ENHANCEMENT OF REGULATORY T CELLS' ACTIVITY USING STATINS

FIELD OF THE INVENTION

This invention relates to methods for treatment of inflammatory and auto immune diseases by enhancing the activity and numbers of regulatory T cells using HMG-CoA reductase inhibitors (statins).

BACKGROUND OF THE INVENTION

Over the past decade, studies have revealed that an inflammatory response is involved in the initiation and progression of the atherosclerotic lesion. Additionally, it has been demonstrated that regulatory T cells (Tregs) actively suppress autoreactive T cells, and thus may play a critical role in the maintenance of self-tolerance (1, 2).

Several lines of evidence support a role for regulatory cells in protection against atherosclerosis: Induction of experimental oral tolerance in mice is associated with attenuation of atherosclerotic lesions (3), whereas transfer of antigen specific lymphocytes leads to enhanced atherosclerosis (4). In addition, in humans with unstable angina cytokines representing regulatory T-cells are reduced (5). The same cytokines are protective against atherosclerosis in experimental mouse models (6).

Very recent evidence suggests that Tregs are capable of attenuating the progression of immune mediated diseases including atherosclerosis in mouse (7). Mor et al (8) suggest that Tregs suppress plaque progression and stabilize the lesions.

Regulatory T-cells are comprised of antigen-specific lymphocytes that act principally via T-helper cytokine secretion, and naturally occurring CD4 ⁺ CD25 T cells (1, 9). The naturally occurring high CD4+CD25+ Treg cells are generated spontaneously in the thymus and constitutively express the transcription factor Foxp3. In addition, a high proportion of these cells constitutively express glucocorticoid induced tumor necrosis factor receptor (GITR). Hence, CD25, foxp3 and GITR are the main markers for the characterization of Tregs.

The number of peripheral Tregs has been shown to be reduced in patients with diabetes, lupus, multiple sclerosis and rheumatoid arthritis and consequent loss of T cell inhibition was suggested to trigger flares in the respective disorders (10-13).

Statins (HMG-CoA reductase inhibitors) are effective lipid lowering agents extensively used in medical practice. Through inhibition of HMG-CoA reductase, they restrict cholesterol synthesis, leading to upregulation of LDL receptors on the cell membrane and a subsequent reduction in atherogenic LDLs. Accumulating evidence indicates that statins exert immunosuppressive effects, independent of their lipid lowering effects (14).

SUMMARY OF THE INVENTION The present invention it based on the discovery that statins increase the numbers and enhance the activity of naturally occurring regulatory T cells (Tregs). Furthermore, it is also based on the realization that patients suffering from atherosclerosis have a compromised T regulatory cell population.

Therefore, in a first of its aspects the present invention provides a method of treatment of a disease characterized by an impaired function of the naturally occurring regulatory T cells by use of statins.

Such a method is suitable for the treatment of any pathology in which Tregs are involved, and in which the activity of these cells is insufficient or dysregulated.

Such pathologies include but are not limited to cardiovascular syndromes e.g. acute coronary syndrome, stroke and atherosclerosis, as well as cancer, inflammatory and autoimmune diseases e.g. rheumatoid arthritis and multiple sclerosis.

According to one embodiment the statins may be administered systemically, namely orally, by injection or by infusion.

Accordingly, the present invention provides a method of treating a disease in which the activity of Tregs is insufficient or dis-regulated, comprising administering to a patient in need thereof a therapeutically effective dose of a statin.

In another embodiment, the statins may be used ex vivo to upregulate a patient's Tregs. These upregulated cells are then infused back into the patient.

The present invention therefore also provides a method of treating a disease in which the activity of Tregs is insufficient or dysregulated, comprising obtaining a

patient's PBMC, incubating the cells ex vivo with a statin, and re-infusing a therapeutically effective amount of the statin-activated cells to said patient. In one embodiment, the statin-activated cells which are re-infused into the patient are administered as a PBMC cell preparation enriched with Tregs. Alternatively, the statin- activated cell preparation undergoes a further step comprising isolating the activated Tregs and administration of a purified Treg cell population.

In another aspect, the present invention provides a pharmaceutical composition for administering to an individual for the purpose of up-regulating Tregs comprising at least one statin as the active ingredient. By another aspect the invention provides a pharmaceutical composition for use in ex vivo up-regulation of Tregs comprising at least one statin. The pharmaceutical composition of the invention may comprise one or more pharmaceutically acceptable additives or excipients.

Any of the following statins can be used in the invention: mevastatin, pravastatin, atorvastatin, rosuvastatin, simvastatin or cerivastatin. In a preferred embodiment the statin is atorvastatin.

The present invention further provides a pharmaceutical composition for use in the above methods. The pharmaceutical composition comprises as an active ingredient a statin along with excipients and pharmaceutical carriers.

SOME SPECIFIC EMBODIMENTS

Some embodiments of the invention are defined in the following numbered paragraphs:

1. A method of treating a disease characterized by impaired T regulatory

(Treg) cell function comprising contacting said Treg cells with a statin. 2. A method according to 1 for the treatment of cardiovascular syndromes, inflammatory or autoimmune diseases.

3. A method according to 2 wherein the inflammatory disease is atherosclerosis.

4. A method of treatment according to any of 1-3 comprising administering to a patient in need thereof a therapeutically effective dose of a statin.

5. A method of treatment according to any of 1-3 comprising: a. Obtaining peripheral blood mononuclear cells (PBMC) from the patient; b. Incubating said PBMC with a statin, at an amount and for a time so as to cause up-regulation of Tregs; and c. Infusing the up-regulated Tregs into the patient.

6. A method according to any of 1-5 wherein the statin is selected from a group consisting of mevastatin, pravastatin, rosuvastatin, simvastatin, cerivastatin and atorvastatin. 7. The method of 6 wherein the statin is Atorva.

8. A method of treatment of inflammatory or autoimmune diseases comprising: a. Obtaining peripheral blood mononuclear cells (PBMC) from the patient; b. Incubating said PBMC with a statin, at an amount and for a time so as to cause up-regulation of Tregs; and c. Infusing the up-regulated Tregs into the patient.

9. A method of treatment of inflammatory or autoimmune diseases comprising administering to. a patient in need thereof an amount of a statin effective in up-regulating Tregs.

10. A method of enhancing the activity and numbers of regulatory T cells (Tregs) using statins.

11. A composition for use in any of the methods of 1-10.

12. A composition for enhancing the numbers and activity of Tregs comprising statin as an active ingredient.

13. A pharmaceutical composition for enhancing the numbers and activity of Tregs, comprising statin as an active ingredient with a pharmaceutically acceptable carrier.

14. A pharmaceutical composition according to 13, for the treatment of inflammatory or autoimmune diseases.

15. Use of statin for manufacturing a medicament for the treatment of diseases associated with impairment of Treg function.

