Autologous transplant for autoimmune disease: optimizing the regulatory T cells

S42

Poster abstracts

our control population, we evaluated Treg reconstitution post HSCT in patients who did not receive IL-2. Nineteen patients (11 matched sibling, 8 alternative donor) have been treated with ULD IL-2 post HSCT (100,000200,000 IU/m2 3
weekly SC), starting before day 30 and continuing for 612 weeks. Median age at time of HSCT was 14 years (range, 6-62). All patients demonstrated an increased percentage of CD4+CD25+FoxP3+ Tregs by flow cytometry from a mean of 4.8% (range, 0-11.0%) pre IL-2 to 11.1% (range, 1.2-31.1%) following 6 weeks of therapy. Compared to controls, a significant rise in mean Treg percentage was found at 1 month post HSCT in IL-2 recipients (10.2 vs. 5.8%, p¼0.02), which remained elevated at 3 months (9.8 vs. 7.1%, p¼0.1). Functional analyses of CD4+CD25bright cells demonstrated suppression of mixed lymphocyte reactions. No grade 3 or 4 toxicities were associated with ULD IL-2. No patients on study developed grade III-IV acute GvHD, compared to 4/33 (12%) of controls. IL-2 recipients retained T cells reactive to viral and tumor antigens, and only 16% of study patients versus 45% of controls developed viral infections. Hence, ULD IL-2 is well-tolerated, expands a CD4+CD25+FoxP3+ Treg population in vivo, and may be associated with lower incidence of GvHD and viral infections. 141 ONE YEAR MULTIPLE INJECTIONS OF ANTIGEN SPECIFIC T REGULATORY LYMPHOCYTES IN REFRACTORY CROHN’S DISEASE PATIENTS, EXTENSION OF CATS1 STUDY P Desreumaux1, M Allez2, L Beaugerie3, X Hebuterne4, Y Bouhnik5, M Nachury6, V Brun7, A Duchange8, N Clerget-Chossat7, A Foussat7, M Forte7, J Colombel1 1 Huriez Hospital, Lille, France, 2St-Louis Hospital, Paris, France, 3St-Antoine Hospital, Paris, France, 4L’Archet Hospital, Nice, France, 5Beaujon Hospital, Clichy, France, 6Minjoz Hospital, Besançon, France, 7TxCell, Valbonne, France, 8Effi-Stat, Paris, France Introduction: CATS1 was an open label phase I/II study with Ovasave, an antigen-specific Treg therapy of Crohn’s Disease (CD). This study was extended to assess the tolerability of several injections for patients who benefited from the initial injection. Methods: CATS1 extension included patients with refractory CD, active inflammation. Ovasave was produced ex vivo from patients’ PBMC exposed to ovalbumin, followed by cell cloning and expansion using feeder cells prior to formulation for infusion. Safety was assessed by clinical and laboratory parameters and efficacy by CDAI. Ovasave’s impact on the proliferative response of patients’ PBMC to ovalbumin was assessed in vitro. Results: Seven patients received a 2nd injection and 2 were treated for up to 1 year. Mean age was 34.5; disease was extensive (duration 12.9 years) with high baseline CDAI; Patients were refractory to standard therapies and 6/7 to immunosuppressors and anti-TNFa. Ovasave was well tolerated with 17 adverse events (AE) post-second injection: 16 not related; 1 related (recovered) and 3 serious AE not related. Upon re-injection of 2 patients who showed immunogenicity to feeder cells after the 1st highest dose injection 1 presented a mild acute reaction and the other was successfully treated with the induction of tolerance protocol. Two patients showed a perceived sustained benefit and were treated for up to 1 year, receiving a total of 4 and 5 injections of 10e6 cells (best dose), respectively with good tolerability. Reduction of in vitro PBMC proliferation to ovalbumin was only observed at the 10e6 cell dose. Conclusion: CATS1 extension indicated good tolerability and patient benefit of multiple injections for up to one year. These data together with the existing ability to produce several years of treatment from a single patient batch are supportive of planned studies to assess chronic treatment of refractory CD with antigen specific Treg cells. 142 NOTCH1 MODULATES MSC-MEDIATED REGULATORY T CELL INDUCTION M Di Ianni1, B Del Papa2, P Sportoletti2, S Baldoni2, D Cecchini2, F Falzetti2 1 University of L’Aquila, L’Aquila, Italy, 2University of Perugia, Perugia, Italy Notch1 signalling has been implicated in regulatory T (Treg) cell differentiation. We previously demonstrated that, when co-cultured with CD3+ cells, mesenchymal stem cells (MSCs) induced a T-cell population with a regulatory phenotype. Here we investigated the molecular mechanism

