Therapeutic Substances, their Preparation and Diagnostic Procedure

Abstract

A method is described for using live mesenchymal stromal cells (MSCs) in a way which allows for identification of patients likely to respond to immunosuppressive treatment using MSCs. The method involves contacting a sample from said patient with live MSCs in vitro, and determining whether the sample is able to induce at least some apoptosis to occur in live MSCs in vitro, or detection of elevated levels of prostaglandin E2 (PGE2). The ability of the sample to induce said apoptosis and/or elevated levels of PGE2 is indicative of responsiveness of said patient to said immunosuppressive treatment and/or indicative of fitness to recover. Also provided are apoptotic MSCs for use in the treatment of immune-mediated disease or conditions, such as allo-immune or autoimmune disease, or for the prevention or treatment of rejection of a transplanted organ; or in regenerative medicine to stimulate tissue repair. Methods for preparing pharmaceutical compositions comprising the apoptotic MSCs are also described and claimed.

Claims

1-45. (canceled)

46. A method for producing apoptotic mesenchymal stromal cells (MSCs) for use in immunosuppression therapy, comprising incubating live MSCs with a pharmaceutically acceptable apoptosis-inducing agent for a period sufficient to form a cell culture in which at least 30% of the MSCs are apoptotic within a period of less than 24 hours.

47. The method of claim 46, wherein the pharmaceutically-acceptable apoptosis-inducing agent is a biological agent.

48. The method of claim 47, wherein the biological agent is selected from at least one of the group consisting of: a serine protease and an anti-FAS antibody.

49. The method of claim 48, wherein the serine protease is human Granzyme B (GrB).

50. A method for producing an immunosuppressive effect in a patient in need thereof, comprising administering to the patient an effective amount of apoptotic MSCs.

51. The method of claim 50, wherein the patient is suffering from an immune-mediated disease or condition or requires regenerative medicine to stimulate tissue repair.

52. The method of claim 51, wherein the disease is allo-immune or autoimmune disease or the condition is organ transplantation rejection.

53. The method of claim 51, wherein the disease is selected from the group consisting of: Graft-vs-Host-Disease, multiple sclerosis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, arthritis, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, and acute respiratory distress syndrome.

54. The method of claim 50, wherein efficacy of the treatment in the patient is monitored by detecting the presence of phagocytes or IDO in a sample taken from the patient after administration of MSCs.

55. A composition comprising apoptotic MSCs configured for use in the treatment or prevention of immune-mediated disease and conditions or in regenerative medicine to stimulate tissue repair.

56. The composition of claim 55, wherein the disease is allo-immune or autoimmune disease or the condition is organ transplantation rejection.

57. The composition of claim 55, wherein the disease is selected from the group consisting of: Graft-vs-Host-Disease, multiple sclerosis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, arthritis, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, and acute respiratory distress syndrome.

58. The composition of claim 55, wherein the composition comprises apoptotic MSCs obtained using a method for producing apoptotic mesenchymal stromal cells (MSCs) for use in immunosuppression therapy, the method comprising: incubating live MSCs with a pharmaceutically acceptable apoptosis-inducing agent for a period sufficient to form a cell culture in which at least 30% of the MSCs are apoptotic within a period of less than 24 hours; wherein the pharmaceutically-acceptable apoptosis-inducing agent is a biological agent; wherein the biological agent is selected from at least one of the group consisting of: human Granzyme B (GrB) and an anti-FAS antibody.

59. The composition of claim 58, wherein the apoptotic MSCs are configured for use in immunosuppression therapy.

60. The composition of claim 58, wherein the apoptotic MSCs comprise a suicide mechanism, optionally in the form of a suicide gene, particularly a caspase-inducible gene, such as caspase 9 or caspase 3.

61. The composition of claim 58, wherein the composition is a pharmaceutical composition.

62. The composition of claim 61, further comprising live MSCs.

63. A method of identification of suitably-potent live MSCs capable of undergoing induced apoptosis and/or of producing PGE2 in at least one sample, the method comprising: contacting the live MSCs in vitro from each sample with at least one biological sample from at least one patient; and detecting either the presence of apoptotic MSCs among the live MSCs in vitro or elevated levels of prostaglandin E2 (PGE2) in each sample; wherein the presence of apoptotic MSCs among the live MSCs or elevated levels of PGE2 in vitro is indicative of suitable potency of the live MSCs.

64. The method of claim 63, wherein each contacting step comprises incubating the biological sample with live MSCs to form an incubate, and wherein each detecting step comprises detecting the presence and/or determining the potency of apoptotic MSCs in the incubate.

65. The method of claim 63, wherein the presence of elevated PGE2 and apoptotic MSCs among the live MSCs in a patient are biomarkers of the patient's fitness to recover from tissue injury and/or responsiveness to immunosuppressive treatment.

Description

FIGURES

[0113] The invention will now be particularly described by way of example with reference to the accompanying drawings which are summarised below.

[0114] FIG. 1. MSC undergo in vivo apoptosis after infusion without affecting delivery of immunosuppression.

[0115] A: luc-MSC were injected i.v. into naïve, BM and GvHD mice 3 days after transplantation. All animals were then injected i.p. with DEVD-aminoluciferin and imaged 1 hour later. N: 6 mice per group, grouped from 3 independent experiments. White lines separate multiple photographs assembled in the final image. B: TLS was measured from the images of mice in FIG. 1A and shown as mean±SD. C, D: Infiltration of GvHD effector cells (CD8+Vβ8.3+) in the spleen (C) and lungs (D) of GvHD mice (black circles) and GvHD mice treated with MSC (black squares), 4 days after MSC injection. N: 15 (GvHD) and 13 (GvHD+MSC) mice, grouped from 4 independent experiments; mean±SD are shown. Statistics in B: one-way ANOVA, with Tukey's Multiple Comparison Test. **: p<0.01, ***: p<0.001, ns: not significant. In C and D: unpaired t-test. **: p<0.01.

[0116] FIG. 2. MSC apoptosis is indispensable for immunosuppression and requires functionally activated cytotoxic cells in the recipient.

[0117] A: The percentage of CD8+Vβ8.3+ cells in lung cell suspensions from naïve C57BL/6 male, BM or GvHD mice was analyzed in the lymphocyte population; mean±SD are shown. N: 12 (GvHD), 3 (BM) and (3 (naïve) mice, grouped from 3 independent experiments. B: CD8+ cells were sorted from the lungs and spleens of naïve female Mh (grey bars) or GvHD mice (white bars) 7 days after transplant and tested for their ability to induce MSC apoptosis in vitro. The results show annexin-V+/7-AAD− MSC (mean±SD) in 3 independent experiments (N=10 per group), black bar represents the level of apoptosis in MSC cultured alone used as control (N: 3) C: luc-MSC were infused in three independent experiments in GvHD (N=7) and GvHDPerf−/− (N=7) mice 3 days after transplantation. 1 hour later mice were injected with DEVDaminoluciferin and imaged. White lines separate multiple photographs assembled in the final image. D: TLS was obtained from FIG. 2C and expressed as mean±SD. E, F: infiltration of effector GvHD cells (CD8+Vβ8.3+) in the spleen (E) and lungs (F) of untreated GvHDPerf−/− (N=16) and GvHDPerf−/− (N=17) mice treated with MSC (mean±SD of 4 independent experiments). Statistics in A and B: oneway ANOVA, with Tukey's Multiple Comparison Test. *: p<0.05; ***: p<0.001. In D, E and F: unpaired t-test. ***: p<0.001. ns: not significant.

[0118] FIG. 3. Cytotoxic activity against MSC predicts clinical responses to MSC in GvHD patients.

[0119] A, B: PBMC obtained from healthy controls (HC) or patients with GvHD receiving MSC in the following 24 hours were incubated in 24-well plates with MSC at a 20/1 PBMC/MSC ratio for 4 hours. The level of apoptosis was measured in MSC assessing the level of annexin-V/7-AAD by flow-cytometry. (A) Representative plots for HC, clinical responders (R) and non-responders (NR). (B) The level of apoptosis was compared among HC (circles, N=5), R (triangles; N=5) and NR (squares; N=12). Statistics: one-way ANOVA and Tukey's Multiple Comparison test. ***: p<0.0001. ns: not significant.

[0120] FIG. 4. MSC apoptosis is mediated by activated CD8+ and CD56+ cytotoxic cells and is the result of a bystander effect.

