SPHINGOLIPIDS FOR GENERATING REGULATORY CD4+ T CELLS

Abstract

The present invention relates to a substance of formula (I), whereby R.sub.1 is an alkyl or alkenyl group having 6 to 20 carbon atoms; R.sub.2 is H or missing, whereby O is bound via a double bond, R.sub.3 is H or an acyl group -C(O)R.sub.5, whereby R.sub.5 is an alkyl or alkylene group having 1 to 10 carbon atoms, and R.sub.4 is H or a phosphate group for use as a medicament and for use in a method of preventing or treating a subject suffering from an autoimmune disease. The present invention further relates to a method for generating regulatory T cells (Treg cells) in vitro comprising the steps of providing precursor CD4.sup.+T cells, cultivating the precursor CD4.sup.+T cells provided in step 1) in the presence of the substance as defined herein, and, optionally, isolating the generated regulatory T cells (Treg cells).

Claims

1. A substance of formula (I) ##STR00004## whereby R.sub.1 is an alkyl or alkenyl group having 6 to 20 carbon atoms; R.sub.2 is H or missing, whereby O is bound via a double bond, R.sub.3 is H or an acyl group -C(O)R.sub.5, whereby R.sub.5 is an alkyl or alkylene group having 1 to 10 carbon atoms, and R.sub.4 is H or a phosphate group, for use as a medicament.

2. A substance of formula (I) ##STR00005## whereby R.sub.1 is an alkyl or alkenyl group having 6 to 20 carbon atoms; R.sub.2 is H or missing, whereby O is bound via a double bond, R.sub.3 is H or an acyl group -C(O)R.sub.5, whereby R.sub.5 is an alkyl or alkylene group having 1 to 10 carbon atoms, and R.sub.4 is H or a phosphate group, for use in a method of preventing or treating a subject suffering from an autoimmune disease.

3. The substance for use according to claim 1 wherein the substance is sphinganine, sphinganine-1-phosphate and/or 3-keto-sphinganine, preferably wherein the substance is sphinganine.

4. The substance for use according to claim 1, wherein the substance is in the erythro-form, preferably erythro-sphinganine, erythro- sphinganine-1-phosphate and/or erythro-3-keto-sphinganine, still more preferably wherein the substance is in the D-erythro-form, still more preferably D-erythro-sphinganine, D- erythro-sphinganine-l-phosphate and/or D-erythro-3-keto-sphinganine, and most preferably D-erythro-sphinganine.

5. The substance for use according to claim 1, wherein the substance is used in combination with an agent, preferably selected from the group consisting of retinoic acid, copaxone, insulin, a molecule capable of interacting with CD3, a molecule capable of interacting with CD28, transforming growth factor β (TGFβ), interleukin-2 (IL-2), a short-chain fatty acid, a bile acid, polysaccharide A, an n3 polyunsaturated fatty acid, retinoic acid, Vitamin D (VitD), Vitamin C (VitC), a polyphenol, quercetin, resveratrol, a non-steroidal anti-inflammatory drug (NSAID), rapamycin and/or a peptide fragment from an autoreactive protein, more preferably selected from transforming growth factor β(TGFβ) and/or interleukin-2 (IL-2).

6. A method for generating regulatory T cells (Treg cells) in vitro comprising the steps of: 1) providing precursor CD4.sup.+T cells, 2) cultivating the precursor CD4.sup.+T cells provided in step 1) in the presence of a substance of formula (I) ##STR00006## whereby R.sub.1 is an alkyl or alkenyl group having 6 to 20 carbon atoms; R.sub.2 is H or missing, whereby O is bound via a double bond, R.sub.3 is H or an acyl group -C(O)R.sub.5, whereby R.sub.5 is an alkyl or alkylene group having 1 to 10 carbon atoms, and R.sub.4 is H or a phosphate group, and, optionally, 3) isolating the generated regulatory T cells (Treg cells).

7. The method of claim 6, wherein the substance of formula (I) is sphinganine, sphinganine-1-phosphate and/or 3-keto-sphinganine, preferably wherein the substance is sphinganine.

