Inflammation ( # 2014) DOI: 10.1007/s10753-014-0082-0
Natural Killer Cell Functional Activity After 4-1BB Costimulation Shadi sadat Navabi,1 Mehrnoosh Doroudchi,1 Ahmad Hosseini Tashnizi,2 and Mojtaba Habibagahi1,3
Abstract—Reports show enhancement of CD8 T cells’ activity through CD137 (4-1BB) signal; however, not all data proved similar effect in natural killer (NK) cells. Here, the impact of 4-1BB signal on NK cells’ function was assessed during short term cultures. To that end, cytokine-activated NK cells were cocultured with adenovirally transduced MCF-7 stimulator cells expressing 4-1BB ligand. Cellular cytotoxicity, cytokine production, and expression of cytotoxicity related genes were assessed after overnight cultures. Sharp decrease of CD56+ and CD56bright NK cells was demonstrated. 4-1BB neither enhanced cellular degranulation nor improved IFN-γ production although it promoted granzyme B, perforin, and FasL gene expression. 4-1BB signal stimulated higher proportions of CD56bright population to degranulate and express CD107a; however, it could not recover killing activity against K562 targets. Our data could not show major promotion in activity of all NK subpopulations. Due to great heterogeneity of NK cells, more investigation is needed to draw a comprehensive conclusion. KEY WORDS: CD137 (4-1BB); CD137 ligand (4-1BBL); costimulation; natural killer cell.
INTRODUCTION Natural killer (NK) cells are known as CD3−TCR− large granular cytotoxic lymphocytes with major immune reactivity against tumors and virus-infected cells without prior activation. Unique features of NK cells have inspired scientists to exploit NK cells for cancer immunotherapy. However, NK cells are not a homogeneous population and several subtypes can be recognized among these cells which might not react similarly against their targets [1]. Unlike T and B lymphocytes, receptors of NK cells do not undergo genetic rearrangement. Instead, innate immune system contains diverse arrays of NK cells with differential expression of a wide variety of receptors and functions [2– 4]. Simply, based on the expression levels of neural cell adhesion molecule (NCAM, CD56), NK cells can be subdivided as CD56bright (high expression) and CD56dim 1
Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran 2 Institute for Cancer Research, Shiraz University of Medical Sciences, Shiraz, Iran 3 To whom correspondence should be addressed at Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran. E-mail: [email protected]
(low expression) which can reflect their differences in function as well [1]. In the current models of NK cell activation, NK cells govern cell activation/inhibition by keeping precise balance between signals transduced from activating and inhibitory receptors [5]. Recent evidence also show adaptive-like immune reactivities of NK cells, similar to Ag specific and memory formation of T cells, which should be incorporated in the activation scenario of NK cells. In fact, in addition to the traditional ideas of existence of “missing self receptors” and the “pathogen recognition receptors,” NK cells express arrays of other surface molecules which can participate in the process of cell activation. It has been demonstrated that NKG2D transduces activation signals to NK cells similar to CD28 to activate p85 subunit of phosphatidylinositol 3-kinase (PI3K) [6]. In this regard, it is being increasingly appreciated that activation of NK cells, like T cells, can take advantage of several costimulatory ligand receptor interactions [3, 7]. In case of T cells, multiple costimulatory interactions during Ag stimulation can shape the ultimate T cell function [8–10]. Among them, signal transduction by CD137 (4-1BB), a member of tumor necrosis factor receptor (TNFR) superfamily, costimulates T cells independent of CD28 for proliferation, and enhances T cell
0360-3997/14/0000-0001/0 # 2014 Springer Science+Business Media New York
Navabi, Doroudchi, Tashnizi, and Habibagahi survival after the initial activation [8, 11]. Like T cells, human and murine NK cells express 4-1BB after activation which can interact with the ligand (4-1BBL) or anti-CD137 antibodies and transduce intracellular signals [12, 13]. While some studies showed negative impact of CD137 signal on cytotoxic activities or cytokine production by human or mice NK cells [13–15], others could demonstrate the enhancement of those activities [16–18]. It means that more investigations are needed to resolve the controversies about the role of 4-1BB costimulation for NK cells’ function. Therefore, in this study, we focused on cytotoxicity function of NK cells in response to short exposure with 41BBL-expressing tumor stimulator cells. To this end, target cell killing, cytokine production, and expression of three cytotoxicity related genes by NK cells from different culture conditions were assessed. Results showed that 4-1BB signal may not be able to enhance the activity of all NK cell subpopulations as it does in T cells although different NK cell subsets reacted differently.
