WO2018206794A1 - Utilisations diagnostiques et thérapeutiques d'un système in vitro de culture de lymphocytes autologues - Google Patents

Utilisations diagnostiques et thérapeutiques d'un système in vitro de culture de lymphocytes autologues Download PDF

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WO2018206794A1
WO2018206794A1 PCT/EP2018/062256 EP2018062256W WO2018206794A1 WO 2018206794 A1 WO2018206794 A1 WO 2018206794A1 EP 2018062256 W EP2018062256 W EP 2018062256W WO 2018206794 A1 WO2018206794 A1 WO 2018206794A1
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cells
regulatory
cell
anyone
lymphopenia
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Isaac ROSADO SÁNCHEZ
Manuel Leal Noval
Yolanda María PACHECO LÓPEZ
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Universidad de Sevilla
Servicio Andaluz de Salud
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Universidad de Sevilla
Servicio Andaluz de Salud
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/22—Immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/416—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/418—Antigens related to induction of tolerance to non-self
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]

Definitions

  • the present invention relates to the field of immunology and medicine, particularly refers to an in vitro system of autologous lymphocytes culture that allows studying the homeostatic proliferation mechanisms of human na ' i ' ve CD4 T-cells.
  • the in vitro system of the present invention can be used in a method for the identification and classification of individuals suffering from a lymphopenia-related disease.
  • the regulatory T cells obtained in the present invention and their use in cell therapy for treating autoimmune diseases, inflammatory diseases, allergic or asthmatic condition, graft versus host disease or for preventing graft rejection.
  • the size and composition of human peripheral T-cell pool is homeostatically kept constant throughout the life.
  • immune insults as several viral infections, autoimmune disorders, chemotherapy against cancer and other drugs can lead to a lymphopenic status.
  • the restoration of the T-cell pool after such aggressions is not uniform. While lymphodepletion of memory T-cell numbers is usually reverted early, the restoration of na ' i ' ve CD4 and CD8 T-cell pools are slower and delayed.
  • the size and functional restoration of the na ' i ' ve T-cell pool is of vital importance in order to recover the immune homeostasis.
  • Several mechanisms are involved in the reconstitution of na ' i ' ve T-cell pool.
  • thymus is a lymphoid organ that supports the maturation of hematopoietic precursors leading to generation of new T-cells and ensuring the preservation of TCR variability.
  • the homeostatic proliferation (HP) process involves a peripheral clonal expansion of T-cells in response to homeostatic stimuli like self- or commensal-antigens and cytokines. Indeed, it is known that different types of homeostatic proliferation exist.
  • the fast or spontaneous HP is defined by a rapid cellular division of na ' i ' ve T-cells mediated by the low avidity TCR-contact (antigen-driven proliferation) that generate memory-like T-cells.
  • the slow HP is defined by a slower cellular division of na ' i ' ve T-cells that do not result in a change of phenotype and carried out by homeostatic cytokines like IL7.
  • homeostatic cytokines like IL7.
  • the homeostatic proliferation has an important role in early stages of immune reconstitution. For this reason, the comprehension of homeostatic proliferation process in human lymphopenic disorders has a pivotal importance.
  • several in vivo models using different species of animals and chimeras, and several in vitro models using generated antigen-presenting cells or anti-CD3/CD28 stimulation have been developed.
  • self- and commensal antigens, that are well-known triggers of the HP show a large variability among different subjects and scenarios and such natural composition is not considered in these experimental approximations.
  • regulatory T cells Although the therapeutic potential of regulatory T cells was envisioned decades ago, clinical implementation of their potent immune regulatory activity by in vivo administration of agents has proven challenging. Therefore, there is a need for a new method for regulatory T cell therapy that is more efficient for treating a patient in need thereof.
  • the present invention relates to the use of antigen-presenting cells (APCs) for studying the homeostatic proliferation mechanisms in humans.
  • the antigen-presenting cells are autologous antigen-presenting cells (aAPCs).
  • a second aspect of the present invention relates to a method to evaluate the potential homeostatic proliferation of T-cells of a human subject using APCs, preferably using their autologous APCs (aAPCs)defined in the first aspecto, hereinafter first method of the invention, comprising: a) Isolation of two cellular subsets and control of purity from a human biological sample, wherein:
  • Cellular subset 1 comprising APCs, preferably aAPCs, and II.
  • Cellular subset 2 comprising na ' i ' ve CD4 T-cells, b) Irradiate the APCs, preferably aAPCs,of the cellular subset 1
  • step (b) Cultivate the na ' ive CD4 T-cells from cellular subset 2 of step (c) in a plate and add the APCs, preferably aAPCs, of the cellular subset 1 and rlL7,
  • step (e) Perform the basal analysis (day 0) in an aliquot of the cell culture of step (e),
  • a third aspect of the present invention relates to the in vitro model for studying homeostatic proliferation process, hereinafter in vitro model of the invention, comprising the two cellular subsets as described in the first method of the invention.
