice with RPMI 1640 culture medium containing 10% FBS. Cells are then resuspended at the desired concentration and subsequently used for proliferation assays.
  • CFSE carboxy- fluorescein succinimidyl ester
  • 7-AAD (7-amino-actinomycin D) staining Apoptosis of stimulated CFSE- labeled or unlabeled nTregs and Tconv was determined using the 7-AAD assay. Briefly, cultured cells are stained with 20 ⁇ g/mL nuclear dye 7-AAD (Sigma- Aldrich) for 30 min at 4 °C. FSC/7-AAD dot plots distinguish living (FSC high /7-AAD " ) from apoptotic (FSC high /7- AAD + ) cells and apoptotic bodies (FSC low /7- AAD + ) and debris (FSC low /7- AAD " ). Living cells are identified as CD3 + 7-AAD " FSC cells.
  • the IOTest Beta Mark TCR-VP Repertoire kit (Beckman Coulter, Miami, FL) was used for the assessment of the following TCR-VP regions: ⁇ 1, ⁇ 2, ⁇ 3, ⁇ 4, ⁇ 5.1, ⁇ 5.2, ⁇ 5.3, ⁇ 7.1, ⁇ 7.2, ⁇ 8, ⁇ 9, ⁇ 11, ⁇ 12, ⁇ 13.1, ⁇ 13.2, ⁇ 13.6, ⁇ 14, ⁇ 16, ⁇ 17, ⁇ 18, ⁇ 20, ⁇ 21.3, ⁇ 22, and ⁇ 23.
  • This kit includes 8 cocktails, each containing antibodies against 3 different TCR- ⁇ regions covering 24 TCR- ⁇ antigens and approximately 70% of the normal human TCR- ⁇ repertoire.
  • the panel included antibody against CD3 (clone SK7), CD4 (clone SK3), CD8 (clone SKI), iNKT (clone 6B11) and Foxp3 + (clone 259D) to allow evaluation of Va24 + CD4 + FOXP3 + , Va24 + CD4 + FOXP3 " , Va24 + CD4 CD8 and Va24 + CD8 + subpopulations. Functional Assays.
  • T-cell proliferation is assessed CFSE dilution assay in RPMI supplemented with 5% FBS, 100 IU/ml penicillin/streptomycin, 1 mM sodium pyruvate, 1 mM nonessential amino acids, glutamax and 10 mM HEPES (RPMI-5 media) in normoxia.
  • stimulated CFSE-labeled Tconv are harvested, costained with anti-CD3 mAb and 7-AAD, and the percentage of living proliferating cells (defined as CFSE low fraction) in gated CD3 + 7-AAD- cells is determined by flow cytometry.
  • Standard polyclonal cell-cell contact Treg suppression assay CFSE-labeled T CO nv (4 ⁇ 10 4 per well), used as responder cells, are cultured with ACD3- feeder (4 ⁇ 10 4 per well) in the presence or absence of defined amounts of Foxp3 T cells (blood Treg or ex vivo generated T cells) for 4 to 5 d. Cultures are performed in round-bottom plates coated with 0.2 ⁇ g/mL anti-CD3 mAb in 200 of complete RPMI medium.
  • Results are expressed as the percentage of proliferating CFSE low T cells or as a percentage of suppression calculated as follows: (100 x [(percentage of T CO nv CFSE low cells - percentage of T CO nv CFSE low in coculture with nTregs)/percentage of T CO nv CSFE low cells,
  • CFSE-labeled T con v CD4 CD25 T cells (5 10 4 ) are stimulated either with 1 x 10 4 pulsed iDC in RPMI-5 media or with 5 x 10 3 pulsed - mDC in IMDM-5 media supplemented with IL-2 (20 IU/ml) IL-lb (10 ng/ml), IL-6 (30 ng/ml), IL-21 (50 ng/ml) and IL-23 (30 ng/ml) in the presence or absence of defined amounts of Foxp3 T cells (blood Treg or ex vivo generated T cells) for 5 to 6 d.
  • IL-2 (20 IU/ml)
  • IL-6 (10 ng/ml)
  • IL-21 50 ng/ml
  • IL-23 (30 ng/ml) in the presence or absence of defined amounts of Foxp3 T cells (blood Treg or ex vivo generated T cells) for 5 to 6 d.
