Novel IL-4-/IL-13-derived peptide compounds for the treatment or prevention of neurodegenerative or neuroinflammatory diseases

Abstract

The present invention relates to a compounds consisting of one or more peptides comprising the structure in the following order: A-L1-B-L2-C wherein A corresponds to a first amino acid sequence that is derived from A or C alpha helical region of human or animal IL-4 or IL-13, B corresponds to a second amino acid sequence that is derived from A or C alpha helical region of human or animal IL-4 or IL-13, C corresponds to a third amino acid sequence that is derived from D or B alpha helical region of human or animal IL-4, or D alpha helical region of human or animal IL-13. L1 and L2 correspond to one or more linking amino acids, wherein said compound is capable of stimulating neuronal axon outgrowth.

Claims

1. A compound consisting of one or more peptides consisting of the structure
A-L1-B-L2-C wherein A corresponds to a first amino acid sequence that is derived from A or C alpha helical region of human or animal IL-4 or IL-13, B corresponds to a second amino acid sequence that is derived from A or C alpha helical region of human or animal IL-4 or IL-13, C corresponds to a third amino acid sequence that is derived from D or B alpha helical region of human or animal IL-4, or D alpha helical region of human or animal IL-13, L1 and L2 correspond to one or more linking amino acids.

2. The compound according to claim 1, wherein A corresponds to the amino acids WNR, RAR, LMR, LIR, EIIKT, or EIIGI B corresponds to the amino acids EIIKT, EIIGI, ELIEELVNIT, ELIEELSNIT, RLDRNLWG, or RLFRAFRC C corresponds to the amino acids KTIMREKY, FVKDLLLHLKK, RAATVLRQFYS, KSIMQMDY, FITKLISYTKQ, or RASKVLRIFYL, or a variant thereof having a different amino acid at one position, wherein said peptide or the variant thereof is capable of stimulating neuronal axon outgrowth.

3. The compound according to claim 1, wherein A corresponds to the amino acids WNR, B corresponds to the amino acids EIIKT, C corresponds to the amino acids KTIMREKY, or a variant thereof having a different amino acid at one position, wherein said peptide or the variant thereof is capable of stimulating neuronal axon outgrowth.

4. The compound according to claim 1, wherein A corresponds to the amino acids LMR, B corresponds to the amino acids ELIEELVNIT, C corresponds to the amino acids FVKDLLLHLKK, or a variant thereof having a different amino acid at one position, wherein said peptide or the variant thereof is capable of stimulating neuronal axon outgrowth.

5. The compound according to claim 1, wherein A corresponds to the amino acids EIIKT, B corresponds to the amino acids RLDRNLWG, C corresponds to the amino acids RAATVLRQFYS, or a variant thereof having a different amino acid at one position, wherein said peptide or the variant thereof is capable of stimulating neuronal axon outgrowth.

6. The compound according to claim 1, wherein L1 and/or L2 correspond to S, GS, SGS, P, GP or PGP.

7. The compound according to claim 1, wherein the peptide is a human IL-4 derivative comprising an amino acid sequence WNRSEIIKTGSKTIMREKY (SEQ ID NO: 1), or a variant thereof having a different amino acid at one or more positions, wherein said peptide or the variant thereof is capable of stimulating neuronal axon outgrowth.

8. The compound according to claim 1, wherein the peptide is a human IL-13 derivative comprising an amino acid sequence LMRSELIEELVNITGSFVKDLLLHLKK (SEQ ID NO: 2), or a variant thereof having a different amino acid at one or more positions, wherein said peptide or the variant thereof is capable of stimulating neuronal axon outgrowth.

9. The compound according to claim 1, wherein the peptide is a human IL-4 derivative comprising an amino acid sequence EIIKTGSRLDRNLWGSGSRAATVLRQFYS (SEQ ID NO: 3), or a variant thereof having a different amino acid at one or more positions, wherein said peptide or the variant thereof is capable of stimulating neuronal axon outgrowth.

10. The compound according to claim 1, wherein the peptide is a murine IL-4 derivative comprising an amino acid sequence RARSEIIGIGSKSIMQMDY (SEQ ID NO: 4), or a variant thereof having a different amino acid at one or more positions, wherein said peptide or the variant thereof is capable of stimulating neuronal axon outgrowth.

