How to regenerate the thymus

Abstract

The present invention relates to a method for treating aging, and in particular immunosenescence, restoring thymic function and regenerating the thymus in a subject in need thereof, comprising the administration of T cell progenitors comprising CD7+CD34- cells.

Description

[Technical Field]

[0001] The present invention relates to the field of treatment, particularly therapeutic treatment, of aging, including immunosenescence. In particular, the present invention relates to a method of restoring polyclonal T cell production by the thymus of a subject by restoring thymic function in the subject. [Background technology]

[0002] Thymic hypoplasia secondary to aging leads to a restricted T cell repertoire and subsequent immunodeficiency. Notably, thymic involution is largely dependent on contraction of the thymic epithelium. Improving intrathymic T cell development and selection is therefore crucial for replenishment of the peripheral T cell compartment.

[0003] Previously, a feeder-free platform for producing clinical-grade, ex vivo human early T cell precursors was disclosed based on the use of immobilized delta-like ligand 4 (DLL4) and cytokines to enable the expansion and differentiation of CD34+ hematopoietic stem and progenitor cells (HSPCs) into human T lymphoid progenitors (HTLPs, also called T cell progenitors).

[0004] In the present invention, Applicants show that these early T cell precursors, when administered to subjects with immunosenescence, induce thymic regeneration and restore thymic function, in particular the ability of the thymus to produce polyclonal T cells. Summary of the Invention

[0005] The present invention relates to a method for treating senescence, particularly immunosenescence, in a subject, comprising administering to the subject T cell progenitors comprising CD7+CD34- cells.

[0006] The present invention also relates to a method of restoring thymic function in a subject, comprising administering to the subject CD7+CD34- T cell progenitors.

[0007] The present invention further relates to a method for regenerating the thymus in a subject.

[0008] In some embodiments, the methods of the present invention are for restoring polyclonal T cell production by the thymus of a subject.

[0009] In some embodiments, the subject is a human.

[0010] In some embodiments, the subject is at least about 50 years old, preferably about 60, 70, 80, or 90 years old.

[0011] In some embodiments, the subject is affected by or has been diagnosed with thymic atrophy or thymic involution.

[0012] In some embodiments, the CD7+CD34- cells include cells that are CD5- and / or CD1a+, preferably at least about 50%, or at least about 60, 70, 80, 90, or 95% of the CD7+CD34- cells are CD5- and / or CD1a-.

[0013] In some embodiments, the T cell progenitors are genetically modified, for example, to express an exogenous protein.

[0014] In some embodiments, the T cell progenitors are transfected or transduced with exogenous nucleic acid encoding a chimeric antigen receptor (CAR) or a recombinant TCR.

[0015] In some embodiments, the T cell progenitors express cytokines, such as RANKL or lymphotoxin A (LTA).

[0016] In some embodiments, T cell progenitors are prepared by culturing CD34+ cells in the presence of immobilized Notch ligand or a fragment thereof, preferably the Notch ligand or a fragment thereof is immobilized on the inner surface of a culture vessel or on beads.

[0017] In some embodiments, the cells are also exposed to fibronectin or a fibronectin fragment, the fragment comprising an RGD motif, a CS-1 motif, and / or a heparin-binding domain. In some embodiments, the fibronectin fragment is Retronectin®.

[0018] In some embodiments, the fibronectin or fibronectin fragments are immobilized on the interior surface of a culture vessel or on beads.

[0019] In some embodiments, CD34+ cells are cultured in a culture medium containing TNF-α and / or an antagonist of the aryl hydrocarbon / dioxin receptor.

[0020] In some embodiments, the aryl hydrocarbon / dioxin receptor antagonist is StemRegenin 1 (SR1).

[0021] In some embodiments, the CD34+ cells are isolated from a human. In some embodiments, the CD34+ cells are isolated from an adult donor. In some embodiments, the CD34+ cells are isolated from umbilical cord blood.

[0022] In some embodiments, the T cell progenitors are autologous. In some embodiments, the T cell progenitors are allogeneic.

[0023] The present invention further relates to CD7+CD34- comprising T cell progenitors for treating or for use in treating aging, and in particular immunosenescence, in a subject.

[0024] The present invention also relates to CD7+CD34- comprising T cell progenitors for restoring or for use in restoring thymic function in a subject.

[0025] The present invention further relates to CD7+CD34- comprising T cell progenitors for regenerating or for use in regenerating the thymus of a subject.

[0026] In some embodiments, the T cell progenitors are for use in restoring or restoring polyclonal T cell production by the thymus of a subject.

[0027] In some embodiments, the subject is a human.

[0028] In some embodiments, the subject is at least about 50 years old, preferably about 60, 70, 80, or 90 years old.

[0029] In some embodiments, the subject is affected by or has been diagnosed with thymic atrophy or thymic involution.

[0030] In some embodiments, the CD7+CD34- cells include cells that are CD5- and / or CD1a+, preferably at least about 50%, or at least about 60, 70, 80, 90, or 95% of the CD7+CD34- cells are CD5- and / or CD1a-.

[0031] In some embodiments, the T cell progenitors are genetically modified, such as to express an exogenous protein. For example, the exogenous protein may be a chimeric antigen receptor (CAR) or a recombinant TCR.

[0032] In some embodiments, T cell progenitors are transfected or transduced with exogenous nucleic acid encoding, for example, a chimeric antigen receptor (CAR) or a recombinant TCR.

[0033] In some embodiments, the T cell progenitors express cytokines, such as RANKL or lymphotoxin A (LTA).

[0034] In some embodiments, T cell progenitors are prepared by culturing CD34+ cells in the presence of immobilized Notch ligand or a fragment thereof, preferably the Notch ligand is immobilized on the inner surface of a culture vessel or on beads.

[0035] In some embodiments, the cells are also exposed to fibronectin or a fibronectin fragment, wherein the fragment comprises an RGD motif, a CS-1 motif, and / or a heparin-binding domain.

[0036] Thus, in some embodiments, the cells are also exposed to immobilized Notch ligand or a fragment thereof and fibronectin or a fibronectin fragment.

[0037] In some embodiments, the fibronectin or fibronectin fragments are immobilized on the interior surface of a culture vessel or on beads.

[0038] In some embodiments, the fibronectin fragment is Retronectin®.

[0039] In some embodiments, the CD34+ cells are cultured in a culture medium containing TNF-α and / or an aryl hydrocarbon / dioxin receptor antagonist, hi some embodiments, the aryl hydrocarbon / dioxin receptor antagonist is StemRegenin 1 (SR1).

[0040] In some embodiments, the CD34+ cells are isolated from a human. For example, the CD34+ cells may be isolated from an adult donor or umbilical cord blood.

[0041] In some embodiments, the T cell progenitors are autologous.

[0042] In some embodiments, the T cell progenitors are allogeneic.

