Methods for monitoring and assessing efficacy of Treg cell therapy

Abstract

The present invention relates to methods of monitoring and efficacy assessment of cell therapy for patients treated with CD4 + Fox P3 + T regulatory cells. Further subject matter relates to methods of cell therapy using CD4 + Fox P3 + T regulatory cells and methods of monitoring of the invention.

Description

[0001] The present invention relates to methods for monitoring and evaluating the efficacy of cell therapy for treating patients with CD4+ Fox P3+ T regulatory cells (hereinafter referred to as "Treg cells" or "Tregs"), respectively. Additional subject matter relates to cell therapy methods using CD4+ Fox P3+ T regulatory cells and monitoring methods of the present invention.

[0002] Both clinical and laboratory data demonstrate the importance of the immune system in the pathogenesis of type 1 diabetes (DM1). Although the trigger may be a viral infection or genetic susceptibility, the disease develops due to an imbalance between an excessive auto-aggressive T cell response and an impaired tolerance mechanism (1, 2). Therefore, the most promising disease modification strategy is built around immunotherapy (3, 4). Only recently has telizumab been introduced, the first drug of this century approved by the FDA for the treatment of presymptomatic DM1 individuals. It is a monoclonal antibody against autoreactive CD3+ T cells that can delay the onset of symptomatic DM1 by at least two years (5). Among the many attempts to prevent or at least delay DM1, cell therapy using FoxP3+T regulatory cells (Treg) seems to be of particular interest. The inventors and others have conducted multiple clinical trials on T regulatory cell products and achieved promising results (5-10).

[0003] The major challenge facing all of these therapies is the search for a good immune marker that can predict treatment response, given the β-cell destruction that causes DM1 (11). The tissue available for sampling is usually peripheral blood, which is far from the local tissue lesions. Therefore, β-cell function remains the only acceptable endpoint for treatment efficacy to date (4). Unfortunately, this monitoring allows us to detect the disease relatively late, when islet destruction is ongoing. It only shows the progression of DM1 and cannot predict treatment efficacy in advance during treatment.

[0004] The technical problem underlying the present invention is to provide elements for evaluating the progress of cell therapy using Treg cells.

[0005] A method for solving the above-mentioned technical problem is provided by the embodiments of the present invention as described in the claims and disclosed in this specification and the drawings.

[0006] The present invention is based on the discovery that certain biomarkers can be used to characterize a cell therapy regimen for a patient in need of such cell therapy using Treg cells, as described herein and in further detail in the Examples below. Biomarkers identified as being particularly useful for this purpose are PD-1 and CD73. In addition, the inventors have determined that certain expression values ​​of PD-1 and / or CD73 indicate that cell therapy treatment using Treg cells is effective.

[0007] Specifically, the present invention provides a method for monitoring cell therapy of a patient treated with CD4+ Fox P3+ Treg cells, comprising the following steps: determining in vitro the expression of at least one protein selected from PD-1 and CD73 on the patient's CD4+ FoxP3+ T cells, preferably CD4+ FoxP3+ Treg cells, and / or determining the expression of PD-1 on the patient's CD4+ FoxP3-T cells, preferably CD4+ FoxP3-T effector cells ("Teff cells" or "Teffs").

[0008] Preferably, determining the expression of at least one protein on CD4+ FoxP3+ T cells and / or determining the expression of PD-1 on CD4+ FoP3- cells is performed on a patient sample (in other contexts of the present disclosure, synonymously "performed with").

[0009] In a preferred embodiment, the monitoring method of the present invention comprises isolating CD4+ FoxP3+ T cells from a patient sample prior to determining the expression of at least one protein, and / or isolating CD4+ FoxP3- cells from a patient sample prior to determining the expression of PCD1 on CD4+ FoxP3- cells. The isolation of cells of interest according to the present invention disclosed herein (particularly from peripheral blood) is generally performed according to methods known in the art, which generally include the isolation of peripheral blood mononuclear cells (PBMCs), which are described in more detail in the Examples below.

[0010] More preferably, the isolated CD4+FoxP3+ T cells and / or isolated CD4+FoxP3- cells are expanded prior to determining the expression of the biomarkers of the present invention as described above. The term "expansion of isolated cells" as used herein refers to the cells being cultured for an appropriate period of time under appropriate conditions known in the art and as shown in the following examples after being isolated from a patient sample to increase the number of cells to be analyzed in a given culture or a test sample taken therefrom, thereby detecting the expression of a protein of interest on the cells.

[0011] Preferably, the patient sample is peripheral blood.

[0012] As described above, the isolated and optionally expanded CD4+ FoxP3+ T cells are preferably CD4+ FoxP3+ T regulatory cells.

[0013] Furthermore, the isolated and optionally expanded CD4+ FoxP3- T cells are preferably CD4+ FoxP3- T effector cells.

[0014] In a preferred embodiment of the present invention, the monitoring method comprises the following steps:

[0015] (i) determining the expression of PD-1 on CD4+ FoxP3+ T regulatory cells; and / or

[0016] (ii) determining the expression of PD-1 on CD4+ FoxP3- T effector cells; and / or

[0017] (iii) Determine the expression of CD73 on CD4+ FoxP3+ T regulatory cells.

[0018] In certain embodiments of the present invention, the above-mentioned monitoring method preferably includes the above-mentioned step (i). In other embodiments of the present invention, the above-mentioned monitoring method preferably includes the above-mentioned step (ii). In other embodiments of the present invention, the above-mentioned monitoring method preferably includes the above-mentioned step (iii). In other embodiments of the present invention, the above-mentioned monitoring method preferably includes the above-mentioned steps (i) and (ii). In other embodiments of the present invention, the above-mentioned monitoring method preferably includes the above-mentioned steps (i) and (iii). In other embodiments of the present invention, the above-mentioned monitoring method preferably includes the above-mentioned steps (ii) and (iii). In other embodiments of the present invention, the above-mentioned monitoring method preferably includes the above-mentioned steps (i), (ii) and (iii). When two or more of the above-mentioned steps (i), (ii) and (iii) are performed, each step may be performed simultaneously or sequentially.

[0019] In a preferred embodiment, the T regulatory cells used in the present invention have the phenotype CD4+FoxP3+CD25highCD127-doublet-.

[0020] In other preferred embodiments, the T effector cells used in the present invention have the phenotype CD4+FoxP3-CD25lowCD127+biocytes-.

[0021] The method according to any of the preceding claims, wherein the method is performed on the patient's cells at least once, preferably at least 2 weeks, preferably 1 month, more preferably 2 months, even more preferably 3 months after administration of the cell therapy.

[0022] In other embodiments of the present invention, the cells (CD4+FoxP3, preferably CD4+FoxP3+CD25 high CD127- double-linked cells-, and / or CD4+FoxP3-CD25 low CD127+ double-linked cells-, T cells) are subjected to the method of the present invention more than once. More preferably, the method of the present invention is performed at intervals, which may be regular or irregular, preferably regular. It is particularly preferred that the monitoring method is performed in a continuous manner to facilitate continuous monitoring of the patient's cell therapy, preferably, as long as the patient's cell therapy continues, the monitoring method of the present invention is performed. The method may be performed until a certain endpoint is reached, such as, for example, until a predetermined threshold value of PD-1 and / or CD73 expression on CD4+ FoxP3+, preferably CD4+ FoxP3+CD25 high CD127- double-linked cells-(Treg) cells is reached, and / or a predetermined threshold value of PD-1 expression on CD4+ FoxP3+, preferably CD4+ FoxP3+CD25 high CD127- double-linked cells-(Teff) cells is reached.

[0023] In the context of the present invention, the "expression threshold value" is preferably a certain percentage value of the corresponding cells expressing the corresponding biomarker of the present invention based on the total number of cells present in the assay, preferably in the test sample, more preferably in the test sample obtained from the patient, more preferably in the test sample after separation from the patient sample, and most preferably in the test sample after amplification of the isolated cells. Such a predetermined threshold value can be the maximum or minimum value of the expression of the corresponding biomarker used in the present invention on the corresponding cells.

[0024] The maximum or minimum value as the endpoint threshold value can be a predetermined absolute maximum or minimum value during the entire cell therapy. In other embodiments, the maximum or minimum value can be a predetermined time window of the duration of the cell therapy, such as about 1 to 6 months, such as about 1, about 2, about 3, about 4, about 5 or about 6 months, preferably the maximum or minimum value in a time window of about 1 year, about 1.5 years or about 2 years.

[0025] A preferred threshold value according to the present invention is based on the total number of cells in the test sample, such as based on the volume of a test sample containing a certain number of CD4+ FoxP3+ T cells (preferably Treg cells), and PD-1 is expressed on at least 16%, more preferably more than 16% of CD4+ FoxP3+ T cells (preferably Treg cells). Another preferred threshold value according to the present invention is based on the total number of cells in the test sample, such as based on the volume of a test sample containing a certain number of CD4+ FoxP3+ T cells (preferably Treg cells), and CD73 is expressed on at least 7%, more preferably more than 7% of CD4+ FoxP3+ T cells (preferably Treg cells). Another preferred threshold value according to the present invention is based on the total number of cells in the test sample, such as based on the volume of a test sample containing a certain number of CD4+ FoxP3-T cells (preferably Teff cells), and PD-1 is expressed on at least 8%, more preferably more than 8% of CD4+ FoxP3-T cells (preferably Teff cells). In certain embodiments of the present invention, at least one of the above-mentioned preferred threshold values ​​of the above-mentioned biomarkers on each cell determines the endpoint. In other embodiments of the present invention, at least two of the above thresholds determine the endpoint. In one embodiment, the endpoint is determined by the above-mentioned preferred threshold of PD-1 on the CD4+FoxP3+T cells (preferably Treg cells) together with the above-mentioned threshold of CD73 on the CD4+FoxP3+T cells (preferably Treg cells). In another embodiment, the endpoint is determined by the above-mentioned preferred threshold of PD-1 on the CD4+FoxP3+T cells (preferably Treg cells) together with the above-mentioned threshold of PD-1 on the CD4+FoxP3-T cells (preferably Teff cells). In another embodiment, the endpoint is determined by the above-mentioned preferred threshold of CD73 on the CD4+FoxP3+T cells (preferably Treg cells) together with the above-mentioned threshold of PD-1 on the CD4+FoxP3-T cells (preferably Teff cells). In other embodiments, the endpoint is determined by all three of the above-mentioned preferred thresholds. In a preferred embodiment, the preferred endpoint as described above is reached when the expression of at least one, or at least two, or three of the biomarkers is below the above value, whereby it should be understood that during the monitoring method, the expression value of the biomarker has reached the threshold value at least once before the expression value reaches the corresponding endpoint value.