11. The compound according to claim 1, wherein the peptide is a murine IL-13 derivative comprising an amino acid sequence LIRSELIEELSNITGSFITKLLSYTKQ (SEQ ID NO: 5), or a variant thereof having a different amino acid at one or more positions, wherein said peptide or the variant thereof is capable of stimulating neuronal axon outgrowth.

12. The compound according to claim 1, wherein the peptide is a murine IL-4 derivative comprising an amino acid sequence EIIGIGPRLFRAFRCSGSRASKVLRIFYL (SEQ ID NO: 6), or a variant thereof having a different amino acid at one or more positions, wherein said peptide or the variant thereof is capable of stimulating neuronal axon outgrowth.

13. A pharmaceutical composition comprising at least one compound according to claim 1 and a pharmaceutically suitable carrier, vehicle or agent.

14. A compound according to claim 1 for use in the treatment or prevention of a neuroinflammatory or neurodegenerative disorder.

15. A compound according to claim 1 for use in the treatment or prevention of neuropathies or traumatic nervous system injuries.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0082] The present invention is further illustrated in the following examples:

Design of IL-4 Derivatives and IL-13 Derivatives

[0083] The IL-4 derivatives and IL-13 derivatives of the invention are compounds that are defined by the structure in the order A-L1-B-L2-C. The inventors have tested various IL-4 derivatives that they derived from different a helical regions of human and murine IL-4 and which are composed in accordance with the above structure. These newly generated IL-4 derivatives are named as Link4 and AvoC herein.

[0084] The inventors also tested various IL-13 derivatives that are derived from the a helical regions of human and murine IL-13 and that are composed in accordance with the above structure. These newly generated IL-13 derivatives are named Link13 herein.

[0085] In FIG. 1, the design of the derivatives Link4 and Link13 is described. Based on the known 3-D structure of IL-4 and the IL-4 receptor, the inventors combined the amino acid stretches that are required to bind to IL-4R. The inventors then identified which amino acids were located on the surface of the IL-4 protein to decide which combinations of the A to D helices to make. A part of the C helix was turned around and connected with one linking amino acid to a part of the A helix which was bound with two linking amino acids to a part of the D helix. The receptor-binding amino acids have been identified and coupled, which yielded IL-4 and IL-13 derivatives in which the co-receptor chains were linked to each other. The derivatives of IL-4 and IL-13 were designed in a similar manner based upon the sequence homology of IL-4 and IL-13.

[0086] The IL-4 derivative AvoC was designed based upon another binding principle of IL-4 to its receptor. The binding sequence of IL-4 is defined by so-called avocado clusters in which a core is surrounded by hydrophobic residues (T. D. Mueller, J. L. Zhang, W. Sebald, A. Duschl, Structure, binding and antagonists in the IL-4/IL-13 receptor system, Biochimica Biophysica Acta 1592, 237-250 (2002)). Stretches of amino acids were arranged in such a way to mimic the surface structure of IL-4. Also this derivative is likely to link the two receptor chains, since the sequence derived from the A helix binds to both receptor chains.

[0087] FIG. 1A shows the amino acid sequences of human IL-4 (SEQ ID NO: 23) and murine IL-4 (SEQ ID NO: 24) aligned with the location of the a-helices A, B, C, D and the -linkers. Black arrowheads indicate binding sites to the IL-4Ra chain, grey arrowheads point to binding sites to the co-chains, either IL-13R1 or common -chain. The amino acids in bold letters are the amino acids that are used for the construction of the IL-4 derivatives of the present invention as shown in FIG. 1B and 10.

[0088] FIG. 1B shows the IL-4 derivative Link4 with the selected amino acids from the C, A and D helical regions in bold letters.

[0089] FIG. 10 shows the IL-4 derivative AvoC with the selected amino acids from the A, C, and B helical regions in bold letters.

[0090] FIG. 1D shows the aligned amino acid sequences of human IL-13 (SEQ ID NO: 25) and murine IL-13 (SEQ ID NO: 26) comprising the -helices A, B, C, D. The amino acids used to design the IL-13 derivative Link13 of the present invention are depicted.

[0091] FIG. 1E shows the IL-13 derivative Link13 with the selected amino acids from the C, A and D helical regions in bold letters.

[0092] FIG. 1F shows the sequence similarities of the mouse and human IL-4 and IL-13 derivatives of the present invention. Whereas full length IL-4 has only approximately 40% homology between the mouse and human variants, mouse and human Link4 share 68.4% similarity, AvoC shares 61%, and Link13 shares 81% similarity.