  • a fourth aspect of the present invention relates to a method for obtaining data that can be used for the characterization and classification of human subject suffering from a lymphopenia- related disease, hereinafter second method of the invention, comprising the steps according to the first method of the invention, and also comprising: i) quantitation of the T-cells that undergo homeostatic proliferations: slow (with partial CFSE dilution and maintenance of the na ' ive phenotype) and fast homeostatic proliferation (with full CFSE dilution and acquisition of memory phenotype)
  • the second method of the invention also comprises: ii) the characterization and, optionally quantification, of different cellular markers in both types of proliferated cells including at least those related with differentiation and maturation (CD45RA,CD27,CCR7), activation (HLA-DR,CD25), survival (BCL-2), apoptosis-prone (CD95) or homing (37-integrin).
  • a fifth aspect of the present invention relates to a method to characterize and classify a human subject suffering from lymphopenia-related disease, hereinafter third method of the invention, comprising the steps according to the first and/or the second method of the invention, and further comprising: iii) assigning the subjects from steps (i-ii) preferably attending to frequencies of slow and fast homeostatic proliferation and to the ratio between these types of homeostatic proliferation.
  • a sixth aspect of the invention relates to a specific/autologous generated regulatory T cell during the HP process, hereinafter regulatory T cell of the invention, obtainable by the steps as described in the first method of the invention, and furthermore comprising the cell sorting of such cells.
  • cellular markers like CD25, CD45RA and the levels of CFSE could be used to get a cellular fraction enriched in such regulatory T-cells, although other cellular marker associated to these cells like CTLA-4, FoxP3, CD39 or CD141 could be also used
  • a seven aspect of the invention relates to a composition, hereinafter composition of the invention, comprising a regulatory T cell or regulatory T cell population of the invention.
  • An eight aspect of the invention relates to the specific/autologous regulatory T cell of the invention, and/or the regulatory T cell population of the invention, for use in therapy.
  • a ninth aspect of the present invention relates to the specific/autologous regulatory T cell of the invention, and/or the regulatory T cell population of the invention, for use in the treatment, amelioration or prevention of autoimmune and inflammatory events associated with lymphopenia-related diseases.
  • a tenth eleventh aspect of the invention relates to use of the in vitro model of the invention for evaluating a pharmacological and/or chemical product.
  • the pharmacological and/or chemical product derived from this approach potentially impacting in the homeostatic proliferation process, and are useful for in the treatment, amelioration or prevention of autoimmune and inflammatory events associated with lymphopenia-related diseases.
  • Figure 1 Schematic representation of purity controls of cellular subsets isolations.
  • Figure 2 Schematic diagram of the gating strategy.
  • Figure 3 T-cell viability in different culture conditions, a) naive CD4 T-cells stimulated with aAPCs (1/1 ratio aAPC/Na ' ive); b) na ' ive CD4 T-cells stimulated with rlL7 (10 ng/ml); c) naive CD4 T-cells stimulated with both, aAPCs (1/1 ratio aAPC/Na ' ive) and rlL7 (10 ng/ml); d) na ' i ' ve CD4 T-cells without stimuli. Data are represented as median value and standard deviation (SD).
  • SD standard deviation
  • FIG. 4 Effect of different homeostatic stimuli on proliferation of human naive CD4 T- cells and representative plots, a) na ' i ' ve CD4 T-cells stimulated with aAPC (1/1 ratio aAPC/Naive); b) na ' i ' ve CD4 T-cells stimulated with rlL7 (10 ng/ml); c) naive CD4 T-cells stimulated with aAPC (1/1 ratio aAPC/Na ' ive) and rlL7 (10 ng/ml).
  • Plots (right panels) are representative of the 10 th , 22 th and 10 th days of culture, respectively. Bars data on left panels represent median value and standard deviation (SD). Fast homeostatic proliferation, slow homeostatic proliferation and non-proliferating T-cells are highlighted in right panels using red, green and blue boxes and lines, respectively.
  • Figure 5 Effect of different aAPC/Naive ratios and rlL7 concentrations on both types of homeostatic proliferation, a) effect of different aAPC/Na ' ive ratios on homeostatic proliferation process (with 2.5 ng/ml rlL7); b) effect of different concentrations of rlL7 (0-10 ng/ml) on homeostatic proliferation process (with 1/1 ratio aAPC/Na ' ive). Bars data are represented as median value Data are representative of at least 3 independent experiments.
  • FIG. 6 Immunophenotyping of the homeostatic proliferation process, a) cycling-marker ( ⁇ 67) upregulation during homeostatic proliferation; b) rlL7-related upregulation of MFI BCL2 during homeostatic proliferation. Data are represented as median value and standard deviation (SD). Fast homeostatic proliferation, slow homeostatic proliferation and non-proliferating T-cells are highlighted using red, green and blue dots, respectively.
  • FIG. 7 CD4 T-cell subsets generation during homeostatic proliferation. Data are represented as median value; a) CD4 T-cell subsets generated by slow homeostatic proliferation; b) CD4 T-cell subsets generated by fast homeostatic proliferation, c) CCR7 expression in memory cells generated by fast homeostatic proliferation, d) CD95 expression in in memory cells generated by fast homeostatic proliferation. Data are representative of 3 independent experiments.
  • Figure 8 OX40 and a4-integrin upregulation during the homeostatic proliferation (HP) of human naive CD4 T-cells. Purified naive CD4 T-cells were culture in presence of rlL7 (10 ng/ml) and irradiated autologous APCs (1/1 ratio).
