METHODS FOR OBTAINING REGULATORY T CELLS AND USES THEREOF

Abstract

Disclosed is a method for obtaining a population of human Treg cells including the steps of: (a) culturing a population of human monocytes with a medium including an amount of an interleukin-34 (IL-34) polypeptide in order to obtain a population of immunosuppressive macrophages; (b) co-culturing a population of human peripheral blood mononuclear cells (PBMCs) and the population of immunosuppressive macrophages obtained at step (a).

Claims

1-13. (canceled)

14. A method for obtaining a population of human Treg cells comprising the steps of: (a) culturing a population of human monocytes in presence of interleukin-34 (IL-34) polypeptide in order to obtain a population of immunosuppressive macrophages; (b) co-culturing a population of human peripheral blood mononuclear cells (PBMCs) and the population of immunosuppressive macrophages obtained at step (a); and (c) optionally isolating the population of human Treg cells obtained at step (b).

15. The method of claim 14 wherein the population of immunosuppressive macrophages are allogeneic to the population of human PBMCs.

16. An isolated population of human Treg cells obtainable by the method of claim 14.

17. The isolated population of human Treg cells of claim 16 wherein said Treg cells are CD4+ Foxp3+ Treg cells and/or CD8+ Foxp3+ Treg cells.

18. The isolated population of human Treg cells of claim 16 wherein said Treg cells are CD4+CD45RClow Treg cells and/or CD8+CD45RClow Treg cells.

19. A method for preventing or treating an immune and/or inflammatory disease or condition in a subject, comprising administering to the subject the isolated population of human Treg cells of claim 16.

20. The method according to claim 19, wherein the immune/inflammatory disease or condition is selected from the group consisting of graft rejection and GVHD.

21. A population of immunosuppressive macrophages obtained at step (a) of the method of claim 14.

22. A method for preventing or treating an immune and/or inflammatory disease or condition in a subject, comprising administering to the subject the population of immunosuppressive macrophages of claim 21.

23. The method according to claim 22, wherein the immune/inflammatory disease or condition is selected from the group consisting of graft rejection and GVHD.

Description

FIGURES

[0104] FIG. 1: Phenotype of IL34-differentiated macrophages. The phenotype of CD14.sup.+ monocytes from healthy individuals was evaluated 6 days after purification and differentiation or not with IL34. Cells were recovered and stimulated or not 24 h with LPS and stained for the expression of several markers. One representative histogram from three independent experiments.

[0105] FIG. 2: Efficient human Foxp3+ Treg expansion and potentiation following IL34-macrophage differentiation. CD14.sup.+ monocytes were differentiated for 6 days with IL34 or not and added to total allogeneic PBMCs for 15 days. The percentage of Foxp3 positive cells was evaluated in PBMCs among CD4.sup.+ or CD8.sup.+ CD45RC.sup.low T cells. A representative plot (A) and graph (B) is shown before and after culture for 3 healthy individuals. (C) Fold expansion was evaluated for Foxp3.sup.+ CD4.sup.+ or CD8.sup.+ Tregs. (D) The percentage of CD45RC.sup.low evaluated among CD4 or CD8 T cells is shown before and after culture for 2 or 3 healthy individuals. (E) Fold expansion was evaluated for CD45RC.sup.low CD4.sup.+ or CD8.sup.+ Tregs before and after culture for 3 healthy individuals. (F-G) Unstimulated, stimulated or IL34-expanded CD4.sup.+ CD25.sup.highCD127.sup.low and CD8.sup.+ CD45RC.sup.low Tregs were tested for suppression of CFSE-labeled CD4.sup.+ CD25.sup.? T cell proliferation in response to allogeneic T-depleted PBMCs and analyzed by flow cytometry for CFSE dilution after 5 days of culture. n=3. The proportion of dividing CD4.sup.+ CD25.sup.? T cells in the control proliferation condition with allogeneic T-depleted PBMCs only represented approximately 60% of the cells on day 5 and was given a value of 100 in each experiment. Results are expressed as mean?SEM of the relative proportion of dividing CD4.sup.+ CD25.sup.? T cells. A representative raw CFSE profile is displayed. Two Way RM ANOVA, *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001