[0093] FIG. 2 shows a toxicity assay for mouse embryonic fibroblasts (Mef) (FIG. 2A) and hippocampal neurons (HT22) (FIG. 2B). Toxicity was checked by subjecting mouse embryonic fibroblasts (Mef) and the hippocampal cell line HT22 to a CellTiter-blue cell viability assay (ctb). Link4 and AvoC, applied in increasing concentrations, had no toxic effects on fibroblasts (FIG. 2A) or hippocampal neurons (FIG. 2B).

[0094] FIG. 3 shows the in vivo effects of the IL-4 derivative Link4. Treatment of mice during the chronic phase of EAE (grey bar) via lumbar (A) intrathecal injection or (B) nasal application showed a significant improvement in the clinical score of IL-4- and Link4-treated animals, as compared to PBS controls that remained at a high sickness level. Also shown is the lumbar treatment with the Ph8 peptide which had no effect on the disease course.

[0095] To test whether Link4 has similar effects to IL-4 in vivo, two pilot experiments were performed. In the first experiment (FIG. 3A), B16 mice were subjected to EAE and treated with IL-4, Link4, Ph8 and PBS (n=6-7 animals per group) via lumbar intrathecal injection directly into the cerebrospinal fluid (CSF) every other day for 14 days, in the chronic phase of the disease starting from day 5 after the first disease peak (grey bar). The treatment with Link4 led to a significant amelioration of the clinical score, similar to IL-4, whereas the known partial agonist Ph8 did not improve clinical signs. For better clinical translation, the treatment was then switched to nasal treatment with IL-4 and Link4 (FIG. 3B; 2 pooled experiments, n=15-16 per group). Nasally applied treatments can enter the brain through the cribriform plate, where olfactory nerve fibers cross through the bone, resulting in a connection between the roof of the nasal cavity and the brain (C. F. Xiao, F. J. Davis, B. C. Chauhan, K. L. Viola, P. N. Lacor, P. T. Velasco, W. L. Klein, and N. B. Chauhan, Brain transit and ameliorative effects of intranasally delivered anti-amyloid-beta oligomer antibody in 5XFAD mice. J Alzheimers Dis 35, 777-788 (2013)). Again, both IL-4 and Link4 treatments were beneficial.

[0096] FIG. 4 shows the effects of the inventive IL-4 and IL-13 derivatives on the immune system. A-B: Effects of incubation with IL-4 and different concentrations of Link4 and Link13 on the differentiation of A: CD11b.sup.+ bone marrow-derived macrophages (BMDMs) into F80.sup.+CD206.sup.+ cells, and B: CD4.sup.+ T cells into Gata3.sup.+ cells. C-D: FACS results for different populations of T cells and CD11b.sup.+MHCII.sup.+ monocytes/macrophages/microglia of immune cells isolated from C: spleen and D: central nervous system (CNS) of EAE mice treated with PBS, IL-4 or Link4.

[0097] In order to investigate whether Link4 and Link13 are able to modulate immune cells, in vitro assays were performed on bone marrow-derived macrophages (BDMBs) and on nave T cells. Treatment of BDMBs with IL-4 increased the expression of the differentiation markers F80 and CD206 on CD11b.sup.+ BMDMs. This differentiation did not occur in response to Link4 or Link13 (FIG. 4A). The expected differentiation into Gata3.sup.+ CD4.sup.+ T cells observed with IL-4 again did not take place when cells were incubated with Link4, or Link13 (FIG. 4B). To rule out that the Link-analogues were needed in a higher concentration than IL-4, the assays were performed with increasing amounts, which did not make a difference.

[0098] Subsequently, the effects of IL-4 and Link4 on immune cells were analysed when applied intranasally, similarly as in FIG. 3B. Since nasally applied treatments can enter the periphery (lungs, blood stream) it was of utmost importance to check whether the substances affected the lymphocyte populations in the spleen. A reduction was found of CD8.sup.+ cytotoxic T-cells for Link4, whereas all CD4.sup.+ T helper cell types were unchanged. The CD11 b+MHCII+ population of monocytes/macrophages was also not changed. For the populations of lymphocytes entering the brain and spinal cord (taken together as CNS, central nervous system), only a significant rise in IL-10.sup.+ cells was observed.

