Polypeptides and uses thereof for reducing CD95-mediated cell motility

Abstract

The present invention relates to polypeptides and uses thereof for reducing CD95-meditated cell motility. In particular, the present invention relates to a polypeptide having an amino acid sequence having at least 70% of identity with the amino acid sequence ranging from the amino-acid residue at position 175 to the amino-acid residue at position 191 in SEQ ID NO:1.

Claims

1. A nucleic acid sequence encoding for a fusion protein comprising a polypeptide having an amino acid sequence having at least 96, 97, 98, 99, or 100% identity with an amino acid sequence ranging from an amino acid residue at position 175 to an amino acid residue at position 209 or 210 as set forth in SEQ ID NO: 1, wherein said polypeptide is fused to a heterologous cell-penetrating polypeptide.

2. A vector and an expression cassette in which the nucleic acid sequence of claim 1 is associated with suitable elements for controlling transcription and, optionally translation.

3. A host cell comprising the vector of claim 2.

4. The host cell of claim 3 which is a prokaryotic or eukaryotic host cell genetically transformed with the vector.

5. A host cell comprising the nucleic acid sequence of claim 1.

6. The host cell of claim 5 which is a prokaryotic or eukaryotic host cell genetically transformed with the nucleic acid sequence.

7. A method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a fusion protein comprising a polypeptide having an amino acid sequence having at least 96, 97, 98, 99, or 100% identity with an amino acid sequence ranging from an amino acid residue at position 175 to an amino acid residue at position 209 or 210 as set forth in SEQ ID NO: 1, wherein said polypeptide is fused to a heterologous cell-penetrating polypeptide.

8. The method of claim 7 wherein the subject suffers from a triple negative breast cancer.

9. A method of treating an auto-immune disease or an inflammatory condition in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a fusion protein comprising a polypeptide having an amino acid sequence having at least 96, 97, 98, 99, or 100% identity with an amino acid sequence ranging from an amino acid residue at position 175 to an amino acid residue at position 209 or 210 as set forth in SEQ ID NO: 1, wherein said polypeptide is fused to a heterologous cell-penetrating polypeptide.

10. The method of claim 9 wherein the subject suffers from systemic lupus erythematosus.

11. A method of treating a Th17 mediated disease condition in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a fusion protein comprising a polypeptide having an amino acid sequence having at least 96, 97, 98, 99, or 100% identity with an amino acid sequence ranging from an amino acid residue at position 175 to an amino acid residue at position 209 or 210 as set forth in SEQ ID NO: 1, wherein said polypeptide is fused to a heterologous cell-penetrating polypeptide.

12. A pharmaceutical composition comprising a fusion protein comprising a polypeptide having an amino acid sequence having at least 96, 97, 98, 99, or 100% identity with an amino acid sequence ranging from an amino acid residue at position 175 to an amino acid residue at position 209 or 210 as set forth in SEQ ID NO: 1, wherein said polypeptide is fused to a heterologous cell-penetrating polypeptide.

13. A method for screening a drug for reducing CD95-mediated cell motility comprising the steps consisting of a) determining the ability of a candidate compound to inhibit the interaction between CD95 and a fusion protein comprising a polypeptide having an amino acid sequence having at least 96, 97, 98, 99, or 100% identity with an amino acid sequence ranging from an amino acid residue at position 175 to an amino acid residue at position 209 or 210 as set forth in SEQ ID NO: 1, wherein said polypeptide is fused to a heterologous cell-penetrating polypeptide and b) positively selecting the candidate compound that inhibits said interaction.

Description

FIGURES

(1) FIGS. 1A-1D: Cleaved-CD95L mobilizes Calcium ions from both extracellular and intracellular compartments by mechanisms involving Ca.sup.2+ channels, PLC, IP3 and Ryanodine Receptors. [Ca.sup.2+].sub.i, was monitored via the ratio F340 nm/F380 nm (relative [Ca.sup.2+]cytosolic) using Fura2 as fluorescent probe. Data represent mean+/SD of at least 60 cells (3 independent experiments). Jurkat cells were stimulated with 100 ng/ml cl-CD95L (black arrow). A. Cells were bathed in a 2 mM Ca.sup.2+-containing medium (black squares) or in a Ca.sup.2+-free medium (open circles). The transient Ca.sup.2+ increase occurring in Ca.sup.2+-free medium (open circles) corresponds to the release of calcium ions stored in intracellular compartments. The plateau phase observed in 2 mM Ca.sup.2+-containing medium, and disappearing in Ca.sup.2+-free medium is mainly due to calcium influx from the extracellular space. B. In PLC1/ Jurkat cells (open circles), cl-CD95L failed to induce the initial, transient increase in [Ca.sup.2+]i, which is restored when PLC1 is reintroduced in the PLC1/ cells (black squares). 1C. Jurkat cells were pretreated (2 M, 20 minutes) with Xestospongin C (XestoC, open circles), a membrane permeable, potent IP3 receptors blocker. Such a treatment again, completely blocked the initial component of the calcium response. D. Jurkat cells were pretreated (20 minutes) with high concentrations (10 M) of Ryanodine (Rya, open triangle), in order to block Ryanodine receptors, or with Ryanodine and Xestospongin C (XestoC, open circle) to block both ryanodine and IP3 receptors. Rya did not reduce the initial peak but blocked the plateau phase. Combination of Rya and XestoC abolished the CD95-mediated Ca.sup.2+ response.

(2) FIGS. 2A-2D: TAT CID 175-210 and TAT CID 175-191 impair the PLC/IP3-dependent calcium response to cl-CD95L. [Ca.sup.2+], was monitored via the ratio F340 nm/F380 nm (relative [Ca.sup.2+]cytosolic) using Fura2 as fluorescent probe. Data represent mean+/SD of at least 60 cells (3 independent experiments). Jurkat (A and B) and activated PBL (C and D) were stimulated with 10 ng/ml Cl-CD95L (black arrow). A. Jurkat were pre-incubated with a TAT peptide control (1 hr, 10 M, open triangle) or with a TAT peptide corresponding to the aa 192-210 of the death receptor CD95 (1 hr, 10 M, open circles) or not (control, black squares). The treatments did not significantly modify the calcium response to cl-CD95L. B. Jurkat were pre-incubated with a TAT peptide corresponding to the aa 175-210 of the death receptor CD95 (1 hr, 10 M, open circles) or with a TAT peptide corresponding to the aa 175-191 of the death receptor CD95 (1 hr, 10 M, open triangles) or not (black squares). Both treatments greatly reduced the calcium response to cl-CD95L, particularly the initial peak. C. Activated PBL were pre-incubated with the TAT peptide control (1 hr, 10 M, open triangles) or with the TAT peptide corresponding to the aa 192-210 of the death receptor CD95 (1 hr, 10 M, open circles) or not (control, black squares). The treatments did not significantly modify the calcium response to Cl-CD95L. D. Activated PBL were pre-incubated with a TAT peptide corresponding to the aa 175-210 of the death receptor CD95 (1 hr, 10 M, open circles) or with a TAT peptide corresponding to the aa 175-191 of the death receptor CD95 (1 hr, 10 M, open triangles) or not (black squares). Both treatments greatly reduced the calcium response to cl-CD95L, particularly the initial peak.

