Does NK cell alloreactivity prevent relapse? Yes!!! Andrea Velardi Bone Marrow Transplant Program University of Perugia
Recognition of missing self HLA triggers lysis NK Inhibitory receptor Activating receptor Self HLA Ligand Autologous cell Protection Mismatched HLA NK Inhibitory receptor Activating receptor Ligand Allogeneic target Lysis
Inhibitory KIR recognition of self HLA class I educates NK cells to become fully functional and alloreactive Donor Recipient HLA NK cell Target HLA-C group 2 Cw2 KIR2DL1 HLA-C group 2 Cw2/Cw4 HLA-C group 1 Cw1 KIR2DL 2/3 missing HLA-C group 1 HLA-Bw4 B27 KIR3DL1 HLA-Bw4 B51
The second round of NK cell education - Ruggeri et al., Blood 1999 - Ruggeri et al., Science 22 - Ruggeri et al., Blood 27 - Stern et al., Blood 28
% alloreactive NK clones cells/cmm Post-transplant regeneration of donor vs recipient alloreactive NK repertoires Phenotype of potentially alloreactive NK subsets 6 45 3 15 KIR2DL2/3/S2+ KIR2DL1+ KIR3DL1+ Donor 1 2 3 4 5 6 7 8 9 1 11 12 months Frequencies of alloreactive NK clones 25 2 15 1 Donor KIR ligands missing on target: HLA-C2 HLA-C1 HLA-Bw4 5 Donor 1 3 6 12 months Ruggeri, Blood 1999, Ruggeri, Blood 27, updated 213
Allo NK-based conditioning: Ablation of recipient targets Donor alloreactive NK cells T NK Lysis T NK NK T Kill recipient T cells = improve engraftment. Reduced-intensity MM-BMT Lysis Kill recipient APCs = protect from GvHD. T-replete mismatched BMT with 4 times the lethal dose of allogeneic T cells DC DC Lysis DC leukemia Kill leukemia = GvL effect Ruggeri et al. Science 22 Klas Kärre: A perfect mismatch Science 22 (Editorial)
Probability AML: Event-free Survival 1. Chemoresistant relapse 1. Any remission.8.8 NK alloreactive (n=32).6.6.4 NK alloreactive (n=21).4 P =.4.2 P =.5 Non-NK alloreactive (n=3).2 Non-NK alloreactive (n=32).. 2 4 6 8 1 12 14 16 Years 2 4 6 8 1 12 14 16 Years Ruggeri et al., Blood 1999; Science 22; Blood 27; updated 213
Survival Donor NK cell allospecificity and survival 1. 1..8.8.6 NK alloreactive (n = 53).6 Missing HLA-C1 (n = 23).4 P =.3.4 Missing HLA-Bw4 (n = 9) Missing HLA-C2 (n = 21).2 NK non-alloreactive (n = 62).2. 2 4 6 8 1 12 14 16 Years. 2 4 6 8 1 12 14 16 Years
Relapse and survival of AML patients transplanted in any remission: Results of a randomised study on NK alloreactivity Cumul. incidence of relapse Probability of survival 1, 1,,8,8 NK alloreactive (n=32),6,6,4 Non-NK alloreactive (n=32),4 P =.4,2 P =.3,2 Non-NK alloreactive (n=32), NK alloreactive (n=32), 5 1 15 2 Years 5 1 15 2 Years Ruggeri Blood 1999; Science 22; Blood 27; Stern Blood 28; updated 213
No role for NK cell alloreactivity in URD (T replete)-bmt T cell alloreactions precede NK development and antagonize/obscure NK cell effects T cell T cell T cell T cell T cell T cell T cell T cell T cell T cell T cell Stem cell T-replete BM graft: 4-log more T cells, 1-log fewer stem cells than haplo Fewer NK cells NK Antagonizing effects of post-transplant GVHD prophylaxis/treatment Overwhelm NK alloreactivity! Davies et al. Blood 22 and many others to date: no advantage for KIR ligand incomp. in URD-BMT!
T cell depleted megadose stem cell transplant Non-NK alloreactive NK alloreactive
Positively selected, megadose CD34+ grafts (Reisner, Martelli) TCR-αβ/CD19 negatively selected grafts = upfront NK cell therapy! (Handgretinger) Non-T cell-depleted (unmanipulated) grafts with post transplant cyclophosphamide (Fuchs; Bacigalupo), or post-transplant rapamycin (Ciceri), G-CSF priming of donor bone marrow, and intensified post transplant immune suppression (Huang; Arcese).
