SUBPOPULATION OF CD8+CD45RCLOW TREGS AND USES THEREOF

Abstract

Disclosed is a new subpopulation of CD8.sup.+CD45RC.sup.low Tregs, namely IFN?.sup.+IL-10.sup.+IL-34.sup.+ secreting population of CD8.sup.+CD45RC.sup.low Treg cells, methods for their isolation and expansion and their use as drug, more particularly for immunotherapy as well as biomarker

Claims

1-19. (canceled)

20. An isolated population of IFN?.sup.+IL-10.sup.+IL-34.sup.+ secreting human CD8.sup.+CD45RC.sup.low T regulatory (Treg) cells.

21. The isolated population according to claim 20, wherein said Treg cells are GITR.sup.+ and/or Foxp3.sup.+.

22. The isolated population according to claim 20, wherein said Treg cells are genetically modified Treg cells comprising a chimeric antigen receptor (CAR).

23. A method for detecting and/or isolating IFN?.sup.+IL-10.sup.+IL-34.sup.+ secreting human CD8.sup.+CD45RC.sup.low Treg cells from a biological sample containing human peripheral blood mononuclear cells (PBMCs) or lymphocytes, comprising the following steps of: (a) contacting said population of human PBMCs or lymphocytes with means useful for isolating a population of human CD8.sup.+CD45RC.sup.low Treg cells; (b) contacting the isolated population of human CD8.sup.+CD45RC.sup.low Treg cells obtained at step (a) with means useful for isolating IFN?.sup.+IL-10.sup.+IL-34.sup.+ secreting human CD8.sup.+CD45RC.sup.low Treg cells.

24. The method according to claim 23, wherein the means useful for isolating the population of human CD8.sup.+CD45RC.sup.low Treg cells are a combination of at least three antibodies consisting of a monoclonal anti-human CD3 antibody, a monoclonal anti-human CD8 antibody and a monoclonal anti-human CD45RC antibody.

25. The method according to claim 23, wherein the means useful for isolating IFN?.sup.+IL-10.sup.+IL-34.sup.+ secreting human CD8.sup.+CD45RC.sup.low Treg cells are a combination of at least two bispecific antibodies selected from the group consisting of a bispecific antibody which binds to IFN? and to a cell surface molecule specific for T cells (e.g. CD3, CD8, CD45), a bispecific antibody which binds to IL-10 and to a cell surface molecule specific for T cells (e.g. CD3, CD8, CD45) and a bispecific antibody which binds to IL-34 and to a cell surface molecule specific for T cells (e.g. CD3, CD8, CD45).

26. The method according to claim 23, wherein the means useful for isolating IFN?.sup.+IL-10.sup.+IL-34.sup.+ secreting human CD8.sup.+CD45RC.sup.low Treg cells are a combination of at least two bispecific antibodies selected from the group consisting of a bispecific antibody which binds to IFN? and to a cell surface molecule selected from the group consisting of CD3, CD8 and CD45, a bispecific antibody which binds to IL-10 and to a cell surface molecule selected from the group consisting of CD3, CD8 and CD45 and a bispecific antibody which binds to IL-34 and to a cell surface molecule selected from the group consisting of CD3, CD8 and CD45.

27. A method for producing a population of IFN?.sup.+IL-10.sup.+IL-34.sup.+ secreting human CD8.sup.+CD45RC.sup.low Treg cells said Treg cells optionally being GITR.sup.+ and/or Foxp3.sup.+, wherein said method comprises: isolating CD8.sup.+GITR.sup.+ Treg cells from a biological sample containing human peripheral blood mononuclear cells (PBMCs) or lymphocytes, and expanding said cells in a culture medium suitable for expanding Treg cells.

28. The method according to claim 27, wherein said culture medium suitable for expanding Treg cells comprises at least an antigen-specific stimulating agent selected from the group consisting of antigens, cells, MHC polymers and antibodies.

29. The method according to claim 27, wherein said culture medium comprises at least one cytokine.

30. The method according to claim 27, wherein said culture medium comprises an amount of interleukin-2 (IL-2) and/or an amount of interleukin-15 (IL-15).

31. A method for preventing or treating transplant rejection, GVHD, chronic inflammatory diseases, autoimmune diseases, unwanted immune response against therapeutic proteins or allergies in a patient in need thereof, comprising administering to the subject an isolated population of IFN?.sup.+IL-10.sup.+IL-34.sup.+ secreting human CD8.sup.+CD45RC.sup.low Treg cells or an isolated population of expanded IFN?.sup.+IL-10.sup.+IL-34.sup.+ secreting human CD8.sup.+CD45RC.sup.low Treg cells.

32. An in vitro method for determining whether a patient is at risk of transplant rejection, GVHD, chronic inflammatory diseases, autoimmune diseases, unwanted immune response against therapeutic proteins or allergies, comprising a step of determining the presence of IFN?.sup.+IL-10.sup.+IL-34.sup.+ secreting human CD8.sup.+CD45RC.sup.low Treg cells in a biological sample obtained from said patient, wherein the presence of a IFN?.sup.+IL-10.sup.+IL-34.sup.+ secreting human CD8.sup.+CD45RC.sup.low Treg cells is indicative of a reduced risk of transplant rejection, GVHD, chronic inflammatory diseases, autoimmune diseases, unwanted immune response against therapeutic proteins or allergies.

33. The method of claim 23, wherein the Treg cells are GITR.sup.+ and/or Foxp3.sup.+.

34. The method of claim 23, wherein step (a) further comprises contacting said population of human PBMCs or lymphocytes with means useful for isolating a population of GITR.sup.+ and/or Foxp3.sup.+ CD8.sup.+CD45RC.sup.low Treg cells.