(3) FIGS. 3A-3C: TAT CID 175-210 is effective in various cell models. [Ca.sup.2+].sub.i was monitored via the ratio F340 nm/F380 nm (relative [Ca.sup.2+]cytosolic) using Fura2 as fluorescent probe. Data represent mean+/SD of at least 60 cells (3 independent experiments). CEM (A), MDA MB 231 (B) and MEF (C) were stimulated with 100 ng/ml cl-CD95L (black arrow). A and B. CEM and MDA MB 231 cells were pre-incubated with a TAT peptide corresponding to the aa 175-210 of the death receptor CD95 (1 hr, 10 M, open circle). The treatment (open circles) completely abolished the calcium response in both cell types. C. MEFs were pre-incubated with the TAT peptide corresponding to the aa 175-210 of the death receptor CD95 (1 hr, 10 M, open circle) or with the TAT peptide control (1 hr, 10 M, open triangles). If the latter did not modify the initial phase, the former blocked it.

(4) FIG. 4: TAT CID 175-219 inhibits migration of triple cell negative cancer cells. The triple negative breast cancer cell line MDA-MB-231 was pre-incubated for 1 hour with 10 M of DID 175-220 and cell migration was assessed using the Boyden chamber assay in the presence or absence of cl-CD95L (100 ng/mL) for 24 h. Migrating cells were stained with Giemsa. For each experiment, five images of random fields were acquired.

(5) FIGS. 5A-5I. High amounts of serum CD95L in SLE patients correspond to a homotrimeric ligand inducing endothelial transmigration of activated T lymphocytes. A. Soluble CD95L was dosed by ELISA in sera of newly diagnosed SLE patients and healthy donors. *** indicates P0.0001 using a two-tailed student's t test. B. Sera from SLE patient were fractionated using size exclusion S-300-HR Sephacryl columns and CD95L was dosed by ELISA. Inset: CD95L was immunoprecipitated in fractions 40-46 and 76-78 and loaded in a 12% SDS-PAGE. Anti-CD95L immunoblot is depicted. C. Activated PBLs from healthy donors were incubated in presence of gel filtration fractions obtained in B and endothelial transmigration was assessed as described in Materials and Methods. Where indicated, fractions 76-78 were pre-incubated 30 minutes with the antagonist anti-CD95L mAb NOK-1 (10 g/ml). D. CD95L, IL17 and CD4 expression levels were analyzed by Immunohistochemistry in inflamed skins of lupus patients or in healthy subjects (mammectomy). Numbers correspond to different patients. E. Densitometric analyses of CD95L and IL17 staining depicted in D revealed that the expression levels of these two markers vary in a correlated manner. F. The indicated human T-cell subsets were subject to transmigration assay in presence of sera taken from SLE patients or healthy donor as controls. Data were analyzed using Mann-Whitney U-test. ***P<0.001 G. Human T-cell subsets were subject to transmigration assay as above except in the lower chamber Fas-Fc was added at increasing concentrations in parallel with cl-CD95L. Data represent the mean of 4-5 individual donors SD and were analyzed using a 2-way Anova. H. Transmigration of CD4 T-cell subsets was analyzed in Boyden chambers in presence or absence of cl-CD95L (200 ng/ml). I. Transmigration of human regulatory T-cells and Th17 cells was assessed by Boyden Chamber assay in presence or absence of cl-CD95L. Data was analyzed using a 2-way Anova. P values <0.05 was considered significant; *P<0.05, ***P<0.001

(6) FIGS. 6A-6I. In vivo administration of cl-CD95L preferentially attracts Th17 cells. Mice were injected once with cl-CD95L (200 ng) or vehicle, and 24 hrs later subject to examination. (A-B). Total cell counts for the peritoneal cavity (A) and spleen (B) were performed. (C-D). Differential white blood cell count was performed 24 hrs post injection. Peritoneal Exudate Cells (PEC) (C) and spleen (D) cells were subject to flow cytometry analysis to identify the percentage of infiltrated CD4.sup.+ cells. (E-I). PEC CD4.sup.+ cells were purified by AutoMACS separation and RNA prepared. Cells were subject to real-time PCR for (E) IL-17A, (F) IL-23R, (G) CCR6, (H) IFN-, and (I) FoxP3. Data presented are averages of groups of 6 mice SD, with experiments repeated twice. Data were analyzed using the students t-Test, P values <0.05 was considered significant; *P<0.05, **P<0.01, ***P<0.001.

(7) FIGS. 7A-7E. CD95 implements a Death Domain-independent Ca.sup.2+ response. A. CEM cells were stimulated with CD95L (100 ng/mL) and CD95 was immunoprecipitated. The immune complex was resolved by SDS/PAGE, and the indicated immunoblottings were performed. Total lysates were loaded as control. B. Parental Jurkat T cells, PLC-1-deficient and its PLC-1-reconstituted counterparts were loaded with the Ca.sup.2+ probe FuraPE3-AM (1 M) and then stimulated with cl-CD95L (100 ng/mL, black arrow). Ratio images (F340/F380, R) were taken every 10 s and were normalized vs pre-stimulated values (R.sub.0). Data represent meanSD of R/R.sub.0 measured in n cells. Inset: PLC1-deficient Jurkat cells or its reconstituted counterpart was lysed and the expression levels of PLC1 and CD95 were evaluated by immunoblotting. Tubulin was used as a loading control. C. Cells were loaded with the Ca.sup.2+ probe FuraPE3-AM (1 M) and then stimulated with cl-CD95L (100 ng/ml). Data were analyzed as described in B. Inset: Parental Jurkat cells (A3) or its counterparts lacking either FADD or caspase-8 were lysed and the expression levels of CD95, FADD and Caspase-8 were evaluated by immunoblotting. D. Representation of the different CD95 constructions. E. CEM-IRC cells expressing GFP alone or GFP-fused CD95 constructs shown in D, were loaded with the Ca.sup.2+ probe fluo2-AM (1M). The cells were then stimulated with cl-CD95L (100 ng/mL; black arrow) and [Ca.sup.2+], was monitored via the ratio F/F.sub.0 (relative Ca.sup.2+.sub.[CYT]). Data represent meanSD of F/F.sub.0 measured in n cells

(8) FIGS. 8A-8F. The CD95-mediated Ca.sup.2+ signal stems from amino-acid residues 175 to 210 in CD95. A. HEK cells were co-transfected with the GFP-fused CD95 constructions and wild type PLC1. Twenty-four hours after transfection, CD95 expression level in these cells was evaluated by flow cytometry. B. Cells in A were stimulated with CD95L (100 ng/mL) and CD95 was immunoprecipitated. The immune complex was resolved by SDS-PAGE, and the indicated immunoblotting was performed. Total lysates were loaded as a control. C. Left Panel: HEK cells were co-transfected with PLC1 and CID-mCherry or mCherry alone. After 24 h, cells were lyzed and PLC1 was immunoprecipitated. The immune complex was resolved by SDS/PAGE, and the indicated immunoblottings were performed. Total lysates were loaded as a control. Right Panel: HEK cells were co-transfected with PLC1 and CID-mCherry or mCherry alone. After 24 h, cells were stimulated in presence or absence of CD95L (100 ng/mL) and CD95 was immunoprecipitated. The immune complex was resolved by SDS-PAGE, and the indicated immunoblotting was performed. Total lysates were loaded as a control. D. Upper panel; protein sequences of TAT-CID and TAT-control. Lower panel; The leukemic T cell line CEM was pre-incubated for 1 h with 10 M of TAT-control or TAT-CID and then stimulated in presence or absence of cl-CD95L (100 ng/mL) for the indicated times. Cells were lysed and CD95 was immunoprecipitated. The immune complex was resolved by SDS-PAGE, and the indicated immunoblotting was performed. Total lysates were loaded as a control. E. Jurkat and CEM were loaded with FuraPE3-AM (1 M), pretreated for 1 h with 10 M of TAT-control or TAT-CID and then stimulated with 100 ng/ml of cl-CD95L (black arrow). Ratio images were taken every 10 s and were normalized vs pre-stimulated values. F. Human PBLs from healthy donors were loaded with furaPE3-AM (1 M) pretreated for 1 h with 10 M of TAT-control or TAT-CID or with the IP3R inhibitor Xestospongin C (positive control, 1 M, 1h) and then stimulated with 100 ng/ml of cl-CD95L (black arrow). Ratio values (R) were normalized vs pre-stimulated values (R0). Data represent meanSD of R/R.sub.0 measured in n cells.