[0106] FIG. 3: Expansion of CD8+ Tregs with IL-34 differentiated macrophages. A. CD8.sup.+ CD45RC.sup.low Tregs were expanded around 115 fold in presence of IL34-differentiated macrophages. B-C. Co-culture of Tregs with IL34-differentiated macrophages resulted in enrichment in cells secreting suppressive cytokines IL34 and TGFb and expressing Tregs-associated markers Foxp3, GITR, PD1 and HLADR. B. Mean+/?SEM of cells expressing Tregs-associated markers among CD8.sup.+ CD45RC.sup.low cells.

[0107] FIG. 4: Effect of the Tregs of the invention on GVHD in humanized mice. A. Co-transfer of Tregs delayed mice weight loss induced by GVHD development in a dose dependent manner. B. Co-transfer of Tregs improved mice survival at ?1:1 PBMC: Tregs ratios.

[0108] FIG. 5: Effect of the Tregs of the invention on allogeneic skin graft rejection in humanized mice. A. Co-transfer of Tregs delayed rejection of allogeneic human skin graft in humanized mice. B. Graft survival was prolonged in humanized mice transferred with Tregs at a 1:2 PBMC:Tregs ratio.

[0109] FIG. 6: Comparison of IL34-differentiated macrophages and MCSF-differentiated macrophages. The percentage of Foxp3 and CD45RC positive cells were evaluated in healthy individuals among CD4.sup.+ or CD8.sup.+ T cells following expansion with undifferenciated, IL34-differenciated or MCSF-differenciated macrophages. A representative plot of healthy individuals is shown.

[0110] FIG. 7: Effect of IL-34 on different subpopulations of monocytes. A. Monocytes were sorted on CD14 and CD16 markers expression by FACS Aria. B. Monocytes are mainly CD14.sup.+ CD16.sup.? in blood of healthy volunteers. CD14.sup.+ CD16.sup.?, CD14.sup.? CD16.sup.+, and CD14.sup.+ CD16.sup.+ represents 20%, 8% and 2.5% of PBMCs respectively. C. Cell survival in presence of IL34 depends on monocytes population. On day 6, 12%, 37% and 22% of seeded CD14.sup.+ CD16.sup.?, CD14.sup.? CD16.sup.+, and CD14.sup.+ CD16.sup.+ respectively were harvested. D. IL34 differentiation from both CD14.sup.+ CD16.sup.? and CD14.sup.+ CD16.sup.+ monocytes resulted in higher number of M2-like macrophages with low expression of M1-associated markers than IL34 differentiation from CD14.sup.? monocytes (CD14.sup.? CD16.sup.+).

[0111] FIG. 8: Effect of 3 days IL-34 treatment on macrophages. A. After 3 days culture with IL-34, 107%, 157% and 91% of seeded CD14.sup.+ CD16.sup.?, CD14.sup.? CD16.sup.+, and CD14.sup.+ CD16.sup.+ monocytes respectively were harvested. After 4 days culture with IL34, 59%, 264% and 275% of seeded CD14.sup.+ CD16.sup.?, CD14.sup.? CD16.sup.+, and CD14.sup.+ CD16.sup.+ monocytes respectively were harvested. B. IL34 differentiation from both CD14.sup.+ CD16.sup.? and CD14.sup.+ CD16.sup.+ monocytes resulted in high expression of M2-associated markers and low expression of M1-associated markers as compared with IL34 differentiation from CD14.sup.? monocytes (CD14.sup.? CD16.sup.+), after 3 or 4 days culture, as observed in 6 days culture.

EXAMPLE

[0112] Material & Methods

[0113] Healthy Volunteer Blood Collection and PBMC Separation:

[0114] Blood was collected from healthy donors, after informed consent was given, at the Etablissement Francais du Sang (Nantes, France). Blood was diluted 2-fold with PBS before PBMC were isolated by Ficoll-Paque density-gradient centrifugation (Eurobio) at 2000 rpm for 30 min at room temperature without braking. Collected PBMC were washed in 50 mL PBS at 1800 rpm for 10 min.

