Modulation de la différenciation lymphocytaire T par thérapie cellulaire et génique dans le thymus. Valérie Zimmermann

Transcription

1 Modulation de la différenciation lymphocytaire T par thérapie cellulaire et génique dans le thymus Valérie Zimmermann

2 Thymopoiesis Bone Marrow 2-28 days Thymus Periphery Hematopoietic progenitor Hematopoietic Stem Cell Lymph nodes edulla ortex

3 CD CD8 Thymopoiesis Bone Marrow 2-28 days Thymus Periphery Double Negative DN: CD-CD8- Double Positive DP: CD+CD8+ Single Positive Single Positive CD CD CD CD8 SP CD+ T cells SP8 Hematopoietic progenitor CD8 CD8 WT CD8+ T cells SP DN DP SP8

4 Severe Combined Immunodeficiency Disease (SCID) Heterogeneous group of genetic diseases T lymphocytes are absent or non-functional Abnormal development of other hematopoietic lineages (B, NK,...) Lethal due to opportunistic infections in infancy

5 Severe Combined Immunodeficiency Disease (SCID) Heterogeneous group of genetic diseases T lymphocytes are absent or non-functional Abnormal development of other hematopoietic lineages (B, NK,...) Lethal due to opportunistic infections in infancy ZAP-70 immunodeficiency

6 ZAP-70: A tyrosine kinase required for T Cell Receptor (TCR) signaling Response to TCR engagement MHC/peptide TCR CD/CD8 T Cell CD3 P P P zeta P ZAP-70 Signal transduction Cytokine secretion Proliferation Apoptosis Cytotoxicity

7 CD CD8 Altered thymopoiesis in ZAP-70-/- mice 2-28 days Bone Marrow Thymus Periphery Double Negative DN: CD-CD8- Double Positive DP: CD+CD8+ Single Positive Single Positive CD CD TCR CD CD8 SP CD+ T cells SP8 Hematopoietic progenitor CD8 CD8 WT ZAP-70 -/- CD8+ T cells SP DN DP SP8

8 Altered thymopoiesis in ZAP-70-/- patient 2-28 days Bone Marrow Thymus Periphery Double Negative DN: CD-CD8- Double Positive DP: CD+CD8+ Single Positive Single Positive CD CD TCR CD CD8 SP Non functional CD+ T cells SP8 Hematopoietic progenitor CD8 CD8 CD8+ T cells

9 SCID treatments Allogeneic HSC transplantation (cell therapy) Donor Immunodeficient patient HSCs Benefits: - high success rate - long term reconstitution Difficulties: - lack of compatible donors Reconstituted patient

10 SCID treatments Genetic correction of patient HSCs (gene therapy) Retroviral and Lentiviral vector Benefits: - no donor required Immunodeficient patient Difficulties: - insertional mutagenesis - loss of HSC potential by ex vivo manipulation Reconstituted patient

11 Common limitations to reconstitution of intravenously injected allogeneic and gene-corrected HSC Site of T cell differentiation Site of hematopoiesis - Time for polyclonal T cell reconstitution >100 days - Duration of progenitor migration to the thymus

12 An innovative therapeutic approach: Targeting the thymus Site of T cell differentiation Site of the block in genetic immunodeficiencies Easily accessible under anesthesia; no surgical intervention required Intrathymic administration: Minimizes loss of cells/vectors during migration Homing «assured» Thymic lobes injected non-injected

13 An innovative therapeutic approach: Targeting the thymus Targeting the thymus: Immunodeficient thymus? Gene transfer Lentivirus/AAV DN DP Allogeneic progenitors Donor HSCs? T cell compartment reconstitution

14 An innovative therapeutic approach: Targeting the thymus Targeting the thymus: Immunodeficient thymus? Gene transfer Lentivirus/AAV DN DP? T cell compartment reconstitution