  • Figure 10 Human homeostatic proliferation generates regulatory T cell (Treg); a) representative gating plot of FoxP3 upregulation during homeostatic proliferation; b) representative gating plot of generated total, resting and activated Treg; c) representative histograms of TGF- ⁇ production by the different Treg subsets; d) negative correlation between the frequencies of generated activated Tregs and the size of fast homeostatic proliferation; e) total Treg generation in different APCs/Na ' i ' ves CD4 T-cell ratios; f) total Treg generation in the different cellular subsets. Data are expressed as mean value and standard deviation (SD).
  • SD standard deviation
  • Figure 11 Generated regulatory T-cells show in vitro suppressive capacity; Sorted Treg subsets were seeded with autologous PBMCs in different Tconv/Treg ratios and then stimulated. The suppression capacity of the in vitro generated regulatory T-cells was assayed evaluating the expression of activation markers (CD69 and CD154) on conventional CD25 " CD4 T-cells after stimulation, a) expression of CD154 on CD25 " CD4 T-cells; b) expression of CD69 on CD25 " CD4 T-cells. Results are representative of 5-6 independent experiments.
  • activation markers CD69 and CD154
  • Figure 12 Scale-up of coculture system; we seeded increased numbers of naive CD4 T-cells and aAPCs with a fixed 1 :1 aAPC/Na ' i ' ve ratio in the presence of 2.5 ng/ml rlL7.
  • the examples of the present invention show a new in vitro model based on a cellular culture of human lymphocytes exposed to natural and autologous homeostatic stimuli, which allows simultaneously studying both types of homeostatic proliferation (fast and slow HP) in response to homeostatic stimuli.
  • the in vitro method allows to reproduce simultaneously both types of homeostatic proliferation process of human na ' ive CD4 T-cells, as well as to reproduce several in vivo observed phenomena related to such homeostatic proliferation.
  • the in vitro model of the present invention can be a useful tool to explore specific features of human homeostatic proliferation process in different lymphopenic human conditions and diseases.
  • the present invention relates to the use of antigen-presenting cells (APCs), preferably autologous antigen-presenting cells (aAPCs), for studying the homeostatic proliferation mechanisms in humans.
  • APCs antigen-presenting cells
  • aAPCs autologous antigen-presenting cells
  • the antigen-presenting cells are cells that display antigens soaked with major histocompatibility complexes (MHCs) on their surfaces; this process is known as antigen presentation. These cells process antigens and present them to T-cells. T cells may recognize these complexes using their T cell receptors (TCRs).
  • TCRs T cell receptors
  • the antigens that present these cells could be self-antigens or exogenous-antigens. Moreover, a fraction of such exogenous antigens are derived from commensal organisms (commensal antigens) and are tolerated by immune system. An important source of these commensal antigens is the microbioma of each individual. Both self- and commensal antigens have been reported as triggers of fast HP.
  • the present invention instead of other types of stimulation like anti-CD3/CD28 and in vitro generated APCs, use autologous APCs that potentially expose the self- and commensal-antigens present in each subject. This is important because: - the relative frequencies in the different host APCs subsets varies,
  • the functionality of the cells is variable (activated, not activated, more or less mature, etc %) which affect to their presentation capacity, - the type of antigens that are presented by these cells, which are highly variable between subjects.
  • the APCs is understood to comprise cells that display antigens soaked with major histocompatibility complexes (MHCs) on their surfaces.
  • MHCs major histocompatibility complexes
  • the APCs fraction comprises all B cells, dendritic cells (DC), myeloid derived suppressor cells (MDSC), plasmacytoid dendritic cells (pDCs), monocites and any other potential antigen-presenting cells speciffically present in each individual (donor for the experiment).
  • DC dendritic cells
  • MDSC myeloid derived suppressor cells
  • pDCs plasmacytoid dendritic cells
  • a second aspect of the present invention relates to a method to evaluate the homeostatic proliferation of T-cells of human subjects using their autologous APCs (aAPCs) as previously described , hereinafter first method of the invention, comprising: a) Isolation of two cellular subsets and control of purity from a human biological sample, wherein:
  • Cellular subset 1 comprising aAPCs
  • Cellular subset 2 comprising na ' i ' ve CD4 T-cells, b) Irradiate the aAPCs of the cellular subset 1 ,
  • step (e) Perform the basal analysis (day 0) in an aliquot of the cell culture of step (e),
  • the aAPCs are obtained after depleting T-cells (CD3+) and NK cells (CD56+).
  • the na ' i ' ve CD4 T-cells are obtained after an enrichment of CD4 T-cells and a subsequent depletion of CD45RO+ cells.
  • the aAPCs are irradiated in a ⁇ -irradiator at dosis of 15 Gy to 35 Gy, preferably 20 Gy to 30 Gy, and more preferably at 25 Gy (25J/kg).
  • the na ' i ' ve CD4 T-cells are stained with carboxyfluorescein succinimidyl ester (CFSE).
  • CFSE carboxyfluorescein succinimidyl ester
  • the concentration of na ' i ' ve CD4 T-cells in the culture medium is about 1x10 5 cells/mL.
  • the irradiated aAPCs and the na ' i ' ve CD4 T-cells in culture ranges in a ratio from 1 :1 to 1 :40, more preferably is about 1 :1.
  • the concentration of rlL7 ranges between 0 to 10 ng/ml, and more preferably is about 2.5 ng/ml.
