Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Immune system wikipedia , lookup
Psychoneuroimmunology wikipedia , lookup
Molecular mimicry wikipedia , lookup
Polyclonal B cell response wikipedia , lookup
Adaptive immune system wikipedia , lookup
Lymphopoiesis wikipedia , lookup
Cancer immunotherapy wikipedia , lookup
Immunosuppressive drug wikipedia , lookup
NK cells Expansion and Activation for Cancer Immunotherapy Arkadi Yakirevitch*, Naama Zabari, Jacob Schachter, Michal J. Besser Ella Institute for Melanoma Research and Department of Otorhinolaryngology-H&NS* Sheba Medical Center, Tel-Hashomer, Israel Background Background ¾Natural ¾Natural Killer Killer (NK) (NK) cells cells are are large large granular granular lymphocytes lymphocytes of of the the innate innate immune immune system, system, comprising comprising ~ ~ 15% 15% of of all all circulating circulating lymphocytes lymphocytes ¾In ¾In contrast contrast to to BB and and TT cells, cells, they they do do not not rearrange rearrange the the TT cell cell receptor receptor or or the the immunoglobulin immunoglobulin genes genes ¾NK ¾NK cell cell killing killing does does not not require require that that the the tumor tumor cells cells express express intact intact self-MHC self-MHC antigens. antigens. In In addition, addition, NK NK cells cells are are able able to to be be catalytic catalytic without without prior prior sensitization. sensitization. Further Further activation activation of of NK NK cells cells by by cytokines cytokines can can increase increase killing killing activity activity and and broaden broaden the the spectrum spectrum of of malignant malignant cells cells that that are are killed. killed. Activated Activated NK NK cells cells kill kill target target cells cells through through various various mechanisms, mechanisms, predominantly predominantly through through the the release release of of cytolytic cytolytic enzymes enzymes ¾Donor ¾Donor NK NK cell cell infusion infusion can can provide provide potent potent antitumour, antitumour, antiviral antiviral and and engraftment-facilitating engraftment-facilitating activity, activity, without without causing causing GVHD GVHD ¾In ¾In order order to to avoid avoid GVHD GVHD in in clinical clinical application, application, alloreactive alloreactive NK NK cells cells need need to to be be expanded expanded in in extensive extensive TT cell-depleted cell-depleted setting setting Materials Materials and and methods methods Study Study Purpose Purpose To To define define the the best best algorithm algorithm of of the the isolation, isolation, activation activation and and expansion expansion of of the the NK NK cells cells for for clinical clinical ¾Cell ¾Cell source: source: human human peripheral peripheral blood blood mononuclear mononuclear cells cells ¾Isolation: ¾Isolation: CD3 CD3 depletion depletion using using magnetic magnetic microbeads microbeads cell cell sorting sorting use use under under good good manufacturing manufacturing practice practice conditions conditions ¾Purity ¾Purity estimation: estimation: flow flow cytometry cytometry analysis analysis Exp. No. Checked parameters 1 Materials and Methods Results Various conditions of incubation Medium types: X-VIVO, CellGro, AIM-V, DMEM IL-2 concentration 62.5-1000 IU/ml PHA concentration: 1-10 μg/ml NK: feeder cell proportion: 1:1, 1:10, 1:10 (2 rounds) OKT-3 +/Incubation of fresh and Fresh and frozen NK and feeder frozen NK and feeder cells cells of the same donor Donor-to-donor variations in Fresh NK cells of two donors incubation results T cells depletion CD3 depletion of PBMCs on 2X LS columns--> 1XLD. Number of feeder rounds 1 or 2 rounds (the 2nd on the 7th day) during expansion 2 3 4 5 Effect of feeder preincubation with OKT3 Preincubation with OKT3 Preincubation with OKT3 -> washing Direct adding of OKT3 to culture PBMC separation on 2 followed columns has a benefit in context of T cell contamination/NK purity Second round of feeders results in high purity Growth Curves, Experiment 5 250 400 6 200 NK Number, X10 NK Fold Expansion No donor dependence of expansion rates Preincubation with OKT3 has a positive effect on both purity and expansion of the NK population Experiment 5, Day 19 150 100 50 0 20% High purity of NK cells with use of CellGro Low purity of NK cells with use of AIM-V Unsatisfying expansion rates with use of IL-2 or PHA alone Optimal IL-2 concentration 500 IU/ml Optimal PHA concentration 5 μg/ml 2nd round of feeder seems of benefit Better expansion rates of the fresh NK cells 40% 60% 80% 100% Purity 300 200 100 0 0 Day 13 19 Medium Feeders OKT3 AIM-V X10x10 direct addition X-VIVO X10x10 direct addition X-VIVO x10x10 feeder preincubation with OKT3 X-VIVO x10x10 feeder preincubation with OKT3 -> wash