(9) FIGS. 9A-9D. TAT-CID is an inhibitor of the cl-CD95L-induced Th17 cell accumulation in organs. A. Mouse Th17 cell transmigration was monitored by Boyden Chamber assay in presence or absence of the indicated concentrations of the TAT-CID peptide. B-D. C57BL/6 mice were injected with 40 mg/kg of TAT-control or TAT-CID two hours prior to IP injection cl-CD95L (200 ng) or vehicle, and 24 hours later subject to examination. B. Total cell counts for the peritoneal cavity was performed. C. Peritoneal Exudate Cells (PEC) were subject to flow cytometry analysis to identify the percentage of infiltrated CD4.sup.+CD62L.sup. T-cells. D. IL-17A levels in the peritoneal cavity were quantified by ELISA. Statistical analysis was performed using a 2-way Anova p-values indicated are **p<0.01, ***p<0.001.

EXAMPLE 1

(10) Cells were loaded with Fura2-AM (1 M) at resting temperature for 30 min in Hank's Balanced Salt Solution (HBSS). After washing with HBSS, the cells were incubated for 15 min in the absence of Fura2-AM to complete de-esterification of the dye. Cells were placed in a thermostated chamber (37 C.) of an inverted epifluorescence microscope (Olympus IX70) equipped with a 40, UApo/340-1.15 W water-immersion objective (Olympus), and fluorescence micrograph images were captured at 510 nm and at 12-bit resolution by a fast-scan camera (CoolSNAP fx Monochrome, Photometrics). To minimize UV light exposure, 44 binning function was used. Fura2-AM was alternately excited at 340 and 380 nm, and ratios of the resulting images (excitations at 340 and 380 nm and emission filter at 520 nm) were produced at constant intervals (5 s or 10 s according to the stimulus). Fura-2 ratio (F.sub.ratio 340/380) images were displayed and the F.sub.ratio values from the regions of interest (ROIs) drawn on individual cells were monitored during the experiments and analyzed later offline with Universal Imaging software, including Metafluor and Metamorph. Each experiment was independently repeated 3 times, and for each experimental condition, we displayed an average of more than 20 single-cell traces. Fluorescent images were pseudocolored using the IMD display mode in MetaFluor and assembled without further manipulation in Photoshop (Adobe). Raw data were acquired with MetaFluor and graphed in Origin (OriginLab). [Ca.sup.2+].sub.i was calculated using the following equation: [Ca.sup.2+].sub.i=K.sub.d(RR.sub.min)/(RR.sub.max)Sf2/Sf1, where K.sub.d is the Fura2-AM dissociation constant at the two excitation wavelengths (F.sub.340/F.sub.380); R.sub.min is the fluorescence ratio in the presence of minimal calcium, obtained by chelating Ca.sup.2+ with 10 mM EGTA; R.sub.max, is the fluorescence ratio in the presence of excess calcium, obtained by treating cells with 1 M ionomycin; Sf2 is the fluorescence of the Ca.sup.2+-free form; and Sf1 is the fluorescence of the Ca.sup.2+-bound form of Fura2-AM at excitation wavelengths of 380 and 340 nm, respectively. In some experiments cells were placed in a Ca.sup.2+-free medium consisting of the HBSS described above in which CaCl.sub.2 was omitted and 100 M EGTA was added in order to chelate residual Ca.sup.2+ ions. This medium was added to the cells just before recording to avoid leak of the intracellular calcium stores. Results are shown in FIGS. 1-3.

EXAMPLE 2

(11) Boyden chambers contained membranes with a pore size of 8 m (Millipore, Molsheim, France). After hydration of the membranes, breast cancer cells (10.sup.5 cells per chamber) were added to the top chamber in low serum (1%)-containing medium. The bottom chamber was filled with low serum (1%)-containing medium in the presence or absence of cl-CD95L (100 ng/mL). Cells were cultured for 24 h at 37 C. To quantify migration, cells were mechanically removed from the top-side of the membrane using a cotton-tipped swab, and migrating cells from the reverse side were fixed with methanol and stained with Giemsa. For each experiment, five representative pictures were taken for each insert, then cells were lyzed and absorbance at 560 nm correlated to the amount of Giemsa stain was measured. Results are shown in FIG. 4.

EXAMPLE 3

Methods

Patients and Ethics Statement

(12) SLE patients fulfilled four or more of the 1982 revised ACR criteria for the disease. All clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. Blood was sampled from patients diagnosed with SLE after written consent was obtained from each individual. This study was approved by institutional review board at the Centre Hospitalier Universitaire de Bordeaux.

Antibodies Other Reagents

(13) PHA, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), protease and phosphatase inhibitors were purchased from Sigma-Aldrich (L'Isle-d'Abeau-Chesnes, France). Anti-CD95L mAb was from Cell Signaling Technology (Boston, Mass., USA). Recombinant IL-2 was obtained from PeproTech Inc. (Rocky Hill, N.J., USA). Anti-PLC1 was purchased from Millipore (St Quentin en Yvelines, France). Anti-CD95 mAbs (APO1-3) came from Enzo Life Sciences (Villeurbanne). PE-conjugated anti-human CD95 (DX2) mAb, anti-human FADD mAb (clone1), neutralizing anti-CD95L mAb (Nok1) were provided by BD Biosciences (Le Pont de Claix, France). Anti-caspase-8 (C15) and anti-Fas (C-20) mAbs were from Santa Cruz Biotechnology (Heidelberg, Germany). CD95-Fc, neutralizing anti-ICAM-1 and E-selectin mAbs.

Plasmids and Constructs

(14) GFP-tagged human CD95 (hCD95) constructs were obtained by PCR and inserted in frame between the Nhe1 and EcoR1 sites of pEGFP-N1 (Clontech). Note that for all CD95 constructs the numbering takes into consideration the subtraction of 16 amino-acid of the signal peptide. Substitution of the cysteine at position 183 by a valine in hCD95.sup.(1-210) was performed using the Quickchange Lightning Site-directed Mutagenesis kit (Agilent Technologies, Les Ulis, France) according to manufacturer instructions. The CID-mCHERRY construct was obtained by PCR amplifying the hCD95 sequence coding for the residues 175 to 210. The resulting fragment was inserted between the EcoR1 and BamH1 site of a pmCHERRY-N1 vector. Mouse full length CD95 (mCD95) was kindly provided by Dr Pascal Schneider (Universite de Lausanne, Lausanne, Switzerland). The mCD95 sequence lacking the signal peptide (SP-residues 1-21) was amplified by PCR. After digestion by BamHI/EcoRI, the amplicon was inserted into pcDNA3.1(+) vector in frame with SP sequence of the influenza virus hemagglutinin protein followed by a flag tag sequence and a 6 amino acid linker. The pTriEx-4 vector encoding for Myc-tagged full-length human PLC1 was a gift from Dr. Matilda Katan (Chester Beatty Laboratories, The Institute of Cancer Research, London, United Kingdom). Plasmids coding for full length CD95L and the secreted IgCD95L have been described elsewhere (Tauzin et al., 2011). All constructs were validated by sequencing on both strands (GATC Biotech, Constance, Germany).