[0121] A: PBMC from healthy donors were activated using phytohemagglutinin (PHA) (PHA-aPBMC) or MLR (MLR-aPBMC). Resting (light grey bars), PHAaPBMC (black bars) or MLR-aPBMC (dark grey bars) were incubated with MSC at the indicated ratios for 4 hours. ND: Not done. B, E, F, I: MLR-aPBMC or PHA-aPBMC were cultivated with MSC in the presence or absence of the pan-caspase inhibitor Z-VAD-FMK (10 μM) (B), GrB inhibitor Z-AAD-CMK (300 μM), perforin inhibitor ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) (4 mM) (E), neutralizing concentrations of FAS-L mAb anti-CD178 (F), or escalating doses (10 to 75 μM) of PKCζ-PS (I). H: MLR-aPBMC were cultivated with MSC in direct contact or separated by a transwell®. C, D: MLR-aPBMC were used unfractionated, positively selected for CD11b+, CD4+, CD8+ or CD56+ cells (C) or depleted of CD56+, CD8+ or both (D). G: apoptosis in MSC after culture with autologous (black bars) or allogeneic (grey bars) PHA-aPBMC in the presence or absence of neutralizing doses of anti-HLA-A-B-C or anti-HLA-DR antibodies. In B-I: the PBMC/MSC ratio was 20/1. Results represent the mean±SD of 3 or 6 (H) independent experiments. Statistics: one-way ANOVA, with Tukey's Multiple Comparison Test. *: p<0.5. **: p<0.01. ***: p<0.001. ns: not significant.

[0122] FIG. 5. MSC apoptosis does not interfere with the antigen-specific cytotoxic cell recognition of the cognate target.

[0123] A: apoptosis in T2-cell after culture with 4D8 cells at a 20/1 4D8:T2 ratio. Where indicated increasing concentrations of MSC (used as cold target) were added. Apoptotic T2 cells were identified as annexin-V+/7-AAD+ cells. B: apoptosis in K562 cultured with NK cells (20/1 NK:K562 ratio). Where indicated increasing concentrations of MSC (used a cold target) were added. C: apoptosis in MSC cultured with 4D8 cells (20/1 4D8:MSC ratio). Where indicated increasing concentrations of T2 cells (used as cold target) were added. D: apoptosis in MSC cultured with NK cells at a 20/1 NK:MSC ratio. Where indicated, increasing dilutions of K562 (used as cold target) were added. In all experiments the level of MSC, T2 or K562 cell apoptosis was assessed after 4 hours of co-culture by flow cytometry. Results represent the mean±SD of 3 independent experiments. Statistics in A, B, C and D: unpaired T-test. *: p<0.05. ns: not significant.

[0124] FIG. 6. Apoptotic MSC exert in vivo immunosuppressive in a Th2-type inflammation model in the absence of cytotoxic cells.

[0125] A: luc-MSC were injected into naïve (N=3) and OVA+MSC (N=6) mice one hour after the last challenge. One hour later, mice received DEVDaminoluciferin and were imaged in 3 independent experiments. White lines separate multiple photographs assembled in the final image. B: TLS was measured from FIG. 6A (mean±SD). C: Eighteen hours after MSC infusion, eosinophil infiltration was assessed in the BAL of naïve (N=3), naïve infused with MSC (N=3), OVA (N=6) and OVA+MSC (N=6) mice in two independent experiments and mean±SD are shown. D: eosinophil infiltration (mean±SD) in BAL of OVAsensitized mice treated with ApoMSC. Groups were: OVA without ApoMSC (N=6), OVA treated with 1×10.sup.6 ApoMSC (N=7); and naïve mice receiving 1×10.sup.6 (N=2) ApoMSC. Results represent the mean±SD of 3 independent experiments. Statistics in B: unpaired t-test. ns: not significant. Statistics in C and D: one-way ANOVA and Tukey's Multiple comparison test. *: p<0.05. ns: not significant.

[0126] FIG. 7. ApoMSC exert immunosuppressive activity in GvHD and are engulfed by recipient phagocytic cells in which they elicit IDO production.

[0127] A-D: Infiltration of GvHD effector cells was assessed in spleen (A, C) and lungs (B, D) of GvHD mice (black circles) and GvHD mice treated with ApoMSC (black squares). ApoMSC were infused i.p. (GvHD mice N=10, GvHD+ApoMSC mice N=8) (A, B), or i.v. (GvHD mice N=9, GvHD+ApoMSC mice N=7) (C, D). Results represent the mean±SD of 3 independent experiments. Statistics: unpaired t-test. *: p<0.05; **: p<0.01. ns: not significant. E-K: MSC were labelled using CellTrace™ Violet and subjected to apoptosis induction using GrB/FAS-L (5 μg/ml and 10 μg/ml, respectively). ApoMSC were injected i.p. (E, F and 3) or i.v. (G, H, I and K) into GvHD mice 3 days after the transplant. After 2 hours, animals were sacrificed and mesenteric lymph nodes (E, F and J) or lungs (G, H, I and K) were harvested. Cells engulfing ApoMSC were identified as Violet+ cells within the CD11b+ (E), CD11c+ (F), CD11bhighCD11cint (G), CD11c+CD11b− (H) and CD11bhighCD11c− (I) subpopulations. The corresponding subpopulations were gated in GvHD mice which had not received violet-labelled ApoMSC. J and K: IDO expression was assessed in CD11c+ and CD11b+ (J) or CD11bhighCD11cint, CD11c+CD11b− and CD11bhighCD11c− (K) cells positive for CellTrace™ Violet (engulfing apoMSC) and compared with the corresponding populations in GvHD mice that had not received ApoMSC. Data are representative of similar results obtained from three mice in 2 independent experiments.

[0128] FIG. 8. Recipient phagocytes and IDO production are required for MSC immunosuppressive activity in GvHD.

[0129] A, B: GvHD mice were treated with liposomal clodronate 10 minutes after the transplant. Where indicated, MSC were infused 3 days later. The infiltration of GvHD effector cells (CD8+Vβ8.3+) in spleen (A) or lungs (B) was quantitated in spleen and lungs after 4 additional days. Mean±SD was obtained grouping three independent experiments with N:12 (GvHD) and 10 (GvHD+MSC) mice per group. C, D: GvHD effector cell infiltration was studied in spleen (C) and lungs (D) of GvHD mice treated with the IDO-inhibitor 1-DMT. In the treated mice, MSC were infused 3 days after the transplant (N=11). Controls consisted of GvHD mice which did not receive MSC (N=9). Results refers to the mean±SD of 3 independent experiments. Statistics: unpaired t-test. *: p<0.05; **: p<0.01. ns: not significant.

[0130] FIG. 9. MSC can be traced in the lungs of mice after infusion.

[0131] A: lethally irradiated C57BL/6 male mice were transplanted with bone marrow (BM) and CD4+-purified cells from female syngeneic donors with or without CD8+ cells purified from Mh mice (CD8+Vβ8.3+) (GvHD and BM groups, respectively). At day +3 post-transplant,luc-MSC were infused and mice imaged one hour later for the analysis of caspase 3 activation after i.p. injection of DEVD-aminoluciferin. At day +7 posttransplant, mice were sacrificed and the infiltration of GvHD effector cells (CD8+Vβ8.3+) in lungs and spleen was analyzed by flow-cytometry. B: in order to confirm the presence of luc-MSC in the lungs of all groups of mice infused with MSC, the same mice imaged in FIG. 1A were injected with D-Luciferin. White lines separate multiple photographs assembled in the final image. C: TLS was measured from the images of mice in FIG. 9B and shown as mean±SD. Statistics: one-way ANOVA, with Tukey's Multiple Comparison Test. ns: not significant.

[0132] FIG. 10. Human MSC immunosuppression is not ‘licensed’ by murine cytokines.

