WO2006111986A1

WO2006111986A1 - Method for the detection and the isolation of immunosuppressive regulatory t cells and uses thereof

Info

Publication number: WO2006111986A1
Application number: PCT/IT2005/000227
Authority: WO
Inventors: Giovanna Borsellino; Luca Battistini; Olaf RÖTZSCHKE; Kirsten Falk
Original assignee: Fondazione Santa Lucia I.R.C.C.S.
Priority date: 2005-04-19
Filing date: 2005-04-19
Publication date: 2006-10-26

Abstract

The present invention provides a mean to identify and to isolate T cells with immunosuppressive function from the peripheral food of mammals, by detecting blood cells expressing NTPDases. This method can be used in vitro and in vivo, and has use for the treatment and the monitoring of autoimmune conditions.

Description

Method for the detection and the isolation of immunosuppressive regulatory T cells and uses thereof

Technical Field

The present invention provides a mean to identify and to isolate T cells with immunosuppressive function from the peripheral blood of mammals, by detecting blood cells expressing NTPDases. This method can be used in vitro and in vivo, and has use for the treatment and the monitoring of autoimmune conditions. Background of the invention Homeostasis in the immune system is maintained by a complex network of control mechanisms which shut down the immune response when responsiveness to a given antigen is no longer required. Regulatory T cells (Tregs)have long been elusive but compelling evidence indicates the existence of specialized CD4+ T lymphocytes with the potential to regulate a variety of immune responses (). Transfer of these regulatory cells has been shown to be effective in ameliorating autoimmune diseases such as thyroiditis, gastritis, insulin-dependent diabetes mellitus and colitis, and of experimentally induced graft versus-host disease, whereas depletion of Tregs cells results in the development of systemic autoimmune diseases.

Although in recent years an impressive amount of effort has been dedicated to the study of regulatory T cells, the mechanisms by which these cells exert their action are still unclear. Moreover, their precise identification is hindered by the lack of specific surface marker(s) uniquely expressed by this subset. Indeed, expression of CD25 (IL-2R⁽()is the only "flag" which permits to fish out T cells with suppressive function. However, activated non-regulatory CD4+ T cells also express CD25, thus hampering the precise identification of Tregs. Recently, expression of the transcription factor FoxP3 has been shown to identify a population of T cells with regulatory function, both in mice and in humans, but this marker cannot be used to track Tregs in vivo. The identification and isolation of regulatory T cells is daunting researchers. To date, no one has identified a surface cell marker which is unique to regulatory T cells. A method to identify and effectively isolate regulatory T cells would be of immense importance. There are many important uses, but one must first have the starting material— the regulatory cells that make these uses possible-before they may be investigated and applied. The invention described herein is the first to provide that starting material.

Description of the invention

In this invention the inventors have identified CD39 as a surface marker which unequivocally identifies T regulatory cells, and whose expression confers unique functions that may explain at least in part the mechanism of suppression. CD39 is a member of the family of ecto-nucleoside triphosphate diphosphohydrolases (E-NTPase family) ectpenzymes, which metabolize ATP/ADP to 5'-AMP, and it is the main enzyme involved in the regulation of extracellular concentration of nucleotides.

Nucleotides regulate inflammatory and immune responses and lymphocytes are known to express purinergic receptors. ATP is present in the cytosol at very high concentrations (5nM), and is released in large amounts during inflammatory reactions. The effects of ATP on cells of the immune system are both direct, through binding of purinergic receptors expressed on all lymphocytes, and indirect, through its catabolites, such as adenosine. In particular, adenosine is known to have powerful immunosuppressive abilities, and signaling through adenosine receptors in lymphocytes has been shown to terminate inflammation. Thus, CD39 on the surface of Tregs may have a role in the generation of molecules with immunosuppressive functions, such as adenosine and in the reduction of the concentration of proinflammatory molecules such as ATP.

Only CD4+CD25^high cells are able to suppress proliferation.

Human CD4 levels express different levels of CD25 (Fig 1).

CD4

Fig. 1. Within CD4+ T cells, three subsets can be identified based on CD25 expression: 0025¹^, CD25^Iow, and CD25^πe8.