underlying MSC induction of human Treg cells. MSCs expressed the Notch1 ligands Jagged1, Jagged2, DLL 1,3-4 mRNAs, with Jagged1 being prevalent. After a 4-day CD4+ cell co-culture with MSCs, Notch1 and HES1 mRNA expression increased 2.070.94 fold and 4.652.7 fold respectively, while FBW7 decreased by up to 0.50.21 fold. Western blot analysis revealed significant over-expression of the Notch1 intracellular domain (IC) and the transmembrane/cytoplasmic portion. Compared with CD25 negative cells (non-Treg cells) the Notch1 signalling was upregulated in the Treg cells, expression of FOXP3 mRNA doubled, Notch1 and HES1 were up-regulated and FBW7 was significantly down-regulated, suggesting the MSC effects are specific to Treg cells. Inhibition of Notch1 signalling through GSI-I or the Notch1 neutralizing antibody reduced expression of HES1 and FoxP3, down-regulated Notch1-IC and TM expression and reduced the percentage of MSC-induced CD4+CD25highFOXP3+ cells in vitro. The suppressive capacity of CD25 enriched Treg cells was significantly reduced after GSI-I and anti-Notch1 treatments (51% 7 and 49.7%12.4 Vs 83%18 untreated cells). Moreover, we demonstrate that FOXP3 is a downstream target of Notch signalling in human cells. No cross-talk between Notch1 and TGF-b signalling pathways was observed in our experimental system. These findings indicate that activation of the Notch1 pathway is a novel mechanism in the human Treg-cell induction mediated by MSCs, ensuring sustained FOXP3 expression and more stable Treg-cell phenotypes. 143 PEDIATRIC THYMIC TISSUE AS A SOURCE OF CD25+FOXP3+ REGULATORY T CELLS (TREGS) FOR CELLULAR THERAPY E Dijke1,2, M Levings3, A McMurchy3, I Larsen1,2, I Rebeyka1,2, D Ross1,2, L West1,2 1 University of Alberta, Edmonton, AB, Canada, 2Alberta Transplant Institute, Edmonton, AB, Canada, 3University of British Columbia, Vancouver, BC, Canada Introduction: Major challenges for development of cellular therapy using Tregs include difficulties isolating pure Tregs populations and expanding these cells to clinically relevant numbers. During infant cardiac surgery, thymectomy is typically performed to gain exposure of the retrosternal operative field. Discarded thymuses may represent a rich source of naïve Tregs. Evidence that therapeutic Tregs do not need to be HLA-identical to the recipient led us to investigate the potential of thymic tissue as a source of Tregs for “off-the-shelf” cellular therapy. Methodology: Thymuses (n¼8) were obtained during pediatric cardiac surgery; thymocytes were recovered through mechanical dissociation. FOXP3+ cells were isolated by automated magnetic cell separation of CD25+ thymocytes and expanded with anti-CD3, IL-2, rapamycin and artificial antigenpresenting cells; CD25-depleted cells were controls. FOXP3 and intracellular cytokine staining were performed to define their phenotype. Suppressive capacity was determined by co-culturing expanded cells with anti-CD3/CD28stimulated autologous and allogeneic peripheral blood mononuclear cells (PBMC) and analyzing proliferative responses by Cell Proliferation ELISA. Results: Large quantities of thymocytes were isolated from thymic tissue (median: 7
109; range: 2-15
109); 2.2 to 3.2% of these thymocytes were FOXP3+CD25+ cells. Isolated CD25+ cells were 72% positive for FOXP3 (median, range: 60-81%). After two weeks culture, we observed 4 to 48-fold expansion of CD25+ cells with >95% viability (control cells: 0.4 to 25-fold expansion with 49-88% viability). Expanded CD25+ cells were more than 90% FOXP3+ and produced no IL-2 or IFN-g (controls: <14% FOXP3+ and 58-65% produced IFN-g). Moreover, in contrast to controls, expanded CD25+FOXP3+ cells efficiently suppressed both autologous and allogeneic proliferating PBMC more than 50% even at a 1:10 ratio of Tregs:PBMC. Conclusion: Expanded CD25+FOXP3+ Tregs isolated from pediatric thymic tissue strongly suppressed proliferation of both autologous and allogeneic PBMC. Our results indicate that explanted thymuses are a potential source of Tregs for “off-the-shelf” cellular therapy. 144 AUTOLOGOUS TRANSPLANT FOR AUTOIMMUNE DISEASE: OPTIMIZING THE REGULATORY T CELLS TD Prestidge1, JH Davis2, J Huang3, M Himmel1, M Levings3 1 Starship Children’s Hospital, Auckland, New Zealand, 2BC Children’s Hospital, Vancouver, BC, Canada, 3Child and Family Research Institute, Vancouver, BC, Canada

