NEW DRUG SCREENING ASSAY USING REGULATORY MACROPHAGES

Abstract

The present invention relates to a method for identifying compounds that are able to modulate the development of certain regulatory T cells which are induced by regulatory macrophages. Since induced regulatory T cells (iTregs) play a crucial role in diseases and pathological conditions, the method is highly suitable for identifying potentially active therapeutics. The method is particularly suitable for the screening of large libraries of candidate drug substances, such as small molecule or antibody libraries.

Claims

1. Method of identifying a compound which is able to modulate the development of regulatory macrophage (Mreg)-induced regulatory T cells (iTregs), comprising the steps of: a) providing DHRS9.sup.+ CD258.sup.+ IDO.sup.+ Mreg cells; b) providing T cells; c) co-culturing the DHRS9+CD258+ IDO+ Mreg cells and the T cells to induce the development of Foxp3.sup.+ CD25.sup.+ iTreg cells; wherein a test compound is added in step a), b) or c), and d) determining whether the test compound influences the generation of Foxp3+CD25+ iTreg cells.

2. Method of identifying a compound which is able to modulate the development of regulatory macrophage (Mreg)-induced regulatory T cells (iTregs), comprising the steps of: a) providing CD14.sup.+ monocytes, b) culturing the CD14.sup.+ monocytes to induce the development of DHRS9.sup.+ CD258.sup.+ IDO.sup.+ Mreg cells; wherein a test compound is added in step a), or b), and c) determining whether the test compound influences the generation of DHRS9.sup.+ CD258.sup.+ IDO.sup.+ Mreg cells.

3. Method of identifying a compound which is able to modulate the development of regulatory macrophage (Mreg)-induced regulatory T cells (iTregs), comprising the steps of: a) providing CD14.sup.+ monocytes, b) culturing the CD14.sup.+ monocytes to induce the development of DHRS9.sup.+ CD258.sup.+ IDO.sup.+ Mreg cells c) providing T cells; d) co-culturing the DHRS9+CD258+ IDO+ Mreg cells and the T cells to induce the development of Foxp3.sup.+ CD25.sup.+ iTreg cells; wherein a test compound is added in step a), b), c) or d), and e) determining whether the test compound influences the generation of DHRS9.sup.+ CD258.sup.+ IDO.sup.+ Mreg cells or Foxp3+CD25+ iTreg cells.

4. Method of claim 1, wherein said Mreg cells are CD11b.sup.+ CD33.sup.+ DHRS9.sup.+ CD258.sup.+ IDO.sup.+ Mreg cells.

5. Method of claim 1, wherein said test compound is part of a drug library.

6. Method of claim 1, wherein said test compound is a small molecule.

7. Method of claim 1, wherein said test compound is selected from the group consisting of a peptide, a polypeptide, a protein, an antibody and an antibody fragment.

8. Method of claim 1, wherein said test compound is selected from the group consisting of a polynucleotide, a DNA, a DNA analog, an RNA and an RNA analog.

9. Method of claim 1, wherein the Mreg cells are co-cultured with the T cells at an Mreg:T cell ratio of 1:1 or 1:2.

10. Method of claim 1, wherein the Mreg cells and are co-cultured with the T cells for at least 48 hours, preferably at least 72 hours.

11. Method of claim 1, wherein the T cells are nave CD4.sup.+ T cells.

12. Method of claim 1, wherein the co-culturing of the Mreg cells and the T cells is performed in the presence of said test compound.

13. Method of claim 1, wherein the amount of Mregs used in the co-culture step (c) will be in the range of 110.sup.4 to 110.sup.7, more preferably 110.sup.5 to 110.sup.6.

14. Method of claim 1, wherein the amount of T cells used in the co-culture step (c) will be in the range of 110.sup.4 to 110.sup.7, more preferably 110.sup.5 to 110.sup.6.

15. Method of claim 1, wherein step (d) is performed by FACS staining.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0081] FIG. 1 shows that tacrolimus inhibited the formation of Foxp3+CD25+ iTreg cells in a dose-dependent manner. Dashed line indicates mean of iTreg frequencies in untreated cultures; dotted line indicates mean of iTreg frequencies in 1% DMSO vehicle-only controls.