  • a third aspect of the present invention relates to the in vitro model for studying homeostatic proliferation, hereinafter in vitro model of the invention, comprising the two cellular subsets as described in the first method of the invention.
  • an "in vitro model for studying homeostatic proliferation” means an in vitro representation of this particular phenomenon making this process easier to understand and to get conclusions.
  • a fourth aspect of the present invention relates to a method for obtaining data that can be used for the characterization and classification of human subjects suffering from a lymphopenia- related disease, hereinafter second method of the invention, comprising the steps according to the first method of the invention, and also comprising: i) quantitation of the T-cells that undergo homeostatic proliferations: slow (with partial CFSE dilution and maintenance of the na ' ive phenotype) and fast homeostatic proliferation (with full CFSE dilution and acquisition of memory phenotype).
  • the second method of the invention also comprises: ii) the characterization of different cellular markers in both types of proliferated cells including, at least, those related with differentiation and maturation (CD45 A,CD27,CCR7), activation (HLA- DR,CD25), survival (BCL-2),apoptosis-prone (CD95) or homing (37-integrin).
  • a "lymphopenia-related disease” means a human disorder characterized or showing an abnormally low level of peripheral lymphocytes in blood.
  • an allergic disease, transplant rejection and an autoimmune disease have one fundamental principle in common, they all are triggered by an imbalanced immune system which reacts hyperactive against a specific exogenic and/or endogenic challenge and therewith contributes significantly to the disease status.
  • Effector T cell-directed immunomodulation therefore is the key to successful treatment of asthma, an autoimmune condition, prevention of graft vs. host disease (GVHD) and prevention of graft rejection.
  • GVHD graft vs. host disease
  • T lymphocytes designated as regulatory T cells control immune responses by suppressing the effector function of CD4 + T cells and CD8 + T cells.
  • the lymphopenia-related disease is selected from the group consisting on: autoimmune diseases like systemic lupus erythematosus (SLE), rheumatoid arthritis or insulin-dependent diabetes mellitus, inflammatory diseases like Crohn's disease, allergic or asthmatic condition, graft versus host disease or graft rejection, or combinations thereof.
  • SLE systemic lupus erythematosus
  • rheumatoid arthritis or insulin-dependent diabetes mellitus
  • inflammatory diseases like Crohn's disease, allergic or asthmatic condition
  • graft versus host disease or graft rejection or combinations thereof.
  • a regulatory T cell means a CD4 T-cell subset which has a suppressive function over other immune cells preventing the excessive cellular activation and the occurrence of inflammation and autoimmune disorders.
  • the lymphopenia-related disease could be caused by the Human Immunodeficiency Virus (HIV) infection, which has been associated with several autoimmune and inflammatory disorders.
  • the lymphopenia- related disease is selected from potential rejection of a solid organ transplant or potential graft versus host disease.
  • a fifth aspect of the present invention relates to a method to characterize and classify a human subject suffering from lymphopenia-related disease, hereinafter third method of the invention, comprising the steps according to the first and/or the second method of the invention, and further comprising: iii) assigning the subjects from steps (i-ii) preferably attending to frequencies of slow and fast homeostatic proliferation and to ratio between these types of homeostatic proliferation.
  • the lymphopenia-related disease is selected from the group consisting on: autoimmune diseases like systemic lupus erythematosus (SLE), rheumatoid arthritis or insulin-dependent diabetes mellitus, inflammatory diseases like Crohn's disease, allergic or asthmatic condition, graft versus host disease or graft rejection, or combinations thereof.
  • SLE systemic lupus erythematosus
  • rheumatoid arthritis or insulin-dependent diabetes mellitus
  • inflammatory diseases like Crohn's disease, allergic or asthmatic condition
  • graft versus host disease or graft rejection or combinations thereof.
  • the lymphopenia-related disease is the Human Immunodeficiency Virus (HIV) infection.
  • HIV Human Immunodeficiency Virus
  • the lymphopenia-related disease is selected from potential rejection of a solid organ transplant or potential graft versus host disease.
  • a sixth aspect of the invention relates to a specific/autologous generated regulatory T cell during the HP process, hereinafter regulatory T cell of the invention, obtainable by the steps as described in the first method of the invention, and optionally comprising the cell sorting of such cells.
  • regulatory T cell of the invention obtainable by the steps as described in the first method of the invention, and optionally comprising the cell sorting of such cells.
  • cellular markers like CD25, CD45RA and the levels of CFSE could be used to get a cellular fraction enriched in such regulatory T-cells, although other marker associated to these cells like CTLA-4, FoxP3, CD39 or CD141 could be also used
  • a seventh aspect of the invention relates to a specific/autologous regulatory T cell population, hereinafter regulatory T cell population of the invention, obtainable by the steps as described in the first method of the invention, and optionally comprising cell sorting.
  • the regulatory T cell population of the invention comprises at least one regulatory T cell of the invention. More preferably, the regulatory T cell population is substantially pure.
  • said population of the invention is a cell population in which at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, of the cells are regulatory T cell of the invention.
  • At least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, of the cells in the population are regulatory T cells of the invention. More preferably the regulatory T cell and/or the regulatory T cell population of the invention are isolated.
  • the term "isolated” indicates that the regulatory T cell or regulatory T cell population to which it refers is not within its natural environment.
  • the regulatory T cell or regulatory T cell population has been substantially separated from surrounding tissue.