Elderly high risk AML patients in morphological (or better) CR after (re-) induction and consolidation chemotherapy, not eligible for stem cell transplantation INDUCTION/CONSOLIDATION CHEMOTHERAPY SELECTION OF HAPLOIDENTICAL NK ALLOREACTIVE DONOR MORPHOLOGICAL OR BETTER CR LEUKAPHERESIS AND CD3-/CD56+ NK SELECTION 1) LYMPHOABLATION BY CY+FLU (FOR EMPTINESS!) 2) NK CELL INFUSION (1-5x1 6 /Kg CD3-/CD56+) FOLLOW UP
% donor chimerism IL-15 (pg/ml) NK cells/ L 12 4 1 8 6 4 2 5 1 15 2 25 3 days after NK cell infusion 3 2 1 3 5 1 17 24 days after NK cell infusion 2 16 12 8 4 PB BM 3 5 1 13 17 2 28 days after NK cell infusion
% lysis cells/ L 2 KIR2DL2/3+/NKG2A- 2 KIR2DL1+/NKG2A- 1 1 3 6 9 12 15 18 21 24 27 3 3 6 9 12 15 18 21 24 27 3 days post NK infusion days post NK infusion 1 1 5 5 HLA-C1+ donor alloreactive NK clones detected in HLA-C1 missing patients HLA-C2+ donor alloreactive NK clones detected in HLA-C2 missing patients
Patients characteristics 16 patients Median Age (yrs) 65 (51-73) Sex (M/F) 8/8 WBC>3x1 9 /L 4/16 (25%) Secondary AML 3/16 (18%) Cytogenetics: Favorable (t8;21;inv16) Intermediate (normal; -Y) Unfavorable (other than favorable and intermediate) 2/16 (12.5%) 12/16 (75%) 2/16 (12.5%) Genotype: NPM+/FLT3- NPM+/FLT3+ NPM-/FLT3- NPM-/FLT3+ /16 (%) /16 (%) 14/16 (87%) 2/16 (13%)
Overall survival of elderly AML patients in CR at the time of NK cell infusion vs non-nk cell treated controls NK tx No NK tx from CR no GvHD, no myeloablation, no toxicity other than chemo itself
Larger alloreactive NK cell repertoires are associated with control of leukemia relapse % alloreactive NK clones 2 * * Patients in remission after NK treatment (8) 15 * Patients in relapse after NK treatment (5) 1 5 * P=.1 * * P=.4 Donors +3 +9 +12 +18 +2 days post NK infusion
Treg and Tcon adoptive immunotherapy in haplo HSCT for patients with high risk AL 2x1 6 /kg 1x1 6 /kg days 1 st clinical trial 8 Gy TBI Thiotepa Fludarabine Cyclophosphamide Conditioning Di Ianni et al., Blood 211; EBMT 213 (A Tabilio, F Falzetti) 2 nd clinical trial Sept 28-Oct 29 May 21-Sept 213 Anti-T cell Abs 8 Gy TBI Thiotepa Fludarabine T regs In vivo activation of donor Tregs specific for recipient allo-antigens CD34 + Tcons 1x1 6 /kg No post-transplant immunosuppression
Days post BMT Days post BMT PRIMARY ENGRAFTMENT 2/2 (1%) 3 6 2 4 1 2 >5 >1 ANC/ l >25 >5 PLT/mm 3 GRAFT vs HOST DISEASE Grade II 4/2 (2%)
Proliferating pathogen specific CD8+ T cells/1 6 cells Proliferating pathogen -specific CD4+ T cells/1 6 cells Long-term reconstitution of pathogen-specific T cells capable of clonal expansion 1 1 1 1 Donors 1 1 1 1 Standard Haplo (n=15) 1 1 1 1 T-reg Haplo (n=2) ASP Cand CMV ADV HSV VZV Toxo 1 1 1 1 1 2 4 6 8 1 12 1 2 4 6 8 1 12 1 1 1 1 1 1 2 4 6 8 1 12 Months after transplant 2 4 6 8 1 12 Months after transplant
GvL? Strange case of NK cell - T cell collaboration
cells/cmm % alloreactive NK clones cells/cmm % alloreactive NK clones 6 45 3 15 6 Treg immunotherapy did not impair regeneration of donor vs recipient alloreactive NK cell repertoires Standard haplo (single) KIR2DL2/3+ (single) KIR2DL1+ (single) KIR3DL1+ Donor 1 2 3 4 5 6 7 8 9 1 11 12 Donor 1 3 6 12 T-reg haplo Lysis of KIR ligand-mismatched targets 25 2 15 1 5 25 C2 mismatch C1 mismatch Bw4 mismatch 45 2 3 15 1 15 5 Donor 1 2 3 4 5 6 7 8 9 1 11 12 months post transplant Donor 1 3 6 12 months post transplant
Survival (%) Clearance of human leukemia by human alloreactive NK clones in SCID mice CML blastic crisis in NOD-SCID mice Human CD45 BCR/ABL non-allo NK 1 allo NK, 1 5 allo NK, 2x1 5 allo NK, 4x1 5 5 control Ruggeri et al. Science 22 3 12 Days
Ruggeri et al. Science 22
CD4+CD25+ regulatory T cells preserve graft versus-tumor-activity while inhibiting graftversus host disease after bone marrow transplantation Edinger et al., Nat Med 23, 9:1144-115 % survival Clearance of human leukemia by human T regs + T cons in immunodeficient mice Leukemia (7x1 6 ) T cells (3x1 6 ) 1 8 6 4 2 Leukemia Leuk. + T cons = no leuk, GvHD Leuk. + T regs = leukemia Leuk. + T regs/tcons = survival w/o leukemia and GvHD 5 1 15 Days
Cumulative incidence Cumulative incidence Strange case of NK cell + T cell collaboration..2.4.6.8 1...2.4.6.8 1...2.4.6.8 1. Standard Haplo ALL NK allo (n=19) Non-NK allo (n=31) T reg Haplo ALL Non-NK allo (n=8) NK allo (n=3) 5 1 15 2 1 2 3 4 5..2.4.6.8 1. AML Non-NK allo (n=32) NK allo (n=32) 5 1 15 2 AML Non-NK allo (n=2) NK allo (n=14) 1 2 3 4 5 Years post Tx
BMT Unit Alessandra Carotti Adelmo Terenzi Rita Felicini Lucia Amico Maria Speranza Massei Luca De Carolis Sara Piccinelli Graft processing Franca Falzetti Mauro Di Ianni Roberta Iacucci Tiziana Zei Immunology Loredana Ruggeri Antonella Mancusi Antonio Pierini Elena Urbani Fabiana Topini Antonella Tosti Franco Aversa Massimo F Martelli Professor Emeritus EU
Issue Highlights: NK Cells: Heroes in Bone Marrow Transplants Editorial Klass Kärre (Karolinska Institutet)