16. The use according to 15 wherein the disease is atherosclerosis.

17. A method for screening HMG-CoA reductase inhibitors (statins) useful for treating diseases associated with impaired Treg function comprising: a. Providing peripheral blood mononuclear cells (PBMC); b. Culturing said PBMC in the presence of a statin, at an amount and for a time suitable to cause up-regulation of Tregs; and c. Measuring the number of upregulated Tregs in the culture.

18. A method according to 17 wherein the number of upregulated Tregs is measured by evaluating the level of FoxP3 expression.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carried out in practice, preferred embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

Fig. 1 Demonstrates the effect of oxLDL on the number and function of Tregs from healthy subjects. (A) A graphic representation of a single sample from a healthy subject. The Y-axis represent 0-100% of the respective cell population (responders or Tregs). (B) Representative FACS pictures. (C) Shown is a representative experiment out of four yielding essentially similar results.

Fig. 2 Demonstrates that the numbers of naturally occurring CD4 ⁺ CD25 ⁺

Regulatory T Cells are reduced in patients with ACS. (A) A graph showing the collective analyses of results from 3 groups: NCA (healthy subjects), patients with stable angina and patients with acute coronary syndrome (ACS) * p<0.001. (B) Representative FACS pictures from a single patient in each group.

Fig.3 Demonstrates a comparative analysis of peripheral blood T-responder cells and Treg proliferative capacity in various patient groups. (A) A graph representing the proliferation of Treg and T responders as measured by radioactive reading of cpm (Counts per minute) for each of the tested groups. (B, C, D) are a graphic representation

of the percent of T responder suppression as a function of the ratio between Tregs and T responder cells in healthy subjects (B), stable angina patients (C) and acute coronary syndrome patients (D).

Fig. 4 Demonstrates the effect OxLDL on the number of Tregs. The graph shows the average reduction in Treg numbers after a 3-day co-culture with oxLDL.* p<0.01.

Fig. 5 Demonstrates the expression of foxp3 and CTLA-4 mRNAs in Tregs. (A) Results represent mean ± SEM of values obtained from densitometric analysis with GAPDH employed as the reference housekeeping gene. B. Purified Tregs from all three groups were assayed for foxp3 protein content by Western blot. C. Cumulative results of the densitometric analysis are represented in a graph, as well as representative pictures from 3 patients in each group.* p<0.05.

Fig. 6A is a schematic representation of FACS analysis of CD4 ⁺ CD25 ^hlgh of total CD4 ⁺ CD25 ⁺ . Cultured cells were stained with FITC-labeled anti-CD4 and PE-labeled anti-CD25. Fig. 6B. is a graph presenting FACS analysis results: %CD4 ⁺ CD25 ^hlgh of total

CD4 ⁺ CD25 ⁺ , relative to control.

Fig. 6C is a schematic representation of FACS analysis of CD4 ⁺ CD25 ⁴ Foxp3 ⁺ of total CD4 ⁺ CD25 ⁺ .

Fig. 6D. is a graph presenting FACS analysis results: %CD4 ⁺ CD25 ⁴ Foxp3 ⁺ of total CD4 ⁺ CD25 ⁺ , relative to control.

Fig. 6E is a photograph of a Western blot demonstrating Foxp3 expression in cultured PBMCs. Protein quantification was performed by Tina-quant assay, and is presented in a graph as %(OD-background)/mm ² , relative to control.

Fig. 6F is a schematic representation of FACS analysis of CD4 ⁺ CD25 ^hlgh of total CD4 ⁺ CD25 ⁺ .

Fig. 6G is a schematic representation of FACS analysis of CD4 ⁺ CD25 ⁺ Foxp3 ⁺ of total CD4 ⁺ CD25 ⁺ .

Fig. 6H is a graph presenting FACS analysis results: Delta %CD4 ⁺ CD25 ^hlgh of total CD4 ⁺ CD25 ⁺ , relative to control. Fig. 61 is a graph presenting FACS analysis results: Delta %CD4 ⁺ CD25 ⁺ Foxp3 ⁺ of total CD4 ⁺ CD25 ⁺ , relative to control.

Fig. 6 J is a photograph of a Western blot for determination of Foxp3 expression, in 48hr and 96hr samples. Protein quantification was accomplished as mentioned in Fig 6E (M- mevastatin, P- pravastatin, A- atorvastatin, *- p< 0.05).

Fig. 7A is a schematic representation of FACS analysis of total CD4 ⁺ CD25 ⁺ before (time 0) and after a 96 hr stimulation with anti-CD3 mAb (control).

Fig. 7B is a schematic representation of FACS analysis of CD4 ⁺ CD25 ⁺ Foxp3 ⁺ of total CD4 ⁺ CD25 ⁺ in the presence and absence of atorvastatin.

Fig. 7C is a graph presenting FACS analysis results %total CD4 ⁺ CD25 ⁺ is increased in the presence of anti-CD3 mAb and decreased in the presence of atorvastatin in a dose-dependent manner.

Fig. 7D is a graph presenting FACS analysis results: %CD4 ⁺ CD25 ⁺ Foxp3 ⁺ of total CD4 ⁺ CD25 ⁺ following treatment with atorvastatin .(A-atorvastatin *- pv<0.05).

Fig. 7E is a graph presenting FACS analysis results: %CD4 ⁺ CD25 ⁺ Foxp3 ^' of total CD4 ⁺ CD25 ⁺ following treatment with atorvastatin .(A-atorvastatin *- pv<0.05). Fig. 8A is a graph presenting results of a suppression assay. Addition of pravastatin to the culture leads to an increase in the suppression rate, in a non-significant manner (*p=0.05, **p=0.08).

Fig. 8B is a graph presenting results of a suppression assay. Addition of atorvastatin to the culture leads to a significant increase in the suppression rate (***p<0.05).

Fig. 9A is a schematic representation of FACS analysis of CD4 ⁺ CD25 ^high of total CD4 ⁺ CD25 ⁺ . PBMCs were isolated from fresh blood specimens of individuals, before and after treatment with statins (pravastatin (P): treatment for 8 weeks, n=5, simvastatin (S): treatment for 4 weeks, n=7). Fig. 9B is a graph presenting FACS analysis results: %CD4 ⁺ CD25 ^high of total

CD4 ⁺ CD25 ⁺ , relative baseline values. Both pravastatin and simvastatin treatment increased the number of CD4 ⁺ CD25 ^high Tregs.

Fig. 9C is a photograph of a Western blot demonstrating Foxp3 expression in fresh PBMCs. Protein quantification was performed as in Fig. 6. (P- pravastatin, S- simvastatin, *- pv< 0.05).

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

The present invention demonstrates for the first time that the number and functional properties of peripheral CD4 ⁺ CD25 ⁺ regulatory T cells (Tregs) in patients with acute coronary syndromes (ACS) are significantly reduced compared to healthy subjects. The expression of foxp3, the transcriptional regulator of Tregs, is also reduced in ACS patients. Thus, events that result in plaque destabilization are associated with a dysregulated peripheral Treg pool.

