Alloreattività e Tolleranza nei Trapianti di Cellule Staminali Emopoietiche Allogeniche

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1 Alloreattività e Tolleranza nei Trapianti di Cellule Staminali Emopoietiche Allogeniche Massimo Fabrizio Martelli Ematologia ed Immunologia Clinica Università degli Studi di Perugia 41 Congresso Nazionale SIE Ottobre 2007 BOLOGNA

2 Alloreattività e Tolleranza nei Trapianti di Cellule Staminali Emopoietiche Allogeniche Massimo Fabrizio Martelli Ematologia ed Immunologia Clinica Università degli Studi di Perugia 41 Congresso Nazionale SIE Ottobre 2007 BOLOGNA

3 E. DONNALL THOMAS 1990 Nobel Laureate in Medicine for their discoveries concerning organ and cell transplantation in the treatment of human disease

4 Allogeneic Hematopoietic Stem Cell Transplant Bone marrow harvest from HLA identical sibling I.V.INFUSION of HSCs Supportive Care Graft versus Host Disease Prophylaxis Preparative Regimens (chemotherapy + TBI) Immunosuppression Malignant Cell Eradication Hematopoietic and Immunological Recovery

5 In the contest of conventional allogeneic HSCT donor T-cell alloresponses are responsible for three major transplant events: Engraftment Graft versus Host Disease Graft versus Leukemia Effect

6 Presentation of recipient antigens to donor T-cells by host antigen presenting cell

7 Donor + Donor Recipient APC Recipient APC

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9

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11 Current immunosuppressive armamentarium Inhibitors of DNA synthesis Methotrexate Azathioprine Mycophenolate mofetil Inhibitors of cytokine production Cyclosporine Tacrolimus Inhibitors of cytokine binding IL-2 receptor-specific monoclonal antibody Inhibitiors of cytokine receptor signal transduction Rapamaycin They target the cascade of events leading from antigen recognition, processing and presentation to clonal proliferation of immune effectors cells

12 GvHD after standard prophylaxis Acute, grade II-IV Chronic <20y y y 20% 30% 79% sibling MUD 10-33% 30-70% sibling 10-35% MUD 20-50%

13 Duration of immunosuppressive treatment for chronic GvHD Time to Discontinuation of Immunosuppression Cumulative incidence of discontinued immunosuppressive treatment without relapse among all patients (panel A), among patients transplanted with mobilized peripheral blood (B), among male recipients with female donors (C) and among patients with HLA disparity (D) Stewart BL Blood, 104, 3501, 2004

14 Development of Tolerance Donor T cells that develop in the recipient thymus after HSCT do not cause acute GvHD Those that express receptors for recipients alloantigens are eliminated through interactions with thymic epithelial cells and also with dendritic cells of the recipient that may survive for several months after transplantation. The presence of donor marrow-derived dendritic cells in the thymus prevents the development of T-cells that could recognize and destroy the graft.

15 T-cell alloresponses against the recipient s lympho-hematopoietic system favor engraftment eradication of minimal residual disease

16 RIC HLA-identical transplant Chimerism analysis of a patient using primers M551 and Posttransplant samples were sorted into myeloid cells (M) and T cells (T) on days 56,70, and 100 and showed donor T-cell engraftment preceding donor myeloid engraftment Childs R et al Blood 94, 3234, 1999

17 Allogeneic HSCT cures leukemia through the concerted action of two mechanisms: 1) the myeloablative effect of a conditioning regimen 2) immune cells in the graft eliminating residual leukemic cells (GvL effect) As a rule the GvL effect is mediated by T-cell alloreactions directed against minor recipient antigens

18 GVL effects in clinical HSC transplants Probability of Relapse Less relapse in patients with agvhd and/or cgvhd than in those without GvHD More relapses after identical twin than allo HSCT High relapse rates after T-cell depleted HSCT Horowitz et al, Blood 1990

19 GVL effects in HSCT Donor lymphocyte infusions induce cytogenetic and molecular remissions after post-hsct relapse Non-myeloablative conditioning which has little anti-tumor activity gradually achieves durable complete remissions months after HSCT