[0115] Graft-Versus-Host Disease and Skin Transplantation Rejection

[0116] 1.5?10.sup.7 PBMCs or FACS-Aria sorted CD45RC.sup.? PBMCs from healthy volunteers (HV) donor were injected intravenously in 10-13 week old NSG SCID mice treated 16 hours earlier with whole-body sublethal irradiation of 1.5 Gy. Expanded Tregs were co-injected with syngeneic PBMCs in mice at a 1:1 or 1:2 PBMC:Tregs ratio. GVHD is characterized by mice weight loss; mice were sacrificed at 20% initial weight loss.

[0117] 9 to 11 week old NSG mice were grafted with human skin. 6 weeks later, mice were i.v. injected with 1.5?10.sup.7 PBMCs allogeneic to the skin to induce allograft rejection (day 0). Tregs syngeneic with PBMCs were expanded with IL34-differenciated macrophages as previously described and co-injected with PBMCs at 1:1 or 1:2 PBMC:Tregs ratio. Allograft rejection is scored from 1 to 3 by histological assessment.

[0118] Recipients were weighted every day and sacrificed when percentage of weight loss was ?20% of their initial weight. Follow up was performed in blind conditions when possible. Groups of 3 to 6 animals were treated. Mice were matched in sex, age and initial weight and randomizely treated or not.

[0119] Treg and Monocyte Differentiation Protocol:

[0120] PBMCs from healthy volunteers (HV) blood were isolated by Ficoll gradient, and monocytes were elutriated according to FSC and SSC morphology parameters. CD14.sup.+ monocytes were then sorted by FACS Aria, washed, and seeded at 1?10.sup.6/ml in medium (RPMI 1640, glutamine 2 mM, penicillin 100 U/ml, streptomycin 0.1 mg/ml, AB serum 10%) supplemented with 50 ng/ml hIL34 (ebiosciences). At day 6, cells were harvested, stimulated with 100 ng/ml LPS for 24 h for phenotype analysis, or seeded at 4?10.sup.5/ml with 1 to 5 allogeneic PBMCs in Iscove medium (IMDM, glutamine 2 mM, penicillin 100 U/ml, streptomycin 0.1 mg/ml, AB serum 5%). IL-2 (25 U/ml) and IL-15 (long/ml) were freshly added at days 10, 13 and 16. Macrophages were removed by successive transfers of floating cells to a new plate at days 19 and 21 for 48 h and 2 h respectively. At day 21, cells were stimulated with PMA and ionomycin in presence of brefeldin A for phenotype analysis, or T cells, CD8.sup.+ CD45RC.sup.low T cells, and CD4.sup.+ CD25.sup.highCD127.sup.low T cells were sorted by FACS Aria for suppression assay. Fresh syngeneic CD4.sup.+ CD25.sup.? T cells were used as responder T cells stimulated with allogeneic APC isolated from the same donor as CD14.sup.+ cells. Proliferation was assessed 5 days later by CFSE dilution, by gating on CD3.sup.+ CD4.sup.+ cells after exclusion of dapi-labeled dead cells and CPD405 labeled CD4.sup.+ Tregs.

[0121] Monoclonal Antibodies and Flow Cytometry:

[0122] Antibodies against CD3-PeCy7 (SKY7), CD4-PercPCy5.5 (L200), CD25-APCCy7 (M-A251), CD127-PE (HIL7-R M21, BD Bioscience), CD45RC-FITC (MT2, IQ Product), Foxp3-APC (236A/E7, ebiosciences) and IL34-PE (578416, R&D) were used to characterize human cell phenotypes. Fluorescence was measured with a Canto II cytometer (BD Biosciences, Mountain View, Calif.), and the FLOWJO software (Tree Star, Inc. USA) was used to analyze data. Cells were first gated by their morphology excluding dead cells by selecting DAPI viable cells.

[0123] CD14.sup.+ monocytes from healthy volunteers were cell-sorted and differentiated in the presence of IL34 for 6 days and the phenotype was analyzed for expression of CD163, CD14, HLA-DR, CD86 and CD80.