  • TSDR Treg specific demethylation region
  • TSDR PCR-amplified using the following reaction: 50 ⁇ reaction volume containing 25 ⁇ of ZymoTaq PreMix buffer (Zymo Research) and 0.5 ⁇ each of the primers FOXP3_TSDRfwd (5'-
  • TSDR real-time PCR was performed with probes that targeted methylated or demethylated target sequences.
  • the reaction was performed in 96-well white trays with a Roche LightCycler 480 system (Roche Diagnostics). Each reaction contained 10 ⁇ LightCycler 480 Probes Master Mix (Roche), 10 ng of bisulfite converted DNA sample or standards, 1 ⁇ of each primer, and 150 nM of each probe with a final reaction value of 20 ⁇ .
  • the probes used for amplification were TSDR-Forward 5'- GGTTTGTATTTGGGTTTTGTTGTTATAGT-3' (SEQ ID NO: 3) and TSDR-Reverse 5 '-CT AT AAAAT AAAAT ATCTACCCTCTTCTCTTCCT-3 ' (SEQ ID NO: 4).
  • the probes for target sequence detection were FAM-labeled methylated probe, FAM- CGGTCGGATGCGTC-MGB-NFQ (SEQ ID NO: 5), or VIC-labeled unmethylated probe, VIC-TGGTGGTTGGATGTGTTG-MGB-NFQ (SEQ ID NO: 6). All samples were tested in triplicate.
  • the protocol for real-time amplification is as follows: after initial denaturation at 95°C for 10 min, the samples were subjected to 50 cycles at 95°C for 15 s and at 61°C for 1 min. Fourteen different ratios of fully methylated and demethylated template were used as real-time standards. A six-order polynomial equation was used to extrapolate the percentage of cells demethylated at the TSDR for each sample.
  • Histone acetylation analysis of the four different sites of FOXP3 gene was evaluated by ChIP assay, as previously described by Ling Lu (Ling Lu et al, PNAS 2014). Briefly, 50,000 cells of each treated nTreg cell sample were harvested and cross-linked with 1% formaldehyde, and then lysed with 120 ⁇ of lysis buffer [50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% (wt/vol) SDS, protease inhibitor mix (1 : 100 dilution; Sigma), 1 mM PMSF, 20 mM Na-butyrate].
  • lysis buffer 50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% (wt/vol) SDS, protease inhibitor mix (1 : 100 dilution; Sigma), 1 mM PMSF, 20 mM Na-butyrate.
  • the chromatin in the lysate was sonicated to 500-800-bp fragments and then diluted with 800 ⁇ of RIPA ChIP buffer [10 mM Tris-HCl, pH 7.5, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% (vol/vol) Triton X-100, 0.1% (wt/vol) SDS, 0.1% (wt/vol) Na-deoxycholate, protease inhibitor mix (1 : 100 dilution; Sigma), 1 mM PMSF, and 20 mM Na- butyrate].
  • RIPA ChIP buffer 10 mM Tris-HCl, pH 7.5, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% (vol/vol) Triton X-100, 0.1% (wt/vol) SDS, 0.1% (wt/vol) Na-deoxycholate, protease inhibitor mix (1 : 100 dilution
  • Dynabeads protein G ( ⁇ ; Invitrogen) was incubated with ⁇ g ofH3K4me3 (Abeam) or H3K9ac (Cell Signaling) or normal rabbit IgG negative control ChlP-grade antibodies for 2 h separately. Then, ⁇ of the sheared chromatin was immunoprecipitated with pretreated antibody-bead complexes and another ⁇ of the sheared chromatin for total input DNA extraction separately.
  • Immunoprecipitated DNA was quantified by real-time PCR with following primers: promoter, 5'-ACC GTA CAG CGT GGT TTT TC-3' (SEQ ID NO: 7) and 5'-CTA CCT CCC TGC CAT CTC CT-3' (SEQ ID NO: 8); CNS1, 5'- CCC AAG CCC TAT GTG TGATT-3' (SEQ ID NO: 9) and 5'-GTG TGT CAG GCC TTG TGC TA-3' (SEQ ID NO: 10) ; CNS2, 5'-GTC CTC TCC ACAACC CAA GA-3' (SEQ ID NO: 11) and 5'-GAC ACC ACG GAG GAA GAG AA -3' (SEQ ID NO: 12); and CNS3, 5'-AGG TGC CGA CCT TTA CTG TG-3' (SEQ ID NO: 13) and 5'- ACA ATA CGG CCT CCT CCT CT-3' (SEQ ID NO: 14). Results a
  • both CD3 + TCRaP + MHCII restricted T cells and CD3 + invTCR Va24 + CD 1 -restricted T cells express Foxp3.