[0043] definition For the purposes of the present invention, the following terms have the following meanings:

[0044] "About," when referring to a measurable value, such as an amount, time, etc., is meant to encompass variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the stated value, as such variations are suitable for practicing the disclosed methods.

[0045] "Cortical thymic epithelial cells (cTECs)" refer to structural components of the thymic cortex. cTECs are known to play essential roles in the commitment of early thymocyte precursors to the T cell lineage and in the positive selection of T cells.

[0046] "Medullary thymic epithelial cells (mTECs)" refer to structural components of the thymic medulla. mTECs are known to play essential roles in thymocyte maturation and the establishment of central tolerance (negative selection of T cells).

[0047] A "pharmaceutically acceptable excipient" refers to an excipient that does not produce adverse reactions, allergic reactions, or other side effects when administered to mammals, preferably humans. Pharmaceutically acceptable excipients include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, and absorption delaying agents. A pharmaceutically acceptable carrier or excipient refers to any type of non-toxic solid, semisolid, or liquid filler, diluent, encapsulating substance, or formulation auxiliary. For human administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards required by regulatory authorities such as the FDA or EMA.

[0048] "Expressed," "positive," or "+," and "not expressed," "negative," or "-" refer to the expression level of the target cell marker, where the expression level of the cell marker corresponding to "+" is high, intermediate, or low (i.e., the cell marker is expressed or present on the cell surface), and the expression level of the cell marker corresponding to "-" is zero (i.e., the cell marker is not expressed or present on the cell surface).

[0049] "Subject" refers to a living organism (e.g., a mammal, particularly a human, primate, dog, cat, horse, sheep, etc.) in which an immune response can be elicited. In some embodiments, the subject is a human. In some embodiments, the subject may be a "patient," i.e., a warm-blooded animal, preferably a human, awaiting or receiving medical care, or receiving medical care, or having been / is / will be the subject of a medical procedure, or being monitored for the development of a target disease or condition. In some embodiments, the subject is male. In other embodiments, the subject is female.

[0050] A "therapeutically effective amount" refers to an amount of cells or compositions described herein effective to achieve a particular biological result. Thus, the term "therapeutically effective amount" refers to a concentration or amount of a composition or number of cells targeted to (1) delay or prevent the onset of a target disease or condition; (2) slow or halt the progression, worsening, or deterioration of one or more symptoms of a target disease or condition; (3) bring about the amelioration of symptoms of a target disease or condition; (4) reduce the severity or prevalence of a target disease or condition; or (5) cure a target disease or condition, without causing significant negative or harmful side effects to the target. A therapeutically effective amount may be administered before the onset of a target disease or condition for prophylactic or preventative effects. Alternatively, or in addition, a therapeutically effective amount may be administered after the onset of a target disease or condition for therapeutic effects.

[0051] "Transfection" or "transduction" refers to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid, and includes the primary subject cell and its progeny.

[0052] "Treating" refers to therapeutic treatment and prophylactic or preventative measures, where the goal is to prevent or slow (alleviate) the target disease or condition, or both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already suffering from the condition, as well as those susceptible to the condition, or those in whom the condition is to be prevented. A subject is successfully "treated" for a disease or condition if, after receiving a therapeutic amount of cells or compositions described herein, the cells show an observable and / or measurable improvement in one or more of the following: an increase in thymus size; an increase in medulla size; restoration of normal thymus structure; an increase in polyclonal T cell production; alleviation, to some extent, of one or more symptoms associated with the particular condition; a reduction in morbidity and mortality; and / or an improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in a condition can be readily measured using routine procedures familiar to physicians. DETAILED DESCRIPTION OF THE INVENTION

[0053] The present invention relates to a method for treating senescence, particularly immunosenescence, in a subject, comprising administering to the subject T cell progenitors comprising CD7+CD34- cells.

[0054] In some embodiments, immunosenescence is a condition associated with aging in which a subject's immune system becomes impaired (also referred to as immunosenescence), resulting in a gradual loss of the subject's ability to protect the body from diseases or conditions, including, for example, infections or cancer. Immunosenescence can be associated, for example, with abnormal thymus structure, resulting in decreased production of naive T cells by the thymus and decreased production of T cell progenitors by the bone marrow. Immunosenescence can also be associated with inflammation (which can be referred to as inflammaging). Inflammaging can be particularly associated with increased concentrations of pro-inflammatory markers in cells and tissues, as well as chronic inflammation.

[0055] The present invention also relates to methods for preventing, reducing, or slowing the progression of thymic atrophy or involution, which may be caused by disease (e.g., DiGeorge syndrome), aging, or treatment (e.g., chemotherapy or radiation therapy), comprising administering to a subject T cell progenitors comprising CD7+CD34- cells.

[0056] In some embodiments, thymic atrophy or involution is age-related, hi some embodiments, thymic atrophy or involution is not age-related but may be caused, for example, by disease or treatment with a thymic toxic agent (e.g., chemotherapy or radiation therapy).

[0057] The present invention also provides a method for restoring thymic function in a subject, comprising: + CD34 - In some embodiments, the method comprises restoring thymopoiesis, i.e., differentiation of thymocytes into mature T cells, proliferation of mature T cells, positive selection of differentiating T cells, and / or establishment of central tolerance (e.g., including negative selection of T cells).

[0058] The present invention also relates to methods for regenerating a subject's thymus, e.g., to restore normal function and / or structure of the thymus that has been impaired by aging or by treatment, e.g., chemotherapy or radiation therapy. In some embodiments, the methods include restoring the normal structure of the thymic epithelium, including the medulla and cortex. For example, restoring the normal structure of the thymic epithelium may be associated with an increase in the number and / or proportion of medullary thymic epithelial cells (mTECs).

[0059] The present invention also relates to a method for populating the empty thymus in patients suffering from severe immunodeficiency or in patients who lack endogenous T cell progenitor production.

[0060] The present invention further relates to T cell progenitors comprising CD7+CD34− cells for treating or for use in the treatment of aging, particularly immunosenescence, in a subject in need thereof.

[0061] The present invention further relates to a composition comprising or consisting essentially of T cell progenitors comprising CD7+CD34− cells for treating or for use in the treatment of aging, particularly immunosenescence, in a subject in need thereof.

[0062] As used herein, the term "consisting essentially of" in reference to a composition, pharmaceutical composition, or medicament means that the T cell progenitor is the only biologically active therapeutic agent or agent in the composition, pharmaceutical composition, or medicament.

[0063] The present invention further relates to the use of T cell progenitors comprising CD7+CD34- cells for treating senescence, particularly immunosenescence, in a subject in need thereof.The present invention further relates to the use of a composition comprising or consisting essentially of T cell progenitors comprising CD7+CD34- cells for treating senescence, particularly immunosenescence, in a subject in need thereof.