35. The method of claim 33, wherein step (a) further comprises contacting said population of human PBMCs or lymphocytes with means useful for isolating a population of GITR.sup.+ and/or Foxp3.sup.+ CD8.sup.+CD45RC.sup.low Treg cells.

36. The method of claim 24, wherein the means useful for isolating the population of human CD8.sup.+CD45RC.sup.low Treg cells further comprises an anti-human GITR antibody.

37. The method of claim 27, wherein the isolating step further comprises isolating CD8.sup.+CD45RC.sup.lowGITR.sup.+ Treg cells from a biological sample containing human peripheral blood mononuclear cells (PBMCs) or lymphocytes.

38. The method of claim 27, further comprising a step of freezing and subsequently thawing the isolated Treg cells prior to the expanding step.

39. The method of claim 37, further comprising a step of freezing and subsequently thawing the isolated Treg cells prior to the expanding step.

Description

FIGURES

[0279] FIG. 1. Human CD8.sup.+CD45RC.sup.low Tregs suppress more efficiently anti-donor immune responses than human CD4.sup.+CD25.sup.+CD127.sup.? Tregs. CD8.sup.+CD45RC.sup.low T cells were compared to classical CD4.sup.+CD25.sup.+CD127.sup.? Tregs for suppressive activity on proliferation of syngeneic effector CD4.sup.+CD25.sup.? T cells stimulated with allogeneic T-depleted PBMCs, in a range from 16:1 to 1:4 effector:suppressor ratio. Two Way RM ANOVA. Proliferation was normalized to proliferation in absence of Tregs. *, p<0.05.

[0280] FIG. 2. The suppressive activity of human CD8.sup.+CD45RC.sup.low Tregs is preferentially increased by interaction with pDCs. Sorted CD8+ Tregs were stimulated 12 h with anti-CD3 and anti CD28 mAbs and analyzed for suppressive activity on proliferation of syngeneic effector CD4.sup.+CD25.sup.? T cells stimulated with allogeneic T-depleted PBMCs (APC), cDCs or pDCs, in a range from 8:1 to 1:1 effector:suppressor ratio. Two Way RM ANOVA. Proliferation was normalized to proliferation in absence of Tregs. *p<0.05, **, p<0.01.

[0281] FIG. 3. A subpopulation of human CD8.sup.+CD45RC.sup.low Tregs expresses GITR. The expression of the surface receptor GITR was analyzed by flow cytometry in isolated CD8.sup.+CD45RC.sup.low Tregs and isolated CD8.sup.+CD45RC.sup.high Tregs. Results are expressed as the mean percentage of cells (+/? SEM) expressing GITR.

[0282] FIG. 4. Expression of TGF?-1, IL34, IFN? and GITR is enriched in Foxp3.sup.+ human CD8.sup.+ Tregs. PBMCs were isolated from the blood of healthy volunteers by Ficoll gradient, and were stimulated for 4 h with 50 ng/ml PMA and 1 ?g/ml ionomycine in presence of 10 ?g/ml Brefeldine A. Isolated CD8.sup.+ cell populations were analyzed by FACS staining for expression of Foxp3, IL-34, IFN?, TGF?-1 and GITR after gating on viable cells. One representative experiment. A. Expression of Foxp3 and CD45RC was analyzed in CD8.sup.+ Tregs. B. Expression of IL-34, IFN?, GITR and TGF?-1 was determined in Foxp3.sup.+CD45RC.sup.low, Foxp3.sup.?CD45RC.sup.low and CD45RC.sup.high CD8.sup.+ cells.

[0283] FIG. 5. Specific subpopulations of human CD8.sup.+CD45RC.sup.low Tregs demonstrate high suppressive capacity in vitro. A. Sorted CD8.sup.+ Tregs were stimulated 24 h with anti-CD3 and anti-CD28 mAbs, sorted on IFN? and/or IL-10 secretion, and tested for suppressive activity in a range of effector:suppressor ratios in a MLR assay where syngeneic CFSE-labeled CD4.sup.+CD25.sup.? T cells were stimulated by allogeneic T-depleted PBMCs. The sorted CD8.sup.+ Tregs were compared to total anti-CD3.sup.+ anti-CD28 mAbs stimulated CD8.sup.+ Tregs. Two Way ANOVA RM. Proliferation was normalized to proliferation in absence of Tregs. **, p<0.01. B. Sorted CD8.sup.+CD45RC.sup.low Tregs were stimulated 12 h with 1 ?g/ml anti-CD3 and anti-CD28 mAbs and newly sorted on GITR expression by FACS Aria. GITR.sup.+CD8.sup.+CD45RC.sup.low Tregs and GITR.sup.?CD8.sup.+CD45RC.sup.low Tregs were analyzed for suppressive activity on proliferation of syngeneic effector CD4.sup.+CD25.sup.? T cells stimulated with allogeneic T-depleted PBMCs, in a range from 32:1 to 1:1 effector:suppressor ratio. Two Way RM ANOVA. Proliferation was normalized to proliferation in absence of Tregs. **, p<0.01

[0284] FIG. 6. IFN?, IL-10 and IL-34 are key mediators of CD8.sup.+CD45RC.sup.low Tregs suppression. 50 ?g/ml of anti-IL-34 (?-IL-34), anti-IL-10 (?-IL-10) or anti-IFN? cytokines (?-IFN?) or their anti-receptor anti-IL-10R (?-IL-10R) or anti-IFN?R (?-IFN?R) purified blocking Abs were added at day 0 of co-culture assay using sorted CD8.sup.+CD45RC.sup.low T cells, and syngeneic CFSE-labeled effector CD4.sup.+CD25.sup.? T cells stimulated with allogeneic T-depleted PBMCs. Proliferation in presence of Tregs was normalized to proliferation in absence of Tregs. Suppression in presence of blocking Abs was normalized to suppression in presence of isotypic control Ab.

