Terapia cellulare con Linfociti T Regolatori Marco Romano, PhD student
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1 Terapia cellulare con Linfociti T Regolatori Marco Romano, PhD student Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale (DIMES) Università di Bologna Ospedale Sant Orsola Malpighi Via Massarenti, Bologna
2 GRAFT REJECTION (A) Alloantigen presentation via the direct, semi-direct and indirect pathways following organ transplantation, and (B) the relative intensity of each antigen-presentation pathway during the post-transplantation (post-tx) period. Sagoo et al 2010.
3 Chronic GRAFT VERSUS HOST DISEASE phatogenesis Bruce R. Blazar et al Nature Reviews Immunology 12, June 2012
4 Common Immunosuppressive Drugs
5 Treg Discovery FOXP3 + regulatory T cells in the human immune system S. Sakaguchi Nature Reviews Immunology 10, (July 2010)
6 How Regulatory T cells work How regulatory T cells work. Vignali D., Lauren W. Collison and Creg J. Workman Nat Rev Immunol 2008
7 Clinical strategy Donor Tregs Ex-vivo expansion Infusion Infusion of freshly isolated cells Infusion In vivo expansion
8 Background First-in-man clinical results of the treatment of patients with graft versus host disease with human ex vivo expanded CD4+CD25+CD127- T regulatory cells. Trzonkowski P. et al Clin Immunol Oct;133(1):22-6. Infusion of ex vivo expanded T regulatory cells in adults transplanted with umbilical cord blood: safety profile and detection kinetics. Brunstein CG et al. Blood Jan 20;117(3): Donor Regulatory T Cells Infusion in Patients With Chronic Graftversus-host Disease ClinicalTrials.gov Identifier: NCT Treg will be administered fresh. A dose of 0.5 x106 Treg/kg should be ideally administered. PI: Baron F. University Hospital of Liege
9 Background Trial of Regulatory T-cells Plus Low-Dose Interleukin-2 for Steroid-Refractory Chronic Graft-versus-Host-Disease. ClinicalTrials.gov Identifier: NCT PI: John Koreth, MD, Dana-Farber Cancer Institute Therapy of type 1 diabetes with CD4 + CD25 high CD127 - regulatory T cells prolongs survival of pancreatic islets Results of one year follow-up. N. Marek-Trzonkowska. Clinical Immunology Volume 153, Issue 1, July 2014, Pages ONE STUDY ongoing Thril (ClinicalTrials.gov Identifier: NCT ) ongoing...
10 Peripheral Blood analysis: Healthy Donors HD Lymphocyte % CD4+CD25+ % CD4+CD25 high % CD4+CD25 high FoxP3+ % 1 38,6 3,1 0,7 0, ,7 0,9 0,5 3 22,5 4,7 0,6 0, ,8 0,9 0, ,8 0, ,7 0,9 0,3 7 22,4 3 0,7 0,6 8 27,6 3,3 0,6 0,4 mean 28,89 4,04 0,76 0,44 St. Dev 5,21 1,06 0,13 0,13 Percentages are expressed as proportion of white blood cells
11 Peripheral Blood analysis: patients in waiting list for liver transplantation Patients Lymphocyte % CD4+CD25+ % CD4+CD25 high % CD4+CD25 high FoxP3+ % ,7 1,5 0, ,4 0,2 3 24,5 3,6 0,7 0,4 4 28, , ,5 0,9 0, ,6 0,3 0, ,7 0,6 0, ,6 0,7 0, ,8 0,8 0, ,9 1 0, ,5 7 0,6 0,5 12 4,9 4 0,8 0,8 mean 21,04 5,45 0,78 0,55 St. Dev 8,86 2,29 0,31 0,23 Percentages are expressed as proportion of white blood cells
12 Tregs/uL WBC/uL Circulating Tregs: HD Vs Patients HD Liver Pts HD Liver Pts
13 Tregs purification Phenotypic evaluation Treg function Cryopreservation
14 Phenotyphic characterization Gating strategy used to characterize Treg cells pre and post thawing
15 % Cryopreservation strategy Medium: 50% Plasma AB; 40% CliniMacs Buffer 10% DMSO Yeld Yield Patients Healthy Donors Post-thaw ing Cells were cryopreserved using Planer Kryo
16 Suppression assay Method A B A: only stimulus B: Treg/Tresp 1/4 C: Treg/Tresp 1/10 C Co-colture (4 days) of autologous Tregs with CD4+ CD25- (Tresp) stimulated with anti-cd3 and anti CD28 (5ug/ml) and labelled with CFSE. ModFit LT TM 3.1Verify software house
17 Proliferation (Fold-change) Proliferation (Fold change) Function of freshly isolated and thawed Tregs from healthly donors CD4+CD25+ FOXP3+ CD4+CD low CD4+CD25+FOXP3+ CD4+CD low 89,60 92,43 % 86,14 % 94,43 % Fresh Thawed * ** * ** 0.6 * stimulus Treg/Tresp 1/25 Treg/Tresp 1/10 Treg/Tresp 1/4 0.0 Stimulus Treg/Tresp 1/25 Treg/Tresp 1/10 Treg/Tresp 1/4 FOLD-CHANGE was calculated as ratio between the upper generation proliferation index of Tresp cultured in the presence of increasing ratios of Tregs and the upper generation proliferation index of CTR colture.
