In-vitro methods 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), Treg resistance, by measuring the expression levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha, PPARGC1A (PGC-1α) in activated CD4+ T-cells, in particular in patients suffering from relapsing remitting multiple sclerosis. The invention relates to an in vitro screening 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 peroxisome proliferator-activated receptor gamma coactivator 1-alpha, PPARGC1A (PGC-1α) 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. PCG-1α or an upstream regulator of Treg-resistant T-cells HNF4A, Hdac, RORA, ESRRA, LPIN1 can be used in a screening system for the detection of impaired responsiveness of CD4+ T-cells to Treg.

Claims

1. A method for treating a regulatory T-cell (Treg)-resistance-associated autoimmune condition or disease by identifying an agonist that can up-regulate peroxisome proliferator-activated receptor gamma coactivator 1-alpha, PPARGC1A (PGC-1α) in activated T-cells isolated from a patient suffering from the Treg-resistance-associated autoimmune condition or disease, by determination of responsiveness of effector CD4+ and/or effector CD8+ T-cells to Treg, in the patient comprising (i) isolating effector CD4+ and/or effector CD8+ T-cells from blood samples of a patient suffering from the Treg-resistance-associated autoimmune condition or disease, and Treg-sensitive healthy individuals as controls, (ii) activating the effector CD4+ and/or effector CD8+ T-cells with anti-CD3 and/or CD28 antibodies, (iii) measuring the expression levels of PGC-1α in the activated effector CD4+ and/or effector CD8+ T-cells, (iv) comparing the expression levels of PGC-1α of the activated effector CD4+ and/or effector CD8+ T-cells of the patient suffering from the Treg-resistance-associated autoimmune condition or disease with the PGC-1α expression levels in the activated effector CD4+ and/or effector CD8+ T-cells of Treg-sensitive healthy individuals to determine Treg resistance, wherein said agonist is identified by inducing an upregulation of PGC-1α within the effector CD4+ and/or effector CD8+ T-cells of the patient suffering from the Treg-resistance-associated autoimmune condition or disease, and (v) administering said agonist to the patient to treat the Treg-resistance-associated autoimmune condition or disease by normalizing PGC-1α expression and restoring Treg responsiveness in the effector CD4+ and/or effector CD8+ T-cells of said patient.

2. The method according to claim 1, wherein Treg responsiveness of T-cells is restored by normalizing PGC-la expression in the activated Treg-resistant T-cells of said patient suffering from the Treg-resistance-associated autoimmune condition or disease by administering IFN-β.

3. The method according to claim 1, wherein in addition to PGC-1α, the expression levels of the upstream regulators of Treg-resistant T-cells HNF4A, Hdac, RORA, ESRRA, LPIN1 are determined within CD4+ T-cells and compared to Treg-sensitive T-cells.

4. The method according to claim 1, wherein step (i) includes isolating said CD4+ and/or CD8+ T-cells from peripheral blood mononuclear cells (PBMCs) from said blood samples of said patient suffering from the Treg-resistance-associated autoimmune condition or disease.

Description

FIGURE LEGENDS

(1) FIG. 1: Functional gene chip analysis identified 16 upstream regulators of Treg-resistant MS T-cells compared to Treg-sensitive T-cells of HC.

(2) A) Schematic overview of the experimental setup used for Affymetrix gene chip analysis. B) Volcano plot of stimulatory response genes that are differently expressed in T-cells of HC and MS and that statistically exhibit a two-fold change. C) Pathway analysis of activated T-cells. Upper panel: numbers of upstream regulators predicted to be activated in activated T-cells of MS, HC or both. Lower panel: numbers of upstream regulators predicted to be inhibited in activated T-cells of MS patients, HC or both. D) Upstream regulators that were statistically more than +1 or −1 (in z-score) significant were examined regarding a connection to the IL-6/PKB/c-Akt signalling pathway (A: Regulator increases production/activity of IL-6/Akt; B: Regulator decreases production/activity of IL-6/Akt; C: Regulator's activity/expression is increased by IL6/Akt, D: Regulator's activity/expression is decreased by IL-6/Akt and E: no information.

(3) FIG. 2: qRT-PCR identifies PGC-1α as a potential molecule linked to Treg-resistance of MS T-cells.