[0285] FIG. 7. The suppressive activity of human CD8.sup.+CD45RC.sup.low Tregs is contact dependent. Transwell experiments were performed as follow: sorted CD8.sup.+ Tregs were stimulated 12 h with anti-CD3 and anti-CD28 mAbs and analyzed for suppressive activity on proliferation of syngeneic effector CD4.sup.+CD25.sup.? T cells stimulated with allogeneic T-depleted PBMCs following 5-day culture in presence of IL-2. Allogeneic T-depleted PBMCs were added in both the upper and the bottom well. The Tregs were added in the upper well and the CD4.sup.+CD25.sup.? T cells in the bottom well, in a range from 1:1 to 1:4 effector:suppressor ratio. Two Way RM ANOVA. Proliferation was normalized to proliferation in absence of Tregs. *, p<0.05.

[0286] FIG. 8. Significant expansion of both freshly sorted and thawed CD8.sup.+CD45RC.sup.low Tregs following 14-day stimulation. A. Sorted CD8.sup.+ Tregs were stimulated at day 0 with allogeneic APCs (APC) optionally with anti-CD3 and anti-CD28 mAbs (poly), and stimulated again or not (noted X) with the same APCs (APC) or APCs and anti-CD3.sup.+ anti-CD28 mAbs (poly). The graph represents fold expansion of cells obtained in 14-day culture. B. Freshly sorted (fresh) or thawed CD8.sup.+CD45RC.sup.low cells were seeded at 3.3?10.sup.5 cells/ml in complete medium with allogeneic T-depleted PBMCs at 1:4 Tregs:APCs ratio and 1 g/ml anti-CD3 and anti-CD28 Abs at day 0. On day 7, the Tregs were stimulated again with 1 g/ml anti-CD3 and anti-CD28. On days 0, 7, 10, and 12, the culture medium was supplemented with 1000U/ml human IL-2 and 10 ng/ml human IL-15. On day 14, the Tregs were analyzed for expansion yield. The graph represents fold expansion of cells obtained in 14-day culture. Thawed cells show significantly increased expansion. Mann Whitney t test ***, p<0.001.

[0287] FIG. 9. Expanded CD8.sup.+ Tregs survive in presence of immunosuppressive drugs. Sorted CD8.sup.+ Tregs were stimulated at day 0 with allogeneic T-depleted PBMCs at 1:4 Tregs:APCs ratio and 1 ?g/ml anti-CD3 and anti-CD28 Abs. On day 7, Tregs were stimulated again with 1 ?g/ml anti-CD3 and anti-CD28. On days 0, 7, 10 and 12, the culture medium was supplemented with human IL-2 (1000U/ml) and human IL-15 (10 ng/ml). On day 14, Tregs were stimulated again with allogeneic APCs, in presence or not (Non TreatedNT) of immunosuppressive drugs (IS): cyclosporine A (CsA), mycophenolate mofetil (MPA), methylprednisolone (MPr), rapamycin (Rapa), or tacrolimus (Tacro) and in presence or not of IL-2/IL-15. Treg survival was analyzed 6 days later. The graph represents the percentage of cell survival obtained following the last 6 days of culture. Percent of survival was normalized to proliferation at day 14 before addition of immunosuppressive drugs (IS).

[0288] FIG. 10. CD8.sup.+ Tregs proliferate more efficiently than CD4.sup.+ Tregs in the same culture conditions. Sorted CD8.sup.+CD45RC.sup.low (CD8.sup.+) and CD4.sup.+CD25.sup.highCD127.sup.low (CD4.sup.+) Tregs were stimulated with allogeneic T-depleted PBMCs at 1:4 Tregs:APCs ratio and 1 ?g/ml anti-CD3 and anti-CD28 Abs at day 0. On days 7 and 14, Tregs were stimulated again with 1 ?g/ml anti-CD3 and anti-CD28. On days 0, 7, 10, 12, 14, 17, and 19 the culture medium was supplemented with 1000U/ml human IL-2 and 10 ng/ml human IL-15. Treg expansion yield was analyzed every 7 days. Two-way RM Bonferroni post test *, p<0.05

[0289] FIG. 11. Expanded CD8.sup.+CD45RC.sup.low Tregs are significantly more efficient at suppressing allogeneic CD4.sup.+CD25.sup.? effector T cell responses. Tregs expanded for 7 days with APCs or anti-CD3+ anti-CD28 mAbs+APCs were tested for suppressive activity on syngeneic CD4.sup.+CD25.sup.? T cells (sorted from the donor of Tregs) stimulated with the allogeneic APCs used for expansion. Thawed, sorted and non-expanded Tregs (noted X) from the same donor defined the initial suppressive activity. Proliferation was normalized to proliferation in absence of Tregs.

[0290] FIG. 12. Expanded CD8.sup.+CD45RC.sup.low Tregs are enriched in IFN?, IL-10 and IL-34-secreting Tregs. The cytokine expression (IL-10, IL-34 and IFN?) of 14-day expanded CD8.sup.+CD45RC.sup.low Tregs (expanded) was analyzed and compared to that of CD8.sup.+CD45RC.sup.low Tregs before expansion (fresh). **p<0.01, ***p<0.001.