15 Treatment of ZAP-70 deficiency by intrathymic injection of a T-cell specific lentiviral vector Why lentiviral vectors? Transduction of quiescent and dividing cells Long term and stable expression of the transgene Lineage-specific transgene expression is feasible T specific lentiviral vector encoding ZAP-70 ZAP-70 -/- mice? T cell compartment reconstitution

16 Intrathymic lentiviral transfer in ZAP-70 -/- mice T specific lentiviral vector encoding for ZAP-70 ZAP-70 -/- mice T cell compartment reconstitution

17 Intrathymic lentiviral transfer in ZAP-70 -/- mice T specific lentiviral vector encoding for ZAP-70 ZAP-70 -/- mice T cell compartment reconstitution But: - Low transduction of thymocytes Limited diversity of the T cell repertoire - Ineffective in macaques Adjali et al., JCI, 2005

18 Promising results need to improve thymocyte transduction: Assessment of other vectors: raav?

19 Adeno-Associated Virus (AAV) Vectors Derived from single-stranded DNA adeno-associated viruses Advantages: - already used in clinic - transduction of quiescent and dividing cells - long term and stable expression of the transgene in non-dividing cells - broad and flexible tropism: different serotypes available (capside modification) - predominantly non-integrative Disadvantages: - predominantly non-integrative - minimal published data on hematopoietic cell transduction by AAV

20 egfp/hprt (AU) raav2/8 promotes efficient intrathymic gene transfer in mice and macaques PGK GFP ss + scaav2/1-pgk-gfp ss + scaav2/2-pgk-gfp ss + scaav2/-pgk-gfp ss + scaav2/5-pgk-gfp ss + scaav2/8-pgk-gfp WT macaque IT injection: endoscopy Analysis of thymocyte transduction Thymus SC SS /1 2/2 2/ 2/5 2/8 Day 7 Moreau/Vicente et al, Mol. Therapy, 2009 Potential use of scaav2/8 for modulation of thymus function

21 Testing AAV8-ZAP-70 gene therapy: ZAP-70 -/- mouse model ZAP-70 -/- mice AAV8 IT injection ssaav8-pgk-zap-70 PGK ZAP Following immune reconstitution scaav8-pgk-gfp PGK GFP

22 IT AAV8 gene transfer induces long term peripheral T cell reconstitution AAV8-ZAP-70 IT transfer: Lymph Nodes Peripheral T cell numbers remain elevated for up to 3 weeks post transplantation

23 Conclusions AAV8-ZAP-70 gene transfer: Rapid but transient formation of a thymic medulla Rapid reconstitution of a functional T cell compartment in ZAP-70 -/- mice Stable expression of ZAP-70-transduced T cells for >0 weeks WT ZAP-/- IT:AAV-ZAP medulla K1/Aire

24 Future Directions Effect of high ectopic ZAP-70 levels on T cell responsiveness Pathological conditions (infection, tumor, ) Evaluate the mechanisms accounting for the persistence of ZAP-70 in AAV2/8-transduced T cells vector copy number in different organs vector integration

25 An innovative therapeutic approach: Targeting the thymus Targeting the thymus: Immunodeficient thymus? DN DP Allogeneic progenitors Donor HSCs? T cell compartment reconstitution

26 T cells (%) T cell reconstitution in ZAP-70 -/- mice following IT administration of BM progenitor cells CD 50 donor HSC ZAP-70 -/- mice T cell compartment reconstitution Spleen ** WT ZAP -/- IV IT WT ZAP -/- IV IT Transplanted ZAP-70 -/- mice Time Post-BMT wks 8 wks 13 wks Adjali et al., PNAS, 2005 Vicente et al., Blood, 2010 De Barros et al., Blood, 2013 ZAP -/- + BMT IV IT #11 3/8/ #8 3/8/ # /8/ # /8/ FL2-H FL2-H OG /8/ /8/ FL2-H FL2-H FL2-H FL2-H CD8 Thymus IT administration of progenitor cells results in: - faster/enhanced T cell reconstitution - long term thymopoiesis