[0026] Preferably, after the start of cell therapy, i.e., after the administration of CD4+FoxP3+ Treg cells, the method is performed on the patient's cells at an interval of at least about 2 weeks, preferably at least about 1 month, more preferably at least about 2 months, and even more preferably at least about 3 months. The "start" of cell therapy is defined herein as day 0, i.e., the first administration of CD4+FoxP3+Treg cells (also referred to as "the first administration of cell therapy" in other contexts of the present disclosure).

[0027] In a preferred embodiment of the present invention, after administration, preferably the first administration of cell therapy, in particular the administration of CD4+FoxP3+Treg cells, preferably CD4+ FoxP3+, preferably CD4+ FoxP3+CD25highCD127-bilinked cells-(Treg) cells, the monitoring method is performed on the patient's cells for at least about 6 months, more preferably at least about 1 year, and more preferably at least about 2 years, wherein the monitoring method is preferably performed within the above-mentioned interval.

[0028] Further preferably, the patient's cells are subjected to at least one monitoring method before the administration of cell therapy, in particular, at least one monitoring method is performed before the administration of CD4+ FoxP3+ Treg cells, preferably CD4+ FoxP3+, preferably CD4+ FoxP3+CD25 high CD127- doublet cells-(Treg) cells. That is, the monitoring method of the present invention includes determining the expression baseline value of each of the above cells before the start of cell therapy.

[0029] As described above, the determination of the expression values ​​of the biomarkers used in the present invention on each cell can be performed by various methods known in the art, such as Western blotting, detecting the mRNA of the protein of interest, cell sorting, etc. The preferred method of the assay, such as Western blotting, cell sorting, etc., is to treat a suitable test sample with a compound, preferably an antibody, more preferably a monoclonal antibody (which also includes a suitable antibody fragment specific for the corresponding biomarker). It is further preferred to use cell sorting, more preferably fluorescence activated cell sorting (FACS), to determine the percentage of the corresponding cells expressing the corresponding biomarker. The specific assay steps that can be used to implement the present invention are further outlined in detail in the following examples.

[0030] The monitoring method of the present invention is particularly suitable for monitoring cell therapy using respective Treg cells for patients suffering from or developing autoimmune diseases. The autoimmune disease in the context of the present invention may be one or more of the following:

[0031] Acromegaly;

[0032] Acquired aplastic anemia;

[0033] Acquired hemophilia;

[0034] Primary agammaglobulinemia;

[0035] Alopecia areata;

[0036] Ankylosing spondylitis (AS);

[0037] Anti-NMDA receptor encephalitis;

[0038] Antiphospholipid syndrome (APS) | Catastrophic antiphospholipid syndrome (CAPS) / Asherson syndrome;

[0039] arteriosclerosis;

[0040] Autoimmune Addison's disease (AAD);

[0041] Autoimmune autonomic ganglionopathy (AAG) / autoimmune autonomic dysfunction|autoimmune gastrointestinal dysmotility (AGID);

[0042] Autoimmune encephalitis | Acute disseminated encephalomyelitis (ADEM);

[0043] Autoimmune gastritis;

[0044] Autoimmune hemolytic anemia (AIHA);

[0045] Autoimmune hepatitis (AIH);

[0046] Autoimmune hyperlipidemia;

[0047] Autoimmune hypophysitis;

[0048] Autoimmune inner ear disease (AIED);

[0049] Autoimmune lymphoproliferative syndrome (ALPS);

[0050] Autoimmune myelofibrosis;

[0051] Autoimmune myocarditis;

[0052] Autoimmune oophoritis;

[0053] Autoimmune pancreatitis (AIP);

[0054] Autoimmune polyglandular syndromes, types I, II, and III (APS1, APS2, APS 3, APECED);

[0055] Autoimmune progesterone dermatitis;

[0056] Autoimmune retinopathy (AIR);

[0057] Autoimmune sudden sensorineural hearing loss (SNHL);

[0058] Barlow's disease;

[0059] Behcet's disease;

[0060] Shotgun choroidopathy / shotgun uveitis;

[0061] Bullous pemphigoid;

[0062] Castleman disease;

[0063] Celiac disease;

[0064] Chagas disease;

[0065] chronic inflammatory demyelinating polyneuropathy (CIDP);

[0066] Chronic autoimmune urticaria;

[0067] Churg-Strauss syndrome / eosinophilic granulomatosis with polyangiitis (EGPA);

[0068] Kogan syndrome;

[0069] Cold agglutinin disease;

[0070] CREST syndrome | Limited cutaneous systemic sclerosis;

[0071] Crohn's disease (CD);

[0072] Cronkhite-Canada syndrome (CSS);

[0073] Cryptogenic organizing pneumonia (COP);

[0074] Dermatitis herpetiformis;

[0075] Dermatomyositis;

[0076] Diabetes mellitus type 1 (DM1)

[0077] Discoid lupus;

[0078] Dressler syndrome / postmyocardial infarction / postpericardiotomy syndrome;

[0079] Eczema / atopic dermatitis;

[0080] Endometriosis;

[0081] Eosinophilic esophagitis;

[0082] Eosinophilic fasciitis;

[0083] Erythema nodosum;

[0084] Essential mixed cryoglobulinemia;

[0085] Evans syndrome;

[0086] Fibrotic alveolitis / idiopathic pulmonary fibrosis (IPF);

[0087] Giant cell arteritis / temporal arteritis / Houghton’s disease;

[0088] Giant cell myocarditis;

[0089] Glomerulonephritis;

[0090] Goodpasture syndrome / anti-GBM / anti-TBM disease;

[0091] Granulomatosis with polyangiitis (GPA) / Wegener's granulomatosis;

[0092] Graves' eye disease / thyroid eye disease;

[0093] Guillain-Barré syndrome (GBS);

[0094] Hashimoto's thyroiditis / chronic lymphocytic thyroiditis / autoimmune thyroiditis; Henoch-Schönlein purpura / IgA vasculitis;

[0095] Hidradenitis suppurativa;

[0096] Hallstein's disease / acute hemorrhagic leukoencephalitis (AHLE);

[0097] Hypogammaglobulinemia;

[0098] IgA nephropathy / Berger's disease;

[0099] immune-mediated necrotizing myopathy (IMNM);

[0100] Immune thrombocytopenia (ITP) / autoimmune thrombocytopenic purpura / autoimmune thrombocytopenia;

[0101] Inclusion body myositis;

[0102] IgG4-related sclerosis (ISD);

[0103] Interstitial cystitis;

[0104] Juvenile idiopathic arthritis / adult-onset Still's disease;

[0105] Juvenile polymyositis | Juvenile dermatomyositis | Juvenile myositis;

[0106] Kawasaki disease;

[0107] Lambert-Eaton myasthenic syndrome (LEMS);

[0108] Leukocytoclastic vasculitis;

[0109] Lichen planus;

[0110] Lichen sclerosus;

[0111] Wood-like conjunctivitis;

[0112] Linear IgA disease (LAD) | Linear IgA bullous dermatopathy (LABD);

[0113] Lupus nephritis;

[0114] Lyme disease / chronic Lyme disease / post-treatment Lyme disease syndrome (PTLDS);

[0115] lymphocytic colitis / microscopic colitis;

[0116] Lymphocytic hypophysitis / autoimmune hypophysitis;

[0117] Meniere's disease;

[0118] Microscopic polyangiitis (MPA) / ANCA-associated vasculitis;

[0119] Mixed connective tissue disease (MCTD);

[0120] Mooren's ulcer;

[0121] Mucha-Habermann disease;

[0122] Multifocal motor neuropathy;

[0123] Multiple sclerosis (MS);

[0124] Myalgic encephalomyelitis (ME) / chronic fatigue syndrome (CFS);

[0125] Myasthenia gravis (MG);

[0126] Narcolepsy;

[0127] Neuromyelitis optica / Devic's disease;

[0128] ocular cicatricial pemphigoid;

[0129] Opsoclonus-myoclonus syndrome (OMS);

[0130] palindromic rheumatism;

[0131] paraneoplastic cerebellar degeneration;

[0132] paraneoplastic pemphigus;

[0133] Parry-Romberg syndrome (PRS) / hemifacial atrophy (HFA) / progressive facial hemiatrophy; paroxysmal nocturnal hemoglobinuria (PNH);

[0134] peripheral uveitis / pars planitis;

[0135] PANS / PANDAS;

[0136] Parsonage-Turner syndrome;

[0137] Pemphigus gestationis / herpes gestationis;

[0138] Pemphigus foliaceus;

[0139] Pemphigus vulgaris;

[0140] Pernicious anemia;

[0141] POEMS syndrome;

[0142] Polyarteritis nodosa;

[0143] Polymyalgia rheumatica;

[0144] Polymyositis;