[0291] FIG. 13. Increased secretion of IFN? can be detected in the supernatant of CD8.sup.+CD45RC.sup.low Tregs. CD8.sup.+CD45RC.sup.low cells were stimulated with 1 ?g/ml anti-CD3 and anti-CD28 Abs overnight and either added in a MLR assay where syngeneic effector CD4+CD25? T cells were stimulated with allogeneic T-depleted PBMCs in a 1:1 effector:suppressor ratio, or seeded at 3.3?10.sup.5 cells/ml in complete medium with allogeneic T-depleted PBMCs at 1:4 Tregs:APCs ratio and 1 ?g/ml anti-CD3 and anti-CD28 Abs for 14-day expansion. On day 7, the Tregs were stimulated again with 1 ?g/ml anti-CD3 and anti-CD28 Abs and the medium culture was changed. On days 0, 7, 10, and 12, the culture medium was supplemented with 1000U/ml human IL-2 and 10 ng/ml human IL-15. IFN? was measured in culture supernatants by ELISA on day 14.

[0292] FIG. 14. Expanded CD8.sup.+CD45RC.sup.low Tregs efficiently suppress allogeneic human skin rejection. A. Human skin was grafted on NSG mice and left 15 days for acceptance. 5?10.sup.6 PBMCs allogeneic to the graft were then transferred intraperitoneally with or without syngeneic 15 days expanded Tregs (eTregs). B. Graft rejection was evaluated and scored. C. The percentage of mice survival was assessed.

[0293] FIG. 15. Expanded CD8.sup.+CD45RC.sup.low Tregs efficiently suppress GVHD in humanized mice. A. For xenogeneic GVHD experiments, 1.5?10.sup.7 syngeneic PBMCs were intravenously transferred with or without polyclonally expanded Tregs (eTregs) at 1:1 and 1:2 PBMCs:expanded Tregs ratio in 12 h-previously irradiated NSG mice. B. Human PBMCs engraftment was monitored in blood, and GVH development was evaluated by weight loss. C. The percentage of mice survival was assessed. *p<0.05, **p<0.01.

EXAMPLE: A NEW POPULATION OF IFN?.SUP.+.IL-10.SUP.+.IL-34.SUP.+ SECRETING HUMAN CD8^{+.CD45RC.SUP.LOW .TREGS EFFICIENTLY SUPPRESSES GRAFT REJECTION}

[0294] Material & Methods

[0295] Healthy Volunteers Blood Collection and PBMC Separation:

[0296] Blood was collected from healthy donors at the Etablissement Fran?ais du Sang (Nantes, France). Approval for this study was obtained from the institutional review boards. Written informed consent was provided according to institutional guidelines. Blood was diluted 2-fold with PBS before PBMC were isolated by Ficoll-Paque density-gradient centrifugation (Eurobio, Courtaboeuf, France) at 2000 rpm for 20 at room temperature without braking. Collected PBMC were washed in 50 mL PBS at 1800 rpm for 10 min and remaining red cells and platelets are eliminated after incubation 5 min in a hypotonic solution and centrifugation at 1000 rpm for 10 min. When indicated, PBMCs were frozen in DMSO:SVF 1:9 and washed twice in medium-10% SVF for thawing.

[0297] Cell Isolation:

[0298] T cells were obtained from PBMCs by negative selection by elutriation (DTC Plateforme, Nantes) and magnetic depletion (Dynabeads, Invitrogen) of B cells (CD19.sup.+ cells) and remaining monocytes (CD14.sup.+ and CD16.sup.+ cells) before sorting of CD3.sup.+CD4.sup.+CD25.sup.? cells as responder cells, CD3.sup.+CD4.sup.?CD45RC.sup.low as CD8.sup.+ Tregs and CD3.sup.+CD4.sup.+CD25.sup.?CD127.sup.low cells as CD4.sup.+ Tregs cells. Tregs sorted from thawed PBMCs were stimulated 24 h with anti-CD3 and anti-CD28 mAbs in presence of 250 U/ml IL-2 before plating. IFN? and/or IL-10 secreting cells were sorted after 24 h polyclonal stimulation of Tregs using secretion assay detection kits from Miltenyi. A FACS ARIA I (BD Biosciences, Mountain View, Calif.) was used to sort cells.

[0299] APCs used as stimulator cells were obtained by magnetic depletion of CD3.sup.+ cells and 35Gy irradiation. pDCs and cDCs were obtained by CD3, CD14, and CD16 positive cells depletion and Nrp1.sup.+ or CD1c.sup.+ cell sorting respectively.

[0300] Monoclonal Antibodies and Flow Cytometry:

[0301] For interleukins, IFN? and Foxp3 analysis, PBMCs were stimulated with PMA (50 ng/ml) and ionomycine (1 ?g/ml) for 7 h in presence of Brefeldine A (10 ?g/ml) for the last 4 h.

[0302] Fluorescence was measured with a LSR II or a Canto II cytometer (BD Biosciences, Mountain View, Calif.) for phenotype and functional analysis respectively, 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 viable cells.

[0303] Mixed Lymphocyte Reaction:

[0304] Tregs suppressive activity was assessed on syngeneic effector CD4.sup.+CD25.sup.? T cells (obtained from the donor of Tregs) stimulated with allogeneic APCs. A range of stimulator: effector ratio was tested to reach significant proliferation of responder cells when stimulated with DCs, other experiments were realized with 1:1 APCs:responder ratio. Blocking mAbs or isotypic control mAbs were added at 50 ?g/ml at day 0 of co-culture. Transwell membrane (0.4 ?M ?) was used to seclude Tregs from responder cells, both in contact with stimulator cells. 1000U/ml IL-2 (Miltenyi) was added to assess cytokine deprivation by Tregs. Suppressive activity of expanded Tregs from thawed PBMCs was assessed on syngeneic effector cells stimulated with the allogeneic APCs from the same donor that was used for expansion, and compared to unstimulated Tregs sorted from the same donor of thawed PBMCs. Proliferation of CFSE-labeled responder cells was analyzed by flow cytometry after 4.5 days of coculture in 5% AB serum medium, by gating on CD3.sup.+CD4.sup.+ living cells (DAPI negative) and excluding of CPD-V450 labeled CD4.sup.+ Tregs when added.