[0064] The present invention further relates to CD7+CD34- comprising T cell progenitors for preventing, delaying or reducing the progression of thymic atrophy or involution, or for use in preventing, delaying or reducing the progression of thymic atrophy or involution.The present invention further relates to compositions comprising or consisting essentially of CD7+CD34- comprising T cell progenitors for preventing, delaying or reducing the progression of thymic atrophy or involution, or for use in preventing, delaying or reducing the progression of thymic atrophy or involution.

[0065] The present invention further relates to the use of CD7+CD34- comprising T cell progenitors to prevent, delay or reduce the progression of thymic atrophy or involution.The present invention further relates to the use of a composition comprising or consisting essentially of CD7+CD34- comprising T cell progenitors to prevent, delay or reduce the progression of thymic atrophy or involution.

[0066] The present invention further relates to T cell progenitors comprising CD7+CD34- cells for use in restoring thymic function in a subject.The present invention further relates to compositions comprising or consisting essentially of T cell progenitors comprising CD7+CD34- cells for use in restoring thymic function in a subject.

[0067] The present invention further relates to the use of T cell progenitors comprising CD7+CD34- cells for restoring thymic function in a subject.The present invention further relates to the use of a composition comprising or consisting essentially of T cell progenitors comprising CD7+CD34- cells for restoring thymic function in a subject.

[0068] In some embodiments, the T cell progenitors comprising CD7+CD34- cells are for use in regenerating or regenerating the thymus of a subject. In some embodiments, a composition comprising or consisting of T cell progenitors comprising CD7+CD34- cells is for use in regenerating or regenerating the thymus of a subject.

[0069] The present invention further relates to T cell progenitors comprising CD7+CD34- cells for use in restoring normal function and / or structure of the thymus, e.g., impaired by aging or by treatments such as chemotherapy or radiotherapy, or for use in regeneration.The present invention further relates to compositions comprising or consisting of T cell progenitors comprising CD7+CD34- cells for use in restoring normal function and / or structure of the thymus, e.g., impaired by aging or by treatments such as chemotherapy or radiotherapy, or for use in regeneration.

[0070] The present invention further relates to the use of T cell progenitors comprising CD7+CD34- cells to regenerate the thymus of a subject.The present invention further relates to the use of a composition comprising or consisting of T cell progenitors comprising CD7+CD34- cells to regenerate the thymus of a subject.

[0071] The present invention further relates to T cell progenitors comprising CD7+CD34- cells for populating or for use in populating an empty thymus in patients suffering from a severe immunodeficiency or in patients who lack endogenous T cell progenitor production.The present invention further relates to compositions comprising or consisting of T cell progenitors comprising CD7+CD34- cells for populating or for use in populating an empty thymus in patients suffering from a severe immunodeficiency or in patients who lack endogenous T cell progenitor production.

[0072] The present invention further relates to the use of T cell progenitors comprising CD7+CD34- cells to populate empty thymuses in patients suffering from severe immunodeficiencies or who lack endogenous T cell progenitor production.The present invention further relates to the use of compositions comprising or consisting essentially of T cell progenitors comprising CD7+CD34- cells to populate empty thymuses in patients suffering from severe immunodeficiencies or who lack endogenous T cell progenitor production.

[0073] In some embodiments, the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable excipient.

[0074] Examples of pharmaceutically acceptable excipients that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, saturated vegetable fatty acids such as protamine sulfate, water, partial glyceride mixtures of salts or electrolytes, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances (e.g., sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0075] In some embodiments, the composition is a drug.

[0076] The present invention further relates to the use of T cell progenitors comprising CD7+CD34− cells for the manufacture of a medicament for treating aging, particularly immunosenescence, in a subject in need thereof.

[0077] The present invention further relates to the use of CD7+CD34- comprising T cell progenitors for the manufacture of a medicament for preventing, delaying or reducing the progression of thymic atrophy or involution.

[0078] The present invention further relates to the use of T cell progenitors, including CD7+CD34- cells, for the manufacture of a medicament for restoring thymic function in a subject.

[0079] The present invention further relates to the use of T cell progenitors, including CD7+CD34− cells, for the manufacture of a medicament for populating the empty thymus in patients suffering from severe immunodeficiency or in patients lacking endogenous T cell progenitor production.

[0080] In some embodiments, a therapeutically effective amount of T cell progenitors is administered to or intended for administration to a subject in need thereof.

[0081] In some embodiments, a therapeutically effective amount of T cell progenitors is administered to or is intended for administration to a subject in need thereof to regenerate the subject's thymus, hi some embodiments, a therapeutically effective amount of T cell progenitors is administered to or is intended for administration to a subject in need thereof to regenerate the subject's thymus, e.g., to restore normal function and / or structure of the thymus that has been impaired by aging or by treatment, e.g., chemotherapy or radiation therapy.

[0082] In some embodiments, a therapeutically effective amount of primitive T cells is about 1 x 10 6 ~about 100~10 6 Cells / kg body weight, preferably about 1 x 10 6 ~About 10×10 6 cells / kg body weight range.

[0083] In some embodiments, the subject is a human. In one embodiment, the subject is male. In some embodiments, the subject is female.

[0084] In some embodiments, the subject is an adult.

[0085] In some embodiments, the subject is at least about 50 years old, preferably about 60, 70, 80, or 90 years old.

[0086] In some embodiments, the subject is a patient with a severe immunodeficiency or a patient lacking endogenous T cell progenitor production. Examples of causes of severe immunodeficiency include, but are not limited to, inherited immunodeficiencies, immunodeficiencies induced by chemotherapy (e.g., to treat leukemia), conditioning, stem cell-only transplants, post-transplant treatments to prevent graft-versus-host disease (GVH), patient age, partial thymectomy, autoimmune diseases, and coexisting conditions such as infections.

[0087] In some embodiments, the subject suffers from, or preferably has been diagnosed with, thymic atrophy (also known as thymic involution). Thymic atrophy is associated with a loss of thymocytes and ultimately, the destruction of thymic architecture. Thymic atrophy results in a decrease in naive T cells and limited T cell receptor diversity. Thus, in some embodiments, the methods of the present invention are for restoring polyclonal T cell production by the subject's thymus. Thymic atrophy / involution is also associated with a decrease in thymic epithelial cells (TECs), i.e., a decrease in cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells (mTECs), as well as the accumulation of adipose tissue in the thymus.

[0088] In some embodiments, the subject is at risk for thymic atrophy. The risk of thymic atrophy may be associated with disease, aging, or treatment toxicity. In some embodiments, the subject is at risk for severe immunodeficiency.

[0089] In some embodiments, the subject does not suffer from and has not been diagnosed with a severe immunodeficiency, hi some embodiments, the subject is not immunosuppressed.

[0090] In some embodiments, the subject is in need of a hematopoietic stem cell transplant, e.g., of CD34+ cells. In some embodiments, the subject has received, is receiving, or will receive a hematopoietic stem cell transplant, e.g., of CD34+ cells.

