Chimerism and DLI in HSCT for non-malignant disorders. Giorgio La Nasa

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1 Chimerism and DLI in HSCT for non-malignant disorders Giorgio La Nasa

2 Fisiopatolologia dell eritropoiesi inefficace nella Talassemia NEJM

3 Talassemia Major

4 Problematiche del trapianto allogenico legate alla componente mielo-ablativa nelle emoglobinopatie Massa eritroide iper-espansa iperplasia della serie eritroide eritropoiesi inefficace metaplasia eritroide Epatomegalia Ipersiderosi metaplasia eritroide pazienti splenectomizzati Splenomegalia Ipersplenismo esaltata emocateresi metaplasia eritroide Paziente immunocompente talvolta iper-immunizzato

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6 Insufficiente Eradicazione Tipi di Rigetto del Trapianto Ricos5tuzione autologa talassemica Insufficiente Immuno- soppressione Rige8o immunologico Chimerismo misto transitorio o persistente Aplasia (rara) Ricos5tuzione autologa talassemica

7 Clinical Chimerism testing HSCT X X X Donorcell NormalRecipientcell X MalignantRecipientcell Donor Patient before HSCT Complete Chimerism Mixed Chimerism Mixed chimerism presence of both donor and recipient lymphohematopoiesis Split chimerism presence within a single compartment of different donor and recipient cells proportion Microchimerism presence of donor cells that are detectable only with very sensitive techniques Patient after HSCT

8 Types of Chimerism Mixed Chimerism presence of both donor and recipient lymphohematopoiesis Split Chimerism presence within a single compartment of different donor and recipient cells proportion Micro Chimerism presence of donor cells that are detectable only with very sensitive techniques

9 Mixed Chimerism in Haemoglobinopathies MC may be defined : Transient (TMC) if present early after HSCT showing an evolution to graft failure or to complete chimerism Persistent (PMC) when donor and recipient cells coexist for more than 24 months after HSCT

10 Mixed Chimerism in Thalassemia after HSCT The main goal of monitoring engraftment is to identify the presence or absence of residual host cells (RHC) after HSCT The prompt determination of Mixed Chimerism (MC) my help to identify patients at high risk of relapse and rejection

11 Strategies for engraftment monitoring evaluation Biological Samples: Bone Marrow Peripheral Blood Lymphoid cell subsets Time of investigation: Patient Pre-transplant profile Donor profile Early Post-Transplant follow-up controls Periodic late Post-Transplant controls Method sensitivity: FISH, STR, Real time PCR

12 Engraftment detection methods after HSCT in haemoglobinopathies in nucleated cells - Peripheral Blood - Lymphid Cells Subsets (CD3, CD19, CD56 etc.) - T regulatory cells (Tr1) - Bone Marrow - Erythroid Precursurs (BFU-E)

13 Markers commonly used for mixed chimerism detection in nucleated cells - VNTR - FISH analysis (if sex mismatched) - STR (2-5%) - Real Time PCR ( %) Increasing Sensitivity

14 Engraftment detection methods after HSCT in haemoglobinopathies in red blood cells Cytofluorimetric analysis of washed RBCs, incubated with anti- ABO or anti-c, c, D, E, e monoclonal antibodies Patient Donor AB0: A A Rh: + + Subgroup: ccdee ccdee Useful Marker: E anti-e anti-e

15 Mixed Chimerism in Thalassemia after HSCT % of pts with MC % 2 months Transient MC represents a risk factor for graft rejection Number of RHCs present early after HSCT Presence in the PB of split chimerism within the CD3+ cells Conditioning regimes used

16 Mixed Chimerism in Thalassemia after HSCT Transient MC evolution Number of RHCs present early after HSCT Presence in the PB of split chimerism within the CD3+ cells Conditioning regimes used Andreani M, Testi M, Lucarelli G. Tissue Antigens. 2014

17 Mixed Chimerism in Thalassemia after HSCT Chimerism status early after HSCT > 25% Transplant outcome 50-60% Rejection PMC Complete Chimerism patient Rejection 10-25% PMC donor 3-12% Complete Chimerism Rejection < 10% PMC Complete Chimerism

18 Patients enrolled only if a clinical control at 1 year after HSCT was performed Engraftment Total N of Patients status early after HSCT Engraftment evolution over the time (minimum 1 year) (152) CC MC level 1 MC level 2 MC level 3 Rejection MC level (5.6%) MC level (20%) MC level (70%) CC (2.7%) 21