[0145] Postural tachycardia syndrome (POTS);

[0146] Primary biliary cirrhosis (PBC) / primary biliary cholangitis;

[0147] Primary sclerosing cholangitis (PSC);

[0148] psoriasis;

[0149] Palmoplantar pustulosis;

[0150] Psoriatic arthritis;

[0151] Idiopathic pulmonary fibrosis (IPF);

[0152] Pure red cell aplasia (PRCA);

[0153] Pyoderma gangrenosum;

[0154] Rasmussen encephalitis;

[0155] Raynaud's syndrome / phenomenon;

[0156] Reactive arthritis / Reiter's syndrome;

[0157] Reflex sympathetic dystrophy (RSD) / complex regional pain syndrome (CRPS); relapsing polychondritis;

[0158] Restless Leg Syndrome (RLS) / Willis-Ekbom Disease;

[0159] Rheumatic fever;

[0160] Rheumatoid arthritis;

[0161] Sarcoidosis;

[0162] Schmidt's syndrome / autoimmune polyendocrine syndrome type II;

[0163] Scleritis;

[0164] scleroderma;

[0165] Sclerosing mesenteritis / mesenteric panniculitis;

[0166] Serpiginous choroidopathy;

[0167] Sjogren syndrome;

[0168] Stiff-person syndrome (SPS);

[0169] Small fiber sensory neuropathy;

[0170] Systemic lupus erythematosus (SLE);

[0171] Subacute bacterial endocarditis (SBE);

[0172] Subacute cutaneous lupus;

[0173] Susac syndrome;

[0174] Sydenham chorea;

[0175] Sympathetic ophthalmia;

[0176] Takayasu's arteritis (vasculitis);

[0177] Testicular autoimmunity (vasculitis, orchitis);

[0178] Tolosa-Hunt syndrome;

[0179] Transverse myelitis (TM);

[0180] Tubulointerstitial nephritis uveitis syndrome (TINU);

[0181] Ulcerative colitis (UC);

[0182] Undifferentiated connective tissue disease (UCTD);

[0183] anterior / middle / posterior uveitis;

[0184] Vasculitis;

[0185] VEXAS syndrome;

[0186] Vitiligo and

[0187] Voigt-Koyanagi-Harada syndrome (VKH)

[0188] Preferably, the autoimmune disease is type 1 diabetes (DM1).

[0189] In a preferred embodiment of the invention, the patient is a child or adolescent, in particular a child or adolescent suffering from or developing DM1. Preferably, the age of the patient suffering from or developing an autoimmune disease, preferably DM1, is from about 7 to about 18 years old, more preferably from about 8 to about 16 years old. In a further preferred embodiment of the invention, with respect to all monitoring, diagnostic, evaluation and treatment methods defined and disclosed herein, in particular in relation to autoimmune diseases, in particular DM-1, the patient, preferably a child or adolescent, preferably of the age as described above, is in the early stage or early onset of the autoimmune disease, preferably DM1. In a preferred embodiment of the invention, "early stage" or "early onset" of the disease, in particular in the context of DM-1, refers to a fasting plasma C-peptide level of more than about 0.7 ng / ml in the patient and / or preferably an increase in the plasma C-peptide level in the GST (glucagon stimulated C-peptide test (42); 1 mg glucagon injected intravenously within 10 seconds) by at least 100%, respectively, compared to the plasma C-peptide level in the fasting state. In other preferred embodiments of the invention, "early stage" or "early onset" of the disease, especially in the context of DM-1, refers to a plasma C-peptide level in the patient's fasting state of more than about 0.7 ng / ml compared to the plasma C-peptide level in the fasting state, and / or preferably, an increase in plasma C-peptide level of at least 100% in a mixed meal tolerance test (MMT) (preferably as disclosed in (41)).

[0190] Another aspect of the invention is a method for evaluating the efficacy of a cell therapy in a patient treated with CD4+ FoxP3+ T regulatory cells, comprising performing the monitoring method of the invention, wherein at least 16%, more preferably more than 16%, of the CD4+ FoxP3+ T regulatory cells express PD-1, and / or at least 7%, more preferably more than 7%, of the CD4+ FoxP3+ T regulatory cells express CD73, and / or at least 8%, more preferably more than 8%, of the CD4+ FoxP3- T cells express PD-1, indicating an effective cell therapy. It should be understood that the percentage of said cells is based on the total number of said cells in the test sample, as described above, in the monitoring method of the invention, respectively, in relation to a preferred threshold or endpoint value.

[0191] The cell therapy may also include the administration of other drugs for treating the corresponding disease, preferably such as the autoimmune diseases mentioned above, most preferably DM1.

[0192] In the case of the above-mentioned cell therapy, it is preferred that the cell therapy is performed with the further administration of at least one anti-CD20 antibody or a fragment thereof having anti-CD20 affinity (i.e., a fragment of the antibody that retains specific binding to CD20). Preferably, the anti-CD20 antibody is a monoclonal antibody, and more preferably, the anti-CD20 monoclonal antibody is a humanized antibody. Most preferably, the anti-CD20 antibody is rituximab (hereinafter also referred to as "RTX"). The administration of Treg cells and anti-CD20 antibodies or fragments thereof (most preferably RTX) can be performed simultaneously or non-simultaneously. Preferably, the administration of anti-CD20 antibodies or fragments thereof, most preferably, requires about 200 mg / m 2 Body surface area ("BSA") to about 400 mg / m 2 The amount per body surface area is preferably about 330 mg / m 2 BSA is about 390 mg / m 2 BSA, most preferably about 375 mg / m 2 BSA. Preferably, the administration of a unit dose of an anti-CD20 antibody or fragment thereof (most preferably RTX) is performed multiple times, wherein the administration is preferably by intravenous administration. The administration of an anti-CD20 antibody or fragment thereof (preferably RTX) is preferably performed at regular or irregular intervals. A preferred regimen is to administer the unit dose, preferably as described above, at intervals of about one week to about two weeks, such as at intervals of about 6 to 15 days, such as 7, 8, 9, 10, 11, 12, 13 or 14 days.

[0193] The BSA value of the patient can be calculated based on the patient's weight and height and (optionally) the patient's age. In a preferred embodiment of the present invention, BSA is calculated according to formulas known in the art, such as the Boyd formula, the Dubois formula, the Gehan-George formula, the Haycock formula, the Mosteller formula or the Takahira formula. According to the present invention, when treating children or adolescents, the calculation of BSA is preferably performed according to the Gehan-George formula and / or the Haycock formula.

[0194] The antibodies used in the present invention may be polyclonal or monoclonal. The antibodies preferably used in the present invention are monoclonal antibodies.

[0195] According to the present invention, "antibody fragments with affinity" and / or "fragments retaining specific binding" for the corresponding antigens (such as CD20, PD-1 and / or CD73) refer to one or more fragments of an antibody that retains the ability to specifically bind to the corresponding antigen. Examples of such antibody fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, heavy chain antibodies, single domain antibodies (sdAb), single chain fragment variables (scFv), fragment variables (Fv), VH domains, VL domains, single domain antibodies, nanobodies, IgNAR (immunoglobulin new antigen receptor), double scFv, bispecific T cell engagers (BITE), dual affinity retargeting (DART) molecules, tribodies, double antibodies, single chain double antibodies, alternative scaffold proteins and fusion proteins thereof.

[0196] Preferably, the term "specific binding" as used herein means that the corresponding entity such as the antibody or its fragment as described above shows a dissociation constant (K) for binding to the target. D ), the target is usually an antigen of the antibody disclosed in the present invention, such as an antibody and its fragment, and its range is less than about 10 -6 M or less, preferably about 10 -7 M or less, more preferably about 10 -8 M or less, even more preferably about 10 -9 M or less, most preferably about 10 -10 Below M.

[0197] A further subject of the present invention is a cell therapy method for a patient in need, preferably a child or adolescent, preferably suffering from or developing an autoimmune disease, such as an autoimmune disease as described above, most preferably DM1, the method comprising administering Treg cells as described above, and performing the monitoring and / or evaluation method of the present invention, preferably further comprising administering rituximab. Preferably, the cell therapy method is performed until at least one threshold and / or endpoint as described above is reached.

[0198] The present invention also relates to the use of Treg cells as described herein to treat patients by cell therapy, wherein the patient is preferably a patient such as a child or adolescent, preferably suffering from or developing an autoimmune disease (such as an autoimmune disease as described above), most preferably a patient with DM1, wherein the cell therapy comprises administering the Treg cells, and optionally an anti-CD20 antibody or fragment thereof as defined herein, most preferably rituximab, and further comprising implementing the evaluation method and / or monitoring method of the present invention. Preferably, the cell therapy of the patient is performed until at least one threshold value and / or endpoint as described above is reached. In a preferred embodiment, the Treg cells for treatment defined herein, particularly CD4+ FoxP3+ Treg cells, most preferably CD4+ FoxP3+CD25 high CD127- doublet cells-Treg cells are preferably as described below.

[0199] In the context of the present invention, a preferred dose of Treg cells is about 10 x 10 6 cells to about 90 x 10 6 cells, preferably about 20 x 10 6 About 70x 10 6 , more preferably about 30 x 10 6 About 60x 10 6 , such as about 30x10 6 , about 40x 10 6 , about 50x 10 6 or about 60x 10 6 , most preferably about 30 x 10 6 A unit dose of 10 Treg cells is generally administered as one dose at a time, preferably intravenously (iv).

[0200] The unit dose of Treg cells used in the present invention, preferably the unit dose as described above, comprises Treg cells in a suitable culture medium, preferably suitable for intravenous administration, such as a physiological NaCl solution, preferably in an amount of about 100 to about 500 ml, preferably about 200 to about 300 ml, such as about 250 ml of culture medium, such as 0.9% (w / v) NaCl in water for injection.