[0082] FIG. 2 shows the effect of an anti-PAEP antibody upon Mreg-mediated iTreg generation. 20 g/ml anti-PAEP goat polyclonal antibody or 20 g/ml non-specific goat polyclonal antibody were added to a co-culture of Mregs with nave CD4+ cells (n=6).

[0083] FIG. 3 shows the effect of a recombinant BTLN8-Fc fusion protein upon Mreg-mediated iTreg generation. 10 g/ml BTNL8-Fc fusion protein or recombinant human Fc (rhFc) were added to a co-culture of Mregs with nave CD4+ cells (n=8).

[0084] FIG. 4 shows the effect of a TGF-RII antibody upon Mreg-mediated iTreg generation. 20 g/ml TGF-RII goat polyclonal antibody or 20 g/ml non-specific goat polyclonal antibody were added to a co-culture of Mregs with nave CD4+ cells (n=6).

[0085] FIG. 5 shows that anti-TGFRII significantly reduced iTreg generation. In addition, antibodies against ALK1 and ALK3 were found to enhance iTreg development.

[0086] FIG. 6 shows the results from the IFN- studies. A linear regression can be observed indicating a significant dose-dependent relationship (p<0.0001) between the IFN- concentration in the co-culture phase and iTreg generation.

EXAMPLES

[0087] The following examples merely describe preferred embodiments of the invention and should not be understood as limiting the invention.

Example 1: Preparation of Mregs in 6-Well Culture Plates

[0088] A preparation of human Mregs (Mreg A) was prepared in 6-well culture plates according to a modified protocol of Hutchinson. Peripheral blood mononuclear cells (PBMC) were obtained from a healthy donor for use as starting material for Mreg generation. The donor was screened for relevant disease markers, including infectious diseases, not more than 30 days prior to collection of PBMC. The donor was re-screened for the same disease markers on the day of leucocyte donation. PBMC were obtained by leukapheresis using the Spectra Optia device. PBMC from leukapheresis products were further purified by Ficoll-gradient centrifugation. CD14.sup.+ monocytes were isolated from Ficoll-separated, washed PBMC by positive magnetic bead selection using research-grade CD14 microbeads (Miltenyi Biotec, GmbH) in accordance with the manufacturer's instructions. The preparation of isolated monocytes was quality-controlled for viability and purity of CD14.sup.+ cells and contamination with CD3.sup.+ T cells by flow cytometry. Isolated monocytes were resuspended in Mreg culture medium at a density of 10.sup.6 viable monocytes/3 ml then plated in Cell+6-well plates (Sarstedt) at 10.sup.6 viable monocytes/well. Mreg medium comprised RPMI-1640 medium without Phenol Red (Lonza) supplemented with 10% human AB serum (ZKT Tiibingen), 2 mM L-GlutaMax (Gibco), 100 U/ml penicillin, 10 g/ml streptomycin (Gibco), and recombinant human M-CSF (R&D Systems) at a final concentration of 25 ng/ml carried on 0.1% human albumin (Octapharm). The cells were cultured for 6 days. On day 6, cultures were stimulated with 25 ng/ml recombinant human IFN- (Millipore). On day 7, adherent cell layers had formed which consisted of human Mregs.

Example 2: Preparation of Mregs in Flasks

[0089] A preparation of human Mregs (Mreg B) was prepared in 75 cm.sup.2 flasks according to a modified protocol of Hutchinson. Peripheral blood mononuclear cells (PBMC) were obtained from a healthy donor for use as starting material for Mreg generation. The donor was screened for relevant disease markers, including infectious diseases, not more than 30 days prior to collection of PBMC. The donor was re-screened for the same disease markers on the day of leucocyte donation. PBMC were obtained by leukapheresis using the Spectra Optia device. Human Mregs can also be generated from leukapheresates collected with alternative devices or from whole blood samples. PBMC from leukapheresis products were further purified by Ficoll-gradient centrifugation. CD14.sup.+ monocytes were isolated from Ficoll-separated, washed PBMC by positive magnetic bead selection using research-grade CD14 microbeads (Miltenyi Biotec, GmbH) in accordance with the manufacturer's instructions. The preparation of isolated monocytes was quality-controlled for viability and purity of CD14.sup.+ cells and contamination with CD3.sup.+ T cells by flow cytometry. Isolated monocytes were resuspended in Mreg culture medium at a density of 7.510.sup.5 viable monocytes/ml then plated in Cell+T75 flasks (Sarstedt) at 210.sup.5 viable monocytes/cm.sup.2 in a total volume of 20 ml medium. Mreg cultures can be scaled to smaller or larger flasks by adjusting cell density and medium volume in proportion to the cell culture area. Mreg medium comprised RPMI-1640 medium without Phenol Red (Lonza) supplemented with 10% human AB serum (ZKT Tiibingen), 2 mM L-GlutaMax (Gibco), 100 U/ml penicillin, 10 g/ml streptomycin (Gibco), and recombinant human M-CSF (R&D Systems) at a final concentration of 25 ng/ml carried on 0.1% human albumin (Octapharm). The cells were cultured for 6 days. On day 6, cultures were stimulated with 25 ng/ml recombinant human IFN- (Millipore). On day 7, the adherent cell fraction was washed then recovered by Accutase treatment (Stemcell Technologies) followed by gentle scraping. Mregs were then washed and resuspended in co-culture medium comprising X-Vivo 10 medium (Lonza) supplemented with 25 ng/ml rhM-CSF (R&D Systems) and 2 mM GlutaMax (Gibco).