8. The method according to claim 6, wherein the substance of formula (I) is in the erythro-form, preferably erythro-sphinganine, erythro- sphinganine-1-phosphate and/or erythro-3-keto-sphinganine, more preferably wherein the substance is in the D-erythro-form, still more preferably D-erythro-sphinganine, D-erythro- sphinganine-1-phosphate and/or D-erythro-3-keto-sphinganine, and most preferably D- erythro-sphinganine.

9. The method according to claim 6, further comprising the step of cultivating the precursor CD4.sup.+T cells in the presence of an additional compound that is capable of inducing the generation of regulatory T cells (Treg cells); preferably in the presence of a molecule capable of interacting with CD3, a molecule capable of interacting with CD28, transforming growth factor β (TGFβ), interleukin-2 (IL- 2), a short-chain fatty acid, a bile acid, polysaccharide A, an n3 polyunsaturated fatty acid, retinoic acid, Vitamin D (VitD), Vitamin C (VitC), a polyphenol, quercetin, resveratrol, a non-steroidal anti-inflammatory drug (NSAID), rapamycin and/or a peptide fragment from an autoreactive protein; more preferably in the presence of TGFβand/or IL-2; still more preferably in the presence of (1) TGFβand/or IL-2; and (2) an anti-CD3 antibody and/or an anti-CD28 antibody; and/or (3) a peptide fragment; yet still more preferably in the presence of TGFβ, an anti-CD3 antibody and an anti-CD28 antibody or in the presence of TGFβ and a peptide fragment; most preferably in the presence of TGFβ, IL-2, an anti-CD3 antibody and an anti-CD28 antibody or in the presence of TGFβ, IL-2 and a peptide fragment.

10. The method according to claim 6, wherein the precursor CD4.sup.+T cells are naïve CD4.sup.+T cells isolated from a subject, preferably from the spleen, lymph node or peripheral blood, or wherein the precursor CD4.sup.+T cells are splenocytes or peripheral blood mononuclear cells (PBMCs) isolated from a subject, preferably isolated from intravenous blood.

11. The method according to claim 6, wherein the precursor CD4.sup.+T cells are isolated using flow cytometry sorting or magnetic cell sorting using cell surface markers, preferably wherein these cell surface markers are CD4.sup.+and CD25.sup.+or CD25.sup.high or are CD4.sup.+and CD25.sup.+or CD25.sup.high and CD127.sup.−or CD127.sup.low.

12. The method according to claim 10, wherein the subject suffers from an autoimmune disease.

13. The method according to claim 6, wherein the substance in step 2) is added to a final concentration of 0.1 to 20 μM, preferably to a final concentration of 1 to 15 μM, more preferably to a final concentration of 3 to 10 μM, most preferably to a final concentration of 5 to 6.25 μM.

14. The method according to claim 6, wherein the precursor CD4.sup.+T cells in step 2) are cultivated for 24 to 144 hours, preferably for 24 hours to 120 hours, more preferably for 48 hours to 96 hours.

15. A regulatory T cell (Treg cell) obtainable by the method according to claim 6, preferably for use as a medicament, more preferably for use in a method of preventing or treating a subject suffering from an autoimmune-disease.

16. The substance of Formula 1 for use according to the method of claim 12, wherein the autoimmune disease is autoimmune encephalitis, autoimmune encephalomyelitis, rheumatoid arthritis, type 1 diabetes, psoriasis, autoimmune kidney disease, systemic lupus erythematosus, celiac disease, inflammatory bowel disease or graft-versus-host disease, preferably wherein the autoimmune disease is multiple sclerosis.