[0201] The administration of Treg cells is carried out at least once. Preferably, the administration is carried out more than 1 time, wherein the intervals between administrations may be equal or may be different. According to the present invention, the preferred intervals in the treatment are about 1 to about 6 months, preferably about 2 to 4 months, and most preferably about 3 months. In some preferred embodiments of the present invention, the administration of Treg cells is preferably carried out in a unit dose as described above, and is administered at least 2 times within the intervals previously outlined, and most preferably administered more than 2 times within an interval of 3 months.

[0202] According to the present invention, it should be understood that the term "treatment of a patient suffering from a disease", such as an autoimmune disease, preferably an autoimmune disease as described above, most preferably DM1, requires treatment of the patient, preferably the disease in a child or adolescent. The term "treatment" as used herein includes delaying the development of a disease (such as a disease as described herein, most preferably DM-1), such as delaying at least about 6 months, preferably at least about 1 year, more preferably at least about 1.5 years, and even more preferably at least about 2 years or more, such as at least about 3 years, at least about 4 years, at least about 5 years, or at least about 6 years or more. "Delay" in the development of a "disease" (such as an autoimmune disease disclosed herein, preferably DM-1) as used herein preferably means that the patient's condition is such that the time for the patient to develop to the clinical stage of the disease according to the general knowledge and accepted clinical criteria of those skilled in the art (particularly doctors) is extended, preferably extended for the time period as described above. In the context of DM1, the delay in progression or development preferably means that the time until the patient develops to an acceptable clinical stage (wherein insulin treatment of the patient becomes necessary according to acceptable clinical criteria) is extended, preferably for the time period as described above.

[0203] A further subject of the invention is a method for diagnosing effective cell therapy in a patient treated with CD4+ FoxP3+ T regulatory cells, comprising the steps of obtaining a patient sample, isolating CD4+ FoxP3+ T cells and / or CD4+ FoxP3- cells from the patient, contacting the isolated CD4+ FoxP3+ T cells and / or isolated CD4+ FoxP3- cells with at least one detectable compound having affinity for (i.e. specifically binding to) PD-1 and / or CD73, preferably detecting in the test sample said compound binding to CD4+ FoxP3+ T cells expressing PD-1 and / or CD73 and / or to CD4+ FoxP3- cells expressing PD-1, respectively, and determining the proportion of CD4+ FoxP3+ T cells expressing PD-1 and / or CD73 and / or the proportion of CD4+ FoxP3- cells expressing PD-1.

[0204] The invention also relates to the use of said detectable compound in the diagnostic method of the invention as defined in the previous paragraph.

[0205] Preferably, the isolated CD4+ FoxP3+ T cells and / or isolated CD4+ FoxP3- cells are expanded prior to contacting the CD4+ FoxP3+ T cells and / or isolated CD4+ FoxP3- cells with said detectable compound.

[0206] Preferably, the detectable compound is an anti-PD1 antibody or a fragment thereof that retains specific binding to PD-1, or an anti-CD73 antibody or a fragment thereof that retains specific binding to CD73.

[0207] It is further preferred that the detectable compound, such as an anti-PD-1 antibody or an anti-CD73 antibody (or a fragment of such an antibody as defined herein) is coupled to a detectable label, preferably a fluorescent label. Preferred fluorescent labels according to the present invention include, but are not limited to, the fluorescent chromophores shown in Table 3A.

[0208] In the diagnostic method defined above, a sample of the patient is obtained, which is preferably peripheral blood. For preferred embodiments and techniques, the above-mentioned in vitro monitoring method is referred to for the separation of cells, the amplification of cells and the detection of the bound detectable compound and the determination of the proportion of cells bound to the detectable compound, respectively. Preferably, the proportion of cells bound to the corresponding detectable compound indicates an effective cell therapy as described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0209] Figure 1 Research flow chart of clinical trials and in vitro model experiments according to an embodiment of the present invention.

[0210] Figure 2 Regulatory T cell phenotype during patient follow-up compared with in vitro models.

[0211] Fig. 3 Expression of PD-1 on regulatory T cells and effector T cells during patient follow-up compared with in vitro models.

[0212] Figure 4 Surface expression of CD73 on regulatory T cells (Tregs FoxP3+) and effector CD4+ T cells from administration to 2-year follow-up. ROC curve for percentage of CD73+Tregs in patients treated with polyclonal regulatory T cells and rituximab.

[0213] Fig. 5 Evolution of the patient's humoral immune response within two years after treatment.

[0214] Figure 6 Clinical relevance of regulatory T cells and effector T cells during patient follow-up.

[0215] Fig. 7 Serum cytokine network during patient follow-up.

[0216] Figure 8 Flow cytometry gating strategy for single cell antigen expression. (A) Flow cytometry gating strategy for single cell antigen expression. Representative examples of CD279 (PD-1) antigen surface expression on regulatory T cells (Tregs FoxP3+), effector CD4+T cells, and CD8+T cells are shown. (B) Flow cytometry gating strategy for single cell antigen expression. Representative examples of lymphocyte B subsets. (C) Flow cytometry gating strategy for single cell antigen expression. Representative examples of CD279 (PD-1) antigen surface expression on regulatory T cells in in vitro culture are shown. (D) Flow cytometry FCS gating strategy and representative examples of proliferation modeling in FlowJo software are shown.

[0217] Figure 9 Basic flow cytometry FCS gating strategy and dimensionality reduction algorithm analysis scheme in FlowJo software. (A) Basic flow cytometry FCS gating strategy in FlowJo software for dimensionality reduction algorithm analysis. (B) Dimensionality reduction algorithm analysis scheme in FlowJo software.

[0218] Fig.10 Surface expression of CD39 on regulatory T cells (Tregs FoxP3+) and effector CD4+ T cells from administration to 2-year follow-up.

[0219] Fig.11 Surface expression of CD304 (NRP-1) on regulatory T cells (Tregs Helios+ FoxP3+) from administration to 2-year follow-up.

[0220] Fig.12 Surface expression of CD134 (OX-40) on regulatory T cells (Tregs Helios+ FoxP3+) from administration to 2-year follow-up.

[0221] Fig.13 Serum IgA concentrations from administration to 2-year follow-up.

[0222] Fig.14 Correlation of IL-1Ra serum concentrations with daily insulin dose per kg body weight (DDI / kg body weight) at 12 months and glycated hemoglobin at 24 months after administration.

[0223] Fig.15 Correlation of IL-17 serum concentrations with surface CD73 expression on effector CD4+ T cells three months after administration.

[0224] Fig.16Serum IL-8 / CXCL8 concentrations from administration to 2-year follow-up, and correlation of IL-8 / CXCL8 serum concentrations with surface CD39 expression on regulatory T cells (Tregs FoxP3+) at six months after administration.

[0225] Fig.17 Serum IL-4 and IL-5 concentrations from administration to 2-year follow-up.

[0226] Fig.18 Serum sCD40L concentrations from administration to 2-year follow-up, and correlation of sCD40L serum concentrations with daily insulin dose per kilogram body weight (DDI / kg bm) at 24 months after administration.

[0227] Fig.19 Lymphocyte B from administration to 2-year follow-up.

[0228] The present invention is further illustrated by the following non-limiting examples. Example

[0229] introduce

[0230] This study according to this embodiment aims to identify and validate biomarker candidates for DM1 immune intervention in the clinical trial TregVAC2.0 (clinical trial registration number ISRCTN37116985). The inventors evaluated the cellular and humoral immunity of newly diagnosed DM1 patients treated with a combination of autologous polyclonal CD3+CD4+CD25 high CD127-T regulatory cells and anti-CD20 antibodies (Tregs+RTX group) and compared them with patients who received polyclonal Treg treatment alone (Tregs group) or standard insulin treatment (control group). Ultimately, we expect to find biomarkers in the immune system that are associated with β-cell function and can be used to predict the response to the immunotherapy used in the trial ( Figure 1 )'s marker.

[0231] Materials and methods

[0232] Study Design

[0233] The inventors conducted a phase 1 / 2 prospective multicenter clinical trial to study the efficacy of autologous Treg administration and anti-CD20 monoclonal antibody combined therapy in children and adolescents newly diagnosed with DM1 and its effects on selected immune parameters. This study was a prospective, open-label, randomized 24-month clinical trial with registration numbers ISRCTN37116985 and EudraCT 2014-004319-35. In this three-group trial, follow-up was performed in a standard care control group (control; 11 patients), a group treated with autologous polyclonal Tregs alone (Tregs; 12 patients), and a group treated with autologous polyclonal Tregs and anti-CD20 antibodies combined (Tregs+RTX; 13 patients). The intervention group consisted of Tregs+RTX and Tregs patients who received two doses of Tregs in an open-label manner, 30x10 6 cells / kg body weight, at intervals of three months starting from day 0. Anti-CD20 antibodies were administered in a blinded manner and placebo-controlled. Patients were randomly assigned to the anti-CD20 antibody or placebo group by chance (coin) and received four doses of rituximab (Tregs+RTX group) or placebo (Tregs group) on days +14, +22, +29 and +36 of the trial, respectively. As shown in the study flow chart, all participants were followed up for two years after treatment and evaluated after treatment, three months, six months, twelve months and twenty-four months. The efficacy and safety report of the combined treatment of this clinical trial has been published. We have demonstrated that the combination of autologous Treg administration and anti-CD20 antibodies is superior to Treg treatment alone in DM1, as assessed by the AUC of the C-peptide mixed meal tolerance test and the percentage of patients in clinical remission at 24 months of the trial. Although the incidence of adverse events in Tregs+RTX patients was 80%, the therapy was safe as no adverse events led to discontinuation of intervention or patient death (7).

