Phosphate and tensin homolog (PTEN) for the detection of autoimmune diseases or conditions

Abstract

A method for the detection of impaired responsiveness of CD4+ T-cells to regulatory T-cells (Treg), referred to as Treg resistance. The method includes measuring the expression levels of phosphatase and tension homolog (PTEN) in activated CD4+ T-cells. Furthermore, a screening method for the detection of an autoimmune disease or a condition, may comprise the steps of generating a functional gene expression profile by measuring the expression levels of phosphatase and tension homolog (PTEN) in Treg-resistant CD4+ T-cells from patients suffering of an autoimmune disease or condition, and comparing the obtained gene expression profile with the expression profile from Treg-sensitive CD4+ T-cells from healthy controls. PTEN can be utilized in a screening system for the detection of impaired responsiveness of CD4+ T-cells to Treg.

Claims

1. A method for the detection of impaired responsiveness of CD4+ T-cells to regulatory T-cells (Treg), referred to as Treg resistance, by measuring the expression levels of phosphatase and tensin homolog (PTEN) in activated CD4+ T-cells.

2. The method according to claim 1, wherein the expression levels of PTEN are compared between activated CD4+ T-cells from patients and activated Treg-sensitive CD4+ and CD8+ T-cells from healthy donors, wherein a downregulation of PTEN within activated CD4+ T-cells as compared to the activated Treg-sensitive CD4+ T-cells is indicative for Treg resistance.

3. The method according to claim 1, wherein an upregulation of PTEN is correlated with a responsiveness of activated CD4+ T-cells to Treg-mediated suppression.

4. The method according to claim 1, wherein the Treg resistance correlates with an accelerated IL-6 production after T cell receptor (TCR) stimulation, enhanced phosphorylation of PKB/c-Akt and/or an increased IL-6 receptor (IL-6R) expression.

5. The method according to claim 1, wherein impaired responsiveness of CD4+ T-cells is restored by normalizing PTEN expression in activated Treg-resistant T-cells with IFN- or dimethyl fumarate (DMF).

6. The method according to claim 1, wherein in addition to the determination of the expression levels of PTEN, the expression levels of IL-6 are determined, wherein an increase of IL-6 expression correlates with PTEN down-regulation.

7. A method for the detection of an autoimmune disease or a condition, comprising the steps of generating a functional gene expression profile by measuring the expression levels of phosphatase and tensin homolog (PTEN) in activated CD4+ T-cells from samples of patients suffering of an autoimmune disease or condition, and comparing the obtained gene expression profile with the expression profile from Treg-sensitive CD4+ T-cells from healthy controls.

8. The method according to claim 7, wherein the generation of a functional gene expression profile comprises the steps of: a. extraction and purification of RNA from T-cells, b. synthesis of cDNA from the RNA templates, c. labelling of cDNA with a marker molecule and hybridization to a gene chip, d. analysis of the gene chips by detecting the intensity of the marker molecule.

9. The method according to claim 7, wherein impaired responsiveness of CD4+ and/or CD8+ T-cells to regulatory T-cells (Treg) is detected by a method wherein the expression levels of PTEN are compared between activated CD4+ T-cells from patients and activated Treg-sensitive CD4+ and CD8+ T-cells from healthy donors, wherein a downregulation of PTEN within activated CD4+ T-cells as compared to the activated Treg-sensitive CD4+ T-cells is indicative for Treg resistance.

10. The method according to claim 7, wherein the autoimmune disease or condition is rheumatoid arthritis, rheumatic fever, systemic lupus erythematosus (SLE), ulcerative colitis, Crohn's disease, autoimmune inflammatory bowel disease, diabetes type I, multiple sclerosis (MS), myasthenia gravis, psoriasis, pemphigus vulgaris, pemphigoid.

11. The method according to claim 7, wherein agonists are identified that up-regulate PTEN expression in activated T-cells isolated from autoimmune patients.

12. The method according to claim 10, wherein therapeutic treatment process of the autoimmune disorder or condition is monitored.

13. The method according to claim 7, wherein in addition to the determination of the expression levels of PTEN, the expression levels of IL-6 are determined, wherein an increase of IL-6 expression correlates with PTEN down-regulation in the sample of patients suffering of an autoimmune disease or condition.

14. The method according to claim 7, wherein a disturbed PTEN expression in T cells is associated with impaired responsiveness of CD4+ T-cells to Treg (Treg resistance).