[0133] A: human MSC were plated overnight at serial dilutions alone or in the presence of hIFN-γ/hTNF-α (20 ng/ml each) or mIFN-γ/mTNF-α (20 ng/ml each) or supernatant obtained from PHA-aPBMC for 72 hours or mSpl activated with ConA (ConA-aSpl) for 72 hours, as indicated. MSC were then tested for the ability to inhibit the proliferation of ConA-stimulated mSpl labelled with carboxyfluorescein succinimidyl ester dye. Proliferation was determined after 72 hours by flow-cytometry. The curve was obtained plotting the percentage of inhibition against the corresponding MSC/mSpl ratio. B, C: human MSC were plated overnight either untreated or exposed to hIFN-γ/hTNF-α (20 ng/ml each) as indicated and then tested for the ability to suppress mSpl proliferation at 1:10 MSC/mSpl ratio. The histogram plot (B) is representative of 3 independent experiments, while bars (C) represent the mean±SD of 3 independent experiments. Statistics: one-way ANOVA and Tukey's Multiple Comparison test. ***: p<0.001. ns: not significant. D: human MSC were incubated alone or in the presence of hIFN-γ/hTNF-α (20 ng/ml each), mIFN-γ/mTNF-α (20 ng/ml each), supernatants obtained from PHA-aPBMC or ConA-aSpl. After 24 hours, IDO, TSG6 and PTSG2 expressions were assessed by real time PCR and calculated as relative expression in comparison to untreated MSC. Representative results of three independent experiments are shown.

[0134] FIG. 11. MSC apoptosis is activated by cytotoxic cells in a non-antigen specific manner.

[0135] A: CD8+ cells isolated from naïve female Mh mice were stimulated for 3 days with anti-CD3/CD28 beads and cultured with MSC at a 20/1 Mh T-cell:MSC ratio. After 4 hours the level of apoptosis was assessed in MSC by annexin-V/7AAD stainings. Results represent the mean±SD of 3 independent experiments. Statistics: one-way ANOVA, with Tukey's Multiple Comparison Test. ***: p<0.001. B: in order to confirm the presence of luc-MSC in the lungs of all groups of mice infused with MSC, the same mice imaged in FIG. 2C were injected with D-Luciferin. White lines separate multiple photographs assembled in the final image. C: TLS was measured from the images of mice in FIG. 11B and shown as mean±SD. Statistics: unpaired t-test. ns: not significant.

[0136] FIG. 12. Cytotoxicity against MSC varies amongst PBMC donor but is independent on the percentage of CD8+ or CD56+ in GvHD patients.

[0137] A: PBMC obtained from 2 different GvHD patients (Patient 1 and Patient 2) were tested for their cytotoxic activity against MSC from two different donors (MSC1 and MSC2). B: apoptosis in MSC obtained from different donors (MSC1, MSC2 and MSC3) after incubation with PBMC from four different MLR responder/stimulator combinations (MLR1, MLR2, MLR3, MLR4). In A and B the level of apoptosis was assessed by flowcytometry after 4 hours of co-culture. C, D: PBMC obtained from 11 GvHD patients (R: 3, NR: 8) were analysed for the percentage of CD8+ (C) and CD56+ (D) cells. Statistics: unpaired t-test. ns: not significant.

[0138] FIG. 13. MSC killing is mediated by caspase 3 and effected by GrB and perforin.

[0139] A: PHA-aPBMC were incubated with MSC at escalating PBMC/MSC ratios. MSC apoptosis was assessed by annexin-V/7-AAD at different time-points by flowcytometry. Results represent the mean±SD of 3 independent experiments. B, C: MSC were transfected with the pECFP-DEVDR-Venus vector (FRET-MSC) and FRET between pECFP and Venus-YFP FRET was studied by flow-cytometry and CAf calculated. FRET-MSC were cultured alone, with PHA-aPBMC, or PHAaPBMC in the presence of Z-VAD-FMK (50 μM) (B), GrB inhibitor Z-AAD-CMK (300 μM) or the perforin inhibitor EGTA (4 mM) (C). Results of 5 (B) or 3 (C) independent experiments are shown. When PBMC were present, the PBMC:MSC ratio was 40/1. Statistics: oneway ANOVA and Tukey's Multiple Comparison test. **: p>0.01. ***: p>0.001. ns: not significant. D: MLR-aPBMC were cultivated with MSC (20/1 ratio) and apoptosis evaluated by flow-cytometry 4 hours later. Where indicated, the TNF-α inhibitor Etanercept or the mAb anti-TRAIL were used at 10 μ/ml or 100 μg/ml. Results represents the mean±SD of 3 independent experiments.

[0140] FIG. 14. Infused MSC can be imaged in the lungs of mice with Th2-type lung inflammation.

[0141] A: Balb/C mice were immunized i.p. with OVA at day 0 and 7 and subsequently challenged with OVA through aerosol at days 14, 15 and 16 (OVA group). Experimental group was treated with MSC one hour after the last challenge (OVA+MSC). When luc-MSC were used, mice were imaged one hour after infusion for the analysis of caspase 3 activation after i.p. injection of DEVD-aminoluciferin. After 18 hours from treatment, eosinophils infiltration in BAL was evaluated. B-E: Percentage (B, D) and absolute numbers (C, E) of different cellular types in the BAL (B, C) and lungs (D, E) of naïve (with bars) (N=3) and OVA-sensitized (black bars) (N=3) mice. Results represent the mean±SD of 3 independent experiments. In OVAsensitized mice, the analysis was performed 1 hour after the last aerosol challenge. F: in order to confirm the presence of luc-MSC in the lungs of all groups of mice infused with MSC, the same mice imaged in FIG. 6A were injected with D-Luciferin. White lines separate multiple photographs assembled in the final image. G: TLS was measured from the images of mice in FIG. 14F and shown as mean±SD. Statistics: unpaired t-test. ns: not significant.

[0142] FIG. 15 are a series of graphs showing (A) the percentage of ApoMSCs obtained using various concentrations of FasL (anti-Fas antibody), and (B) the percentage of ApoMSCs obtained using various concentrations of FasL (anti-Fas antibody) over various time periods.

[0143] FIG. 16 is a graph showing how PBMC from patients suffering from inflammatory bowel disease (IBD) induce MSC apoptosis ex vivo. PBMC from IBD patients (IBD, n=82) or healthy controls (HC, n=8) were co-cultured with MSC for 4 hours at a 20:1 PBMC:MSC ratio and apoptosis assessed by Annexin-V/7-AAD staining by flow cytometry. Statistics: Unpaired T-test. ***p<0.001.

[0144] FIG. 17 is a series of graphs showing how IBD patients induce PGE2 upregulation in MSC and correlate with the proportion of annexinV.sup.+ MSC. (A) MSC (5×10.sup.4) and patients PBMC (1×10.sup.6) were co-cultured for 24 hours, then the supernatant was collected and PGE2 measured by ELISA. Cells were either left untreated or pre-treated with the pan-caspase inhibitor Z-VAD-FMK (50 μM). Statistics: Paired T-test. *p<0.05. (B) Correlation between Annexin-V+ MSC and PGE2 levels in the supernatant.

[0145] FIG. 18 is a series of graphs showing the ability of patients' PBMC to induce MSC to undergo apoptosis predicts clinical responses to MSC infusions in GvHD. 32 patients affected by steroids-resistant GvHD were treated with intravenous MSC (1-3×10.sup.6 cells/Kg) (A). Before treatment, their peripheral blood mononuclear cells (PBMC) were incubated with MSC for 4 hours and MSC apoptosis was assessed by annexinV staining. The graph reports the proportion of annexinV+MSC in responders and non-responders (B). ROC calculated according to 11.5% MSC killing (annexinV+MSC) by patients PBMC (C). Overall survival of patients after MSC in responders and non-responders. Amongst the potential factors investigated, cytotoxicity (11.5% threshold) was the only factor influencing clinical response in multivariate analysis.

[0146] FIG. 19 shows MSC from different sources exhibiting varying sensitivity to undergo apoptosis. Different batches of MSC isolated from bone marrow (BM) or umbilical cord (UC) were incubated with PHA-activated PBMC from healthy individuals. After 4 hours, MSC were assessed for apoptosis (annexinV+7-AAD−).

[0147] FIG. 20 shows PBMC from patients with other inflammatory conditions harbour anti-MSC cytotoxic cells. PBMC were freshly isolated from patients with active inflammatory bowel disease (IBD) or who underwent liver transplantation (Ltx) and co-cultured with bone marrow-derived MSC (20:1 ratio) for 4 hours prior to FACS analysis. Statistic analysis by unpaired t-test (*p<0.05, **p<0.01, ***p<0.001).

[0148] FIG. 21 shows that skin fibroblasts are also susceptible to undergo apoptosis when exposed to cytotoxic cells. PBMC isolated from healthy donors were activated for 3 days with PHA and then co-cultured with dermal fibroblasts at different ratios. Apoptotic cells, identified as AnnexinV+7AAD− cells, were quantified after 4 hours by FACS analysis.