The present invention further provides evidence that administration of statins results in upregulation of several cellular markers indicative of regulatory T cells (Tregs). The term "up-regulation " as used herein denotes the ability of a compound, in this case statins, to increase the number of cells, and/or their activity, and/or the expression of certain cellular markers which are indicative of an increase in regulatory activity of the cells. These markers include membranal CD25, the transcriptional activator foxp3, and the glucocorticoid induced tumor necrosis factor receptor (GITR). Furthermore, statins not only increase the expression of these markers but also enhance the suppressive effect of the Tregs, namely their ability to inhibit proliferation of CD4+CD25- T-cells.

Therefore, statins may be protective against atherosclerosis and other inflammatory and autoimmune diseases by upregulation of the Treg population.

The determination of an impaired or disregulated Treg function, or the existence of a disease associated with such an impaired or disregulated Treg function in a patient can be assessed by means well known in the art. A non-limiting example for a method of determining impaired or disregulated Treg function is provided below in the examples section.

The term "treating", "treat" ox "treatment" as used herein includes preventative (e.g., prophylactic) and palliative treatment.

By "pharmaceutically acceptable" it is meant the carrier, diluent, excipients, and/or salt must be compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.

The term "statin ", "vastatin ", or as used interchangeably herein "3-hydroxy-3- methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor" refers to a pharmaceutical agent which inhibits the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG- CoA) reductase. This enzyme is involved in the conversion of HMG-CoA to mevalonate, which is one of the steps in cholesterol biosynthesis. Such inhibition is readily determined according to standard assays well known to those skilled in the art.

Preferred statins which may be used in accordance with this invention include atorvastatin, disclosed in U.S. Pat. No. 4,681,893, atorvastatin calcium, disclosed in U.S. Pat. No. 5,273,995, cerivastatin, disclosed in U.S. Pat. No. 5,502,199, dalvastatin, disclosed in European Patent Application Publication No. 738,510 A2, fluindostatin, disclosed in European Patent Application Publication No. 363,934 Al, fluvastatin, disclosed in U.S. Pat. No. 4,739,073, lovastatin, disclosed in U.S. Pat. No. 4,231,938, mevastatin, disclosed in U.S. Pat. No. 3,983,140, pravastatin, disclosed in U.S. Pat. No. 4,346,227, simvastatin, disclosed in U.S. Pat. No. 4,444,784 and velostatin, disclosed in U.S. Pat. No. 4,448,784 and U.S. Pat. No. 4,450,171. Especially preferred 3-hydroxy-3- methylglutaryl coenzyme A reductase inhibitors include atorvastatin, atorvastatin calcium, also known as Liptor.RTM., lovastatin, also known as Mevacor.RTM., pravastatin, also known as Pravachol.RTM., and simvastatin, also known as Zocor.RTM..

Statins are preferably administered in amounts ranging from about 0.1 mg/kg to about 1000 mg/kg/day in single or divided doses, preferably about 1 mg/kg/day to about 200 mg/kg/day for an average subject, depending upon the statin and the route of administration. However, some variation in dosage will necessarily occur depending on the condition of the subject being treated. The individual responsible for dosing will, in any event, determine the appropriate dose for the individual subject. The pharmaceutical compositions of this invention may be administered to a subject in need of treatment by a variety of conventional routes of administration, including orally, topically, parenterally, e.g., intravenously, rectally, subcutaneously or intramedullary. Further, the pharmaceutical compositions of this invention may be administered intranasally, as a suppository or using a "flash" formulation, i.e., allowing the medication to dissolve in the mouth without the need to use water.

The pharmaceutical compositions of this invention may be administered in single (e.g., once daily) or multiple doses or via constant infusion.

The pharmaceutical composition of this invention may be administered alone or in combination with pharmaceutically acceptable carriers, vehicles or diluents, in either single or multiple doses. Suitable pharmaceutical carriers, vehicles and diluents include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents. The pharmaceutical compositions formed by combining the statins and the pharmaceutically acceptable carriers, vehicles or diluents are then readily administered in a variety of dosage forms such as tablets, powders, lozenges, syrups, injectable solutions and the like. These pharmaceutical compositions can, if desired, contain additional ingredients such as flavorings, binders, excipients and the like. Thus, for purposes of oral administration, tablets containing various excipients such as sodium citrate, calcium carbonate and/or calcium phosphate may be employed along with various disintegrants such as starch, alginic acid and/or certain complex silicates, together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and/or acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often useful for tabletting purposes. Solid compositions of a similar type may also be employed as fillers in soft and hard filled gelatin capsules. Preferred materials for this include lactose or milk sugar and high molecular weight polyethylene glycols. When aqueous suspensions or elixirs are desired for oral administration, the active pharmaceutical agent therein may be combined with various sweetening or flavoring agents, coloring matter or dyes and, if desired, emulsifying or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin and/or combinations thereof.

For parenteral administration, solutions of the compounds of this invention in sesame or peanut oil, aqueous propylene glycol, or in sterile aqueous solutions may be employed. Such aqueous solutions should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration, m this connection, the sterile aqueous media employed are all readily available by standard techniques known to those skilled in the art.

Generally, a composition of this invention is administered orally, or parenterally

(e.g., intravenous, intramuscular, subcutaneous or intramedullary). Topical administration may also be indicated, for example, where the patient is suffering from gastrointestinal disorders or whenever the medication is best applied to the surface of a tissue or organ as determined by the attending physician.

Buccal administration of a composition of this invention may take the form of tablets or lozenges formulated in a conventional manner.

For intranasal administration or administration by inhalation, the compounds of the invention are conveniently delivered in the form of a solution or suspension from a pump spray container that is squeezed or pumped by the patient or as an aerosol spray presentation from a pressurized container or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurized container or nebulizer may contain a solution or suspension of a compound of this invention. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of a compound or compounds of the invention and a suitable powder base such as lactose or starch. For purposes of transdermal (e.g., topical) administration, dilute sterile, aqueous or partially aqueous solutions (usually in about 0.1% to 5% concentration), otherwise similar to the above parenteral solutions, are prepared.

Methods of preparing various pharmaceutical compositions with a certain amount of active ingredient are known to those skilled in this art. For examples of methods of preparing pharmaceutical compositions, see Reminciton's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 19th Edition (1995).

Example 1: Assessment of Regulatory T cell numbers and function in ACS patients .

Methods

Patients

Institutional ethics committee approved the study and informed consent was obtained from all patients. Three groups of subjects were selected: group 1- ACS patients admitted to the intensive coronary care unit (n= 32), group 2- Patients with stable angina pectoris and atherosclerosis documented by angiography (n= 28), and group 3- Subjects with normal coronary arteries (NCA) on angiography (n= 28) (Table I).