  • the cell or cell population is substantially separated from surrounding tissue if the sample contains at least about 75%, in some embodiments at least about 85%, in some embodiments at least about 90%, and in some embodiments at least about 95% regulatory T cells.
  • the sample is substantially separated from the surrounding tissue if the sample contains less than about 25%, in some embodiments less than about 15%, and in some embodiments less than about 5% of materials other than the regulatory T cells.
  • Such percentage values refer to percentage by weight or by cell number.
  • the term encompasses cells which have been removed from the organism from which they originated, and exist in culture.
  • the term also encompasses cells which have been removed from the organism from which they originated, and subsequently re-inserted into an organism.
  • the organism which contains the re-inserted cells may be a different organism, i.e. a different individual of the same species, or even still more preferable the same individual or organism from which the cells were removed.
  • composition of the invention comprising a regulatory T cell or regulatory T cell population of the invention.
  • the composition of the invention may comprise at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, of the regulatory T cells of the invention, either calculated by number, or by weight or by volume of the composition.
  • composition of the invention also comprises a pharmaceutically acceptable vehicle and/or a pharmaceutically acceptable excipient.
  • composition of the invention is a pharmaceutical composition.
  • active ingredient means any component which potentially provides a pharmacological activity or another different effect in diagnosing, curing, mitigating, treating, or preventing a disease, or which affects the structure or function of the human body or body of other animals.
  • active ingredients of biological origin include growth factors, hormones, and cytokines.
  • a variety of therapeutic agents is known in the art and may be identified by their effects. Certain therapeutic agents are capable of regulating cell proliferation and differentiation.
  • chemotherapeutic nucleotides examples include chemotherapeutic nucleotides, drugs, hormones, non-specific (non-antibody) proteins, oligonucleotides (e.g., antisense oligonucleotides that bind to a target nucleic acid sequence (e.g., m NA sequence)), peptides, and peptidomimetics.
  • compositions of the present invention can be used in a treatment method in an isolated manner or together with other pharmaceutical compounds.
  • pharmaceutically acceptable excipient refers to the fact that it must be approved by a regulatory agency of the federal government or a national government or one listed in the United States Pharmacopoeia or the European Pharmacopoeia, or some other pharmacopoeia generally recognized for use in animals and in humans.
  • vehicle relates to a diluent, excipient, carrier or adjuvant with which the regulatory T cells of the invention, as well as the cells of the cell population of the invention, must be administered; obviously, said vehicle must be compatible with the cells.
  • Illustrative, non-limiting examples of said vehicle include any physiologically compatible vehicle, for example isotonic solutions (e.g.
  • sterile saline solution (0.9% NaCI), phosphate -buffered saline solution (PBS), Ringer-lactate solution, etc.
  • serum preferably with autologous serum
  • culture media e.g. DMEM, RPMI, McCoy, etc.
  • a solid, semisolid, gelatinous or viscous support medium such as collagen, collagen-glycosamine-glycan, fibrin, polyvinyl chloride, poly- amino acids, such as polylysine, or polyornithine, hydrogels, agarose, dextran sulphate silicone.
  • the support medium can, in special embodiments, contain growth factors or other agents.
  • the cells can be introduced in a liquid phase of the vehicle that is treated subsequently so that it is converted into a more solid phase.
  • said vehicle in which the vehicle has a solid structure, said vehicle can be configured according to the form of the lesion.
  • the pharmaceutical composition of the invention can, if desired, also contain, when necessary, additives for increasing and/or controlling the desired therapeutic effect of the cells, e.g. buffering agents, surface-active agents, preservatives, etc.
  • the pharmaceutically acceptable carrier may comprise a cell culture medium which supports the cells' viability.
  • the medium will generally be serum-free in order to avoid provoking an immune response in the recipient.
  • the carrier will generally be buffered and/or pyrogen free.
  • chelating agents of metals for stabilizing the cellular suspension.
  • the stability of the cells in the liquid medium of the pharmaceutical composition of the invention can be improved by adding additional substances, such as, for example, aspartic acid, glutamic acid, etc.
  • Said pharmaceutically acceptable substances that can be used in the pharmaceutical composition of the invention are generally known by a person skilled in the art and are normally used in the production of cellular compositions.
  • suitable pharmaceutical vehicles are described in "Remington's Pharmaceutical Sciences” by E.W. Martin. Additional information on said vehicles can be found in any manual of pharmaceutical technology (that is, galenical pharmacy).
  • the pharmaceutical composition of the invention will be administered in a suitable pharmaceutical form of administration.
  • the pharmaceutical composition of the invention will be formulated according to the chosen form of administration.
  • the formulation will be adapted to the method of administration.
  • the pharmaceutical composition is prepared in a liquid, solid or semisolid dosage form, e.g. in the form of suspension, in order to be administered by implanting, injection or infusion to the subject needing treatment.
  • a pharmaceutically acceptable excipient e.g. an isotonic solution, for example, phosphate-buffered saline solution (PBS), or any other suitable, pharmaceutically acceptable vehicle, for administration to a subject parenterally, although other routes of administration can also be used.
  • PBS phosphate-buffered saline solution
  • composition of the invention to the subject who needs it will be carried out using conventional means.
  • said pharmaceutical composition of the invention can be administered to the subject parenterally using suitable devices such as syringes, catheters, trocars, cannulas, etc.