MATERIALS AND METHODS Antibodies and Reagents Recombinant human interleukin (IL)-2 and IL-15 proteins and PerCP-Cy5-labeled anti-CD56 antibody were obtained from Abcam, UK. The fluorescent-conjugated monoclonal antibodies including CD16-FITC, CD137PE, CD137L-PE, CD107a-APC, and cocktail of FITClabeled anti-CD3 with PE-labeled anti-CD16/CD56 were purchased from BD (BD Pharmingen, USA). The protein transport inhibitor (brefeldin A) and GolgiStop (monensin) were also obtained from BD (BD Pharmingen, USA). Carboxyfluorescein diacetate succinimydyl ester (CFSE) (Molecular Probes), TRIzol RNA extraction reagent (Invitrogen), Master Mix for real-time PCR (Applied Biosystems) were purchased from Life Technology, USA. RevertAid First-Strand cDNA Synthesis Kit (Fermentas, Lithuania) was used to produce cDNA samples. Interferon-γ (IFN-γ) enzyme-linked immunosorbent assay (ELISA) kit was purchased from eBioscience (Bendermed Systems), Austria. Magnetic beads for separation of NK cells were provided by Miltenyi Biotec, Germany. Purifications of Human NK Cells Peripheral blood samples were obtained by venipuncture from healthy lab donors. All volunteers gave their written informed consent, and the study was approved by
the ethics committee of the university. Using magnetic beads, NK cells were purified from PBMCs by negative selection according to the manufacturer’s instructions. The purity of the enriched NK cells was evaluated by staining for CD3, CD16, and CD56 and flow cytometry using FACSCalibur instrument (BD, USA) with CellQuest Pro platform for acquisition of the data. FlowJo software (Tree Star, Inc., USA) was used for analysis and presentation of the data. A minimum purity and viability of 90–95 % for the enriched NK cells was considered in all experiments.
Production of NK Cell Stimulators by Adenoviral Transduction in MCF-7 Cells E1- and E3-deleted recombinant Ad-4-1BBL and AdGFP adenoviral vectors expressing human 4-1BBL and the green fluorescent protein (GFP), respectively, were courtesy provided by Dr. P.F. Searle (Gene Therapy laboratory, Birmingham University, UK). MCF-7 human breast cancer cell line was used to overexpress the natural ligand for 41BB (or GFP as control) after infection with one MOI of the viruses. The infected cells were seeded into 24-well plates at 1×105 cells/ml and used as NK cell stimulator after 48 h. The expressions of the transgenes by the infected cells were verified by flow cytometry.
Activation of NK Cells in Coculture with MCF-7 Stimulators For the initial activation of NK cells and to induce the expression of CD137, the purified cells were cultured in complete medium containing 10 % bovine serum (Biosera, UK), 50 IU/ml of IL-2, and 25 ng/ml of IL-15 and kept for 48 h at the standard culture condition. Afterward, NK cells (2×105) were dispensed into the wells already contained MCF-7 cells with or without 4-1BBL or GFP expression. Coculture of the NK and target cells were incubated for further 12 h; then, NK cells were used for downstream experiments.
Measurement of IFN-γ Cytokine Secretion Cell-free culture supernatant from 12-h cultures of MCF-7 cells alone or NK cells with MCF-7 stimulators with or without the expression of the transgenes were collected, and the concentration of IFN-γ was measured by high-sensitivity ELISA kits in duplicates according to the manufacturer’s instruction.
4-1BB Signal Not Always Costimulate NK Cells Flow-Cytometry-Based Cytotoxicity Assay with CFSE Dye Cytotoxic activity of the cultured NK cells was investigated by flow cytometry using CFSE-labeled K562 cells as target. MCF-7 cells were infected to express 4-1BBL or GFP and used to stimulate purified NK cells in cultures as described. Subsequently, NK cells were harvested and cocultured with CFSElabeled K562 target cells at 2:1 ratio (2 × 105 NK cells: 1 × 105 K562 cells). Cultures were incubated for 5 h, then harvested, washed, and stained with propidium iodide (PI, 2.5 μg/ml). At last, cells were analyzed by flow cytometry.