[0099] FIG. 5 shows the effects of the IL-4 and IL-13 derivatives of the invention on neurons. A: Cortical outgrowth in response to 50 ng/ml IL-4, Link4, or Link13, compared to PBS control. The increase in growth at 48 h was calculated over the basal (untreated) growth at 24 h. B: Effects on phosphorylation of signalling molecules after 10 min treatment of dissociated cortical neurons with IL-4, Link4 or PBS. Link4 showed the same neuronal signalling effects as IL-4.

[0100] FIG. 6 shows that human Link4 is equally effective as mouse Link4 at reducing clinical symptoms in a mouse EAE model. The inventors performed EAE experiments and observed that human Link4 is equally effective as mouse Link4 and mouse full length IL-4 in reducing disease symptoms in the mouse. These results are indicative that the derivatives of the present invention can be used across species. Treatment of mice during the chronic phase of EAE (grey bar) via lumbar injection showed a significant improvement in the clinical score.

[0101] FIG. 7 shows the neuroregenerative actions of Link4 in mouse and human

[0102] A: Histological analysis of corticospinal tract axons in spinal cords of EAE mice treated during the chronic phase of the disease. Mouse Link4 significantly reduced the number of axon swellings.

[0103] B: Human Link4 induced neurite outgrowth in human H9 cell-derived neurons. Human neurons were cultivated with commercially available human neural stem cells (H9 cells), which were then differentiated to become neurons. Human Link4 was able to increase neurite outgrowth of these cells in a similar way as full length IL-4. The growth assay for human H9 cells described herein can be used to easily detect effects of the derivative peptides of the present invention on human neurons. In particular, this assay can be used to quickly test whether derivative peptides falling within the scope of the present invention are functional.

[0104] Description of Preferred Embodiments:

Materials and Methods

Experimental Autoimmune Encephalomyelitis (EAE)

[0105] For active EAE, female 9-10-weeks-old C57BI6 mice were immunized as previously described (M. Paterka, J. O. Voss, J. Werr, E. Reuter, S. Franck, T. Leuenberger, J. Herz, H. Radbruch, T. Bopp, V. Siffrin, and F. Zipp, Dendritic cells tip the balance towards induction of regulatory T cells upon priming in experimental autoimmune encephalomyelitis. J Autoimmun 67, 108-114 (2017)) by subcutaneous injection of 200 g myelin oligodendrocyte protein 35-55 (MOG.sub.35-55) mixed with complete Freund adjuvant (CFA). Following the MOG.sub.35-55 immunization 400 ng pertussis toxin (PTX) was administered intraperitoneally at the day of immunizaton and after 24 h. Clinical signs were scored using the following parameters:

TABLE-US-00001 Score Signs of EAE 0 no detectable signs 0.5 tail weakness 1 complete tail paralysis 2 partial hind limb paralysis 2.5 unilateral complete hind limb paralysis 3 complete bilateral limb paralysis 3.5 complete hind limb paralysis and partial forelimb paralysis 4 total paralysis of forelimbs and hind limbs 5 death

[0106] Treatment with rIL-4 (1 g, Peprotech), Ph8 (1 g, custom synthesized, Schafer N), Link4 (1 g, custom synthesized, Schafer N) or vehicle (PBS) was performed during the chronic phase of the disease models by lumbar intrathecal injection (R. Lu and A. Schmidtko, Direct intrathecal drug delivery in mice for detecting in vivo effects of cGMP on pain processing. Methods Mol Biol 1020, 215-221 (2013)) or nasal application of IL-4 and Link4 was performed according to published procedures (C. F. Xiao, F. J. Davis, B. C. Chauhan, K. L. Viola, P. N. Lacor, P. T. Velasco, W. L. Klein, and N. B. Chauhan, Brain transit and ameliorative effects of intranasally delivered anti-amyloid-beta oligomer antibody in SXFAD mice. J Alzheimers Dis 35, 777-788 (2013)). Mice were pre-trained to avoid stress and held at a 45 degree angle to apply IL-4 solution (1 g, Peprotech) to the nostrils using a pipette tip.