EXAMPLE 1

MSC Undergo Apoptosis in Recipient GvHD Animals

[0149] In order to explain the mechanism by which MSC are rapidly cleared after injection, the applicants tested the hypothesis that MSC undergo apoptosis in a mice model of GvHD. C57BL/6 (H2b) mice were purchased from Harlan Laboratories (Bicester, UK). Mh (C57Bl/6 background, CD8+Tg, H-2b, CD45.2+, H-2Db-restricted) mice are transgenic for a T-cell receptor specific for the male antigen UTY presented in the context of H-Db, and were bred in-house. All mice were used between 6 and 12 weeks of age.

[0150] Acute GvHD was induced as previously described (T. Toubai et al. Blood, 119, 3844-3853 (2012)). Briefly, after lethal irradiation (11 Gy), recipient C57BL/6 male mice were transplanted with 1×10.sup.6 purified CD8+ cells transgenic for a T-cell receptor specific for the male HY-antigen Uty (Matahari, Mh) from female Mh mice as GvHD effectors (FIG. 9A), 5×10.sup.6 unfractionated bone marrow (BM) and 2×10.sup.6 purified polyclonal CD4+ cells from female syngeneic donors (C57BL/6 wild-type donors). The control group received BM and purified CD4+ cells only. CD4+ and CD8+ T cells were obtained by positive selection using magnetic beads (Miltenyi Biotec Ltd, Bisley, UK). Live MSC (1×10.sup.6) were injected i.v. at day +3, whilst apoMSC (1×10.sup.6) were administered i.v. or i.p. at day +1, +3 and +6 from the transplant. Unless otherwise specified, animals were euthanized for analysis at day +7. The infiltration of GvHD effector cells was assessed by flow-cytometry and the percentage was expressed as proportion of cells in the lymphocyte gate, based on the physical characteristics of the cells.

[0151] In this model, the expansion of the T cells effecting GvHD (CD8+Vβ8.3+) can be precisely enumerated.

[0152] In vivo MSC caspase activation was evaluated using MSC that were transfected with the pGL3-control vector for the expression of firefly luciferase (Luc+) (luc-MSC). Caspase activation was measured as luciferase activity using DEVD-aminoluciferin. In this system, caspase 3 activation could be quantified on the basis of emitted light since DEVD is cleaved upon activation of caspase 3, leading to release of aminoluciferin which in turn can be metabolized by the firefly luciferase expressed in MSC. Luc-MSC were injected into recipients of BM transplant with CD8+ Mh T cells (GvHD group) and one hour later caspase activity was measured as total luminescence signal (TLS). Control mice consisted of naïve males (naïve group) and a group of mice which were irradiated and received CD4+ and BM cells (BM group) without the transgenic T cells to reproduce the condition of MSC infusion in the absence of activated cytotoxic T cells (FIG. 9A). High caspase activity was observed only in MSC injected into GvHD mice (FIGS. 1, A and B). High signal could be detected from the lungs of all animals when the control D-luciferin (firefly luciferase substrate) was used (FIGS. 9, B and C), thus confirming that luc-MSC can be tracked in the lungs also when caspase activity could not be detected.

[0153] The evidence that MSC undergo apoptosis after infusion prompted the question of whether they are still capable of suppressing antigen-driven T cell expansion. Therefore, their immunosuppressive effect was analysed by enumerating CD8+Vβ8.3+ Mh T cells (GvHD effector cells) in MSC treated or -untreated GvHD mice. MSC produced a significant reduction in GvHD effector cell infiltration in both spleen and lungs (FIGS. 1, C and D). These results indicate that, despite the presence of MSC apoptosis after infusion (FIGS. 1, A and B), MSC immunosuppression still occurs.

[0154] The possibility that the observed immunosuppressive activity could be the consequence of the recipient inflammatory cytokines can be excluded because in our xenogeneic combination murine inflammatory cytokines will not cross-react with the corresponding human receptors and will not activate immunosuppressive molecules in human MSC, whilst retaining the ability to expand murine effector cells mediating GvHD. Accordingly, human MSC were not able to inhibit concanavalin-A (ConA) induced proliferation of murine splenocytes (mSpl) unless pre-activated by human cytokines (FIGS. 10, A, B and C). Furthermore, exposure of human MSC to murine inflammatory cytokines did not upregulate IDO, TNF-stimulated gene 6 protein (TSG-6) or prostaglandin-endoperoxide synthase-2 (PTSG2), considered major effectors of human MSC mediated in vitro immunosuppression (FIG. 10D).

EXAMPLE 2

In Vivo MSC Apoptosis Depends on Activated Recipient GvHD Effector Cells

[0155] Our results show that MSC rapidly undergo apoptosis after infusion, providing the long-sought after explanation for the rapid clearance of transplanted MSC in the recipient. The absence of in vivo MSC apoptosis in naïve and BM mice clearly demonstrates that MSC apoptosis is not the result of xenogeneic recognition of human MSC, because it is detected only in GvHD mice. When we enumerated GvHD effector cell infiltrate (CD8+Vβ8.3+) in the lungs of mice, where MSC apoptosis occurs, we found that only the lungs of GvHD but not naïve and BM mice contained a large proportion of CD8+Vβ8.3+cells (FIG. 2A), thus confirming the correlation between caspase activation in MSC and the presence of GvHD effector cells.

[0156] To test the hypothesis that GvHD effector cells were responsible for MSC apoptosis, MSC were cultivated with CD8+ T cells purified from the lungs or spleens of GvHD (in vivo activated) or naïve Mh (in vivo resting) mice. Activated, but not resting, Mh CD8+ cells induced MSC apoptosis (FIG. 2B). In further support of the lack of antigen-specificity in the induction of the killing activity, high levels of cytotoxicity could be elicited by naïve Mh CD8+ cells stimulated in vitro by CD3/CD28 beads (FIG. 11A).

[0157] The requirement of cytotoxic cells in the induction of MSC apoptosis and the consequent immunosuppression was evaluated using Mh/Perforin Knock-Out mice (Mh/Perf−/−) as donors of defective cytotoxic GvHD effector cells (GvHDPerf−/− group). C57BL/6-Prf1tm1Sdz/J (Perforin−/−) mice were purchased from J Jackson labs, bred with Matahari Rag2−/− mice and the resulting offspring intercrossed for 2 generations to obtain the Mh Rag2−/−. Perf KO F3 mice.

[0158] Luc-MSC were infused into GvHDPerf−/− or control GvHD mice which had received Mh CD8+ T cells. Mice were imaged 1 hour later and caspase activation measured as described before. We observed much lower caspase activity in GvHDPerf−/− mice compared to GvHD controls (FIGS. 2, C and D). High signal was detected in the lungs of all animals when the control D-luciferin was used (FIGS. 11, B and C), thus confirming that luc-MSC were in the lungs also when caspase activity could not be detected. Importantly, the infiltration of GvHD effector cells in the spleen and lungs of mice receiving MSC was not reduced in GvHDPerf−/− receiving MSC (FIGS. 2, E and F). We conclude that MSC apoptosis is indispensable for immunosuppression and requires functionally activated cytotoxic cells in the recipient.

EXAMPLE 3

Cytotoxic Activity against MSC is a Biomarker Predictive of Clinical Response to MSC in GvHD Patients

[0159] Based on these findings, we inferred that the presence of cytotoxic cells in the recipient could be predictive of MSC therapeutic activity.

[0160] 16 patients (mean age 40.5 years (range: 10-69), with severe steroid resistant grade 3-4 GvHD were treated with MSC in various hospitals over a period of years. MSC were administered for compassionate use (according to Regulation (EC) No 1394/2007). Patients had received a myeloablative or reduced-intensity conditioning prior to hematopoietic stem cell transplantation. All patients received GvHD prophylaxis with 3 or 4 doses of methotrexate combined with cyclosporine. T-cell depletion with alemtuzumab or ATG was performed in all adult patients transplanted in the UK centers. Of the 16 patients included in the study, 13 developed GVHD following hematopoietic stem cell transplantation, and the remaining 3 after DLI. 12 patients were affected by acute GvHD, 3 by late onset acute GvHD and 1 by chronic GvHD. The diagnosis of GvHD was made on histological criteria and GvHD staged according to standard criteria. Patients were considered to be steroid-refractory if: (a) those with aGVHD failed to respond to high-dose methylprednisolone after 6 days; (b) the one with cGVHD failed to respond to high-dose steroids after 2-4 weeks, with the addition of Mycophenol Mycophenolate Mofetil (MMF) and cyclosporine at 1 and 4 weeks respectively. Clinical responses to MSC were assessed 1 week after MSC infusion and defined as an improvement of at least 50% in at least one organ affected by GvHD. Patient characteristics are summarized in Table 1 hereinafter.

