Unfolded protein response activator compounds and their use in thymic regeneration

Abstract

The present invention concerns Unfolded Protein Response (UPR) activating compounds and their use as a regenerative therapy for promoting thymic regeneration.

Description

[0001] FOXN1 -activating compounds and their use in thymic regeneration

[0002] The present invention concerns FOXN1 -activating compounds and their use in thymic regeneration. In particular, the present invention relates to Unfolded Protein Response (UPR) activating compounds which induce the activation of FOXN1, and to their use as a regenerative therapy for promoting thymic regeneration.

[0003] It is well known that the thymus plays a vital role in generating and sustaining T cell immunity. Thymopoiesis is a highly complex process involving crosstalk between developing thymocytes and their supporting non-hematopoietic stromal microenvironment, which includes thymic epithelial cells (TECs), endothelial cells, mesenchymal cells and hematopoietic cells1.

[0004] However, thymus function is extremely sensitive to acute injuries and declines with age, which leads to weakened immune function. In particular, the thymus is particularly sensitive to injuries due to common cancer therapies such as chemotherapy, immune or radiation therapy, infections, auto-immunity, steroids and GVHD2. These deleterious effects lead to quantitative and qualitative changes to the thymus, including disruption of stromal architecture and reduction of naive T cell output2.

[0005] Impaired thymic recovery has important clinical consequences, especially in recipients of allogeneic hematopoietic cell transplantation (allo-HCT). Posttransplant T cell deficiency is associated with an increased risk of infections, relapse of malignancy and impairment in the application of immunotherapeutic approaches. Previous studies demonstrated that patients with delayed or defective recovery of thymic function have a significantly higher risk of leukemia relapse and adverse clinical outcome3. More recent studies demonstrated that genetic factors, which influence thymic output, adversely impact disease-free survival and outcome in allo-HCT recipients4, further advocating that optimal thymic function is crucial for the success of the therapy. While the thymus is severely damaged by several injuries, the persistence of residual thymus tissue can be targeted by regenerative therapies2.

[0006] Some studies have previously shown that thymic epithelial cells (TECs) play a fundamental role in the recovery of thymic function by sensing damage signals and producing cytokines and growth factors that drive thymic regeneration. Inparticular, it has been demonstrated that thymic endothelial cells promote thymic regeneration and immune reconstitution through the upregulation of Foxnl5, a critical factor for TEC development and function6. Some groups demonstrated that in vivo delivery of recombinant Foxnl or its genetic overexpression induces thymic regeneration in mouse models78. In humans, while loss-of-function mutations in F0XN1 are well known to cause Nude Severe Combined Immunodeficiency, more recent studies showed that patients carrying a heterozygote mutation in the coding region of F0XN1 show decreased thymic output and impaired T cell immunity9. Together, these results demonstrate that the levels of FOXN1 tightly correlate with thymic function and its T cell output.

[0007] Currently, several approaches have been explored in preclinical mouse models for thymus regeneration and some of them advanced into the clinic. These approaches include: the administration of keratinocyte growth factor (KGF)10, growth hormone (GH)11, Ghrelin12, interleukin-2213, sex hormone inhibitors14, Receptor Activator of Nuclear Factor-KB Ligand (RANKL)15and bone morphogenetic protein 4 (BMP4)5. However, the approaches that have been tested in clinical studies have shown limited efficacy on thymic recovery. In addition, several of the previously proposed approaches are based on recombinant proteins which may not completely recapitulate the functional capacities of their endogenous counterparts and also pose challenges for their cost of production. Thus, there is an urgent need to identify new therapies for treating thymic insufficiency and promoting thymic regeneration.

[0008] In the light of the above, it is therefore apparent the need to provide new therapies for treating thymic insufficiency and promoting thymic regeneration.

[0009] According to the present invention a novel thymic-regenerative approach has now been found based on the targeting of FOXN1 by induction of UPR triggered, for instance, by proteasome inhibitors or by inducers of endoplasmic reticulum (ER) stress.

[0010] In particular, according to the invention, it has been found that the activation of UPR results in Foxnl expression and, consequently, in thymic regeneration.

[0011] Specifically, the activation of the unfolded protein response pathway occurs through the activation of one or more of the following transcription factors: ATF4, ATF6 and / or Xbpls. According to the present invention it has been surprisingly found that by directly or indirectly activating UPR, thymic regeneration is induced.In particular, activation of UPR can be a direct activation, for example through the use of specific compounds able to activate ATF4, ATF6 and / or Xbpls or can be an indirect activation, for example by triggering ER proteotoxic stress, or by proteasome inhibition. In fact, proteasome inhibition induces endoplasmic reticulum (ER) stress, which in turn induces the unfolded protein response (UPR). The proteasome is a multi-subunit protease complex whose primary function is degrading unneeded, damaged, or misfolded proteins. Proteasome is critical in controlling many cellular processes, including the cell cycle, signaling, and responses to stress. The 26S proteasome is the central form of the proteasome in eukaryotic cells and it consists of two main parts: the 20S and 19S. The 20S is formed by 4 rings: 2 rings are formed by a-subunits (a1-7) and 2 rings form the [3-subunits ([31- 7). The 19S particle consists of 19 individual proteins including the ATPase subunits (Rpt1-Rpt6), the Rpn subunits (e.g. Rpn1 and Rpn2) and the Ubiquitin receptors (e.g. Rpn10 and Rpn13).

[0012] As shown in the experimental data reported below, according to the present invention 1530 FDA-approved compounds were scrutinized, by using a drug screening system developed by the inventors, for their capacity to induce FOXN1 activity in TECs. As a result of the screening process, a group of drugs that induced Foxnl in the tested system has been identified. Then, it was found that among the above-mentioned compounds, proteasome inhibitors, as well as other compounds known to activate ER stress and UPR signaling, strongly induced Foxnl expression in murine and human primary TECs. In particular, mechanistically, it has been observed that the inhibition of the proteasome activates the unfolded protein response (UPR) which correlates with the induction of Foxnl expression. In particular, as shown in the experimental data below, a significant upregulation of the transcription factor ATF4 was found after treatment with proteasome inhibitors.

[0013] In this regard, it was also found that a direct activation of UPR through the activation of ATF4 by a specific compound, i.e. salubrinal, also correlates with the induction of Foxnl expression.

[0014] Therefore, according to the present invention, it was surprisingly found that by directly or indirectly activating UPR, for example by upregulating the expression and activity of ATF4, ATF6 and / or Xbpls or by inducing ER stress or proteasome inhibition, an induction of Foxnl expression is obtained, resulting in thymic regeneration.Regarding the proteasome inhibitors compounds, as a further evidence of the involvement of proteasome inhibition in thymic regeneration, the analysis carried out according to the invention was extended to include bortezomib, a well-known FDA-approved proteasome inhibitor that was not present in the initial screened group of drugs. In line with the previous data, the analysis revealed that also bortezomib promoted Foxnl transcriptional synthesis and the transactivation of Foxnl downstream target genes.

[0015] Interestingly, it has also been found that other compounds with proteasome inhibition activity, such as nitazoxanide (NTZ), could induce Foxnl. As shown in the example below, mice treated with NTZ showed accelerated thymic recovery following a sub-lethal total body irradiation (SL-TBI), showing the thymic regenerative effects of a proteasome inhibitor in a preclinical mouse model of immunological injury.

[0016] Therefore, according to the present invention it has been found that inhibition of the proteasome triggers ER stress and UPR, which in turn leads to the activation of ATF4 and ultimately of FOXN1, thereby resulting in thymic regeneration, which can be achieved through pharmacological induction of this pathway.

[0017] It is therefore specific object of the present invention an Unfolded Protein Response (UPR) activator compound, or a pharmaceutical composition comprising said compound together with one or more pharmaceutically acceptable excipients and / or adjuvants, for use in the treatment of thymic insufficiency or thymic disfunction, wherein said UPR activator compound is chosen from a direct UPR activator compound, which is able to activate at least one of ATF4, ATF6 and Xbp1 s, and an indirect UPR activator compound chosen from a proteasome inhibitor compound and an inducer of endoplasmic reticulum (ER) stress.

[0018] According to the present invention, a person skilled in the art can select a UPR activator compound by routine methods. In particular, the activation of UPR can be verified by detecting the expression of at least one of ATF4, ATF6 and Xbpls, for example by measuring the protein amount by Western blot or by measuring the related mRNA, wherein the protein expression is higher than the basal expression.

[0019] According to the present invention:

[0020] - thymic insufficiency or disfunction can be characterised by a reduced numbers of T cells or Naive T cells with respect to a healthy subject.Alternatively or in addition, thymic insufficiency can be characterised by a reduced number of recent thymic emigrating (RTE) T cells with respect to a healthy subject, for example assessed by measurement of T cell receptor excision circles (TRECs);

[0021] - thymic insufficiency can be associated with acute or chronic viral or bacterial infection, patient treatment with immunomodulating or cytoreductive drugs such as glucocorticoids, cyclosporine, anti -thymocyte globulin, anti-T lymphocyte globulin, chemotherapeutic agents;

[0022] - thymic insufficiency can be associated with trauma to the thymus as consequence of surgical procedures that impact its function or mass (such as patients receiving cardiothoracic surgery) and partial or total thymectomy;

[0023] - thymic insufficiency can be observed in patients receiving cellular therapies, such as CAR-T or hematopoietic stem cell transplantation as a consequences of pre-transplant preparative conditioning regimen, lymphodepletion, graft-versus host disease and infections; and / or

[0024] - thymic insufficiency can be associated with the decline in immunity associated with aging.

