Rejuvenation of the ageing T cell compartment. Marcel R.M. van den Brink Departments of Medicine and Immunology

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1 Rejuvenation of the ageing T cell compartment Marcel R.M. van den Brink Departments of Medicine and Immunology

2 Conclusions IL-7, KGF and Leuprolide show promise in early clinical trials to enhance post-transplant T cell recovery PreT cells can be used as an off-the-shelf adoptive cell therapy across MHC barriers to enhance posttransplant T cell recovery (preclinical data) Extrathymic T cell development (esp. MLN) occurs in HSCT recipients IL-22 can enhance post-transplant thymic recovery

3 Strategies to enhance T cell reconstitution following allo-hsct

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5 Interleukin-7: the Elusive Lymphoid Growth Factor? Human IL-7: 25 kda glycoprotein secreted by fetal liver and stromal cells in bone marrow and thymus IL-7 receptor: IL-7Rα chain and the common cytokine receptor γ-chain expressed on T precursors, thymocytes, naïve T cells, activated T cells, pre-b cells and BM macrophages Non-redundant cytokine for B and T cell development has proliferative and anti-apoptotic effects Administration can increase T and B cell cellularity in normal, and cyclophosphamide-treated or irradiated mice Increased thymopoiesis in SIV-infected primates after syngeneic, autologous or allogeneic BMT increases thymocyte, T and B cell numbers and function Human Phase I studies in solid tumor, HIV and Hepatitis B and C: low toxicity and increases in T cell numbers and function

6 Conclusions from Phase I trial with Interleukin-7 in recipients of CD34+ allografts Administration of rhil-7 (CYT107) post TCD allo-hsct results in: No significant toxicities Increased CD4+ and CD8+ T cell counts No effect on NK, CD19+, or Tregs Expansion of naïve + effector CD4+ T cells, and naïve CD8+ T cells Increased CD4+ and CD8+ RTE in some patients Increased TRECs in 5/6 patients Increased mitogen responses Increased CMV-specific responses in CMV-seropositive patients Perales, EBMT 2011

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8 Keratinocyte Growth Factor (KGF) Fibroblast growth factor-7 (FGF-7) produced by thymic stroma and thymocytes FGFR2IIIb: epithelial tissues including thymic epithelial cells, hepatocytes, gut epithelium and skin keratinocytes. FGFR2IIIb -/- mice: decreased thymic cellularity and abnormal T cell development. Palifermin is FDA approved for mucositis KGF prevents GVHD in mouse models KGF had no effect on GVHD in phase II trial

9 KGF enhances CD4 recovery in recipients of CD34+ allografts after TBI-based conditioning Retrospective review of 114 recipients of CD34+ allograft and TBI from at MSKCC Regimen Survival Parameter Longitudinal Model, effect of KGF on CD4 Survival Model CD4 Slope (of log transformed CD4 count) 95% CI P-value HR 95% CI P-value TBI OS ~ ~ EFS ~ ~ Chemo OS NS NS NS ~ EFS NS NS NS ~ Goldberg/ Perales, unpublished

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11 Leuprolideenhances IgM/G1 responses in autologoushsct recipients 25 adult patients receiving auto-hsct for NHL, HD or myeloma Randomized to Leuprolide3-month depot x3 (sex steroid inhibition from day 0 to +60) vsplacebo No significant toxicity Improved IgM/G1 responses after KLH vaxcomparing 8 Leuprolide vs 5 placebo patients Trend towards increased naïve and TREC+ CD4 T cells in Leuprolide group Champlin et al., ASH 2010

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14 Stages in Early T cell Development

15 T cell development on OP9-DL1 cells OP9-DL1: murine BM stromal cell line retrovirally transduced with Notch 1 ligand Delta-like 1 culture of HSC on OP9-DL1 cells with IL-7 (5 ng/ml) and FLT3L (5 ng/ml) 97% Expansion: 3-4 log in 3-4 weeks Schmitt TM, Zúñiga-Pflücker JC. Immunity2002;17:

16 Adoptive therapy with off-the-shelf precursor T cells can enhance post-transplant T cell recovery across MHC barriers Administration of T cell precursors to allogeneic HSCT recipients: Enhances thymocyte, T cell and NK cell reconstitution Generates single wave of functional host-mhc restricted and tolerant T cells that persist for several months Improves antimicrobial and anti-tumor effects Can be optimized by lentivirally transducing T cell precursors in vitro Can be performed with T cell precursors from any donor to any recipient (off-the-shelf) Thymus, d14 Spleen, d28 Zakrzewski JL. Nat Med, Zakrzewski JL. Nat Biotechnol, 2008.

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18 Targeted immunotherapy of hcd19 + A20-TGL lymphoma using adoptive transfer of pret transduced to express 19z1 BALB/c BALB/c (850 cgy), 0.33x10 6 A20-TGL-hCD19 1x10 3 HSCs + 8x10 6 B6 T cell precursors (50% 19z1 + ), n = 7-8 Zakrzewski JL

19 Targeted immunotherapy of hcd19 + A20-TGL lymphoma using adoptive transfer of pret transduced to express 19z1 BALB/c BALB/c (850 cgy), 0.33x10 6 A20-TGL-hCD19 1x10 3 HSCs + 8x10 6 B6 T cell precursors (50% 19z1 + ), n = 7-8 Zakrzewski JL

20 Targeted immunotherapy of hcd19 + A20-TGL lymphoma using adoptive transfer of pret transduced to express 19z1 BALB/c BALB/c (850 cgy), 0.33x10 6 A20-TGL-hCD19 1x10 3 HSCs + 8x10 6 B6 T cell precursors (50% 19z1 + ), n = 7-8 Zakrzewski JL