[0161] Clinical responses to MSC were defined by an improvement of at least 50% in at least 1 organ affected by GvHD as previously described. Five patients obtained a clinical response.

[0162] Peripheral blood mononuclear cells (PBMC) were freshly collected within the 24 hours preceding the MSC infusion and tested directly for their ability to induce MSC apoptosis ex vivo in a 4-hour cytotoxic assay (see below). One patient received two doses of MSC and the cytotoxic assay was performed before each dose independently. MSC were sourced from the same donor used for the infusion (N=8) or from a different donor (N=9). At the time of performing the assay and cytofluorimetric analysis the operator was blind to patients' clinical details. PBMC from healthy donors (N=5) were used as controls.

[0163] Overall, PBMC from GvHD patients exhibited an average cytotoxic activity against MSC higher than that detected in control PBMC but without reaching a significant difference (mean±SD were: 10.63±8.76% and 3.82±2.50%, respectively; p=0.10). However, the level of cytotoxicity between clinical responders and non-responders to MSC was markedly different, with the proportion of apoptotic MSC (annexin-V+/7AAD−) exhibiting a four-fold difference (FIGS. 3, A and B). The discrimination threshold of apoptotic MSC between responders and non-responders calculated using the receiver-operating characteristic curve revealed that a 14.85% cut-off was predictive of clinical response with the highest sensitivity and specificity. The level of cytotoxicity did not vary amongst MSC preparations, because when we tested patients' PBMC against the MSC used for the infusion as compared to another preparation obtained from an unrelated donor, no difference in apoptosis induction could be detected (FIG. 12A). To further confirm the irrelevance of the specific MSC preparation, we evaluated the susceptibility of MSC sourced from different unrelated donors to be killed by 4 different mixed lymphocyte reaction (MLR) combinations. The proportion of apoptotic MSC was similar amongst the different MSC preparations when the same MLR was tested. Conversely, the cytotoxic activity against the same MSC varied amongst different MLR (FIG. 12, B).

[0164] Finally, we ruled out the possibility that different proportions of CD8+ and CD56+ cells could account for the differing cytotoxic activity because the average frequency in the PBMC of responders and non-responders was similar (FIGS. 12, C and D). Therefore, we conclude that the presence of activated cytotoxic cells in the recipient is predictive of MSC therapeutic activity.

EXAMPLE 4

MSC Apoptosis Induced by Cytotoxic Cells is the Result of a Bystander Effect

[0165] To define the mechanisms that drive apoptosis in MSC, we used in-vitro-activated PBMC as effector cells. We found that activated but not resting PBMC induced extensive early apoptosis (annexin-V+/7AAD−) in MSC (FIG. 4A), which peaked at 4 hours and shifted towards late apoptosis (annexin-V+/7AAD+) by 24 hours (FIG. 13, A). In accord with our in vivo observations (FIGS. 1, A and B), only activated PBMC induced caspase activation in MSC with a peak at 90 minutes, and this was completely abrogated by the pan-caspase inhibitor Z-VAD-FMK (FIG. 4B and FIG. 13B).

[0166] In order to identify the cells inducing apoptosis in MSC, we performed selective enrichment and depletion experiments amongst activated PBMC. We found that CD56+ natural killer (NK) and CD8+ populations were the only cells responsible for initiating MSC apoptosis (FIGS. 4, C and D). To characterize the mechanisms mediating MSC apoptosis induced by activated cytotoxic cells, we studied potential factors involved in caspase 3 activation. Inhibition of either Granzyme B (GrB) or perforin completely abolished the ability of activated PBMC to kill MSC (FIG. 4E) and activate caspase 3 (FIG. 13C). We also observed reduced PBMC mediated cytotoxicity when CD95 ligand (CD95L, also known as FasL or APO-1L) was neutralized (FIG. 4F), but not when Tumor Necrosis Factor-α (TNF-α) or TNF-related apoptosis-inducing ligand (TRAIL) were inhibited, even in the presence of very high concentrations of their respective inhibitors (FIG. 13D).

[0167] We then interrogated the nature of the MSC-cytotoxic cell interaction. We observed that apoptosis was not affected by the presence of anti-HLA class I- or anti-HLA class II neutralizing antibodies. Consistently, the cytotoxic activity of activated PBMC on autologous or allogeneic MSC did not differ (FIG. 4G). However, although PBMC required physical contact with MSC to induce apoptosis (FIG. 4H), blocking immunological synapse formation by inhibiting the polarization of microtubule organizing center (FIG. 4I) had no effect. These results demonstrate that MSC killing by activated cytotoxic cells is a bystander effect that does not involve the immunological synapse.

EXAMPLE 5

MSC Apoptosis does not Interfere with the Recognition of the Specific Target of Cytotoxic Cells

[0168] Having determined that the MSC apoptosis induced by cytotoxic cells is MHC-independent and not antigen-specific, we asked whether MSC could exert their immunosuppressive effects by competing with and antagonizing antigen-specific recognition. NY-ESO1-specific CD8+ T cell clone (4D8) or IL2-activated polyclonal CD56+ purified NK cells were used as effector cells against NY-ESO-1 peptide pulsed T2 or K562 cells, respectively. Two different sets of experiments were performed. In the first set, 4D8 or NK cells were tested against fixed numbers of putative (susceptible) target cells in the presence of escalating numbers of MSC used as a cold target. The alternative condition consisted of escalating the numbers of the putative target cells—now used as cold targets—in the presence of a fixed number of MSC then considered as the susceptible target. MSC did not compete with antigen-specific T cell cytotoxicity, since the killing of peptide-pulsed T2 cells was not affected by the presence of MSC (FIG. 5A). The same results were obtained using NK cells (FIG. 5B). In contrast, the presence of the putative target cells markedly reduced MSC killing in a dose dependent manner in both systems (FIGS. 5, C and D). Our data show that MSC killing does not interfere with the primary recognition of the cognate antigen.

EXAMPLE 6

Apoptotic MSC are Immunosuppressive in a Th2-Type Inflammation Model

[0169] Our data imply that, since MSC killing does not interfere with the primary recognition of the cognate antigen, induction of apoptosis must be prominently involved in the immunosuppressive activity.

[0170] Accordingly, in the GvHD model described, MSC apoptosis produced by recipient cytotoxic cells is required for immunosuppression. Therefore, we asked whether this causative relationship remains valid in a different disease model associated with non-cytotoxic Th2-type inflammation. We selected the model of ovalbumin (OVA)-induced allergic airway inflammation summarized in FIG. 14A.

[0171] OVA-induced airway inflammation was induced as previously described (Y. Riffo-Vasquez et al., 2012, Am 3. Respir. Cell Mol Biol 47, 245-252). Briefly, female Balb/C mice (Harlan Laboratories, Bicester, UK) were injected intraperitoneally with 30 μg of chicken egg albumin (OVA type V) (Sigma-Aldrich Company Ltd, Dorset, UK) on day 0 and 7. Controls received vehicle (aluminum hydroxide) only. On day 14, 15 and 16 animals were challenged with an aerosolized solution of OVA (3%) for 25 minutes. MSC or ApoMSC were injected 1 hour after the last challenge. After additional 18 hours, mice were terminally anaesthetized, a cannula inserted into the exposed trachea and three aliquots of sterile saline were injected into the lungs. The total number of cells in the lavage fluid was counted. For differential cell counts, cytospin preparations were stained with Diff Quick (DADE Behring, Germany) and cells counted using standard morphological criteria.

[0172] Although cytotoxic immune cells have been implicated in the induction of this condition, CD8+ and NK1.1+ cells infiltrating bronchoalveolar lavage (BAL) and lung tissues were less than 2% one hour after the last challenge, when MSC were infused (FIGS. 14, B, C, D and E). To confirm the absence of MSC killing, mice received luc-MSC to assess caspase activation after infusion and imaged one hour later. No caspase activation was detected in any of the mice (FIGS. 6, A and B).