(4) CD4.sup.+ or CD8.sup.+ T-cells from MS patients (red) or HC (black) were left unstimulated or were activated with plate-bound anti-CD3 and anti-CD28 mAb. Expression levels of FGF19, HNF4A, Hdac, RORA, IRF8 and PPARGC mRNA were detected by qRT-PCR. β-Actin was used as housekeeping gene. Four independent experiments are shown as median with interquartile range, p-values relative to HC T-cells. B) Total T-cells (left panel) or either CD4.sup.+ or CD8.sup.+ T-cells (right panel) from MS patients (red) or HC (black) were left unstimulated or were activated with plate-bound anti-CD3 and anti-CD28 mAb. Expression levels of PPARGC1A mRNA were detected by qRT-PCR. β-Actin was used as housekeeping gene. Six to eleven independent experiments are shown as median with interquartile range, p-values relative to HC T-cells. Left: shown are expression levels of PPARGC1A in activated CD4.sup.+ and CD8.sup.+ T-cells normalized to resting T-cells. Right: shown are expression levels of PPARGC1A in resting CD3.sup.+ T-cells normalized to the housekeeping gene β-Actin.

(5) FIG. 3: IL-6 induces down regulation of PGC-1α in activated T-cells and thereby mediates Treg resistance.

(6) A) Upper panel: CD3.sup.+ T-cells and Treg were coculture 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 mean±SEM of triplicate measurements. One of four independent experiments is shown. Lower panel: PBMC from HC were cultured for 24 h with (grey) or without (black) IL-6, washed extensively and CD4.sup.+ or CD8.sup.+ T-cells were isolated. Cells were left unstimulated or were activated with plate-bound anti-CD3 and anti-CD28 mAb for four hours. Shown are expression levels of (PPARGC1A) PGC-1α mRNA in activated CD4.sup.+ and CD8.sup.+ T-cells (TC) normalized to resting T-cells. β-Actin used as housekeeping gene. Bars represent mean of four experiments, p-values relative to TC without IL-6. B) PKB/c-Akt phosphorylation was analyzed by flow cytometry within CD3.sup.+ T-cells after 24 h of culture with (black) or without IL-6 (grey). C) Flow cytometric determination of PKB/c-Akt phosphorylation. T-cells from HC were incubated for 24 h in presence (grey) or absence of IL-6 (black) or in the presence of IL-6 and IL-6R mAb (grey, white striped). Shown is the mean fluorescence intensity (MFI) of pPKB/c-Akt in T-cells, one representative result of five independent experiments. D) PBMC from HC were cultured for 24 h in presence of IL-6 (grey) or in presence of IL-6 and VIII inhibitor (striped, 0.01, 0.1 and 1 μM). PKB/c-Akt phosphorylation was analyzed by flow cytometry within CD3.sup.+ T-cells. Shown are the arithmetic means of MFI values including the standard deviation of one representative experiment (n=6).

(7) FIG. 4: Presence of PPARα agonists in cocultures induces PGC-1α upregulation and thereby restores T cell sensitivity also in presence of IL-6.

(8) A) PBMC from HC were cultured for 24 h with media (black), PPARα antagonist (blue) or PPARα agonist (green), washed extensively and expression level of PGC-1α mRNA in activated T-cells was detected by qRT-PCR. β-Actin used as housekeeping gene. Bars represent mean of PGC-1α expression of three different experiments. B) PKB/c-Akt phosphorylation was analysed by flow cytometry within CD3.sup.+ T-cells after 24 h of culture with media (black), PPARα agonist (green) or PPARα antagonist (blue). Shown is one representative of four independent experiments. C) T-cells and Treg were coculture in presence (grey) or absence (black) of IL-6 or in presence of IL-6 and PPARα agonist (green) and stimulated with anti-CD3 mAb. Proliferation was determined by .sup.3H-Tdr incorporation on day three and displayed as mean±SEM of triplicate measurements. One of three independent experiments is shown. D) T-cells and Treg were coculture in presence (blue) or absence (black) of PPARα antagonist and stimulated with anti-CD3 mAb. Proliferation was determined by .sup.3H-Tdr incorporation on day three and displayed as mean±SEM of triplicate measurements. One of three independent experiments is shown.

(9) FIG. 5: Treg resistance of T-cells from MS patients is ameliorated after IFN-β therapy and correlated with upregulation of PGC-1α.

(10) A) Treg-depleted PBMC from therapy-naïve (black), IFN-β-treated MS patients (blue) or HC (white) were cocultured with allogeneic Treg and stimulated with anti-CD3 mAb. T cell proliferation was determined by 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-naïve 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.sup.+ T-cells from therapy-naïve (red), IFN-βtreated 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-naïve (red), or IFN-β-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-naïve MS or HC are shown. D) CD3.sup.+ T-cells from therapy-naïve, IFN-β-treated MS patients, or HC were left unstimulated or were activated for 4 h with plate-bound anti-CD3 and anti-CD28 mAb. Expression level of PGC-1α mRNA in activated T-cells was detected by qRT-PCR. β-Actin was used as housekeeping gene. Six independent experiments are shown as median with interquartile range (n=6), p-values relative to expression level of HC or therapy-naïve MS TC are shown, to avoid familywise error rate bonferroni correction was used indicated as p*.