Example 3: Co-Culturing Mregs with T Cells in 6-Well Plates

[0090] Human Mreg prepared in 6-well plates (Mreg A) according to Example 1 form an adherent monolayer at the bottom of the cell culture well. Culture supernatants were removed and the adherent cell layer was washed with Dulbecco's Modified Phosphate Buffered Saline without Ca.sup.2+ or Mg.sup.2+ (DPBS; Sigma). The medium was replaced with Co-culture Medium comprising X-Vivo 10 medium (Lonza) supplemented with 25 ng/ml rhM-CSF (R&D Systems) and 2 mM GlutaMax (Gibco). Freshly isolated CD4.sup.+ T cells were quality-controlled for viability, and the purity and CD4+/CD8.sup.+ ratio was checked by flow cytometry. Isolated CD4.sup.+ T cells were resuspended in co-culture medium then distributed at 10.sup.6 cells/well into 6-well plates containing Mregs. The final volume of co-cultures in 6-well plates was 3 ml, including test substances. Co-cultures were incubated for 5 days at 37 C. 5% CO.sub.2 prior to analysis of iTreg enrichment.

Example 4: Co-Culturing Mregs with T Cells in FACS Tubes

[0091] Human Mreg prepared in T75 flasks (Mreg B) according to Example 2 were harvested on day 7 of culture as described above and then re-suspended in co-culture Medium comprising X-Vivo 10 medium (Lonza) supplemented with 25 ng/ml rhM-CSF (R&D Systems) and 2 mM GlutaMax (Gibco). Mregs were distributed to sterile, lidded FACS tubes (Becton Dickinson) at 210.sup.5 viable Mregs/tube and then returned to the incubator for 1-hour. Freshly isolated CD4.sup.+ T cells were quality-controlled for viability, and the purity and CD4+/CD8.sup.+ ratio was checked by flow cytometry. Isolated CD4.sup.+ T cells were resuspended in co-culture medium then distributed at 410.sup.5 cells/well into tubes containing Mregs. The final volume of co-cultures in lidded FACS tubes was 1 ml, including test substances. Co-cultures were incubated for 3 to 5 days at 37 C. 5% CO.sub.2 prior to analysis of iTreg enrichment.

Example 5: Co-Culturing Mregs with T Cells in 96 Well Plates

[0092] Human Mreg prepared in T75 flasks (Mreg B) according to Example 2 were harvested on day 7 of culture as described above and then re-suspended in co-culture Medium comprising X-Vivo 10 medium (Lonza) supplemented with 25 ng/ml rhM-CSF (R&D Systems) and 2 mM GlutaMax (Gibco). Mregs were distributed to sterile, tissue cultured-treated 96-well plates (Costar) at 410.sup.4 viable Mregs/well and then returned to the incubator for 1-hour. The assay is performed in a round-bottomed 96-well plate. Freshly isolated CD4.sup.+ T cells were quality-controlled for viability, and the purity and CD4+/CD8.sup.+ ratio was checked by flow cytometry. Isolated CD4.sup.+ T cells were resuspended in co-culture medium then distributed at 810.sup.4 viable T cells/well (range 410.sup.4 to 1.610.sup.5 T cells/well) into wells containing Mregs. The final volume of co-cultures in one well of a 96-well plate was 200 l, including test substances. Co-cultures were incubated for 3 to 5 days at 37 C. 5% CO.sub.2 prior to analysis of iTreg enrichment.

