Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

Regulatory T Cells and GVHD

1,959 views

Published on

Adoptive Therapy with Regulatory T-Cells for Prevention of GVHD. Simrit Parmar.

Published in: Education
  • Be the first to comment

Regulatory T Cells and GVHD

  1. 1. Adoptive Therapy with Regulatory T-Cells for Prevention of Graft versus Host Disease
  2. 2. Regulatory T cell CD127lo
  3. 3. Busy Life of Tregs
  4. 4. G V H D
  5. 5. In vivo tracking of light emitting donor cells Allogeneic HCT B T M BM BM BM B T Bone Marrow Splenocytes FVB/N WT luc+ Balb/c H-2q/Thy1.1H-2d/Thy1.2 CD4+ CD8+ B220+ NK1.1+ Gr-1/Mac-1+ 2x105 cells/well Absolute light emission 0.00 0.05 0.10 0.15 Luciferase 2A eGFPbAct luc+ reporter mouse
  6. 6. Acute Graft-vs-Host Disease Development Beilhack, A. et al. Blood. 2005. 106:1113
  7. 7. d15 Death from GVHD 100 5000 1000 20000 1 10 100 1000 10000 0 20 40 60 0 20 40 600 20 40 60 Time [d] post BMT RelativeSignalIntensity 0 25 50 75 100 0 20 40 60 Time [d] post BMT Survival[%] TCD BM only, n = 14 TCD BM + Tcon, n = 15 TCD BM + Tcon + Treg n = 9 Control of GVHD with Retention of GVL TconBM only Tcon + Treg 500 5000 d5 Edinger et al. Nature Medicine 9:1144, 2003
  8. 8. Tregs and GVHD • Allogeneic HCT recipients with aGVHD had Treg frequencies 40% less than those without aGVHD. • Treg frequencies decreased linearly with acute GVHD severity • The frequency of Tregs at acute GVHD onset predicted response to therapy. Magenau et al. BBMT. 2010.
  9. 9. Magenau et al. BBMT. 2010. 38% 63% Circulating Tregs predict OS Non Relapse Mortality Overall Survival
  10. 10. NO POST-SCT IST
  11. 11. Outcomes – U. of Perugia • Deaths: 12/26 • Regimen Related Toxicities: – Veno-occlusive disease (3) – Multi-organ failure (1) • Acute GVHD grade III-IV (2) • Serious infections (7) • Relapse (AML 1) D’Ianni et al. Blood 2011
  12. 12. • N=43 • High Risk AML =33; ALL = 10 • Conditioning: TBI based regimen • Grafts included - CD34+ cells= 9.7 × 106/kg - Tregs= 2.5 × 106/kg) - Tcons=1.1 × 106/kg • NO post-transplant immunosuppression was given • Full-donor type engraftment = 95% • ≥grade 2 acute GVHD =15% • DFS @ median FU 46 mos = 56% • Cumulative incidence of relapse = 5% Blood 2014 Jul 24;124(4):638-44
  13. 13. MDACC EXPERIENCE
  14. 14. 4. Magnetic Activated Cell Sorting, MidiMACS 3. Anti-CD25 microbeads (CD25 Reagent, MiltenyiBiotec) 1. CB unit 5a. CD25 positive cells “Treg”. Cultured at 1x106 cell/ml in T-cell expansion medium. IL-2 @ 200 u/ml Initiated with *CD3/28 beads at 3 beads: 1 cell ratio. + - 6. Cellularity monitored every 2 days& re-adjusted to ~1x106 cell/ml (as required) IL-2 added to maintain 200 u/ml. (No new beads added) 7. Flow cytometry: to reveal CD3/4/8/25 (19/56) and