20 Graft-versus-leukemia effect of DLT: EBMT-95 survey Kolb HJ et al Blood 103, 767, 2004

21 Multivariate analyses of allosct in patients(>50yrs) with AML Reduced Intensity (n=315) versus Myeloablative Conditioning (n=407) RIC versus MA Relative risk P-value TRM 0.48 <0.001 Relapse 1.78 <0.001 LFS Aoudjhane et al Leukemia 2005

22 GvL effect and GvHD mhas dominate allogeneic T-cell recognition of leukemic cells which is not entirely specific for leukemia-associated antigens. Therefore, any specific GvL response is largely masked by GVHD-related GVL effect.

23 Graft T-Cell Depletion for GvHD Prevention in HLA-Matched HSCT Overcoming graft rejection and post-hsct leukemia relapse

24 T-Cell Depleted HSCT from HLA-Identical Sibling Adverse Effects Graft Rejection Graft composition Recipient immunity after T-cell depletion after standard of the HSCs conditioning regimen T T CD34 CD34 CD34 GvHR HvGR T T CD34 T T T Donor Recipient Immunological Balance

25 T-Cell Depleted HSCT from HLA-Identical Sibling Adverse Effects Rejection Leukemia Relapse Lack of the Graft versus Host Disease Related Graft versus Leukemia Effect

26 T-Cell Depleted HSCT from HLA-Identical Sibling Adverse Effects Graft Rejection Leukemia Relapse Counter Measures Conditioning Myeloablation Immunosuppression Conditioning Myeloablation

27 CONDITIONING REGIMEN HFTBI TT CY CY 14.4 Gy HFTBI TT : thiotepa 10 mg/kg CY: cyclophosphamide 50 mg/kg/day

28 The effects of adding myeloablative agents to TBI in a mouse model of stem cell competition Treatment Survival 30 days Chimerism status 2 mo post-transplant post-transplant (donor %) TBI 44/47 (93%) 3/44 (6.8%) TBI + CY 46/47 (97%) 5/46 (10.8%) TBI + TT 26/33 (78%) 21/26 (80%) TBI + BU 28/34 (82%) 27/28 (96%) TBI + DMM 14/18 (77%) 13/14 (93%) Terenzi A. et al. Transplantation 1990,58,717

29 CONDITIONING REGIMEN HFTBI TT CY CY SBA-E- BM ATG No post-transplant immunosuppression 14.4 Gy HFTBI TT : thiotepa 10 mg/kg CY: cyclophosphamide 50 mg/kg/day ATG Merieux

30 T-Cell-Depleted HLA-Matched Bone Marrow Transplantation in Acute Leukemia Adult Patients Conditioning: 14.4 HfTBI, cyclo, ATG, TT Inoculum: SBA-E- BM No post-transplant immunosuppression Graft rejection 0%; GvHD 0% Disease-free Survival 74% Leukemia Relapse % Aversa F. et al. JCO 1999;17:1545

31 T-cell-depleted HLA-matched Bone Marrow Transplantation in acute myeloid leukemia adult patients Papadopoulos et al. Blood 1998;91:1083 Disease-free Survival Relapse

32 These studies showed : the drawbacks of T-cell depletion need to be counterbalanced appropriate modifications to the conditioning regimen must not significantly increase extrahematological toxicity

33 T-Cell-Depleted HSCT from HLA-Identical Sibling Is it still alive?

34 Present strategy in T-cell depleted HSCT from HLA-identical sibling Graft Processing Peripheral blood CD 34+ cells positively immunoselected using the Milteny device Graft Content CD34+ 10x10 6 /kg b.w.; CD3+ 1x10 4 /kg b.w. Highly Myeloablative Conditioning Regimen No post-hsct Immunosuppression

35 T-cell depleted HLA-identical HSCT Conditioning Regimen and Graft Composition TBI TT FLUDARABINE CD34+ ATG No post-transplant immunosuppression TBI: 8 Gy in a single fraction at 16 cgy/m TT : thiotepa 10 mg/kg Fludarabine: 40 mg/sqm/day ATG Merieux Graft content CD34+ 10x10 6 /kg b.w. CD3+ 1x10 4 /kg b.w.