[0025] More in detail, the UPR activator compound or pharmaceutical composition according to the present invention are for use as a regenerative therapy, in particular for promoting thymic regeneration.

[0026] As stated above, according to the present invention, for UPR activator compound is intended a direct inducer of UPR, a proteasome inhibitor compound able to inhibit the activity of the 26s proteasome or compounds able to induce ER stress. The proteasome inhibitor compound can inhibit the activity of said proteasome by a direct or an indirect mechanism. In particular, for direct mechanism is intended that said inhibitor directly binds to the proteasome. The binding between the inhibitor and the proteasome can be reversible, irreversible, covalent or non-covalent.

[0027] More in detail, according to the invention, said proteasome inhibitor, when acting through a direct mechanism, can target one or more subunits of said 26s proteasome complex, such as one or more a subunits (chosen from a1 , a2, a3, a4, a5, a6 and / or a7) and / or one or more [3 subunits (chosen from (31 , [32, [33, [34, [35,(36 and / or (37), preferably one or more [3 subunits, such as one or more [3 subunits chosen from [31 , [32 and [35 subunit, more preferably [35 subunit.

[0028] A compound according to the present invention, through the induction of UPR mechanism, for example by proteasome inhibition or induction of ER stress, is able to enhance F0XN1 expression and, consequently, to cause thymic regeneration.

[0029] According to the present invention, said proteasome inhibitor compound can be chosen from the group consisting of:

[0030] an acylsalicylamide, such as Nitazoxanide,

[0031] Ixazomib, in particular MLN9708 (Ixazomib citrate) or, MLN2238 (Ixazomib), Carfilzomib (PR-171),

[0032] Delanzomib (CEP-18770),

[0033] Bortezomib,

[0034] Daunorubicin, such as Daunorubicin HCI,

[0035] Doxorubicin, such as Doxorubicin HCI,

[0036] Epirubicin, such as Epirubicin HCI,

[0037] Idarubicin, such as Idarubicin HCI,

[0038] Mitoxantrone, such as Mitoxantrone HCI,

[0039] Zinc Pyrithione

[0040] BC-05,

[0041] BMG-431,

[0042] Cerpegin,

[0043] Chymostatin,

[0044] CNN-1,

[0045] CUDC-907,

[0046] D Boronophenylalanine,

[0047] Degrasyn,

[0048] DHP (dihydroisoxazole analogue),

[0049] GSK3494245,

[0050] KZR-616,

[0051] Lactacystin,

[0052] LONP1-IN-2,

[0053] Marizomib,

[0054] MG132,

[0055] NPI-0052,ONX-0912,

[0056] Oprozomib,

[0057] Pepstatin A,

[0058] PI-1840,

[0059] PR-104,

[0060] PR-619,

[0061] PS-341,

[0062] PZ-265,

[0063] RA375,

[0064] Roxadustat,

[0065] S-THIQ,

[0066] Sabarubicin,

[0067] Sodium N-ethylmaleimide,

[0068] or a pharmaceutically acceptable salt, substituent, analogue, tautomer, isomer, active metabolite, or prodrug thereof.

[0069] According to a preferred embodiment of the present invention, said proteasome inhibitor compound is chosen from the group consisting of Nitazoxanide, Ixazomib, Delanzomib, Carfilzomib and Bortezomib or a pharmaceutically acceptable salt or substituent thereof or an analogue, tautomer, isomer, active metabolite or prodrug thereof, more preferably said compound being Nitazoxanide or a pharmaceutically acceptable salt, substituent, analogue, tautomer, isomer, active metabolite or prodrug thereof.

[0070] For example, thiazolide prodrug of Nitazoxanide can be RM-4848, whereas a thiazolide analogue of Nitazoxanide can be RM-5038.

[0071] For example, according to the invention, an active metabolite of Nitazoxanide can be tizoxanide or tizoxanide glucuronide; the analogue of Carfilzomib can be chosen from MG132, Epoxomicin, Celastrol Oprozomib, ONX-0914 (PR-957), Delanzomib, VR23, Marizomib (Salinosporamide A), PI-1840.

[0072] According to the present invention, pharmaceutically acceptable salts or substituents of said proteasome inhibitor compound can be chosen from the group consisting of acetate, acid citrate, acid phosphate, ascorbate, benzenesulfonate, benzoate, besylate, bisulfate, bitartrate, bromide, chloride, citrate, ethanesulfonate, formate, fumarate, gentisinate, gluconate, glucaronate, glutamate, lactate, methanesulfonate, nitrate, iodide, isonicotinate, maleate, oleate, oxalate, p-toluenesulfonate, pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)), pantothenate, phosphate, saccharate, salicylate, succinate, sulfate, tannate and tartrate salts.

[0073] For example, for Nitazoxanide possible salts or substituents may include: Phenol ester - Benzamide - Phenoxy compound - Nitroaromatic compound -Benzoyl - Nitrothiazole - 2,5-disubstituted 1,3-thiazole - Azole - Thiazole -Heteroaromatic compound - Organic nitro compound - Secondary carboxylic acid amide - Carboxamide group - Carboxylic acid ester - C-nitro compound -Organoheterocyclic compound - Organic 1 ,3-dipolar compound - Propargyl-type 1 ,3-dipolar organic compound - Allyl-type 1 ,3-dipolar organic compound - Azacycle - Carboxylic acid derivative - Organic oxoazanium - Monocarboxylic acid or derivatives - Organic nitrogen compound - Organic oxide - Organonitrogen compound - For Nitazoxanide, possible salts or substituents may include: Phenol ester - Benzamide - Phenoxy compound - Nitroaromatic compound - Benzoyl -Nitrothiazole - 2,5-disubstituted 1,3-thiazole - Azole - Thiazole - Heteroaromatic compound - Organic nitro compound - Secondary carboxylic acid amide -Carboxamide group - Carboxylic acid ester - C-nitro compound - Organoheterocyclic compound - Organic 1 ,3-dipolar compound - Propargyl-type 1,3-dipolar organic compound - Allyl-type 1,3-dipolar organic compound - Azacycle - Carboxylic acid derivative - Organic oxoazanium - Monocarboxylic acid or derivatives - Organic nitrogen compound - Organic oxide - Organonitrogen compound - Organooxygen compound - Hydrocarbon derivative - Carbonyl group - Organopnictogen compound - Organic oxygen compound - Organic zwitterion - Aromatic heteromonocyclic compound.

[0074] For Ixazomib, possible salts or substituents may include: N-acyl-alpha amino acid or derivatives - Alpha-amino acid amide - N-substituted-alpha-amino acid -Alpha-amino acid or derivatives - 2-halobenzoic acid or derivatives - 3-halobenzoic acid or derivatives - Halobenzoic acid or derivatives - 1 ,4-dichlorobenzene - Benzoyl - Chlorobenzene - Halobenzene - Aryl halide - Aryl chloride - Vinylogous halide -Boronic acid derivative - Secondary carboxylic acid amide - Carboxamide group -Boronic acid - Organic metalloid salt - Carboxylic acid derivative - Organic nitrogen compound - Organohalogen compound - Organoboron compound - Organochloride - Organonitrogen compound - Organooxygen compound - Carbonyl group - Organic salt - Hydrocarbon derivative - Organic oxide - Organopnictogen compound -Alkylborane - Monoalkylborane - Organic oxygen compound - Aromatic homomonocyclic compound.

[0075] For Carfilzomib, possible salts or substituents may include: Alphaoligopeptide - Phenylalanine or derivatives - Leucine or derivatives - N-acyl-alpha amino acid or derivatives - Alpha-amino acid amide - N-substituted-alpha-amino acid - Alpha-amino acid or derivatives - Amphetamine or derivatives - N-acyl-amine - Morpholine - Oxazinane - Benzenoid - Fatty amide - Fatty acyl - Monocyclic benzene moiety - Tertiary aliphatic amine - Ketone - Amino acid or derivatives -Secondary carboxylic acid amide - Carboxamide group - Tertiary amine - Oxacycle - Azacycle - Dialkyl ether - Oxirane - Ether - Organoheterocyclic compound - Organic oxygen compound - Carbonyl group - Organic nitrogen compound -Organopnictogen compound - Amine - Organic oxide - Hydrocarbon derivative -Organooxygen compound.

[0076] For Delanzomib, possible salts or substituents may include: N-acyl-alpha amino acid or derivatives - Alpha-amino acid amide - 2-phenylpyridine - Pyridine carboxylic acid or derivatives - 2-heteroaryl carboxamide - Monocyclic benzene moiety - Fatty amide - N-acyl-amine - Pyridine - Benzenoid - Fatty acyl -Heteroaromatic compound - Boronic acid derivative - Secondary carboxylic acid amide - Secondary alcohol - Boronic acid - Carboxamide group -Organoheterocyclic compound - Azacycle - Organic metalloid salt - Organic oxide -Organic metalloid moeity - Organic oxygen compound - Organic nitrogen compound - Organonitrogen compound - Carbonyl group - Organooxygen compound -Monoalkylborane - Alkylborane - Alcohol - Hydrocarbon derivative - Aromatic heteromonocyclic compound.