  • the pharmaceutical composition of the invention will be administered using equipment, apparatus and devices suitable for the administration of cellular compositions and known by a person skilled in the art.
  • direct administration of the pharmaceutical composition of the invention to the site that is intended to benefit may be advantageous.
  • direct administration of the pharmaceutical composition of the invention to the desired organ or tissue can be achieved by direct administration (e.g. by injection, etc.) on the external surface of the affected organ or tissue by inserting a suitable device, e.g. a suitable cannula, by infusion (including reverse flow mechanisms) or by other means described in this patent or known in the art.
  • the pharmaceutical composition of the invention can be stored until the moment of its application by the conventional methods known by a person skilled in the art.
  • the pharmaceutical composition of the invention can be stored at or below room temperature in a sealed container, supplemented or not with a nutrient solution.
  • Medium-term storage (less than 48 hours) is preferably carried out at 2-8 °C, and the pharmaceutical composition of the invention includes, in addition, an iso-osmotic, buffered solution.
  • Longer-term storage is preferably carried out by means of suitable cryopreservation and storage in conditions that promote the retention of cellular function.
  • the pharmaceutical composition of the invention can be used in combination therapy.
  • Said additional medicinal products can form part of the same pharmaceutical composition or can, alternatively, be supplied in the form of a separate composition for simultaneous or successive (sequential in time) administration relative to the administration of the pharmaceutical composition of the invention.
  • a ninth aspect of the invention relates to the specific/autologous generated regulatory T cell of the invention, the regulatory T cell population of the invention, and/or the composition of the invention, for use in therapy.
  • the present invention is able to generate specific/autologous regulatory T cell from non-differentiated na ' ive CD4 T cells through a homeostatic process, which constitutes a important advantage in comparison to others.
  • a tenth aspect of the present invention relates to the specific/autologous generated regulatory T cell of the invention, the regulatory T cell population of the invention, and/or the composition of the invention, for use in the treatment, amelioration or prevention of a lymphopenia-related disease.
  • a lymphopenia-related disease is a human disorder characterized or showing an abnormally low level of peripheral lymphocytes in blood.
  • the lymphopenia-related disease is selected from the group consisting on: autoimmune diseases, inflammatory diseases, allergic or asthmatic condition, graft versus host disease or for preventing graft rejection, or combinations thereof.
  • the lymphopenia-related disease is the Human Immunodeficiency Virus (HIV) infection.
  • Human Immunodeficiency Virus (HIV) infection is characterized by a progressive loss of CD4 T-cells that hampers the function of the immune system. As a result, those afflicted with the syndrome are susceptible to a variety of opportunistic infections.
  • the etiologic agent is a cytopathic retrovirus designated the human immunodeficiency virus (HIV).
  • T helper cells CD4+ cells. The infection of T helper cells by HIV results in a profound dysregulation of the immune system including both depleted numbers and impaired functioning of T lymphocytes.
  • the specific regulatory T cell population according to claim that could be obtained using APCs and the naive CD4 T-cells from the donor of the solid organ for transplantation, or using APCs and the naive CD4 T-cells from the recipient of the solid organ for transplantation.
  • the lymphopenia-related disease is the inflammatory bowel disease.
  • the lymphopenia-related disease is selected from potential rejection of a solid organ transplant or potential graft versus host disease.
  • the specific regulatory T cell population could be obtained using APCs and the naive CD4 T- cells from the donor of the solid organ for transplantation, or using APCs and the naive CD4 T- cells from the recipient of the solid organ for transplantation.
  • a preferred embodiment of this aspect of the invention refers to the regulatory T cell or a regulatory T cell population of the invention for use in the treatment, amelioration or prevention of transplant rejection, wherein the regulatory T cells are obtained from the host.
  • a preferred embodiment of this aspect of the invention refers to the regulatory T cell or a regulatory T cell population of the invention for use in the treatment, amelioration or prevention of graft versus host disease, wherein the regulatory T cells are obtained from the donor.
  • the in vitro model of the invention is useful for developing and testing of drugs or any therapeutic intervention in the global scenario of lymphopenia-related diseases, and more particularly, for HIV infection, trying to regulate the homeostatic proliferation process, since some immune alterations found in such scenario are congruent with an exacerbation of such process.
  • HIV-infected subjects known as immunodiscordants to antiretroviral treatment who show a chronic and severe lymphopenic state despite therapeutic control of viremia.
  • an eleventh aspect of the invention relates to use of the in vitro model of the invention for evaluating a pharmacological and/or chemical product.
  • Buffy coat from HIV-1 and hepatitis C virus (HCV)-seronegative blood bank donors were obtained from the Centro Regional de Transfusion Sanguinea de Sevilla-Huelva y Banco de Tejidos (Seville, Spain).
  • HCV hepatitis C virus
  • peripheral blood mononuclear cells were firstly obtained from buffy coats using a ficoll gradient centrifugation (2500 rpm, 30 min), and then were washed and placed in cold supplemented medium (RPMI 1640 supplemented with 10% fetal bovine serum, 1.7 mM glutamine, 100 ⁇ /ml streptomicine and 100 U/ml peniciline). Magnetic negative isolation (StemCell, Vancouver, Canada) was used according to manufacturer's instructions to isolate cellular fractions from previously obtained PBMCs.