17. A kit comprising transforming growth factor beta (TGF-β) and/or interleukin-2 (IL-2), and a substance as defined in claim 1, and optionally an additional compound that is capable of inducing the generation of regulatory T cells (Treg cells), preferably a molecule capable of interacting with CD3, a molecule capable of interacting with CD28, a short-chain fatty acid, a bile acid, polysaccharide A, an n3 polyunsaturated fatty acid, retinoic acid, Vitamin D (VitD), Vitamin C (VitC), a polyphenol, quercetin, resveratrol, a non-steroidal anti-inflammatory drug (NSAID), rapamycin and/or a peptide fragment from an autoreactive protein; more preferably the kit comprises (1) TGFβand/or IL-2; and (2) an anti-CD3 antibody and/or an anti-CD28 antibody; and/or (3) a peptide fragment; still more preferably the kit comprises TGFβ, an anti-CD3 antibody and an anti-CD28 antibody or the kit comprises TGFβ and a peptide fragment; most preferably the kit comprises TGFβ, IL-2, an anti-CD3 antibody and an anti-CD28 antibody or the kit comprises TGFβ, IL-2 and a peptide fragment.

Description

FIGURES

[0100] FIG. 1. Sptic2 deficiency in T cells increased tumor growth but decreased Treg cell formation. The Spfic2.sup.Flox/FloxCd4-Cre (FI/FI, 8 mice) and Sptic2.sup.+/+Cd4-Cre (+/+, 11 mice) mice were subcutaneously implanted with 2 ×10.sup.5 melanoma B16 cells. The tumor sizes were measured with a caliper and calculated as length×width×width / 2. The line graph shows the tumor growth over time (A). The tumors were smashed through a 70 μM cell strainer to make single cell suspension, spun down and resuspended in 40% percoll. The 40% percoll containing tumor cells and tumor-infiltrating cells was loaded onto 80% percoll and spun at 2000 rpm for 15 min. The tumor-infiltrating leukocytes were found in the middle layer between the 40% and 80% percoll after centrifugation and collected for FACS staining and cell counting. The bar graph shows the density of FOXP3 protein- expressing Treg cells in the tumor, calculated as the number of Treg cells divided by the weight of tumors (B). Data are expressed as mean ±SEM and cumulative of 3 (A) and 2 (B) independent experiments. *, p<0.05, Student's t-test.

[0101] FIG. 2. Sptic2 deficiency in Treg cells affected the immunosuppressive function of Treg cells. YFP-positive CD4.sup.+Treg cells and YFP-negative CD4.sup.+non-Treg cells were FACS-sorted. 2 ×10.sup.4 non-Treg cells were cultured in 250 μl complete medium in the presence of T cell stimuli anti-CD3 and anti-CD28-coated microbeads (4 x 10.sup.4 microbeads per cell culture; 16 ng/ml anti-CD3 and 16 ng/ml anti-CD28. Non-Treg cells were co- cultured with the Treg cells at the indicated ratios (e.g. “8:1” means 2 ×10.sup.4 non-Treg cells plus 2.5 ×10.sup.3 Treg cells). The line graph shows suppression% over non-Treg:Treg cells. Data are cumulative of 3 independent experiments. Results are expressed as mean±SEM. *, p<0.05, Student's t-test.

[0102] FIG. 3. Sptic2 deficiency in Treg cells enhanced autoimmunity in the EAE mouse model. The line graph shows the EAE clinical scores over days post EAE induction. Five pairs of mice were used. Results are expressed as mean±SEM. *, p<0.05, Student's t- test.

[0103] FIG. 4. Sphinganine promoted the Treg cell in vitro generation and suppressed the inflammatory Th17 cell formation. The YFP-negative CD4.sup.+non-Treg naïve T cells were FACS-sorted (1 ×10.sup.5 cells in 250 μl complete medium) from the Foxp3Cre-YFP mice. The naive T cells were activated with the anti-CD3 and anti-CD28 for three days, in the presence or absence of cytokine TGFβ (5 ng/ml) and IL-2 (10 ng/ml) for Treg cells and TGFβ (5 ng/ml), IL-6 (20 ng/ml) and anti-IFNy (10 μg/ml) for Th17 cells. A: flow cytometry analysis of Foxp3 protein expression after induction of Treg cell formation with sphinganine (5 μM) in the presence of cytokine TGFβ (5 ng/ml) or vehicle control DMSO (no TGFβ). B: Flow cytometry analysis of IL-17 protein expression after induction of Th17 cell formation with TGFβ (5 ng/ml), IL-6 (20 ng/ml) and anti-IFNy (10 μg/ml) in the presence or absence of sphinganine (5 μM). C: Flow cytometry analysis of Foxp3 protein expression after induction of Treg cell formation in the presence of TGFβ (5 ng/ml) and L- serine (5 μM), 3-KDS (5 μM), shinganine (5 μM), dihydroceramide (50 nM), ceramide (50 nM), sphingosine (1 μM), sphingosine-1-phosphate (1 μM), shinganine-1-phosphate (1 μM), or vehicle control DMSO (TGFβ only). D: Structures of sphingolipids, as indicated, and sphingolipid biosynthetic pathway are depicted. The numbers in each FACS plot show the percentages of the indicated cell population within the total population of cells shown in the FACS plot.