[0305] Expansion of Tregs:

[0306] Tregs were seeded at about 3?10.sup.5/ml in 5% AB serum-medium supplemented with IL-2 (1000U/ml) and IL-15 (10 ng/ml) and were stimulated with coated anti-CD3 mAb (1 ?g/ml), soluble anti-CD28 mAb (1 ?g/ml) and/or allogeneic APCs at 1:4 Tregs:APCs ratio. At day 7, expanded cells were diluted at 1?10.sup.5/ml and stimulated again or not. IL-2 and IL-15 cytokines were freshly added at days 0, 7, 10 and 12. At day 14, expanded Tregs were washed with PBS before use.

[0307] Humanized Mice Models:

[0308] 8-12 week old NOD/SCID/IL-2R?.sup.?/? (NSG) mice were bred in our own animal facilities in SPF conditions. This study was carried out in strict accordance with the protocol approved by the Committee on the Ethics of Animal Experiments of Pays de la Loire (permit number CEEA. 2012.155).

[0309] For xenogeneic GVHD experiments, 1.5?10.sup.7 syngeneic PBMCs were intravenously transferred with or without polyclonally expanded Tregs at 1:1 and 1:2 PBMCs:expanded Tregs ratio in 12 h-previously irradiated NSG mice. Human PBMCs engraftment was monitored in blood, and GVH development was evaluated by weight loss and histopathological analysis of lung, livers, intestines, spleen, kidneys.

[0310] For transplantation model, human skin was provided by CHU Nantes from healthy volunteers and graft was accepted in 15 days by NSG mice. 5?10.sup.6 PBMCs allogeneic to the graft were transferred intraperitoneally with or without syngeneic 15 days expanded Tregs. Graft rejection score was evaluated.

[0311] Results

[0312] Human CD8.sup.+CD45RC.sup.low Tregs are More Efficient Suppressors of Anti-Donor Immune Responses than Human CD4.sup.+CD25.sup.+CD127.sup.? Tregs.

[0313] Previous studies have described the involvement of CD8.sup.+CD45RC.sup.low T regulatory cells in graft acceptance induced by treatment with CD40Ig in a MHC-mismatched heart allograft rat model (Guillonneau et al., 2007; Li et al., 2010). Both studies have underlined the key role of IFN? in the CD40Ig-induced allograft acceptance.

[0314] Phenotypic analysis of the CD45RC marker in healthy individuals showed that the CD45RC marker is differentially represented in healthy individuals as revealed by the overlay of several individuals. This differential expression was not related to age or gender.

[0315] First, the suppressive activity of the total human CD8.sup.+CD45RC.sup.low T cell subset was assessed and compared to the suppressive activity of the well-known CD4.sup.+CD25.sup.+CD127.sup.? Tregs when added at different ratios in a MLR assay where CFSE-labeled CD4.sup.+CD25.sup.? T cells were stimulated by allogeneic T-depleted PBMCs (FIG. 1). Interestingly, human CD8.sup.+CD45RC.sup.low Tregs display a significantly superior suppressive capacity on anti-donor CD4.sup.+CD25.sup.? effector T cells proliferation compared to CD4.sup.+CD25.sup.+CD127.sup.? Tregs at all ratios.

[0316] In the rat, a preferential interaction of CD8.sup.+CD45RC.sup.low Tregs with pDCs was identified for CD8.sup.+CD45RC.sup.low Tregs to exert an optimal suppressive activity. Thus, the suppressive potential of human CD8.sup.+CD45RC.sup.low was tested in presence of different ratios of cDCs (CD3.sup.?CD19.sup.?CD1c.sup.+Nrp?1.sup.?), pDCs (CD3.sup.+CD19.sup.?CD1c.sup.?Nrp?1.sup.+) in comparison to T-depleted PBMCs as stimulators (FIG. 2). Interestingly, a lesser suppressive activity of CD8.sup.+CD45RC.sup.low Tregs was observed in presence of cDCs compared to pDCs and T-depleted PBMCs, suggesting that in human, as it was the case in rat, pDCs are necessary to mediate CD8.sup.+CD45RC.sup.low Tregs suppressive activity on CD4.sup.+ effector T cells.

[0317] In conclusion, human CD8.sup.+CD45RC.sup.low Tregs in presence of pDCs suppress more efficiently anti-donor CD4.sup.+CD25.sup.+ effector T cells responses than CD4.sup.+CD25.sup.+CD127? Tregs.

[0318] A Subpopulation of CD8.sup.+CD45RC.sup.low Tregs Expresses IFN?, IL-34 and IL-10.

[0319] To characterize human CD8.sup.+CD45RC.sup.low Treg, a deep phenotypic analysis was performed using 13-colour flow cytometry. Several markers were analyzed to assess the existence of subpopulations within the total CD8.sup.+CD45RC.sup.low Treg subset: markers previously described on Tregs such as CD103, CTLA-4, GITR or cytokines such as IFN?, IL-34 or IL-10, as well as the known marker of human CD8.sup.+ Tregs, i.e. CD28 and CD122.

[0320] Screening of markers revealed a significant upregulation of CD69, CD71, CD103, Foxp3, Tbet, HLA-DR, CD154, IFN?, IL-2 and IL-34 expression and a decreased CD38 expression among CD8.sup.+CD45RC.sup.low Tregs compared to CD8 CD45RC.sup.high T cells, altogether suggesting a profile of activated pro-tolerogenic cells.