[0234] The present study aimed to evaluate the immune signature of DM1 individuals. During the two-year follow-up, we set up a multicolor immunophenotyping of CD4+Treg and CD4+T Teff and CD8+T cells, trying to correlate them with clinical and laboratory outcomes. Similarly, immune correlations were analyzed in the humoral immune and cytokine context. The results were then matched to an in vitro model of Treg and CD4 Teff cell culture in DM1 patients and healthy volunteers, as shown in the study flow chart ( Figure 1 ). Finally, the laboratory results were correlated with the clinical outcomes of the trial. The study was approved by an independent institutional review board (NKBBN / 374 / 2012-NKBBN / 374-7 / 2014 for clinical trials and NKBBN / 414 / 2018 for in vitro studies), and all participants signed an informed consent form.

[0235] patient

[0236] All participants were recruited according to the detailed inclusion and exclusion criteria of the clinical trial and the in vitro model (Tables 1a, 1b, and 2). A 20% difference in the geometric mean ratio (α5%) of C-peptide AUC (0-240 min) in the randomized treatment groups of 13 patients was satisfactory by power analysis of sample size, which is a significant result for the clinical trial. For the in vitro model, 12 DM1 patients and 12 healthy controls were recruited. The inclusion / exclusion criteria for DM1 patients were the same as for the clinical trial, and healthy controls were blood donors from the Gdańsk City Blood Bank, leaving their buffy coat after the preparation of blood products. Detailed baseline demographic data for the clinical trial and the in vitro model are given in Tables 1A and 1B.

[0237] method

[0238] Cell separation

[0239] Ficoll Paque Plus (GE Healthcare, Chicago, IL) is a density gradient separation method used to isolate PBMCs from EDTA whole blood of DM1 patients or buffy coats prepared from blood products of healthy volunteers. The isolated cells were then counted and tested for viability using the Bio-Rad TC20 (Hercules, CA), an automated trypan blue cell viability analyzer. The minimum viability cutoff for testing was 90%.

[0240] Cell sorting and culture for in vitro models of Treg stimulation

[0241] CD4+ T cells were isolated from PBMCs using a negative immunomagnetic selection kit (EasySep Human CD4 Negative Selection Kit, StemCell Technologies; Vancouver, BC, Canada) and then stained with fluorescently conjugated monoclonal antibodies (BD Biosciences, Poland): anti-CD3 (clone UCHT1), anti-CD4 (clone SK3), anti-CD25 (clone M-A251), and anti-CD127 (clone HIL-7R-M21). Finally, cells were sorted into regulatory T cells (Treg) with a phenotype of (CD4+ / CD25high / CD127- / bi-cells-) and effector T cells (Teff) with a phenotype of (CD4+ / CD25low / CD127+ / bi-cells-) using a FACS Aria II sorter or a FACS Influx sorter (BD Bioscience, Franklin Lakes, NJ). The sorted cells were then cultured according to previously published protocols (34,35). Briefly, cells were suspended in X-Vivo medium (Lonza, Belgium) and supplemented with heat-inactivated autologous serum (DM1 patients) or AB human male serum (healthy controls) and 1×10 4 UI / ml IL-2 (Proleukin, NOVARTIS, Germany) for 12 days. On days 0 and 5, Treg and Teff were activated for proliferation using beads coated with anti-CD3 and anti-CD28 antibodies (MACS GMP ExpAct Treg Kit, Miltenyi Biotec, Germany) at a 1:1 beads to cell concentration ratio.

[0242] Flow cytometry

[0243] Several immune parameters of the clinical trial group were investigated using an expanded peripheral lymphocyte flow cytometry analysis method, as shown in Table 3. 22 markers were analyzed using three 16-color fluorescent dye panels. We used minimal backbone markers for gating, namely CD3, CD4, FoxP3, Helios, CD45RA, and CD62L markers. At this time, Treg, CD4+Teff, and CD8+T cells were gated, as shown in Table 3. Fig. 8A shown.

[0244] Peripheral blood B lymphocytes were divided into CD19 / CD20 double-positive lymphocytes (skeleton markers) and further analyzed for antigens associated with cell maturation, memory development, and class switching ( Figure 8B ), as shown in Table 3.

[0245] Appropriate isotype controls and fluorescence minus one (FMO) were used to gate the population of interest for each analysis. The minimum cell count per flow cytometer tube was 200,000 ± 20,000 viable cells, of which at least 80,000 viable cells were collected for flow cytometry using a BD LSRFortessa cell analyzer (BD Bioscience, Franklin Lakes, NJ, USA).

[0246] Inhibition test

[0247] Eight DM1 and five healthy control cultures were randomly selected and subjected to suppression assays on days 7 and 12 of culture. The suppressive potential of Tregs was assessed as the inhibition of Teff proliferation in the presence of Tregs. 4 Each Teff / culture was stained with 1 μM / ml CFSE proliferation dye (BD Bioscience, Franklin Lakes, NJ) and co-cultured with titrated concentrations of Treg, as shown below (Teff:Treg) 1:2, 1:1, 1:0.5, 1:0.25 and 1:0.125. Cells were then activated with magnetic beads coated with anti-CD3 and anti-CD28 antibodies (MACS GMP ExpAct Treg Kit, Miltenyi Biotec, Germany) at a 1:1 ratio of magnetic beads to Teff and cultured for 72 hours. Teff cultures stimulated with magnetic beads without Tregs were used as positive controls, and unstimulated Teff and Treg cultures were used as negative controls. CFSE fluorescence in samples was collected using a BD LSR Fortessa cell analyzer (BD Bioscience, Franklin Lakes, NJ). The results were analyzed using the proliferation modeling tool in FlowJo (Ashland, OR) and expressed as a proliferation index (PI), Fig.8D A representative gating example is given in .

[0248] Flow cytometry data analysis

[0249] Several immune parameters of Treg, CD4+Teff, CD8+T cells, and B lymphocytes were analyzed. First, we screened the follow-up data of each cohort up to two years using a heat map method ( Figure 2A ). Next, further evaluation was performed by performing randomized analysis of the cell phenotypes to find the best fitting parameters that differentiated the treated and control patients. Finally, ANOVA statistics were performed on the selected parameters to find statistical significance.

[0250] Flow cytometry data were analyzed using Kaluza software (Beckman Coulter, Brea, CA) and FlowJo software (V10, Ashland, OR, USA). First, significant T-cell and B-cell subsets were identified using backbone markers and the expression of different antigens was recorded as percentages. Next, the data were analyzed using FlowJo's software dimensionality reduction algorithm to find significant cell subsets between the test cohorts. We used t-distributed stochastic neighbor embedding (tSNE) and TriMap algorithms as well as the following PhenoGraph algorithm, a clustering method for identifying phenotypically distinct subsets. (37,38,39)

[0251] In FlowJo software, starting with the row FCS file, down-sampling was performed to reduce the number of events to normalize population size, and then gating was performed on Treg, CD4+Teff, and CD8+T cells ( Fig. 9A ). We periodically downsampled 30,000 events based on the chronological order of the collected events. Next, the standardized Treg, CD4+Teff, and CD8+T cell subset events from individual samples were aggregated into an FCS file (specific conditions) for further dimensionality reduction algorithm analysis. tSNE was performed using the opt-SNE configuration with the following settings: number of iterations = 1000, perplexity = 30, k nearest neighbor algorithm as vantage point tree, and Barnes-Hut as gradient algorithm (37). TriMap analysis was performed using the Euclidean distance function, number of nearest neighbors = 10, and number of outliers = 5. (38) Then, the PhenoGraph algorithm was calculated using the k number (number of nearest neighbors for the nearest neighbor graph) recommended by the plugin based on the FCS file structure. (39) Finally, the Cluster Explorer plugin was used to select the population of interest. The detailed analysis scheme is as follows. Fig. 9B shown.

[0252] Serum immunoglobulins and autoantibodies

[0253] Serum concentrations of IgA, IgM immunoglobulins, and IgG subclasses: IgG1, IgG2, IgG3, and IgG4 were measured using the Bio-PlexPro Human Isotyping Panel kit (Bio-Rad, Hercules, CA) according to the manufacturer's protocol and read on a Luminex MAGPIX analyzer (Merck Millipore; Burlington, MA, USA).

[0254] Autoantibodies

[0255] Anti-GAD65 (glutamic acid decarboxylase antibody) and anti-IAA (insulin autoantibody) antibodies were detected by ELISA (Euroimmun, Germany). Anti-ICA ​​(anti-islet cell antibody) was detected by indirect immunofluorescence (IIF) using primate pancreas as antigen substrate (Euroimmun, Germany).

[0256] Serum cytokines

[0257] Serum concentrations of 38 cytokines were measured using a bead-based multiplex assay - Human Cytokine / Chemokine Magnetic Bead Panel Milliplex (Merck Millipore; Burlington, MA) according to the manufacturer's protocol and read on a Luminex MAGPIX analyzer (Merck Millipore; Burlington, MA).