  • FOXP3 expressing CD3 + TCRaP + MHCII restricted T cells represents approximately 9% (1-12 %) of these CD3 + TCRa- ⁇ T cells ( Figure 1; upper right panel).
  • Figure 1 shows that these Foxp3 + T cells express high levels of CD4, CD25 and CTLA4 and low level of CD127 while FOXP3- T cells are mainly CD127 + T cells and express low levels of CD25 and CTLA4.
  • Foxp3 expressing CD3 + invTCR Va24 + CD 1 -restricted T cells represent almost 9% (3- 15 %) of the CD3 + invTCR Va24 + CD 1 -restricted T cells population (see Figure 1, upper right panel).
  • These Foxp3 + T cells present a same phenotypic profile as Foxp3 + CD3 + TCRaP, with respect to the CD4, CD25 and CTLA4 markers, with, however, an increased intensity of expression (as measured by the MFI value) for CD25 and CTLA markers (see Figure 1 , lower panel) while FOXP3 " CD3 + invTCR Va24 + exhibit a similar phenotypic profile as FOXP3 " CD3 + TCRap.
  • FOXP3 + CD3 + invTCR Va24 + CD 1 -restricted T cell subset display low CD161 and CD56 levels, cell membrane phenotypic markers of invariant NKT, while expressing Treg cell phenotypic markers (CD25, CTLA4).
  • TCR-VP repertoire kit (Beckman) is performed. TCR-VP families examined by the panel of available antibodies covered a mean of 70.18% (6 standard deviation of 8.268) of all analyzed cells, representing the majority of cells within each subset investigated.
  • TCR-VP families are investigated in CD3 + Va24 + T-cell subsets: CD3 + Va24 + CD4 + FOXP3 + and CD3 + Va24 + CD4 + FOXP3 " .
  • the repertoire analysis shows that CD3 + Va24 + CD4 + subset cells expressing FOXP3 + exhibit a wide variety of TCR-V chains.
  • This TCR-VP repertoire is relatively comparable to the TCR- ⁇ repertoire of CD3 + Va24 + CD4 + FOXP3 " subset with an increased ⁇ usage involving ⁇ , ⁇ 2, ⁇ 4, ⁇ 8, or ⁇ 5.1 ( Figure 3).
  • Optimal conditions for inducing Foxp3 expression in invariant Tcells are investigated in CD3 + Va24 + T-cell subsets: CD3 + Va24 + CD4 + FOXP3 + and CD3 + Va24 + CD4 + FOXP3 " .
  • nTreg polarizing medium were assessed for their capacity to induce the expression the differentiation of Foxp3 + cells with suppressive function.
  • Figure 5 shows that cultured naive CD3 + invTCR Va24 + T cells exhibit a variable level of Foxp3 dependent on their culture condition of stimulation.
  • Polarizing medium comprising the combination of IL-2, ⁇ , PGE2 and rapamycin results in a higher Foxp3 expression over combinations of IL-2, ⁇ and rapamycin, IL-2 and PGE2, or IL-2 alone.
  • the combination of IL-2, ⁇ , PGE2 and rapamycin results in an optimal intensity of Foxp3 expression in the invTCR Va24 + T cells, as compared to the other combinations.
  • naive CD3 + invTCR Va24 + T cells stimulated with the polarizing medium comprising the combination of IL-2, ⁇ , PGE2 and rapamycin express level and intensity of Foxp3 similar or higher to those of blood naive regulatory T cells (CD3 + TCR ⁇ CD4 + CD127 /low CD45RA + CD25 + ), corresponding to our positive control.
  • Tumor Ag-specific CD3 + TCR Va24 + T cells maintain their ability to perform suppressive function in pro -inflammatory conditions
  • Figure 6A shows that tumor Ag-specific memory invTCR Va24 + T cells ex vivo generated and expanded in the presence of the nTreg polarizing medium above described are endowed of a higher suppressive activity than fresh Foxp3 expressing CD3 + CD4 + TCRc$ + MHCII restricted T cells when using an autologous MLR coculture assay.
