METHODS AND PHARMACEUTICAL COMPOSITIONS FOR REPROGRAMING IMMUNE ENVIRONMENT IN A SUBJECT IN NEED THEREOF

Abstract

The present invention relates to methods and pharmaceutical compositions for reprograming immune environment in a subject in need thereof. The inventors demonstrated that DDA induces differentiation of tumor cells and stimulates the secretion and the production of modified exosomes with anti-tumor properties (DDA-exosomes) via a mechanism dependent of the expression of the LXRbeta in the parental cells. In particular, one object of the present invention relates to a method of promoting Th1 differentiation and functionality and CD8+ cytotoxicity in a subject in need thereof comprising administering to the subject a therapeutically effective amount of DDA or DDA-exosomes.

Claims

1. A method of treating cancer in a subject in need thereof comprising administering a therapeutically effective amount of a population of DDA-exosomes, wherein the population of DDA-exosomes is obtained by a method comprising the steps of contacting a population of tumor cells with an amount of DDA for a time sufficient to induce exosomes releasing by the population of tumor cells.

2. The method according to claim 1, wherein the subject is administered with a vaccine composition comprising the population of DDA-exosomes, as an immunoadjuvant, together with one or more antigens for inducing an immune response against said one or more antigens.

3. The method according to claim 2, wherein the one or more antigens of the vaccine composition is a tumor associated antigen.

4. The method according to claim 1, wherein the subject is administered with a composition enriched by the population of DDA-exosomes.

5. The method according to claim 4, wherein the enriched composition comprises at least 10% of the population of DDA-exosomes.

6. The method according to claim 1, wherein the exosomes are tumor exosomes.

7. A method of treating cancer in a subject in need thereof comprising i) determining the presence or absence of LXRβ in a tumor tissue sample obtained from the subject and ii) administering to the subject a therapeutically effective amount of a population of DDA-exosomes when LXRβ is absent in the tumor tissue sample, wherein the population of DDA-exosomes is obtained by a method comprising the steps of contacting a population of tumor cells with an amount of DDA for a time sufficient to induce exosomes releasing by the population of tumor cells.

8. The method according to claim 7, wherein the population of DDA-exosomes is administered when LXRβ is present in the tumor tissue sample.

9. The method according to claim 7, wherein the subject is administered with a vaccine composition comprising the population of DDA-exosomes, as an immunoadjuvant, together with one or more antigens for inducing an immune response against said one or more antigens.

10. The method according to claim 9, wherein the one or more antigens of the vaccine composition is a tumor associated antigen.

11. The method according to claim 7, wherein the subject is administered with a composition enriched by the population of DDA-exosomes.

12. The method according to claim 11, wherein the enriched composition comprises at least 10% of the population of DDA-exosomes.

13. The method according to claim 7, wherein the exosomes are tumor exosomes.

Description

FIGURES

[0078] FIGS. 1A-E. Sorted naive CD4 T cells were isolated from the spleen of C57BL/6 mice and activated in the indicated Th1, Th2, Treg, or Th17 polarizing conditions as described in the “Materials and methods” section”. These cells were cultured in the presence of increasing concentrations of DDA. After 96 hours, the percentage of (A) Th1: CD4+Tbet+IFNg+, (B) Th2: CD4+GATA3+IL6+, (C) Treg: CD4+Foxp3+IL10+ and (D) Th17: CD4+RORgt+IL6+, cells was measured by flow cytometry. (E) Sorted naive CD8 T cells were isolated from the spleen of the C57BL/6 mice and activated with anti-CD3, anti-CD28 and recombinant IL2. After 96 hours, the percentage of cytotoxic T CD8+GranzymB+IFNg+ cells was measured by flow cytometry.

[0079] FIGS. 2A-E. Sorted naive CD4 T cells isolated from the spleen of C57BL/6 mice were activated in Th1, Th2, Treg, or Th17 polarizing conditions as indicated in the “Materials and methods” section”. After 96 h, increasing concentrations of DDA was added on polarized CD4 T cells for 24 h. Then the percentage of (A) CD4+Tbet+IFNg+, (B) CD4+GATA3+IL6+, (C) CD4+Foxp3+IL10+ and (D) CD4+RORgt+IL6+ cells was measured by flow cytometry. (E) Sorted naïve CD8 T cells were isolated from the spleen of the C57BL/6 mice and activated with anti-CD3, anti-CD28 and recombinant IL2. After 96 h, increasing concentrations of DDA was added on activated CD8 T cells for 24 h. Then the percentage of cytotoxic T CD8+GranzymB+IFNg+ cells was measured by flow cytometry.

[0080] FIGS. 3A-D. Unpolarized Th0 (prepared from naive CD4 T cells isolated from the spleen of C57BL/6 mice were cultivated in presence of anti-CD3, anti-CD28 and recombinant IL-2 as indicated in the “Materials and methods” section”) and with gradual concentration of DDA added at day 0 of culture (condition #1) or Day 4 of culture (condition #2). At the end, for each condition, the percentage of Th1 (CD4+Tbet+IFNg+) (A,B) and Treg CD4+Foxp3+IL10+ (C,D) was measured by flow cytometry.

[0081] FIGS. 4A-C. Tumor growth analysis (A) Exponentially growing E0771 cells were collected, washed twice in PBS and resuspended in PBS (300,000 cells in 100 μl PBS). E0771 tumors were prepared by subcutaneous transplantation into the flanks of C57BL/6 mice. When tumor measured 50 mm3, the mice were treated every 5 days with 0.37 μg/kg or 20 mg/kg DDA or with the solvent vehicle (control). (B) The tumor volume was determined by direct measurement with a caliper and was calculated using the formula (width2×length)/2. (C) The Kaplan-Meier method was used to compare the percentage of animal with tumor<2000 mm3.