[0258] Statistical data analysis

[0259] Data are presented as median plus standard deviation. All statistical tests were performed using only cleaned data after Grubbs test. All comparisons between groups were performed using nonparametric Mann-Whitney U test, while multiple data sets were analyzed using Kruskall-Wallace or Welch ANOVA. Brown-Forsythe ANOVA was used if data were skewed and not Gaussian. Relationships between data sets were tested using Spearman rank correlation, and frequencies were assessed using chi-square test. Correlations calculated for multiple data sets were visualized using color-coded correlation matrix plots and XY data point plots with 95% confidence intervals for the best fit line. Monte Carlo simulations were performed for principal component analysis (PCA). Receiver operating characteristic (ROC) curves were calculated using Wilson / Brown method with 95% confidence intervals (95% CI). Data are presented as median and interquartile range and visualized as bar graphs or individual values. The top of each bar represents the mean and the line represents the standard deviation. Significance was set at p < 0.05. The significance codes for p values ​​are as follows: "***" (0,000-0,001), "**" (0,001-0,010), and "*" (0,010-0,050). All analyses were performed in Prism 9 (GraphPad Software; Boston, MA). Heat maps were generated by Heatmapper (http: / / www.heatmapper.ca / ), using average linkage as the clustering method and Euclidean as the distance measure. (40)

[0260] result

[0261] Treg number, FoxP3+ and Helios transcription factor expression

[0262] No significant differences were found between the groups during the entire follow-up period ( Figure 2A There were no significant changes in the percentage of FoxP3+ or FoxP3+Helios+ Tregs in either the control or treatment groups. No differences were found between the groups during the 2-year monitoring period ( Figure 2B , 2C ). By principal component analysis of FoxP3+ or FoxP3+Helios+ double-positive Tregs, only 11%-20% difference between treatment and control groups was tested from recruitment to up to two years of monitoring ( Figure 2D ).

[0263] In contrast, we found a decrease in the percentage of FoxP3+Tregs and FoxP3+Helios+Tregs between days 7 and 12 in cell culture from DM1 individuals in our in vitro Treg stimulation model, compared with stable percentages in healthy controls (HC) ( Figure 2E , G). Similarly, FoxP3 expression (measured by MFI) decreased throughout the culture, whereas Helios expression decreased only in the DM1 group ( Figure 2F , H).

[0264] PD-1, immune checkpoint antigen

[0265] In-depth analysis showed that the percentage of PD-1+ T cells changed significantly throughout the study ( Figure 3A -C). In the control group, the percentage of PD-1+T effector cells (Teff) gradually decreased from +6 to +24 months of follow-up (p=0.02). In contrast, the percentage of PD-1+CD4+Teff and PD-1+CD8+T cells increased in the treatment groups throughout the trial; however, it was only significant in the combined treatment Tregs+RTX group ( Figure 3B , 3C ). Therefore, the percentage of PD-1+Teff in the control group and the combination treatment group at +24 months (p=0,009) ( Figure 3B , black arrows) and the percentage of PD-1+CD8+T cells at +12 months (p=0,04) ( Figure 3C , brown arrows). PD-1+Treg levels showed a similar trend during follow-up, but neither the decrease in the control group nor the increase in the treatment group reached statistical significance. The only between-group difference in Treg was between the control group and the combination-treated patients at +24 months (p=0,001)( Figure 3A, black arrows). In vitro, the percentages of PD-1+Treg and PD-1+Teff were higher in the DM1 group at day 0 compared with the healthy control group (p=0,032 and p=0,006), and then the PD-1+Treg in both groups of cultures increased, reaching comparable levels at day +7 (p=0,932) ( Figure 3D , F). Interestingly, at day +12, the percentage of PD-1+Treg in DM1 patient cultures dropped dramatically and was significantly lower than that in the healthy group ( Figure 3D ). Meanwhile, the expression of PD-1 per cell, measured by MFI, was significantly higher in cultures from DM1 patients than in cultures from healthy controls at days 0, +7, and +12 ( Figure 3E No such difference was observed in CD4+ Teff cultures ( Figure 3G ).

[0266] Treg suppressive activity was correlated with the percentage of PD-1+Treg in the DM1 group.

[0267] The suppressive activity of Tregs was tested by the inhibition of Teff proliferation in co-cultures of stimulated Teffs and autologous Tregs. Interestingly, only in the DM1 group on day +7, there was a correlation between the percentage of PD-1+Tregs and suppression (r=-0,552; p=0,005) ( Figure 3H ). While the percentage of PD-1+Tregs decreased significantly in the DM1 group, this effect disappeared on day 12 ( Figure 3D For the healthy control group, no correlation was observed on day +7 or day +12 ( Figure 3H , I).

[0268] Expression of other biomarker candidates during follow-up

[0269] For both Treg and Teff, a gradual decrease in the percentage of CD73+ cells was found in control DM1 individuals (p=0,003 and p=0,007, respectively). This decrease was not seen in patients receiving Treg or combined therapy (p>0.05)( Figure 4 There were no differences in the percentage of CD39+ Tregs or Teffs (another enzyme involved in nucleotide metabolism) across the trials ( Fig.10 ). Similar to CD73+ Tregs, the percentage of CD304+ (NRP-1 antigen) Helios+ Tregs decreased in the control group only during follow-up (p=0.02). Nevertheless, the expression of this biomarker on Tregs was very low, rarely exceeding 2% of all Tregs ( Fig.11Finally, the percentage of CD134+ (OX-40 antigen) Helios+ Tregs increased significantly in the control group and both intervention groups throughout the follow-up, reaching the highest level in the control group ( Fig.12 ).

[0270] B cell subsets and immunoglobulins

[0271] Significant changes were observed only in the Tregs+RTX group ( Figure 5A ). B cells were almost completely depleted within the first six months after injection of anti-CD20 antibodies. At +12 months, B cells recovered and the percentage of CD27+ B memory cells was significantly reduced compared to baseline (p<0,001)( Figure 5B ). At the same time, the percentage of Breg-like cells (CD38++CD24++) and transitional (CD38+CD24+) B cells almost doubled (p<0,001). At +24 months, the percentage of the former subset decreased to baseline, but the percentage of the latter subset remained folded ( Figure 5C , D).

[0272] In addition, characteristic changes in the IgG1 / IG2 index were found in the Tregs+RTX group. During the entire follow-up, serum IgG1 concentration decreased significantly (p=0.003), while IgG2 concentration increased ( Figure 5E , F). IgM levels decreased significantly throughout the study period until +24 months (p=0.001), reaching the lowest level at +6 months of the study ( Figure 5G There was no change in IgA serum concentration ( Fig.13 No late hypogammaglobulinemia was observed after one to two years of rituximab treatment for IgA (<0.42 mg / ml) and IgG (IgG1 <3.16 mg / ml; IgG2 <0.86 mg / ml; IgG3 <0.14 mg / ml; IgG4 <0.01 mg / ml) subclasses. (12)

[0273] There was also a partial reduction in serum autoantibodies, such as anti-GAD-65 autoantibodies, which decreased significantly only in Tregs+RTX patients (p<0,001). In contrast, anti-IAA autoantibodies decreased in all patients at +24 months, while anti-ICA ​​autoantibodies did not change during the 24-month monitoring period ( Figure 5H , I, J).

[0274] Correlation between immune markers and disease progression

[0275] The presented immune parameters were then correlated with clinical outcome results, such as mixed meal tolerance test (MMTT) assessed by area under the curve of plasma C-peptide concentration (AUC), glycated hemoglobin (HbA1C), serum C-peptide, and daily dose of insulin per kilogram of body weight (DDI / kg body weight). At two years of follow-up, there was a general correlation between clinical outcomes and the percentage of PD-1+Treg and PD-1+Teff in both the control and treatment groups ( Figure 6 ).

[0276] Less commonly, there was a correlation between clinical parameters and the percentage of CD73+Treg and CD73+Teff at two years of follow-up. Interestingly, there was also a correlation between CD304+Treg and better clinical outcomes ( Figure 6 ).

[0277] The area under the curve (AUC) of the receiver operating characteristic (ROC) curve was calculated, and for Tregs+RTX patients only, significant markers were identified as PD-1+Treg, PD-1+Teff, and CD73+Treg (Table 4). For cutoff values ​​of >16% PD-1+Treg and >8% PD-1+Teff, the sensitivity was 73% (43,44% to 90,25%; 95% CI) and 72% (45,25% to 89,50%; 95% CI), respectively, while the specificity was 92% (64,61% to 99,57%; 95% CI), 90% (70,54% to 96,28%; 95% CI) ( Figure 3A -C). For CD73+Treg percentages >7% cutoff, the sensitivity was 82% (52.30% to 96.77%; 95% CI) and the specificity was 83% (55.20% to 97.04%; 95% CI), ( Figure 4 ).

[0278] Serum cytokine environment

[0279] Using the heat map method to screen serum cytokines, no constant pattern was observed between the groups during the 2-year follow-up period ( Fig. 7A ). Notably, serum IL-10 concentrations were two-fold and three-fold higher in Tregs and Tregs+RTX patients, respectively, compared with control patients at three months follow-up (p<0,001)( Figure 7B , dark grey arrows). This phenomenon persisted until 6 months after recruitment ( Figure 6 B, brown arrows). In addition, in the Tregs+RTX group, IL-10 concentration was positively correlated with the percentage of FoxP3+Helios+ double-positive Tregs six months after recruitment (r=0,772; p=0,013) ( Figure 7CIL-1 receptor antagonist (IL-1Ra), another anti-inflammatory cytokine, was upregulated in the serum of Tregs (p<0,001) and Tregs+RTX (p<0,001) patients compared with controls (p=0,040) throughout the follow-up period. Fig.7D ). It is worth noting that six months after recruitment, the serum IL-1Ra concentration of Tregs patients was 14 times higher than that of the control group, and the serum IL-1Ra concentration of Tregs+RTX patients was 12 times higher than that of the control group (p<0.001). This phenomenon of Tregs+RTX patients continued until the end of follow-up, while this phenomenon almost disappeared in the Tregs group at 24 months of follow-up ( Figure 6 D, black arrows). Then, we found a positive weak correlation between IL-1Ra concentration at +12 months and DDI / kg body weight (r=0,671; p=0,028) and glycated hemoglobin at +24 months (r=0,834; p=0,009) in the Tregs+RTX group ( Fig.14 ).