Table I: Characteristics of the study patients

Acute Stable Healthy

Coronary Angina subjects

Syndrome (n=28) (n=28)

(n=32)

Males 16 (50) 16 (50) 15 (54)

CAD extent (n x vessels) 2±0.7 1.8±0.9 0

Risk factors

Hypertension, n (%) 16 (50) 12(43) 5 (18)

Diabetes, n (%) 7 (22) 8 (29) 2 (7)

Current smoker, n (%) 8 (25) 8 (29) 2 (7)

Past smoker, n (%) 2 (6) 2 (7) 2 (7)

Hyperlipidemia, n (%) 18 (56) 17 (61) 5 (18)

Medications

Beta-blockers, n (%) 10 (31) 12 (43) 5 (18)

ACEI, n (%) 16 (50) 15(53) 3 (11)

Aspirin, n (%) 18 (56) 17 (61) 3 (11)

Statins, n (%) 18 (56) 12 (43) 3 (11)

Calcium blocker, n (%) 2 (6) 2 (7) 0 (0)

Nitrates, n (%) 5 (16) 3 (11) 0 (0)

Diuretics, n (%) 0 (0) 3 (11) 0(0)

Data is presented as mean ± SD.

CAD- coronary artery disease; MI- myocardial infarction; CABG coronary artery bypass grafting; PTCA percutaneous transluminal coronary angioplasty; CVA cerebrovascular accident ACEI angiotensin converting enzyme inhibitors; ARB angiotensin receptor blocker.

ACS was defined as chest pain accompanied in all patients by definite ischemic electrocardiography changes (ST segment changes and/or T wave inversions). Myocardial infarction was diagnosed if there was also elevation of troponin I (>0.1U) or CPK MB (>) or definite (>2mm) ST segment elevations in at least 2 consecutive leads. Patients with stable angina were recruited from the outpatient clinic and a recent angiography exhibited coronary affliction of a similar extent to that found in ACS patients. Healthy subjects were selected on a basis of a recent angiography showing normal coronary arteries (NCA). Ten of the patients with ACS and elevation of cardiac troponin were followed up after 2 months with a repeat evaluation of their Tregs.

Cell separation and flow cytometry

Peripheral blood mononuclear cells (PBMC) were prepared by Ficoll density gradient and stained with combinations of the following monoclonal antibodies: fluorescein (FITC)-labeled anti-CD4- (RPA-T4), phycoerythrin (PE)-labeled anti-CD25 (BC96), FITC-labeled Mouse IgGl K (MOPC-21/P3), or PE-labeled mouse IgGl K isotypic control (P3) from eBioscience. Stained cells were analyzed on a FACSCalibur (Becton Dickinson, San Jose, CA). CD4 ⁺ CD25 ⁺ T cells were isolated from PBMCs by a first step of negative sorting using a cocktail of hapten-conjugated CD8, CDlIb, CD16, CD19, CD36, and CD56

antibodies and microbeads coupled to an antihapten monoclonal antibody (CD4 ⁺ T-cell isolation kit; Miltenyi Biotec, Bergisch Gladbach, Germany). This was followed by a step of positive selection of CD25 ⁺ cells by microbead separation (CD25 microbeads; Miltenyi Biotech), a procedure yielding > 95% purity as assessed by flow cytometric counting of CD4 ⁺ CD25 ⁺ cells.

Functional suppression assays

Costar 96-well plates (Corning, NY) were incubated with 1 μg/mL anti-CD3 monoclonal antibody (UCHTl from R&D systems) overnight at 4°C, and washed. Then, CD4 ⁺ CD25 ^~ (responders) and CD4 ⁺ CD25 ⁺ (Tregs) T cells (10 ⁴ cells/well) were cultured in RPMI medium supplemented with 10% fetal calf serum in different responder/suppressor ratios (1:1, 1: 2, 1: 4 and 1:8). All cells were cultured in a final volume of 200 μl in the presence of 10 ⁵ T cell-depleted and irradiated accessory cells/well. After 72 hours, ³ H~thymidine (1 μCi/well) was added for 16 hours before proliferation was assayed by scintillation counting (β counter). Percent inhibition of proliferation was determined as follows: 1 - (median ³ H-thymidine uptake of 1:1 CD4 ⁺ CD25 ⁺ :CD4 ⁺ CD25- coculture/median ³ H-thymidine uptake of CD4 ⁺ CD25 ⁺ cells). The coculture/proliferation assay for assessment of the functional suppressive properties of Tregs was repeated in the presence or absence of oxidized LDL (1 mcg/ml).

Foxp3 and CTLA-4 expression determined by reverse transcription-polymerase chain reaction (RT-PCR).

RNA was extracted from 10 ⁶ T cells using an RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The amount and purity of the obtained RNA was determined by measurements of optical density at 260 nm and 280 nm. RT-PCR was performed according to the protocol of Reverse-iT 1st. Strand synthesis kit (ABgene, UK). The integrity of the RNA, the efficiency of RT reaction and the quality of cDNA subjected to the RT-PCR was controlled by amplification of transcript of Glucose-3-Phosphate Dehydrogenase (GAPDH). GAPDH was analyzed using the following primers: GAPDH forward 5'-ACCACAGTCCATGCCATCAC -3'

(SEQ ID NO: 1) and GAPDH reverse 5'-TCCACCACCCTGTTGCTGTA -3' (SEQ ID NO: 2).

PCR was carried out with Readymix PCR mastermix (ABgene, UKon PTC (programmable thermal controller) device (MJ Research Inc) at gene specific conditions. Primer sequences for Foxp3 were: Foxp3 forward: 5 ^I -CACTTGCAGACACCATTTGC-3 ^I (SEQ ID NO: 3) and Foxp3 reverse: 5'-CTCTTCTTCCTTGAACCCCA-S' (SEQ ID NO: 4) and for cytotoxic T-lymphocyte antigen-4 (CTL A-4) forward 5'- GCCTATGCCCAGGTAGTATG-3' (SEQ ID NO: 5) and CTLA-4 reverse 5'- CTGTCTTCTGCAAAGCAATG-3' (SEQ ID NO: 6). The PCR products were subjected to electrophoresis on 1.5% agarose gel stained with ethidium bromide. The optical density (OD) of the amplified PCR product was measured by densitometry and was analyzed using "TINA" software. Semiquantitative analyses presented comparison of OD of FoxP3 and CTLA-4 PCR products normalized to OD of co-amplified GAPDH-PCR product.

Western blot analysis of foxp3 protein content in Tregs from patients with ACS, stable angina and healthy subjects.