19th Annual ISCT Meeting

Backgound: Deficiencies in regulatory T cells (Tregs) have been implicated in the physiology of autoimmune disease (AID). Autologous transplant can ameliorate AID, a proposed mechanism is optimization of Tregs. We sought to develop a conditioning protocol specifically to improve Tregs and therefore clinical outcome using post-transplant cyclophosphamide. Method: An 18yr old with uncontrolled Crohn disease was mobilized with cyclophosphamide and granulocyte colony stimulating factor. Conditioning was with rabbit anti-thymocyte globulin 5 mg/kg days -11, -10, -9 and -8; cyclophosphamide 50 mg/kg days -7 and -6 and at days +3 and +4; fludarabine 30 mg/kg days -5, -4, -3, -2 and -1; . Lymphocyte analysis was performed at days -11, +30, 45, 60 and 90. Results: Mobilization and conditioning were well tolerated. Opiates were required for an infected wisdom tooth. No other grade III or IV toxicities. Tregs (as %age of CD4cells) pre-transplant were below matched control at 0.87%. Post transplant a relative expansion of Tregs to 14.8% on day +45, 4.73% on day +90 and 2.04% by day +120 was observed. Clinically she entered a rapid remission and has remained well, off all therapy 2 years post transplant. Conclusions: Treg recovery post transplant is variable but correlates with improvement in autoimmune disease in retrospective analysis. Post-transplant cyclophosphamide has been studied in allogeneic transplant to ameliorate graft-versus-host-disease and is associated with improvement in Treg reconstitution. To date there are no reports of its use in autologous transplant but this case supports the feasibility and its association with optimized Treg recovery and clinical effectiveness. Treg recovery as %age of CD4 cells

Patient

Fold Change cf. Pre-Transplant

Age Matched

Pre-

Control

Transplant

Day +45

Day +60

Day +120

Day +45

Day +60

Day +120

FOXP3+ CD25+

1.40

0.87

14.80

4.73

2.07

17.01

5.44

2.38

FOXP3+

0.68

0.42

7.64

2.33

0.97

18.14

5.53

2.31

FOXP3+ CD45RA+

0.42

0.30

5.76

0.67

0.60

19.20

2.23

2.00

FOXP3+ CD45RA-

1.85

1.18

29.50

8.68

2.47

25.00

7.36

2.09

CD4+ve Cells

CD25+ CD127low

145 IMPACT OF MTOR INHIBITOR, EVEROLIMUS ON INDUCED REGULATORY T CELLS DERIVED FROM CORD BLOOD T Nagamura-Inoue1, Y Yamamoto1, S Kobayashi2, M Yuzawa1, H He2, H Tsunoda3, A Tojo4 1 Dep. of Cell Processing and Tranfusion, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan, 2Dep. of Hematology/Oncology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan, 3Department of Obstetrics, NTT Kanto Hospital, Tokyo, Japan, 4Dep. of Cell Processing and Tranfusion, and Dep. of Hematology/Oncology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan Regulatory T cells (Tregs) play an important role in immune-tolerance to allograft. Unbalance between Tregs and effector T cells is involved in graftversus-host disease (GvHD) and other autoimmune disorders. Cord blood (CB) is rich in naïve T cells and is a promising source of inducible Tregs (iTregs). Here we studied the impact of mTOR inhibitors, rapamycin (Rap) and everolimus (Eve), on ex vivo expansion of iTregs. Isolated CB-CD4+ T cells were cultured in an anti-CD3/CD28 monoclonal antibodies-coated flask with IL-2 and TGF-b in the presence or absence of Rap or Eve. Both Rap and Eve significantly increased the incidence of CD25+Foxp3+ Tregs on in CD4+ T cells. But Rap or Eve in the absence of TGF-b did not induce Tregs. Furthermore, the expression of CD26 on CD4+ T cells was inversely correlated to Foxp3 expression and significantly down-regulated by TGFb with or without mTOR inhibitor. The iTreg-rich population cultured with IL2/ TGFb, IL2/TGFb/Rap, and IL2/TGFb/Eve, but not IL2 alone, efficiently inhibited MLR triggered by allogeneic DCs. These iTregs were also active in MLR using allogeneic responder T cells. Interestingly, IL2/Eve-treated CBCD4+ T cells also inhibited MLR, irrespective of the low or moderate iTreg ratio. Interestingly, mTOR Inhibitor, Eve is an efficient co-inducer in TGF-biTregs from CB-CD4+Cells, although the direct mechanism of mTOR inhibitors on iTreg induction remains to be elucidated.