Analysis of Axon Swellings

[0107] EAE experiments were performed by immunizing YFP-H mice, that express yellow fluorescent protein in corticospinal neurons, with MOG.sub.35-55. Mice were treated with IL-4 or PBS as described above. At the last treatment day, the mice were sacrificed with an overdose of anaesthesia and transcardially perfused with 4% paraformaldehyde. Spinal cords were dissected and further processed for cryosectioning. YFP-labelled corticospinal tract axons were photographed using the Keyence BZ-9000 microscope. For quantification of axon swellings, thresholds were set in ImageJ so that stained pixels were highlighted. Thresholded swellings were counted using the ImageJ plug-in particle analysis.

Flow Cytometry (FACS)

[0108] CNS and spleens were removed from EAE mice treated with nasal IL-4, Link4 or PBS at d35 and immune cells were isolated as previously described (M. Paterka, J. O. Voss, J. Werr, E. Reuter, S. Franck, T. Leuenberger, J. Herz, H. Radbruch, T. Bopp, V. Siffrin, and F. Zipp, Dendritic cells tip the balance towards induction of regulatory T cells upon priming in experimental autoimmune encephalomyelitis. J Autoimmun 67, 108-114 (2017)). For in vitro stimulation of T cells, anti-CD3 (145-2C11) and anti-CD28 (37.51) were used. The following antibodies were used for flow cytometry with the BD FACSCanto II (BD Bioscience): anti-CD4 PEc7 (RM4-5), anti-CD8 FITC (53-6.7), anti-CD3APC (145-2c11), anti-CD11 b bio (M1/70), anti-MHCII PE (AF6-120.1), anti-CD11 b V450 (HL311B), anti-CD45 AF605 (30-F11), anti-GM-CSF PE (MP1-22E9), anti-IL17 APC (eBio17B7), anti-TNF AF700 (MP6-XT22), anti-IFN-V450 (XMG1.2), anti-GATA-3 PE (TWAZ), anti-FOXP3 PEcy7(FZK-16s), anti-IL-10 APC (JESS-16E3), anti-CD4 V450 (RM4-5). All antibodies were purchased from eBioscience or Biolegend.

[0109] Isolation of Murine Lymphocytes (CD4+CD62L+)

[0110] For the isolation of T lymphocytes 5-8 week old C57BL/6 mice were sacrificed by cervical dislocation and the spleens and lymph nodes (LN) were isolated and rubbed trough a 100 m cell strainer into a 50 ml tube with washing medium (5% FCS, 1% P/S,1% HEPES in PBS). After washing, the cells were centrifuged for 5 min at 550 g at 4 C. To remove the red blood cells the supernatant was re-suspended in lysis buffer followed by centrifugation (5 min, 550 g, 4 C.). The cells were resuspended in MACS buffer and magnetic lymphocyte sorting was performed on ice, using the MidiMACS and QuadroMACS Seperators. A CD4 untouched and CD8 touched cell sort was performed a washing step in ml MACS buffer (5 min, 550 g, 4 C.). Cells were incubated with CD4 T cell biotin antibody cocktail (Miltenyi Biotec) in MACS buffer for 5 min at 4 C. Subsequently, anti-biotin microbeads and CD8 microbeads in MACS buffer was added. CD8 microbeads were added to reduce the possible contamination of CD8.sup.+ lymphocytes. After 10 min of incubation at 4 C. the cells were washed again and resuspended in MACS buffer. MACS columns were pre equilibrated and topped with pre-separation filters (30 m). A maximum of 30010.sup.6 cells were added to each column followed by 3 consecutive washing steps with MACS buffer. The CD3.sup.+CD4.sup.+CD8.sup. enriched flow-through was collected for the following CD62L touched sort. After centrifugation (5 min, 550 g, 4 C.) CD62L microbeads in MACS buffer were added to the cells and incubated for 15 min at 4 C. After washing the cells were resuspended in MACS buffer and loaded on a new column. The positive labeled CD4.sup.+CD62L.sup.+ cells were enriched in the column and collected in MACS buffer. The purity of the magnetic cells sorts was assessed by comparing pre- and post-sort samples by flow cytometry. The cells were stained with CD4-Horizon (1:400), CD3-APC (1:600) and CD62L-APC (1:200). CD4.sup.+CD62L.sup.+ purity out of the CD4.sup.+ cells was usually about >97%.