  • aAPCs were obtained after depleting T-cells (CD3+) and NK cells (CD56+) (human CD3 and CD56 positive selection kits), whereas naive CD4 T-cells were obtaining after an enrichment of CD4 T-cells and a subsequent depletion of CD45RO+ cells (human NAIVE CD4+ T-cell enrichment kit).
  • Treg- depleted fraction was obtained by depletion of CD25+ cells (human CD25 positive selection kit) in the naive T-cell fraction.
  • a purity control was always performed after each isolation procedure by flow cytometry and only cellular fractions with a purity >95% were used in experiments.
  • a representative example is shown in Figure 1. CFSE staining
  • na ' ive CD4 T-cells were stained with carboxyfluorescein succinimidyl ester (CFSE) (Sigma-Aldrich, Missouri, USA) to determine lymphocyte proliferation. Briefly, up to 1 x10 7 cells/mL of na ' i ' ve CD4 T-cells were stained in PBS (Phosphate-buffered saline) with an excess of CFSE (2.5 ⁇ ) during 8 minutes at 37°C. Finally, cells were washed and placed in cold supplemented medium.
  • CFSE carboxyfluorescein succinimidyl ester
  • APCs antigen-presenting cells
  • a v- irradiator IBL437C, Cis biolnternational, France
  • DMSO dimethil Sulfoxide
  • na ' ive CD4 T-cells per well were seeded in a 96-well plate (Thermo Fissher, Massachusetts, USA) with supplemented medium at a concentration of 1 x10 6 cells/mL. Then, the homeostatic stimuli (autologous APCs and rlL7) were provided. Specifically, 200.000 irradiated aAPCs were added, stablishing a 1 :1 ratio of aAPCs/naive CD4 T-cells (aAPC/Naive ratio), although other aAPC/Naive ratios were also tested, varying the amount of aAPC fraction.
  • rlL7 was used at a concentration between 1 -10 ng/ml, whereas the condition without rlL7 only included the rlL7-carrier solution (PBS with 2% fetal bovine serum and 1 mM EDTA). All these components were placed at the 96-well plate and an aliquot was therefore used to perform the basal analyses (day 0). The culture was then placed in a culture incubator at 37°C and 5% CO 2 atmosphere. After each 5 days of culture, the half of medium volume was refreshed with new supplemented medium and thawed irradiated aAPCs and rlL7.
  • PBS 2% fetal bovine serum and 1 mM EDTA
  • Immunophenotyping was carried out using multiparametric flow cytometry in the general condition of culture. Briefly, cells were incubated at 4°C with surface antibodies for 20 minutes, followed by fixation and permeabilization according to the manufacturer's instructions (Foxp3/Transcription Factor Staining Buffer, Ebioscience), and subsequently incubated with intracellular antibodies for 30 minutes. Viable cells were identified using LIVE/DEAD fixable Aqua Blue Dead Cell Stain (Life Technologies, USA).
  • Extracellular and intracellular antibodies were: anti-CD3, anti-CD27, anti-CD197/CCR7, anti-CD25, anti-HLADR, anti-FoxP3, anti- CD45RA, anti-TGF- ⁇ , anti-Ki67, anti-BCL2, anti-CD134/OX40, anti-CD95, anti-37-integrin (BD Biosciences, USA); anti-CD49d/a4-integrin (Biolegend, USA). Fluorescence Minus One (FMO) and isotype controls were included when necessary. A schematic diagram of the gating strategy used is shown in Figure 2.
  • CD4 T-cell subsets were defined as follows: naive (T N ; CD45RA + CD27 + ), central memory (T CM; CD45RA " CD27 + CCR7 + ), transitional memory (T TM ; CD45RA " CD27 + CCR7 “ ), effector memory (T EM ; CD45RA CD27 “ ) and terminally differentiated (TEMRAI CD45RA + CD27 " ).
  • Fast homeostatic proliferation was defined as viable CD4 T-cells which fully diluted CFSE and acquired memory phenotype (CD45RA " ), while slow homeostatic proliferation was defined as viable CD45RA + CD4 T-cells which partially diluted CFSE, without acquiring memory phenotype.
  • Treg subsets were defined as follows: total Treg (CD25 hi9h FoxP3 hi9h ), resting Treg (CD45RA + FoxP3 + ) and activated Treg (CD45RA " FoxP3 high ).
  • Flow cytometry was performed on a LSR Fortessa (BD, USA). Analysis was performed using FlowJo version 9.2 (Tree Star) and data is expressed as mean values and standard deviation (o) of the frequencies and MFI (Mean Fluorescence Intensity). Graphs were generated using Prism version 5.0 (GraphPad Software, USA). Cell sorting of Regulatory T -cells
  • Tregs We sorted generated Tregs to determine their suppression capacity. After 10 days of culture, cells were washed and stained at 4°C with surface antibodies for 20 minutes. Antibodies used were: anti-CD3, anti-CD25, anti-CD45RA, anti-CD4 (BD Biosciences, USA). Treg subsets were defined and sorted as follows: resting Treg (CD45RA + CD25 + ) and activated Treg (CD45RA " CD25 hl9h ). Sorted was performed in a Facs Aria Fusion (BD Bioscience, USA).