[0124] Results

[0125] IL34-Differentiated Macrophages Efficiently Induce Foxp3.sup.+ CD45RC.sup.low Tregs and Potentiate Human CD4.sup.+ CD25.sup.+ CD127.sup.? and CD8.sup.+ CD45RC.sup.low Tregs.

[0126] CD14.sup.+ monocytes from healthy volunteers were cell-sorted and differentiated in the presence of IL34 for 6 days and the phenotype was analyzed (FIG. 1). We observed that IL34-differenciated macrophages expressed higher levels of CD163, CD14, HLA-DR, CD86 and CD80 than fresh monocytes. The differentiated macrophages were then added to allogeneic PBMCs for 15 days, then the proportion, number and suppressive capacity of Tregs were analyzed (FIG. 2). Interestingly, we observed that, following culture with IL34-differentiated macrophages, Foxp3.sup.+ CD45RC.sup.lowCD4.sup.+ and Foxp3.sup.+ CD45RC.sup.lowCD8.sup.+ T cells increased as a percentage of CD4.sup.+ or CD8.sup.+ T cells by 5 and 8.2 fold respectively (FIGS. 2A, 2B and 2D). This increase in percentage was also accompanied by an increase in the number of Foxp3.sup.+ CD45RC.sup.lowCD4.sup.+ and Foxp3.sup.+ CD45RC.sup.lowCD8.sup.+ T cells, by 83.4 and 100.6 fold respectively, as seen in the fold expansion (FIGS. 2B and 2C). Accordingly, we observed a significant increase in percentage and number of CD4+CD45RClow and CD8+CD45RClow Tregs (FIGS. 2D and 2E) Most importantly, we observed that the IL34-expanded CD4.sup.+ CD25.sup.highCD127.sup.low Tregs and CD8.sup.+ CD45RC.sup.low Tregs displayed a highly potent suppressive capacity up to a 16:1 effector:suppressor ratio, with around 50% of the suppression compared to unstimulated and polyclonaly stimulated CD4.sup.+ CD25.sup.highCD127.sup.low Tregs and CD8.sup.+ CD45RC.sup.low Tregs (FIGS. 2F and 2G).

[0127] Altogether, these results demonstrate that IL34-differenciated monocytes have the capacity to selectively expand and, not only to maintain, but potentiate Foxp3.sup.+ CD45RC.sup.low Tregs suppressive capacity.

[0128] Expansion of CD8+ Tregs with IL-34-Differentiate Macrophages Improved their Tolerogenic Profile

[0129] Monocytes were isolated by Ficoll gradient, CD3.sup.+, CD19.sup.+ and CD16.sup.+ depletion and FACS Aria sorting on CD14 marker expression. CD14.sup.+ monocytes were seeded at 10.sup.6 cells/ml in complete medium (RPMI1640, 1% penicilline-stretpmycine, 1% glutamine, 10% FBS) supplemented with 50 ng/ml human IL34 protein and cultured for 6 days. On day 6, CD8.sup.+ Tregs were isolated from blood of a different HV (healthy volunteer) donor by Ficoll gradient, CD14.sup.+, CD16.sup.+ and CD19.sup.+ cells depletion and FACS Aria sorting on CD3, CD8 and CD45RC markers expression. CD3.sup.+ CD8.sup.+ CD45RC.sup.low cells were seeded at 10.sup.6 cells/ml in complete medium (RPMI1640, 1% penicilline-stretpmycine, 1% glutamine, 10% AB serum, 1% Hepes, 1% non essential amino acids, 1% sodium pyruvate) with allogeneic IL34-differenciated macrophages at 1:4 Tregs:monocytes ratio at day 0 and 1 ?g/ml anti-CD3 and anti-CD28 Abs. On day 13, Tregs were stimulated again with 1 ?g/ml anti-CD3 and anti-CD28. On days 6, 13, 16 and 18, culture medium was supplemented with 1000 U/ml human IL-2 and 10 ng/ml human IL-15. On day 20, Tregs were analyzed for expansion yield and were stimulated for 4 h with 50 ng/ml PMA and 1 ?g/ml ionomycine in presence of 10 ?g/ml Brefeldine A for Tregs associated markers expression analysis as compared to before expansion.