19 Transient Mixed Chimerism in Thalassemia after HSCT Number of RHCs present early after HSCT Presence in the PB of split chimerism within the CD3+ cells Conditioning regimes used

20 Mixed Chimerism in Thalassemia after HSCT % chimerism of donor cells UPN: days after BMT WBCs CD3+

21 Mixed Chimerism in Thalassemia after HSCT Transient MC evolution Number of RHCs present early after HSCT Presence in the PB of split chimerism within the CD3+ cells Conditioning regimes used

22 Mixed Chimerism in Thalassemia after HSCT incidence of mixed chimerism pts 76 pts 26 pts BU14 - CY200 BU14 - CY160 BU14 - CY120 2 months 24 months

23 Mixed Chimerism in Thalassemia after HSCT incidence of mixed chimerism months 24 months BU14/CY200 BU14/CY160 BU14/CY rejection /1552/26 11/76 7/28 5/56 10/210

24 Mixed Chimerism in Thalassemia after HSCT incidence of mixed chimerism 100 BU14 - CY200 BU14 - CY160 BU14 - CY pts 76 pts 28 pts Mixed Chimerism 10/155 5/56 2/26 2 months 24 months

25 Persistent Mixed Chimerism in Thalassemia after HSCT Clinical conditions of patients with PMC no red blood cells infusions produce high levels of hemoglobin no evolution to graft failure no evolution to complete chimerism Functional graft

26 Status of MC characterized by the presence of large amount of RHCs After 2 months from HSCT After 2 years from HSCT High probability of Rejection NO Rejection Andreani M, Testi M, Lucarelli G. Tissue Antigens. 2014

27 Status of MC characterized by the presence of large amount of RHCs After 2 months from HSCT After 2 years from HSCT High probability of Rejection NO Rejection Time after HSCT Andreani M, Testi M, Lucarelli G. Tissue Antigens. 2014

28 Persistent Mixed Chimerism MC is defined persistent when donor and recipient cells coexist for more than 24 months after HSCT no evolution to graft failure? no evolution to complete chimerism

29 Persistent Mixed Chimerism Functional graft % MC % of mixed Chimerism 32-46% 10-15% 2 months 24 months In many cases the proportion of cells of recipient origin is extremely large

30 Mixed Chimerism in Thalassemia after HSCT G. H. BMT PB BM % Donor Days after BMT

31 Persistent Mixed Chimerism Split chimerism between PBMCs and RBCs

32 Mixed Chimerism in Thalassemia after HSCT G. H. BMT PB RBC BM % Donor RBC: 80% and 73% donor Days after BMT

33 Clinical Chimerism testing Persistent Mixed Chimerism Control of the erythroid expansion Immunological Tolerance

34 Mixed Chimerism in Thalassemia after HSCT Control of the erythroid compartment expansion BFU-E: donor recipient origin DNA extraction STR analysis

35 Mixed Chimerism in Thalassemia after HSCT G. H. BMT PB RBC BM BFU-E % Donor RBC: 80% and 73% donor BFU-E in BM: 23% donor Days after BMT

36 Split chimerism between RBCs and peripheral blood (PB) bone marrow (BM) and erythroid precursors (BFU-E) in a patient with PMC at 60 months after HSCT 100 PB BM BFU-E RBCs Donor Recipient

37 Bone marrow of a thalassemic patient before HSCT transplant 100% RHCs 70% RHCs < 30% Donor Bone marrow of a thalassemic patient with PMC after HSCT transplant X Normal erythropoiesis Erythroid expansion

38 Mixed Chimerism in Thalassemia after HSCT Control of the erythroid compartment expansion Primary BFU-E: donor recipient origin Secondary BFU-E: Self renewal capability of donor and recipient BFU-E Erythroblasts subpopulations sorting

39 Exp 1- Day 0 P1- gated cells P2- gated cells P2- gated cells P3- gated cells Not P5- gated cells Not P4- gated cells

40 Mixed Chimerism in Thalassemia after HSCT Preliminary results for Teyab: days after HSCT Chimerismo Misto in BM - Teyab Sorting precursosri eritroidi Days BM % DON Popol. Control CD34+ Proer. Er. Bas Er. Poly Er. Ort RBC %DON NV 100

41 Clinical Chimerism testing Persistent Mixed Chimerism Control of the erythroid expansion Immunological Tolerance

42 Tolleranza immunologica dopo trapianto di CSE nelle emoglobinopatie

43 Tolleranza immunologica dopo trapianto di CSE nelle emoglobinopatie

44 Tolleranza immunologica dopo trapianto di CSE nelle emoglobinopatie 100 Sviluppo della tolleranza immunologica attraverso la produzione di cellule T regolatorie % Donor Mantenimento della tolleranza immunologica Months after HSCT