[0173] High signal could be detected in all animals receiving control D-luciferin (FIGS. 14, F and G). The therapeutic activity, assessed by quantitating the eosinophil infiltration in the BAL showed no difference between MSC-treated and untreated mice (FIG. 6C). Together, these results indicate that also in this model MSC immunosuppression relies on the presence of recipient cytotoxic cells that mediate MSC apoptosis.

[0174] Therefore, we decided to test whether in vitro generated apoptotic MSC (apoMSC) could bypass the need of cytotoxic cells and ameliorate eosinophil infiltration.

[0175] Conditions under which a cell culture containing a significant (>30%) number of apopotic cells was investigated. MSCs were plated at a concentration of 5×10.sup.5 cells per well in a 96 round-bottom well plate in the presence of synthetic human GrB (5 μg/ml) (Enzo Life Sciences, Exeter, UK) with various concentrations of anti-FAS human (activating, clone CH11) for various times ranging from 15 minutes to 24 hours. After that, the percentage of apoptotic cells were determined by flow cytometry using annexin V staining. The results are summarised in FIG. 9.

[0176] Following this, ApoMSC were obtained for the test by plating 5×10.sup.5 cells per well in a 96 round-bottom well plate in the presence of synthetic human GrB (5 μg/ml) (Enzo Life Sciences, Exeter, UK) and anti-FAS human (activating, clone CH11) (10 μg/ml) (Merk Millipore, Watford, UK) for 24 hours in complete RPMI. The concentration of GrB and FasL was chosen to produce at least 80% of MSC apoptosis.

[0177] When apoMSC were administered to recipient mice a significant reduction of the eosinophil infiltration in BAL was observed (FIG. 6D).

EXAMPLE 7

Apoptotic MSC Infused in GvHD are Immunosuppressive and Induce IDO Production in Recipient Phagocytes

[0178] We subsequently investigated whether apoMSC could be immunosuppressive also in the GvHD model. ApoMSC were administered either intravenously (i.v.). or intraperitoneally (i.p.). and the infiltration of CD8+Vβ8.3+ Mh T cells was assessed and compared to untreated GvHD mice. ApoMSC produced a significant reduction in GvHD effector cell infiltration in both spleen and lungs, but this could only be observed in those mice treated with ApoMSC infused i.p. (FIGS. 7, A, B, C and D). It has been reported that the injection of irradiated thymocytes into animals results in their phagocytosis by recipient macrophages and induction of IDO.

[0179] We therefore tested whether apoMSC followed the same destiny by eliciting in vivo efferocytosis by recipient phagocytes and inducing IDO production. For this purpose, labelled apoMSC were traced in recipient phagocytes after injection.

[0180] Specifically, MSC were first labelled using CellTrace™ Violet labelling (ThermoFisher Scientific, Paisley, UK) at a final concentration of 5 μM and then made apoptotic (ApoMSC) as described above, using synthetic human GrB (5 μg/ml) and anti-FAS human (10 μg/m1) for 24 hours. 10×10.sup.6 labelled apoMSC were then injected i.p. or i.v. and mice sacrificed after 2 hours post-injection. Spleen, lungs, peritracheal, paratracheal, pericardial, mesenteric, periportal and celiac lymph nodes were collected and analysed by flow-cytometry. Positivity of CellTrace™ Violet was assessed as measure of ApoMSC engulfment in CD11b+ and CD11c+ gated subpopulations of phagocytic cells. Cells positive for the CellTrace™ Violet were then assessed for their expression of IDO.

[0181] Following i.p. administration, apoMSC were largely identified inside CD11b+ (FIG. 7, E) and CD11c+ (FIG. 7, F) phagocytes in the peritoneal draining lymph nodes (FIG. 7, E) but absent when searched for in the lungs and spleen. When the i.v. route was used, amongst the several phagocytic populations investigated, CD11bhighCD11cint, CD11bhighCD11c− and CD11b-CD11c+ were detected as engulfing apoMSC in lungs (FIGS. 7, G, H and I, respectively). The analysis of IDO expression in the phagocytes engulfing ApoMSC both in the i.v. and i.p. groups revealed that only the phagocytes in the i.p. group were able to increase IDO expression at a significantly higher level in comparison with their counterparts in untreated GvHD mice (FIGS. 7, J and K). These findings strongly suggest that the immunosuppressive effect of apoMSC involve recipient phagocytes and IDO as crucial effector mechanisms.

EXAMPLE 8

Recipient Derived IDO-Producing Phagocytes are Indispensable for MSC Immunosuppression in GvHD

[0182] To directly test the importance of recipient-derived phagocytes and recipient-produced IDO in MSC immunosuppressive activity, we depleted phagocytes and inhibited IDO activity in GvHD mice before MSC treatment and evaluated the effect of live MSC on the expansion of GvHD effectors. To deplete phagocytes, liposome clodronate (1 mg) was given to mice 72 hours before MSC injection. The treatment, dramatically impaired the ability of MSC to suppress Mh T cell infiltration (FIGS. 8, A and B).

[0183] Finally, animals were given the IDO inhibitor 1-methyl-D-tryptophan (1-DMT) (2 mg/ml) in the drinking water starting 6 days prior to MSC injection until the animals were sacrificed. Also in this case, the beneficial effect of MSC on Mh T cell infiltration was much reduced in mice receiving 1-DMT compared to controls (FIGS. 8, C and D). We therefore conclude that the immunosuppressive effect of MSC requires the presence of recipient phagocytic cells or IDO production.

EXAMPLE 9

[0184] PBMC from inflammatory bowel disease (IBD) patients (IBD, n=82) or healthy controls (HC, n=8) were co-cultured with MSC for 4 hours at a 20:1 PBMC:MSC ratio and apoptosis assessed by Annexin-V/7-AAD staining by flow cytometry. The results are shown in FIG. 16.

[0185] It is clear that in patients affected by IBD, there is a cohort of high and low killers. Similarly to what was observed for GvHD patients, the cut-off of 13% seems to distinguish the 2 cohorts of patients.

[0186] Further investigations into the molecular basis of the difference were carried out.

[0187] In particular MSC (5×10.sup.4) and patients PBMC (1×10.sup.6) were co-cultured for 24 hours, either with or without addition of a pan-caspase inhibibtor, Z-VAD-FMK (50 μM).

[0188] Supernatant was collected and PGE2 levels were measured by ELISA. Results are shown in FIG. 17.

[0189] These results show that after physical contact with patients' PBMC, MSC are induced to secrete high levels of prostaglandin E2 (PGE2). The induction of PGE2 is entirely dependent on caspase, because the addition of a caspase inhibitor in culture largely reduce PGE2 levels (FIG. 17A). Thus, this indicates that the activity correlates with the ability of PBMC to induce MSC apoptosis. The levels showed good correlation with the induction of annexinV+ cells in those samples (see FIG. 17B).

[0190] Thus PGE2 provides a good marker for the apoptosis inducing activity of PBMCs to MSCs which may be used as an alternative to annexinV+ cell detection. This marker has the advantage that it can be measured more simply and objectively by an independent operator, using for example a simple ELISA method.

Materials and Methods

[0191] This study aimed to verify whether MSC undergo apoptosis after infusion and to test the role played by MSC apoptosis in the initiation of recipient-derived tolerogenic immune response.

[0192] A mouse model of GvHD, in which the disease is mediated by the expansion and activation of Mh CD8+ cells in the recipient, was chosen for three important reasons: it recapitulates a minor mismatch between donor and recipient; T cells effecting GvHD can be precisely enumerated; there is proof-of-principle that MSC are effective in treating GvHD. Furthermore, human MSC were used in order to avoid the confounding effects of recipient cytokines on MSC immune-modulating function. In this system, murine inflammatory cytokines will not cross-react with the corresponding human receptors and will not activate immunosuppressive molecules in human MSC, whilst retaining the ability to expand murine effector cells mediating GvHD. Depletion of phagocytes and inhibition of IDO production were conceived as loss of function experiments to assess the requirement of these factors in the delivery of MSC apoptosis-dependent immunosuppression.

[0193] A mouse model of ovalbumin (OVA)-induced allergic airway inflammation was used to assess whether the causative relationship between cytotoxic cells and MSC apoptosis in the delivery of MSC immunosuppression is valid in a disease associated with Th2-type inflammation.