FoxP3 and CTLA4 5b. CD25 negative cells “Non-Treg”. Cultured at 1x106CD3+cell/mlin T-cell expansion medium. IL-2 @ 200 u/ml Initiated with *CD3/28 beads at 3 beads: 1 cell ratio. Current CB “Treg” ex vivo expansion technique 2. Ficoll to isolate CB MNC 8. Functional in vitro assay: Mixed Lymphocyte Reaction (MLR) Notes: (a) IL-2: Proleukin®, Chiron Corp., Emeryville, CA. (b) *CD3/28 beads: (i) Carl H. June MD (University of Pennsylvania), or (ii) invitrogen Dynabeads® ClinExVivoTM CD3/CD28 (c) T-cell expansion medium: RPMI, 5% heat inactivated human serum, antibiotics and L-glutamine (2mM)
  15. 15. Day 7 Day 0 Day 0 Day 7 CD25 Positive Fraction CD25 Negative Fraction
  16. 16. Expanded CB Tregs are Functional Vβ 1.0 Vβ 2.0 Vβ 3.0 Vβ 4.0 Vβ 5.1 Vβ 5.2 Vβ 6.0 Vβ 7.0 Vβ 8.0 Vβ 9.0 Vβ 11.0 Vβ 12.0 Vβ 13.0 Vβ 14.0 Vβ 15.0 Vβ 16.0 Vβ 17.0 Vβ 18.0 Vβ 20.0 Vβ 21.0 Vβ 22.0 Vβ 23.0 Vβ 24.0 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 Countsperminute CD25 FOXP3 CD4 CD127 CD25 94.9% 96.79% 97.59% 95.49% CD4
  17. 17. Successful ex vivo expansion of UCB Tregs
  18. 18. Ex vivo Expanded CB Tregs Maintain Polyclonal Vβ Repertoire
  19. 19. BASELINE FLOW (WHOLE CB)
  20. 20. Expanded CB Tregs Phenotype: CD4+CD25+FOXP3+CD127lo
  21. 21. Expanded CB Tregs: CD4+CD25+FOXP3+CD127lo
  22. 22. DeMethylation status of FOXP3 gene regions represents expanded CB Treg Baron et al, EMBO
  23. 23. Expanded CB Tregs show FOXP3 Demethylation
  24. 24. FOXP3 demethylation correlates with CB Treg Phenotype and Function 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 percentgatedlymphocytes 0 5000 10000 15000 20000 25000 D1+D2 D1+D2+TREG(1:1) CPM 0.00 20.00 40.00 60.00 80.00 100.00 120.00 FOXP3 (7-1) 2CpG FOXP3 (9-1) 2CpG FOXP3 (10-1) 2CpG FOXP3 (11-1) 2CpG Tregs Baseline Methylation% N=3
  25. 25. 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 DONOR 1 DONOR 2 TREG D1+D2 D1+TREG D2+TREG 10:1 1:1 1:10 CB Tregs significantly suppresses 2-way Allogeneic MLR cpm
  26. 26. 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 DONOR 1 DONOR 2 TREG D1+D2 D1+TREG D2+TREG 10:1 1:1 1:10 cpm CB Tregs significantly suppresses 2-way Allogeneic MLR
  27. 27. 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 DONOR 1 DONOR 2 TREG D1+D2 D1+TREG D2+TREG 10:1 1:1 1:10 cpm CB Tregs significantly suppresses 2-way Allogeneic MLR
  28. 28. 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 DONOR 1 DONOR 2 TREG D1+D2 D1+TREG D2+TREG 10:1 1:1 1:10 cpm Treg:MNC ratio CB Tregs significantly suppresses 2-way Allogeneic MLR
  29. 29. Expanded CB Tregs Suppress Two-Way Allogeneic MLR
  30. 30. Xenogenic GVHD Mouse Model