[0130] As shown in FIG. 3, IL-34-differentiated macrophages induce a 115 fold expansion of CD8+CD45RClow Tregs. Moreover, co-culture of Tregs with IL-34-differentiated macrophages induce an enrichment in cells secreting suppressive cytokines IL34 and TGF? and expressing Tregs-associated markers Foxp3, GITR, PD1 and HLADR. Therefore, IL34-differentiated macrophages increased expression of Tregs-associated markers suggesting an increased suppressive activity for CD8.sup.+ CD45RC.sup.low Tregs.

[0131] CD8.sup.+ CD45RC.sup.low Tregs Expanded with IL34-Differentiated Macrophages Delayed Graft Versus Host Disease in Humanized Mice

[0132] 10 to 13 week old NSG mice were 2Gy-irradiated 16 h before i.v. injection of 1.5?10.sup.7 PBMCs to induce xenogeneic GVH reaction. CD8.sup.+ CD45RC.sup.low Tregs were sorted and expanded in presence of IL34-differentiated CD14.sup.+ monocytes. Expanded Tregs were co-injected with syngeneic PBMCs in mice at a 1:1 or 1:2 PBMC:Tregs ratio. GVHD is characterized by mice weight loss; mice were sacrificed at 20% initial weight loss.

[0133] As shown in FIG. 4A, co-transfer of Tregs of the invention delayed mice weight loss induced by GVHD development in a dose dependant manner. Moreover, co-transfer of Tregs of the invention improved mice survival at ?1:1 PBMC:Tregs ratios (FIG. 4B).

[0134] This result thus demonstrate that CD8.sup.+ CD45RC.sup.low Tregs expanded with IL34-differentiated macrophages efficiently delayed GVHD and can be used in cell therapy.

[0135] CD8.sup.+ CD45RC.sup.low Tregs Expanded with IL34-Differenciated Macrophages Delayed Allogeneic Skin Graft Rejection in Humanized Mice

[0136] 9 to 11 week old NSG mice were grafted with human skin. 6 weeks later, mice were i.v. injected with 1.5?10.sup.7 PBMCs allogeneic to the skin to induce allograft rejection (day 0). Tregs syngeneic with PBMCs were expanded with IL34-differenciated macrophages as previously described and co-injected with PBMCs at 1:1 or 1:2 PBMC:Tregs ratio. Allograft rejection is scored from 1 to 3 by histological assessment.

[0137] As shown in FIG. 5A, co-transfer of Tregs of the invention delayed rejection of allogeneic human skin graft in humanized mice. Moreover, graft survival was prolonged in humanized mice transferred with Tregs at a 1:2 PBMC:Tregs ratio (FIG. 5B).

[0138] This result thus demonstrate that Tregs expanded with IL34 differentiated macrophages can control alloimmune responses against the graft.

[0139] IL34-Differenciated Macrophages are More Efficient than MCSF-Differenciated Macrophages to Expand Foxp3.sup.+ CD45RC.sup.low Tregs

[0140] Monocytes were isolated from blood by Ficoll gradient, then CD3.sup.+ and CD19.sup.+ cells were depleted and FACS Aria sorted on CD14 marker expression. CD14.sup.+ monocytes were seeded at 10.sup.6 cells/ml in complete medium (RPMI1640, 1% penicilline-stretpmycine, 1% glutamine, 10% FBS) supplemented with 50 ng/ml human IL34 protein or 25 ng/ml human MCSF protein for 6 days. On day 6, macrophages were seeded at 4?10.sup.5 cells/ml in complete medium (IMDM, 100 U/ml penicilline, 0.1 mg/ml stretpmycine, 2 mM glutamine, 5% AB serum) with allogeneic PBMCs at 5:1 PBMCs:macrophages ratio. 25 U/ml human IL2 and 10 ng/ml human IL15 were freshly added on days 10, 13 and 16. On day 21, T cells were stimulated for 4 h with 50 ng/ml PMA and 1 ?g/ml ionomycine in presence of 10 ?g/ml Brefeldine A for Foxp3 and CD45RC marker expression analysis.