[0194] No randomization method was used. In all experiments, animals were randomly allocated to control or experimental groups. No blinding approach was adopted. No statistical method was used to predetermine sample size, which was estimated only on previous experience with assay sensitivity and the different animal models. Unless otherwise specified, three independent experimental replicates were performed.

[0195] To demonstrate that the presence of cytotoxic cells against MSC in GvHD patients could be predictive of MSC therapeutic activity, samples from GvHD patients were collected and tested for their ability to induce MSC apoptosis in a cytotoxic assay within 24 hours before MSC infusion. At the time of performing the assay and cytofluorimetric analysis, the operator was blind to patients' clinical details. All patients were affected by steroid-resistant GvHD and received MSC for compassionate use. PBMC from healthy donors were used as controls. All samples were collected after informed consent was obtained in accordance with the local ethics committee requirements.

MSC Preparations

[0196] Clinical grade BM-derived human MSC were generated from BM aspirates collected from the iliac crest of healthy donors. Briefly, 2 ml of BM aspirate were collected in a tube with 100 μl preservative-free heparin. The cells were plated within 24 hours at a density of 10-25 million/636 cm2 by using alpha modified Eagle's medium (ThermoFisher Scientific, Paisley, UK), conservative-free heparin (1 UI/ml) (Wockhardt UK Limited, Wrexham, UK) and 5% platelet lysate and then incubated for 3 days at 37 ° C. and 5% CO2 ambience. Non-adherent cells were discarded using phosphate buffered saline (ThermoFisher Scientific, Paisley, UK). When cell confluence of 90-100% was achieved cells were detached with Trypsin-EDTA (0.05% trypsin, 0.5 γmM EDTA.Math.4Na) (ThermoFisher Scientific, Paisley, UK) and reseeded at a density of 5000 cells/cm2. MSC were used at passage 2 for all in vivo experiments, whilst they were used by passage 8 for the in vitro experiments. In the latter case we did not observe any difference in terms of apoptosis susceptibility between different passages. Released criteria were based on positivity (>80%) for CD105, CD90, CD73, negativity (<2%) for CD3, CD14, CD19, CD31, CD45.

Mice and Disease Models

[0197] No randomization method was used. In all experiments animals were randomly allocated to control or experimental groups. No blinding approach was adopted.

Cell Preparations and Media

[0198] Cultures were carried out in complete RPMI 1640 medium containing GlutaMAX™, HEPES (25 mM), Penicillin 5000 U/ml and Streptomycin 5000 μg/ml (ThermoFisher Scientific, Paisley, UK), foetal bovine serum 10% (Labtech.com, Uckfield, UK).

[0199] Human peripheral blood samples from healthy donors were procured by the National Blood Service (Colindale, UK) as leukocyte cones. Samples from GvHD patients were collected within 24 hours before MSC injection. PBMC were isolated by density gradient separation on Histopaque-1077 (Sigma-Aldrich Company Ltd, Dorset, UK). mSpl were isolated through a cell strainer (BD Falcon, Oxford, UK), whilst lungs were cut into small pieces and incubated with Collagenase type IV (250 U/ml) (Lorne Laboratories, Reading, UK), DNAse I from bovine pancreas (250 U/ml) (Merk Millipor, Watford, UK) and foetal bovine serum 6.25% at 37° C. for 1 hour.

Imaging of MSC

[0200] Luc-MSC were transfected with the pGL3-Control vector containing the SV40 promoter for the expression of Luc+ (Promega, Southampton, UK) or with pECFPDEVDR-Venus (Addgene, Teddington, UK) using electroporation (Gene Pulser Xcell, BioRad, Kidlington, UK). Cells were suspended in a total volume of 250 μl of buffer and electroporated in 0.4 cm gap cuvettes using 10 μg of DNA at 250 volts and 950 F. When pECFP-DEVDR-Venus was used, the donor fluorophore pECFP and the acceptor Venus-YFP were linked through the flexible linker DEVDR which is recognized and cleaved by the active form of caspase 3. In this system caspase 3 activity can be monitored through the analysis of the Förster Resonance Energy Transfer (FRET) between pECFP and Venus-YFP. When caspase 3 is not active, the flexible linker DEVDR remains intact and energy transfer from pECFP is allowed with emission of YFP signal. Conversely, in the presence of caspase 3 activation DEVDR is cleaved, thus energy transfer is lost and the pECFP signal increases.

[0201] For confocal imaging, pECFP-DEVDR-Venus transfected MSC were plated in complete RPMI at a concentration of 1×10.sup.5 cells in a 30 mm×10 mm dish (Corning, Flintshire, UK) and let adhere overnight. The following day PHA-aPBMC were added at a PBMC:MSC ratio of 40/1. Where indicated, pan caspase inhibitor Z-VAD-FMK (50 μM), perforin inhibitor EGTA (4 mM), GrB inhibitor Z-AAD-CMK (300 μM) were used.

[0202] Living cell imaging was acquired every 3 minutes for 180 minutes using a Leica TCS SP5 II Confocal Microscope, with 488 nm and 407 nm lasers. The images were processed and analyzed by using the software “R” and EBImage package. In vivo imaging was performed injecting i.v. 1×10.sup.6 luc-MSC into naïve C57BL/6, BM or GvHD mice 3 days after the transplant in the GvHD model. In the airway inflammation model, luc-MSC were infused i.v. in naïve Balb/C or OVA-treated mice 1 hour after the last OVA challenge. After one additional hour, mice were anesthetized with isoflurane (1.5% isofluorane, 98.5% Oxygen), injected i.p. with 3 mg of VivoGlo™ Casp 3/7 Substrate Z-DEVD Aminoluciferine (Promega, Southampton, UK) and imaged using IVIS® Lumina III (PerkinElmer, Waltham, USA) system for a total time of 5 minutes. Images were analyzed by using the software “R” and EBlmage package to obtain mean TLS. Confirmation of the presence of transfected MSC was obtained injecting mice with VivoGlo™ Luciferin (Promega, Southampton, UK).

Pre-Activation of Human PBMC and Murine CD8+ Cells

[0203] PHA-aPBMC were obtained plating 5×10.sup.6 human PBMC in 24-well plate in the presence of PHA (5 μg/ml) (Sigma-Aldrich Company Ltd, Dorset, UK) in a final volume of 2 ml of complete RPMI for 72 hours. MLR-aPBMC were obtained using one-way MLR in which PBMC from one donor (stimulators) were irradiated (30 Gy) and cocultured with the PBMC of an unrelated donor (responder) at a stimulator:responder ratio of 1/1 in complete medium at a density of 0.75×10.sup.6 cells/cm2. Cells were then incubated at 37° C., 5% CO2 for 5 days. NK cells were purified by positively selecting CD56+ cells from healthy donor PBMC (Miltenyi Biotec Ltd, Bisley, UK) and activated with recombinant human-IL-2 (1000 U/ml). NY-ESO1-specific CD8+ T cell clone (Clone 4D8) was kindly supplied by Prof. Vincenzo Cerundolo (Institute of Molecular Medicine, Oxford university, UK). The clone was expanded in complete RPMI 1640 with Sodium Pyruvate (1 mM), 2-Mercaptoethanol (0.05 mM) (ThermoFisher Scientific, Paisley, UK), recombinant human-IL-2 (400 U/ml) (Peprotec EC Ltd, London, UK) and PHA (5 μg/ml) (Sigma-Aldrich Company Ltd, Dorset, UK).

[0204] Mh CD8+ were stimulated using the following protocol: 5×10.sup.6 purified CD8+ Mh cells were plated in 24-well plates in the presence of CD3/CD28-coated beads (Dynabeads®) (ThermoFisher Scientific, Paisley, UK) in a final volume of 2 ml of complete RPMI and incubated for 72 hours.

Immunosuppressive assay

[0205] Serial dilutions of human MSC were plated in a flat bottom 96-well plate and let adhere overnight in 100 μl of complete RPMI. Where indicated, MSC cultures were exposed to human Interferon-γ (hIFN-γ) and human TNF-α (hTNF-α), murine IFN-γ (mIFN-γ) and murine TNF-α (mTNF-α) (20 ng/ml each) (all cytokines were from Peprotec EC Ltd, London, UK), supernatant from PHA-aPBMC or from ConA-aSpl. The following day, 5×10.sup.5 Balb/C mSpl were labelled with Carboxyfluorescein Diacetate Succinimidyl Ester dye (ThermoFisher Scientific, Paisley, UK) and plated with MSC at escalating MSC/mSpl ratios. Culture controls consisted of mSpl plated without MSC in the presence (positive control) or in the absence of ConA (negative control). Proliferation of mSpl was then assessed by flow-cytometry after 72 hours and expressed as the percentage of the proliferation obtained at each MSC/mSpl dilution in comparison with the one obtained in the positive control culture. Results were expressed as percentage of inhibition.