It has already been suggested that the suppressive ability resides mainly within the CD25^hl9h population, which is also characterized by the lack of proliferation following in vitro stimulation. Thus, the different populations of CD4+CD25^hlgh, CD4+CD25^l0W, and CD4+ CD25^neg can be sorted (Fig.2) and tested for their ability to suppress proliferation of activated autologous CD4+CD25- T cells. Indeed CD4+CD25^hl9h cells efficiently inhibited proliferation of CD3-stimulated responder CD4+ cells; on the contrary, CD25^|OW cells proliferated vigorously along with the responder cells, confirming that they represent a population of activated T cells not endowed with suppressive abilities. As expected, CD25^πeg, which are mainly composed of naϊve non effector cells, did not inhibit proliferation (Fig. 3).

Fig.3 0025*"^ CD4+ are able to inhibit proliferation of CD4+ cells following stimulation with anti CD3.

Suppressive CD4+CD25^h'^9h cells express CD39. Surprisingly, CD39, an ectoATPase which sequentially degrades ATP to ADP and AMP, is exclusively expressed by a subset of CD4+CD25high cells (Fig.4), while CD25^|OW and CD25^πeg express low or undetectable levels. CD39 is not expressed by CD8 cells, by NK cells, 'VtTTCR cells, while it is present at high levels in B cells and in CD14+ monocytes. Thus, we investigated whether expression of CD39 is associated with regulatory function, and whether it defines a subset of activated regulatory cells with particularly potent suppressive abilities. To this aim, CD25^hιgh CD39+, CD25^hιgh CD39-, CD25^|OW CD39+ and CD25^|OW CD39- cells were sorted and tested for their ability to suppress proliferation of activated CD4+ cells. As shown in Fig. 5, both CD25^h'^9h CD39+ and CD25^|OW CD39+ cells efficiently suppressed CD4+ T cell proliferation (80% and 72% inhibition at a 1 :1 ratio_μ respectively), contrary to their CD39-counterparts. CD25^hlgh/low CD39+ cells were also anergic, and did not proliferate following stimulation with anti-CD3 or with allogenic feeder cells (show or not show?), whilst c^D25^h,g^h/iow_CD3g_ _ce||s proliferated vigorously, as expected by activated T cells.

Fig.4. CD39 expression by CD25¹"⁸¹¹, CD25^low, and CD25^neg cells. Data is gated on CD4+ cells. Fresh

Fig.5. Inhibition of proliferation of CD4+ responder T cells by sorted CD4+CD25^ω8h CD39+/-, CD4+CD25^l0W CD39+/-, and 004+0025¹^⁸ ceUs.

Human CD39+CD4+ express molecules associated with regulatory cells. To better characterize CD39+CD4+ T cells, we - examined expression of several markers which have been shown to be present in regulatory T cells. Glucocorticoid-induced TNF receptor (GITR) has been suggested to play a major role in the maintenance of tolerance¹, and is known to be expressed constitutively ay high levels by CD4+CD25^high regulatory cells. Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) has been implicated in the control of immune responses, and although it is upregulated in conventional activated T cells, its expression is higher and persistent in regulatory T cells. FOXP3, a transcription factor which has been shown to be involved in an autoimmune disorder in mice and humans, seems to be specifically expressed by cells with regulatory function. Expression of these markers was indeed higher in CD25^hιgh cells, in agreement with data obtained in other laboratories. Interestingly, highest levels of expressions were detected in correlation with CD39. Thus, CD39 is a cell surface marker which can be used to define regulatory T cells.

Fig. 6. Expression of CTLA4 (a), GITR (b) and FOXP3 (c) by different subsets of CD4+ T cells.

ATPase activity is present in CD39+ cells. We then extended our studies to investigate whether CD39 is functionally relevant. CD39 is an ecto-ATPase which hydrolyzes ATP to ADP and AMP₁ and in fact it is the main enzyme regulating the extracellular metabolism of ATP. Thus, we measured ecto-nucleotidase activity in sorted CD4+CD25^hl9h, CD4+CD25^|OW, and CD4+CD25^πeg cells. The amount of liberated inorganic phosphate was measured (Fig.%). The highest levels of ecto- nucleotidase activity was indeed confined to the CD25^highCD39+ subset of cells, and this activity was diminished in the presence of ARL-67156, a selective ecto-ATPase inhibitor. CD25^highCD39- and CD25^πegCD39^' cells hydrolyzed ATP at comparably low levels. Potato apyrase, an ectoATPase similar to CD39, hydrolyzed ATP completely.

Fig. 7 Ectonucleotidasβ activity of sorted subsets of CD4+ cells.