[0104] FIG. 5. Sphinganine treatment ameliorated EAE development. The line graph shows the EAE clinical scores over days post EAE induction. Results are expressed as mean ±SEM. *, p<0.05, **, p<0.01, Student's t-test.

EXAMPLES

[0105] Methods

[0106] Mice. Sptic2.sup.FI/FIand Foxp3.sup.Cre mice on a C57BL/6 background were provided by Professor Xian-cheng Jiang (SUNY Downstate Medical Center, New York) and Professor Alexander Rudensky (Memorial Sloan Kettering Cancer Center, New York), respectively. Cd4.sup.Cre mice on a C57BL/6 background were purchased from the Jackson Laboratory. All mice were maintained in the DKFZ specific pathogen-free facility. Age- and gender-matched littermates (5-10 weeks old) were used as control mice in all experiments. All the studies were performed in accordance with DKFZ regulations after approval by the German regional council at the Regierungsprasidium Karlsruhe.

[0107] B16 melanoma implantation and tumor-infiltrating immune cell preparation. B16-F10 melanoma cells were subcutaneously injected to the Sptic2.sup.Flox/FloxCd4-Cre, Sptic2.sup.+/+Cd4- Cre, Sptic2.sup.Flox/FloxFoxp3-Cre-YFP and Sptic2.sup.+/+Foxp3-Cre-YFP mice (2×10.sup.5 cells per mouse). Tumors were measured every 2-3 days using a caliper. Mice were sacrificed at the indicated time points and tumors were harvested using forceps and scissors. Tumors were smashed through 70-μm cell strainers to generate single-cell resuspension. The tumor cells were centrifuged and resuspended in 40% percoll and loaded to 80% percoll for gradient centrifugation. The immune cells were found in the middle layer between the 40% and 80% percoll after centrifugation. The immune cells were then sucked out to a new tube for flow cytometry staining.

[0108] EAE induction and monitoring. Each mouse was subcutaneously immunized with 200 μg MOG35-55 peptide emulsified in Freund's Complete Adjuvant. Pertussis toxin (400 ng per mouse) was injected intraperitoneally. Where indicated, mice were intraperitoneally injected with sphinganine (every other day from the day of immunization to the end of the experiments). EAE symptoms were scored every day using the following scoring standard: 0, no sign; 1, limp tail; 2, paraparesis (incomplete paralysis of 1 or 2 hind limbs); 3, paraplegia (complete paralysis of 2 hind limbs); 4, paraplegia with forelimb weakness or paralysis; 5, moribund state or death. For the mouse welfare reason, we sacrificed the mice if the score reached 3.