[0321] Co-expression analysis of CD45RC, CD28 and CD122 revealed that each-subpopulations existed inside the CD45RC marker but that the CD8.sup.+CD28.sup.? Tregs and the CD8.sup.+CD122.sup.+ Tregs were not all contained within the CD45RC.sup.low marker, suggesting that each of these markers identifies distinct Tregs sub-populations.

[0322] Of particular interest, a significant expression of IFN? and IL-34 was observed among CD8.sup.+CD45RC.sup.low Tregs (about 60% and 20% respectively), with IL-34 being mostly co-expressed by IL-10-secreting cells.

[0323] The expression of cell the surface receptor GITR was assessed in CD8.sup.+CD45RC.sup.low Tregs and CD8.sup.+CD45RC.sup.high Tregs (FIG. 3). Flow cytometry analysis showed that GITR is significantly more expressed in the population of CD8.sup.+CD45RC.sup.low Tregs (about 15% of the cells express GITR) than in the population of CD8.sup.+CD45RC.sup.high Tregs (about 5% of the cells express GITR). Thus, among the CD8.sup.+CD45RC.sup.low Tregs there is a subpopulation of GITR.sup.+CD8.sup.+CD45RC.sup.low Tregs.

[0324] Finally, CD3.sup.+CD8.sup.+CD45RC.sup.low Tregs were analyzed by FACS staining for expression of Foxp3, IL-34, IFN?, TGF?-1 and GITR after gating on viable cells (FIG. 4). The CD3.sup.+CD8.sup.+CD45RC.sup.low Tregs were obtained from PBMCs stimulated for 4 h with 50 ng/ml PMA and 1 ?g/ml ionomycine in presence of 10 ?g/ml Brefeldine A. Only Foxp3.sup.+CD8.sup.+CD45RC.sup.low Tregs expressed GITR, TGFb1 and IL-34. Foxp3.sup.+CD8.sup.+CD45RC.sup.low Tregs also expressed higher levels of IFN? than Foxp3.sup.?CD8.sup.+CD45RC.sup.low Tregs.

[0325] In conclusion, expression of IL-34, TGFb1 and GITR tightly correlates with Foxp3 expression in CD8.sup.+CD45RC.sup.low Tregs.

[0326] The Subpopulation of IL-10.sup.+IL-34.sup.+IFN?.sup.+-Secreting CD8.sup.+CD45RC.sup.low Tregs Displays a High Suppressive Capacity In Vitro.

[0327] The suppressive potential of the subpopulation of human CD8.sup.+CD45RC.sup.low Tregs expressing IFN? and IL-34 was assessed. First, subpopulations of CD8.sup.+CD45RC.sup.low Tregs were sorted based on the expression of IFN? and IL-10 using the IFN?- or IL-10-secretion assaydetection kit allowing sorting of live cells. From the observation that IL-34 is mostly co-expressed by IL-10-secreting cells, it was assumed that IL-10.sup.+ cells were also IL-34.sup.+ cells. Thus, four subpopulations of CD8.sup.+CD45RC.sup.low Tregs were sorted: IL-10.sup.?(IL-34.sup.?)IFN?, IL-10.sup.? (IL-34.sup.?)IFN?.sup.+, IL-10.sup.+(IL-34.sup.+)IFN?.sup.?, and IL-10.sup.+(IL-34.sup.+)IFN?.sup.+CD8.sup.+CD45RC.sup.low Tregs. Such sorted cells were then added in the MLR assay as described above (FIG. 5A). Importantly, the IL-10.sup.+(IL-34.sup.+)IFN?.sup.+CD8.sup.+CD45RC.sup.low Tregs were significantly more suppressive than the IL-10.sup.?(IL-34.sup.?)IFN?.sup.+CD8.sup.+CD45RC.sup.low Tregs, suggesting a key role for IL-10 and IL-34.

[0328] In conclusion, the subpopulation of IL-10.sup.+IL-34.sup.+IFN?.sup.+-secreting CD8.sup.+CD45RC.sup.low Tregs displays a high suppressive capacity in vitro.

[0329] GITR.sup.+ Sorted CD8.sup.+CD45RC.sup.low Tregs are More Efficient Suppressor than GITR.sup.? CD8.sup.+CD45RC.sup.low Treg

[0330] Similar assays were performed by subdividing the total CD8.sup.+CD45RC.sup.low Treg population with cell-surface markers: GITR, CD38, HLA-DR, CD45RA, CD127, CD197, CD27, CD28, CD25. Although a small loss of suppressive activity could be observed with some other markers such as CD45RA and CD28, there were no significant differences. With one exception, there was no increased suppressive activity, suggesting that these markers cannot be used to further discriminate CD8.sup.+CD45RC.sup.low Tregs with suppressive activity.

[0331] However, most interestingly, GITR.sup.+CD3.sup.+CD8.sup.+CD45RC.sup.low Tregs more efficiently suppressed anti-donor immune responses than GITR.sup.?CD3.sup.+CD8.sup.+CD45RC.sup.low Tregs (FIG. 5B). In conclusion, GITR expression identifies a subpopulation of CD8+ Tregs with a higher suppressive capacity on allogeneic effector CD4.sup.+CD25.sup.? T cell proliferation.

[0332] CD8.sup.+CD45RC.sup.low Treg Mediated Suppressive Activity Depends on IL-34, IL-10 and IFN? Secretion.

[0333] The different mechanisms of action that could underlie the suppressive activity of the CD8.sup.+CD45RC.sup.low Tregs were then reviewed. Such mechanisms of action include cytokine secretion, contact dependent activity, IL-2 deprivation or killing to suppress effector T cell proliferation.