  • the suppression capacity of the in vitro generated regulatory T-cells was assayed following the manufacturer instructions of the Regularoty T-Cell Functon Kit (BD bioscience, USA). Briefly, frozen autologous PBMCs previously isolated from Buffy coat were thawed and resting overnight in a culture incubator at 37°C and 5% CO 2 atmosphere. Then, PBMCs were stimulated with Dynabeads Human T-Activator CD3/CD28 (Life Technologies, USA), and seeded at 1 :2 and 1 :4 sorted Treg/PBMCs and placed in a culture incubator at 37°C and 5% CO 2 atmosphere.
  • HLA-DR and CD25 we also explored whether the HP was associated with an increase in the expression of cellular activation markers.
  • HLA-DR and CD25 we observed that cells undergoing fast HP strongly upregulated HLA-DR expression, whereas no effect was observed in non- proliferating na ' i ' ve T-cells and a negligible upregulation seemed to occur in cells undergoing slow HP (Figure 9. a). As it can be observed, this effect was independent of the rlL7 concentration.
  • CD25 expression we also found that cells undergoing fast HP strongly upregulated such activation marker in a rlL7-independent manner ( Figure 9.b). In contrast with HLA-DR expression, we observed a rlL7-dependent upregulation of CD25 in non- proliferating cells and cells undergoing slow HP.
  • Homing molecules are upregulated during the homeostatic proliferation process
  • Treg regulatory T-cells
  • Treg were able to produce suppressive cytokines as TGF- ⁇ , showing the activated Treg (CD45RA " Foxp3 high ) the highest TGF- ⁇ production, whereas resting Treg (CD45RA + Foxp3 + ) showed the lowest (Figure 10.c).
  • Figure 10. d we also found an interesting negative association between the frequency of activated Treg generated by HP, and the size of fast homeostatic proliferation (Figure 10. d). Such association was not present with other types of Treg (data not shown).
  • Treg generation during HP was highly dependent on cellular contact with aAPC ( Figure 10.e), but no dependence on rlL7 concentrations was observed ( Figure 10.f). Similar results were found with the other phenotypes of Treg.
  • Treg subsets were resting Treg (CD45RA + CD25 + ) and activated Treg (CD45RA " CD25 high ). Both Treg subsets, resting and activated Treg showed suppressive capacity (Fig. 1 1 a-b), which showed a dose-dependent effect depending on the Treg/Tconv ratio used in each condition. Scale-up of coculture system

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Abstract

L'invention concerne un système in vitro de culture de lymphocytes autologues, une méthode permettant d'identifier et de classifier des individus souffrant d'une maladie liée à une lymphopénie, et l'utilisation des lymphocytes T régulateurs obtenus par une méthode de la présente invention pour traiter des maladies auto-immunes, des maladies inflammatoires, un état allergique ou asthmatique, ou une maladie du greffon contre l'hôte, ou pour prévenir un rejet de greffe.
PCT/EP2018/062256 2017-05-11 2018-05-11 Utilisations diagnostiques et thérapeutiques d'un système in vitro de culture de lymphocytes autologues Ceased WO2018206794A1 (fr)

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WO2013076268A1 (fr) * 2011-11-23 2013-05-30 INSERM (Institut National de la Santé et de la Recherche Médicale) Population de cellules t immunorégulatrices spécifiques d'un antigène étranger et ses utilisations pour prévenir ou traiter des maladies immunitaires
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Publication number Priority date Publication date Assignee Title
WO2013076268A1 (fr) * 2011-11-23 2013-05-30 INSERM (Institut National de la Santé et de la Recherche Médicale) Population de cellules t immunorégulatrices spécifiques d'un antigène étranger et ses utilisations pour prévenir ou traiter des maladies immunitaires
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A. GRUBER ET AL: "MHC Class I-Positive Dendritic Cells (DC) Control CD8 T Cell Homeostasis In Vivo: T Cell Lymphopenia as a Prerequisite for DC-Mediated Homeostatic Proliferation of Naive CD8 T Cells", THE JOURNAL OF IMMUNOLOGY, vol. 175, no. 1, 1 July 2005 (2005-07-01), US, pages 201 - 206, XP055492016, ISSN: 0022-1767, DOI: 10.4049/jimmunol.175.1.201 *
BEQ STEPHANIE ET AL: "IL-7 induces immunological improvement in SIV-infected rhesus macaques under antiviral therapy", JOURNAL OF IMMUNOLOGY, vol. 176, no. 2, January 2006 (2006-01-01), pages 914 - 922, XP002782995, ISSN: 0022-1767 *
BOYMAN ONUR ET AL: "Homeostatic proliferation and survival of naive and memory T cells", EUROPEAN JOURNAL OF IMMUNOLOGY, vol. 39, no. 8, August 2009 (2009-08-01), pages 2088 - 2094, XP002782997, ISSN: 0014-2980 *
CIMBRO RAFFAELLO ET AL: "IL-7 induces expression and activation of integrin alpha 4 beta 7 promoting naive T-cell homing to the intestinal mucosa", BLOOD, vol. 120, no. 13, September 2012 (2012-09-01), pages 2610 - 2619, XP002782994, ISSN: 0006-4971 *
DO JEONG-SU ET AL: "Both exogenous commensal and endogenous self antigens stimulate T cell proliferation under lymphopenic conditions", CELLULAR IMMUNOLOGY, vol. 272, no. 2, 2012, pages 117 - 123, XP002782996, ISSN: 0008-8749 *
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