[0077] For Bortezomib, possible salts or substituents may include: Phenylalanine or derivatives - N-acyl-alpha amino acid or derivatives - Alpha-amino acid amide -Amphetamine or derivatives - Pyrazine carboxylic acid or derivatives -Pyrazinecarboxamide - 2-heteroaryl carboxamide - Monocyclic benzene moiety -Fatty amide - Pyrazine - Benzenoid - Fatty acyl - Heteroaromatic compound -Boronic acid derivative - Boronic acid - Carboxamide group - Secondary carboxylic acid amide - Azacycle - Organic metalloid salt - Organoheterocyclic compound -Hydrocarbon derivative - Organic oxide - Organooxygen compound -Organonitrogen compound - Organoboron compound - Organopnictogen compound - Alkylborane - Carbonyl group - Organic oxygen compound - Organicnitrogen compound - Monoalkylborane - Organic salt - Aromatic heteromonocyclic compound.

[0078] According to the present invention, said inducer of endoplasmic reticulum (ER) stress can be chosen from homoharringtonine, verteporfin, diosmetin and abacavir or any pharmaceutically acceptable salt, derivative, substitute, analogue, tautomer, or prodrug thereof.

[0079] Moreover, according to the present invention, said direct UPR activator compound can be salubrinal or a pharmaceutically acceptable derivative or analogue thereof.

[0080] According to the present invention, the above-UPR activator compounds can be used alone or in combination with each other.

[0081] According to the present invention said compound or pharmaceutical composition can be administered orally, by intramuscular injection, by subdermal injection, by intraperitoneal injection, by intravenous injection, by subcutaneous injection, or by inhalation. Moreover, according to the present invention, said compound or pharmaceutical composition can be administered in a dosage from 0.01 pg / kg to 500 mg / kg, for example from 0.01 to 500 pg / kg or from 0.01 to 500 mg / kg, depending on the selected compound, formulation, route of administration, and therapeutic indication.

[0082] In particular, for achieving a therapeutically effective amounts, single or multiple doses of said compounds can be administered during the course of a treatment regimen (e.g., daily, every other day, weekly, monthly, every 6 months, or yearly).

[0083] More in detail, according to the present invention:

[0084] - nitazoxanide can be provided in a dosage from 100 mg to 1000 mg per single administration.

[0085] - Carfilzomib can be provided in a dosage from 20 mg / m2 to 70 mg / m2 per single administration.

[0086] - Bortezomib can be provided in a dosage from 1 mg to 3.5 mg per single administration.

[0087] - Ixazomib can be provided in a dosage from 1mg to 10mg per single administration.

[0088] - Delanzomib can be provided in a dosage from 1 mg / m2 to 5 mg / m2 persingle administration.

[0089] According to the present invention, said pharmaceutical composition can further comprise one or more compounds chosen from:

[0090] compounds that can promote thymus function such as sex hormone inhibitors (which include GnRH-antagonist, GnRH-agonist, androgen or estrogen receptor inhibitors, CYP17A1 Inhibitors, 5a-Reductase Inhibitors); Keratinocyte Growth Factor (KGF); Interleukin 7; Insulin-like Growth Factor (IGF); Ghrelin; Growth Hormone (GH); Interleukin-22; Interleukin-33; Thymosin alpha-1; Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL); Adenosine Triphosphate (ATP); Growth Differentiation Factor 11 (GDF11); Beta-2 Microglobulin (B2M); or Bone Morphogenetic Protein 4 (BMP4);

[0091] compounds for promoting the recovery of bone marrow or hematopoietic function such as Parathyroid hormone (PTH), stem cell factor (SCF), caloric restriction; nutritional support; thrombopoietin (TPO), fms-like tyrosine kinase 3 ligand (Flt3L), Granulocyte Colony-Stimulating Factor (G-CSF), Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Androgens, hormonal ablation.

[0092] According to the invention, said sex hormone inhibitors can also be chosen from Androgen Receptor antagonists, such as Bicalutamide, Enzalutamide, Apalutamide, Flutamide, Nilutamide; 5a-Reductase Inhibitors, such as Finasteride, Dutasteride; GnRH (LHRH) Agonists, such as Leuprolide, Goserelin, Triptorelin, Buserelin; GnRH Antagonists, such as Degarelix, Relugolix; and CYP17A1 Inhibitors, such as Abiraterone.

[0093] The present invention also concerns a combination or kit of parts of i) a UPR activator compound or a pharmaceutical composition as defined above and ii) one or more compounds chosen from

[0094] compounds that can promote thymus function such as sex hormone inhibitors, Keratinocyte Growth Factor (KGF); Interleukin 7; Insulin-like Growth Factor (IGF); Ghrelin; Growth Hormone (GH); Interleukin-22; Interleukin-33; Thymosin alpha-1; Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL); Adenosine Triphosphate (ATP); Growth Differentiation Factor 11 (GDF11); Beta-2 Microglobulin (B2M); or Bone Morphogenetic Protein 4 (BMP4);

[0095] compounds for promoting the recovery of bone marrow function such as Parathyroid hormone (PTH), stem cell factor (SCF), thrombopoietin (TPO), fms-liketyrosine kinase 3 ligand (Flt3L), Granulocyte Colony-Stimulating Factor (G-CSF), Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Androgens, hormonal ablation; and / or

[0096] T cell progenitors (e.g. adoptive transfer of in vitro generated precursor T (pre-T) cells), thymic-like organoids and scaffolds or bone marrow-like organoids and scaffolds

[0097] for separate or sequential use in the treatment of thymic insufficiency or disfunction.

[0098] As stated above, according to the invention, said sex hormone inhibitors can also be chosen from Androgen Receptor antagonists, such as Bicalutamide, Enzalutamide, Apalutamide, Flutamide, Nilutamide; 5a-Reductase Inhibitors, such as Finasteride, Dutasteride; GnRH (LHRH) Agonists, such as Leuprolide, Goserelin, Triptorelin, Buserelin; GnRH Antagonists, such as Degarelix, Relugolix; and CYP17A1 Inhibitors, such as Abiraterone.

[0099] According to the present invention, said i) UPR activator compound or a pharmaceutical composition as defined above and ii) one or more other treatment modalities discussed above can be administered in the same or separate or sequential or different pharmaceutical composition and at substantially the same time or administered sequentially.

[0100] According to the invention, “separate use” is understood as meaning the administration, at the same time, of the compounds of the combination or kit according to the invention in distinct pharmaceutical forms, whereas “sequential use” is understood as meaning the successive administration of the compounds of the combination or kit according to the invention, each in a distinct pharmaceutical form. A person skilled in the art is able to determine a suitable timing for administering the compounds of the combination for sequential use; in particular the administrations of the compounds of the combination should be close enough to achieve a complete or partial overlapping of the desired therapeutic effects.

[0101] Moreover, the present invention concerns a method of treating a subject suffering from thymic insufficiency or disfunction and / or a subject in need of promoted thymic function comprising administering a therapeutically effective amount of a UPR activator compound or a pharmaceutical composition as defined above.

[0102] The present invention now will be described by an illustrative, but notlimitative way, according to preferred embodiments thereof, with particular reference to the examples and the enclosed drawings:

[0103] Figure 1. Drug screening identifies proteasome inhibitors as FOXN1 inducers in mouse TECs. (A) Validation of the use of the already described ATP16to enhance Foxnl. Stimulation of the FOXN1 reporter cells in luciferase assay after treatment with bzATP 300 pM for 24 h (n=7). (B) Nucleofection of the Foxnl reporter cell line cells with a third plasmid codifying for Foxnl open reading frame (ORF) and evaluation of Firefly / Renilla (F / R) ratio at 6 h and 24 h (n=2). (C) The Foxnl reporter cells were incubated with a drug library of 1530 compounds (all tested at a concentration of 5 pM), and F / R ratio, related to cell confluence, is reported in the graph, where each dot represents a compound. (D) Upon retesting under the same conditions, 36 of the 53 initial hits identified in C were confirmed. Among these, two compounds were excluded due to precipitation and aggregate formation (Fig. S1), resulting in a final list of 34 validated hits that were considered for further analyses.

[0104] Figure 2. Half maximal effective concentration (ECso - A) and half maximal growth inhibition (Glso- B) were performed on Foxnl reporter cell line cells for the proteasome inhibitors Carfilzomib, Delanzomib (CEP-18770), and Ixazomib (MLN9708 / MLN2238), identified in the screening.

[0105] Figure 3. Proteasome inhibitors induce Foxnl and D / / 4 expression in TECs. Proteasome inhibitors Ixazomib (MLN9708), Delanzomib, Carfilzomib and Bortezomib, were tested at sub-toxic concentrations (0.5 pM) on ANV41.2 cells to quantify Foxnl (A - n=3) and DII4 (B - n=4) by qPCR. For all data, graphs represent the mean and SEM of at least three independent experiments, *p < 0.05; **p < 0.01.

[0106] Figure 4. Nitazoxanide interferes with the activity of proteasome subunits and leads to accumulation of ubiquitinated proteins. (A) ANV41.2 cells were stimulated with NTZ and ubiquitinated proteins were assessed by WB.

[0107] (B) Densitometry was performed by normalizing UB-proteins on GAPDH and fold induction was calculated by comparing NTZ treatment to the respective control for each time point (n=3). The graph represents the mean and SD of three independent experiments, **p < 0.01. (C) Evaluation of (31 , [32 and [35 proteasome subunit activity following NTZ stimulation for 2 hours (n=2 to 4). Data are expressed as luminescence intensity (arbitrary units). The graph represents the mean and SD of two independent experiments, *p < 0.05.