Example 6: Analysis of iTreg Cells

[0093] The cocultured Mreg and T cells were harvested. Briefly, cells were centrifuged at 300g for 6 min, and the cell pellets were resuspended in 1 ml DMPS. 510.sup.5 cells were used for each staining reaction. After adding 1 l Fixable Live-Dead Viability DyeFluor506, the resuspended cells were incubated at 4 C. in dark for 30 min. After incubation, the cells were washed one time with 10 ml DPBS followed by centrifugation at 300g for 6 min and resuspended in 100 l FACS buffer+10% FcR block for 15 min. The cell suspensions were transferred into FACS tubes (100 l/tube) and antibodies against CD45RA, CD8, CD25, TGIT, FoxP3, CD4, CD3 and Helios were added.

[0094] Cell suspensions were incubated with said antibodies at 4 C. in the dark for 30 min. Afterwards, the samples were washed one time in 2 ml DPBS followed by centrifugation at 300g for 6 min. Then, the cells were gently vortexed and the supernatant was removed. The cells were fixed and permeabilized at 4 C. in the dark for 30 min. Samples were washed in 2 ml Perm Buffer followed by centrifugation at 300g for 6 min, and incubated with intracellular staining antibodies (i.e. against FoxP3, or Helios) for 30 min at RT in the dark. After incubation, the samples were washed 3 times in 2 ml Perm Buffer followed by centrifugation at 300g for 6 min. Finally, cells were resuspended in 300 l Perm Buffer for analysis by flow cytometry.

Example 7: Co-Culturing in the Presence of Tacrolimus

[0095] An experiment was performed to determine whether tacrolimus affected Mreg-mediated iTregs. For this purpose, Mregs from n=3 independent donors were prepared in 75 cm.sup.2 flasks as described in Example 2 (Mreg B). These Mregs were then set in co-culture in lidded FACS tubes with nave CD4.sup.+ T cells from n=3 independent donors according to the method described in Example 4. The co-culturing step was performed in the presence of different concentrations of tacrolimus (1 nM to 10 M). The results are shown in FIG. 1. It can be seen that tacrolimus potently suppressed iTreg generation in a dose-dependent manner. This demonstrates that the screening method of the invention can identify inhibitors of Mreg-induced Treg generation.

Example 8: Co-Culturing in the Presence of Anti-PAEP Antibody

[0096] An experiment was performed to investigate the involvement of PAEP in Mreg-mediated iTreg generation. For this purpose, Mregs from n=6 independent donors were prepared in 75 cm.sup.2 flasks as described in Example 2 (Mreg B). These Mregs were then set in co-culture in lidded FACS tubes with nave CD4.sup.+ T cells from n=6 independent donors 3 independent donors according to the method described in Example 4. The co-culturing step was performed in the presence of 20 g/ml of a goat polyclonal antibody against the progestagen-associated endometrial protein (PAEP). This protein, which is also commonly known as glycodelin or placental protein 14, is a 28-kDa glycoprotein produced by secretory and decidualized endometrium in women and by seminal vesicle epithelium in men. The results are shown in FIG. 2. It can be seen that the addition of the anti-PAEP antibody to allogeneic co-cultures of Mregs and nave CD4+ T cells led to a small, but consistent decrease in iTreg generation compared to addition of 20 g/ml control goat polyclonal antibody.

Example 9: Co-Culturing in the Presence of BTNL8-Fc Protein

[0097] Butyrophilin-like 8 (BTNL8) is a Ig-superfamily (IgSF) receptor belonging to the four-member butyrophilin-like (BTNL) gene family in humans, which is closely related to the butyrophilin (BTN) family, both of which families are structurally and functionally related to the B7-costimulatory molecules. Experiments show that the 37-kD form of BTNL8 is selectively expressed in activated iTregs. An experiment was performed to investigate the involvement of BTNL8 in Mreg-mediated iTreg generation. Mregs from n=8 independent donors were prepared in 75 cm.sup.2 flasks as described in Example 2 (Mreg B). These Mregs were then set in co-culture in lidded FACS tubes with nave CD4.sup.+ T cells from n=8 independent donors according to the method described in Example 4. The co-culturing step was performed in the presence of 10 g/ml BTNL8-Fc fusion protein. The results are shown in FIG. 3. It can be seen that the addition of the BTNL8-Fc fusion protein to allogeneic co-cultures of Mregs and nave CD4.sup.+ T cells led to a small, but consistent increase in iTreg generation compared to addition of 10 g/ml control recombinant human Fc protein. This demonstrates that the screening method of the invention can also identify enhancers of Mreg-induced Treg generation.