[0109] Mouse primary T cell culture. Complete medium was used for cell culture and was prepared by supplementing RPMI 1640 plain medium with 10% fetal calf serum, penicillin/streptomycin antibiotics and non-essential amino acids. For Treg and Th17 cell in vitro differentiation, naïve splenic T cells were purified from the Sptic2.sup.+/+Foxp3Cre-YFP wild type mice using the flow cytometry sorter. Foxp3 is expressed in the cell nucleus and FACS staining of FOXP3 protein requires cell fixation and permeabilization, which kills cells and is not suitable for subsequent cell culture. In this Foxp3Cre-YFP mouse strain, the FOXP3 protein expression is reported by the YFP protein expression. FOXP3- expressing cells could not directly be FACS-sorted without fixing and permeabilizing cells and thus preserve cell viability. YFP-positive CD4.sup.+Treg cells and YFP-negative CD4.sup.+non- Treg cells were FACS-sorted. The naïve CD4.sup.+T cells (1 ×10.sup.5 cells in 250 μl medium) were activated with the T cell stimuli anti-CD3 and anti-CD28 for three days, in the presence or absence of cytokine TGFβ (5 ng/ml) and IL-2 (10 ng/ml) for Treg cells and TGFβ (5 ng/ml), IL-6 (20 ng/ml) and anti-IFNy (10 μg/ml) for Th17 cells. Sphinganine or other sphingolipids were added as indicated (1 or 5 μM). The Foxp3 and interleukin-17(IL-17) protein expression was examined by flow cytometry assay.

[0110] Flow Cytometry. FACS buffer (PBS with 0.5% FCS) was used to stain cell surface antigen. To stain intracellular antigens, cells were fixed with Biolegend fixation buffer (for cytokines) or eBioscience Fixation/permeabilization buffer (for transcription factors). Dead cells were excluded using the LIVE/DEAD Fixable Dead Cell Stains (Thermo Fisher Scientific). For Treg cell co-culture suppression assay, responder cells were labeled with Celltrace Violet (37° C., 20 minutes) and washed with RPMI 1640 medium with 1% FBS for 3 times. Samples were run on the LSR II and analyzed using the Flowjo software (FlowJo, LLC, BD).

[0111] Antibodies and cytokines. The antibodies were ordered from Biolegend, eBioscience and BD Biosciences for detecting the following antigens using flow cytometry: CD4 (GK1.5), IL-17 (TC11-18H10.1), and Foxp3 (FJK-16s). Anti-CD3 (17A2) and anti-CD28 (37.51) were used for cell culture.

[0112] Example 1

[0113] Sptic2 deficiency in T cells increased tumor growth but decreased Treg cell formation. The Sptic2.sup.Flox/FloxCd4-Cre (FI/FI, 8 mice) and Sptic2.sup.+/+Cd4-Cre (+/+, 11 mice) mice were implanted with 2 ×10.sup.5 melanoma B16 cells. Results are shown in FIG. 1. We observed that the genetic deficiency of Sptic2 in T cells impaired anti-tumor immunity (FIG. 1A) and we further observed that a subset of T cells, called regulatory T cells (Treg cells), were reduced by the SPTLC2 deficiency (FIG. 1B).

[0114] Example 2

[0115] Sptic2 deficiency in Treg cells affected the immunosuppressive function of Treg cells. We purified the Treg and non-Treg cells from the Sptic2.sup.Flox/FloxFoxp3Cre-YFP mice or Sptic2.sup.+/+Foxp3Cre-YFP mice using the flow cytometry sorter. Foxp3 is expressed in the cell nucleus and FACS staining of FOXP3 protein requires cell fixation and permeabilization, which kills cells and is not suitable for subsequent cell culture. In this Foxp3Cre-YFP mouse strain, the FOXP3 protein expression is reported by the YFP protein expression. We could not directly FACS-sort FOXP3 xpressing cells without fixing and permeabilizing cells and thus preserve cell viability. YFP-positive CD4.sup.+Treg cells and YFP-negative CD4.sup.+non-Treg cells were FACS-sorted. The non-Treg cells were labeled with a fluorescent dye Celltrace Violet (CTV, to determine cell proliferation) and co- cultured with or without the Sptic2-deficient or -sufficient Treg cells for three days in the presence of T cell stimuli anti-CD3 and anti-CD28. The suppression % was calculated as [(T cell proliferation rate without Treg cells - T cell proliferation rate with Treg cells)/ T cell proliferation rate without Treg cells %]. We found that SPTLC2 was required for the immunosuppressive function of Treg cells. Results are shown in FIG. 2.

[0116] Example 3

[0117] Sptic2 deficiency in Treg cells enhanced autoimmunity in the EAE mouse model.