[0334] First, since IFN?, IL-34 and IL-10 discriminate the CD8.sup.+CD45RC.sup.low Treg subpopulation with the best regulatory activity, cytokine production was tested in the MLR supernatant. Analysis of cytokine production in the MLR's supernatant by day 5 demonstrated a significant production of IFN? in presence of CD8.sup.+CD45RC.sup.low Treg versus no Tregs and CD8.sup.+CD45RC.sup.high T cells (FIG. 13).

[0335] To further assess the role of IFN?, IL-34 and IL-10 in CD8.sup.+CD45RC.sup.low Treg-mediated suppression, the following blocking antibodies were added in a suppressive MLR assay in presence of CD8.sup.+CD45RC.sup.low Tregs: anti-IL-34, anti-IL-10, anti-IL-10R, anti-IFN? and anti-IFN?R (FIG. 6). Interestingly, blockade of IL-34, IL-10 and IL-10R, IFN? and IFN?R resulted in a similar loss of suppressive activity of the CD8.sup.+CD45RC.sup.low Tregs on the CD4.sup.+CD25.sup.? T cell anti-donor immune response.

[0336] CD8.sup.+CD45RC.sup.low Treg Mediated Suppressive Activity Also Depends on Contact.

[0337] To determine the importance of a contact for CD8.sup.+CD45RC.sup.low Tregs mediated suppression, transwell experiments were performed (FIG. 7). The proliferation of CFSE-labeled CD4.sup.+CD25.sup.? T cells stimulated with allogeneic T-depleted PBMCs in the lower chamber was assessed in presence or not of CD8.sup.+CD45RC.sup.low Tregs in the lower or upper chamber. A complete loss of suppressive activity was observed when CD8.sup.+CD45RC.sup.low Tregs were added separately in the upper chamber, demonstrating the requirement for a contact.

[0338] As this could suggest suppressive mechanisms through killing, next increasing ratios of CD8.sup.+CD45RC.sup.low Tregs-mediated cytotoxicity were tested towards syngeneic CD4.sup.+CD25.sup.? effector T cells or allogeneic T-depleted PBMCs following 15 h incubation. There was no necrosis or late apoptosis induction toward syngeneic or allogeneic targets, however a small early apoptosis induction was observed toward both targets at high target:effector ratios (data not shown). The absence of end-stage apoptosis induction suggests that cytotoxic mechanisms play little role in the suppressive effect mediated by the CD8.sup.+CD45RC.sup.low Tregs.

[0339] CD8.sup.+CD45RC.sup.low Tregs can be Efficiently Expanded, Even after Thawing.

[0340] As cell therapy using CD4.sup.+ Tregs is a promising issue to prevent allograft from rejection, and as CD8.sup.+CD45RC.sup.low Tregs are more efficient than CD4.sup.+CD25.sup.highCD127.sup.? Tregs to inhibit anti-donor immune response ex vivo (FIG. 1), a protocol of expansion of CD8.sup.+CD45RC.sup.low Tregs was set up.

[0341] First the most efficient conditions for expansion of total CD8.sup.+CD45RC.sup.low Tregs were identified using different ratios of APCs (from 1:1 to 1:16 Treg:APCs ratios), different sources of sera (AB serum, autologous plasma, Tex Mecs or albumine 2%) and different stimulations (polyclonal anti-CD3.sup.+ anti-CD28 vs pooled allogeneic T-depleted APCs).

[0342] It was thus found that a 1:4 Treg:APC ratio was necessary and sufficient for efficient 7-days Tregs expansion and that the use of AB serum in the culture media led to efficient CD8.sup.+CD45RC.sup.low Tregs expansion at such ratio. It was next observed that CD8.sup.+CD45RC.sup.low Tregs can be efficiently expanded until 1000-fold in 14 days, even after thawing, when stimulated indifferently with allogeneic T-depleted APCs with or without polyclonal anti-CD3+ anti-CD28 mAbs from day 0 to day 7 of culture, in presence of high dose of IL-2 and IL-15, and re-stimulated or from day 7 to day 14 (FIG. 8A).

[0343] The expansion of freshly sorted CD8.sup.+CD45RC.sup.low Tregs and thawed CD8.sup.+CD45RC.sup.low Tregs was compared after 14 days of culture (FIG. 8B). On day 0, the cells were stimulated with allogeneic T-depleted PBMCs at 1:4 Tregs:APCs ratio and with polyclonal anti-CD3+ anti-CD28 Abs. On day 7, the cells were stimulated again with anti-CD3+ anti-CD28 Abs. On days 0, 7, 10 and 12 the culture medium was supplemented with IL-2 and IL-15. After 14 days, freshly sorted CD8.sup.+CD45RC.sup.low Tregs were expanded 135-fold and thawed CD8.sup.+CD45RC.sup.low Tregs were expanded 612-fold. Thus, thawed CD8.sup.+CD45RC.sup.low Tregs proliferated more efficiently than freshly sorted CD8.sup.+CD45RC.sup.low Tregs.

[0344] In conclusion, CD8.sup.+CD45RC.sup.low Tregs can efficiently be expanded, even more so after thawing.