Cell Lines and Peripheral Blood Lymphocytes

(15) All cells were purchased from ATCC (Molsheim Cedex, France). T leukemic cell lines CEM, H9 and Jurkat were cultured in RPMI supplemented with 8% heat-inactivated FCS (v/v) and 2 mM L-glutamine at 37 C. in a 5% CO2 incubator. CEM-IRC cell expressing a low amount of plasma membrane CD95 was described in (Beneteau et al., 2007; Beneteau et al., 2008). HEK293 cells were cultured in DMEM supplemented with 8% heat-inactivated FCS and 2 mM L-glutamine at 37 C. in a 5% CO.sub.2 incubator. PBMCs (peripheral blood mononuclear cells) from healthy donors were isolated by Ficoll centrifugation, washed twice in PBS. Monocytes were removed by a 2 hours adherence step and the naive PBLs (peripheral blood lymphocytes) were incubated overnight in RPMI supplemented with 1 g/ml of PHA. Cells were washed extensively and incubated in the culture medium supplemented with 100 units/ml of recombinant IL-2 for 5 days. Human umbilical vein endothelial cell (HUVEC) (Jaffe et al., 1973) were grown in human endothelial serum free medium 200 supplemented with LSGS (Low serum growth supplement) (Invitrogen, Cergy Pontoise, France). CEM-IRC cells were electroporated using BTM-830 electroporation generator (BTX Instrument Division, Harvard Apparatus) with 10 g of DNA. 24 hours after electroporation, cells were treated for one week with 1 mg/mL of neomycin and then clones were isolated using limiting dilution.

Immunohistocytology

(16) Skins from lupus patients were embedded in paraffin and cut into 4 m sections. For CD4, CD8 and IL17 detection, Immunohistochemical staining was performed on the Discovery Automated IHC stainer using the Ventana OmniMap detection kit (Ventana Medical Systems, Tucson, Ariz., USA). The slides were rinsed with Ventana Tris-based Reaction buffer (Roche). Following deparaffination with Ventana EZ Prep solution (Roche) at 75 C. for 8 min, antigen retrieval was performed using Ventana proprietary, Tris-based buffer solution CC1 (pH8) antibody, at 95 C. to 100 C. for 48 min. Endogen peroxidase was blocked with Inhibitor-D 3% H2O2 (Ventana) for 10 min at 37 C. After rinsing, slides were incubated at 37 C. for 60 min with IL17 (Bioss), CD4 and CD8 (Dako), and secondary antibody: OmniMap HRP for 32 min (Roche). Signal enhancement was performed using the Ventana ChromoMap Kit Slides (biotin free detection system). For CD95L (BD Pharmigen) detection, antigen retrieval was performed using antigen unmasking solution pH 9 (Vector) at 95 C. for 40 min and endogenous peroxidase was blocked using 3% w/v hydrogen peroxide in methanol for 15 min. Slides were incubated in 5% BSA for 30 min at RT and then stained overnight at 4 C. Tissue sections were incubated with Envision+ system HRP-conjugated secondary antibodies for 30 min at RT and labeling was visualized by adding liquid DAB+. Sections were counterstained (hematoxylin) and mounted with DPX mounting medium. Using ImageJ software (IHC toolbox), densitometry analysis was undertaken on scanned slides to evaluate the amount of the different markers. The mean area for each marker was assessed and we determined if a correlation existed between the quantities of IL17-expressing cells and CD8.sup.+ T cells and the expression level of CD95L.

Mouse and Human CD4+ T-cell Subset Generation

(17) Animal experiments were subject to ethical review by the University of Nottingham were appropriate and conducted using PPL 40/3412 in accordance with the UK Home Office guidance and under ASPA (1986). For the generation of murine T-cell subsets, spleens were removed from C57B1/6 mice and single cell suspensions prepared. CD4.sup.+CD62L.sup.+ nave cells were isolated using Miltenyi Biotec microbeads. Nave human CD4.sup.+ T-cells were prepared using the Miltenyi Biotec nave CD4.sup.+ T-cell isolation kit II, which are sorted produced a 99% pure population of CD4.sup.+CD45RA.sup.+ cells. Purified cells were cultured in complete IMDM media all with -CD3 (1 g/ml), -CD28 (2 g/ml), and as follows; Th1 cells IL-12 (10 ng/ml) with -IL-4 (10 g/ml), Th2 cells IL-4 (10 ng/ml) and -IFN- (10 g/ml), Th17 cells I1-6 (10 ng/ml), TGF-1 (2 ng/ml), -IFN- (10 g/ml) with -IL-4 (10 g/ml), and Tregs IL-2 (10 ng/ml) TGF-1 (5 ng/ml), -IFN- (10 g/ml) and -IL-4 (10 g/ml). Cells were converted to T-cell subsets over five days as outlined above. All cytokines were supplied by PeproTech (London, UK). Mouse CD3 (clone 2C11); human CD3 (UCHT1); mouse CD28 (37.51); human CD28 (CD28.2); mouse IL-4 (11B11); human IL-4 (MP4-25D2); mouse IFN- (XMG1.2); human IFN- B27 came from BD Pharmigen.

In Vivo Administration of cl-CD95L

(18) Female C57BL/6 mice (Harlan UK) aged between 8-10 weeks were placed in groups of 6 and administered IP. Twenty-four hours following injection, mice were sacrificed and periteonial cavities were washed with 5 ml of PBS/2% FCS, blood smears were prepared, and spleens were collected. Blood smears and cytospins of periteonal cells (PECs) were stained with Giemsa and differential counts performed. Single cell suspensions of spleens and PECs were prepared, cell counts performed, CD4+CD62-T-cells were isolated with Miltenyi microbeads and number of cells determined by trypan blue exclusion. For experiments where animals received TAT-mCID or control peptides, 800 g (40 mg/kg) was injected IP 2 hrs prior to administration of cl-CD95L. All mouse experiments were performed under ethical approval from the University of Nottingham local animal ethics committee and adhering to UK Home Office guidelines under the Project License 40/3412.

Metalloprotease-Cleaved and Ig-fused CD95L Production

(19) Ig-CD95L was generated in the laboratory as described in (Tauzin et al., 2011). HEK 293 cells maintained in an 8% FCS-containing medium were transfected using Calcium/Phosphate precipitation method with 3 g of empty plasmid or wild type CD95L-containing vector. 16 hours after transfection, medium was replaced by OPTI-MEM (Invitrogen) supplemented with 2 mM L-glutamine and 5 days later, media containing cleaved CD95L and exosome-bound full length CD95L were harvested. Dead cells and debris were eliminated through two steps of centrifugation (4500 rpm/15 minutes) and then exosomes were eliminated by an ultracentrifugation step (100000 g/2 hours).

Size Exclusion Chromatography

(20) Sera from 4 different SLE patients (5.10.sup.7 cells) were filtrated using a 0.2 m filter and then 5 ml was resolved using a mid range fractionation S300-HR Sephacryl column (GE Healthcare) equilibrated with PBS (pH 7.4). Using an AKTAprime purifier apparatus (GE Healthcare), fractions were harvested with a flow rate of 0.5 mL/min. Fifty fractions were harvested and analyzed by ELISA to quantify CD95L.