[0108] Figure 5. Nitazoxanide promotes Foxnl expression in mouse andprimary human TECs. Half maximal effective concentration (ECso)(A) and half maximal growth inhibition (Glso) (B) were performed on Foxnl reporter cell line cells after NTZ treatment at 24 h and 72 h, respectively. The graph represents the mean of three independent experiments). Foxnl and DII4 mRNA levels were quantified by qPCR on ANV 41.2 cells (C; n=4 to 6) or primary human TECs derived from the thymus of pediatric patients undergoing cardiac surgery (D; n=6) stimulated with NTZ 10 pM for 24 h. For the graphs in C and D data represent the mean and SD of two to four independent experiments, **p < 0.01 , *p < 0.05.

[0109] Figure 6. Proteasome inhibition leads to unfolded protein response (UPR) activation. cDNA derived from ANV 41.2 cells stimulated for 24 h with NTZ 10 pM was tested on a gene expression array for UPR genes. In the graph, only the genes with a fold induction of 2 or higher and with significant increase are listed (n=3). The graph represents the mean and SD of three independent experiments, *p < 0.05 compared to DMSO.

[0110] Figure 7. NTZ and other proteasome inhibitors upregulate Atf4.

[0111] Evaluation of Atf4 at mRNA (A) and protein (B) level on ANV41.2 cells treated with the proteasome inhibitors at 0.5 pM and NTZ 10 pM, for 24 h (n=3). For graphs in A, bars represent the mean and SD of three independent experiments. *p < 0.05. WB is representative of 2 independent experiments.

[0112] Figure 8. Proteasome inhibition NTZ-mediated activates UPR through ER stress. (A) ER staining with Concanavalin A after treatment with NTZ for 24 hours with quantification of mean fluorescence intensity (B) and vesicle number (C) per cell. Magnification 40x; scale bar = 100 pm. The graphs in B and C and fluorescent images in A are representative of two independent experiments (n=4 to 6). *p < 0.05.

[0113] Figure 9. Activation of UPR induces Foxnl. Figure shows the effects of salubrinal, a well-characterized inhibitor of elF2a dephosphorylation that activates UPR, on the induction of Foxnl as well as Atf4 transcript levels. Foxnl and Atf4 mRNA levels were quantified by qPCR in ANV41.2 cells treated for 24 h with Salubrinal (n=4). Bars represent the mean ± SD of three independent experiments.

[0114] Figure 10. NTZ promotes autophagy. (A) WB analysis for LC3B-I and II performed on ANV41.2 cells treated for 24 h with NTZ 10 pM + / - NH4CI. (B) Densitometry was performed by normalizing LC3B-II protein abundance on GAPDH (n=5). The graph represents the mean and SD of five independent experiments; *p< 0.05.

[0115] Figure 11. NTZ increases the number of LC3BII aggregates within the cells. (A) LC3B-II immunofluorescence in ANV41.2 cells treated with NTZ 10 pM + / - NH4CI. (B) Quantification of puncta was performed by counting the number of LC3B-II aggregates within the cells. Magnification 60x; cale bar = 20 pm (n=4). The graph represents the mean and SD of four independent experiments; *p < 0.05.

[0116] Figure 12. NTZ promotes thymic regeneration after radiation in mice.

[0117] C57BL / 6J young female mice received a sub-lethal dose of total body irradiation (SL-TBI) and were treated with NTZ or vehicle by oral gavage (OG). Thymi were collected 8 days after SL-TBI and total organ weight were evaluated (A). Absolute numbers of whole thymus cellularity at 8 and 15 days post SL-TBI (B). The graphs represent the mean and SD of two to four independent experiments (n=7-15). ***p < 0.001, **P < 0.01.

[0118] Figure 13. NTZ increases the number of hematopoietic and stromal cells in vivo. (A) Absolute numbers of DN (double negative CD4-CD8-), DP (double positive CD4+CD8+) and CD4+ and CD8+ single positive thymocytes gated on CD45+ cells. (B) Absolute numbers of cTEC (UEA1l0Ly51hi), mTEC (UEA1hiLy51l0), mTECl0and mTEChi. TEC populations were gated on CD45- / EPCAM+ / MHCII+ cells. The graphs represent the mean and SD of two to four independent experiments (n=7-15). *p < 0.05, ***p < 0.001.

[0119] Figure 14. NTZ increases the number of regulatory T cells (Treg) in vivo.

[0120] After NTZ treatment, thymic regulatory T cells (CD4+CD25+Foxp3+) increased in absolute number following SL-TBI. Figure shows representative flow cytometry plots and Treg counts. The graphs represent the mean and SD of two independent experiments (n=7-8). *p < 0.05.

[0121] Figure 15. NTZ protects thymic epithelial cells from damage induced by irradiation. Representative flow cytometry plots of Annexin V / viability staining of cTEC and mTEC at day 8 post-SLTBI and frequencies of live, early apoptotic and late apoptotic cells within each subset. Data represent mean ± SD from two independent experiments (n = 8). *p < 0.05, **p < 0.01.

[0122] Figure 16. NTZ promotes expression of Foxnl and its downstream genes in TECs in vivo. mRNA expression of Foxnl, DII4, Psmb11, Atf4, Xbp1, Ccl25 and Aire in sorted cTEC and mTEC 4 days post SLTBI from mice treated with vehicle or NTZ. Expression was normalized to housekeeping genes and expressedas fold to vehicle-treated mice. For sorting of TECs, thymi were pooled two-by-two prior to sorting, resulting in four pooled samples across two independent experiments (n = 4, representative of 8 mice). Data represent mean ± SD from two independent experiments (n = 8). *p < 0.05. (B) Quantification of ATF4 expressing TECs (ATF4 / pan-keratin double-positive cells) in thymic sections stained for DAPI (nuclei), pan-keratin (epithelium) and ATF4 in vehicle- or NTZ-treated and control mice at day 8 after irradiation. Data represent mean ± SD of four biological replicate per group from two independent experiments; 4 to 5 fields were analyzed per thymus (n=8-9). *p < 0.05; **p < 0.01; ***p < 0.001; ***p < 0.0001.

[0123] Materials and methods

[0124] All animals were purchased from Charles River and maintained in the Plaisant Castel Romano animal facility in Rome. Mice experiments were conducted in compliance with the EU and national ethical requirements and were approved by the Italian Health Ministry.

[0125] The origin of animal cell lines used in the experiments are reported below in “Cell cultures” paragraph.

[0126] Human thymic samples for human TEC culture were obtained from pediatric patients undergoing corrective cardiac surgery. All samples were anonymously collected in accordance with local ethical guidelines, written informed consent was obtained, and the protocol was approved by the Ethics Committee of OPBG Hospital.

[0127] Reagents

[0128] For western blot analysis, proteins were detected using the following primary antibodies: rabbit monoclonal anti-GAPDH (mAb #BK5174S, Cell Signaling), rabbit monoclonal anti-ubiquitin (mAb #BK43124S, Cell Signaling), rabbit polyclonal anti-LC3B (Ab #NB 100-2220, Novus Biologicals), mouse monoclonal anti-HSP90 (mAb #sc-13119, Santa Cruz), and rabbit monoclonal anti-ATF4 (mAb #11815, Cell Signaling). The following HRP-conjugated secondary antibodies were used: goat anti-rabbit IgG (Cat. #1706515, Bio-Rad) and horse anti-mouse IgG (Cat. #7076, Cell Signaling). For immunofluorescence assays targeting LC3B, a rabbit monoclonal anti-LC3B-ll antibody (mAb #38685, Cell Signaling) and a goat antirabbit IgG secondary antibody conjugated to Alexa Fluor 488 (Cat. #A27034, Thermo Fisher Scientific) were used. A plasmid containing the murine Foxnl coding sequence (NM_008238) was purchased from OriGene (Clone MR226744).Nitazoxanide (Cat. #N0290, Merck), carfilzomib (Cat. #HY-10455, DBA), bortezomib (Cat. #A2614, APExBio) and Salubrinal (#S2923-5MG, Aurogene) were reconstituted in 100% DMSO (Sigma-Aldrich). NH4CI was obtained from Sigma-Aldrich (Cat. #A9434).

[0129] Cell cultures

[0130] The murine cortical TEC ANV41.2 cell line previously described by Prof. Andrew G. Farr (University of Washington, Seattle, USA)17(Country of origin: USA) was maintained in DMEM (Euroclone) supplemented with 10% Fetal Bovine Serum (FBS, Euroclone), 2 mM glutamine (Euroclone), 100 units / mL penicillin and 0.1 mg / mL streptomycin (Euroclone). Cell cultures of primary human TECs (hTECs) were established following the procedure previously described. Briefly, hTECs were maintained on a feeder layer of lethally irradiated 3T3-J2 murine fibroblasts (#EF3003; Kerafast, USA) and cultured in a humidified atmosphere of 5% CO2 in growth medium composed of DMEM:Ham’s F-12 (3:1 mixture), 10% fetal calf serum (FCS) (ThermoFisher Scientific), insulin (5 pg / ml, Eli Lilly), adenine (0.18 mM, Sigma), hydrocortisone (0.4 pg / ml, Sigma), cholera toxin (0.1 nM, List Labs), triiodothyronine (2 nM, Sigma), glutamine (4 mM, ThermoFisher Scientific), and antibiotics. Epidermal growth factor (10 ng / ml, Austral Biologicals) was added to the medium after 48 h of culture. 24 hours before the start of the experiments, hTECs were plated in KGM-Gold-defined medium (Lonza) in the absence of a feeder layer.