Isolation of Murine Antigen-Presenting Cells (APCs)

[0111] Antigen-presenting cells were isolated by lysis of adult C57BU6 mice spleen and magnetic immune cell sorting with MidiMACS and QuadroMACS Separators. The cell suspension was washed with MACS buffer and centrifuged (5 min, 550 g, 4 C.). Subsequently the cells were resuspended in 95 l MACS buffer and CD90.2 untouched microbeads and incubated for 15 min at 4 C. After washing with MACS buffer and centrifugation (5 min, 550 g, 4 C.) the cells were resuspended in MACS buffer, loaded on the columns and collected in MACS buffer. To stop their proliferation cycle the purified APCs were irradiated at 30 Gy/3000 rad.

T.SUB.H.2 Differentiation

[0112] For the initial stimulation the CD4.sup.+CD62L.sup.+ cells were co-cultured with antigen-presenting cells (APCs) in a 1:5 ratio in a concentration of 6 million cells in 2 ml mouse medium (10% FCS, 1% P/S, 1% L-glutamine, 0.1% -mercaptoethanol, 1% HEPES in RPMI buffer) per well on a 24-well plate. For unspecific T cell receptor activation 2 g/ml anti-CD3 was added to the culture. For differentiation into T.sub.H2 cells, IL-4 (10 ng/ml), -IL-12 (10 g/ml) and a-IFN (10 g/ml) were added. At day 3 and 5 the cells were split and plated in fresh medium containing 10 g/ml IL-2 and 10 ng/ml IL-4. At day 7 cells were stimulated with anti-CD3/anti-CD28 and treated with Brefeldin A to block secretion. After 4 hours incubation, a cytokine check of the different T lymphocyte cultures was performed by flow cytometry using CD4-PECy7 (1:1000) for extracellular staining, Fc-receptor-block (1:100) and IFN-g-Horizon (1:200), TNFa-AF700 (1:200) and IL-10-APC (1:200) for intracellular staining. T.sub.H2 cells were additionally stained for intranuclear Gata3-PE (1:100).

Generation of Bone Marrow Derived Macrophages (BMDMs)

[0113] Murine BMDMs were isolated from adult C57BL/6 mice. The tibia and femur bones were flushed with sterile PBS and the bone marrow was collected in washing medium. After filtering the cell suspension trough a 100 m nylon mesh, the cells were centrifuged (5 min, 550 g, 4 C.) and resuspended in mouse medium (10% FCS, 1% P/S, 1% L-glutamine, 0.1% -mercaptoethanol, 1% HEPES in RPMI buffer). For the in vitro generation of BMDMs cells were plated in 6 well-plates and exposed to 20 ng/ml Macrophage stimulating factor (M-CSF) for 4 days. For the activation of the cells, Dexametason (510.sup.7M), LPS (10 g/ml) and IL-4 (10 ng/ml) were added to the culture for the following 3 days.

CellTiter-Blue Cell Viability Assay (ctb)

[0114] Mouse embryonic fibroblasts (Mef) (Sigma-Aldrich) and hippocampal HT22 (ThermoScientific) cells were cultured according to the manufacturer's protocols. Ctb assays were performed according to the manufacturer's protocols (Promega).

Cortex Growth Assay and Dissociated Cortical Neurons

[0115] Neonatal (P1-3) cortex explants were dissected from 250 m thick vibratome (HM650V, Thermo Fisher) sections from Bregma 0 to 1.5 (motor cortex). Cortical layer V was micro-dissected, plated on poly-D-lysine (0.5 mg/ml) and laminin (1 mg/ml)-coated glass coverslips and grown for 24 h in neurobasal medium with 2% horse serum, 2% B27, 1% glutamax and 0.5% penicillin/streptomycin. Explants were treated for 48 h with rIL-4 (50 ng/ml), Link4 (50 ng/ml), Link13 (50 ng/ml) or PBS. Axon length was assessed using Adobe Photoshop by measuring the distance of the 40 longest axons, corrected for the initial growth at 24 h. Dissociated cortical neurons were prepared as previously described (J. T. Walsh, S. Hendrix, F. Boato, I. Smirnov, J. Zheng, J. R. Lukens, S. Gadani, D. Hechler, G. Golz, K. Rosenberger, T. Kammertons, J. Vogt, C. Vogelaar, V. Siffrin, A. Radjavi, A. Fernandez-Castaneda, A. Gaultier, R. Gold, T. D. Kanneganti, R. Nitsch, F. Zipp, and J. Kipnis, MHCII-independent CD4+ T cells protect injured CNS neurons via IL-4. J Clin Invest 125, 699-714 (2015)). Cortices of embryonic day 18 (E18) mice were incubated with Trypsin/DNase solution and dissociated by trituration. Cells were resuspended in plating medium (lx MEM-Glutamax, 20% glucose, 10% HS, 1 PenStrep), filter-sterilize 600,000 cells per well in a 6-well-plate. At 5 h after plating, medium was aspirated, wells were washed 2 with warm PBS and cells were cultured in NB-medium. Cultures were allowed to expand for 3 days, before treatment with 50 ng/ml IL-4, 50 ng/ml Link4 or equivalent volumes of PBS. After 10 min, cells were harvested in lysis buffer and processed for Western blotting. Cell culture reagents were obtained from Fisher Scientific and Sigma. For the signaling experiments, cells were incubated with 50 ng/ml IL-4 (Peprotech) or equivalent volumes of PBS for 10 min.