Cytotoxic Assay

[0206] 1×10.sup.5 MSC were plated overnight in a total volume of 500 μl. The day after preactivated immune cells were plated at different concentrations (2.5 to 40/1 effector:MSC ratios). MSC apoptosis was then tested at different time points using flow-cytometry or confocal microscopy analysis. Eventually, the assay was performed for 4 hours in the vast majority of the cases. At flow-cytometry MSC were identified as CD45− cells.

[0207] Antigen-specific cytotoxic activity of clone 4D8 was tested using T2 cells pulsed with NY-ESO-1 antigen (epitope SLLMWITQC) at a concentration of 0.1 μM for 1 hour. In the competition assay, T2 (from Hans Stauss, University College London) and K562 cells (from Junia Melo, Imperial College London) were discriminated from effector cells by CellTrace™ Violet labelling. The tracer concentration was optimized for the T2 (1 μM) and K562 (2.5 μM) cells. Cell lines were tested for mycoplasma contamination before use.

[0208] When flow-cytometry was used, the level of apoptosis was assessed using the PE annexin-V apoptosis detection kit (BD Biosciences, Oxford, UK). Unless specified, apoptotic cells were identified as annexin-V+/7-AAD− cells.

Inhibitors

[0209] Where indicated, cultures were supplemented with pan-caspase inhibitor Z-VAD-FMK (10 μM in the flow-cytometry experiments or 50 μM in the living cell confocalexperiments) (R&D System, Oxon, UK), perforin inhibitor EGTA (4 mM) (Sigma-Aldrich Company Ltd, Dorset, UK), GrB inhibitor Z-AAD-CMK (300 μM) (Merk Millipor, Watford, UK), neutralizing antibodies against HLA-DR (clone L243) (50 μg/ml), human HLA-A,B,C (clone W6/32) (100 μg/ml) (BD Biosciences, Oxford, UK), TNF-α antagonist Etanercept (Enbrel®) (10 μg/ml or 100 μg/ml) (Amgen, Cambridge, UK). Each reagent was incubated with MSC 1 hour before the culture with effector killer cells. In all cases, the concentration of the corresponding inhibitor was kept for the duration of the cytotoxic assay.

[0210] The neutralizing anti-CD178 (Clone NOK-1) (10 μg/ml or 100 μg/ml) (BD Biosciences, Oxford, UK), anti-TRAIL (clone 2E2) (10 μg/ml or 100 μg/ml) (Enzo Life Sciences, Exeter, UK) antibodies, MYR Protein Kinase-Cζ Pseudosubstrate (PKCζ-PS) (10 μM, 25 μM or 75 μM) (ThermoFisher Scientific, Paisley, UK) and Etanercept (10 μg/ml or 100 μg/ml) were incubated with effector killer cells for 2 hours before the cultures with MSC. In all cases, the concentration of the corresponding inhibitor was kept for the duration of the cytotoxic assay.

Flow-Cytometry

[0211] The following antibodies specific for murine molecules were used: anti-CD45 (FITC, Clone 30-F11) (eBiosciences Ltd, Hatfield, UK), anti-Vβ8.3 (FITC, Clone 1B3.3), antiCD8 (APC, Clone 53-6.7), antiCD4 (PE, Clone H129.19), anti-CD19 (APC-H7, Clone 1D3), anti-NK1.1 (PerCP-Cy5.5, Clone PK136) (BD Biosciences, Oxford, UK), anti-CD11b (PerCP-Cy5.5, clone M1/70), anti-CD11c (APC-Cy7, clone n418), anti-Ido1 (Alexa Fluo647, clone 2e2) (BioLegend, London, UK). For human specific molecules, the following antibodies were used: anti-CD45 (FITC, clone 2D1), anti-CD8 (APC, Clone SK1), anti-CD4 (PE, Clone SK3), anti-CD11b (PerCP-Cy5.5, clone M1/70), anti-CD56 (FITC, clone HCD56) (BD Biosciences, Oxford, UK). All samples were acquired using BD FACS Canto II using the software FACS Diva and analyzed with Flow-jo software. FRET and Caspase activity (CAf) were assessed by flow-cytometry as previously described (He et al. Am 3. Pathol 164, 1901-1913 (2004)).

Real Time Quantitative PCR

[0212] MSC RNA was obtained from TRIzol® (ThermoFisher Scientific, Paisley, UK) lysates and extracted using RNeasy Mini Kit (Qiagen, Manchester, UK). Real Time quantitative PCR (qRT-PCR) was performed following TaqMan® RNA-to-CT™ 1-Step Kit instructions (ThermoFisher Scientific, Paisley, UK), using 20 ng of RNA template per reaction. Assays were carried out in duplicates on an StepOnePlus® PCR system thermal cycler (Applied Biosystem, UK) using TaqMan primers (all purchased from ThermoFisher Scientific, Paisley, UK). The human primers used were the following: IDO2 (Hs01589373_m1), TSG6 (Hs01113602_m1) and PTSG2 (Hs00153133_m1) and HPRT1 (Hs02800695_m1) as housekeeping gene. Data were then analysed using StepOne™ software version 2.1 and relative quantification obtained with ΔΔCt method, considering untreated MSC as reference.

Statistics

[0213] Results were expressed as mean±SD. The unpaired Student t test was performed to compare 2 mean values. One-way ANOVA and Tukey's Multiple Comparison test was used to compare 3 or more mean values. Probability of null hypothesis less than 5% (p>0.05, two-sided) was considered statistically significant. No statistical methods were used to predetermine sample size, which was estimated only on previous experience with assay sensitivity and the different animal models.

TABLE-US-00001 TABLE 1 Clinical features of GvHD patients Concomitant MSC Donor Organs therapy for Dose Re- Diagnosis type GvHD Grade involved GvHD (×10.sup.6/Kg) sponse DLBCL SIB Late 3 skin, Steroid, MMF, 1.60 NR onset liver Infliximab CLL SIB Late 4 gut Steroid, MMF, 2.80 R onset Infliximab, Alemtuzumab HL VUD Acute 3 skin, Gut Steroid  3.00.sup.†  NR.sup.†  7.40.sup.‡  R.sup.‡ CML VUD Late 3 Skin, MMF 3.00 NR onset* Liver AML VUD Chronic* N/A Skin Steroid, CSA 2.70 NR AML SIB Acute 3 Gut Steroid, CSA 2.10 R CML VUD Acute* 4 Gut Steroid, CSA 2.90 R AML VUD Acute 4 Skin, gut Steroid, CSA 3.10 NR FL SIB Acute 4 Skin, Steroid, CSA, 1.60 R gut, MMF Liver MM SIB Acute 4 Gut Steroid, 2.10 NR Infliximab AML VUD Acute 4 Gut Steroids, 1.28 NR Budenofalk, CsA pre-B UUD Acute 4 Skin Steroids, Topic 1.03 NR ALL glucorticoids MDS/ VUD Acute 4 Gut Steroids, 1.55 NR RAEB-2 Tacrolimus, MMF, Etanercept, Ruxolitinib, MTX, Alemtuzumab, CSA Mixed VUD Acute 4 Gut Steroids, CSA 1.33 NR AML/ T-ALL MM VUD Acute 3 Gut Steroids, CSA 1.01 NR B-ALL, SIB Acute 3 Skin, Steroids, ECP, 1.11 NR BCRABL+ Liver CSA .sup.†first dose .sup.‡second dose *GvHD post-Donor Lymphocyte Infusion AML: Acute Myeloid Leukemia; CML: Chronic Myeloid Leukemia; CLL: Chronic Lymphocytic Leukemia; CSA: Cyclosporine; DLBCL: Diffuse Large B-Cell Lymphoma; FL: Follicular Lymphoma; HL: Hodgkin Lymphoma; NR: no response; MM: Multiple Myeloma; MMF: Mycophenolate Mofetil; R: response; SIB: HLA-identical sibling; VUD: Volunteer Unrelated Donor.