[0345] Furthermore, the survival of expanded CD8.sup.+CD45RC.sup.low Tregs in presence of immunosuppressive drugs was assessed (FIG. 9). On day 0, the cells were stimulated with allogeneic T-depleted PBMCs at 1:4 Tregs:APCs ratio and with polyclonal anti-CD3+ anti-CD28 mAbs. On day 7, the cells were stimulated again with anti-CD3+ anti-CD28 mAbs. On days 0, 7, 10 and 12 the culture medium was supplemented with IL-2 and IL-15. On day 14, the Tregs were stimulated again with allogeneic APCs, in presence or not of immunosuppressive drugs (cyclosporine A, mycophenolate mofetil, methylprednisolone, rapamycin, or tacrolimus) and in presence or not of IL-2/IL-15. On day 20, the Tregs were analyzed for survival. CD8.sup.+CD45RC.sup.low Tregs survival was not altered in presence of immunosuppressive drugs. Furthermore, CD8.sup.+CD45RC.sup.low Tregs proliferated in presence of the immunosuppressive drugs tested, with the exception of mycophenolate mofetil. Proliferation was more efficient with IL-2/IL-15 supplementation, suggesting that both cytokines could be added in vivo in supplementation of CD8.sup.+ Tregs to maintain their proliferation.

[0346] Finally, the proliferation of CD8.sup.+CD45RC.sup.low Tregs and CD4.sup.+CD25.sup.highCD127.sup.low Tregs was compared in the same culture conditions (FIG. 10). On day 0, both subpopulations of Tregs were stimulated for 21 days with allogeneic T-depleted PBMCs at 1:4 Tregs:APCs ratio and with polyclonal anti-CD3+ anti-CD28 mAbs. On days 7 and 14, the Tregs were stimulated again with anti-CD3+ anti-CD28 mAbs. On days 0, 7, 10, 12, 14 and 19 the culture medium was supplemented with IL-2 and IL-15. CD8.sup.+CD45RC.sup.low Tregs were expanded 354-fold whereas CD4.sup.+CD25.sup.high Tregs were only expanded 11-fold in response to polyclonal stimulation. Furthermore, efficient and exponential CD8.sup.+CD45RC.sup.low Treg proliferation was maintained until at least day 21 in contrast to CD4.sup.+CD25.sup.high Treg proliferation that reached a plateau on day 14.

[0347] 14-Days Expanded CD8.sup.+CD45RC.sup.low Tregs Upregulates IFN?, IL-10 and IL-34 to Inhibit Immune Rejection.

[0348] Importantly, both allogeneic and polyclonally expanded CD8.sup.+CD45RC.sup.low Tregs were significantly more efficient at suppressing allogeneic CD4.sup.+CD25.sup.? effector T cell responses than thawed, sorted and non-expandedCD8.sup.+CD45RC.sup.low Tregs (FIG. 11).

[0349] In addition, such 14-day expanded CD8.sup.+CD45RC.sup.low Tregs displayed comparable cytotoxic activity than non-expanded or 7-day expanded CD8.sup.+CD45RC.sup.low Tregs, demonstrating that the superior suppressive capacity acquired upon expansion was not due to increased killing activity of the CD8.sup.+CD45RC.sup.low Tregs.

[0350] Most importantly, such protocol of expansion resulted in significant enrichment of IFN?.sup.+IL-34.sup.+IL-10-secreting CD8.sup.+CD45RC.sup.low Tregs (FIG. 12).

[0351] IFN? Likely Plays a Crucial Role in CD8.sup.+CD45RC.sup.low Treg Function

[0352] Detection of IFN? was assessed in the supernatant of different CD8.sup.+CD45RC.sup.low Treg cultures (FIG. 13). Isolated CD8.sup.+CD45RC.sup.low Tregs were stimulated with polyclonal anti-CD3 and anti-CD28 Abs overnight and either added in a MLR assay where syngeneic effector CD4.sup.+CD25.sup.? T cells were stimulated with allogeneic T-depleted PBMCs in a 1:1 effector:suppressor ratio, or stimulated with allogeneic T-depleted PBMCs at 1:4 Tregs:APCs ratio at day 0 and polyclonal anti-CD3 and anti-CD28 Abs for 14-day expansion. On day 7, cells were stimulated again with anti-CD3 and anti-CD28 Abs. On days 0, 7, 10, and 12, the culture medium was supplemented with IL-2 and IL-15. IFN? was measured in culture supernatant by ELISA on day 14. IFN? secretion was detectable in presence of effector CD4.sup.+CD25.sup.? T cells and APCs (mean 2.5 ng/ml), but increased following 6-day incubation with CD8.sup.+ Tregs (mean 3.9 ng/ml) or 14-day expansion of CD8+ Tregs (mean 8.7 ng/ml) with anti-CD3 and anti CD28 mAbs.

[0353] In conclusion, high levels of IFN? are detectable in the supernatants of CD8.sup.+CD45RC.sup.low Treg culture, suggesting a crucial role for this cytokine in CD8.sup.+CD45RC.sup.low Treg function.

[0354] Expanded IFN?.sup.+IL-10.sup.+IL-34.sup.+ Secreting CD8.sup.+CD45RC.sup.low Tregs Display a Therapeutic Effect In Vivo

[0355] Finally, the suppressive potential of such expanded CD8.sup.+CD45RC.sup.low Tregs was assessed using two distinct models of transplantation using NSG mice, a model of allograft rejection of human skin following injection of allogeneic PBMCs (FIG. 14) and a model of xenogeneic GVHD following injection of human PBMCs (FIG. 15).

[0356] In these models, syngeneic expanded CD8.sup.+CD45RC.sup.low Tregs were injected at the same time than PBMCs to assess their potential at inhibiting anti-donor immune responses by measuring percentage of survival, skin histology and weight loss.

[0357] Interestingly, in both cases the co-transfer of expanded CD8.sup.+CD45RC.sup.low Tregs significantly inhibited graft rejection, demonstrating the potential of the CD8.sup.+CD45RC.sup.low Tregs as a cellular therapy.

REFERENCES

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