[0082] FIGS. 5A-D. Infiltration of immune cells inside E0771 tumor. (A-D) These bar graphs represent the ratio between (A) Th1 (CD4+ Tbet+) and Treg (CD4+ Foxp3+), (B) CTL cells (CD8+ Granzym B+) on No CTL cells (CD8+ Granzym b−), (C) macrophage type M1 (CD14+ CCR7+ IFNg+) and type M2 (CD14+ CD206+ IL10+) (D) dendritic cells (CD11c+) and myeloid derived suppressive cells (MDSC: CD11b+CD11clow, LY6C+ly6Gint) infiltrated inside the tumor. The tumors were removed at day 15 post treatment with DDA at 0.37 μg/kg (b) or with the solvent vehicle (control) (a). To observe the cytotoxic CD8 T cells, the tumor suspension was prealably stimulated 2 h in vitro with a cocktail of PMA (50 ng/ml), ionomycine (500 ng/ml) and golgistop (concentration from manufacture BD Pharmagen), then the cells were stained with specific antibodies.

[0083] FIGS. 6A-B. The analysis of exosomes secreted from B16F10 cells after DDA (called DDA-exosomes) or the solvent vehicle (called control-exosomes) treatments demonstrates that DDA modifies their composition. (A) DDA-exosomes are enriched in proteins with antigen-presentating properties such as CD1d, MHC-II and Hsp70, with differentiation antigenes such as tyrosinase. (B) DDA-exosomes present also a decrease level in PGE2, an immunosuppressive lipid, compared with control-exosomes. DDA-exosomes may thus activate the immune system against the tumor. It has to be noted that the exosomes produced from tumor cells are described in the literature as immuno-suppressor and pro-tumor. This is due to the fact that tumor exosomes are enriched in immuno-suppressive molecules, such as PGE2.

[0084] FIG. 7. A single intra-dermal injection of DDA-exosomes (1 μg/mouse) purified from the media of B16F10 cells treated with 1 μM DDA for 24 h into the flank of mice grafted with a B16F10 tumor, inhibits tumor growth and increases mice survival compared with injection of control-exosomes in same conditions. DDA-exosomes inhibit tumor growth and increase mice survival. DDA is the first molecule, to our knowledge, to be able to stimulate the production of anti-tumor exosomes from tumor cells. We have a pharmacological modification of the phenotype and activity of tumor exosomes by DDA.

[0085] FIG. 8. DDA-exosomes purified from human SKMEL-28-shCTRL cells media increase cell surface markers of mature human dendritic cells.

[0086] FIGS. 9A-B. Sorted naive CD4 T cells were isolated from the spleen of WT or LXRαβKO mice (collaboration with Hervé Guillou, INRA, Toulouse) and activated in the indicated Th1 or Treg polarizing conditions. These cells were cultured in the presence of increasing concentrations of DDA. After 96 hours, the percentage of (A) Th1: CD4+Tbet+IFNg+ and (B) Treg: CD4+Foxp3+IL10+ cells was measured by flow cytometry and expressed relative to the control.

[0087] FIG. 10. Sorted naive CD4 T cells were isolated from the spleen of WT or LXRαβKO mice and activated in Th2 polarizing conditions. These cells were cultured in the presence of increasing concentrations of DDA. After 96 hours, the percentage of Th2: CD4+GATA3+IL4+ cells was measured by flow cytometry and expressed relative to the control.

[0088] FIGS. 11A-B. Bone marrow was isolated from the tibia and femur of WT or LXRαβKO mice and cultivated with 20 ng/ml GM-CSF and in presence of increasing concentrations of DDA. At day 3 and 5 half of medium was replaced by fresh medium containing GM-CSF. After 7 days of culture, the percentage of (A) differentiated CD11c+CD8a dendritic cell (B) mature CD11c+CD8a+ dendritic cells (CD11c+CD8a+CD86hi CCR7hi) was measured by flow cytometry and expressed relative to the control.

[0089] FIG. 12. Bone marrow was isolated from the tibia and femur of WT or LXRαβKO mice and cultivated with 20 ng/ml GM-CSF and in presence of increasing concentrations of DDA. At day 3 and 5 half of medium was replaced by fresh medium containing GM-CSF. After 7 days of culture, the mean of florescence (MFI) of MHC class II expressed on the surface of CD11c dendritic cells: CD11c+H2db+ was measured by flow cytometry and expressed relative to the control.

[0090] FIGS. 13A-B. Tumor growth analysis (A) Exponentially growing E0771 sh control or E0771 sh LXR cells were collected, washed twice in PBS and resuspended in PBS (300,000 cells in 100 μl PBS). C57BL/7 mice were grafted subcutaneously with 300 000 E0771 sh control or E0771 sh LXR tumor cells. When the tumors reached a volume of 50 mm.sup.3, the mice were treated every 2 days with 0.37 μg/kg DDA or treated with the solvent vehicle (untreated). (B) The tumor volume was determined by direct measurement with a caliper and was calculated using the formula (width2×length)/2. At day 15 of treatment with DDA or the solvent vehicle, the tumors were removed and the immune cells infiltrated into the tumors were analyzed by flow cytometry (see FIG. 14).

[0091] FIGS. 14A-D. C57BL/7 mice were grafted subcutaneously with 300 000 E0771 tumor cells knocked down for the LXR (E0771 sh LXR) or with control cells (E0771 Sh control). When the tumors reached a volume of 50 mm.sup.3, the mice were treated or not with 0.37 μg/kg of DDA every 2 days. 15 days post DDA treatment, the tumor cells were collected to assess by flow cytometry the CD4 Th1 cells (CD4+ Tbet+), CD4 T regulatory (Treg: CD4+ Foxp3+), CD8 T cells cytotoxic (CTL: CD8+ granzyme +)) or not (non CTL: CD8+ Granzyme −), macrophage M1 (F4/80+ CD206− CD86+) and M2 (F4/80+ CD206+ CD86−), dendritic cells CD11c+, CD11c+CD8α+ and myeloid derived suppressive cells (MDSC: CD11b+CD11clow, LY6C+ly6Gint) infiltrated into the tumors. To observe the cytotoxic CD8 T cells, the tumor suspension was prealably stimulated 2 h in vitro with a cocktail of PMA (50 ng/ml), ionomycine (500 ng/ml) and golgistop (concentration from manufacture BD Pharmagen), then the cells were stained with specific antibodies. The bars graphs represent the ratio between (A) Th1 and Treg, (B) CTL and non CTL, (C) M1 and M2 and (D) Alls DCs (CD11c+ and CD11c+ CD8α+) and MDSC infiltrated inside the tumors: E0771 sh control or E0771 Sh LXR (knocked down for LXRβ) from mice treated or not with DDA at 0.37 ug/kg. In cells expressing the LXRβ (E0771 shcontrol cells), DDA increases the infiltration of activated immune cells (Th1, CTL, macrophages M1 and DC) and decreases the infiltration of immunosuppressives cells (Treg, non CTL, M2 and MDSC). In cells knocked down for the LXRβ (E0771 shLXR cells), the effect of DDA is significantly decreased.