18 Proliferation (Fold-change) Proliferation (Fold-change) Function of freshly isolated and thawed Tregs from patients in waiting list for liver transplantation CD4+CD25+ FOXP3+ CD4+CD low CD4+CD25+FOXP3+ CD4+CD low 91,93 % 95,43 % 85,93 % 92 % Fresh Thawed ** ** STIMULUS Treg/Tresp 1/25 Treg/Tresp 1/10 Treg/Tresp 1/4 0.0 STIMULUS 1/10 Treg/Tresp 1/4 Treg/Tresp 1/2 Treg/Tresp FOLD-CHANGE was calculated as ratio between the upper generation proliferation index of Tresp cultured in the presence of increasing ratios of Tregs and the upper generation proliferation index of CTR colture.
19 Tregs expansion CD3 Treg cell + MACSiBead rhil-2 (1000 U/mL) Rapamicyn (100nM) CD28 Treg Expansion is based on Beads pre-loaded with CD3 and CD28 antibodies (Miltenyi, Germany) at a bead-to-cell ratio of 4:1. Best expansion is achieved using medium supplemented with recombinant Interleukin-2 and Rapamicyn CD8+ depleted CD25+ enriched 1 x 10 6 Tregs IL-2 (Miltenyi, Germany) Second stimulation Third stimulation Infusion/ criopreservation DAY
20 CD8+ depleted CD25+ enriched Manufacture Protocol One Study Stimul 1 Stimul 2 Stimul 3 Final Harvest 5 Feedings Medium 50% increase Rapa 5 Feedings Medium 50% increase Rapa 5 Feedings Medium 50% increase Rapa Cryopreservation IL-2 feed 3-4 IL-2 feed 1-4 IL-2 feed 1-4 TexMACS +5%hAB 100nM Rapamycin 1000U/ml IL-2 ExpAct Treg beads CD3/28 Phenotype >60% Bead removal <100b/3x10 6 cells Viability >70% Potency >60% Sterility Endotoxin Mycoplasma
21 Manufacture Timelines Day -49 Day -13 Day 0 Day +5 Transport Max 24h DP manufacture Storage of frozen DP Kidney Manufacture Day -1 Final harvest DP infusion Blood procurement Manufacture Day 0 Tx Day 0 Day +37 Day +92 Month 3 Transport Max 24h DP manufacture Storage of frozen DP Starting material procurement Manufacture Day 0 Final harvest Liver DP infusion Tx
22 Why RAPAMYCIN Immunoregulatory functions of mtor inhibition. Nat Rev Immunol 2009; Angus W. et al.
23 Cell number FOLD INCREASE Tregs Expansion from Healthy Donors Donor A Donor B Donor C DAY 0 DAY 7 DAY 14 DAY 21 DAY 0 DAY 7 DAY 14 DAY 21
24 % % Phenotypic evaluation of rapamycin expanded Tregs from HD CD4+CD25+ CD4+ CD25+ FOXP3+ CD4+CD25+CD DAY 0 DAY 7 DAY 14 DAY DAY 0 DAY 7 DAY 14 CD8+ cells Th17 cells CD19+ cells CD14+ cells CD3+ CD56+ cells DAY 21
25 Proliferation (Fold-change) Function of rapamycin expanded Tregs from healthy donors * ** Stimulus 1/10Treg/Tresp 1/4 Treg/Tresp 1/2 Treg/Tresp FOLD-CHANGE was calculated as ratio between the upper generation proliferation index of Tresp cultured in the presence of increasing ratios of Tregs and the upper generation proliferation index of CTR colture.