  • Figure 6B shows that these tumor Ag-specific invTCR Va24 + T cells still maintain their suppressive activity, when the autologous MLR coculture assay are performed in presence of a high inflammatory medium containing IL-2 IL-1 IL-6, IL-21 IL-23 cytokines, while fresh Foxp3 expressing CD3 + CD4 + TCRaP + MHCII restricted T cells lose their suppressive activity.

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Abstract

La présente invention concerne une nouvelle population de lymphocytes T régulateurs Foxp3+ invariants, une méthode ex vivo pour la génération et la multiplication de ces cellules, et leurs utilisations thérapeutiques. Les auteurs de l'invention ont cherché à déterminer les conditions optimales permettant d'induire l'expression de Foxp3 dans les lymphocytes T invariants en évaluant la capacité de différents milieux polarisants nTreg à induire l'expression de la différenciation des cellules Foxp3+ avec une fonction suppressive. Les auteurs de l'invention ont montré que les lymphocytes T mémoires invTCR Vα24+ spécifiques de l'Ag tumoral, générés et multipliés ex vivo en présence du milieu polarisant nTreg, conservent leur capacité de fonction suppressive dans des conditions pro-inflammatoires. La présente invention concerne en particulier une population isolée de lymphocytes T régulateurs Foxp3+ invariants ayant le phénotype suivant : CD3+ Vα24+ Foxp3+.
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US11384336B2 (en) 2016-12-07 2022-07-12 East Carolina University Compositions and methods for in vitro cultivation and/or expansion of regulatory T cells

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130195919A1 (en) * 2010-03-05 2013-08-01 President And Fellows Of Harvard College Induced dendritic cell compositions and uses thereof
WO2014116908A1 (fr) * 2013-01-24 2014-07-31 University Of Florida Research Foundation, Incorporated Procédé de génération de lymphocytes t régulateurs à l'aide de facteurs secrétés par les cellules inkt

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130195919A1 (en) * 2010-03-05 2013-08-01 President And Fellows Of Harvard College Induced dendritic cell compositions and uses thereof
WO2014116908A1 (fr) * 2013-01-24 2014-07-31 University Of Florida Research Foundation, Incorporated Procédé de génération de lymphocytes t régulateurs à l'aide de facteurs secrétés par les cellules inkt

Non-Patent Citations (27)

* Cited by examiner, † Cited by third party
Title
A. PLUCKTHUN: "Antibody engineering; Advances from use of E. coli expression systems", BIO/TECHNOLOGY, vol. 9, no. 6, 1991, pages 545 - 51
BERNARDO ET AL: "Decreased circulating iNKT cell numbers in refractory coeliac disease", CLINICAL IMMUNOLOGY, ACADEMIC PRESS, US, vol. 126, no. 2, 31 October 2007 (2007-10-31), pages 172 - 179, XP022415739, ISSN: 1521-6616, DOI: 10.1016/J.CLIM.2007.09.005 *
BETTER ET AL., SCIENCE, vol. 240, 1988, pages 1041
BIRD ET AL., SCIENCE, vol. 242, 1988, pages 423
BORREBAECK; CARLSSON, CURR. OPIN. PHARMACOL., vol. 1, 2001, pages 404 - 408
CHEN W ET AL: "Conversion of Peripheral CD4+CD25- Naive T cells to CD4+CD25+ Regulatory T cells by TGF-beta Induction of Transcription Factor Foxp3", THE JOURNAL OF EXPERIMENTAL MEDICINE, ROCKEFELLER UNIVERSITY PRESS, US, vol. 198, no. 12, 15 December 2003 (2003-12-15), pages 1875 - 1886, XP003010702, ISSN: 0022-1007, DOI: 10.1084/JEM.20030152 *