[0091] In some embodiments, administration of the T cell progenitor occurs immediately before, immediately after, or concomitantly with hematopoietic stem cell transplantation, such as with CD34+ cells, in the subject.

[0092] In some embodiments, the subject is not in need of a hematopoietic stem cell transplant, e.g., of CD34+ cells. In some embodiments, the subject has not received, has not received, or will not receive a hematopoietic stem cell transplant, e.g., of CD34+ cells.

[0093] In some embodiments, the T cell progenitors are genetically modified, e.g., to express an exogenous protein. In some embodiments, the T cell progenitors are transfected or transduced with an exogenous nucleic acid, which can, for example, encode an exogenous protein.

[0094] In some embodiments, the T cell progenitors are genetically modified to silence a gene of interest or reduce expression of the gene of interest by the cells.

[0095] In some embodiments, the T cell progenitor is genetically modified with a viral vector, a nucleic acid fragment, a plasmid, or a plasmid RNA or DNA sequence. In some embodiments, the T cell progenitor is genetically modified using gene editing, a base editing system, or a prime editing system.

[0096] In some embodiments, the T cell progenitor comprises a nucleic acid sequence encoding an element suitable for gene editing. In some embodiments, the element suitable for gene editing is a meganuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector-based nuclease (TALEN), a CRISPR-Cas nuclease, or a transposase (e.g., Sleeping Beauty (SB)).

[0097] In some embodiments, the T cell progenitors are transfected or transduced with exogenous nucleic acid encoding a chimeric antigen receptor (CAR) or a recombinant TCR.

[0098] In some embodiments, the CD7+CD34- cells comprise cells that are CD5- and / or CD1a+, preferably at least about 50%, or at least about 60, 70, 80, 90, or 95% of the CD7+CD34- cells are CD5- and / or CD1a-. In some embodiments, the CD7+CD34- cells comprise cells that are CD5-, preferably at least about 50%, or at least about 60, 70, 80, 90, or 95% of the CD7+CD34- cells are CD5-. In some embodiments, the CD7+CD34- cells comprise cells that are CD1a-, preferably at least about 50%, or at least about 60, 70, 80, 90, or 95% of the CD7+CD34- cells are CD1a-. In some embodiments, the CD7+CD34- cells comprise cells that are CD5-CD1a-, preferably at least about 50%, or at least about 60, 70, 80, 90, or 95% of the CD7+CD34- cells are CD5-CD1a-.

[0099] In some embodiments, the T cell progenitors express at least one homing marker, hi some embodiments, the T cell progenitors express at least one homing marker selected from the group including or consisting of CXCR4 (also known as CD184), CCR9, CD62L (also known as L-selectin), and CCR7.

[0100] In some embodiments, the T cell progenitors comprise at least about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 57, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70%, preferably at least about 50%, of cells expressing the marker CXCR4.

[0101] In some embodiments, the T cell progenitors comprise at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50%, preferably at least about 20%, of cells expressing the marker CCR9.

[0102] In some embodiments, the T cell progenitors comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25%, preferably at least about 10%, of cells expressing the marker CD62L.

[0103] In some embodiments, the T cell progenitors comprise at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35%, preferably at least about 15%, of cells expressing the marker CCR7.

[0104] In some embodiments, the T cell progenitors express at least one thymic juvenating / regenerating molecule, examples of which include, but are not limited to, RANKL and lymphotoxin A (LTA).

[0105] In some embodiments, the T cell progenitors comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25%, preferably at least about 10%, of cells expressing the marker RANKL.

[0106] In some embodiments, the T cell progenitors comprise at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, or 65%, preferably at least about 30%, of cells expressing marker LTA.

[0107] In some embodiments, the T cell progenitors express cytokines, particularly cytokines involved in thymus regeneration, such as, for example, RANKL or LTA. In some embodiments, the T cell progenitors are genetically modified to express cytokines, particularly cytokines involved in thymus regeneration, such as, for example, RANKL or LTA.

[0108] In some embodiments, the T cell progenitors are autologous.

[0109] In some embodiments, the T cell progenitors are allogeneic.

[0110] In some embodiments, T cell progenitors are prepared by culturing CD34+ cells in the presence of immobilized Notch ligand or a fragment thereof. In some embodiments, the Notch ligand or a fragment thereof is immobilized on the inner surface of a culture vessel or on beads.

[0111] In some embodiments, the CD34+ cells are isolated from a human.

[0112] In some embodiments, the CD34+ cells are isolated from an adult donor. In some embodiments, the CD34+ cells are isolated from a bone marrow aspirate or from peripheral blood from an adult donor that has been mobilized using, for example, G-CSF.

[0113] In some embodiments, the CD34+ cells are isolated from umbilical cord blood.

[0114] Methods for isolating CD34+ cells are well known in the art and include, but are not limited to, the use of beads coated with an antibody that recognizes CD34.

[0115] In some embodiments, the CD34+ cell populations used in the methods of the invention are at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% pure.

[0116] In some embodiments, the CD34+ cells are from about 10 6 ~about 10 7 Cells are seeded in culture medium at concentrations ranging from 1000 to 10000 cells / mL.

[0117] In some embodiments, the culture vessel is selected from conventional culture vessels, including (but not limited to) culture dishes, 6-96 wells, Petri dishes, flasks, stir bottles, microtiter plates, test tubes, hollow fiber devices, cellular foams, and bags. The amount of cells to be seeded can be adapted by one skilled in the art depending on the culture vessel used.

[0118] In some embodiments, the medium is adapted for the culture of CD34+ cells. Examples of culture media adapted for the culture of CD34+ cells include, but are not limited to, α-MEM, DMEM, RPMI 1640, IMDM, BME, McCoy's 5A, SFII (StemCell Technologies) medium, and Fisher's medium.

[0119] In some embodiments, the culture medium is feeder cell-free.

[0120] In some embodiments, the culture medium is serum-free. In one embodiment, the culture medium is supplemented with fetal bovine serum (FBS) or fetal calf serum (FCS), preferably containing at least 15% or 20% v / v FBS or FCS.

[0121] Notch proteins are transmembrane receptors that regulate cellular responses to numerous environmental signals. Four Notch receptors (Notch1-4) and five ligands (Delta-like-1, Delta-like-3, Delta-like-4, Jagged-1, and Jagged-2) have been described in mammals (Weinmaster Curr Opin Genet Dev 2000:10:363-369).

[0122] In some embodiments, the Notch ligand is Dela-like-4, preferably human Dela-like-4 (also known as DL-4; Uniprot Accession Number: Q9NR61, SEQ ID NO: 2), or a fragment thereof.