[0092] FIGS. 15A-D. DDA treatment decreases the percentage of T regulatory CD4 T cells and increases the activated CD4+ and CD8+ T cells inside tumors. Immunocompetent C57BL/6 mice (Janvier Laboratory) were implanted subcutaneously with 300 000 E0771 (ER+) mouse mammary tumor cells expressing the LXRb (wild type cells). When tumors were palpable (50 mm3), the animals were treated with the vehicle (empty symbol) or s.c. 0.37 μg/kg of DDA (full symbol) every 2 days, once a day. Fifty days post treatment, the animals were sacrified to collect the tumors. The tumors were dissociated by using gentlemac technology (Miltenyi) and then the suspension of tumor cells were stimulated with 50 ng/ml of PMA (Sigma), 500 ng/ml Ionomycin (Sigma) and 1/1000 of golgi stop (ebiosicence) during 3 h at 37° C. Tumor infiltrated Lymphocytes (TIL) isolated from E0711 tumors were stained with antibodies against CD45, CD8, CD4, PD-1, Foxp3, T-bet, IFN-g, Granzym B, PD-1 as well as live/dead stain. By gating on CD45+CD4+ (A-B) or CD45+CD8+ T cells populations (C-D), the tumor-infiltrating (A) T regulatory cells (Treg; Foxp3+), (B) effector CD4+ cells (Th1; T-bet+) and (C) cytotoxic CD8 T cells (CTL; IFN-γ+Granzym B+) were analyzed by flow cytometry. The vehicle condition is normalized at 1 and the graphs are representative of three independent experiments. (D). The graph represents the percentage of PD-1 negative CD8+ cells inside tumors of mice treated with vehicle or DDA and is representative of three independent experiments.

[0093] FIGS. 16A-C. The control of tumor growth and the increase in animal survival upon DDA treatment are dependent on the LXRβ expressed in tumor cells. Immunocompetent C57BL/6 mice (Janvier Laboratory) were implanted subcutaneously with 300 000 E0771 (ER+) mouse mammary tumor cells expressing the LXRb (E0 shC, square symbol) or knockdown for the LXRb expression with shRNA (shLXRb, circle symbol). When the tumor reached a volume of 50-100 mm3, animals (n=12 mice per group) were treated intraperitoneally once per day, every two days with 0.37 μg/kg of DDA (Affichem). The animals were monitored over time for (A) tumor growth and (B) animal survival. (C) Tumor weights were measured at the end of the experiment. The tumor volume was determined by direct measurement with a caliper and was calculated using the formula (width2×length)/2. The mean tumour volume±s.e.m is shown. The Kaplan-Meier method was used to compare mice survival.

[0094] FIGS. 17A-D. The silencing of LXRβ in tumor affects the effects of DDA on T regulatory and cytotoxic CD8+ T cells population infiltrated inside tumor. Animals grafted with E0711 tumors expressing the LXRβ (square symbol) or knockdown for the LXRβ expression (circle symbol) treated in FIG. 2 were sacrified to collect tumors, 15 days post treatment with the vehicle (empty symbol) or 0.37 μg/kg of DDA (full symbol). The tumor were dissociated by using gentlemac technology (Miltenyi), and the suspension of tumor cells obtained were stimulated with 50 ng/ml of PMA (Sigma), 500 ng/ml Ionomycin (Sigma) and 1/1000 of golgi stop (ebiosicence) during 3 h at 370 to analyze by flow cytometry the phenotype of tumor-infiltrated lymphocytes. By gating on CD45+CD4+ (A-B) or CD45+CD8+ T cells populations (C-D) as well as live/dead stain, the tumor-infiltrating (A) T regulatory cells (Treg; Foxp3+), (B) effector CD4+ cells (Th1; T-bet+) and (C) cytotoxic CD8 T cells (CTL; IFN-γ+Granzym B+) were determined. The dots in the graphs indicate the relative number of cell subpopulations (A) T reg, (B) Th1 and (C) CTL present into tumor. The vehicle condition was normalized to 1. (D) measure of the PD-1 expression on the surface of CTL cells infiltrated inside tumors. The dots in the graph represents the percentage of PD-1 negative CD8+ cells inside the tumors.

[0095] FIGS. 18A-B. The silencing of LXRβ in tumor cells modifies the effect of DDA treatment on the ratio of macrophage infiltrated inside tumor. Animals were grafted with tumor cells and treated as described in FIG. 2 and the suspensions of tumor cells were stained for macrophage phenotype. By gating on F4/80+CD11b+ cells, the percentage of macrophage (A) M1 (CD86+CD206−) and (B) M2 (CD86−CD206+) was determinated. The dots in the graphs represent the relative number of macrophage M1 and M2 infiltrated inside the tumors. The vehicle condition was normalized to 1.

[0096] FIGS. 19A-I. The silencing of LXRβ in tumor cells modifies the effect of DDA treatment on dendritic cells infiltrated inside tumor. Animals were grafted with tumor cells and treated as described in FIG. 2 and the suspensions of tumor cells were stained for dendritic cell phenotype. By gating on live cells, the relative number of (A) MDSC (CD11b+Ly6G+ Ly6Cint), (B) dendritic cells CD11c+ and (C) CD11c+CD8α+ (CD86−CD206+) was assessed. (D-E) The dots in the graphs represent the ratio between MDSC and either (D) dendritic cells CD11c+CD8α+ or (E) CD11c+. The vehicle was normalized to 1 and graphs are representative of three independent experiments. (E-H) The level of the migratory receptor CCR-7 (E, G) and the mature marker (F, H) expressed on the surface of dendritic cells CD11c+ (E-F) and CD11c+CD8α+ (G-H) were measured by flow cytometry and the mean of fluorescence (MFI) is indicated by bars in the graphs. MDSC: myeloid suppressive cells. CD11c+CD8α+: antigen presenting dendritic cells.