26 Next step Treg stability in the presence of 2 pro-inflammatory milieu: A: IL-2, IL-1β, IL-6, TGF-β. B: IL-2, IL-21, IL-23, TGF-β. Evaluation of IL-17 and IFN-γ production CTLA-4, GITR and CD39 TSDR analysis Differential effects of rapamycin and retinoic acid on expansion, stability and suppressive qualities of human CD4+CD25+FOXP3+ T regulatory cell subpopulations. Scottà et al. Haematologica Aug;98(8):
27 Impact of immunosuppressive regimen on ex vivo expanded human regulatory T cells Cristiano Scottà, Giorgia Fanelli, Julie Hoong, Marco Romano, Mitalee Sukthankar, Giuliana Guggino, Henrieta Fazekasova, Ratnasothy Kulachelvy, Pablo Becker, Ben Afzali, Robert Lechler and Giovanna Lombardi Question: Can immunosuppressive drugs affect Treg viability,function and trafficking?
28 Immunesuppressive Drugs Tacrolimus Tacrolimus is used after allogeneic organ transplant to reduce the activity of the patient's immune system and lower the risk of organ rejection. Tacrolimus inhibits calcineurin and prevents the dephosphorylation of NF-AT. Thus it negatively affects both T-lymphocyte signal transduction and IL-2 transcription. Prednisolone Prednisolone irreversibly binds with glucocorticoid receptors. Its interaction with DNA modifies gene transcription. It induces synthesis of some proteins, and inhibit synthesis of others. Regulation of gene suppression leads to systemic suppression of inflammation and immune response. Mycophenolate Mofetil (MMF) - MPA MMF reversibly inhibits inosine monophosphate dehydrogenase (IMPDH) in purine biosynthesis which is necessary for the growth of T cells and B cells. It is extensively used in transplant medicine to suppress T and B cells from attacking donor s graft.
29 Viable cell (%) Treg Viability in the Presence of Immunosuppressants 100 Reagent Amount :256 1:128 1:64 1:32 1:16 1:8 1:4 Dilution from highest conc 1:2 Max IL-2 anti-cd3/cd28 beads Rapamycin In vivo drug distribution Methyl-Prednisolone (mpred) Mycophenolate (MPA) Tacrolimus (Tacro) 20 IU/ml 1:2 (Stimulation) 100 nm 0-4 ug/ml ug/ml 0-20 ng/ml Tacrolimus * : Erythrocyte fraction 70-80% Plasma fraction 20% Leukocytes 1% Mycophenolate * * : Albumin bound ~ 97% * Zahir et al. The Drug Monit 2004, * * Bullingham et al. Clinical Pharm 1998
30 Viable cell (%) Viable cell (%) Viable cell (%) Dose Response Curves to Derive the Concentration of Immunosuppressant Tacrolimus ng/ml EC 50 : 2ng/ml methyl-prednisolone MPA 0.31 ug/ml 0.62 EC 50 : 1ug/ml EC 50 : 0.5ug/ml ug/ml
31 Number of cells (x10 3 ) Immunosuppressants Can Reduce the Proliferative Ability of Treg Preparations after 5 Days of Culture * * * ** CTR TAC MPA mpr IS Mix 10 1 CTR TAC MPA mpr IS Mix
32 Proliferation (%) Proliferation (%) Tregs Maintain Full Suppressive Capacity after 5 days of Culture with Drugs :10 1:3 1: CTR TAC MPA mpr IS Mix
33 CD25 MFI MFI Immunosuppressants Do Not Affect FOXP3 Expression after 5 Days of Culture HD % IS Mix (5d) 88.4% FOXP3 Expression HD2 98.8% IS Mix (5d) 87.4% CTR TAC MPA mpr IS Mix CD25 Expression FOXP3
34 Number of cells (%) Functional Treg Molecules are not Affected by Immunosuppressants (5 days) CTR TAC MPA mpr IS Mix CD39 GITR HLA-DR CD95 PD-L1 CTLA4 ICOS TGFbeta-LAP
35 Acknowledgement Laboratorio Terapia cellulare, istituto Seragnoli: Francesca Ulbar L. Catani S. Rizzi, M. Motta, E. Dan D. Sollazzo, M. Arpinati Prof. RM Lemoli Immuneregulation Lab Cristiano Scottà Prof. Giovanna Lombardi Prof. Robert Lechler Giorgia Fanelli Julie Hoong Estefania Nova Lamperti Ratnasothy Kulachelvy Giuliana Guggino Ben Afzali Reuben McGregor Pablo Becker
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