[0123] SEQ ID NO: 2 MAAASRSASGWALLLLVALWQQRAAGSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSY RVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQ EDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPNFTGSNCEKKVDRCTSNPCANGGQCLNRGPSRMCRCRPGFTGTYCELHVSDCARNPCAHGGTCHDLENGLMCTCPAGFSGRRCEVRTSIDACASSPCFNRATCYTDLSTDTFVCNCPYGFV GSRCEFPVGLPPSFPWVAVSLGVGLAVLLVLLGMVAVAVRQLRLRRPDDGSREAMNNLSDFQKDNLIPAAQLKNTNQKKELEVDCGLDKSNCGKQQNHTLDYNLAPGPLGRGTMPGKFPHSDKSLGEKAPLRLHSEKPECRISAICSPRDSMYQSVCLISEERNECVIATEV

[0124] In some embodiments, the Notch ligand or fragment thereof is a soluble domain of at least one Notch ligand. In some embodiments, the soluble domain of a Notch ligand represents the extracellular portion of said ligand.

[0125] In some embodiments, the Notch ligand or a fragment thereof (preferably the soluble domain of the Notch ligand) is fused to a protein that allows the Notch ligand to be immobilized on a support.

[0126] In some embodiments, the Notch ligand or a fragment thereof (preferably, the soluble domain of the Notch ligand) is fused to the Fc region of an IgG protein, such as, for example, a human IgG protein. In some embodiments, the Notch ligand or a fragment thereof (preferably, the soluble domain of the Notch ligand) is fused to the Fc region of an IgG2 protein, such as, for example, a human IgG2 protein (NCBI Accession Number: 4HAF_A, SEQ ID NO: 3).

[0127] SEQ ID NO: 3 VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKT ISKTKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0128] In some embodiments, the culture medium comprises DL-4 or a fragment thereof, preferably a fragment comprising or consisting of the soluble domain of DL-4.

[0129] In some embodiments, the soluble domain of DL-4 comprises or consists of amino acids 1-526 of SEQ ID NO: 2. In another embodiment, the soluble domain of DL-4 comprises or consists of amino acids 1-525 of SEQ ID NO: 2. In another embodiment, the soluble domain of DL-4 comprises or consists of amino acids 1-524 of SEQ ID NO: 2.

[0130] In some embodiments, DL-4 or a soluble domain thereof is fused to the Fc receptor region of an IgG protein (such as a human IgG protein), particularly an IgG2 protein, and preferably human IgG2. An example of a protein comprising the soluble domain of DL-4 fused to the Fc receptor region of a human IgG2 protein is SEQ ID NO:4.

[0131] SEQ ID NO:4 MAAASRSASGWALLLLVALWQQRAAGSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLC KKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSC RERNQGANYACECPPNFTGSNCEKKVDRCTSNPCANGGQCLNRGPSRMCRCRPGFTGTYCELHVSDCARNPCAHGGTCHDLENGLMCTCPAGFSGRRCEVRTSIDACASSPCFNRATCYTDLSTDTFVCNCPYGFVGSRCEFPVGLPPSTMVRSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVQFNWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0132] An example of a protein containing the soluble domain of DL-4 fused to the Fc receptor region of an IgG1 protein (e.g., human IgG1 protein) is a commercially available product (Sino Biologicals) that contains the extracellular domain (Met 1 to Pro 524) of human DLL4 (full-length DLL4, accession number NP_061947.1) fused at its C-terminus to the Fc region of human IgG1.

[0133] In some embodiments, the Notch ligand or fragment thereof is immobilized (i.e., attached to a solid support) on the culture vessel used for culturing, although it is possible that certain elements may be found in solution. In some embodiments, the Notch ligand or fragment thereof is immobilized on a surface, preferably the inside surface, of the culture vessel. Without wishing to be bound by any theory, Applicants suggest that immobilizing the Notch ligand or fragment thereof may stabilize the Notch ligand or fragment thereof to facilitate interaction with CD34+ cells, thereby enabling activation of the Notch receptor on the CD34+ cells. In another embodiment, the Notch ligand or fragment thereof is immobilized on the surface of beads, preferably microbeads, or polymeric or magnetic beads (generally having a diameter of 1 to 5 μm), present in the culture medium.

[0134] The Notch ligand or fragment thereof may or may not be covalently bound (e.g., to a bead or the surface of a culture vessel). Binding of the Notch ligand or fragment thereof can be non-covalently achieved by adsorbing the Notch ligand or fragment thereof to the surface of a culture vessel or to a bead. Methods for attaching proteins or peptides to beads or culture vessels are well known in the art and include, but are not limited to, fragment crystallizable (Fc) regions of immunoglobulin molecules (e.g., human IgG).

[0135] Methods for coating culture vessels or beads with a Notch ligand or fragment thereof are disclosed in WO2016 / 055396, which is incorporated herein by reference. In some embodiments, according to WO2016 / 055396, about 75% of the Notch ligand or fragment thereof, particularly DL-4, adheres to the surface of the culture vessel or bead when used at 5 μg / mL. In some embodiments, the composition used to coat the culture vessel or bead with the Notch ligand or fragment thereof comprises a Notch ligand or fragment thereof at a concentration of 1.25 μg / mL or greater, preferably in the range of about 2.5-5 μg / mL.

[0136] In some embodiments, the culture medium comprises fibronectin or a fibronectin fragment (fibronectin can have a sequence corresponding to Uniprot Accession Number P02751, SEQ ID NO: 5). In some embodiments, the fibronectin fragment comprises or consists of an RGDS motif, a connecting segment 1 (CS-1) motif, and / or a heparin-binding domain. Preferably, the fibronectin fragment comprises or consists of an RGDS motif, a CS-1 motif, and a heparin-binding domain.

[0137] Fibronectin is a protein that, in its native form, is a large, V-shaped dimer 100 nm long and 460 kDa. Two monomers are connected at their C-termini by two disulfide bridges. The terms "fibronectin" or "fibronectin fragment" are understood to mean the native fibronectin protein (i.e., any isoform produced by alternative splicing), but also a monomer of this protein, or a fragment of this protein (where specified, the RGDS motif, the CS-1 motif, and the heparin-binding site).

[0138] An example of a fibronectin fragment particularly suitable for carrying out the process disclosed herein is Retronectin®. This protein corresponds to a fragment of human fibronectin (CH-296 fragment, Kimizuka et al., J. Biochem., 1991 Aug. 110(2):284-91, Chono et al., J. Biochem., 2001 Sep. 130(3):331-4) and contains a cell-binding C domain (including the RGDS motif, heparin-binding domain, and CS-1 motif). This protein is sold, among others, by the companies Takara Bio Inc. (Shiga, Japan), Clinisciences (Nanterre, France, also known as NovoNectin®), and Fisher Scientific (Hampton, United States).

[0139] In some embodiments, the fibronectin fragments are immobilized on the interior surface of a culture vessel or on beads.