Example 10: Co-Culturing in the Presence of Anti-Cytokines Antibodies

[0098] Cytokines are secreted proteins that act as mediators of signals between leucocytes. An experiment was performed to investigate the involvement of TGF- in Mreg-mediated iTreg generation. Mregs from n=6 independent donors were prepared according to the method described in Example 2 (Mreg B). These Mregs were then set in co-culture in lidded FACS tubes with nave CD4.sup.+ T cells from n=6 independent donors according to the method described in Example 4. The co-culturing step was performed in the presence of neutralizing antibodies against TGF-RII. The results are shown in FIG. 4. It can be seen that the blockade of TGF RII significantly decreased iTreg generation. These results show that the method of the invention can be used for screening the biological activity of potential drug substances, in this case neutralizing antibodies.

Example 11: Screening Assay

[0099] The following experiment was performed to assess whether the Mreg-induced iTreg assay is capable of detecting a known active substance in the context of a true screening. Example 10 and FIG. 4 establish that neutralization of TGFRII leads to a substantial reduction in iTreg generation. Mregs were prepared from n=8 independent donors according to the method described in Example 2 (Mreg B). These Mregs were placed in co-culture with allogeneic CD3+ T cells from a further n=8 independent donors according to the method described in Example 5. This co-culturing step was performed in the presence of antibodies with known specificities for receptors of the TGF-3 superfamily (TGF-SF) or isotype control antibodies. When this screening experiment was performed, it was not explicitly known whether the TGF- superfamily receptors (except for TGFRII) had any biological effect in the system or indeed that said receptors are expressed by Mregs and/or T cells. Furthermore, it was not known whether the antibodies (except for anti-TGFRII) tested were antagonistic, agonistic or had no biological effect on said receptors. The results are shown in FIG. 5. Values represent iTreg frequency (%) in the treated samples indexed against iTreg frequency (%) in the no-treatment control. Values were log.sub.2-transformed and centered upon the mean of the relevant isotype control. A one-way ANOVA was performed to identify significantly increased or decreased iTreg generation. It can be seen that anti-TGFRII significantly reduced iTreg generation, so the positive control was shown to be positive. It can also be seen that antibodies against ALK1 and ALK3 enhanced iTreg development; therefore, ALK1 and ALK3 were identified by the Mreg-induced iTreg assay as novel molecular targets that can be further investigated as potential drug targets. ALK1 is a TGF-SF Type I receptor that dimerises with TGFRII to form a functional receptor for TGF-31. ALK3 is a TGF-SF Type I receptor that dimerises with BMPRII/IIB or ActRII/IIB to form a functional receptor for BMPs.

Example 12: Dose Dependency Study

[0100] The following experiment was performed to assess whether the Mreg-induced iTreg assay is capable of revealing a dose-response relationship between concentration of a test substance and iTreg development. Differentiation of human Mregs with iTreg-inducing capacity is dependent upon stimulation of Mregs with IFN- for 18-24 hours before T cell co-culture. Here, the biological effect of adding IFN- to Mreg-T cell co-cultures was investigated. Mregs were prepared from n=8 independent donors according to the method described in Example 2 (Mreg B). These Mregs were placed in co-culture with allogeneic CD3+ T cells from a further n=8 independent donors according to the method described in Example 5. This co-culturing step was performed in the presence of recombinant human IFN- at concentrations ranging from 0-100 ng/ml. When this experiment was performed, it was not explicitly known whether IFN- affects iTreg generation when added during the co-culture phase; furthermore, prior to this experiment, the range of concentrations over which IFN- might affect iTreg generation was not known. The results are shown in FIG. 6. Values represent log.sub.2-transformed iTreg frequency (%) in treated samples indexed against iTreg frequency (%) in the untreated control sample. A linear regression shows a significant dose-dependent relationship (p<0.0001) between IFN- concentration in the co-culture phase and iTreg generation.