[0097] FIGS. 20A-C. The priming of T cells inside tumor side lymph node is dependent of the LXR expressed by tumor cells. Animals were grafted with tumor cells and treated as described in FIG. 2. At the end of the experiments, tumor side lymph nodes (mesenteric, auxiliary and brachial) were collected, dissociated and stimulated in vitro with 50 ng/ml of PMA (Sigma), 500 ng/ml Ionomycin (Sigma) and 1/1000 of golgi stop (ebiosicence) during 3 h at 370 to analyze by flow cytometry the phenotype of T cells. (A-C) The dots in the graphs represent the relative number of (A) T regulatory cells (CD4+ Foxp3+), (B) Th1 CD4+ cells (CD4+ T-bet+) and (C) cytotoxic CD8+ T cells (CD8+ Granzym B+ IFNγ+). The vehicle was normalized to 1. Graphs are representative of three independent experiments.

[0098] FIGS. 21A-E. DDA-exosome treatment controls tumor growth and animal survival. Immunocompetent C57BL/6 mice were implanted subcutaneously (s.c) with 300 000 E0771 (ER+) mouse mammary cancer cells expressing the LXRβ (E0 ShC, full symbol) or silenced for the LXRβ expression (E0 ShLXRβ, empty symbol). When the tumor reached a volume of 50-100 mm.sup.3, animals (n=8-10 mice per group) were treated at time indicated with either DDA at 0.37 μg/kg (Affichem) (.square-solid.,□) or vehicle (.circle-solid.,∘) once per day, every two, or with 5 μg exosomes purified from the media of cell treated with 2.5 μM DDA for 24 h (DDA-exo, .Math.,∇) or with vehicle (C-exo .box-tangle-solidup.,Δ), or with a combo treatment DDA+DDA-exo () or DDA+C-exo (⋄,.diamond-solid.). Animals were monitored over time for tumor growth, the dots in the graphs show tumor volumes 20 days post-treatment. The tumor volume was determined by direct measurement with a caliper and was calculated using the formula (width.sup.2×length)/2. The mean tumour volume±s.e.m is shown. The Kaplan-Meier method was used to compare the mice survival. Data are representative of 2 experiments.

[0099] FIGS. 22A-B. DDA-exosome treatment protect against a rechallenge with tumor cells. The mice, grafted with E0771 shC or E0771 shLXRβ and treated as indicated, which exhibited complete tumor eradication from previous experiments (FIG. 7), were injected in the tail vein (i.v.) with 300 000 E0771 tumor cells. Since no mice treated with vehicle have survived from the experiments of FIG. 7, we have used as control mice, healthy mice that have not been injected previously with tumor cells. These control mice were injected in the tail vein with E0771 tumor cells (n=3), as the survival mice of the experiments of FIG. 7. Seven days later, all the mice were killed and their lungs were isolated and stained intratracheally with 15% India Black Ink solution and tumor surface (not stained with the black ink) relative to healthy surface of the lungs (stained with the black ink) was measured with the Image J software. The bar graphs represent the mean of tumor-free total lung surface (in %) from mice having been grafted subcutaneously with (A) E0711shC or (B) E0711 shLXRβ (except control mice), and rechallenged with E0771 tumor cells and treated as indicated. Data are representative of 2 experiments.

[0100] FIGS. 23A-C. The cytokine gradient modified by DDA and DDA-exosome treatment is dependent on the LXRβ expressed in the tumor cells. Measurement of cytokines by multiplex cytokine bead array (CBA) in plasma of mice grafted with E0771 ShC or E0711 ShLXRβ as previously described in FIG. 7. Seven days after different treatments, the blood was collected and the plasma were separated by high speed centrifugation. The results are expressed as pg/mL concentration, the bar grafts (A, B) show anti-tumor cytokines, (C) pro-tumor cytokine.

EXAMPLE 1

[0101] Methods

[0102] Cell Culture. E0771, B16F10 and SKMEL28 tumor cells were from the American Type Culture Collection (ATCC, USA). Cells were grown at 37° C. in humidified atmosphere with 5% C02 in media containing 2 mM L-glutamine, 50 U/ml of penicillin/streptomycin and 10% fetal bovine serum (FBS) (for SKMEL-28, FBS was heated for 1 h at 56° C.). E0771 cell were cultured in RPMI 1640 medium supplemented 1% Hepes. B16F10 (passages did not exceed 20) were grown in DMEM 4 g/l sucrose plus 2 mM glutamine and SKMEL28 in RPMI 1640. The cells were splitted at 80% confluence.

[0103] Obtention of LXRβ knock-down cells. SKMEL28 cells (5×105) or E0771 (3×10.sup.6) were transfected with the Neon Transfection System (Invitrogen) with 1 μg or 3 μg of small hairpin RNA targeting human LXRβ or mouse LXRβ (two different shRNA were used) or with 1 μg control ShRNA. Transfected cells were selected in multiwell plates (10 000 cells/well) with puromycin ranging from 1-10 μg/ml. Two clones transfected with two different shRNA against LXRβ with LXRβ expression knocked-down by 70% and 80% (for SKMEL28) and by 90% and 95% (for E0771) and two control clones were selected.

[0104] Exosome preparation. Cells were seeded in complete medium at 50% confluence with exosome-free FBS, obtained after ultracentrifugion overnight at 110 000×g to eliminate serum exosomes and other microvesicles, and sterilized through a 0.2 μm filter. Human SKMEL28 cells were incubated with 2.5 μM DDA or vehicle (ethanol 1/1000 v/v final) for 24 h and mouse B16F10 cells were incubated with 1 μM DDA for 24 h. After this time, cell culture medium was collected and exosomes were purified from the cell culture medium by differential centrifugations. Briefly, cell culture medium was sequentially centrifuged at 4° C. at 1200×g for 5 min and 10 000×g for 30 min. Exosomes were then pelleted at 110 000×g for 70 min, resuspended in 5 ml PBS and centrifuged again at 110 000×g for 70 min. Final exosome pellet was diluted in PBS. For in vivo experiments, exosome were prepared in sterile conditions or sterilized by filtration through a 0.2 μm culture sterilization filter before injection into mice.