[0140] The term "RGDS motif" is intended to refer to any peptide or protein containing the RGDS (SEQ ID NO: 6) pattern and therefore capable of binding to the integrin VLA-5. The ability of such peptides or proteins to bind to VLA-5 integrin can be tested by methods known and described in the art. The RGDS motif binds to the integrin VLA-5 (Very Late Antigen-5), a dimer composed of CD49e (α5) and CD29 (β1).

[0141] Heparin-binding domains are well known in the art and are present in many proteins that bind to heparin. The sequence is generally XBBXBX or XBBBXXBX (B = basic amino acid; X = hydrophilic amino acid; Cardin and Weintraub, Arterioscler Thromb Vasc Biol. 1989;9:21-32). The presence of such a heparin-binding domain is particularly preferred when CD34+ cells are exposed to a viral (especially retroviral) vector to transduce the viral (especially retroviral) vector and obtain T cell progenitors that express the transgene.

[0142] The CS-1 motif is a 25-amino acid peptide (DELPQLVTLPHPNLHGPEILDVPST, SEQ ID NO: 7) as described by Wayner et al., 1989, J. Cell Biol. 109: 1321. The CS-1 motif binds to the VLA-4 (Very Late Antigen-4) receptor. VLA-4 is a dimeric integrin composed of CD49d (α4) and CD29 (β1).

[0143] In some embodiments, the fibronectin or fibronectin fragment is immobilized (i.e., bound to a solid support). The binding of the fibronectin or fibronectin fragment to (e.g., beads or the surface of a culture vessel) may or may not be covalent. In some embodiments, the fibronectin or fibronectin fragment is immobilized on the inner surface of a culture vessel (although it is possible that certain elements may be found in solution). In another embodiment, the fibronectin or fibronectin fragment is immobilized on the surface of beads, preferably microbeads, or polymeric or magnetic beads (generally having a diameter of 1-5 μm). In some embodiments, the Notch ligand or fragment thereof and the fibronectin or fragment thereof are immobilized on the same beads. In another embodiment, the Notch ligand or fragment thereof and the fibronectin or fragment thereof are immobilized on different beads.

[0144] In some embodiments, the immobilization of fibronectin or fibronectin fragments is non-covalent, by adsorbing the fibronectin or fragments to the interior surface of a culture vessel or to the surface of beads. Methods for attaching proteins or peptides to beads or surfaces of culture vessels are well known in the art and are described herein above.

[0145] Methods for coating culture vessels or beads with fibronectin or fragments thereof are disclosed in WO 2016 / 055396. In some embodiments, the composition used to coat culture vessels or beads with fibronectin or fragments thereof comprises fibronectin or fragments thereof at a concentration in the range of 10-100 μg / mL, preferably about 25 μg / mL.

[0146] In some embodiments, the culture medium comprises at least one, two, or three (e.g., one, two, or three) cytokines selected from the group comprising or consisting of SCF (stem cell factor), Flt3-L (Flt3 ligand), and IL-7. In some embodiments, the culture medium comprises at least one, two, or three (e.g., one, two, or three) cytokines selected from the group comprising or consisting of human SCF, human Flt3-L, and human IL-7.

[0147] In some embodiments, the culture medium comprises at least one, two, three, or four (e.g., one, two, three, or four) cytokines selected from the group comprising or consisting of SCF (stem cell factor), Flt3-L (Flt3 ligand), TPO (thrombopoietin), and IL-7 (interleukin 7). In some embodiments, the culture medium comprises at least one, two, three, or four (e.g., one, two, three, or four) cytokines selected from the group comprising or consisting of human SCF, human Flt3-L, human TPO, and human IL-7.

[0148] For example, Peprotech offers hSCF, hFlt3-L, hTPO, and hIL-7.

[0149] In some embodiments, the concentration of SCF, preferably hSCF, ranges from about 2 to about 300 ng / mL, preferably from about 40 to about 300 ng / mL, or from about 40 ng / mL to about 200 ng / mL, and more preferably is about 100 ng / mL.

[0150] In some embodiments, the concentration of Flt3-L, preferably hFlt3-L, ranges from about 2 to about 300 ng / mL, preferably from about 40 to about 300 ng / mL, or from about 40 ng / mL to about 200 ng / mL, and more preferably is about 100 ng / mL.

[0151] In some embodiments, the concentration of TPO, preferably hTPO, ranges from about 2 to about 300 ng / mL, preferably from about 40 to about 300 ng / mL, or from about 40 ng / mL to about 200 ng / mL, and more preferably is about 100 ng / mL.

[0152] In some embodiments, the concentration of IL-7, preferably hIL-7, ranges from about 2 to about 300 ng / mL, preferably from about 40 to about 300 ng / mL, or from about 40 ng / mL to about 200 ng / mL, and more preferably is about 100 ng / mL.

[0153] In some embodiments, the medium comprises IL-3, preferably human IL-3, hi some embodiments, the medium does not comprise IL-3.

[0154] In some embodiments, CD34+ cells are cultured in a culture medium containing TNF-α and / or an antagonist of the aryl hydrocarbon / dioxin receptor.

[0155] In some embodiments, CD34+ cells are cultured in a culture medium comprising a Notch ligand or a fragment thereof, TNF-α, and fibronectin or a fragment thereof. In some embodiments, CD34+ cells are cultured in a culture medium comprising a Notch ligand or a fragment thereof, an aryl hydrocarbon / dioxin receptor antagonist, and fibronectin or a fragment thereof. In some embodiments, CD34+ cells are cultured in a culture medium comprising a Notch ligand or a fragment thereof, TNF-α, an aryl hydrocarbon / dioxin receptor antagonist, and fibronectin or a fragment thereof.

[0156] In some embodiments, the TNF-α is human TNF-α, for example, having the sequence of SEQ ID NO: 1 (Uniprot accession number: P01375).

[0157] SEQ ID NO: 1 MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANG VELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL

[0158] TNF-α is produced primarily as a type II transmembrane protein arranged in stable homodimers, with each monomer containing 233 amino acids in humans. In humans, the soluble portion of human TNF-α consists of amino acids 77-233 of SEQ ID NO:1.

[0159] In some embodiments, the culture medium comprises full-length TNF-α or a soluble fragment thereof.

[0160] In some embodiments, TNF-α or a fragment thereof is added starting on day 0 of culture. In some embodiments, TNF-α or a fragment thereof is present in the culture medium for at least about 1, 2, 3, 4, 5, 6, or 7 days starting on day 0. In some embodiments, TNF-α or a fragment thereof is present in the culture medium from day 0 until the end of culture.

[0161] In some embodiments, TNF-α or a fragment thereof is used at a concentration ranging from about 10 to about 300 ng / mL, such as at least about 10, 20, 30, 40, 50, 100, 200, or 300 ng / mL. In some embodiments, TNF-α or a fragment thereof is used at a concentration of about 10 ng / mL. However, other concentrations, such as about 5, 10, 20, or 50 ng / mL, are also suitable.