CD25^high cells generally represent 2-3% of the total peripheral blood cell population, and 12-16% of the CD4+ cell population. Within the CD4+CD25^high T cells, a variable fraction of cells also expresses CD39 (32±13, range 2-54%). These cells can be sorted from the leukapheresis of an individual, expanded in vitro, and re-injected as a therapy for autoimmune diseases or to prevent transplant rejection. Indeed these cells proliferate when stimulated with anti-CD3, provided that lnterleukin 2 (IL-2) is added to the culture medium (see methods section). As shown above, the CD39+ fraction of CD25⁺ cells correspond to the only population of cells which are endowed with suppressive abilities, and probably represent the true effectors which down-modulate immune responses.

It is interesting to note that in patients with autoimmune disorders of the nervous system, it has been described that T regulatory cells may display loss of functional suppression. Indeed we have found that in these patients, in the acute phase of the disease the fraction of CD39+ cells within the CD25^high subsets is significantly diminished (16±15, range 2-44%). On the contrary, patients with no symptoms or signs of disease display a percentage of CD25^hιgh CD39+cells comparable to that of healthy individuals. Thus, the measurement of CD39 expression by CD4+CD25^h'^9h cells provides a means to monitor disease activity.

The reduced frequency of CD39+ CD25^high T cells in patients with autoimmune disease immediately suggests therapy with injection of said cells as a possible means of restoring the immunological equilibrium of the individual, in which autoreactive cells fail to be downregulated by the immune system.

Similarly, patients undergoing transplantation may benefit from the administration of CD25^high CD39+ cells, which may prevent rejection and ameliorate engraftment.

Methods.

Isolation and immunostaining of blood: PBMC will be isolated from blood samples by discontinuous density centrifugation over Ficoll- hypaque according to standard procedures. Briefly, heparinized blood will be diluted with 1 volume of RPMI media and gently layered over the Fycoll. Following centrifugation at 66Og for 30 minutes, cells at the interface of the gradient will be collected and washed 3 times in media. After the final wash cells will be resuspended in PBS with 1 % human serum.

For each staining 5 x 10⁶ PBLs will be used, in a final volume of 100 μl Monoclonal antibodies (conjugated with the appropriate fluorochrome) will be added to predetermined optimal concentrations and cells will be incubated for 10 minutes at 4 ⁰C in the dark. Cells will be then washed twice, resuspended in 250 μl. of PBS contaning 0,5% FCS, and analyzed at the flow cytometer. A minimum of 2 x 10⁶ events will be acquired per sample, in order to enable the precise evaluation of each cell subset.

For in vitro proliferation assays, 500 x10⁶ cells will be stained for CD4, CD25, and CD39, and cells will be sorted in sterile conditions on a high-speed cell sorter. Reagents: For the characterization of effector regulatory T cells, the following reagents are used: CD4, CD25, CD39, CTLA4 (from Pharmingen), and GITR (R&D Systems)

Cytofluorimetric Analysis and cell sorting: Immunofluorescence analysis will be performed on a Cytomation MoFIo Cytofluorimeter equipped with three lasers, and data will be analyzed using Summit software (Cytomation, Boulder, CO), or FlowJo software (Treestar Inc.,

San Diego, CA). Single-stained cells for each fluorochrome will be acquired and used as controls to set compensation. Compensation matrix will be determined using FlowJo software. Subsets of T cell will be sorted and used for functional assays. 4-way sorting will allow for simultaneous sorting of 4 subsets of lymphocytes in each sample.

In vitro suppression assays. 5x10⁴/well irradiated (3,000 Rad) BPMC were used together with 2,5x10⁴/well CD4+ responder cells and titrated amounts of suppressor cells in 96 well V-bottom plates (Costar). Cells were stimulated with 10 μg/ml soluble αCD3 (Becton Dickinson) and incubated for 72 h at 37 ⁰C in RPMI/10 % FCS. In some experiments 20 U/ml hlL-2 (Roche) were added.1 μCi/well ³H- Thymidine was added for the last 18 h of incubation and proliferation was determined using a beta-plate reader (Wallac).