[0280] Regarding proinflammatory cytokines, we found a significant decrease in IL-17 concentrations throughout the study. While IL-17 levels in control samples remained stable throughout the 2-year follow-up (p = 0,970), they gradually decreased in the Tregs (p = 0,003) and Tregs + RTX (p = 0,006) groups ( Fig. 7E In addition, a negative correlation between CD73+CD4+Teff and IL-17 concentrations was observed in Tregs+RTX patients at +3 months (r=-0,694, p=0,016) ( Fig.15 ).

[0281] Another upregulated cytokine in Tregs and Tregs+RTX was the chemokine IL-8 / CXCL8. In control samples, IL-8 / CXCL8 was comparable during the 2-year follow-up (p=0,590), but serum concentrations increased in Tregs (p=0,040) and Tregs+RTX (p=0,010). Fig.16 In the Tregs group, CD39+ FoxP3+ Treg was positively correlated with IL-8 / CXCL8 serum concentration (r=0,615, p=0,038) at +6 months ( Fig.16 ).

[0282] The B cell class switching process was accompanied by an increase in the serum concentrations of IL-4, IL-5 and soluble CD40 ligand (sCD40L) in the Tregs group and the Tregs+RTX group. From the third month to the second year of follow-up, IL-4 was highly upregulated in the Tregs group. As for IL-5, the serum concentration of Tregs+RTX patients reached a peak at +6 months (p<0,001)( Fig.17 ). At follow-up +12 months, IL-4 in the Tregs+RTX group was positively correlated with serum IgG2 (r=0,767, p=0,014). Similarly, at +12 months after recruitment, IL-5 in the Tregs+RTX group was positively correlated with serum IgG1 (r=0,753, p=0,019) ( Figure 7F , G).

[0283] Unlike the control or Tregs groups, sCD40L serum concentrations were upregulated in Tregs+RTX (p=0,040), with the highest concentrations at +12 months ( Fig.18 In addition, at the end of the trial, sCD40L was positively correlated with DDI / kg body weight (r=0,812, p=0,021) ( Fig.18 ).

[0284] discuss

[0285] The study according to this embodiment attempts to determine the most accurate immune biomarker for the efficacy of Treg treatment of DM1. In the TregVAC2.0 trial (clinical trial ISRCTN37116985), we tracked the immune parameters of cellular and humoral immunity and cytokine networks in newly diagnosed DM1 patients who received combined therapy with autologous polyclonal Treg and anti-CD20 antibodies. These data were compared with monotherapy with polyclonal Treg and control standard care patients treated with insulin alone. We found that the increase in the percentage of PD-1+ cells in CD4+Treg, CD4+Teff and CD8+T cells in peripheral blood was associated with good treatment results. In vitro, the higher the percentage of PD-1+Treg, the better the inhibitory activity in the functional inhibition test after 7 days of stimulation. This effect disappeared on the 12th day, and the percentage of PD-1+CD4+Treg in the culture was significantly reduced. These correlations were not found in the cultures of healthy controls. Furthermore, in the combination-treated group, the B-cell compartment was remodeled to increase the proportion of regulatory B cells at the expense of reduced memory B cells and to increase the proportion of serum IgG2 at the expense of reduced serum IgG1 concentrations. These changes in treated subjects were accompanied by a reduction in proinflammatory potential, as evidenced by increased serum IL-10 and IL-1Ra concentrations and decreased IL-17 concentrations, compared with control patients.

[0286] A very important finding of this example study is that the administration of polyclonal Tregs retains the expression of PD-1, and the combination therapy improves it further, which may be a significant therapeutic effect of the treatment. At the end of the trial, the percentage of PD-1 in CD4+Treg, CD4+Teff, and CD8+T cells in DM1-treated patients was higher than that in the DM1 control group. Compared with the baseline, the PD-1 expression in the DM1 control group decreased, while the PD-1 expression in the Tregs group was not affected, and the PD-1 expression in the Tregs+RTX group increased. The PD-1+ cutoff value of regulatory T cells and effector T cells can be calculated to monitor the treatment outcome. The cutoff value of PD-1+Treg is higher than 16%, and the cutoff value of PD-1+Teff is higher than 8%, indicating that the Tregs+RTX group is in a state of remission. Similarly, the cutoff value of CD73+Treg is higher than 7%, which is also associated with better treatment outcomes. These calculations prove that Treg and Teff phenotypes can be used for individual treatment response monitoring. We then verified this observation in an in vitro model, comparing DM1 patients and healthy controls. This part of the study highlights the importance of PD-1 expression on Tregs in DM1. We found that in cultures from DM1 patients, the expression of this receptor on Tregs and the percentage of PD-1+Tregs increased significantly on day +7, while this percentage decreased significantly on day +12. ( Figure 3D -G). Interestingly, in cultures from DM1 patients, the suppressive potential of Tregs in the suppression assay was mainly correlated with the percentage of PD-1+ Tregs, as it was enhanced on day +7 and significantly reduced on day +12. No significant differences in the stimulated expression of the PD-1 receptor were found in CD4+ Teff cultures, nor was any association found in healthy control cultures. This may suggest that the expression of the PD-1 receptor on T cells protects against autoimmunity in DM1. Disease is a stimulus that, similar to in vitro stimulation, upregulates the expression of PD-1. Unfortunately, Tregs from DM1 patients can only transiently upregulate PD-1; therefore, the suppressive effect is quickly lost and the disease may progress ( Figure 3H-I). This is an exciting observation in DM1, as it is known that expression of the PD-1 antigen on activated T cells and B cells can control T cell function and proliferation. (13,14) This study demonstrates a link between this mechanism and protection against autoimmunity such as DM1. It has been reported that approximately one-third of cancer patients treated with PD-1 / PD-L1 blocking antibodies develop immune-related adverse events that resemble autoimmune-like syndromes, such as autoimmune insulin-dependent diabetes. (15,16) Moreover, in non-obese diabetic mice (DM1 model), blockade of the PD-1 pathway leads to rapid progression of diabetes. (17) In addition, studies have shown that the PD-1 / PD-L1 axis only controls the early stages of diabetogenic effector T cells in the pancreas, indirectly demonstrating that only early intervention can slow disease progression. (18) Similar observations have been observed in systemic lupus erythematosus (SLE) patients treated with rituximab, where the percentage of PD-1 high CD4+ T cells decreased during follow-up, which was associated with disease progression. (19) Finally, in our study, PD-1 expression was most strongly associated with clinical markers of β-cell function, and we found multiple associations in both treated and control patients. Furthermore, PD-1 associations were widespread across cell types (Treg, CD4+Teff, and CD8+T cells) and follow-up time points ( Figure 6 ). PD-1 upregulation is beneficial to β-cell function, and its high expression is associated with better suppressive function of Tregs as confirmed by in vitro models.

[0287] Another important finding of this example is that PD-1 expression is highest in the Tregs+RTX population, which may be attributed to anti-CD20 treatment. In autoimmunity, such as idiopathic thrombocytopenic purpura (ITP), rituximab has been shown to increase the number of Tregs and the expression of Fas ligand, the mRNA levels of proteins BAX and BCL2 involved in apoptosis, and restore the Th1 / Th2 ratio and TCR Vβ clonality. (29, 21) From a functional perspective, it is speculated that the depletion of CD20+B cells will change T cell activation in several pathways, among which reduced antigen presentation may be crucial. (22, 23) Similar to rheumatoid arthritis (RA), CD20+B cell depletion reduces the pool of antigen presenting cells (APCs) and delays autoimmunity, but the effect is limited. Once B cells are restored, antibody production and T cell activation will resume, and the disease will relapse. (24, 25) In mouse models of arthritis and autoimmune diabetes, B cell depletion inhibited antigen-specific CD4+T cell expansion, which once again demonstrated that B cells are essential for T cell responses. (26) We noted that in the Tregs+RTX group, B cell depletion resulted in an increase in the proportion of naive, transitional, and regulatory-like B cells. In addition, when disease-specific autoantibodies were measured, anti-GAD65 levels persisted in the control and Tregs groups but were significantly reduced in the Tregs+RTX group. Figure 5H ). In addition, the Tregs+RTX group was characterized by increased levels of anti-inflammatory cytokines, especially IL-10 and IL-1Ra, while serum IL-17 concentrations were reduced. The same was true for the Tregs group, but only IL-17 continued to decrease, while IL-1Ra and IL-10 did not increase over time ( Figure 7B -D). This is consistent with other reports where reconstitution of the B cell compartment with rituximab resulted in fewer autoreactive clones and more B cell subsets capable of producing IL-10, primarily transitional B cells. (22)

[0288] From this perspective, the therapeutic strategy of combining rituximab with regulatory T cell administration in this example is reasonable because B cell depletion reduces antigen presentation, inducing a pro-tolerant B cell phenotype and an anti-inflammatory cytokine environment. At the same time, Tregs reduce T cell proliferation and promote anti-inflammatory responses. PD-1 expression can be used as a biomarker for this immunomodulatory therapy because its upregulation can predict the therapeutic effect ( Figure 3A , B).