[0162] In some embodiments, the aryl hydrocarbon / dioxin receptor antagonist is StemRegenin 1 (SR1, 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol, CAS 1227633-49-10).

[0163] In some embodiments, the aryl hydrocarbon / dioxin receptor antagonist is present in the culture medium from day 0 of the culture.

[0164] In some embodiments, the aryl hydrocarbon / dioxin receptor antagonist is added to the culture medium at a concentration of about 1 ng / mL to about 300 ng / mL, and preferably 1 ng / mL or more, or 3 ng / mL or more, or 10 ng / mL or more, and preferably less than 200 ng / mL or less than 150 ng / mL, and generally in the range of 3 ng / mL to 100 ng / mL.

[0165] In some embodiments, the CD34+ cells are cultured for up to 10 days, preferably 3-7 days. [Brief explanation of the drawings]

[0166] [Figure 1] 1 is a plot showing CD34 and CD7 expression by HTLP cells obtained after culturing CD34+ cells for 7 days in the presence of IL-7, Flt3-L, SCF, TPO, TNFα, and in the presence of immobilized DLL4 / RetroNectin. [Figure 2A] A and B are a combination of UMAP plots showing the expression of homing markers: CXCR4, CCR9, CD62L, and CCR7 by HTLP cells obtained after culturing CD34+ cells for 7 days in the presence of IL-7, Flt3-L, SCF, TPO, TNFα, and immobilized DLL4 / RetroNectin. The area around the plot indicates cells expressing the marker of interest (i.e., CXCR4, CCR9, CD62L, or CCR7). (A) A combination of UMAP plots showing the expression of CXCR4, CCR9, and CD62L transcripts, as characterized by single-cell RNAseq (scRNAseq). The grayer the color, the greater the gene expression. (B) A combination of UMAP plots showing the expression of CXCR4, CCR9, CD62L, and CCR7 at the protein level, as characterized by mass cytometry. The grayer the color, the lower the protein abundance. [Figure 2B]A and B are a combination of UMAP plots showing the expression of homing markers: CXCR4, CCR9, CD62L, and CCR7 by HTLP cells obtained after culturing CD34+ cells for 7 days in the presence of IL-7, Flt3-L, SCF, TPO, TNFα, and immobilized DLL4 / RetroNectin. The area around the plot indicates cells expressing the marker of interest (i.e., CXCR4, CCR9, CD62L, or CCR7). (A) A combination of UMAP plots showing the expression of CXCR4, CCR9, and CD62L transcripts, as characterized by single-cell RNAseq (scRNAseq). The grayer the color, the greater the gene expression. (B) A combination of UMAP plots showing the expression of CXCR4, CCR9, CD62L, and CCR7 at the protein level, as characterized by mass cytometry. The grayer the color, the lower the protein abundance. [Figure 3] This is a composite of UMAP plots showing the expression of thymic juvenile molecules: lymphotoxin alpha (LTA) and RANKL by HTLP cells obtained after culturing CD34+ cells for 7 days in the presence of IL-7, Flt3-L, SCF, TPO, TNFα, and immobilized DLL4 / RetroNectin. The area around the plot indicates cells expressing the protein of interest (i.e., lymphotoxin alpha (LTA) or RANKL). The grayer the color, the lower the amount of protein. [Figure 4](A and B) are combined confocal microscopy and histology images showing HTLP cells (CD45+, light gray) infiltrating the thymic epithelial stroma, which is composed of cortical thymic epithelial cells (cTEC) (keratin 8+) and medullary epithelial cells (mTEC) (keratin 14+). (A) A combined confocal microscopy and histology image showing HTLP cell (CD45+, light gray) infiltration at 2 weeks. The grayer the color, the less colony formation by human CD45+ cells. (B) A combined confocal microscopy and histology image showing HTLP cell (CD45+, light gray) infiltration at 10 weeks. The light gray area corresponds to CD45+ cells (upper panel). The surrounding surface corresponds to medullary islands composed of keratin 14+ TECs (mTEC) and CD45+ clusters (lower panel). [Figure 5] Graph showing the ratio of medullary to cortical TECs (mTEC / cTEC) after HTLP cell engraftment or CD34+ HSPC engraftment. [Figure 6] Figures A-C show a combination of schematics and flow cytometry plots demonstrating the reconstitution of the complete T cell compartment by HTLP cells after administration in irradiated adult immunodeficient mice. (A) Schematic of the HTLP cell injection protocol in irradiated adult immunodeficient mice. (B) Flow cytometry plot showing the expression of human CD4 and human CD8 on cells present in the thymus of mice 12 weeks after injection. (C) Flow cytometry plot showing the expression of human CD3 and TCRαβ on cells present in the thymus of mice 12 weeks after injection. [Figure 7] This is a combination of a diagram and histograms showing human cell engraftment in the thymus of irradiated senescent mice after administration of HTLP cells. (A) A schematic diagram of the HTLP cell injection protocol in senescent mice after irradiation. (B) A histogram showing the percentage of human CD45 cells in the thymus of irradiated senescent mice injected with CD34+ cells or co-injected with CD34+ and HTLP cells. [Example]

[0167] The present invention is further illustrated by the following examples. Example 1: Materials and Methods Generation and characterization of human T lymphoid progenitors.

[0168] CD34+ HSPCs isolated from umbilical cord blood (C) were cultured for 7 days in α-MEM supplemented with 20% defined fetal bovine serum and the following human cytokines: IL-7, Flt3-L, SCF, TPO, and TNFα on DLL / NetroNectin-coated culture plates. The resulting human TEC lymphoid progenitors (HTLPs; also known as T cell progenitors) were then analyzed by single-cell RNA sequencing (scRNAseq) and mass cytometry.

[0169] In vivo characterization of human T lymphoid progenitors The generated HTLP cells were cultured in a 24-well plate at a dose of 1 M cells per recipient in a culture of neonatal NSG (NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ) mice (1-4 days old) were intrahepatically injected with IgG. T cell engraftment and thymic juvenation were investigated by flow cytometry and histology (confocal).

[0170] result The generated HTLPs express thymus homing markers and thymus regeneration markers. The resulting HTLP cells were characterized as CD34-CD7+ cells (see Figure 1). As shown in Figures 2A and 2B, these cells express various chemokine receptors, such as CXCR4, CCR9, and CCR7 (the periphery of the plot indicates cells expressing the gene or protein of interest), which are known to be essential for thymic entry. These progenitors are also defined by the expression of adhesion molecules, including CD62L, which has been implicated in homing to lymphoid organs and has been shown to correlate with thymic engraftment in progenitors. HTLP cells also strongly express lymphotoxin a (LTA), which plays an important role in the physiological regulation of lymphoid tissues and thymic epithelial regeneration. The resulting HTLP cells also express RANKL, which acts in thymic development and regeneration by specifically promoting the maturation of mature medullary TECs (thymic epithelial cells).