In vitro expansion of regulatory cells. CD4+CD25highCD39+ cells are sorted in sterile conditions. Sorted cells are placed in 96 well U-bottom plates (100.000 cells/well), with each well containing 100.000 irradiated (3000 rad) feeder cells obtained from another donor. Medium is RPMI supplemented with 10% human serum, non essential aminoacids, antibiotics (pen/strep). Cells are stimulated with soluble anti CD3 (10 ug/ml) and with hr IL-2 (10 U/ml, Boehringer). Fresh medium is added every 3 days, and growing cells are split and expanded twice a week, or as needed. Every 14 days cells are restimulated. Cells for in vitro experiments are used 10-14 days following restimulation (in the resting phase). The phenotype of the growing cells is monitored by FACS analysis.

Claims

1. A method to unequivocally identify cells with immunosuppressive abilities in healthy mammals and in mammals with an autoimmune condition, said method comprising the immunofluorescent labelling of peripheral blood cells with antibodies specific for NTPDases in combination with antibodies specific for the CD4 molecule.

2. The method of claim 1 , where the autoimmune disease is alopecia areata, Addison's disease, autoimmune hypoparathyroidism, autoimmune hypophysitis, autoimmune oophoritis, autoimmune orchitis, Graves's disease, Hashimoto's thyroiditis, Polyglandular

Autoimmune Syndrome type 1 (PAS-1), Polyglandular Autoimmune Syndrome type 2 (PAS-2), , Polyglandular Autoimmune Syndrome type 3 (PAS-3), diabetes mellitus, autoimmune hepatitis, celiac disease, inflammatory bowel disease, primary biliary cirrhosis, Autoimmune Myocarditis, Churg-Strauss Syndrome, Giant Cells Arteritis, Kawasaki's Disease, Polyarteritis Nodosa, Takayasu's Arteritis, Wegener's Granulomatosis, ankylosing spondilitis, rheumatoid arthritis, rheumatic fever, polymyositis/dermatomyositis, Meniere's syndrome, Mooren's ulcer, Reiter's syndrome, autoimmune uveitis, Bullous Pemphigous, Epidermolysis Bullosa Acquisita, Pemphigous vulgaris, Pemphigous Foliaceous, Vitiligo, Chronic inflammatory demyelinating neuropathy, Guillan-Barre Syndrome, Multiple Sclerosis, Myasthenia Gravis, Antiphospholipid Syndrome, Autoimmune Polyendocrinopathy, Bechet's Disease, Goodpasture's Syndrome, Rheumatoid Arthritis, Sarcoidosis, Scleroderma, Sjogren's Syndrome, Systemic Lupus Erythematosus.

3. The method of claim 1 , wherein said NTPDase is CD39.

4. A method to monitor disease activity in individuals with an autoimmune condition, said method comprising the measurement of extracellular protein levels of NTPDases in CD4+ lymphocytes.

5. The method of claim 4, where the autoimmune disease is alopecia areata, Addison's disease, autoimmune hypoparathyroidism, autoimmune hypophysitis, autoimmune oophoritis, autoimmune orchitis, Graves's disease, Hashimoto's thyroiditis, Polyglandular Autoimmune Syndrome type 1 (PAS-1), Polyglandular Autoimmune Syndrome type 2 (PAS-2), , Polyglandular Autoimmune Syndrome type 3 (PAS-3), diabetes mellitus, autoimmune hepatitis, celiac disease, inflammatory bowel disease, primary biliary cirrhosis, Autoimmune Myocarditis, Churg-Strauss Syndrome, Giant Cells Arteritis, Kawasaki's Disease, Polyarteritis Nodosa, Takayasu's Arteritis, Wegener's Granulomatosis, ankylosing spondilitis, rheumatoid arthritis, rheumatic fever, polymyositis/dermatomyositis,

Meniere's syndrome, Mooren's ulcer, Reiter"s syndrome, autoimmune uveitis, Bullous Pemphigous, Epidermolysis Bullosa Acquisita, Pemphigous vulgaris, Pemphigous Foliaceous, Vitiligo, Chronic inflammatory demyelinating neuropathy, Guillan-Barre Syndrome, Multiple Sclerosis, Myasthenia Gravis, Antiphospholipid Syndrome, Autoimmune Polyendocrinopathy, Bechet's Disease, Goodpasture's Syndrome, Rheumatoid Arthritis, Sarcoidosis, Scleroderma, Sjogren's Syndrome, Systemic Lupus Erythematosus.

6. A method to treat patients with autoimmune diseases, by injecting autologous T regulatory cells which have been previously expanded in vitro.

7. A method to treat patients undergoing organ transplantation, by injecting autologous regulatory T cells which have been previously expanded in vitro.