Purified Tregs from all patients were lysed, and protein concentration in lysates was determined using BCA protein kit (PierceUSA). Cell lysates were resolved on 8% SDS-PAGE and transferred onto a nitrocellulose membrane. Western blot was performed using a rat serum anti-foxP3 (eBioscience, USA) at a dilution of 1:1,000 and a secondary antibody-peroxidase-conjugated AffmiPure donkey anti rat IgG (H+L) (Jackson Laboratories) for detection with chemiluminescent substrate (Santa-Cruz, USA). Comparative analysis was performed by quantitative densitometry.

Statistical Analysis

Comparison between the 3 patient groups was carried out employing the one-way ANOVA test. Statistical significance was set at p<0.05. Results are reported as mean ± SEM unless otherwise specified.

Results

OxLDL influences Tregs number and function.

OxLDL is considered an instrumental factor that promotes atherosclerosis initiation, progression and possibly, plaque destabilization (6). We studied the effects of 1 mcg/ml oxLDL (a concentration that is in the range, reported to be present in human plasma) on relative CD4 ⁺ CD25 ⁺ Tregs and CD4 ⁺ CD25 ^" cell numbers after in vitro incubation. PBMC were incubated with oxLDL (lmcg/ml) for 48 hrs. CD4+CD25+ Tregs and T-responder numbers were studied by FACS with the labeled antibodies (see methods). Tregs were significantly more sensitive to oxLDL as their relative number was reduced by 40 ±8% in comparison to a negligible effect on CD4 ⁺ CD25 ^" cells (15 + 5%) (Figures: IA, IB). When the assay was repeated in the presence, or in the absence of the caspase inhibitors DEVD-CMK (Calbiochem, La Jolla, CA ₅ USA) or NAC (Sigma, St. Louis, MO, USA), we have found that the effect of oxLDL on reduction of Tregs was attenuated producing a 17±6% or 20±6% reduction in regulatory and effector T cells, respectively suggesting that apoptosis was responsible for the effect.

We than investigated whether Tregs from healthy subjects are compromised in their suppressive properties when exposed to oxLDL. Tregs were incubated with OxLDL (1 mcg/ml) in the presence of irradiated antigen presenting cells and responder T cells activated by plate bound anti-CD3 (as described in methods). Proliferation was evaluated by thymidine incorporation. Indeed, incubation of Tregs with 1 mcg/ml oxLDL, resulted in a significant attenuation of their ability to suppress CD4 ⁺ CD25 ^' proliferation at all Treg-T-responder ratios (Fig. 1C).

Circulating CD4^CD25* T cell numbers are reduced inpatients with ACS. Freshly drawn fϊcoll-eluted blood mononuclear cells from patients with ACS, stable angina pectoris and healthy subjects were stained with different combinations of labeled anti-CD4-FITC and anti-CD25-PE antibodies as described in methods (Fig. 2A and B). The cells were gated on lymphocytes via their forward and side scatter features. We have found that in patients with ACS, the number of CD4 ⁺ CD25 ⁺ Tregs was significantly reduced (1.9%±0.1) as compared to patients with stable angina with a similar extent of coronary affliction (3.8%±0.6; P<0.001) and healthy individuals with

NCA (4.4±0.7; P<0.001). Treg numbers were not significantly different in patients with stable atherosclerotic disease and healthy subjects with NCA. This favors the conclusion that the dysregulation in the Tregs' population in associated with events culminating in plaque destabilization rather than with stable atherosclerosis per se.

CD4 ⁺ CD25 ⁺ Regulatory T Cells are functionally compromised in Patients with ACS

As the functional suppressive properties of Tregs may be as important as their numbers, we isolated highly pure CD4 ⁺ CD25 ⁺ regulatory and CD4 ⁺ CD25 ^~ responder cell populations by magnetic bead sorting (> 95% purity). Purified Tregs from patients with ACS, stable angina pectoris and healthy subjects were incubated in the presence of irradiated antigen presenting cells with anti-CD3 activated responder T cells in different Treg/T responder ratios for three days as described in methods. Assay was performed for each of the patients in all three groups. CD4 ⁺ CD25 ^~ responder cells from patients with ACS, stable angina and healthy individuals exhibited similar proliferation, evident by thymidine incorporation to plate-bound anti-CD3 antibodies (Fig. 3A). CD4 CD25 T cells isolated from all groups were anergic to stimulation at all doses by plate-bound anti- CD3 and did not differ (1889±378 cpm-NCA, 2394±306cpm-stable angina and 1703±215 cpm in ACS patients)

Quantitative analysis of the regulatory function of CD4 ⁺ CD25 ⁺ Tregs was performed by co-culturing them with autologous T-responder cells (10 ⁴ cells/ well) at different ratios (responder/suppressor ratios: 1:1, 1:2, 1:4, and 1:8). The assay was repeated for all subjects. Similar to other investigators we found that in healthy individuals, CD4 ⁺ CD25 ⁺ T cells suppressed responder T-cell proliferation at a 1:1 ratio and the effect was diluted by reducing the relative numbers of Tregs. Tregs isolated from the circulation of patients with ACS exhibited hampered inhibition of responder CD4 ⁺ CD25 ^~ T cell proliferation when compared with Tregs from patients with stable angina pectoris or healthy individuals (Fig. 3). For example, at a 1:8 Tregs-T-responder ratio, mean inhibition of proliferation of Tregs from ACS patients was 11.8%±7.3% as compared with 29.6%±9.2% achieved for stable angina pectoris and 58%±2.6% for healthy individuals (Fig. 3). Interestingly, the functional suppressive

properties were significantly compromised in patients with stable atherosclerotic disease as compared with healthy subjects (Fig. 3).

OxLDL induces a differential effect on Treg numbers in patients with ACS, stable angina and healthy subjects

We then investigated the hypothesis that differential sensitivity to oxLDL mediated Treg depletion exists between patients with ACS, stable angina and healthy individuals. We have found that CD4 ⁺ CD25 ⁺ T cells from patients with ACS were significantly more sensitive to oxLDL-mediated depletion (a mean of 31% depletion) as compared with Tregs from patients with stable angina (a mean of 16.2%) and healthy subjects (a mean of 18.7%; P<0.01) (Fig. 4). A co-culture assay was performed as described in the "methods" section above in the presence of lmcg/ml oxLDL.

The numbers of Treg cells were evaluated in ten of the patients with ACS 2 months after discharge. We have found a significant increase in the number of circulating Tregs from 2.1±0.5 to 3.4±0.5 (p<0.05) in the 10 studied patients.