[0118] EAE was induced in the Sptic2.sup.Flox/FloxFoxp3Cre-YFP mice or Sptic2.sup.+/+Foxp3Cre-YFP mice. Briefly, each mouse was subcutaneously immunized with 200 μg MOG.sub.35-55 peptide emulsified in Freund's Complete Adjuvant. Pertussis toxin (400 ng per mouse) was injected intraperitoneally. EAE symptoms were scored every day. Sptic2.sup.Flox/FloxFoxp3Cre- YFP mice developed more severe EAE compared with the wildtype control mice. Results are shown in FIG. 3.

[0119] Example 4

[0120] Sphinganine promoted the Treg cell in vitro generation and suppressed the inflammatory Th17 cell formation. Naïve splenic T cells were purified from the Sptic2.sup.+/+Foxp3Cre-YFP wild type mice using the flow cytometry sorter. The naïve CD4.sup.+T cells were activated with the T cell stimuli anti-CD3 and anti-CD28 and IL-2 for three days, in the presence or absence of cytokine TGF-β(FIG. 4A, to induce Treg cell formation) or TGF-βplus interleukin-6 (IL-6) (FIG. 4B, to induce Th17 cell formation). Sphinganine (5 μM) or vehicle control DMSO was added. The Foxp3 and interleukin-17 (IL-17) protein expression was examined by flow cytometry assay. Alternatively, naïve CD4.sup.+T cells were cultured under the Treg cell-inducing condition (the same as to FIG. 4A) in the presence of sphinganine or other sphingolipids or vehicle control before flow cytometry analysis of Foxp3 protein expression (FIG. 4C). The structures and the sphingolipid biosynthetic pathway are depicted (FIG. 4D). The cytokine TGF-βinduced Foxp3-expressing Treg cells as reported (Chen et al., 2003). It was found that sphinganine or other sphingolipids increase Foxp3 protein expression, induce Treg cell formation and reduce IL-17 protein expression.

[0121] Example 5

[0122] Sphinganine treatment ameliorated EAE development. C57BL/6 mice were subcutaneously immunized with 200 μg MOG.sub.35-55 peptide emulsified in Freund's Complete Adjuvant to induce EAE development. Pertussis toxin (400 ng per mouse) was injected intraperitoneally. Mice were intraperitoneally injected with sphinganine (1000 μg/kg body weight, one injection every two days, from day 1 to day 15). EAE symptoms were scored every day. Results are shown in FIG. 5.

REFERENCES

[0123] Berod L. et al., Nature Medicine 20:1327-1333, 2014.

[0124] Bezie S. et al., Frontiers in Immunology 8, Article 2014, 2018.

[0125] Blackley S. et al., Journal of Virology 81: 13325-13334, 2007.

[0126] Bluestone J. A. et al., Science Translational Medicine 7: 315ra189, 2015.

[0127] Chen W. et al., The Journal of experimental medicine 198: 1875-1886, 2003.

[0128] Goyvaerts C. et al., Gene Therapy 19: 1133-1140, 2012.

[0129] Hanada K. et al., Biochim Biophys Acta 1632:16-31, 2003.

[0130] Hang S. et al., Biorxiv doi: https://doi.org/10.1101/465344, 2018.

[0131] Hod S. et al., Science 299:1057-1061, 2003.

[0132] Ishida Y. et al., EMBO Journal 11:3887-3895 1992.

[0133] Javeed A. et al., Transplantation Immunology 20:253-260, 2009.

[0134] Klages K. et al., Cancer research 70: 7788-7799, 2010.

[0135] McGeachy M. J. et al., J Immunol. 175: 3025-3032, 2005.

[0136] Park H. et al., Nature immunology 6: 1133, 2005.

[0137] Park B. et al., Cancer Discovery 6:1366-1381 2016.

[0138] Simonetta F. et al., Frontiers in Immunology 4, Article 215, 2013.

[0139] Tivol et al., Blood. 2005 Jun. 15; 105(12): 4885-4891.

[0140] Wong C. et al., Immunology Letters 139: 7-13, 2011.

[0141] Wu J. et al., Immunity 50: 1-14, 2019.