[0105] Exosome quantification. 1) Protein content in exosomes was quantified by the spectrophotometric method of Lowry in presence of 0.1% w/v sodium dodecyl phosphate. 2) Exosomes were also quantified by flow cytometry following labeling with the fluorescent lipid Bodipy-ceramide (Invitrogen-Molecular Probes) for 1 hour at 37° C. Excess of Bodipy-ceramide was removed by filtration and washing through the 1000 kDa Vivaspin filter and exosomes were quantitated by FACS. 3) Numeration of exosomes vesicles was performed either by nano tracking analysis (Nanosight equipment, Malvern, France) or by TRPS (tunable resistive pulse sensing) technology (qNano equipment, Izon, UK).

[0106] Exosome characterization. By flow cytometry: (tetraspanin analysis), exosomes (10 μg) were bound onto 10 μl of latex beads (Interfacial Dynamics/Invitrogen) in 200 μl PBS for 1 hour at 25° C. with gentle periodical shaking. Free sites on latex beads were saturated with 100 μl vesicle-free FBS for 30 min at 25° C. Beads with bound exosomes were centrifuged for 5 min at 5000 rpm, washed in 200 μl PBS, and diluted in 100 μl FACS buffer. Specific primary antibody or control isotype (1:50) were added and incubated at room temperature for 30 min. After centrifugation and washing, secondary antibody (1:100) was added and incubated for 30 min at room temperature. Beads with bound antibody-labelled exosomes were diluted in 1 ml FACS buffer and analyzed by flow cytometry (FACScalibur, Becton-Dickinson). By western-blot analysis: 5-20 μg exosomes were directly diluted in sample buffer and denaturated by heating at 60° C. for 10 min. Equal amounts of proteins were deposited in each well and proteins were resolved in SDS-PAGE and transferred onto PVDF membranes, saturated with 5% w/v non-fat milk in TBS-Tween 0.1%. Antibodies were added in 1% w/v non-fat milk in TBS-Tween 0.1% at the indicated dilutions according to the manufacturer. Revelation from immunoblotting was performed by enhanced chemiluminescence and analysed by ChemiDoc imager (BioRad) or by Pxi imager (Ozyme). By sucrose gradient: the density of exosomes was measured through a sucrose gradient. 50 μg exosomes in 100 μl PBS were deposited on top of a discontinuous gradient constituted by 9 layers of increasing sucrose concentration from 0.25 M to 2.25 M and a cushion of 2.5 M sucrose, and centrifugated at 160 000 g for 16 hours in swinging buckets. Fractions of 1 ml were harvested, diluted in 10 ml PBS and centrifugated for 2 h at 110 000×g. Pellets were recovered in Laemli buffer and their protein content resolved through SDS-PAGE, then probed for expression of CD9, Alix, Hsp70 and tyrosinase as indicated.

[0107] Prostaglandin determination. PGE2 in exosomes from SKMEL-28 was determined at the lipidomic facility of IMBL/INSA-Lyon from 70 μg protein. Briefly lipids were extracted with ethylacetate, samples were spiked with 10 ng of deuterated prostaglandins standards (Cayman), lipids separated by UHPLC and characterized by MS/MS. PGE2 in exosomes from B16F10 cells were determined from samples extracted by methanol/water, spiked with standards and analyzed by LC/ESI-MS.

[0108] Generation and treatment of DC: Peripheral blood mononuclear cells were isolated from human peripheral blood of healthy donors by standard density gradient centrifugation on Ficoll-Hypaque (GE Healthcare). Mononuclear cells were separated from peripheral blood lymphocytes (PBL) by centrifugation on a 50% Percoll solution (GE Healthcare). Monocytes were purified by immunomagnetic depletion (Life technologies, Rockville, Md., USA) using a cocktail of monoclonal antibodies (Ab) anti-CD19 (4G7 hybridoma), anti-CD3 (OKT3, ATCC, Rockville, Md., USA) and anti-CD56 (NKH1, Beckman Coulter, Fullerton, Calif., USA). Monocytes (purity>90%) were differentiated to immature DC (iDC) during 7 days with human rGM-CSF and rIL-4 (Human DC cytokine package, Peprotech) in RPMI 1640 supplemented with 2 mM glutamine, 10 mM Hepes, 40 ng/ml gentamycin (Life Technologies) and 10% FBS. Cells were treated at day 6 for 24 h with 20 μg exosomes. All cells and supernatants were collected at day 7. Control mature DC (mDC) were obtained by adding 1 μg/ml LPS (from Escherichia coli 0127:B8) at day 6 for 24 h. All DC were more than 95% pure as assessed by CD14 and CD1a labeling. DC Phenotyping: DC phenotype was analyzed on a FACSCanto (BD Biosciences, Le Pont de Claix, France) using FITC-conjugated anti-CD14, -HLA-DR, -CD80, -CD54, and PE-conjugated anti-CD1a, -CD86, -CD83 and -CD40 (Beckman Coulter). Mixed Lymphocyte Reaction (MLR): T lymphocytes were purified from PBL, after Ficoll-Hypaque and Percoll gradient centrifugation as described above, by immunomagnetic depletion using a cocktail of monoclonal Ab anti-CD19 (4G7), anti-CD56 (NKH1), anti-CD16 (3G8), anti-CD14 (RMO52) and anti-glycophorin A (11E4B7.6) (Beckman Coulter). T lymphocytes were more than 95% pure as assessed by CD3 labeling. Primary MLR were conducted in 96-well flat-bottom culture with various DC/T lymphocyte ratios (1/10; 1/20; 1/40).

[0109] Healthy C57BL/6 mice treatment with DDA: 6 weeks healthy C57BL/6 mice (from Janvier laboratory) were injected intraperitoneally (IP) with 100 μl of DDA (synthesized by Affichem) (0.37 μg/kg, 5 mg/kg or 20 mg/kg in sterile water) or with the solvent vehicle (control) every 5 days. Mice were killed at day 20 and single-cell suspension was prepared from spleen for flow cytometry analysis.