CD95L ELISA

(21) Anti-CD95L ELISA (Diaclone, Besanon, France) was performed to accurately quantify the cleaved-CD95L present in sera following the manufacturer's recommendations.

Immunoprecipitation

(22) T-cells (510.sup.7 cells per condition) were stimulated with Ig-CD95L or cl-CD95L (100 ng/mL) for indicated times at 37 C. Cells were lysed, incubated with APO1.3 (1 ug/mL) for 15 min at 4 C. and CD95 was immunoprecipitated using A/G protein-coupled magnetic beads (Ademtech, Pessac, France) for 1 h. After extensive washing, the immune complex was resolved by SDS-PAGE and immunoblotting was performed with indicated antibodies.

Immunoblot Analysis

(23) Cells were lyzed for 30 minutes at 4 C. in lysis buffer (25 mM HEPES pH 7.4, 1% v/v Triton X-100, 150 mM NaCl, 2 mM EGTA supplemented with a mix of protease inhibitors (Sigma-Aldrich)). Protein concentration was determined by the bicinchoninic acid method (PIERCE, Rockford, Ill., USA) according to the manufacturer's protocol. Proteins were separated on a 12% SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare, Buckinghamshire, England). The membrane was blocked 15 minutes with TBST (50 mM Tris, 160 mM NaCl, 0.05% v/v Tween 20, pH 7.8) containing 5% w/v dried skimmed milk (TBSTM). Primary antibody was incubated overnight at 4 C. in TBSTM. The membrane was intensively washed (TBST) and then the peroxydase-labeled anti-rabbit or anti-mouse (SouthernBiotech, Birmingham, Ala., US) was added for 45 minutes. The proteins were visualized with the enhanced chemiluminescence substrate kit (ECL, GE Healthcare).

Transendothelial Migration of Activated T Lymphocytes

(24) After hydration of the Boyden chamber membranes containing 3 pore size membranes (Millipore, Molsheim, France), activated T-lymphocytes (10.sup.6) were added to the top chamber on a confluent monolayer of HUVEC in a low serum (1%)-containing medium. The bottom chamber was filled with low serum (1%)-containing medium in presence or absence of 100 ng/ml of cl-CD95L. In experiments using human sera, 500 l of serum from either healthy donors or SLE patients was added in the lower reservoir. Cells were cultured for 24 h at 37 C. in a 5% CO2, humidified incubator. Transmigrated cells were counted in the lower reservoir by flow cytometry using a standard of 2.510.sup.4 fluorescent beads (Flow-count, Beckman Coulter).

Endothelial Cell Adhesion Assay

(25) Blocking antibodies were used against E-selectin and ICAM-1 in the CHEMICON endothelial cell adhesion assay (Millipore). Briefly, after activation of the endothelial cell layer with TNF-, anti-mouse Ig controls, anti-E-selectin or anti-ICAM-1 were added at final concentrations of 10 g/ml. Thereafter calcein-AM-stained T-cell subsets were incubated for 24 hours and unbound cells are washed. Cells attached to the endothelium were assessed using fluorescence plate reader.

Real-Time qPCR

(26) Single cell suspensions of spleens and PECs were prepared as described above. RNA was extracted from CD4 T-cells using phenol/chloroform. cDNA was prepared using the Promega GO-Script Reverse Transcription Kit and used in Real-Time PCR. Briefly, cDNA samples were subject to Taqman assay performed on a Roche Lightcycler. Results are reported as expression levels were calculated using the ct method relative to HPRT.

Video Imaging of the Calcium Response in Living Cells

Experiments on Parent Cell Lines

(27) T cells were loaded with Fura-PE3-AM (1 M) at room temperature for 30 min in Hank's Balanced Salt Solution (HBSS). After washing, the cells were incubated for 15 min in the absence of Fura-PE3-AM to complete de-esterification of the dye. Cells were placed in the temperature controlled chamber (37 C.) of an inverted epifluorescence microscope (Olympus IX70) equipped with an 40 UApo/340-1.15 W water-immersion objective (Olympus), and fluorescence micrograph images were captured at 510 nm and 12-bit resolution by a fast-scan camera (CoolSNAP fx Monochrome, Photometrics). To minimize UV light exposure, a 44 binning function was used. Fura-PE3 was alternately excited at 340 and 380 nm, and the ratios of the resulting images (emission filter at 520 nm) were produced at constant intervals (10 seconds). The Fura-PE3 ratio (F.sub.ratio 340/380) images were analyzed offline with Universal Imaging software, including Metafluor and Metamorph. F.sub.ratio reflects the intracellular Ca.sup.2+ concentration changes. Each experiment was repeated 3 times, and the average of more than 20 single-cell traces was analyzed.

Experiments on GFP-Expressing Cell Lines

(28) Fluo2-AM was used, instead of Fura-PE3-AM for experiments with GFP-expressing cells, because GFP fluorescence disturbs Ca.sup.2+ measurement with Fura-PE3. As for Fura-PE3-AM, T cells were loaded with Fluo2-AM (1 M) for 30 min in Hank's Balanced Salt Solution (HBSS) and then incubated for 15 min in the Fluo2-AM free HBSS to complete de-esterification of the dye.Ca.sup.2+ changes were evaluated by exciting Fluo2-AM-loaded cells at 53535 nm. The values of the emitted fluorescence (60550 nm) for each cell (F) were normalized to the starting fluorescence (F.sub.0) and reported as F/F.sub.0 (relative Ca.sup.2+.sub.[CYT]). Only GFP-positive cells were considered.

Results

Serum CD95L in Lupus Patients Promotes Endothelial Transmigration of Activated Th17 Cells