Expression offoxp3 and CTLλ-4 in Tregs from patients with ACS

Foxp3 is a master transcriptional regulator of naturally occurring CD4 ⁺ CD25 ⁺ Tregs. We thus set out to investigate whether compromised suppressive function of Tregs observed in ACS patients was associated with down regulated foxp3 message. Total RNA was isolated from Purified Tregs of patients with ACS, stable angina pectoris and healthy subjects. The RNA was reverse-transcribed (as described in Methods) and assayed for foxp3 and CTLA-4 mRNAs expression levels (Fig. 5A). We have found that foxp3 expression was significantly reduced in purified Tregs from patients with ACS as compared with healthy subjects (a mean of 68% reduction; p<0.05) and with patients having stable angina (a mean of 56% decrease; p<0.05). We also assayed expression of

CTLA-4, an additional potential phenotypic marker of CD4 ⁺ CD25 ⁺ . We have found that the expression of CTLA-4 was also significantly reduced in Tregs from ACS patients as compared to both healthy subjects and patients with stable angina (a 64% reduction and a

39% reduction, respectively, p<0.05).

Western blot analysis of foxp3 protein content in Tregs from the three groups disclosed a significant reduction in Tregs from ACS patients (-49% expression compared with Tregs from NCA patients, p<0.05) and from stable angina patients (-40% expression compared with Tregs from NCA patients; P<0.05) (Fig. 5B and 5C).

Example 2: Assessment of the effect of statins on Regulatory T cell numbers andfunction

Materials and Methods

Study Population For in vitro experiments, peripheral blood mononuclear cells (PBMCs) were isolated from 5 healthy donors at the age range: 27-41 yrs. For ex-vivo experiments PBMCs were isolated from subjects with hypercholesterolemia, starting a treatment with either 20mg simvastatin (n=7), or 10-40 mg pravastatin (n=5) from (Teva Pharmaceutical Industries Ltd. Israel). AU experiments were approved by the institutional ethics committee and informed consent was obtained from all patients.

Cell Culture

PBMCs were prepared by Ficoll-Paque density gradient (Lymphoprep™, Nycomed

Pharma AS, Oslo, Norway). PBMCs were cultured (1.5 x 10 ⁶ /ml) at 37 ⁰ C in an atmosphere of 5% CO ₂ in RPMI 1640 medium (Gibco-BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco-BRL), 1% penicillin-streptomycin and 1% glutamine

(Biological Industries, Kibbutz Beit Haemek, Israel).

Mevastatin (0.5 and I /M), Pravastatin (20, 50, 100, 250 and 500 μM) (Sigma-

Aldrich Inc. St. Louis, MO, USA) and Atorvastatin (Pfizer Inc. prescription formulation) (2, 5, 10, 25 and 50//M) were added separately to cultured PBMCs, and were incubated for 96 hr. These concentrations were selected since they have been previously shown to promote immunomodulatory effects of statins (17-19). PBMCs cultured with media only were used as controls.

Flow Cytometry

Human PBMCs were stained with FITC-labeled anti-CD4 (L3T4) and PE-labeled anti-CD25 (BC96). FITC-labeled Mouse IgGl K (MOPC-21/P3) and PE-labeled mouse IgGl K (P3) from eBioscience were used as isotypic controls. CD4 ⁺ CD25 ⁺ Foxp3 ⁺ cells were detected by Foxp3 intracellular staining, using Human

Regulatory T Cell Staining Kit (eBioscience, USA) according to manufacturer's protocol. Briefly, cells were counterstained with FITC-labeled anti-C25 (BC96) and APC-labeled anti-CD4 (RPA-T4). After incubation of 30 min, cells were incubated with a fixation solution, washed, and resuspended in a permeabilization solution. Fixated and permeabilized cells were stained with PE-labeled anti Foxp3 (PCHlOl). FITC-labeled Mouse IgGl K (MOPC-21/P3), PE-labeled rat IgG2a (eBR2a) and APC labeled mouse IgGl K (P3) were used as isotypic controls (eBioscience, USA.). Stained cells were analyzed on a FACScac flow cytometer, using CellQuest software (Becton Dickinson).

Western blot analysis of Foxp3 protein content

Cultured or fresh PBMCs were lysed, and protein concentration in lysates was determined using BCA protein kit (Pierce, USA). Cell lysates were resolved on 8% SDS- PAGE and transferred onto a nitrocellulose membrane. Western blot was performed using a polyclonal rat serum anti-FoxP3 (eBioscience, USA) at a dilution of 1:1,000 and a secondary antibody-peroxidase-conjugated AffiniPure donkey anti rat IgG (H+L) (Jackson Laboratories) for detection with chemiluminescent substrate (Santa-Cruz, USA). Comparative analysis was performed by quantitative densitometry.

Cell separation CD4 ⁺ CD25 ⁺ T cells were isolated from PBMCs by a first step of negative sorting using a cocktail of hapten-conjugated CD8, CDlIb, CD 16, CD 19, CD36, and CD56 antibodies and microbeads coupled to an anti-hapten monoclonal antibody (CD4 ⁺ T-cell isolation kit; Milteny Biotech, Bergisch Gladbach, Germany). This was followed by a step of positive selection of CD25 ⁺ cells by microbead separation (CD25 microbeads; Miltenyi Biotech), a procedure yielding 92-98% purity as assessed by flow cytometric counting of CD4 ⁺ CD25 ⁺ cells.

Functional suppression assays

Costar 96-well plates (Corning, NY) were incubated with 5 μg/ml anti-CD3 monoclonal antibody (UCHTl from R&D systems) overnight at 4°C ₅ and washed. Then, CD4 ⁺ CD25 ^~ (responder T cells) and CD4 ⁺ CD25 ⁺ (Tregs) were cultured (2*10 ⁴ cells/well) in RPMI medium supplemented with 10% fetal calf serum in different responder/suppressor ratios (1:1, 1:1/2 and 1:1/4). All cells were cultured in a final volume of 200 μl in the presence of 10 ⁵ mitomycin- C treated CD4 ^" cells/well (40 min of incubation, 50μg/ml) (Sigma-Aldrich Inc. USA), serving as antigen presenting cells (APCs). After 72 hours, ³ H- thymidine (1 μCi/well) was added for 16 hours before proliferation was assayed by scintillation counting (β counter). Percent inhibition of proliferation was determined as follows: 1 - (median ³ H-thymidine uptake of CD4 ⁺ CD25 ⁺ :CD4 ⁺ CD25 ^" co-culture / median ³ H-thymidine uptake of CD4 ⁺ CD25 ^" cells). The suppression was repeated in the presence and absence of pravastatin (20 and 100 μM) and atorvastatin (2 and 10 μM).

Statistical Analysis

Data are presented as mean±SEM. Significance between each two groups was examined by a one way Anova test. P value < 0.05 was considered significant.

Results

The effect of mevastatin, pravastatin and atorvastatin on the number of human CD4+CD25+ Tregs, in vitro

Human peripheral blood mononuclear cells (PBMC) were prepared by Ficoll density gradient. The cells were plated at a density of 1.5 X 10 ⁶ cells/ml and co-cultured with either mevastatin (0.5 and 1 μM), pravastatin (20, 50, 100 and 250 μM) or atorvastatin (2, 5, 10 and25/^M) for 96 hours. Cultures were incubated in 5% CO2 at 37°C. After 96 hours, cells were harvested and stained with combinations of the following monoclonal antibodies: fluorescein (FITC)-labeled anti-CD4, phycoerythrin (PE)-labeled anti-CD25. FITC-labeled Mouse IgGl or PE-labeled mouse IgGl were used as isotypic control (eBioscience, USA). Stained cells were analyzed on a FACScac flow cytometer, using CellQuest software (Becton Dickinson).