  31. 31. Xenogenic GVHD Mouse Model Lymphoid follicle Extra- medullary hematopoeisis mixed with histiocytes Spleen Small Intestine 400x (Apoptosis with Enteritis) Apoptotic bodies diagnostic for GvHD (arrows) Apoptotic bodies Interstitial edema Skin vein B.D. A B.D. Yellow arrows represent dead hepatocytes. Outlined area is a combination of necrotic hepatocytes, fibrosis, and some lymphocytes. Small Intestine Liver
  32. 32. PBMC + Tregs PBMC ONLY EDEMA LYMPHOCYTES APOPTOTIC BODIES DIAGNOSTIC FOR GVHD (arrows) Small Intestine 400x (Apoptosis with Enteritis)
  33. 33. 3rd Party CB Tregs injection on day -1 at 1:1 ratio Day -1 CB Tregs 1x107
  34. 34. 3rd Party CB Tregs Prevent GVHD when infused on day -1 at 1:1 ratio NO Treg WITH Treg NO Treg WITH Treg LUNGLIVER PBMC 1 x107 PBMC+ Treg 1 x107
  35. 35. CHALLENGE! • Limitation in generating clinically relevant doses of Tregs from a single CB unit
  36. 36. CB Hematopoieitic Cells have Decreased Ability to bind Selectins • P- and E-selectin are expressed by the microvasculature of the hematopoietic microenvironment and mediate hematopoietic cell homing and adhesion Katayama et al. Blood. 2003;102:2060-7
  37. 37. Fucosylation can Restore Selectin Binding Xia et al. Blood. 2004 ;104:3091-6 Hidalgo and Frenette Blood. 2005;105:567-75
  38. 38. Fucosyltransferase (FT) VI CB Hematopoieitic Cell GDP-Fucose Glycoprotein Fucosylated glycoprotein √ X P- & E-Selectin BM endothelium Impact on homing & engraftment?Bone marrow microvasculature Enhancing Selectin Binding through Ex Vivo Fucosylation
  39. 39. Fucosylation of CB CD34+ cells enhances engraftment in NSG mice 4.5x104 CD34+ cells/mouse Fucosylated Untreated 100 101 102 103 104 CLA FITC CB FTVI 40.008100 101 102 103 104 CLA FITC washed cb.008 CLA Untreated Fucosylated CD34
  40. 40. Preparative Therapy Days -8 to -2 0 Thaw and infuse CB#1 Thaw, treat with FT-VI and GDP-fucose (30 minutes), wash and infuse. CB#2 Preparative Therapy Days -8 to -2 0 Thaw and infuse CB#1 Thaw and infuse CB#1 Thaw, treat with FT-VI and GDP-fucose (30 minutes), wash and infuse. CB#2 Thaw, treat with FT-VI and GDP-fucose (30 minutes), wash and infuse. CB#2 CB Fucosylation Study MDACC #2010-0658, IND#14897 High-Risk Hematologic Malignancies
  41. 41. MDACC CB Fucosylation Trial N=23 FT-VI treated CB Time to Neutrophil Engraftment (ANC ≥ 500) Median: 17 Days Range: 12 – 34 Time to Platelet Engraftment (plts ≥ 20,000) Median: 33 Days Range: 18 -100 Popat et al., ASH 2013
  42. 42. Is there Impact of Fucosylation on CB Treg Homing?
  43. 43. Fucosyltransferase-VI-GDP Fucose (FT-VI) Treatment Increases CB Treg Fucosylation CD4 Fucosylation (CLA/sLex) Control + FT-VI
  44. 44. Fucosylated CB Tregs show Increased E-selectin Ligand Binding Ability Fig 1C Fig 1D Fig 1E Control + FT-VI 0 10 20 30 40 50 60 70 80 90 100 CLA (Fuco) E-Selectin P-Selectin L-Selectin percentbinding untreated FTVI treated P=0.008 P=0.0001 P=0.004 P=0.8