2−ΔΔCt, was used to combine gene quantification and normalization into a single calculation where GFP control cultures were used as baseline [19]. Statistical Analysis The differences between results from various experimental conditions were analyzed by non-parametric Wilcoxon signed rank test using SPSS version 16 (IBM SPSS, USA) and GraphPad Prism version 5 software (GraphPad, USA). In all statistical analysis, p
Natural Killer Cell Functional Activity After 4-1BB Costimulation Shadi sadat Navabi,1 Mehrnoosh Doroudchi,1 Ahmad Hosseini Tashnizi,2 and Mojtaba Habibagahi1,3
Abstract—Reports show enhancement of CD8 T cells’ activity through CD137 (4-1BB) signal; however, not all data proved similar effect in natural killer (NK) cells. Here, the impact of 4-1BB signal on NK cells’ function was assessed during short term cultures. To that end, cytokine-activated NK cells were cocultured with adenovirally transduced MCF-7 stimulator cells expressing 4-1BB ligand. Cellular cytotoxicity, cytokine production, and expression of cytotoxicity related genes were assessed after overnight cultures. Sharp decrease of CD56+ and CD56bright NK cells was demonstrated. 4-1BB neither enhanced cellular degranulation nor improved IFN-γ production although it promoted granzyme B, perforin, and FasL gene expression. 4-1BB signal stimulated higher proportions of CD56bright population to degranulate and express CD107a; however, it could not recover killing activity against K562 targets. Our data could not show major promotion in activity of all NK subpopulations. Due to great heterogeneity of NK cells, more investigation is needed to draw a comprehensive conclusion. KEY WORDS: CD137 (4-1BB); CD137 ligand (4-1BBL); costimulation; natural killer cell.
INTRODUCTION Natural killer (NK) cells are known as CD3−TCR− large granular cytotoxic lymphocytes with major immune reactivity against tumors and virus-infected cells without prior activation. Unique features of NK cells have inspired scientists to exploit NK cells for cancer immunotherapy. However, NK cells are not a homogeneous population and several subtypes can be recognized among these cells which might not react similarly against their targets [1]. Unlike T and B lymphocytes, receptors of NK cells do not undergo genetic rearrangement. Instead, innate immune system contains diverse arrays of NK cells with differential expression of a wide variety of receptors and functions [2– 4]. Simply, based on the expression levels of neural cell adhesion molecule (NCAM, CD56), NK cells can be subdivided as CD56bright (high expression) and CD56dim 1
Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran 2 Institute for Cancer Research, Shiraz University of Medical Sciences, Shiraz, Iran 3 To whom correspondence should be addressed at Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran. E-mail: [email protected]
(low expression) which can reflect their differences in function as well [1]. In the current models of NK cell activation, NK cells govern cell activation/inhibition by keeping precise balance between signals transduced from activating and inhibitory receptors [5]. Recent evidence also show adaptive-like immune reactivities of NK cells, similar to Ag specific and memory formation of T cells, which should be incorporated in the activation scenario of NK cells. In fact, in addition to the traditional ideas of existence of “missing self receptors” and the “pathogen recognition receptors,” NK cells express arrays of other surface molecules which can participate in the process of cell activation. It has been demonstrated that NKG2D transduces activation signals to NK cells similar to CD28 to activate p85 subunit of phosphatidylinositol 3-kinase (PI3K) [6]. In this regard, it is being increasingly appreciated that activation of NK cells, like T cells, can take advantage of several costimulatory ligand receptor interactions [3, 7]. In case of T cells, multiple costimulatory interactions during Ag stimulation can shape the ultimate T cell function [8–10]. Among them, signal transduction by CD137 (4-1BB), a member of tumor necrosis factor receptor (TNFR) superfamily, costimulates T cells independent of CD28 for proliferation, and enhances T cell
0360-3997/14/0000-0001/0 # 2014 Springer Science+Business Media New York
Navabi, Doroudchi, Tashnizi, and Habibagahi survival after the initial activation [8, 11]. Like T cells, human and murine NK cells express 4-1BB after activation which can interact with the ligand (4-1BBL) or anti-CD137 antibodies and transduce intracellular signals [12, 13]. While some studies showed negative impact of CD137 signal on cytotoxic activities or cytokine production by human or mice NK cells [13–15], others could demonstrate the enhancement of those activities [16–18]. It means that more investigations are needed to resolve the controversies about the role of 4-1BB costimulation for NK cells’ function. Therefore, in this study, we focused on cytotoxicity function of NK cells in response to short exposure with 41BBL-expressing tumor stimulator cells. To this end, target cell killing, cytokine production, and expression of three cytotoxicity related genes by NK cells from different culture conditions were assessed. Results showed that 4-1BB signal may not be able to enhance the activity of all NK cell subpopulations as it does in T cells although different NK cell subsets reacted differently.