As shown in Fig. 6 A and 6B, mevastatin did not significantly alter the percentage of CD4+CD25 ^high of total CD4+CD25+ (17.8 ±2.1% in the presence of 1 μM mevastatin versus 17.1 ±2.3% in control). Pravastatin increased the percentage of CD4+CD25 ^hlgh cells but this effect was found to be non significant (19 ±3.4% in the presence of 100 μM pravastatin versus 17.1 +2.3% in control). However, atorvastatin significantly increased the percentage of CD4+CD25 ^high of total CD4+CD25+ cells (27.6±3.4% and 28.2± 5.4% in the presence of 5 μM and 10 μM atorvastatin, respectively). Higher concentrations of atorvastatin did not result in additional elevation.

The effect ofatorvastatin on the level ofFoxp3 expression on human PBMC, in vitro

The effect of atorvastatin on the level of Foxp3 expression was measured by Western blot and FACS analysis. PBMC were co-cultured with atorvastatin, harvested and analyzed for CD4, CD25 and Foxp3 expression.

For Western blot analysis, samples were lysed, and protein concentration in lysates was determined using BCA protein kit (PierceUSA). Cell lysates were resolved on 8% SDS- PAGE and transferred onto a nitrocellulose membrane. Western blot was performed using a rat serum anti-Foxp3 (eBioscience, USA) at a dilution of 1:1,000 and a secondary antibody- peroxidase-conjugated AffiniPure donkey anti rat IgG (Jackson Laboratories) for detection with chemiluminescent substrate (Santa-Cruz, USA). Comparative analysis was performed by quantitative densitometry.

Intracellular staining of Foxp3 for FACS analysis was performed on cultured PBMC with PE Anti-Human Foxp3 Staining Set (eBioscience, USA) according to manufacturer's protocol. Briefly, cells were incubated with a fixation solution, washed, and resuspended in permeabilization solution. Before fixation, the cells were counterstained with fluorescein (FITC)-labeled anti-CD4 and allophycocyanin (APC)-labeled anti-CD25. FITC- labeled Mouse IgGl, PE-labeled mouse IgGl, or APC labeled mouse IgGl were used as isotypic controls (eBioscience, USA.).

The observed effect of atorvastatin correlated closely with the results of Foxp3 expression by FACS (Fig. 6C and 6D). Indeed, 10 μM atorvastatin increased the number of CD4+CD25+Foxp3+ cells of total CD4+CD25+ by 48.7 ± 22.2% relative to control, and

again, this effect was not evident with the other statins. Similar results were obtained when Foxp3 expression was examined by Western blotting (Fig. 6E). Ten μM atorvastatin led to an increase of 66.8 ± 4.5% in Foxp3 expression. Kinetic analysis demonstrated that the most pronounced effects of atorvastatin on the number of CD4 ⁺ CD25 ^hlgh , as well as on the level of Foxp3 expression were evident after 96 hr of incubation, in comparison to a 48hr treatment (Fig 6F-6J).

Atorvastatin promotes the conversion of peripheral human CD4 ⁺ CD25 ^~ Foxp3 ^~ T cells to CD4 ⁺ CD2S ^¥ Foxp3 ⁺ Treg cells The following experiment was performed in an attempt to examined whether the source of the "newly formed" regulatory T cells, induced in vitro by atorvastatin, is the CD4 ⁺ CD25 ^~ Foxp3 ^' T cells subset, in which Foxp3 and CD25 expression is upregulated.

Human CD4 ⁺ CD25 ^" T cells were purified from healthy individuals by magnetic bead separation and exposed to 2 μM and 10 μM atorvastatin, in the presence of anti-CD3 mAb, for responders stimulation, and mitomycin-C treated CD4 depleted cells, serving as APCs. Previous studies have shown that in humans, during anti-CD3 mediated activation of CD4 ⁺ CD25 ^~ T cells, two populations of cells may arise, effector CD4 ⁺ CD25 ⁴ Foxp3 ^" and CD4 ⁺ CD25 ⁺ Foxp3 ⁺ with regulatory activity (15). Indeed, stimulation of CD4 ⁺ CD25 ^" cells with anti-CD3 alone for 96 hr led to the generation of a new CD4 ⁺ CD25 ⁺ T cells subset which constituted 36.2 ±0.7% of the total number of cells. 16.1 + 1.3% of these newly generated CD4 ⁺ CD25 ⁺ cells expressed Foxp3, and the rest did not express foxp3 signifying activated T responder cells (Fig 7 A and 7B).

Addition of 2 μM and 10 μM atorvastatin to the culture led to a significant decrease in the number of these anti-CD3 induced CD4 ⁺ CD25 ⁺ cells, in a dose dependent manner (Fig 7C). Despite the decrease in the total CD4 ⁺ CD25 ⁺ cells number, the percentage of foxp3 expressing cells of those CD4 CD25 ⁺ remaining cells, increased in the presence of atorvastatin, and 10 μM atorvastatin led to the appearance of 22.1 ± 1.3% CD4 ⁺ CD25 ⁺ Foxp3 ⁺ cells (Fig. 7B, TD or 7E). These findings indicate that atorvastatin promoted the conversion of CD4 ⁺ CD25 ^" Foxp3 ^" cells to CD4 ⁺ CD25 ⁺ Foxp3 ⁺ regulatory T cells, accompanied by the inhibition of the anti-CD3 mediated T cells activation.

The possibility that the source of these induced by atorvastatin

CD4 ⁺ CD25 ⁺ Foxp3 ⁺ cells were rare CD4 ⁺ CD25 ^' Foxp3 ⁺ that were activated by atorvastatin and as a consequence regained CD25 expression is ruled out, since only 1.6% of the purified CD4 ⁺ CD25 ^" cells expressed Foxp3 and this population remained stable in the presence of anti-CD3 mAb and atorvastatin (data not shown).

Atorvastatin upregulates the regulatory function ofTregs in humans in vitro

A thymidine incorporation assay was conducted in order to determine whether the newly generated statin inducible Treg population possesses improved functional suppressible properties.

A quantitative analysis of the regulatory function of CD4 ⁺ CD25 ⁺ Tregs was performed by co-culturing them with autologous T-responder cells (2*10 ⁴ cells/well) at different ratios (Treg/responder ratios: 1:1, 1: 2 and 1: 4), in the presence of APCs (10 ⁵ cells/well) and plate-bound anti-CD3 mAb. As presented in Fig.8 A, addition of 20 μM and 100 μM pravastatin to the co- cultured Tregs and T-responder cells at a 1:1 ratio led to a non significant increase of 7.8 ± 2% and 11.5+ 4.6% in the inhibition rate, correspondingly. This observation supports our previous findings that the effect of pravastatin on the Tregs pool is minor.