15. The method according to claim 14, wherein the T cells are MS T cells.

Description

FIGURE LEGENDS

[0029] FIG. 1: Treg resistance is directly linked to IL-6 and PKB signaling pathway

[0030] A) PBMC from therapy-nave MS patients (red) or HC (black) were cocultured with Treg and stimulated with anti-CD3 mAb. T cell proliferation was determined by .sup.3H-Tdr incorporation on day three. Box plots show percentage of suppression in presence of Treg (ratio 4:1) normalized to proliferation of T cells alone as median with interquartile range (n=15), P-values relative to T cells of HC are shown, to avoid familywise error rate bonferroni correction was used p*. B) C) IL-6R expression within CD3+ T cells of PBMC from HC or MS was determined by flow cytometry. One representative of eight independent experiments is shown. D) PKB/c-Akt phosphorylation was determined by flow cytometry within CD3.sup.+ T cells of MS (red) or HC (black). Six different experiments are shown, p-values relative to MFI of HC. E) MS T cells with Treg were stimulated with anti-CD3 mAb without (circle) or with (quadrats) anti-IL-6R mAb or (triangles) Akt-VIII inhibitor. Each dot represents percentage of proliferation with Treg normalized to T cells alone (n=4), p-values to MS T cells.

[0031] FIG. 2: Dysregulated PTEN expression in Treg resistant MS T cells

[0032] A) Left: Relative PTEN mRNA expression in CD4.sup.+ T cells from healthy donors (black) and therapy-naive MS patients (red) 2 hours after activation with 0.5 g/ml anti-CD3 and 1 g/ml anti-CD28 mAb. Every dot represents one individual donor (healthy n=9, MS n=13). Right: Relative PTEN mRNA expression in resting CD4.sup.+ T cells from healthy donors (black) and therapy-naive MS patients (red). Bars show mean PTEN mRNA expression of n=12 individuals. B) Western blot depicts total PTEN protein expression of CD4.sup.+ T cells from healthy donors (left) and therapy-nave MS patients (right) at different time points after activation with 0.5 g/ml anti-CD3 and 1 g/ml anti-CD28 mAb.

[0033] FIG. 3: PTEN knockdown results in an accelerated IL-6 mRNA production and activation of PKB

[0034] A) Relative PTEN mRNA expression in CD4.sup.+ T cells 24 h after electroporation with 1 M PTEN siRNA normalized to scrambled control. Every dot represents one individual knockdown experiment (n=9). Knockdown efficiency ranges from 70-80%. B) Western blot depicts total PTEN protein expression of electroporated T cells at different time points after electroporation. C) After electroporation with PTEN siRNA and scrambled control, CD4.sup.+ T cells were activated and PKB/c-Akt phosphorylation was analyzed. Western Blot shows phosphorylation state of PKB/c-Akt (Ser473) at different time points (0-45 min) after activation. D) 24 h after electroporation CD4+ T cells were activated with 0.5 g/ml anti-CD3 and 1 g/ml anti-CD28 mAb for 2 hours. Bars show relative IL-6 mRNA expression in CD4+ T cells treated with scrambled control (black) and PTEN siRNA (red) 2 h after activation normalized to unstimulated controls.

[0035] FIG. 4: PTEN knockdown induces Treg resistance in CD4.sup.+ T cells

[0036] A) 24 h after electroporation CD4.sup.+ T cells were cocultured with freshly isolated Treg and activated with 0.1 g/ml anti-CD3 mAb. CD3-depleted PBMC served as costimulus. Proliferation of T cells was measured via .sup.3H-Tdr incorporation. Left graph shows suppression of electroporated T cells of one representative experiment (n=5). Box plots show percentage of suppression in presence of Treg (T cell:Treg ratio 4:1) of all 5 experiments, black: scrambled control, grey: PTEN siRNA.

[0037] FIG. 5: IL-6 mediates Treg resistance by inducing the phosphorylation of PKB/c-Akt and downregulation of PTEN in activated T cells A) T cells and Treg were cocultured in presence (grey) or absence (black) of IL-6 and stimulated with anti-CD3 mAb. Proliferation was determined by .sup.3H-Tdr incorporation on day three and displayed as meanSEM of triplicate measurements, n=4. B) PKB/c-Akt phosphorylation was analyzed by flow cytometry within T cells after 24 h of culture with (black) or without IL-6 (grey). C) T cells from HC were incubated for 24 h without IL-6 (black) or with IL-6 (grey) or IL-6 and anti-IL-6R mAb (striped). Shown is the mean fluorescence intensity (MFI) of pPKB/c-Akt in T cells (n=5). D) PBMC from HC were cultured for 24 h in the presence or absence of 500 ng/ml IL-6. CD4.sup.+ T cells were isolated, left unstimulated or were activated with anti-CD3 and anti-CD28 mAb for two hours. Total proteins were isolated and PTEN protein expression was analyzed via western blot.