36 T-cell-depleted HSCT from a HLA-matched donor after TBI-Thiotepa-Fludarabine Patients Age, median in yrs (range) Disease: AML/ALL NHL/HD Disease status at Tx: CR Relapse Graft composition CD34+ (x 10 6 /kg) CD3+ (x 10 4 /kg) (26-63) 10/2 2/ ( ) 1 ( ) Aversa F. et al

37 RICOSTITUZIONE POST-TRAPIANTO Results 5 4,5 4 3,5 3 2,5 2 1,5 1 0,5 0 Globuli Bianchi Neutrophils Piastrine Platelets Median 12 (range( 10-17) Median 14 (range( 9-26) Engraftment 15 (100%) Acute GvHD II Chronic GvHD Overall / Event-free Survivors Relapse Non-relapse mortality 1/15 (DRB1 #) 0/11 10 / 9 (1-25 months after Tx) 4 (3,6,12,13 months after Tx) 2(1GvHD,1IP) Aversa F. et al

38 HLA-identical CD34+ cell transplantation CD4+ cell recovery Without ATG in the conditioning months With ATG in the conditioning Aversa F. et al

39 CMV Antigenemia No ATG Evaluable CMV-positivity gg TMO ATG Aversa F. et al gg TMO

40 T-Cell Depleted HSCT across MHC Barriers

41 PROBABILITY OF HAVING A BONE MARROW DONOR No Donor Matched sibling 30% HSCT from an alternative donor is the only option matched unrelated donor unrelated cord blood unit mismatched family donor

42 Matched unrelated donor HSCT Disadvantages 30% of patients do not find a matched donor Many high-risk acute leukemia patients who might find an appropriate donor often relapse while the HLA typing is in progress or while waiting to start the transplant procedures More accurate matching inevitably reduces the probability of identifying a suitable donor Other drawbacks: Age > 50 yrs, advanced stage disease

43 UNRELATED CORD BLOOD TRANSPLANT Advantages Disadvantages Median search time <1month Rare haplotypes 20% Low incidence of GvHD HLA mismatches No risk to donor Low stem cell dose Slow hematological recovery High risk of graft rejection in adults Risk of congenital disease Naif status of cord blood lymphocytes

44 HLA-haploidentical three loci mismatched family members Overall incompatibility : 3 loci (A+B+D) Recipient Donor A1 B8 Dw3 A2 B12 Dw4 A3 B7 Dw2 A3 B7 Dw2 ONE HAPLOTYPE MISMATCHED HSCT Obvious Advantages a family donor for almost every patient no undue delay

45 HLA-Haploidentical Transplants HLA-haploidentical three loci mismatched family members Overall incompatibility : 3 loci (A+B+D) Recipient A1 A3 B8 B7 Dw3 Dw2 A2 B12 Dw4 A3 B7 Dw2 Donor In the setting of MHC disparity, GvH and HvG alloresponses are the strongest Recipient Antigen Presenting Cell Donor T-lymphocyte 2% or more of the total T-cells may be reactive with an allogeneic MHC determinant. Only one in of the same T-cell pool is reactive with an exogenous protein.