Atorvastatin, however, increased the extent of thymidin uptake inhibition in a significant dose-dependent manner (30. l± 5.4% and 49.7± 0.3% in the presence of 2 μM and 10 μM atorvastatin, respectively, at a 1:1 ratio), and this effect repeated itself in all Treg/responder ratios (Fig. 8B).

Treatment with pravastatin and simvastatin increases the number and functional properties of CD4+CD25+ ^high cells, in h umans

The effect of statins on the number of Tregs in humans was examined ex-vivo by comparing the number of CD4+CD25 ^hlgh and Foxp3 expression before (time 0) and after oral treatment with statins for a period of 4/8 weeks. Since there is a considerable variability in the number of Tregs between individuals (16), baseline levels in a given individual represented the referenced value. Thus, we evaluated subjects with hypercholesterolemia, initiating a treatment with either pravastatin or simvastatin. These

subjects appear to have mean levels of Tregs that are similar to non hyperlipidemic subjects (data not shown). As shown in Fig. 9A and 9B, 8 weeks of treatment with pravastatin led to a median 3.7-fold increase in the percentage of CD4 ⁺ CD25 ^hlgh of total CD4 ⁺ CD25 ⁺ relative to baseline values. Similar effects were evident in patients treated with simvastatin, which led to a median 2.4-fold increase of circulating CD4 ⁺ CD25 ^hlgh cells after 4 weeks.

Western blot analysis of Foxp3 expression revealed a similar trend: pravastatin and simvastatin upregulated Foxp3 expression, an effect that was more pronounced 8 weeks treatment of pravastatin (Fig. 9C)

REFERENCES

1. Sakaguchi S. Naturally arising CD4 ⁺ regulatory T cells for immunologic self- tolerance and negative control of immune responses. Annu Rev Immunol 2004; 22: 531

5 62.

2. Jonuleit H, Schmitt E, Stassen M, Tuettenberg A, Knop J, Enk AH. Identification and functional characterization of human CD4 ⁺ CD25 ⁺ T cells with regulatory properties isolated from peripheral blood. J Exp Med 2001; 193: 1285 94.

3. George J, Yacov N ₅ Breitbart E, Bangio L, Shaish A, Gilburd B, Shoenfeld Y, Harats 10 D. Suppression of early atherosclerosis in LDL-receptor deficient mice by oral tolerance with beta 2-glycoprotein I. Cardiovasc Res 2004; 62: 603-9.

4. George J, Harats D, Gilburd B, Afek A, Shaish A, Levy Y, Kopolovic J, Shoenfeld Y. Adoptive transfer of β2-glycoprotein I reactive lymphocytes promotes early atherosclerosis in LDL-receptor deficient mice. Circulation 2000; 102: 1822-7.

155. Heeschen C ₅ Dimmeler S, Hamm CW, Fichtlscherer S, Boersma E, Simoons ML, Zeiher AM; CAPTURE Study Investigators. Serum level of the antiinflammatory cytokine interleukin-10 is an important prognostic determinant in patients with acute coronary syndromes. Circulation. 2003; 107: 2109-14.

6. Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J 20 Med. 2005; 352: 1685-95.

7. Ait-Oufella H, Salomon BL, Potteaux S, Robertson AK, Gourdy P, ZoIl J, Merval R, Esposito B, Cohen JL, Fisson S, Flavell RA, Hansson GK, Klatzmann D, Tedgui A, Mallat Z. Natural regulatory T cells control the development of atherosclerosis in mice. Nat Med. 2006; 12: 178-80.

258. Mor A, Planer D, Luboshits G ₅ Afek A., Metzger S, Chajek-Shaul T, Keren G,

George J. The role of naturally occurring CD4+CD25+ regulatory T cells in experimental atherosclerosis. Arterioscler Thromb Vase Biol. In press. 9. O'Garra A, Vieira P. Regulatory T cells and mechanisms of immune system control.

Nat Med. 2004; 10: 801-5. 3010. Crispin JC ₅ Martinez A, Alcocer-Varela J. Quantification of regulatory T cells in patients with systemic lupus erythematosus. J Autoimmun 2003; 21 : 273 6.

11. Kukreja A ₅ Cost G, Marker J ₅ Zhang C ₅ Sun Z ₅ Lin-Su K, Ten S ₅ Sanz M ₅ Exley M, Wilson B ₅ Porcelli T ₅ Maclaren N. Multiple immuno-regulatory defects in type-1 diabetes. J Clin Invest 2002; 109: 131 40.

12. Ehrenstein MR ₅ Evans JG, Singh A ₅ Moore S ₅ Warnes G, Isenberg DA ₅ Mauri C. 5 Compromised function of regulatory T cells in rheumatoid arthritis and reversal by anti-

TNFalpha therapy. J Exp Med. 2004; 200: 277-85.

13. Viglietta V ₅ Baecher- Allan C ₅ Weiner HL ₅ Hafler DA. Loss of functional suppression by CD4+CD25+ regulatory T cells in patients with multiple sclerosis. J Exp Med. 2004; 971: 9-19.

1014. Greenwood J ₅ Steinman L, Zamvil SS. Statin therapy and autoimmune disease: from protein prenylation to immunomodulation. 2006; 6: 358-70. 15. Walker MR ₅ Kasprowicz DJ, Gersuk VH, Benard A, Van Landeghen M, Buckner JH et al. Induction of FoxP3 and acquisition of T regulatory activity by stimulated human

CD4+CD25- T cells. JCHn Invest 2003; 112: 1437-1443. 1516. Gregg R ₅ Smith CM, Clark FJ, Dunnion D ₅ Khan N, Chakraverty R et al. The number of human peripheral blood CD4+CD25high regulatory T cells increases with age. CHn

Exp Immunol 2005; 140: 540-546.

17. Weitz-Schmidt G, Welzenbach K ₅ Brinkmann V ₅ Kamata T, Kallen J ₅ Bruns C et al. Statins selectively inhibit leukocyte function antigen- 1 by binding to a novel regulatory

20 integrin site. Nat Med 2001 ; 7: 687-692.

18. Rasmussen LM, Hansen PR, Nabipour MT, Olesen P, Kristiansen MT, Ledet T. Diverse effects of inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase on the expression of VCAM-I and E-selectin in endothelial cells. Biochem J 2001; 360: 363- 370.

2519. Grip O, Janciauskiene S, Lindgren S. Pravastatin down-regulates inflammatory mediators in human monocytes in vitro. Eur J Pharmacol 2000; 410: 83-92.