(29) Recent reports suggest that a soluble form of CD95L increases in bronchoalveolar lavage fluid of patients suffering from acute respiratory distress syndrome (ARDS). Surprisingly, this soluble CD95L conserves its amino-terminal extracellular stalk region (amino acid residues 103 to 136), a region normally eliminated after shedding by metalloprotease of the membrane-bound CD95L (Herrero et al., 2011). Additionally under native conditions, this ARDS CD95L exhibited a hexameric stoichiometry and exerted a cytotoxic activity towards alveolar epithelial cells in lungs (Herrero et al., 2011). These results encouraged us to evaluate the stoichiometry of serum CD95L in SLE patients. First, we confirmed that soluble CD95L was significantly increased in the sera of 34 SLE patients as compared to 8 age-matched healthy donors (360224.8 pg/ml in SLE patients vs 30.0428.52 pg/ml in healthy subjects, P<0.0001) (FIG. 5A). Second, these sera were fractionated using size-exclusion chromatography and CD95L concentration was quantified in each eluted fraction (FIG. 5B). CD95L was detected in fractions 76 to 78, that contained proteins whose native molecular mass ranged between 75 and 80 kDa. This CD95L was next immunoprecipitated and resolved under denaturing/reducing conditions (SDS-PAGE) at 26 kDa (FIG. 5B) indicating that the serum CD95L accumulated in lupus patients corresponded to a homotrimeric ligand. Upon examination, we noted that functionally this serum CD95L retained the previously reported activity of cleaved-CD95L (cl-CD95L), as it promoted the transmigration of T lymphocytes across an endothelial monolayer (FIG. 5C). Specifically, significantly more activated T lymphocytes isolated from healthy donors exposed to fractions 76-78 crossed endothelial monolayers in comparison to lymphocytes exposed to fractions 42-44. These latter fractions, which contain exosome-bound CD95L (data not shown) failed to exert any pro-migratory effect (FIG. 5C). Furthermore, T-cell transmigration induced by fractions 76-78 was inhibited by up to 50% using a neutralizing anti-CD95L mAb (FIG. 5C) confirming that soluble CD95L in SLE patients plays a role in the endothelial transmigration of T lymphocytes. If T-cell infiltration is involved in tissue damage and Th17 cells contribute to this clinical outcome through a CD95-driven recruitment, we assumed that CD95L-expressing cells should be detected in the inflamed organs. Using immunohistochemistry, we evaluated the distribution of CD95L and IL17-expressing cells in lupus patients with skin lesions. Of note, CD95L and IL17 staining were observed in skin biopsies of lupus patients while they were undetectable in control skins (i.e., skins from breast reconstruction) (FIG. 5D). Moreover, CD95L was mainly detected on endothelium of blood vessels, which were surrounded by immune cell infiltration (FIG. 5D). Moreover, a densitometric analysis of lupus patients (n=10) highlighted that the amount of CD95L was correlated with the quantity of tissue-infiltrating IL17-expressing immune cells suggesting that this ligand may represent a chemoattractant for CD4.sup.+ Th17 cells (FIG. 5E). To further investigate if after cleavage by metalloprotease, CD95L exerted a chemoattractant activity toward all T-lymphocytes or selectively promoted migration of a sub-population, endothelial transmigration of nave CD4.sup.+ T-cells isolated from healthy donors and subjected to in vitro differentiation was evaluated in presence or absence of healthy or SLE sera. As compared to healthy sera, sera from SLE patients triggered a moderate increase in Th1 transmigration while they dramatically enhanced endothelial transmigration of Th17 cells (FIG. 5F). More importantly, this transmigration process relied on CD95 signaling because pre-incubation of SLE sera with a decoy receptor (CD95-Fc) prevented Th17 cell migration in a dose-dependent manner (FIG. 5G).

(30) Both Th 1 and Th17 T-cells have been reported to accumulate in enflamed organs of lupus patients and lupus-prone mice contributing to disease pathogenesis. To eliminate a putative role played by other serum components in the observed phenomenon, we hereafter used a recombinant and homotrimeric version of CD95L. To this end, HEK 293 cells were transfected with a full-length CD95L-encoding vector and we used the metalloprotease-cleaved CD95L (cl-CD95L) contained in this supernatant (Tauzin et al., 2011). Similarly to serum CD95L in lupus patients, cl-CD95L was more efficient to promote the transmigration of Th1 and Th17 lymphocytes as compared to undifferentiated Th0 and differentiated Th2 cells (FIG. 5H). As imbalance of the Th17/T-regulatory (Treg) cell ratio in enflamed organs has been suggested to participate in autoimmune disorders and specifically lupus pathogenesis (Yang et al., 2009), we next evaluated the effect of cl-CD95L on the transmigration of Treg cells. As shown in FIG. 5I, cl-CD95L enhanced endothelial transmigration of Th17 T cells but failed to induce significant Treg transmigration indicating that the accumulation of Th17 cells at the expense of Treg cells in the inflamed tissues of lupus patients. These findings revealed that the higher levels of serum CD95L in SLE patients as compared to healthy donors could contribute to the accumulation of Th17 cells in inflamed organs.

(31) Cellular recruitment and trafficking can be controlled by expression levels of adhesion molecules on lymphocytes and their molecular partners on endothelial cell surfaces. The expression of these molecules during an inflammatory response is a dynamic process, which increases or decreases the extravasation of immune cells into tissues. Recently, Th17 cells have been shown to accumulate in organs as a result of their interaction with E-selectin during rolling and ICAM-1-dependent arrest on activated endothelium (Alcaide et al., 2012). To address if these molecules contributed to the CD95-mediated endothelial T-cell migration of Th17 cells, we evaluated the expression level of key adhesion molecules on endothelial cells and differentiated Th cells in presence or absence of cl-CD95L. Of note, while an important amount of E-selectin was observed at the surface of HUVECs, no P-selectin was detected in these cells. Moreover, cl-CD95L did not alter the expression level of different adhesion molecules on HUVEC. By contrast, in presence of cl-CD95L, Th17 cells underwent up-regulation of P-selectin glycoprotein (PSGL-1), a ligand of E- and P-selectin, and ICAM-1 binding partner LFA-1. The expression level of these ligands remained unaffected in Th1 cells and tended towards a down-regulated state in Treg cells. Functionally the impact of PSGL-1 up-regulation in cl-CD95L-stimulated Th17 cells was evaluated by use of an E-selectin neutralizing mAb. Anti-E-selectin inhibited more efficiently Th17 cell transmigration when compared to similarly treated Th1 cells. Conversely blockade of ICAM-1/LFA-1 interactions by anti-ICAM-1 mAb impaired to a lesser extent both Th1 and Th17 cell migration across endothelial cells. These findings suggested that cl-CD95L promoted CD95-mediated Th17 cell transmigration by enhancing PSGL-1/E-selectin interaction.

Cl-CD95L Causes In Vivo a Rapid Accumulation of Th17 Cells

(32) To confirm in vivo the chemoattractant ability of cl-CD95L towards Th17 cells, mice were injected intraperitoneally with a single dose of cl-CD95L or vehicle and 24 hours later, composition of T-cells infiltrating the peritoneal cavity (peritoneal exudate cells-PECs) and the spleen was examined. Total cell counts from the PEC and spleen revealed a significant increase in the number of lymphocytes in these compartments as compared to vehicle-injected mice (FIG. 6A-B). Loss of CD62L expression is associated with T-cell receptor engagement. Using this marker, we evaluated the amount of activated CD4.sup.+ T-cells (CD4.sup.+CD62L.sup.) recruited into the spleen and the peritoneal cavity of mice injected with or without cl-CD95L. We observed an increased amount of T cells recruited in the peritoneal cavity and the spleen upon injection of cl-CD95L as compared to control medium (FIG. 6C-D). Moreover, Q-PCR analyses of key markers of the Th17 lineage including IL-17 (FIG. 6E), IL-23R (FIG. 6F), and CCR6 (FIG. 6G), performed on these activated CD4+ T cells showed that cl-CD95L induced the recruitment of Th17 cells in these tissues. Furthermore, there was no increase in levels of IFN- (Th1 cells) and FoxP3 (Treg) levels upon examination (FIG. 6H-I) strongly supporting that cl-CD95L acted primarily as a potent chemotactic ligand to Th17 T cells.