  45. 45. Suppression of in vitro Allogeneic MLR by Fucosylated CB Tregs is comparable to Untreated Ex vivo Expanded CB Tregs 0 10000 20000 30000 40000 50000 60000 70000 80000 90000 Countsperminute
  46. 46. 4. Magnetic Activated Cell Sorting, MidiMACS 3. Anti-CD25 microbeads (CD25 Reagent, MiltenyiBiotec) 1. CB unit 5a. CD25 positive cells “Treg”. Cultured at 1x106 cell/ml in T-cell expansion medium. IL-2 @ 200 u/ml Initiated with *CD3/28 beads at 3 beads: 1 cell ratio. + 2. Ficoll to isolate CB MNC Experimental Procedure: Imaging Tregs Day 3: eGFP-FFluc retro-viral transduction Continually culture for 11 additional days 30 min incubation w FTVI enzyme Treg FTVI-Treg
  47. 47. Longer in-vivo Persistence of Fucosylated CB Tregs Day 7 Day 10 Fig 2F T-Tre Day -1 Day 0 FT-Treg + PBMC Treg+PBMC Fig 2A Fig 2B Fig 2D FT-Treg 1 x107 + PBMC Treg 1 x107 + PBMC
  48. 48. Are Fucosylated Tregs more Potent? PBMC: 1x107 Group 1 PBMC: 1x107 Treg: 1x106 Group 2 PBMC: 1x107 Fucosylated Treg: 1x106 Group 3
  49. 49. Fucosylated CB Tregs Preserve Recipient Weight at 1-log less Dose P =0.03 70 75 80 85 90 95 100 105 110 Day 0 Day 1 Day 3 Day 7 Day 10 Day 13 Day 14 Day 17 Day 18 Day 19 Day 20 Day 21 Day 26 Day 31 Actualweight/baselineweightx 100 Treg + PBMC FT-Treg + PBMC PBMC alone PBMC+ Treg: 1x106 PBMC + FT-Treg: 1x106 PBMC : 1x107
  50. 50. Fucosylated CB Tregs Render Lower GVHD Score at 1-log less Dose P 0.009 0 1 2 3 4 5 6 7 Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 17 Day 18 Day 19 Day 20 Day 21 Day 26 Day 31 GVHDSCORE Treg + PBMC FT-Treg+ PBMC PBMC alone PBMC+ Treg: 1x106 PBMC + FT-Treg: 1x106 PBMC : 1x107
  51. 51. Fucosylated CB Tregs Improve Survival at 1-log less Dose P<0.0001 PBMC + FT-Treg x106 PBMC + Treg x106 PBMC 1 x107 FTVI-Treg 106 cell dose
  52. 52. Are Fucosylated Tregs more Potent? PBMC: 1x107 Group 1 PBMC: 1x107 Treg: 1x105 Group 2 PBMC: 1x107 Fucosylated Treg: 1x105 Group 3
  53. 53. Fucosylated CB Tregs Improve Survival at 2-log less Dose PBMC + FT-Treg x105 PBMC + Treg x105 PBMC 1 x107 P=0.01 FTVI-Treg 105 cell dose
  54. 54. PILOT STUDY OF INFUSION OF FUCOSYLATED REGULATORY T CELLS TO PREVENT GRAFT VS. HOST DISEASE Protocol Number: 2014-0150 Principal Investigator: Simrit Parmar, MD
  55. 55. Pilot study of CB Fucosylated Tregs to prevent GVHD Day -8 Day -7 Day -6 Day -5 Day -4 Day -3 Day -1 Day 0 Flu 40mg/m2 Flu 40mg/m2 Flu 40mg/m2 Flu 40mg/m2 TBI FTVI- Treg infusion SCT Cytoxan 50mg/kg + MESNA IST: Sirolimus + MMF ‘Off the Shelf’ CB Fucosylated Tregs
  56. 56. CB Treg Dose Levels Dose Cohort Treg Dose Dose Level 1 1 × 106 FTVI-Tregs/kg Dose Level 2 1 × 107 FTVI-Tregs/kg
  57. 57. Thank You!!

×