46 ONE HAPLOTYPE MISMATCHED HSCT Obstacles T-replete BMT T-depleted BMT High incidence of severe GvHD* *mediated by the high frequency of anti-host alloreactive T cells in unmanipulated grafts High incidence of rejection* *mediated by residual anti-donor CTL-p s which survive the conditioning

47 T-cell Depleted Mismatched HSCT Experimental Data* Escalating doses of T-cell-depleted mismatched marrow cells ensure full donor type engraftment in mice: presensitized with donor lymphocytes partially reconstituted with graduated number of host T cells before the transplant pretreated with sublethal doses of TBI * Lapidot et al Blood 73, 1989 *Bachar-Lustig et al Nat. Med. 1,1995 *reviewed in Reisner Y and Martelli MF Immunol Today 1999;20:

48 Megadose of T-Cell Depleted CD34+ Cells G-CSF Mobilized Peripheral Blood Hematopoietic Progenitor Cells T-Cell Depletion Procedures Soybean agglutination and E-rosetting ( ) E-rosetting and CD34+ selection ( ) One Step Automated CD34+selection ( ) CliniMacs CD 34+ Cells Purity 95% Recovery 78% After Before CD3+cells log depletion 4.5 Aversa F. et al., Blood 1994; 84: Aversa F. et al., N Engl J Med 1998;339: Aversa F. et al., J Cln Oncol. 2005;23:

49 Factors involved in engraftment of T-cell depleted Haploidentical HSCs stbi Thiotepa Fludara ATG Conditioning No post-transplant immunosuppression Graft T cell Stem Stem Stem Stem Stem Stem Stem Stem Stem Stem Median Dose dose of CD3+ CD34+Cells cells 12,8x x 10 6 /kg 4 /kg b.w. Median Dose of CD3+ Cells 1x10 4 /kg b.w. Median MedianDose dose of CD34+ CD20+ cells Cells 4.1x10 12 x 10 4 /kg 6 /kg b.w.

50 CONDITIONING REGIMENS March August 1995 October 1995 TBI TT CY CY HSC TBI TT HSC ATG days ATG FLUDARABINE TBI: 8 Gy in a single fraction at 16 cgy/m TT : thiotepa 10 mg/kg ATG (Fresenius): 5 mg/kg/day Cyclophosphamide 50 mg/kg/day Fludarabine 40 mg/sqm/day Martelli MF et al ASH 1995

51 Fludarabine as an Immunosuppressor for HSCT Conditioning Cyclophosphamide, at high dosages combined with chemo and/or radiotherapy induces: haemorrhagic cystytis interstitial pneumonia cardiotoxicity VOD Fludarabine is highly immunosuppressive and does not induce notable extra-hematological complications

52 1500 Fludarabine An alternative to Cyclophosphamide in the conditioning regimen for allo HSCT Clonable cells TBI FLU + TBI TBI + CY TBI + FLU Terenzi A. et al. Transplant Proc 1996;28:3101

53 P= Engraftment & GvHD 255 Patients with Acute Leukemia Primary Engraftment 96% 95% G-CSF+ 84% Acute GvHD grade II G-CSF % SBA n=36 Cellpro n=44 Clinimacs n=175 P= % 0 Days to engraft (>500 N) Days after transplantation Overall Engraftment =98% Aversa F. et al., Blood 1994 Aversa F. et al., N Engl J Med 1998 Aversa F. et al., J Clin Oncol. 2005

54 Haplo-Transplant in 255 Patients With Acute Leukemia EVENT FREE SURVIVAL ALL (n=108) AML (n=147) CR 1 (n=34) 0.50± 0.09 CR 2 (n=39) 0.27± 0.10 CR 2 (n=49) 0.35± 0.07 CR 1 (n=23) 0.25± 0.08 Relapse (n=64) 0.14± 0.04 P= Relapse (n=46) 0.05± 0.04 P= Aversa F. et al., Blood 1994; 84: Aversa F. et al., N Engl J Med 1998;339: Aversa F. et al., J Cln Oncol. 2005;23:

55 Comparison of outcomes after Unrelated Cord Blood or Haploidentical T-cell depleted Peripheral Blood Stem Cells in Adults with High Risk Acute Leukemia V Rocha, F Aversa, M Labopin, G Sanz, F Ciceri, W Arcese, D Bunjes, J Rowe, P Di Bartolomeo, F Frassoni, M Martelli and E Gluckman on behalf of the Eurocord Group and Acute Leukemia Working Party EBMT 5 Workshop on Haploidentical Stem Cell Transplantation Catania October 4-6,2007

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