45 Induzione della tolleranza periferica tramite cellule T regolatorie CD4-8 - γδ + CD4 + NK-T CD8 +

46 INDUCTION OF PERIPHERAL TOLERANCE BY REGULATORY T CELLS CD4-8 - γδ + CD4 + NK-T CD8 + Tr1 ntr (CD4 + CD25 + FOXP3 +) COMMON MECHANISMS: Ø Suppression of proliferation/cytokine production in vitro Ø Suppressive cytokines and/or inhibitory receptors

47 MORE RECENT ACHIEVEMENTS Granzyme B CD49b and LAG-3 Definition of new tolerogenic markers for Tr1 cells:

48 MORE RECENT ACHIEVEMENTS GranzymeB CD49b and LAG-3 Definition of new tolerogenic markers for Tr1 cells:

49 Identificazione di cellule Tr1 umane Blood PBMC FACS analysis Ficoll grandient T T T Staining with CD4, CD45RO, CD49b and LAG- 3 Tr1 cells

50 Identificazione e selezione delle cellule Tr1 dopo trapianto di CSE nella talassemia origine sia del donatore che del ricevente Clonaggio in vitro Origine n cloni n Tr1 Ricevente 12 5 Donatore 16 7 clone IL10 IL2 IL4 IFNg IL17 IL10/IL4 origin producono alti livelli di IL-10 se confrontati con cloni Tr1 di individui normali # ,9 D # ,9 D # ,4 H # ,5 D # ,6 H # ,0 H # ,5 D # ,0 D Serafini G, Andreani M, Testi M et al. Haematologica. 2009

51 CD45RA - CD49b + LAG- 3 + Tr1 cells are highly represented in PMC Control Pt CC Tolerant Pt PMC Tolerant patients after BMT Gagliani N., Magnani C., and Huber S:, Nat Med 2013

52 Primo paziente talassemico sottoposto a trapianto di midollo osseo (Seattle 1981) Primo paziente talassemico sottoposto a trapianto di midollo del 1981

53 Evoluzione (BMT 1981) Rigetto del trapianto documentato nel 2013 Ripresa terapia di supporto trasfusionale Ripresa terapia ferrochelante

54 3 x 10 7 CD3+/kg

55 1 x 10 7 CD3+/kg 3 x 10 7 CD3+/kg

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57 DLI = 1.5 x 10 7 CD3+/kg median n 2 (1-6) 7 early- DLI group (group 1) 4 defferred-dli group (group 2) 10 late- DLI group (group 3) Results : CC = 3 (16%) MC= 9 (47%) GR= 7 (37%) Group 1: GR = 57% Group 2: GR = 75% Group 3: GR = 20%

58 G.D. TMO 28/12/2005 Outcome : PMC 0.5 x x 10 6 chimerismo 10 x x ,0 12/05 05/06 10/06 03/07 08/07 01/08 06/08 11/08 04/09 09/09 02/10 07/10 12/10 05/11 10/11 03/12 08/12 01/13 06/13 11/13 04/14 09/14 02/15 07/15 12/15 05/16 10/16 12,0 11,0 10,0 9,0 8,0 7,0 6,0 5,0 12/05 12/06 12/07 12/08 12/09 12/10 12/11 12/12 12/13 12/14 12/15 12/16 Hb (g/dl)

59 R.M. 12/02/2008 chimerismo 100% TMO 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 1 DLI 4 DLI 0.5 x x e ultima DLI 3 x 10 6 GF Paziente trasfusione dipendente A+ in 0+ 11% fabbisogno trasfusionale (ml/kg/anno) 03/08 06/08 09/08 12/08 03/09 06/09 09/09 12/09 03/10 06/10 09/10 12/10 03/11 06/11 09/11 12/11 03/12 06/12 09/12 12/12 03/13 06/13 09/13 12/13 03/14 06/14 09/14 12/14 03/15 06/15 09/15 12/15 03/

60 C.M. 22/12/2015 DLI PCM Tx dipendente A+ in % 90% 1 x x x 10 7 DLI DLI DLI DLI DLI DLI DLI DLI 80% 70% 60% chimerismo 50% 40% 30% 20% 10% emotrasfusioni 0% giorno post TMO

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63 Grazie per l attenzione Adriana Vacca Eugenia Piras MariaGrazia Orofino Antonio Piroddi