[0131] Foxnl reporter cell line generation

[0132] To generate the FOXN1 dual luciferase reporter system, 675 bp of the DII4 promoter containing putative FOXN1 binding motifs was amplified by PCR and inserted into the Firefly Luciferase Vector PGL4.26 (Promega). The same sequence was then subjected to site-directed mutagenesis using modified primers to abolish the FOXN1 binding motifs. ANV 41.2 cells were nucleofected with the plasmids carrying the dual luciferase reporter gene system and were maintained in antibiotic selection with puromycin dihydrochloride (1.5 pg / ml, Merck Millipore) and hygromycin B (650 pg / ml, Merck Millipore).

[0133] Luciferase assay

[0134] To measure firefly and Renilla luciferase activities, the Dual-Luciferase® Reporter 1000 Assay System (Promega) was used according to the manufacturer's instructions. In brief, the Foxnl reporter cell line was seeded in a white, clear-bottom 96-well plate (Perkin Elmer). Following stimulation with the drug library, the mediumwas removed, and the cells were washed twice with PBS 1X (Euroclone). Cells were then lysed with 20 pL of Passive Lysis Buffer (Promega) diluted 1:5 in H2O and incubated in agitation for 15 minutes at room temperature. Subsequently, 100 pL of Luciferase Assay Reagent II (LAR-II) was added to each well, and the firefly luciferase signal was measured using a Synergy plate reader (BioTek). To quantify the Renilla luciferase signal, 100 pL of Stop & Gio® Reagent was added to each well, and the luminescence was measured.

[0135] Drug screening

[0136] To identify drugs capable of inducing FOXN1 activity, a drug screening with 1530 small molecules from the Discovery Probe™ FDA-approved Drug Library (ApexBio) was performed. Foxnl reporter cells seeded in a 96-well plate at a density of 5x103cells per well in 100 pL of DMEM were used. Each of the 1530 compounds was tested at a concentration of 5 pM. After 20 hours of stimulation, cell count and viability were assessed by incubating cells with the viable HOECHST stain for 20 minutes, followed by nuclear counting using Operetta (Perkin Elmer) and Celigo (Nexcelom Bioscience) cytometers. Scans of the total area of each well were acquired in two channels: fluorescence for nuclei count and brightfield for assessing cell morphology and viability. Confluency was calculated as (n° of nuclei of treated cells / n° of nuclei of control cells) X 100. Subsequently, the medium was removed, and the Firefly / Renilla (F / R) signals were quantified in the luciferase assay.

[0137] After the first round of screening, the following parameters for each of the 1,530 compounds were calculated to evaluate FOXN1 trans-activation in the reporter gene system: absolute F / R ratio, fold induction relative to DMSO, and F / R ratio corrected for internal controls. For each plate of the drug screening, the 71-factor was calculated to determine the quality and reliability of the assay (Z’ -factor > 0.5).

[0138] Compounds were then ranked by efficacy (from highest to lowest F / R ratio), and the three parameters were compared to establish a threshold of corrected F / R equal to 6, based on an absolute F / R ratio >2 and a fold induction >1.5. Cell confluence was not a determinant in defining this threshold. The same parameters were then applied to screen the initially identified 53 compounds in the second round of screening.

[0139] Quantitative PCR and reverse transcription

[0140] Total RNA was extracted from ANV 41.2 cells or primary human TEC treatedwith drugs / vehicle by using the RNeasy Plus micro kit (Qiagen). Reverse transcription was performed on 2 pg of cell-derived RNA with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). For mRNA extracted from sorted TECs, reverse transcription was carried out with SuperScript Vilo MasterMix (Invitrogen) For RNA quantification, quantitative PCR (qPCR) was performed in duplicate on 50 ng of retro transcribed cDNA with a QuantStudio 7 Pro instrument (Applied Biosystems). qPCR reactions were set up by using Luna® Universal qPCR Master Mix (New England Biolabs), containing TaqMan probes for the following genes: human Foxnl (Hs00919266_m1), human DH4 (Hs00184092_m1), human Hprt (Hs02800695_m1), Foxnl (Mm00433948_m1), DII4 (Mm00444619_m1), Psmb11 (Mm004212161_s1), Atf4 (Mm00515324_m1), Xbp1 (Mm00457357_m1), Ccl25 (Mm00436443_m1), Aire (Mm00477461_m1), Gapdh (Mm99999915_g1) and Hprt (Mm03024075_m1); all purchased from Life Technologies (Carlsbad, CA). For the analysis of genes involved in the UPR stress response, TaqMan® Array Mouse Unfolded Protein Response 96-well plate (ThermoFisher) was used. Relative amounts of mRNA were calculated by the comparative C(t) method.

[0141] Western Blot analysis

[0142] For analysis of protein expression, ANV41.2 cells were detached from 6 multi-well plates and collected in 1.5 mL tubes. Cells were lysed on ice for 20 minutes in a H2O solution containing RIPA buffer (Cell Signaling) diluted 1:10 and Protease and Phosphatase Inhibitor Cocktail (Merck) diluted 1:100. Cell lysates were centrifuged at 14000 rpm for 20 min at 4 °C, and the supernatants were then collected. After protein extraction and quantification, performed with Pierce™ BCA Protein Assay Kits (Thermo Fisher Scientific), 20-50 pg of proteins per sample were subjected to denaturation in LDS sample buffer (Thermo Fisher Scientific) at 100 °C for 10 and electrophoresis separation by SDS-PAGE. Proteins were then transferred to a 0.45-pm Immobilon-P PVDF membrane (Millipore Sigma) for western blotting (WB), and transfer efficiency was verified by Ponceau (Sigma-Aldrich) staining. Membranes were blocked for 1 h in a PBS-0.05% Tween solution (Merck KgaA, Darmstadt, Germany) supplemented with 5% (wt / vol) of nonfat dry milk (Cell Signaling) and then incubated overnight at 4 °C with the primary antibodies listed in the Reagents paragraph properly diluted in a PBS-0.05% Tween solution supplemented with 5% (wt / vol) of dry milk. The membranes were finally incubated with Immobilon Western chemiluminescent HRP substrate (Merck Millipore), andthe immunoreaction was revealed by Chemidoc™ XRS+ Imaging System (Bio Rad). Immunoblots were analyzed by densitometry using Fiji Imaged software. Data are analyzed as a ratio of the band intensity of the target protein to the reference GAPDH.

[0143] Evaluation of Proteasome activity

[0144] To evaluate proteasome activity, the Proteasome-Glo™ Cell-Based kit (Promega) was used according to the manufacturer's instructions. In brief, ANV 41.2 cells were seeded in a white, clear-bottom 96-well plate (Perkin Elmer) at a cell density of 5x103cells / well. Cells were then stimulated with the compounds and then 100 pl the Suc-LLVY-Glo™ or Z-LRR-Glo™ or Z-nLPnLD-Glo™ substrates were added. After 10 minutes of incubation, luminescent signal derived from proteasome cleavage of specific substrates, was measured using a Synergy plate reader (BioTek).

[0145] Fluorescence imaging

[0146] For LC3B immunofluorescence, ANV 41.2 cells were seeded on sterile glass coverslips in a 24 multi-well plate at a cell density of 1x105cells / well in 500 pl. After NTZ treatment, cells were washed in PBS 1X, fixed, and permeabilized with cold methanol for 10’ at -20°C. Coverslips were then incubated for 1 h with 3% goat serum (Sigma-Aldrich) for blocking of non-specific signals and then overnight at 4°C with primary antibody for LC3B-II (Rabbit mAb #38685, Cell Signaling) in 1% goat serum. Cells were then washed with PBS and labeled for 1 h with a goat anti-rabbit 488-conjugated secondary antibody (#A27034, Thermo Fisher Scientific) diluted in 1% goat serum. Nuclei were stained with 1 pg / ml DAPI for 20 min at room temperature, and images were acquired with a Leica TCS-SP8X laser-scanning confocal microscope equipped with a 60x (NA 1.4) oil immersion objective (Leica Biosystems). Puncta analysis was performed using “Fiji” open-source software.

[0147] Pheno Vue™ Cell Painting Kit (Revvity) was used to stain the endoplasmic reticulum (ER) and nuclei. In brief, ANV41.2 cells were seeded in a 96-well black, clear-bottom CellCarrier Ultra microplate (Perkin Elmer) at a density of 5 x 10scells / well. After a 24-hour treatment with 10 pM NTZ, cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with PBS containing 0.1% (v / v) Triton X-100. Subsequently, ANV 41.2 cells were stained with a cell painting mix containing PhenoVue Hoechst 33342 to stain nuclei and PhenoVue Fluor 488 (Concanavalin A) for ER staining. Image acquisition was then performed using the Operetta high-content screening system (Perkin Elmer). Image analysis andfluorescence quantification were performed with Harmony® software.

[0148] Mice and animal procedures

[0149] C57BL / 6J female mice (Charles River Laboratories) between 8 and 12 weeks of age were used. To model thymic damage and lymphoid depletion, C57BL / 6J received SL-TBI with no hematopoietic rescue. C57BL / 6J mice received NTZ (1 mg / kg in PBS, 1% DMSO; oral gavage - OG) or vehicle (PBS with 1% DMSO) two hours before and on days 2, 5, and 6 post-TBI, for a total of 4 administrations. All animal procedures were carried out at Plaisant animal facility.