MATERIALS AND METHODS Antibodies and Reagents Recombinant human interleukin (IL)-2 and IL-15 proteins and PerCP-Cy5-labeled anti-CD56 antibody were obtained from Abcam, UK. The fluorescent-conjugated monoclonal antibodies including CD16-FITC, CD137PE, CD137L-PE, CD107a-APC, and cocktail of FITClabeled anti-CD3 with PE-labeled anti-CD16/CD56 were purchased from BD (BD Pharmingen, USA). The protein transport inhibitor (brefeldin A) and GolgiStop (monensin) were also obtained from BD (BD Pharmingen, USA). Carboxyfluorescein diacetate succinimydyl ester (CFSE) (Molecular Probes), TRIzol RNA extraction reagent (Invitrogen), Master Mix for real-time PCR (Applied Biosystems) were purchased from Life Technology, USA. RevertAid First-Strand cDNA Synthesis Kit (Fermentas, Lithuania) was used to produce cDNA samples. Interferon-γ (IFN-γ) enzyme-linked immunosorbent assay (ELISA) kit was purchased from eBioscience (Bendermed Systems), Austria. Magnetic beads for separation of NK cells were provided by Miltenyi Biotec, Germany. Purifications of Human NK Cells Peripheral blood samples were obtained by venipuncture from healthy lab donors. All volunteers gave their written informed consent, and the study was approved by
the ethics committee of the university. Using magnetic beads, NK cells were purified from PBMCs by negative selection according to the manufacturer’s instructions. The purity of the enriched NK cells was evaluated by staining for CD3, CD16, and CD56 and flow cytometry using FACSCalibur instrument (BD, USA) with CellQuest Pro platform for acquisition of the data. FlowJo software (Tree Star, Inc., USA) was used for analysis and presentation of the data. A minimum purity and viability of 90–95 % for the enriched NK cells was considered in all experiments.
Production of NK Cell Stimulators by Adenoviral Transduction in MCF-7 Cells E1- and E3-deleted recombinant Ad-4-1BBL and AdGFP adenoviral vectors expressing human 4-1BBL and the green fluorescent protein (GFP), respectively, were courtesy provided by Dr. P.F. Searle (Gene Therapy laboratory, Birmingham University, UK). MCF-7 human breast cancer cell line was used to overexpress the natural ligand for 41BB (or GFP as control) after infection with one MOI of the viruses. The infected cells were seeded into 24-well plates at 1×105 cells/ml and used as NK cell stimulator after 48 h. The expressions of the transgenes by the infected cells were verified by flow cytometry.
Activation of NK Cells in Coculture with MCF-7 Stimulators For the initial activation of NK cells and to induce the expression of CD137, the purified cells were cultured in complete medium containing 10 % bovine serum (Biosera, UK), 50 IU/ml of IL-2, and 25 ng/ml of IL-15 and kept for 48 h at the standard culture condition. Afterward, NK cells (2×105) were dispensed into the wells already contained MCF-7 cells with or without 4-1BBL or GFP expression. Coculture of the NK and target cells were incubated for further 12 h; then, NK cells were used for downstream experiments.
Measurement of IFN-γ Cytokine Secretion Cell-free culture supernatant from 12-h cultures of MCF-7 cells alone or NK cells with MCF-7 stimulators with or without the expression of the transgenes were collected, and the concentration of IFN-γ was measured by high-sensitivity ELISA kits in duplicates according to the manufacturer’s instruction.
4-1BB Signal Not Always Costimulate NK Cells Flow-Cytometry-Based Cytotoxicity Assay with CFSE Dye Cytotoxic activity of the cultured NK cells was investigated by flow cytometry using CFSE-labeled K562 cells as target. MCF-7 cells were infected to express 4-1BBL or GFP and used to stimulate purified NK cells in cultures as described. Subsequently, NK cells were harvested and cocultured with CFSElabeled K562 target cells at 2:1 ratio (2 × 105 NK cells: 1 × 105 K562 cells). Cultures were incubated for 5 h, then harvested, washed, and stained with propidium iodide (PI, 2.5 μg/ml). At last, cells were analyzed by flow cytometry.
2−ΔΔCt, was used to combine gene quantification and normalization into a single calculation where GFP control cultures were used as baseline [19]. Statistical Analysis The differences between results from various experimental conditions were analyzed by non-parametric Wilcoxon signed rank test using SPSS version 16 (IBM SPSS, USA) and GraphPad Prism version 5 software (GraphPad, USA). In all statistical analysis, p