[0289] In a previous clinical trial (TN-05), results showed that four doses of rituximab could maintain β-cell function within one year, but when extended to 30 months, no significant improvement was found. (27, 29) However, surprisingly, the present example shows that the combination of rituximab with polyclonal Tregs can achieve better results in MMTT and fasting C-peptide levels, DDI / kg body weight, HbA1c, remission and insulin independence at two years of follow-up, thereby controlling DM1. The time to B cell repopulation between TN-05 and TregVAC2.0 was between six and twelve months after depletion ( Fig.19 ) and the duration of the decrease in serum IgM levels was similar ( Figure 5G ). Previously, there have been conflicting reports on the effects of B cell depletion on serum immunoglobulin levels: in the TN-05 trial, DM1 patients treated with rituximab were characterized by similar or increased serum total IgG concentrations in long-term follow-up compared with control patients (27, 28), while some reports showed that serum IgG1, IgG2, IgG3, and IgG4 concentrations remained unchanged after rituximab (29) or only serum IgG4 subclasses were selectively reduced (30). However, in the study according to the present example, once B cells were re-implanted in Tregs+RTX patients, an increase in IgG2 and a decrease in IgG1 were observed ( Figure 5E , F), which is consistent with concentrations of class-switched cytokines (31), as IgG1 serum concentrations were positively correlated with peripheral IL-4 and IgG2 with IL-5 approximately 12 months after rituximab treatment (the time of B cell re-engraftment). This switch may directly affect IgG function, as IgG1 binds more to complement and FcR receptors on monocytes and neutrophils than IgG2. (32) Therefore, higher levels of IgG2 combined with a higher proportion of regulatory-like B cells may contribute to the better clinical outcomes in the Tregs+RTX group. Interestingly, serum IgG2 levels have been reported to be negatively correlated with systemic and muscle insulin sensitivity and to insulin-stimulated glucose disposal in healthy individuals. Given factors known to alter insulin sensitivity, such as age, sex, and BMI (33), changes in antibody levels after B cell depletion with rituximab may also affect infectious immunity. For example, patients with rheumatoid arthritis have reduced levels of IgM, IgG1, and IgG3 in the humoral response after influenza vaccination. This phenomenon was time-dependent and was only observed in individuals who were depleted approximately one month before vaccination, but disappeared in individuals who had received rituximab treatment six to ten months before. (34) Although approximately 60% of the Tregs+RTX group in the study according to this example became infected, their infection rates were comparable to those of the Tregs and control groups. (7)

[0290] In conclusion, the efficacy of the combination therapy can be attributed to several factors. This is mainly related to the increase in the percentage of PD-1+ T cells (Teff and CD8+ T cells in vivo and Treg in vivo and in vitro) and the reconstitution of the B cell compartment towards a tolerogenic phenotype. PD-1 expression on T cells may be a promising biomarker for the efficacy of this therapy. Our data provide a solid background for immune monitoring in future clinical trials and shed light on the immunopathogenesis of DM1.

[0291] References

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[0335] surface

[0336] Table 1A : Baseline characteristics of patients participating in the clinical trial

[0337]

[0338] The P values ​​in Table 1A are based on the one-way ANOVA F statistic for continuous data and the Kruskal-Wallis statistic for multilevel categorical data; adapted from (7).

[0339] Table 1B : Baseline characteristics of patients in the DM1 in vitro model

[0340]

[0341] p values ​​are based on Fisher's exact test for categorical data and t-test for continuous data. Healthy controls were anonymous blood donors and all characteristics were assumed to be within normal ranges. n / a – not applicable

[0342] Table 2 : Inclusion and exclusion criteria for clinical trial and in vitro model participants

[0343]

[0344]

[0345] Adapted from (41).

[0346] Table 3A :Flow cytometry staining chart - whole blood lymphocyte T flow cytometry antigen panel

[0347]

[0348]

[0349]

[0350] For surface / intracellular staining, the Foxp3 / Transcription Factor Staining Buffer Kit from eBioscience (Thermo Fisher Scientific, Waltham, MA, USA) was used strictly according to the protocol.

[0351] Table 3B: Flow cytometry staining chart - whole blood lymphocyte B flow cytometry antigen panel

[0352]

[0353] Table 3C :Flow cytometry staining chart - in vitro model lymphocyte T flow cytometry antigen panel

[0354]

[0355] For surface / intracellular staining, the protocol was strictly used according to eBioscience's Foxp3 / Transcription Factor Staining Buffer Kit, Thermo Fisher Scientific (Waltham, MA, USA).

[0356] Table 4 : Area under the ROC curve for the selected parameter

[0357]

Claims

1. A method for monitoring cell therapy for treating a patient with CD4+Fox P3+T regulatory cells, comprising the following steps: The expression of at least one protein selected from the group consisting of PD-1 and CD73 on the patient's CD4+FoxP3+T cells is determined in vitro, and / or the expression of PD-1 on the patient's CD4+FoxP3- cells is determined in vitro.

2. The method of claim 1, wherein: Determining the expression of said at least one protein on CD4+FoxP3+ T cells and / or determining the expression of PD-1 on CD4+FoxP3- cells is performed on a sample from said patient.

3. The method according to claim 1 or 2, wherein: The method comprises isolating CD4+FoxP3+T cells from a patient sample before determining the expression of the at least one protein, and / or isolating CD4+FoxP3- cells from a patient sample before determining the expression of PCD1 on CD4+FoxP3- cells.

4. The method of claim 2 or 3, further comprising expanding the isolated CD4+FoxP3+T cells and / or the isolated CD4+FoxP3- cells.

5. A method as claimed in any one of the preceding claims, wherein: The sample is peripheral blood.

6. A method as claimed in any one of the preceding claims, wherein: The isolated and optionally expanded CD4+FoxP3+ T cells are CD4+FoxP3+ T regulatory cells.

7. A method as claimed in any one of the preceding claims, wherein: The isolated and optionally expanded CD4+FoxP3- T cells are CD4+FoxP3- T effector cells.

8. A method as claimed in any one of the preceding claims, comprising the steps of: (i). Determining the expression of PD-1 on CD4+FoxP3+T regulatory cells; and / or (ii). Determining the expression of PD-1 on CD4+FoxP3- T effector cells; and / or (iii). Determine the expression of CD73 on CD4+FoxP3+ T regulatory cells.

9. The method of any one of the preceding claims, wherein the T regulatory cells have the phenotype CD4+FoxP3+CD25highCD127-bipartite cells-.

10. The method of any one of the preceding claims, wherein the T effector cells have the phenotype CD4+FoxP3-CD25lowCD127+biocytes-.

11. A method as claimed in any one of the preceding claims, wherein: The method is performed one or more times on the patient's cells at least 2 weeks, preferably 1 month, more preferably 2 months, and even more preferably 3 months after administration of the CD4+Fox P3+ T regulatory cells.

12. The method of claim 11, wherein: After administration of the CD4+Fox P3+ T regulatory cells, the method is performed on the patient's cells at intervals of at least 2 weeks, preferably at least 1 month, more preferably at least 2 months, even more preferably at least 3 months.

13. The method according to claim 11 or 12, wherein: The method is performed on the patient's cells at least 6 months, more preferably at least 1 year, more preferably at least 2 years after administration of the CD4+Fox P3+ T regulatory cells.

14. The method according to any one of claims 11 to 13, wherein: The method is performed on the patient's cells at least once prior to administration of the CD4+Fox P3+ T regulatory cells.

15. A method as claimed in any one of the preceding claims, wherein: The patient suffers from an autoimmune disease.

16. The method of claim 15, wherein: The autoimmune disease is type 1 diabetes.

17. The method according to claim 15 or 16, wherein: The patient is a child or adolescent.

18. A method as claimed in any one of the preceding claims, wherein: The patient is also treated with an anti-CD20 antibody or a fragment thereof that retains specific binding to CD20.

19. The method of claim 18, wherein: The anti-CD20 antibody is rituximab.

20. A method for evaluating the efficacy of cell therapy in a patient treated with CD4+Fox P3+ T regulatory cells, comprising performing the method of any one of the preceding claims, wherein: Expression of PD-1 on at least 16% of CD4+FoxP3+ T regulatory cells, and / or expression of CD73 on at least 7% of CD4+FoxP3+ T regulatory cells, and / or expression of PD-1 on at least 8% of CD4+FoxP3- T cells indicates that the cell therapy is effective.

21. CD4+Fox P3+ T regulatory cells for use in treating an autoimmune disease in a patient suffering from or developing an autoimmune disease, respectively, by cell therapy, said cell therapy comprising administering said T regulatory cells and further comprising performing a method as defined in any one of claims 1 to 14.

22. The CD4+Fox P3+T regulatory cells for use according to claim 21, further comprising administering an anti-CD20 antibody or a fragment thereof that retains specific binding to CD20.

23. The CD4+Fox P3+T regulatory cells for use according to claim 21, wherein: The anti-CD20 antibody is rituximab.

24. The CD4+Fox P3+T regulatory cells for use according to claim 21, wherein: An autoimmune disease as defined in claim 15 or 16.

25. A detectable compound having high affinity for a protein selected from the group consisting of PD-1 and CD73, for use in a method for diagnosing effective cell therapy for a patient treated with CD4+Fox P3+ T regulatory cells, the method comprising the following steps: Obtain a patient sample, isolate CD4+FoxP3+T cells and / or CD4+FoxP3- cells from the patient, contact the isolated CD4+FoxP3+T cells and / or the isolated CD4+FoxP3- cells with the detectable label, detect the label bound to CD4+FoxP3+T cells expressing PD-1 and / or CD73 and / or bound to CD4+FoxP3- cells expressing PD-1, and determine the proportion of CD4+FoxP3+T cells expressing PD-1 and / or CD73 and / or the proportion of CD4+FoxP3- cells expressing PD-1.

26. The detectable compound of claim 25, wherein The isolated CD4+FoxP3+T cells and / or the isolated CD4+FoxP3- cells are expanded before contacting the cells with the detectable label.

27. The detectable compound of claim 25 or 26, wherein The detectable label is an anti-PD1 antibody or a fragment thereof that retains specific binding to PD-1, or an anti-CD73 antibody or a fragment thereof that retains specific binding to CD73.

28. A detectable compound for use as claimed in any one of claims 25 to 27, wherein The compound is coupled to a detectable label.

29. The detectable compound for use as claimed in claim 28, wherein The label is a fluorescent label.

Images