[0150] Immunofluorescence on thymic sections

[0151] Thymus sections (2.5 pm) were deparaffinized, rehydrated, and subjected to antigen retrieval in citrate buffer (pH 6.0) for 20 minutes at 95 °C. After cooling, sections were permeabilized with 0.1% Triton X-100 in PBS and blocked for 1 hour in PBS containing 5% normal goat serum. Slides were then incubated overnight at 4 °C with primary antibodies against mouse ATF4 (#11815, Cell Signaling Technology) and pan-Keratin (#ab8068, Abeam). After washing, sections were incubated for 1 hour with Alexa Fluor 488-conjugated secondary antibody (#A11070, Thermo Fisher Scientific) for ATF4 and Alexa Fluor 555-conjugated secondary antibody (#A21425, Thermo Fisher Scientific) for pan-Keratin, followed by nuclear counterstaining with Hoechst. Images were acquired using a Leica TCS-SP8X confocal microscope equipped with 20* and 60* oil immersion objectives. Quantification of ATF4+epithelial cells was performed on 4-5 fields per section using Fiji (Imaged).

[0152] Flow cytometry

[0153] Individual single-cell suspensions of freshly dissected thymi were obtained by either mechanical dissociation or enzymatic digestion. Briefly, 2 x 106cells were incubated for 20 min at 4°C with antibodies in the dark and washed twice with FACS buffer (PBS 1X + FBS 2%). Flow cytometric analysis was performed on a Fortessa (BD Biosciences) using FACSDiva (BD Biosciences) or FlowJo (Treestar Software). The list of antibodies and clones is reported in Table S.1. For TEC sorting, thymic samples were processed by mechanical dissociation followed by enzymatic. CD45-cells were enriched by depletion of CD45+ cells using LS magnetic separation columns (Miltenyi Biotec) and CD45 microbeads (Miltenyi Biotec). Purity of the depletion was verified by FACS. Cells were then stained for 20 min at 4° C with TEC-specific antibodies (Table S2) and sorting of cTEC and mTEC subsets wasperformed on a BD FACSAria™ III Cell Sorter (BD Biosciences).

[0154] Statistics

[0155] For each experiment, at least two separate independent experiments are represented, unless otherwise indicated. For multiple comparisons, statistical analysis was employed using the nonparametric one-way ANOVA with Dunn’s correction. Statistical analysis between the two groups was performed with the nonparametric, unpaired Mann-Whitney test. A p-value less than or equal to 0.05 was considered statistically significant. Data analysis and relative graphs were generated using GraphPad software.

[0156] Results

[0157] 1. Reporter system setup and in vitro validation

[0158] To identify pharmaceutical approaches to enhance Foxnl transcription and activity, a high-throughput drug screening was set up. While recent studies have provided important insights into the DNA regulatory regions of Foxnl, combining all Foxnl DNA regulatory sequences in a reporter system for a drug screen would be extremely challenging and would still not completely recapitulate the endogenous chromatin complexity. To overcome this issue, a system in which a luciferase reporter gene is under the control of Foxnl transcriptional activity was generated. To do so, a fragment of the murine DII4 promoter containing recently identified FOXN1 DNA binding sequences was cloned upstream of the Firefly luciferase reporter gene. To discriminate between stimuli that activate the DII4 promoter in a FOXN1 -independent manner, site directed mutagenesis was performed to delete FOXN1 DNA binding sequences from the DII4 promoter upstream of a different luciferase gene (Renilla). Thus, drugs inducing Foxnl expression and / or its transactivation activity will translate into a firefly-to-ren il la (F / R) ratio greater than 1. Conversely, drugs promoting the expression of the reporter gene in a FOXN1-independent manner will translate into an F / R ratio close to 1. The murine cortical TEC line ANV 41.2, previously described18, was chosen for the generation of the FOXN1 dual-luciferase reporter cell line for drug screening. To select the clones that correctly integrated the report system after antibiotic selection, the F / R ratio following bzATP treatment was evaluated, which was shown to induce Foxnl expression (Fig. 1A)16. Among the tested clones, we picked clone 13 that effectively doubled the luciferase signal (F / R > 2) following bzATP stimulation. To verify the specificity of the system in detecting and responding to changes in FOXN1 activity,cells were transfected with a third plasmid carrying the Foxnl ORF under a constitutive promoter and measured the luciferase activity. Exogenous expression of FOXN1 resulted in a significant increase in the F / R ratio at 24 h compared to the control group, confirming the functionality and specificity of the reporter assay (Fig.

[0159] 1 B). Thus, a comprehensive assessment of the effectiveness and reliability of the reporter system was ensured for downstream applications in drug screening.

[0160] 2. Drug screening and candidate refinement

[0161] The Foxnl reporter cell line was used to screen a drug library of 1530 small molecules (Discovery ProbeTM FDA-approved Drug Library) and 53 compounds that significantly promoted FOXN1 activity were identified (Fig. 1C). Together with luciferase detection, cell count and viability for each tested drug were also assessed. This approach allowed the inventors to establish a correlation between luciferase data and the impact of the compounds on cell confluency, which is closely linked to the degree of cell proliferation and vitality. Single-well image acquisition was conducted for each drug in two channels: brightfield for the assessment of cell viability and morphology, and fluorescence to directly visualize and count cell nuclei. Confluency and luciferase activity were calculated according to the equations described in the Methods section. After the initial screening, which revealed 53 compounds, drugs were “cherry-picked” and re-tested in the same assay for confirmation (Fig. 1 D).

[0162] To further refine the selection of compounds, a thorough visual inspection of scans acquired during the second round of screening was undertaken to identify and eliminate compounds retrieving false positive results (i.e., the presence of auto-fluorescent drug precipitates). The analysis identified 34 compounds. 12 of them could be grouped based on their capacity to inhibit the proteasome. The 12 compounds are the following: Mitoxantrone HCI (Novantrone), Doxorubicin (Adriamycin), MLN9708 (Ixazomib), MLN2238 (Ixazomib citrate), PR-171 (Carfilzomib), CEP-18770 (Delanzomib), Zinc Pyrithione, Doxorubicin HCI, Daunorubicin HCI, Epirubicin HCI, Idarubicin HCI, Nitazoxanide. The list also contained four compounds known to activate proteotoxic stress through activation of ER stress and UPR signaling: homoharringtonine, verteporfin, abacavir sulfate and abacavir. The 16 compounds are listed in Table 1, which provides information regarding their efficacy in inducing FOXN1 transactivation (defined as F / R).Table 1

[0163]

[0164]

[0165] It was observed that both well-known proteasome inhibitors (MLN9708-Ixazomib citrate, MLN2238-lxazomib, PR-171-Carfilzomib, CEP-18770-Delanzomib) and also the other compounds known to have effects on proteasome inhibition induced significantly elevated levels of FOXN1 activity. Therefore, the relationship between proteasome inhibition and FOXN1 activity was investigated. To this purpose, further characterization was performed by assessing pharmacological parameters such as the half-maximal effective concentration (EC50) at 24 h and the half-maximal growth inhibition (GI50) at 72 h by cell nuclei counting of the well-known proteasome inhibitors (Ixazomib, Delanzomib and Carfilzomib) using the luciferase reporter assay, (Fig. 2). All drugs analyzed demonstrated a dose-dependent modulation of FOXN1 activity with comparable EC50 values. Similarly, vitality curves demonstrated analog patterns.

[0166] To assess whether the proteasome inhibitors induced Foxnl at the gene level, qPCR was performed on the wild-type cTEC ANV 41.2 after stimulation with the compounds. The analysis was extended to include bortezomib, another well-known FDA-approved proteasome inhibitor that was not present in the initial screening. All the analyzed drugs induced elevated levels of Foxnl mRNA and ofits downstream target gene DII4 (Fig. 3A and B respectively), corroborating the observed increase in FOXN1 biological activity as assessed in the luciferase assay. Taking these results together, it was revealed that proteasome inhibition promotes Foxnl transcriptional synthesis and its transactivation activity on downstream target genes.

[0167] 3. The FOXN1 inducer Nitazoxanide promotes proteasome inhibition, ER stress, and UPR.

[0168] The inhibition of proteasome was identified as a key mechanism for FOXN1 induction. It was also found that the tested proteasome inhibitors induced significant cell toxicity (GI50 > EC50). However, these compounds may still be used as effective drugs by modifying their dosage or by using non-cytotoxic analogues thereof. The list of drugs was re-evaluated aiming to identify other drugs with proteasome inhibitory activity but with minimal cytotoxic profiles.