S43

146 DEVELOPMENT OF POLYAMIDE-GELATIN COMPOSITE SCAFFOLD FOR DELIVERY OF ENDOMETRIAL MESENCHYMAL STROMAL CELLS FOR THE TREATMENT OF PELVIC ORGAN PROLAPSE K Su1, S Edwards1, J White1, D Ulrich2,3, KS Tan2,3, J Ramshaw1, C Gargett2,3, J Werkmeister1 1 CSIRO Materials Science and Engineering, Clayton, Australia, 2Monash Institute of Medical Research, Clayton, Australia, 3Monash University Department of Obstetrics and Gynaecology, Clayton, Australia Pelvic organ prolapse (POP) is the herniation of bladder, bowel and/or uterus into the vagina causing urinary incontinence and sexual dysfunction. The outcome of POP reconstructive surgery treatment is poor and the use of synthetic polypropylene meshes has caused further complications. Our aim is to produce a tissue engineering construct using Endometrial Mesenchymal Stromal Cells (eMSCs) from the highly regenerative uterine lining to be delivered in new scaffold designs for effective treatment of POP. We have developed a warp-knitted polyamide (PA) mesh-gelatin composite (PA+G) scaffold1. eMSCs were isolated from endometrial biopsy, with minimum pain and morbidity. The cells were retrieved from collagenase digests and magnetic bead sorting using the novel W5C5 marker2. Passage 3 W5C5+ eMSCs were cultured onto PA scaffolds that had been coated in gelatin (5% - 12%) with varying degrees of cross linking with glutaraldehyde (0.025%, 0.05%, 2%). The growth rate of eMSC was examined by MTS assay. The optimal concentration of gelatin was 10% by optical observation of PA+G scaffolds. The growth rate of W5C5+ cells on PA+G scaffolds was almost twice that on PA constructs. A 0.025% glutaraldehyde concentration was sufficient to allow accelerated gelatin breakdown over 7 days with minimal cytotoxicity. eMSC phenotype was validated over time. Induced differentiation into smooth muscle cells (SMCs) in medium containing TGFb/PDGF-BB and new tissue formation was evaluated for 4 weeks. Immunofluorescence for SM22a and SMMHC showed that W5C5+ eMSCs differentiated into SMCs, necessary for new tissue formation. In summary, PA+G mesh is an appropriate seedbed for W5C5+ eMSC adhesion, proliferation and differentiation, indicating the biocompatibility of this new scaffold. This tissue engineering construct may be suitable for repairing the vaginal endopelvic fascia to treat POP. 1. Ulrich et al. (2012). PLoS ONE 7(11): e50044. 2. Rajaraman et al. (2012). Tissue Engineering Part C: Methods DOI:10. 1089/ten.tec.2011.0718.

147 AN APPROACH TOWARDS THE PROLIFERATION AND DIFFERENTIATION OF STEM CELLS ON CHITOSAN BASED SOLUBLE POLYMER T Debnath1, LK Chelluri1, S Sultana Beevi1, V Verma1, P Usha Shalini1, K Lakshmi2, KS Ratnakar3, K Ravindranath3 1 Transplant Biology & Stem Cell lab, Global Hospitals, Hyderabad, India, 2Department of Gasteroenterology & Bariatric surgery, Global Hospitals, Hyderabad, India, 3Global Hospitals, Hyderabad, India Mesenchymal stem cells (MSCs) derived from various tissue sources were characterized by their surface marker expression using Flow cytometry. The tri-lineage potential of MSC was determined by chondrogenesis, osteogenesis and adipogenesis. MSC are assumed to possess differential capabilities on synthetic bio-polymers like hydrogels. Hydrogels are highly hydrated Chitosan-based polymer described to be maintained as a soluble polymer. The study addresses impact of inducing factors such as TGF-b1 and BMP-2 on chondrogenesis of stem cells from tissues of various sources on hydrogel. The parameter under study includes viability, proliferation, cytotoxicity (MTT Assay) of stromal cells on the hydrogel. The rate of proliferation of MSCs was studied by their growth kinetics. The morphological evaluation (spindle shape) was done using geimsa staining and their potential viability by the formation of JC-1 aggregates. Histological staining indicated glycosamines in chondrocytes by alcian blue staining. MTT Assay revealed minimal cells (8000cells/well) required for significant proliferation (Stimulation index >¼2). Cytotoxixicty gradually decreased as cell number increased from 8000 cells/well and above. Thus, it was observed that these synthetic biomaterials have minimal impact on the study parameters and their cross linking structure provides a favourable matrix for cell growth and tissue regeneration.