21 Expansion of prot1 and prot2 subsets from cord blood CD34+ cells cultured on OP9-DL1 layers Day 28 Day 35 Day % 38% 49.4% 38.7% 45.1% 19.3% CD7 Gated on CD4/CD8 Double Negative Cells CD5 Stem cell population: CD34+ cord blood cells Media: MEM alpha with 20% human AB serum Cytokines: hil-7 (10ng/ml) hflt3l (10ng/ml) hscf (20ng/ml) htpo (20ng/ml) until D14 of culture

22 Phase I clinical trial enrolling patients with high risk ALL/NHL PreT derived from cord blood CD34+ cells Transduced to express 19z1 chimeric antigen receptor In collaboration with Miguel Perales, Isabelle Riviere (Gene Transfer and Somatic Cell Engineering Core), Michel Sadelain (Molecular Pharmacology), and Renier J. Brentjens (Leukemia Service)

23 PreT enhance T cell counts in athymic BMT recipients Spleen F1 CD45.1/CD45.2 B6nu 1: no BMT 2: lin- BM 3: lin- BM + Ly5.1 pret 1100 cgy * p<0.05 ** p<0.01

24 DN2 PreT can engraft in abdomen and develop into mature T cells Spleen d28

25 MLN support extrathymic development of BM-derived CD4+CD8+ cells d21 MLN

26 MLN support extrathymic development of BM-derived CD4+CD8+ cells in BMT recipients d21

27 ExtrathymicT cell development in HSCT recipients results in functional T cells Extrathymic pret-derived T cells: have a diverse T cell repertoire are primarily naïve CD8 T cells are host-tolerant respond against allogeneic stimulation respond to LCMV

28 Critical role for IL-22 in regeneration of thymic function following immune depletion

29 IL-22 is largely redundant for normal thymopoiesis In mice deficient for IL-22 there was no change in total thymus cellularity or within all developing thymocyte subsets

30 IL-22 deficiency leads to profoundly impaired thymic recovery following immune depletion Following immune insult the role for IL-22 is pronounced: In several models of immune depletion/regeneration Sublethal irradiation alone Lethal Irradiation + syn-bmt Lethal irradiation + allo-bmt Significantly impaired regeneration in all developing thymocyte subsets Treatment Recipient TBI dose BM TBI Alone B6 1 x 550 cgy N/A syn-bmt B6 2 x 550 cgy 5 x 10 6 Ly5.1 allo-bmt BALB/c 2 x 425 cgy 5 x 10 6 B6

31 IL-22 deficiency leads to profoundly impaired thymic recovery following immune depletion Supporting stromal microenvironment also significantly impaired by IL- 22 deficiency Cortical and medullary thymic epithelial cells (ctec and mtec) Notably also impacts on non- TECs primarily endothelium Treatment Recipient TBI dose BM TBI Alone B6 1 x 550 cgy N/A syn-bmt B6 2 x 550 cgy 5 x 10 6 Ly5.1 allo-bmt BALB/c 2 x 425 cgy 5 x 10 6 B6

32 What are the intrathymic targets of IL-22?

33 IL-22 receptor is expressed by most thymic stromal cells IL-22 could be mediating its effects through thymic epithelial cells Consistent with findings in mucosal tissue Notably could also act through non-epithelial cells including endothelium

34 Does IL-22 play a role in the endogenous regeneration of thymic tissue?

35 Yes: Intrathymic levels of IL-22 are significantly increased in periods of regeneration Absolute intrathymic levels of IL-22 were significantly increased in all models of immune damage despite significant decline in total thymic size

36 and intrathymic levels of IL-22 inversely correlate with thymic size There appears to be a threshold of thymic size which triggers the production of IL-22

37 Thymic IL-22 expression is reduced during GVHD ELISA 1 week post-bmt B6 BALB/c

38 Administration of recombinant IL-22 leads to significantly enhanced thymic recovery following immune depletion Recombinant IL-22 can be administered systemically (ip) to rapidly boost thymopoiesis

39 Other (preliminary) findings regarding IL-22 and thymopoiesis - TECs respond to IL-22 with activation of STAT3 and STAT5 - TECs can produce IL-22 - IL-22 can maintain TECs in vitro - IL-22 is not required for the thymopoietic effect of sex steroid ablation

40 Summary IL-7, KGF and Leuprolide show promise in early clinical trials to enhance post-transplant T cell recovery PreT cells can be used as an off-the-shelf adoptive cell therapy across MHC barriers to enhance posttransplant T cell recovery (preclinical data) Extrathymic T cell development (esp. MLN) occurs in HSCT recipients IL-22 can enhance post-transplant thymic recovery

41 Combination strategies to enhance T cell reconstitution following allogeneic HSCT

42 Acknowledgements Van den Brink Laboratory Arnab Ghosh Alan Hanash Natalie Singer Mallory West Lauren Young Jarrod Dudakov Steffen Hartrempf Amanda Holland Robert Jenq M. Lia Palomba Kelly Piersanti Marsinay Smith Jennifer Tsai Andrea Tuckett Johannes Zakrzewski Collaborators MSKCC: Miguel Perales Jenna Goldberg Michel Sadelain Isabelle Riviere University of Minnesota Bruce Blazar University of Florida College of Medicine Chen Liu Cytheris Michel Morre Therese Croughs SOBI Jack Bradley Hans Siltberg