[0171] Taken together, these results suggest that the generated HTLP cells may be able to migrate to the thymus and play a role in thymic recovery.

[0172] Thymic medullary epithelium expands in HTLP grafts The thymic epithelial stroma of NSG mice is composed predominantly of cortical thymic epithelial cells (cTECs) (keratin 8+) with only a few medullary thymic epithelial cells (mTECs). After successful seeding with HTLP cells, the thymus of NSG recipients exhibits larger thymi containing distinct medullary islands composed of clusters of keratin 14+ TECs and CD45+ progeny of the transplanted progenitors (see Figures 4A and 4B). Indeed, Figure 4B, showing the thymus at 10 weeks, shows a larger peripheral surface, a larger proportion of medullary tissue (peripheral region, lower panel), and extensive colonization by human CD45+ cells (light gray, upper panel). These morphological traits, characteristic of efficient thymopoiesis, are typically absent in non-transplanted mice and are slowed / reduced in mice injected with CD34+ HSPCs.

[0173] As shown in Figure 5, after engraftment of HTLP cells, the ratio of medullary to cortical TECs (mTEC / cTEC) tended to increase in NGS recipients, correlating with thymic regeneration and progression of thymocytes toward developmental stages located in the thoracic medulla. This result highlights the superior effect of HTLP cells on the intrathymic structure and composition of the epithelial stroma and the faster kinetics of thymic reconstitution after engraftment of these progenitors.

[0174] Taken together, these results demonstrate the regenerative attributes of the resulting HTLP cells, along with their engraftment potential. The presence of medullary islands also demonstrates the regenerative potential of HTLP cells for thymic epithelial regeneration.

[0175] Example 2: Materials and Methods

[0176] Transplantation of HTLP cells in adult immunodeficient mice 6-8 week old NSG ((NOD.Cg-Prkdcscid Il2rg tm1Wjl / SzJ) mice were irradiated with 1.5 Gy on day 0. Twenty-four hours after irradiation, mice received a single dose of 6.25 x 10 6 Fixed peripheral blood (mPB)-derived HTLP cells equivalent to 100 CD7+-expressing cells were administered intravenously (Figure 6A). At 12 weeks post-injection, thymi were harvested and analyzed by flow cytometry to characterize thymocytes with various anti-human antibodies (hCD45, hCD4, hCD8, hCD3, hTCRab) (BD Biosciences) (Figures 6B and 6C).

[0177] result As shown in Figure 6B, 12 weeks after injection of HTLP cells, human CD4 + CD8+ double positive (DP), CD4 + Simple positive (CD4 SP) and CD8 +Simple positive (CD8 SP) thymocytes were characterized in the thymus of adult immunodeficient mice. The generation of these diverse cell populations indicates active and efficient human thymopoiesis. As shown in Figure 6C, human cells also express CD3 and TCRab in the thymus.

[0178] Taken together, these results demonstrate the reconstitution of the complete T cell compartment by HTLP cell administration in adult immunodeficient mice after irradiation. These results demonstrate the ability of HTLP to restore thymic function.

[0179] Example 3: Materials and Methods Co-transplantation of human hematopoietic stem cells (CD34+) and HTLP cells in aged immunodeficient mice

[0180] NSG mice (over 9 months old) were irradiated with 1.5 Gy on day 0. (Figure 7A) Twenty-four hours after irradiation, mice were treated with 0.3 x 10 6 CD34 + cells, and 1 × 10 6 CD7 + HTLPs derived from mPB equivalent to the expressing cells were co-injected intravenously. In a control group, 0.3 × 10 6 CD34 + Twelve weeks after injection, thymuses were harvested and the chimeric status of human cells in the thymus was analyzed by flow cytometry using hCD45 and mCD45 antibodies (BD Biosciences).

[0181] result As shown in Figure 7B, 12 weeks after injection, the percentage of human CD45+ cells was significantly increased in the thymus of co-injected mice compared to the thymus of mice receiving only CD34+ cells (46% and 7.6%, respectively). These results indicate that human cell engraftment and chimeric status are significantly increased in co-injected aged mice. The results also demonstrate the effect of HTLP cells on the thymus and human cell engraftment.

[0182] These results suggest that HTLP cells contribute to the reconstitution of the T cell compartment in aging subjects.

Claims

1. T cell progenitors comprising CD7+CD34- cells for use in treating aging, and preferably immunosenescence, in a subject.

2. T cell progenitors comprising CD7+CD34- cells for use in restoring thymic function in a subject.

3. T cell progenitors comprising CD7+CD34- cells for use in regenerating the thymus of a subject.

4. A T cell progenitor for use according to any one of claims 1 to 3 for use in restoring polyclonal T cell production by the thymus of said subject.

5. The T cell progenitor for use according to any one of claims 1 to 4, wherein the subject is a human.

6. The T cell progenitor for use according to any one of claims 1 to 5, wherein the subject is at least about 50 years old, preferably about 60, 70, 80 or 90 years old.

7. The T cell progenitor for use according to any one of claims 1 to 6, wherein the subject is affected by or has been diagnosed with thymic atrophy or thymic involution.

8. 8. The T cell progenitor for use according to any one of claims 1 to 7, wherein the CD7+CD34- cells comprise cells that are CD5- and / or CD1a+, and preferably at least about 50%, or at least about 60, 70, or 80% of the CD7+CD34- cells are CD5- and / or CD1a-.

9. 9. The T cell progenitor for use according to any one of claims 1 to 8, wherein the T cell progenitor is genetically modified to express an exogenous protein, preferably wherein the exogenous protein is a chimeric antigen receptor (CAR) or a recombinant TCR.

10. The T cell progenitor for use according to any one of claims 1 to 9, wherein said T cell progenitor expresses a cytokine, preferably RANKL or lymphotoxin A (LTA).

11. 11. The T cell progenitor for use according to any one of claims 1 to 10, wherein the T cell progenitor is prepared by culturing CD34+ cells in the presence of immobilized Notch ligand or a fragment thereof, and preferably fibronectin or a fragment thereof, more preferably the fibronectin fragment comprises an RGDS motif, a CS-1 motif, and / or a heparin-binding domain.

12. 12. The T cell progenitor for use according to claim 11, wherein the CD34+ cells are cultured in a culture medium comprising TNF-α and / or an antagonist of the aryl hydrocarbon / dioxin receptor, preferably wherein the antagonist of the aryl hydrocarbon / dioxin receptor is SR1.

13. The T cell progenitor for use according to any one of claims 11 to 12, wherein said CD34+ cells are isolated from humans, preferably from adult donors or umbilical cord blood.

14. The T cell progenitor for use according to any one of claims 1 to 13, wherein the T cell progenitor is autologous.

15. The T cell progenitor for use according to any one of claims 1 to 13, wherein the T cell progenitor is allogeneic.

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