27 CD8 Donor CD3 Targeting the thymus: enhanced T cell reconstitution Donor HSC ZAP-70 -/- mice T cell compartment reconstitution Total thymocytes ZAP -/- + BMT WT ZAP -/- IV IT 19% 0.1% 0.% 15% 81% 0% CD5.1 donor 0.1% 25% CD5.1 donor cells 2.% 2.8% 8% 10% CD 39% 5% 6% 50% 11%.5% 62% 23% Thymus 25 weeks Vicente et al., Blood 2009 IT injection of HSCs results in enhanced T cell reconstitution in the absence of conditioning

28 Can long-term thymopoiesis be achieved by the IT administration of HSCs? Role of conditioning

29 Role of conditioning in the outcome of IV and IT HSC transplantation donor HSC ZAP-70 -/- mice or T cell compartment reconstitution donor Thymus 26 weeks long-term donor-derived thymopoiesis: dependent on conditioning following IV HSC administration independent of conditioning after IT HSC administration

30 Can long-term thymopoiesis be achieved by the IT administration of HSCs? Fate of HSCs in a semi-allogeneic setting <20% of patients have a histocompatible donor

31 Both IV and IT injection of semi-allogeneic HSCs leads to long-term thymopoiesis in conditioned mice HSC semi-allogeneic ZAP-70 -/- mice T cell compartment reconstitution Transplant + conditioning Thymus 26 weeks

32 In the absence of conditioning, only IT injection of semi-allogeneic HSCs leads to long-term thymopoiesis HSC semi-allogeneic ZAP-70 -/- mice T cell compartment reconstitution Transplant - conditioning Thymus 26 weeks

33 Conclusions IT administration of HSCs: Long-term thymopoiesis in the presence and even in the absence of conditioning - Irrespective of donor origin (histocompatible/ semi-allogeneic) Long-term thymopoiesis following IT transplantation: Associated with the persistence of donor-etps - Extended myeloid potential as compared to WT ETPs

34 Future Directions Effect of IT HSC transplantation on thymic architecture restoration --> medulla generation --> T reg differentiation Elucidate the mechanisms resulting in thymus autonomous long term thymopoïesis Feasibility of the thymic injection in SCID models with remnant thymus --> RAG-/- mice Feasibility of the thymic injection in humanized mouse models --> NSG mice + CD3+ transplantation Concomitant injection of progenitors by IT and IV routes

35 Conclusions: AAV gene transfer: Medulla formation Reconstitution of a functional T cell compartment >0 weeks HSC transfer: Rapid and efficient engraftment Thymopoiesis by 2 weeks Long term thymopoiesis independent of donor origin and conditioning Potential translation to the clinic: Development of innovative therapeutic strategies for enhancing T cell development

36 Thank you!! IGMM : Animal Facility Sarah COUDERC Vera DESPRAT Cédric ORFEO MRI : Cytometry Platform Myriam BOYER-CLAVEL RHEM Collaboration : IRS2, Nantes Biotech Oumeya ADJALI Mickaël GUILBAUD Philippe MOULLIER CIML, Marseilles Magali Irla Sarah GAILHAC Alice MACHADO Marie POUZOLLES Naomi TAYLOR Valérie ZIMMERMANN Valérie DARDALHON Marie DAUMUR Anne-Sophie DUME Pedro GONZALEZ Sandrina KINET Maria MATIAS Cédric MONGELLAZ Manuela ROMANO Zoï VAHLAS Carmen YONG Former members : Rita VICENTE Stéphanie de BARROS

37

38

39 Treg generation requires the presence of a functional medulla WT thymus Immunodeficient thymus medull a mtec DN DP SP DN DP SPFoxp3+ SP SP8 Cortex Medulla HSCs DN DP medull a? SP SP8? What is the diversity of the cells that make up the thymic medulla