[0038] FIG. 6: Treg resistance of T cells from MS patients is ameliorated after IFN- therapy

[0039] A) Treg-depleted PBMC from therapy-naive (red), IFN--treated MS patients (blue) or HC (black) were cocultured with allogeneic Treg and stimulated with anti-CD3 mAb. T cell proliferation was determined by .sup.3H-Tdr incorporation on day three. Box plots show percentage of suppression in presence of Treg (ratio 1:1) normalized to proliferation of PBMC alone as median with interquartile range (n=15), p-values relative to suppression of HC or therapy-nave MS, to avoid familywise error rate bonferroni correction was used indicated as (p*). B) PKB/c-Akt phosphorylation was determined by flow cytometry within CD3+ T cells from therapy-naive (red), IFN--treated MS patients (blue) or HC (black). Grey histogram depicts isotypic control of MS. Lower panel shows MFI of PKB/c-Akt phosphorylation of six different experiments, p-values relative to MFI of MS. C) IL-6R expression within PBMC from HC (black), therapy-naive (red), or IFN--treated (blue) MS patients was determined by flow cytometry. Box plots show percentage of IL-6R.sup.+ cells within CD3.sup.+ T cells of six independent donors, p-values relative to IL-6R expression of therapy-nave MS or HC are shown. D) Analysis of PTEN expression in CD4.sup.+ T cells from healthy donors, therapy-nave MS patients and IFN--treated (or DMF) MS patients.

MATERIALS AND METHODS

[0040] Isolation and Culture of Human Immune Cells

[0041] PBMC from either MS patients or healthy donors were isolated from buffy coats or heparinized syringes within 12 h after blood collection as described before using density gradient centrifugation. Blood was kept at room temperature before PBMC enrichment. After isolation, human cells were cultured in X-VIVO-15 (Lonza, Belgium).

[0042] Flow Cytometry

[0043] Flow cytometric analysis was performed using the following antibodies: anti-human CD3 (UCHT1), anti-human CD4 (RPA-T4), anti-human CD8 (SK1), anti-human CD14 (M5E2), anti-human CD19 (HIB19), all from BD Pharmingen, anti-human CD25 (4E3), anti-human CD127 (MB15-18C9), all from Miltenyi Biotec. Fluorokine biotinylated human Interleukin-6 (R&D systems) was used to analyze IL-6R expression on T cells. Cell viability during flow cytometric analysis was determined using 7-AAD and eFluor506 (eBioscience). For blockade experiments, cells were incubated with a neutralizing antibody against anti-IL-6R (Tocilizumab). For surface staining of PBMC or T cells indicated antibodies were incubated for 20 min at 4 C. and washed twice with PBS supplemented with 0.5% HSA+1 mM EDTA+10 g/ml Sandoglobulin (CSL Behring). Stained cells were measured on LSRII with FACS Diva Software (BD Bioscience) or FACSVia and data was analyzed with BD FACSVia Research Software (BD Biosciences). To detect phosphorylated PKB/c-Akt, cells were fixed at 37 C. with BD Cytofix Buffer for 10 min, then permeabilized with BD Phosflow Perm Buffer III for 30 min on ice, washed twice with BD Stain Buffer and stained with anti-Akt pS473 (BD Phosflow) according to manufacturer's instructions.

[0044] Isolation of T Cell Subsets

[0045] Untouched CD4.sup.+CD25.sup. T cells were isolated using CD4.sup.+ T cell isolation kit (Miltenyi Biotec) according to manufacturer's instructions. For some experiments, CD4.sup.+ T cells were enriched by positive selection using anti-CD4 MicroBeads (Miltenyi Biotec). Isolated T cells were then depleted of intrinsic Treg with anti-CD25 Dynabeads (Invitrogen). CD4.sup.+CD25.sup.+Foxp3.sup.+ Treg were isolated from PBMC using anti-CD25 MicroBeads (Miltenyi Biotec) and depleted of contaminating CD8.sup.+, CD14.sup.+ and CD19.sup.+ cells with Dynabeads (Invitrogen) as described previously. Purity was routinely >85%, Treg functionality was ensured in standard suppressor assays. For some experiments PBMC were depleted of CD3 or CD25 using corresponding Dynabeads (1 bead/cell; Invitrogen).