CD95 Triggers a Death Domain-Independent Ca2+ Response

(33) We recently showed that CD95 engagement evoked a Ca.sup.2+ response in activated T lymphocytes that transiently inhibited the apoptotic signal (Khadra et al., 2011) and promoted cell motility (Tauzin et al., 2011). These observations raised the question of whether inhibition of this CD95-mediated Ca.sup.2+ response can simultaneously inhibit cell migration and enhance or at least unalter the apoptotic signal. T-cells exposed to cl-CD95L rapidly formed a molecular complex containing the phospholipase C1 (PLC1) (FIG. 7A). Of note, the lack of this lipase in the T-cell line Jurkat caused a loss of the CD95-mediated Ca.sup.2+ signal, while reconstitution of these cells with wild type PLC1 restored a calcium response similar to that of the parental T-cell line (FIG. 7B). Next, we investigated if the main components of the DISC were instrumental in the CD95-mediated calcium signal. To this end, the calcium signal was assessed in FADD- and caspase-8-deficient Jurkat cells stimulated with cl-CD95L (FIG. 7C). Interestingly, although elimination of these molecules blocked the transmission of the apoptotic signaling pathway, it did not affect the CD95-mediated Ca.sup.2+ signal (FIG. 7C) indicating that PLC1 activation occurred independently of the DISC formation and the implementation of cell death signal. These data prompted us to analyze if the CD95-DD itself was necessary to trigger the Ca.sup.2+ response. CD95 constructs devoid of either the entire intracellular domain (CD95.sup.1-175), the DD (CD95.sup.1-210) or only the last 15 amino acids involved in the FAP-1 recruitment (CD95.sup.1-175) were generated (FIG. 7D). Protein-tyrosine phosphatase FAP-1 is reported to interact with the carboxyl terminal 15 amino acids of CD95 (Sato et al., 1995) and prevent its export from the cytoplasm to the cell surface (Ivanov et al., 2003). These constructs were expressed in a T-cell line selected for its low expression level of CD95 namely CEM-IRC ((Beneteau et al., 2008). While CEM-IRC cells showed a trivial sensitivity to cytotoxic CD95L, expression of CD95.sup.1-303 or wild type CD95 in CEM-IRC cells to a level similar to that of endogenous CD95 in parental CEM cells restored the transmission of the apoptotic signaling pathway. By contrast, high levels of CD95.sup.1-175 or CD95.sup.1-210 failed to induce cell death and as previously observed behave as dominant-negative receptors (Siegel et al., 2000). Also, reconstitution of CEM-IRC cells with wild type CD95 and CD95.sup.1-303 restored the CD95-mediated Ca.sup.2+ signal (FIG. 7E). Strikingly, while the loss of the death domain in the CD95.sup.1-210 construct prevented the implementation of the apoptotic signal, it did not affect the induction of the Ca.sup.2+ signal (FIG. 7E). Given that a CD95 construct devoid of its whole intracellular region failed to evoke a Ca.sup.2+ response, we concluded that the Ca.sup.2+ response stems from the first 36 amino acids in the CD95 intracellular region. To confirm that amino acid residues 175 to 210 of CD95 were responsible for the Ca.sup.2+ response, we determined if this domain was capable to interact with PLC1. To this end, GFP-fused CD95 constructs and wild type PLC1 were first transiently transfected in HEK cells, cells were stimulated with CD95L, lyzed and the immune complex associated with CD95 was analyzed by immunoblotting. Although cells expressed similar levels of the different CD95 chimeric constructs (FIG. 8A), the presence of PLC1 in the CD95 immunoprecipitate was only lost with the CD95.sup.1-175 construct (FIG. 8B), while both CD95.sup.1-210 and CD95.sup.1-175 lost their capacity to recruit the adaptor protein FADD. Interestingly, a CD95 construct devoid of DD showed a higher binding capacity for PLC1 as compared to wild type CD95 (FIG. 8B) suggesting that this region may structurally or functionally interfere with the PLC1 binding to the 175-210 domain. Second, we generated a construct consisting of amino acids 175 to 210 that we designated calcium-inducing domain (CID) fused to mCherry. Unlike mCherry alone, CD95.sup.(175-210)-mCherry interacted with PLC1 and inhibited its recruitment to CD95 (FIG. 8C) indicating that interference with this juxtamembrane domain may represent a way to prevent the CD95-mediated Ca.sup.2+ signal. Finally, to confirm this hypothesis, we synthesized a cell penetrating peptide linking the 36-amino acid-stretch of CID to the 9-amino acid HIV-TAT sequence (FIG. 8D), which serves as carrier to translocate the whole protein across plasma membrane (Vives et al., 1997). Pre-incubation of the T cell lines Jurkat and CEM with the TAT-CID peptide impaired PLC1 recruitment (FIG. 8) and abolished the induction of the CD95-mediated Ca.sup.2+ signal (FIG. 8E). Similarly, pre-incubation of activated T lymphocytes from healthy subjects with the TAT-CID inhibited the PLC1 binding to CD95 (FIG. S4A) and abrogated the CD95-mediated Ca.sup.2+ response, in a similar way to xestospongin C, an antagonist of the calcium-releasing action of inositol-1,4,5-trisphosphate (IP3), the substrate generated by PLC1 activation (FIG. 8F). Moreover, TAT-CID pre-incubation inhibited Akt phosphorylation at its serine 473 (a hallmark of the PI3K signaling pathway activation) in PBLs exposed to cl-CD95L. Of note, although TAT-CID treatment inhibited the CD95-mediated Ca.sup.2+ and PI3K signals, it did not affect the execution of the apoptotic signaling pathway. In conclusion, we mapped a novel domain in CD95 designated calcium-initiating domain that recruited PLC1 and elicited the Ca.sup.2+ response.

(34) Because cysteine at position 183 is subject to palmitoylation promoting CD95 aggregation (Feig et al., 2007) and its redistribution into lipid raft (Chakrabandhu et al., 2007), we wondered whether this amino acid was instrumental in the implementation of the CD95-mediated Ca.sup.2+ signal. To address this question and yet avoid any interference of the apoptotic signaling in the CD95-mediated Ca.sup.2+ response, we reconstituted CEM-IRC cells with a CD95.sup.1-210 (no death domain) in which cysteine 183 was replaced by a valine. Both CD95.sup.1-210 and CD95.sup.1-210(C183V) failed to trigger cell death in presence of Ig-CD95L, but they evoked a similar Ca.sup.2+ response suggesting that the mechanism of palmitoylation was not instrumental in inducing this cue. To confirm this observation, a TAT-CID peptide was synthesized in which cysteine was replaced by a valine. Pre-incubation of Jurkat cells and activated PBLs with this mutated peptide still inhibited the CD95-mediated Ca.sup.2+ response confirming that this cysteine did not contribute to the Ca.sup.2+ response in cells exposed to cl-CD95L. Finally, we evaluated if the inhibitory effect of the TAT-CID was selective of the CD95-mediated Ca.sup.2+ signal. Of note, although TCR stimulation led to a PLC1-dependent Ca.sup.2+ response, TAT-CID pretreatment did not alter this signal. Similarly, the PLC-driven Ca.sup.2+ response evoked by carbachol, a cholinergic agonist known to evoke a Ca.sup.2+ response through activation of G-protein-coupled receptors, was not affected by TAT-CID treatment. These findings indicated that the TAT-CID peptide exerted a selective inhibition of the CD95-mediated calcium signal.

Inhibition of the CD95-mediated Ca2+ Signal Prevents Th17 Cell Transmigration and Alleviates Clinical Signs in Lpr Mice

(35) To address if TAT-CID regimen may represent a therapeutic strategy in lupus, we first evaluated its effect in Th17 cell transmigration. As shown in FIG. 9A, TAT-CID inhibited the CD95-mediated endothelial transmigration of human Th17 cells in a dose-dependent manner. Alignment of human and mouse CD95 proteins indicated a sequence divergence in the CID region suggesting that the human CID (TAT-hCID) may turn out to be inefficient to prevent the Ca.sup.2+ response induced in mouse T cells. To determine the inhibitory activity of TAT-hCID on the Ca.sup.2+ response induced by murin CD95, we first reconstituted CEM-IRC cells with wild type mouse CD95. Both CD95-mediated apoptotic and Ca.sup.2+ signals were restored in these cells as compared to parental CEM-IRC cells. Importantly, TAT-hCID failed to inhibit the CD95-mediated Ca.sup.2+ response in mouse CD95-expressing CEM-IRC cells. By contrast, replacement of the human CID sequence by its mouse ortholog (TAT-mCID) abolished the CD95-mediated Ca.sup.2+ response in these cells. Similarly, TAT-mCID also inhibited the CD95-mediated Ca.sup.2+ signal in mouse T lymphocytes confirming that despite the divergence between human and mouse CD95-CID sequences (48.9% of sequence identity over the complete human and mouse CD95 sequences vs 21.2% over the two CIDs), these domains retained the property to trigger the Ca.sup.2+ signal in these species.