ELAINE LONG ET AL: "Understanding FOXP3: Progress Towards Achieving Transplantation Tolerance", TRANSPLANTATION, vol. 84, no. 4, 1 August 2007 (2007-08-01), GB, pages 459 - 461, XP055347543, ISSN: 0041-1337, DOI: 10.1097/01.tp.0000275424.52998.ad *
FUHRMAN ET AL.: "Divergent Phenotypes of Human Regulatory T Cells Expressing the Receptors TIGIT and CD226", JOURNAL OF IMMUNOLOGY, 2015
H ZOLA: "Monoclonal Antibodies; A manual of techniques", 1988, CRC PRESS
H. TUOVINEN ET AL: "Most human thymic and peripheral-blood CD4+CD25+ regulatory T cells express 2 T-cell receptors", BLOOD, vol. 108, no. 13, 15 December 2006 (2006-12-15), US, pages 4063 - 4070, XP055348655, ISSN: 0006-4971, DOI: 10.1182/blood-2006-04-016105 *
HUSTON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 5879
J. HUSTON ET AL.: "Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single chain Fv analogue produced in E. coli", PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 5879 - 5883, XP000872837, DOI: doi:10.1073/pnas.85.16.5879
KARL O.A. YU ET AL: "The diverse functions of CD1d-restricted NKT cells and their potential for immunotherapy", IMMUNOLOGY LETTERS, vol. 100, no. 1, 1 August 2005 (2005-08-01), pages 42 - 55, XP055199919, ISSN: 0165-2478, DOI: 10.1016/j.imlet.2005.06.010 *
KETTLEBOROUGH ET AL., PROTEIN ENGINEERING, vol. 14, no. 7, 1991, pages 773 - 783
LING LU ET AL., PNAS, 2014
LIPING SONG ET AL: "V[alpha]24-invariant NKT cells mediate antitumor activity via killing of tumor-associated macrophages", JOURNAL OF CLINICAL INVESTIGATION, vol. 119, no. 6, 1 June 2009 (2009-06-01), US, pages 1524 - 1536, XP055347582, ISSN: 0021-9738, DOI: 10.1172/JCI37869 *
MONTOYA CARLOS J ET AL: "Characterization of human invariant natural killer T subsets in health and disease using a novel invariant natural killer T cell-clonotypic monoclonal antibody, 6B11", IMMUNOLOGY, WILEY-BLACKWELL PUBLISHING LTD, GB, vol. 122, no. 1, 1 September 2007 (2007-09-01), pages 1 - 14, XP009175292, ISSN: 0019-2805, DOI: 10.1111/J.1365-2567.2007.02647.X *
MONTOYA CJ ET AL., IMMUNOLOGY, vol. 122, no. 1, September 2007 (2007-09-01), pages 1 - 14
MORRISON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 81, 1984, pages 6851 - 6855
NAGANARI OHKURA ET AL: "FOXP3regulatory T cells: control of FOXP3 expression by pharmacological agents", TRENDS IN PHARMACOLOGICAL SCIENCES, vol. 32, no. 3, 1 March 2011 (2011-03-01), pages 158 - 166, XP028170472, ISSN: 0165-6147, [retrieved on 20101217], DOI: 10.1016/J.TIPS.2010.12.004 *
PETER T LEE ET AL: "Brief Definitive Report Distinct Functional Lineages of Human V _ 24 Natural Killer T Cells", J. EXP. MED, 4 March 2002 (2002-03-04), pages 637 - 641, XP055347737, Retrieved from the Internet <URL:http://jem.rupress.org/content/195/5/637/tab-pdf> [retrieved on 20170220] *
S G R HURRELL: "Monoclonal Hybridoma Antibodies: Techniques and Application", 1982, CRC PRESS
SKERRA ET AL., SCIENCE, vol. 240, 1988, pages 1038
TAKAHASHI T ET AL: "Analysis of human V alpha 24+ CD4+ NKT cells activated by alpha-glycosylceramide-pulsed monocyte-derived dendritic cells", THE JOURNAL OF IMMUNOLOGY, THE AMERICAN ASSOCIATION OF IMMUNOLOGISTS, US, vol. 164, no. 9, 1 May 2000 (2000-05-01), pages 4458 - 4464, XP002364638, ISSN: 0022-1767 *
VERHOEYEN ET AL., SCIENCE, vol. 239, 1988, pages 1534 - 1536
WARD ET AL., NATURE, vol. 341, 1989, pages 544
WINTER; MILSTEIN, NATURE, vol. 349, 1991, pages 293 - 299

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* Cited by examiner, † Cited by third party
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