[0110] Tumor growth analysis. All of the animal procedures for the care and use of laboratory animals were conducted according to the guidelines of our institution and followed the general regulations governing animal experimentation. Exponentially growing cells were harvested, washed two times in PBS, and resuspended in PBS at the indicated concentrations. B16F10 tumors were obtained by subcutaneous transplantation of 35 000 cells in 150 μl into the flank of C57BL/6 or Balb/c female mice respectively. Then 1 μg of exosomes isolated from culture medium of cells treated with DDA or vehicle were injected once intra-dermally into the opposite flank. E0771, E0771 sh control and E0771 sh LXR tumors were prepared by subcutaneous transplantation of 300 000 cells in 100 μl PBS into the flank of C57B16 mice (6 week-old from Janvier laboratory). When the tumors reached a volume of 50 mm.sup.3 (around 10 days), the mice were injected intraperitoneally (IP) with 100 μl of DDA (0.37 μg/kg or 20 mg/kg in sterile water) or with the solvent vehicle (control). The treatment was repeated every 2 or 5 days as indicated until the end of experiment. The tumor volume was determined every 2-3 d by direct measurement with calipers and calculated using the formula [width.sup.2×length]/2. The Kaplan-Meier method was used to compare mice survival.

[0111] Tumor dissociation: Freshly excised tumors were trimmed of skin, fat, and necrotic tissue and minced in cold Hanks' medium. The minced tumor pieces were placed in an enzyme solution consisting of collagenase type D at 1 mg/ml and DNase type 1 at 20 μg/ml in Hanks' medium at 37° C. After 30 min of dissociation, the cell suspension was collected, washed with Hank's medium, and then suspended in PBS 1×, 0.5% BSA, 0.02% azide and 200 mM EDTA (Facs medium).

[0112] Analysis of immune cells by flow cytometry. Immune cells from the tumors were stained with the indicated fluorescent-labelled antibodies: anti mouse α-CD4, α-CD8, α-T-bet, α-Foxp3, α-granzyme B, α-PD-1, α-CD44, α-Ly6C, α-Ly6G, α-CD11b, α-CD11c, α-CD206, α-CD86, α-IL10, α-IL-6, α-IL-4 purchased from eBioscience or Biolegend. Intracellular staining for T-bet, Foxp3, IFNg, Granzyme B, IL-10, IL-4 and IL-6 was performed according the manufacturer's protocol from Biolegend. To observe the cytotoxic CD8 T cells, the tumor suspension was prealably stimulated 2 h in vitro with a cocktail of PMA (50 ng/ml), ionomycine (500 ng/ml) and golgistop (concentration from manufacture BD Pharmingen), then the cells were stained with specific antibodies. To set the gates, flow cytometry dot plots were based on comparison with isotype control. Flow cytometry measurements of single-cells suspension were performed on a Fortessa 20X (BD pharmingen) and data were analyzed using FlowJo software.

[0113] Cells isolation. Single-cells leukocyte suspensions were obtained from spleens of C57BL/6 mice. Naive CD4 or CD8 T cells are isolated by depletion of memory CD4 or CD8 T cells and non-CD4 or non-CD8 T cells according the manufacturer's protocol from Miltenyi kit (Miltenyi biotec). Purities of CD4+CD44l.sup.ow CD62L.sup.high or CD8+CD44l.sup.ow CD62L.sup.high T cells after isolation were >98%

[0114] Immune cell culture. Isolated CD4+ or CD8+ T cells were cultures in 96-well flat bottom plates (0.25×10.sup.6 cells per wells) in 0.25 ml of complete RPMI 1640 media (10% FBS, 1% penicillin/Streptomycin, 1% sodium pyruvate, 1% HEPES and 50 μM b-mercaptoethanol) in the presence of 10 μg/ml plate-bound anti-mouse CD3 (2C11) and 2 μg/ml soluble α-CD28 (LEAF) in addition to 50 ng/ml of recombinant IL-2 (e-bioscience). DDA (synthesized by Affichem) diluted in the solvent vehicle was added at increasing concentration (0-1-10-100 and 1000 nM). Cells were cultured in polarizing Th1 (20 ng/ml of recombinant IL-2 and 10 μg/ml of anti-IL4), Th2 (50 ng/ml of recombinant IL-4, 10 μg/ml of anti-IFNg), Th17 (10 ng/ml of recombinant TGF-b, 100 ng/ml of recombinant IL-6, 10 μg/ml of anti-IFN-g and 10 μg/ml of anti-IL4) or Treg (10 ng/ml of recombinant TGF-b, 10 μg/ml of anti-IFNg and 10 μg/ml of anti-IL4) conditions. All recombinant cytokines were purchased from Peprotech and antibodies were purchased from eBioscience. After 5 days of culture, cells were collected and analyzed by flow cytometry. To investigate the impact of DDA on polarization of CD4 or CD8 naive T cells, DDA or the solvent vehicle was added at the beginning of culture at Day 0 or at Day 4 and cells was analyzed at day 5 by flow cytometry.

[0115] Statistical analyzes. Tumor growth curves in animals were analyzed for significance by the analysis of variance (ANOVA). In other experiments, significant differences in the quantitative data between the control and the treated group were analysed using the Student's t-test for unpaired variables (Graph Pad Prism software). In all figures, *, ** and *** refer to P<0.05, P<0.01 and P<0.001 compared with the control (vehicle), unless otherwise specified.

[0116] Results

[0117] Results depicted in FIG. 1 show clearly that DDA increases the differentiation of Th0 into Th1 from 1 nM concentrations, the differentiation of Th0 into Th17 from 100 nM and the differentiation of naïve CD8 T cells into functional cytotoxic CD8 T cells from 10 nM. In contrast, DDA treatment has no effect on Th2 and Treg differentiation. When DDA was added at day 4, the differentiation of Th0 into Th1 and naïve CD8 T cells into functional cytotoxic CD8 T cells is also increased from 1 nM. In addition, DDA has no effect on the differentiation into Th17 and Th2. Importantly, DDA inhibits the differentiation of Th0 into Treg (FIG. 2). The results depicted in FIG. 3 show that DDA does not activated Th0 into Th1 differentiation but inhibits the differentiation of Th0 into Treg phenotype (more impressive in condition #2, Day 4).

[0118] Whatever DDA concentrations, DDA treatment inhibits tumor growth and increases mice survival (FIG. 4). DDA treatment increases the infiltration of CD4 Th1 cells, activated CD8 (CTL), dendritic cells (DC: CD11c+), and macrophage type M1 inside the tumor. Inversely, DDA treatment decreases the infiltration into the tumor of the regulatory CD4 Treg cells (Treg), inactivated CD8 (No CTL), myeloid derived suppressive cells (MDSC) and macrophage type M2 (FIG. 5). Collectively these data show that DDA treatment allows activation of the immune system against the tumor resulting in the control of tumor growth. The CD4 T cells acquire an activated phenotype which is underlined by the upregulation of CD44 at their surface at day 4 (data not shown). Inversely, DDA has no significant effect on CD8 T cells phenotype (data not shown).