(36) To further investigate the putative therapeutic activity of TAT-CID and determine whether this peptide exerted an inhibitory effect on Th17 T-cell recruitment in vivo, we injected C57B1/6 mice with 40 mg/kg of TAT-control or TAT-CID two hours prior to the intraperitoneal injection of cl-CD95L and the amount of T-cells infiltrating the peritoneal cavity was evaluated 24 hours later. Total cell counts from the PEC revealed that TAT-CID regimen abolished the CD95-mediated accumulation of T lymphocytes in this compartment (FIG. 9B). In agreement with data shown in FIG. 2, cl-CD95L injection triggered an increased IL17 production in the peritoneal cavity that was prevented by the TAT-CID treatment (FIG. 9C). Furthermore, no differences were noted in levels of IFN- between mice injected with control peptide and TAT-CID peptide highlighting that the preferential recruitment of IL-17 secreting CD4 T-cells by cl-CD95L was abolished in vivo by administration of TAT-CID.

(37) In conclusion, the selective inhibition of the CD95-mediated Ca.sup.2+ signal turned out to be a novel and promising therapeutic strategy to reduce Th17 cell accumulation in inflamed tissues of lupus patient without altering the transmission of the apoptotic signal.

Discussion

(38) Our study provides new insights into the cellular and molecular mechanisms by which metalloprotease-cleaved CD95L enhances inflammation in SLE patients. We show that transmembrane CD95L is ectopically expressed by endothelial cells covering blood vessels in the inflamed skins of lupus patients. More importantly, these CD95L.sup.+ vessels are surrounded by a massive immune infiltrate strongly suggesting that these structures may serve as open doors for pro-inflammatory cells among which Th17 cells. Exposed to cl-CD95L, these IL17-expressing cells up-regulate PSGL-1 and LFA-1, two adhesion molecules involved in rolling and tethering of leukocytes to endothelial cells. Of note, T cells with the highest levels of functional PSGL-1 also show the greatest capacity for effector cytokine secretion and for cytotoxic activity (Baaten et al., 2013). Therefore, cl-CD95L may fuel the inflammatory process not only by promoting the recruitment of activated Th1 and Th17 cells in inflamed tissues but also by altering the pattern of cytokine release in these organs.

(39) Recently, Coukos and colleagues demonstrated that CD95L is present in blood vessels of certain cancer tissues (i.e., ovary, colon, prostate, kidney) (Motz et al., 2014) and they associated this staining with scarce CD8.sup.+ infiltration. These authors showed that membrane-bound CD95L on endothelial cells eliminated T cells and by doing so, prevented effective anti-tumor immunity (Motz et al., 2014). We evaluated the CD8.sup.+ T-cell infiltration around CD95L-positive blood vessels in lupus patients and densitometric analysis revealed no inverted correlation between the amounts of CD95L and the quantity of infiltrating CD8.sup.+ T cells. Given that CD95L exerts its chemottractant activity only after its cleavage by metalloproteases (Tauzin et al., 2011), we assume that at least in part, the discrepancy in the magnitude of immune infiltrates surrounding CD95.sup.+-blood vessels observed in certain cancers and lupus patients may be caused by the absence or the presence, respectively of a CD95L-processing metalloprotease that remains to be identified.

(40) Our study also uncovers the CD95 residues involved in the implementation of the Ca.sup.2+ signaling pathway. Even if our data show that CID interacts with PLC1 in unstimulated cells (FIG. 8C) suggesting that a direct interaction may occur between CD95 and this lipase, we can not rule out that a third partner participates in this association. For example, a recent study showed that TRIP6 over-expressed in glioblastoma links the CID domain to the NF-kB signaling pathway and thereby promotes CD95-mediated cell migration in these cells (Lai et al., 2010). Nonetheless, the same authors did not detect TRIP6 in Jurkat T-cells and precluded its participation in the non-apoptotic signal triggered in T cells suggesting a tissue specific activity of this molecule (Lai et al., 2010). Within neuronal cells, the juxtamembrane domain of CD95 (amino acid residues 175 to 188) interacts with ezrin an adaptor molecule linking CD95 to the actin network and thereby promotes neurite outgrowth via Rac1 activation and cytoskeletal remodeling (Desbarats et al., 2003). Cl-CD95L induces PLC1 recruitment rapidly (in the order of the minute) and transiently. Given that this signal stems from CID, this juxtamembrane domain of CD95 will require further analysis of its structure activity relationship to understand how it can evoke the Ca.sup.2+ response without implementing the death domain-dependent and caspase-driven apoptotic signaling pathway.

(41) In this regard, the 175-210 amino acid residues of CD95 involved in the execution of the Ca.sup.2+ response has never been crystallized probably due to the fact that this region corresponds to an intrinsically disordered region (IDR) lacking a unique three dimensional structure. Using different molecular dynamic experiments, we confirmed that this peptide has a very faint folding propensity. Computer simulation also showed that the peptide shares another property of IDR: switches between order and disorder states are frequent. Therefore, we surmise that the peptide (or a part of it) may stably fold in the presence of binding partners, starting from a pre-structured region such as helical segments observed by atomistic simulations (Sugase K. et al., Nature, 447, 1021-1025, 2007; Wright & Dyson, Curr. Opin. Struct. Biol., 19, 31-38, 2009). Significantly, IDRs in proteins tend to take a central role in protein interaction networks (Cumberworth et al., 2013). Indeed, these disordered regions can transiently interact with a large number of partners and thereby modulate cell signaling in a dynamic manner. This molecular feature is consistent with the participation of this domain in inducing a rapid and transient Ca.sup.2+ response promoting cell migration.

(42) Also an analysis of mutations within CD95 found in different pathologies revealed that this region exhibits a lower amount of mutations as compared to the adjacent death domain suggesting that in contrast to the DD, accumulation of mutations in this region may not confer a selective advantage in carcinogenesis or contribute to the inflammatory process in ALPS patients. Of note, before the etiology of ALPS type Ia was associated with mutations in CD95 gene, these patients were erroneously diagnosed as SLE patients.

(43) A recent Phase I/II clinical trial found that a decoy receptor (known as APG101) capable of blocking the CD95/CD95L interaction did not show any toxicity in humans suffering from glioblastoma (Tuettenberg et al., 2012). We may envision that this therapeutic agent may, in a short-term period, benefit lupus patients. However, given that this inhibitor does not discriminate between the anti-tumor/infective functions of CD95L (i.e., the apoptotic signal) and its pro-inflammatory activity, it may leads to deleterious side effects precluding its use in these SLE patients. Because the apoptotic and the calcium signals stem from two separate and distant domains in CD95 and that inhibition of the CD95-mediated Ca.sup.2+ response does not prevent the apoptotic signaling pathway (Khadra et al., 2011), we propose that selective inhibition of the CD95-mediated Ca.sup.2+ response will provide an excellent opportunity to block the pro-inflammatory activity of cl-CD95L in certain chronic inflammatory disorders without affecting the anti-tumor and infectious roles of its membrane-bound counterpart.

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