[0169] Nitazoxanide (NTZ) was identified as a potential candidate, which is currently used in clinical practice as antiviral and antiprotozoal medication. Recent research demonstrated that NTZ and related thiazolides target the 20S proteasome subunit28. Given that no information on the effects of NTZ on thymic epithelial cells is available, ANV41.2 cells were stimulated with NTZ and proteasome modulation over time was assessed by analyzing the accumulation of ubiquitinated proteins (UB-proteins). Treatment with NTZ significantly induced rapid but transient proteasome inhibition, with an effect observed within 2 hours (Fig. 4A and B). To investigate the correlation between UB-proteins accumulation and the inhibition of proteasomal activity, it was conducted a cell-based assay to assess the ability of NTZ to specifically inhibit (31 , [32 and [35 proteasome subunits (also known as the caspase-like, trypsin-like, chymotrypsin-like domains, respectively) within the catalytic core of the 20S complex, as described in literature28. For this purpose, specific substrates for each of the three subunits were employed (Suc-LLVY, Z-LRR and Z-nLPnLD) which produce luminescence upon proteasomal cleavage. As shown in Figure 4C, a 2-hour treatment of ANV 41.2 cells with NTZ 10 pM resulted in a complete inhibition of all three domains activity as evidenced by a sharp decline in luminescence emission. This finding supports the observed accumulation of ubiquitinated proteins obtained after 2h of NTZ stimulation, as demonstrated by Western blot analysis (Fig 4A). Once proteasome inhibition on TEC was confirmed, NTZ effects on FOXN1 were further investigated by performing dose-response curves to evaluate efficacy(ECso) and toxicity profiles (Glso) on Foxnl reporter cell line. The drug demonstrated a dose-dependent induction of luciferase emission, directly proportional to FOXN1 activity, with minimal cell toxicity (Fig. 5A and B). Consistent with an increase in reporter activity, exposure of murine ANV 41.2 cells to NTZ resulted in an increase in both Foxnl and DII4 mRNA levels (Fig. 5C). Importantly, the treatment induced robust upregulation of F0XN1 and DLL4 also in primary human TECs freshly isolated from pediatric thymus tissue, demonstrating that the drug activates the same transcriptional program in physiologically relevant human cells and confirming the conservation of this pathway across species (Fig. 5D).

[0170] Taking together these findings, it was hypothesized that proteasome inhibition could modulate cellular pathways resulting in Foxnl regulation. Among the consequential downstream outcomes associated with proteasome inhibition, the unfolded protein response (UPR) becomes activated in response to the accumulation of misfolded proteins in the endoplasmic reticulum (ER) and activates master transcription factors accountable for adaptive responses. To seek a connection between the proteasome inhibition and UPR, ANV41.2 cells were stimulated with NTZ and a set of UPR-associated genes was evaluated. Among these genes, significant upregulation of Ddit3, Eroll, Amfr, and Atf4 was found after NTZ treatment (Fig 6). Similarly to NTZ, the other proteasome inhibitors tested in the initial screening (Ixazomib, Delanzomib, Carfilzomib and Bortezomib) induced upregulation of ATF4 at both mRNA (Fig. 7A) and protein levels (Fig. 7B).

[0171] As these genes are associated with disruption of ER homeostasis, it was posited that ER stress could serve as a plausible functional link between proteasome inhibition and UPR activation induced by NTZ. To investigate this, the morphological status of ER in ANV41.2 cells after NTZ treatment was evaluated (Fig. 8A). Staining of the cells with Concanavalin A, a lectin specific for the ER, revealed a significant increase in fluorescent signal intensity and intracellular vesicle-like structures, which is indicative of ER stress and altered intracellular trafficking dynamics (Fig. 8B and C).

[0172] To further explore the relationship between Foxnl induction and the ER stress response, it was tested whether pharmacological activation of the UPR could modulate Foxnl expression. Treatment with salubrinal, a well-characterized inhibitor of elF2a dephosphorylation that activates UPR, significantly increased Foxnl as well as Atf4 transcript levels (Fig. 9). Moreover, revisiting the original listof 34 drugs that significantly upregulated Foxnl, the presence of four compounds known to activate ER stress and UPR signaling was observed: homoharringtonine, verteporfin, diosmetin, abacavir sulfate and abacavir (Fig.1 D). All together this data supports the notion that Foxnl induction is not unique to proteasome inhibition and may represent a broader stress-related mechanism.

[0173] 4. NTZ induces Foxnl and autophagy.

[0174] Given that ATF4 is known to regulate key factors associated with autophagy, a critical process responsible for the removal of damaged organelles and misfolded proteins, thereby promoting cellular homeostasis after injuries, a link was hypothesized between NTZ, ATF4, and autophagy. The induction of autophagy in ANV41.2 cells was demonstrated by detecting a significant increase in the lipidation of LC3B (Fig. 10A and B) and in the number of LC3B-II aggregates (referred to as “puncta”) within the cellular cytoplasm 24-hour after NTZ stimulation (Fig. 11 A and B).

[0175] 5. Nitazoxanide accelerates thymus regeneration following SL-TBI challenge.

[0176] After validating NTZ as a potent non-toxic FOXN1 activator and conducting an extensive examination of its mechanism of action in vitro assay, NTZ ability to promote thymus regeneration in vivo was evaluated using a mouse model of acute thymic damage and regeneration by exposing C57BL / 6J (B6) mice to a sub-lethal dose of total body irradiation (SL-TBI, 550 cGy).

[0177] Mice exposed to NTZ treatment showed accelerated recovery of overall thymic mass (Fig. 12A) 8 days after SL-TBI, when compared to vehicle-treated mice. Consistent with this, NTZ treated mice showed increased total thymic cellularity at 8 and 15 days post SL-TBI (Fig. 12B), when compared to vehicle-treated mice. Comprehensive characterization of thymocyte populations revealed a significant increase of the absolute numbers of double-negatives (DN), double-positives (DP) and CD4+ or CD8+ single-positive (SP4 and SP8) thymocytes in NTZ-treated mice compared to the control group (Fig. 13A). Notably, when compared to vehicle administration, NTZ also significantly enhanced the recovery of mTEC and cTEC counts after SL-TBI (Fig. 13B). Interestingly, within the mTEC population, NTZ administration results in significant elevations in MHCH-low TECs (mTECIo) cells compared to vehicle-treated mice, whereas more mature MHCH-high mTECs (mTEChi) appeared unaffected by the drug (Fig. 13B).Thymic regulatory T cells (CD4+CD25+Foxp3+) increased in absolute number while maintaining stable frequency within the CD4 compartment, demonstrating preserved Treg differentiation after NTZ treatment (Fig.14).

[0178] To gain mechanistic insight into how NTZ treatment supported thymic regeneration following SLTBI, apoptosis and proliferation in cTECs and mTECs at day 7 post-irradiation were first assessed. While Ki67 analysis showed no significant differences in TEC proliferation between groups, Annexin V staining revealed a marked reduction in apoptotic TECs in NTZ treated animals, accompanied by a proportional increase in AnnexinV” viable cells (Fig. 15). This protective effect was detected in both TEC compartments, with a more pronounced impact in mTECs (Fig. 15).

[0179] To investigate transcriptional changes triggered by NTZ treatment, cTECs and mTECs were next sorted from treated and control mice at day 4 post-irradiation. In cTECs, in agreement with findings in vitro and in primary human TECs, a significant upregulation of Foxnl, of its downstream target genes DII4 , Ccl25 Psbm11, and of UPR associated genes ATF4 and Xbp1 (Fig. 16) was observed. mTECs displayed a significant increase in Foxnl. Importantly, NTZ treatment did not affected expression of Aire (Fig. 16). Together, these data demonstrate that NTZ treatment induced Foxnl expression in vivo and activated its downstream transcriptional program in cTECs, while providing TEC protection during early phases of thymic regeneration after damage.

[0180] References

[0181] 1 Takahama, Y. Journey through the thymus: stromal guides for T-cell development and selection. Nat. Rev. Immunol. 6, 127-135 (2006).

[0182] 2 Velardi, E. et al. T cell regeneration after immunological injury. Nat Rev Immunol. 21:277-291 (2020).

[0183] 3 Clave, E. et al. Thymic function recovery after unrelated donor cord blood or T-cell depleted HLA-haploidentical stem cell transplantation correlates with leukemia relapse. Front. Immunol. 4,(2013).

[0184] 4 Tsamadou, C. et al. Donor genetic determinant of thymopoiesis rs2204985 impacts clinical outcome after single HLA mismatched hematopoietic stem cell transplantation. Bone Marrow Transplant. 57, 1539-1547 (2022).

[0185] 5 Wertheimer, T. et al. Production of BMP4 by endothelial cells is crucial for endogenous thymic regeneration. Sci. Immunol. 3, eaal2736 (2018).6 Vaidya, H. J., Briones Leon, A. & Blackburn, C. C. FOXN1 in thymus organogenesis and development. Eur. J. Immunol. 46, 1826-1837 (2016).

[0186] 7 Song, Y. et al. FOXN1 recombinant protein enhances T-cell regeneration after hematopoietic stem cell transplantation in mice. Eur. J.

[0187] Immunol. 46, 1518-1528 (2016).

[0188] 8 Bredenkamp, N., Nowell, C. S. & Blackburn, C. C. Regeneration of the aged thymus by a single transcription factor. Development 141, 1627-1637 (2014).

[0189] 9 Bosticardo, M. et al. Heterozygous FOXN1 Variants Cause Low TRECs and Severe T Cell Lymphopenia, Revealing a Crucial Role of FOXN1 in Supporting Early Thymopoiesis. Am. J. Hum. Genet. 105, 549-561 (2019).

[0190] 10 Min D., et al. Sustained thymopoiesis and improvement in functional immunity induced by exogenous KGF administration in murine models of aging. Blood. 2007;109(6):2529-2537.

[0191] 11 Goya R.G. et al. In vivo effects of growth hormone on thymus function in aging mice. Brain Behav Immun. 1992;6(4):341-354.

[0192] 12 Dixit V.D., et al. Ghrelin promotes thymopoiesis during aging. J Clin Invest. 2007 Oct; 117(10):2778-90.

[0193] 13 Dudakov J. A., et al. Interleukin-22 drives endogenous thymic regeneration in mice. Science. 2012;336(6077):91-95.

[0194] 14 Velardi, E. et al. Sex steroid blockade enhances thymopoiesis by modulating Notch signaling. J. Exp. Med. 211, 2341-2349 (2014).