40 Peripheral T cells (%) TG CD+8+ cells (% in P3) TG CD+8+ CD+8+ CD+8+ cells cells (% cells in (% P3) (% in in P3) P3) IT AAV8-ZAP-70 results in long-term maintenance of peripheral T cells AAV-2/8-ZAP70 KO AAV-2/8-GFP AAV-2/8-ZAP70 IT AAV-2/8-ZAP70 KO: AAV8-ZAP-70 KO IT AAV-2/8-ZAP70 AAV-2/8-GFP : AAV8- AAV-2/8-GFP GFP KO AAV-2/8-GFP weeks post gene transfer AAV8-ZAP-70 IT transfer: Rapid and efficient T cell reconstitution T cells maintained for > 10 months

41 SS C Rapid engraftment and expansion of donor HSCs following IT administration in ZAP-70 -/- mice Donor thymocytes (fold expansion) Donor thymocytes fold expansion WT KO 1w IT transplant: w 3w w 21 CD5.1 (donor cells) w 1w 2w 3w w 2w 3w w 100-fold donor thymocyte expansion by 3 weeks Selective advantage of donor progenitors IT Thymus

42 IT HSCT results in a rapid kinetic of thymocyte maturation CD SP donor thymocytes %SP thymocytes (donor) (%) SP8 donor thymocytes %SP8 thymocytes (%) (donor) CD CD8 WT 1w w IT transplant: 2 3w 2 1 w WT 1w 2w 3w w WT 1w 2w 3w w wt 1w 2w 3w IT Transplant w wt 1w 2w 3w IT Transplant SP thymocyte differentiation by 3 weeks SP8 thymocyte differentiation by weeks 6 w Thymus

43 Delayed differentiation of SP Foxp3+ thymocytes following intrathymic HSC transplantation CD2 5 SP Foxp3+ donor thymocytes (%) SPFoxp3+ SP IT transplant: W 2w 3w T w 1.6 Foxp3 SP total WT 2w 3 w IT Transplant wt 2w 3w w w Thymus

44 The kinetic signaling model MHCI MHCII CD8 CD +8+ TCR signaling Persistent TCR signaling + Signal TCR Stop IL-7 MHCI 8+ + Singer et al., NRI, 2008

45 CD SS C IT AAV8 gene transfer induces long term peripheral T cell reconstitution T cells (x10 7 ) CD/CD8 IT : IT : AAV8-ZAP-70 (weeks) WT AAV8-GFP CD 3 0 W T KO * IT: AAV8-ZAP CD 8 0 WT AAV8-ZAP-70 IT transfer: IT: AAV8-ZAP- 70 CD/CD8 ratio significantly skewed to a CD lineage fate Lymph Nodes

46 CD W T W T W T W T W T W T % Cells % Cells CD AAV8-ZAP-70-transduced lymphocytes differentiate into effector cells CD 10 WT IT: AAV8- GFP 56 3w IT: AAV8-ZAP-70 10w 17w w CD EM CD62 L CM Naive CD CD8 CD62L IT: ZAP- 70 N IT: ZAP- 70 EM IT: ZAP- 70 CM IT: ZAP- 70 N IT: ZAP- 70 EM IT: ZAP- 70 CM Significant increase in CM and EM T cells as compared to the majority of N T cells in WT mice

47 Proliferative capacity of ZAP-70 transduced T cells Cell division: CFSE dilution 100% 50 % 25% LN cells + CFSE TCR stimulation (α-cd3 + α-cd28) Proliferation Flow cytometry Transduced ZAP-70 -/- mice WT AAV2/8-ZAP70 AAV2/8-GFP Unstimulated TCR-stimulated Proliferation (CFSE) High proliferation in ZAP-70 transduced T cells following TCR stimulation