[0141] Percentage of Foxp3 and CD45RC positive cells were evaluated in healthy individuals among CD4.sup.+ or CD8.sup.+ T cells following expansion with undifferenciated, IL34-differenciated or MCSF-differenciated macrophages. A representative plot of healthy individuals is shown in FIG. 6.

[0142] The obtained results show that IL34-differenciated macrophages are more efficient than MCSF-differenciated macrophages at expanding Tregs for cell therapy.

[0143] CD14.sup.? CD16.sup.+, CD14.sup.+ CD16.sup.? and CD14.sup.+ CD16.sup.+ Behave Differently In Vitro in Presence of IL-34

[0144] Monocytes were isolated from blood by Ficoll gradient, CD3.sup.+ and CD19.sup.+ cells depletion and FACS Aria sorting on CD16 and CD14 markers expression. CD14.sup.+ CD16.sup.?, CD14.sup.+ CD16.sup.+ and CD14.sup.? CD16.sup.+ monocytes were seeded at 10.sup.6 cells/ml in complete medium (RPMI1640, 1% penicilline-stretpmycine, 1% glutamine, 10% FBS) supplemented with 50 ng/ml human IL34 protein for 7 days. At day 7, cells were analyzed for macrophages associated markers expression as compared with M1 and M2 macrophages differentiated with 10 ng/ml GMCSF and 50 ng/ml IFNg or 25 ng/ml MCSF, 20 ng/ml IL4 and 20 ng/ml IL10 respectively.

[0145] As shown in FIG. 7, cell survival in presence of IL34 depends on monocytes population. On day 6, 12%, 37% and 22% of seeded CD14.sup.+ CD16.sup.?, CD14.sup.? CD16.sup.+, and CD14.sup.+ CD16.sup.+ respectively were harvested (see FIG. 7C). Moreover, as shown in FIG. 7D, IL34 differentiation from both CD14.sup.+ CD16.sup.? and CD14.sup.+ CD16.sup.+ monocytes resulted in higher number of M2-like macrophages with low expression of M1-associated markers than IL34 differentiation from CD14.sup.? monocytes (CD14.sup.? CD16.sup.+).

[0146] Therefore, IL34 preferentially differentiate CD14+ monocytes towards M2-like cells.

[0147] Yield and Phenotype of 3 Days IL34-Differenciated Macrophages

[0148] Monocytes were isolated from blood by Ficoll gradient, CD3.sup.+ and CD19.sup.+ cells depletion, and FACS Aria sorting on CD14 marker expression. CD14.sup.+ monocytes were seeded at 10.sup.6 cells/ml in complete medium (RPMI1640, 1% penicilline-stretpmycine, 1% glutamine, 10% FBS) supplemented with 50 ng/ml human IL34 protein and analyzed 3 days later for M1 and M2 associated markers expression.

[0149] Results are shown in FIG. 8. After 3 days culture with IL34, 107%, 157% and 91% of seeded CD14.sup.+ CD16.sup.?, CD14.sup.? CD16.sup.+, and CD14.sup.+ CD16.sup.+ monocytes respectively were harvested. After 4 days culture with IL34, 59%, 264% and 275% of seeded CD14.sup.+ CD16.sup.?, CD14.sup.? CD16.sup.+, and CD14.sup.+ CD16.sup.+ monocytes respectively were harvested. IL34 differentiation from both CD14.sup.+ CD16.sup.? and CD14.sup.+ CD16.sup.+ monocytes resulted in high expression of M2-associated markers and low expression of M1-associated markers as compared with IL34 differentiation from CD14.sup.? monocytes (CD14.sup.? CD16.sup.+), after 3 or 4 days culture, as observed in 6 days culture.

[0150] These results demonstrate that 3 days culture of CD14.sup.+ monocytes with IL34 are sufficient to induce a M2-like profile with a higher yield.

REFERENCES

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