[0046] Suppressor Assays

[0047] CD4.sup.+CD25.sup. T cells (10.sup.5 cells/well, 96 well plate, flat bottom) were stimulated with 0.1 g/ml anti-CD3 mAb (OKT-3) and cultured in presence or absence of different Treg ratios. CD3-depleted PBMC (510.sup.4/well) were used as costimulus. For some experiments, CD25-depleted PBMC (10.sup.5 cells) were stimulated with 0.1 g/ml anti-CD3 mAb (OKT-3) and co-cultured with or without different Treg ratios. On day 3, .sup.3H-Tdr was added to each well (37 kBq/well) and cells were cultured for an additional 16 h. T cell proliferation was measured by .sup.3H-Tdr incorporation using a liquid 6-scintillation counter. Some experiments were performed by supplementing cultures with neutralizing mAb against IL-6R (30 ng/ml; Tocilizumab; Roacterma; Roche) or supplemented with IL-6 (500 ng/ml, ImmunoTools)

[0048] RNA Isolation, cDNA Synthesis and qRT-PCR

[0049] RNA was extracted from 1-210.sup.6 cells using peqGOLD Micro RNA Kit (VWR) according to manufacturer's instructions. cDNA was synthesized from 100 ng of the isolated RNA by reverse transcription with iScript cDNA synthesis kit (Bio-Rad) and the supplied random hexamer primers. All quantitative RT-PCR reactions were performed in triplicates on a Rotor-Gene Q cycler (Qiagen) using SYBR Green (Bimake). PTEN and IL-6 mRNA expression levels were measured with the appropriate QuantiTect Primer Assay (Qiagen) according to manufacturer's protocol. The mRNA levels of the housekeeping gene EEF1A were used for normalization and relative expression levels were calculated with 2.sup.CT method.

[0050] PTEN Knockdown Experiments

[0051] For a successful knockdown of PTEN protein, PTEN GeneSolution siRNA kit (Qiagen) was used that includes Hs_PTEN_6, Hs_PTEN_9, Hs_PTEN_8, and Hs_PTEN_4 siRNAs.

[0052] AllStars Negative Control siRNA (Qiagen) was employed as scrambled control. Purified CD4.sup.+ T cells were transfected with AMAXA Human T cell Nucleofector kit (Lonza) according to manufacturer's protocol. Briefly, 510.sup.6 T cells were resuspended in 100 l Nucleofector Solution (per sample) supplemented with 1 M PTEN siRNA mix or negative control siRNA. Nucleofector program U-014 was used to efficiently transfect T cells. After nucleofection, T cells were cultured overnight in X-VIVO-15 in a humidified 37 C./5% CO.sub.2 incubator and knockdown efficiency was analyzed at indicated time points using qRT-PCR and Western blot.

[0053] SDS-PAGE and Western Blotting

[0054] Cell pellets were lysed in Cell Extraction Buffer (Invitrogen) supplemented with 1 mM PMSF (Invitrogen) and Protease Inhibitor Cocktail (Sigma) for 30 min on ice with vortexing at 10 min intervals. Total protein concentration of cell lysates was determined with Micro BCA Protein Assay Kit (Thermo Fisher Scientific) according to manufacturer's protocol. Protein separation was performed using the NuPAGE Bis-Tris electrophoresis system (Thermo Fisher Scientific). Briefly, up to 15 g of total protein were mixed with NuPAGE LDS Sample Buffer and NuPAGE Reducing Agent and loaded onto NuPAGE 4-12% (gradient) Bis-Tris gels (denaturing conditions, 200 V, 60 min, with NuPAGE MOPS SDS Running Buffer). The separated proteins were transferred onto PVDF membranes (0.45 m pore size, Novex) using the semi-wet XCell II Blotting system (30 V, 60 min, Invitrogen). For immunoblot analysis, membranes were blocked in TBST buffer/5/0 BSA for at least 1 h at room temperature. The incubation with primary antibodies was performed as suggested by the antibody providers. For primary incubation, the following antibodies were used: rabbit anti-PTEN mAB (138G6), rabbit anti--Actin mAb (13E5), rabbit anti-pan-Akt mAb (C67E7), all from Cell Signaling Technology, mouse anti-PKB/Akt pSer437 mAb (11E6, Nanotools). Purified goat anti-rabbit IgG antibody (Cell Signaling Technology) and rabbit anti-mouse IgG antibody (Abcam) conjugated to horseradish peroxidase were employed for chemiluminescent detection with Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences). Western blots were analyzed and quantified with ImageJ software.