H9 Human Neural Stem Cell Culture

[0116] H9 human neural stem cells (H9 hNSCs) were purchased from Gibco and expanded according to the manufacturer's instructions. For consecutive neural differentiation hNSCs were harvested and reseeded on glass coverslips coated with Matrigel (High Concentration, Growth Factor Reduced; Corning; diluted 1:100 in Knockout DMEM [Gibco]) at a density of 25.000 cells/cm.sup.2. hNSCs were allowed to rest for 48 hours in a basal neuronal medium (Neurobasal, 1X B27, 1x Glutamax, 1x Penicillin/Streptomycin, all from Gibco) before changing to EnStem A Neural Differentiation Medium (SCM017, Millipore) for a further 4 days. Cultures were treated with recombinant human IL-4 (Peprotech) or human Link4 at 50 ng/ml or equal volumes of PBS for 24 h. Following fixation cultures were immunofluorescently stained for III-Tubulin (clone Tuj1; BioLegend) and imaged with a Keyence BZ-X710 all-in-one fluorescent microscope. Analysis was performed in a blinded manner using the Simple Neurite Tracker plugin of ImageJ (NIH) by tracking longest neuritic extensions from at least 5 images per condition.

Western Blotting

[0117] For Western blotting, the following primary antibodies were used: anti-IRS1 (EMD Millipore), anti-phospho-IRS1 (Cell Signaling Technology), anti-MAPK and anti-phospho-MAPK (Cell Signaling Technology), anti-PKC (Santa-Cruz), anti-phospho-PKC (Biozol). DyLight 800/600-coupled secondary antibodies were used for quantitative analysis of the proteins using the Li-Cor Odyssey FC imaging system (Li-Cor Bioscience). Blots probed with the phospho-antibodies were stripped after visualization, to allow incubation with the total antibodies for parallel detection in the same samples.

Statistical Analysis

[0118] Statistical analysis was performed using Graphpad Prism 5 (GraphPad Software Inc). Clinical scores were analyzed using repeated-measures two-way ANOVA with posthoc Bonferroni correction. Data obtained from in vitro assays and Western blots were subjected to unpaired t-test or one-way ANOVA with Tukey's test for multiple comparison.

[0119] Amino Acid Sequences of human (hu) and murine (mu) Link4, Link 13, AvoC The following amino acid sequences were determined for the IL-4 and IL-13 derivatives:

TABLE-US-00002 huLink4(SEQIDNO1): TrpAsnArgSerGluIleIleLysThrGlySer LysThrIleMetArgGluLysTyr huLink13(SEQIDNO2): LeuMetArgSerGluLeuIleGluGluLeuVal AsnIleThrGlySerPheValLysAspLeu LeuLeuHisLeuLysLys huAvoC(SEQIDNO3): GluIleIleLysThrGlySerArgLeuAspArg AsnLeuTrpGlySerGlySerArgAlaAla ThrValLeuArgGlnPheTyrSer muLink4(SEQIDNO4): ArgAlaArgSerGluIleIleGlyIleGlySer LysSerIleMetGln muLink13(SEQIDNO5): LeuIleArgSerGluLeuIleGluGluLeuSer AsnIleThrGlySerPheIleThrLysLeuLeu SerTyrThrLysGln muAvoC(SEQIDNO6): GluIleIleGlyIleGlyProArgLeuPheArg AlaPheArgCysSerGlySerArgAlaSerLys ValLeuArgIlePheTyrLeu