[0119] We show that DDA stimulates the amount of multivesicular bodies (MVB) which contain the exosomes in B16F10 cells, observed by electronic microscopy. The vesicles purified from B16F10 cell culture media after treatment with 1 μM DDA for 24 h or the solvent vehicle were characterized as being exosomes considering their size analysed by electronic microscopy, their density and the presence of specific markers of exosomes such as CD9, CD81 and Lamp2 (data not shown). DDA stimulates the production of exosome secreted into the media by 1.5 to 2-fold in B16F10 cells (data not shown). This effect was also observed in human and murine mammary tumor cells (data not shown). Exosomes modified by DDA (DDA-exosomes) display a differentiated and immunogenic phenotype compared with control-exosomes (FIG. 6). More particularly a single injection of DDA-exosomes controls tumor growth and increases mice survival (FIG. 7). We performed similar experiments with SKMEL-28 cells and we demonstrated that DDA stimulates exosome secretion from human melanoma cells (data not shown). DDA-exosomes from human SKMEL-28 melanoma cells display a differentiated and immunogenic phenotype compared with control-exosomes (data not shown).

[0120] We then determined whether the liver X receptors (LXR), the receptors of DDA which are known to modulate of the immune system, were involved in the secretion and the phenotypic modification of DDA-exosomes in SKMEL-28 cells. The LXRbeta is the only subtype expressed in these cell type. We knocked-down the expression of the LXRbeta in SKMEL-28 by using specific shRNA against the LXRbeta (SKMEL-28-shLXRbeta) compared with control sh (SKMEL-28-shCTRL). SKMEL-28-shLXRbeta and SKMEL-28-shCTR cells were stimulated with 2.5 μM DDA for 24 h or with the solvent vehicle. Then, the exosomes were purified from the cell media, quantified and analysed. DDA (2.5 μM for 24 h) significantly increases the production of exosomes from SKMEL-28-shCTRL cells by about 2-fold while DDA does not stimulate the production of exosomes from SKMEL-28-shLXRbeta, indicating that LXRbeta mediates DDA-induced exosome secretion. DDA produces exosomes from SKMEL-28-shCTRL cells enriched in molecules involved in MVB trafficking (rab27a and b), antigen presentation (HSP70), antigen of differentiation (Melan A, tyrosinase, TRP2) and DC «eat-me» signal (calreticuline). In contrast, DDA produces exosomes from SKMEL-28-shLXRbeta cells that are not enriched in molecules involved in MVB trafficking (rab27a and b), antigen presentation (HSP70), antigen of differentiation (Melan A, tyrosinase, TRP2) and DC «eat-me» signal (calreticuline). These data indicate that the LXRbeta mediates DDA-induced the phenotypic modification of exosome. To determine the immunogenic properties of DDA-exosomes and the implication of LXRbeta in these effects we studied the impact of DDA-exosomes purified from SKMEL-28-shCTRL or SKMEL-28-shLXRbeta cells on dendritic cell maturation. DDA-exosomes purified from human SKMEL-28-shCTRL cells media increase cell surface markers of mature human dendritic cells (FIG. 8). DDA-exosomes purified from SKMEL-28-shCTRL cells media stimulate the secretion of immunoactivating cytokines which are secreted by mature dendritic cells. The IL12/IL10 ratio is strongly increased. This effect is not observed with DDA-exosomes purified from SKMEL-28-shLXRbeta cells media indicating that DC maturation by DDA-exosomes is dependent on the expression LXRbeta in the parental cells (data non shown). Dendritic cells maturated by DDA-exosomes purified from the media of SKMEL-28-shCTRL cells stimulate naive T lymphocytes to produce interferon gamma indicating that DDA-exosomes activate the functionality of naive T lymphocytes toward a immunostimulator Th1 phenotype (INFg production>>IL13, IL6 production). These effects are abolished when similar experiments were realized with DDA-exosomes purified from the media of SKMEL-28-shLXRbeta cells (data non shown). These data indicate that the effect of DDA-exosomes on DC-functionality depends on LXRbeta expression in the parental cancer cells. In conclusion, LXRb expressed in cancer cells drives the effect of DDA on exosome secretion, phenotype modification and immunogenicity.

EXAMPLE 2

[0121] FIG. 9 shows that DDA increases the differentiation of Th0 into Th1 and decreases the differentiation of Th0 into Treg. Moreover, the effect is dependent of LXR expression, because on its absence the DDA effect on Th1 and Treg differentiation is abrogated.

[0122] FIG. 10 shows that that DDA has no impact on Th2 differentiation and is independent of LXR.

[0123] FIG. 11 shows that DDA increases the differentiation of CD11c into CD11c CD8a+ and their maturation, and that this effect is dependent of LXR expression since it is abolished in absence of LXR.

[0124] FIG. 12 shows that 1 μM DDA increases MHC II expression at the surface of CD11c dendritic cells and this effect is dependent of the expression of the LXR since it is abrogated in absence of LXR.

[0125] FIG. 13 shows that DDA significantly controls the growth of tumors expressing the LXRβ (E0771 sh control) while this effect is abolished in tumors knocked down for the expression of the LXRβ (E0771 sh LXR), indicating that the LXRβ mediates the control of tumor growth by DDA.

[0126] FIG. 14 shows that the activation of an immuno-active microenvironment inside the tumors under DDA treatment is dependent of the expression of the LXRβ in the tumors.

EXAMPLE 3

[0127] Material and Methods

[0128] Exosome preparation. Mouse mammary E0771 cells (ATCC) were seeded in DMEM with 10% exosome-free FBS at 50% confluence. Exosome-free FBS were obtained after ultracentrifugion overnight at 110 000×g to eliminate serum exosomes and other microvesicles, and sterilized through a 0.2 μm filter. E0711 cells were incubated with 1.5 μM DDA or vehicle (ethanol 1/1000 v/v final) for 24 h. After this time, cell culture medium was collected and exosomes from cells treated with DDA (DDA-exo) or with the vehicle (C-exo) were purified from the cell culture medium by differential centrifugations. Briefly, cell culture medium was sequentially centrifuged at 4° C. at 1200×g for 5 min and 10 000×g for 30 min. Exosomes were then pelleted at 110 000×g for 70 min, resuspended in 5 ml PBS and centrifuged again at 110 000×g for 70 min. Final exosome pellet was diluted in PBS. For in vivo experiments, exosomes were prepared in sterile conditions.