[0195] 15 Lopes, N., H. Vachon, J. Marie, and M. Irla. Administration of RANKL boosts thymic regeneration upon bone marrow transplantation. EMBO Mol Med 9:835-851 (2017)

[0196] 16 Kinsella, S. et al. Damage-induced pyroptosis drives endogenous thymic regeneration via induction of Foxnl by purinergic receptor activation.

[0197] Preprint at https: / / doi.org / 10.1101 / 2023.01.19.524800 (2023).

[0198] 17 Wang, R., Nelson, A., Kimachi, K., Grey, H. M. & Farr, A. G. The role of peptides in thymic positive selection of class II major histocompatibility complex-restricted T cells. Proc Natl Acad Sci U S A 95, 3804-3809 (1998).

[0199] 18 Kupperman, E. et al. Evaluation of the proteasome inhibitor MLN9708 in preclinical models of human cancer. Cancer Res. 70, 1970-1980 (2010).19 Larsson, P. et al. Repurposing proteasome inhibitors for improved treatment of triple-negative breast cancer. Cell Death Discov. 10, 57 (2024).

[0200] 20 Maione, F. et al. Preclinical efficacy of carfilzomib in BRAF -mutant colorectal cancer models. Mol. Oncol. 18, 1552-1570 (2024).

[0201] 21 Demo, S. D. et al. Antitumor activity of PR-171 , a novel irreversible inhibitor of the proteasome. Cancer Res. 67, 6383-6391 (2007).

[0202] 22 Berkers, C. R. et al. Probing the Specificity and Activity Profiles of the Proteasome Inhibitors Bortezomib and Delanzomib. Mol. Pharm. 9, 1126-1135 (2012).

[0203] 23 Costa, V. M. et al. Mitoxantrone impairs proteasome activity and prompts early energetic and proteomic changes in HL-1 cardiomyocytes at clinically relevant concentrations. Arch. Toxicol. 94, 4067-4084 (2020).

[0204] 24 Sishi, B. J. N., Loos, B., Van Rooyen, J. & Engelbrecht, A.-M.

[0205] Doxorubicin induces protein ubiquitination and inhibits proteasome activity during cardiotoxicity. Toxicology 309, 23-29 (2013).

[0206] 25 Fekete, M. R., McBride, W. H. & Pajonk, F. Anthracyclines, proteasome activity and multi-drug-resistance. BMC Cancer 5, 114 (2005).

[0207] 26 Kiyomiya, K.-l., Kurebe, M., Nakagawa, H. & Matsuo, S. The role of the proteasome in apoptosis induced by anthracycline anticancer agents. Int. J. Oncol. (2002).

[0208] 27 Zhao, C. et al. Repurposing an antidandruff agent to treating cancer: zinc pyrithione inhibits tumor growth via targeting proteasome-associated deubiquitinases. OncotargetS, 13942-13956 (2017).

[0209] 28 Lu, Z. et al. Nitazoxanide and related thiazolides induce cell death in cancer cells by targeting the 20S proteasome with novel binding modes. Biochem. Pharmacol. 197, 114913 (2022).

[0210] 29 Liu Y, Hua H, Cao Y, Li M, Zhang H, Du S et al. Mechanism by which the molecular glue-like verteporfin induces IRE1a dimerization and activation to synergize with AKT inhibition in breast cancer. Cell Chem Biol 2025; 32: 854-871. e6.

[0211] 30 Shimomura I, Watanabe N, Yamamoto T, Kumazaki M, Tada Y, Tatsumi K et al. Selective targeting of KRAS-driven lung tumorigenesis via unresolved ER stress. JCI Insight 2021; 6: e137876.

[0212] 31 Wang M, Zhao S, Yu S, Zhou Z. Investigating The Impact OfHomoharringtonine On K562 Cell Viability And ER Stress Pathways. BIO Web Conf 2025; 163: 03002.

[0213] 32 Cai Z, Hu J, He D, Wu W, Gao L, Bao H. Homoharringtonine Induced Apoptosis of Leukemic Cell Line MUTZ-1 Cells Is Mediated by the Endoplasmic Reticulum Stress Pathway. Blood 2005; 106: 4917-4917.

[0214] 33 Nooka S, Ghorpade A. HIV-1 -associated inflammation and antiretroviral therapy regulate astrocyte endoplasmic reticulum stress responses. Cell Death Discov 2017; 3: 17061.

Claims

CLAIMS1) An Unfolded Protein Response (UPR) activator compound, or a pharmaceutical composition comprising said compound together with one or more excipients and / or adjuvants, for use in the treatment of thymic insufficiency or thymic disfunction, wherein said UPR activator compound is chosen from a direct UPR activator compound, which is able to activate at least one of ATF4, ATF6 and Xbp1 s, and an indirect UPR activator compound chosen from a proteasome inhibitor compound and an inducer of endoplasmic reticulum (ER) stress.2) UPR activator compound or pharmaceutical composition according to claim 1 for use according to claim 1, wherein said proteasome inhibitor compound is chosen from the group consisting ofan acylsalicylamide, such as Nitazoxanide,Ixazomib,Carfilzomib (PR-171),Delanzomib (CEP-18770),Bortezomib,Daunorubicin,Doxorubicin,Epirubicin,Idarubicin,Mitoxantrone,Zinc PyrithioneBC-05,BMG-431,Cerpegin,Chymostatin,CNN-1,CUDC-907,D Boronophenylalanine,Degrasyn,DHP,GSK3494245,KZR-616,Lactacystin,LONP1-IN-2,Marizomib,MG132,NPI-0052,ONX-0912,Oprozomib,Pepstatin A,PI-1840,PR-104,PR-619,PS-341,PZ-265,RA375,Roxadustat,S-THIQ,Sabarubicin,Sodium N-ethylmaleimide,or a salt, substituent, analogue, tautomer, isomer, active metabolite, or prodrug thereof.3) UPR activator compound or a pharmaceutical composition according to any one of the preceding claims for use according to any one of the preceding claims, wherein said proteasome inhibitor compound is chosen from the group consisting of Nitazoxanide, Ixazomib, Delanzomib, Carfilzomib and Bortezomib ora salt or substituent thereof or an analogue, tautomer, isomer, active metabolite or prodrug thereof, preferably Nitazoxanide or a salt, substituent, analogue, tautomer, isomer, active metabolite or prodrug thereof.4) UPR activator compound or pharmaceutical composition according to claim 1 , for use according to claim 1 , wherein said inducer of endoplasmic reticulum (ER) stress is chosen from homoharringtonine, verteporfin, diosmetin and abacavir or a salt, derivative, substitute, analogue, tautomer, or prodrug thereof.5) UPR activator compound or pharmaceutical composition according to claim 1, for use according to claim 1, wherein said direct UPR activator compound is salubrinal or a derivative or analogue thereof.6) UPR activator compound or a pharmaceutical composition according toany one of the preceding claims, for use according to any one of the preceding claims, wherein said compound or pharmaceutical composition is administered orally, by intramuscular injection, by subdermal injection, by intraperitoneal injection, by intravenous injection, by subcutaneous injection, or by inhalation.7) UPR activator compound or a pharmaceutical composition according to any one of the preceding claims, for use according to any one of the preceding claims, wherein said compound or pharmaceutical composition is administered in a dosage from 0.01 pg / kg to 500 mg / kg.8) UPR activator compound or a pharmaceutical composition according to any one of the preceding claims, for use according to any one of the preceding claims, wherein said pharmaceutical composition further comprises one or more compounds chosen fromcompounds for promoting thymus function such as sex hormone inhibitors; Keratinocyte Growth Factor (KGF); Interleukin 7; Insulin-like Growth Factor (IGF); Ghrelin; Growth Hormone (GH); Interleukin-22; Interleukin-33; Thymosin alpha-1; Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL); Adenosine Triphosphate (ATP); Growth Differentiation Factor 11 (GDF11); Beta-2 Microglobulin (B2M); or Bone Morphogenetic Protein 4 (BMP4);compounds for promoting the recovery of bone marrow or hematopoietic function such as Parathyroid hormone (PTH), stem cell factor (SCF), caloric restriction; nutritional support; thrombopoietin (TPO), fms-like tyrosine kinase 3 ligand (Flt3L), Granulocyte Colony-Stimulating Factor (G-CSF), Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Androgens, hormonal ablation.9) Combination of i) a UPR activator compound or a pharmaceutical composition as defined in any one of claims 1-8 and ii) one or more compounds chosen fromcompounds for promoting thymus function such as sex hormone inhibitors; Keratinocyte Growth Factor (KGF); Interleukin 7; Insulin-like Growth Factor (IGF); Ghrelin; Growth Hormone (GH); Interleukin-22; Interleukin-33; Thymosin alpha-1; Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL); Adenosine Triphosphate (ATP); Growth Differentiation Factor 11 (GDF11); Beta-2 Microglobulin (B2M); or Bone Morphogenetic Protein 4 (BMP4);compounds for promoting the recovery of bone marrow function such asParathyroid hormone (PTH), stem cell factor (SCF), thrombopoietin (TPO), fms-like tyrosine kinase 3 ligand (Flt3L), Granulocyte Colony-Stimulating Factor (G-CSF), Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Androgens, hormonal ablation; and / orT cell progenitors, thymic-like organoids and scaffolds or bone marrow-like organoids and scaffoldsfor separate or sequential use in the treatment of thymic insufficiency or disfunction.

Images