48 wt ko AAV-3W AAV-10W AAV-17W AAV-3W wt ko AAV-3W AAV-10W AAV-17W AAV-3W Cytokines (pg/ml) IL17 CTV (pg/ml) IFNg CTV (pg/ml) wt ko AAV-3W AAV-10W AAV-3W Cytokines (pg/ml) TNFa sera (pg/ml) IT AAV8-ZAP-70 gene transfer generates functional T cells able to secrete cytokines upon TCR engagement CBA sera TG TNFa last IL-2 WT KO w w w IT: AAV2/8-ZAP-70 IL-17 WT KO w w w IT: AAV2/8-ZAP TNF-α WT KO w w w IT: AAV2/8-ZAP-70 IFN-γ WT KO w w w IT: AAV2/8-ZAP-70 sera CBA CTV TG IL17 last CBA CTV TG IFNg last T cell stim WT KO w w w w IT: AAV2/8-ZAP-70 0 WT KO w w w w IT: AAV2/8-ZAP-70 WT KO w w w w IT: AAV2/8-ZAP-70 0 WT KO w w w w IT: AAV2/8-ZAP-70 Increase TNF-α and IFN-γ levels in sera at 3w Secretion of IL-17, TNF-α and IFN-γ by TCR stimulated-t cells

49 Nab activity Induction of a T cell-independent humoral response to AAV8 capsid epitopes following IT vector administration WT KO AAV-GFP WT KO IT : 3wAAV-ZAP-70 AAV-ZAP w 10w AAV-ZAP-70 17w 19w AAV8-GFP w 1/1000 1/101/100 1/1000 1/101/100 1/1000 1/101/100 1/1000 1/101/100 1/1000 IT: AAV8-ZAP-70 1/101/100 1/1000 1/101/ of 16 AAV8-ZAP-70-treated ZAP-70 -/- mice positive Similar level with AAV8-GFP

50 Anti-ZAP-70 (ng/ml sera) ng of anti-zap / ml of serum Production of anti-transgene following IT vector administration WT WT KO KO IT : AAV8-GFP GFP 3W-ZAP 3w 10 17w w IT: AAV8-ZAP-70 10W-ZAP 19W-ZAP 3w 3W-ZAP Dilution anti-zap-70 antibodies at early time points (10w) Decrease during time and low so no elimination of gene-transduced T lymphocytes

51 Foxp3 + CD (%) AAV8-ZAP-70-transduced thymocytes differentiate into regulatory T cells WT IT: AAV8-ZAP-70 (d) Early development in the thymus

52 IT HSCT results in a dramatic increase in early thymic progenitors (ETP) CD ETP donor progenitors (%) W T 1w 2w IT transplant: 3w w CD c-kit % ETP (CD5.1) 1w 2w 3w w wt 1w 2w 3w w WT 1w 2w 3w w IT Transplant High % of donor ETP by 1w

53 SC C CD IT HSCT results in a rapid kinetics of thymocyte maturation IT W KO 1w 2w transplant: 3w 8 T w 79 CD CD CD3 Single Positive CD CD3 SP CD Lymphoid Tissue inducer LTi: CD+CD8- CD3- SP thymocyte differentiation by 3w

54 %SP thymocytes (donor) SP donor thymocytes (%) SP8 %SP8 donor thymocytes (donor) (%) SC C SC C CD IT HSCT results in a rapid kinetics of thymocyte maturation IT W KO 1w 2w transplant: 3w 8 T w 79 CD CD CD3 CD CD wt 1w 2w 3w w 0 0 WT 1w 2w 3w w WT 1w 2w 3w w IT Transplant IT Transplant DP thymocyte differentiation by 2w wt 1w 2w 3w w

55 Heterogeneity of TEC compartment: new mtec subtypes IV III II De novo map of thymic stromal populations I Identification of mtec subsets

56

57 Thymus targeting Alternative strategy applicable for both cell and gene therapy Potential treatment for immunodeficiencies and other clinical conditions requiring rapid T cell reconstitution Clinical application (non-traumatic, rapid and feasible)