[0129] Animal experiments. All of the animal procedures for the care and use of laboratory animals were conducted according to the guidelines of our institution and followed the general regulations governing animal experimentation. E0771 exponentially growing cells were harvested, washed two times in PBS, and resuspended in PBS at the indicated concentrations. E0771 Shcontrol (shC) or E0711 ShLXRβ (shLXRβ) tumors were prepared by subcutaneous transplantation of 300 000 cells in 100 μl PBS into the flank of C57B16 mice (6 week-old from Janvier laboratory). When the tumors reached a volume of 50 mm3 (around 10 days), the mice were injected intraperitoneally (IP) with 100 μl of DDA (0.37 μg/in sterile water) or with the solvent vehicle (control) once per day and every two days. Depending of the experiments, the mice were also treated subcutaneously with 5 ug exosomes from E0711 tumor cells treated or not with DDA (DDA-exo versus C-exo) as described above or with 5 ug exosomes (DDA-exo versus C-exo) in combination with DDA (0.37 ug/kg). For the latter, the exosomes were injected 24 h after the first DDA treatment, then DDA treatment was maintained every two days once a day. The tumor volume was determined every 2-3 d by direct measurement with calipers and calculated using the formula [width2×length]/2. The Kaplan-Meier method was used to compare mice survival.

[0130] Organ dissociation and flow cytometry. The tumor-side lymph nodes were dissociated manually while for the tumor, gentlemac technology (Miltenyi) was used. Then, the suspension of tumor cells or lymph node were stimulated with 50 ng/ml of PMA (Sigma), 500 ng/ml Ionomycin (Sigma) and 1/1000 of golgi stop (ebiosicence) during 3 h at 37° C. After that, the single cell suspension were stained with the indicated fluorescent-labelled antibodies: anti mouse α-CD4, α-CD8, α-T-bet, α-Foxp3, α-granzyme B, α-PD-1, α-CD44, α-Ly6C, α-Ly6G, α-CD11b, α-CD11c, α-CD206 IL10, α-CD86 as well as live/dead stain purchased from eBioscience or Biolegend. Intracellular staining for T-bet, Foxp3, and Granzyme B, was performed according the manufacturer's protocol from Biolegend. To set the gates, flow cytometry dot plots were based on comparison with isotype control. Flow cytometry measurements of single-cells suspension were performed on a Fortessa 20X (BD pharmingen) and data were analyzed using FlowJo software.

[0131] Tumor rechallenge: Mice exhaling a complete eradication of E0771 shcontrol (shC) or E0771 Sh LXRβ (shLXRβ) tumors following treatment with DDA combined or not with control-exosomes or DDA-exosomes, were rechallenged with 300 000 E0771 cells injected into the tail vein of mice. Seven days later, their lungs were isolated and stained intratracheally with 15% India Black Ink solution and fixated in Fekete's solution (100 mL of 70% alcohol, 10 mL formalin, and 5 mL glacial acetic acid). The percentage of lung surface invaded by metastatic nodules was analyzed using NIH Image J software. Briefly, lung photographs were converted in gray scale; metastatic nodules (white staining) and healthy lung tissue (black staining) were defined using the threshold color parameter and the respective area measured.

[0132] Measurement of cytokine in plasma: Cytokine plasma levels were determined using commercially available kits, Cytometric Beads Array—CBA (BD Biosciences Pharmingen, USA) to quantify IFN-γ, IL-12 and RANTES. The CBA immunoassay was carried out according to the manufacturer instructions. Flow cytometry measurements were performed on a LSR II (BD pharmingen) and data were analyzed using FCAP array software (BD pharmingen).

[0133] Results

[0134] FIG. 15 shows that DDA decreases the number of T regulatory CD4 T cells and increases the activated CD4+ and CD8+ T cells inside tumors.

[0135] FIG. 16 shows that DDA inhibits tumor growth and increases animal survival by acting through the LXRβ expressed in tumor cells.

[0136] FIG. 17 shows that DDA decreases the number of T regulatory cells and increases the number of effector Th1CD4+ cells infiltrated into the tumors and increases the number of activated cytotoxic CD8+ T cells infiltrated into the tumors. The decrease expression of LXRB into the tumors abolished the effect of DDA on T regulatory and activated cytotoxic CD8+ T cells infiltrated into the tumors but had no effect on the effector Th1CD4+.

[0137] FIG. 18 shows that DDA increases the number of macrophages M1 infiltrated into the tumors and this effect is dependent of the LXRβ expressed in the tumors. DDA decreases the number of macrophages M2 infiltrated into the tumors. This effect is independent of the LXRβ expressed in the tumors.

[0138] FIG. 19 shows that DDA decreases the number of MDSC infiltrated into the tumor and increases the ratio of dendritic cells CD11+ and CD11+ CD8α+ versus MDSC. These effects are abolished in tumors knocked-down for the LXRβ.

[0139] FIG. 20 shows that DDA decreases the number of Treg cells and increases the number of Th1CD4+ cells and cytotoxic CD8 T cells infiltrated into tumor side lymph nodes. The priming of T cells inside tumor side lymph nodes is dependent of the LXRβ expressed by tumor cells.

[0140] FIG. 21 shows that DDA-exosome treatment significantly decreases tumor growth and increases animal survival. Treatment with DDA-exosomes compensates the silencing of LXRβ on tumor cells and the loss of DDA response and increases animal survival and tumor-free mice.

[0141] FIG. 22 shows that DDA-exosomes protect against a rechallenge with tumor cells expressing or not the LXRβ.

[0142] FIG. 23 shows that DDA-exosome treatment increases the anti-tumor cytokines, IFNγ and IL-12, in the blood of mice grafted with tumor expressing the LXRβ. These effects are abolished when animals were grafted with tumor silenced for the LXRβ. No increase was observed for the pro-tumor cytokine Rantes.

REFERENCES

[0143] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.