Effects of mycophenolate mofetil and cyclosporin A on cord blood and peripheral blood natural killer cells

EFFECTS OF MYCOPHENOLATE MOFETIL AND CYCLOSPORIN A ON CORD BLOOD AND PERIPHERAL BLOOD NATURAL KILLER CELLS JULIANA RODRIGUES PERAZZO Dissertação de Mestrado em Bioquímica Universidade do Porto Faculdade de Ciências Instituto de Ciências Biomédicas Abel Salazar 2012 JULIANA RODRIGUES PERAZZO EFFECTS OF MYCOPHENOLATE MOFETIL AND CYCLOSPORIN A ON CORD BLOOD AND PERIPHERAL BLOOD NATURAL KILLER CELLS Dissertação de Candidatura ao grau de Mestre em Bioquímica da Universidade do Porto Orientador – Doutora Aurore Saudemont Categoria – Investigadora Sénior Afiliação – Anthony Nolan Research Institute Co-orientador – Doutora Sophie Derniame Categoria – Investigadora de Pós-doutoramento Afiliação – Anthony Nolan Research Institute 2012 Acknowledgments V Acknowledgments First, I would like to thank Professor Alejandro Madrigal for allowing me to do my master training at the Anthony Nolan Research Institute and for all the comments he made about my work, they were very constructive and encouraging. I would like to thank my supervisor Dr. Aurore Saudemont for her invaluable guidance, for her support and encouragement throughout the time I spent at Anthony Nolan. I am also especially grateful to my co-supervisor Dr. Sophie Derniame for recognising my efforts and encouraging me to pursue the project presented in this thesis. Her constant guidance was very much appreciated. I also wish to express my appreciation to the Anthony Nolan Research Institute staff for being helpful, friendly and welcoming. I would like to thank in particular Michelle Escobedo-Cousin, PhD student, for all her help, support and friendship. I thank the Anthony Nolan Research Institute for providing access to all facilities. Last, but not least, I wish to express my gratitude to all my friends and family for their support and friendship. Table of Contents VII Table of Contents Acknowledgments ............................................................................................... V Table of Contents .............................................................................................. VII List of Figures .................................................................................................... XI List of Tables ....................................................................................................XIII Abbreviations .................................................................................................... XV Summary .......................................................................................................... XIX Resumo ............................................................................................................ XXI Introduction ........................................................................................................ 1 1. Human Natural Killer Cells ................................................................ 1 1.1. NK Cell Subsets ...................................................................... 2 1.2. NK Cell Development ............................................................. 2 1.3. NK Cell Recognition and NK Cell Receptors ............................ 3 2. CB NK Cells ...................................................................................... 5 3. Haematopoietic Stem Cell Transplantation ....................................... 6 3.1. Type of Donor and Source of Stem Cells ................................ 7 3.2. Preparative Regimens ............................................................. 7 3.3. GvL versus GvHD ................................................................... 8 4. Immunosuppressive Drugs ............................................................... 8 4.1. Mycophenolate Mofetil ........................................................... 9 4.1.1. Mechanism of Action ............................................................ 9 4.1.2. Effects on B and T Lymphocytes .......................................... 10 4.1.3. Effects on NK Cells ............................................................. 11 4.1.4. Effects on Dendritic Cells .................................................... 11 4.1.5. Effects on Adhesion Molecules ............................................ 11 4.2. Cyclosporine A ..................................................................... 11 4.2.1. Mechanism of Action .......................................................... 12 Table of Contents VIII 4.2.2. Pharmacokinetic ................................................................. 12 4.2.3. Toxic Effects ....................................................................... 12 4.2.4. Effects on Lymphocytes and other Cell Types ..................... 12 5. Aim ................................................................................................ 13 Materials and Methods ...................................................................................... 15 1. Collection of Blood Samples ........................................................... 15 2. Cell Isolation .................................................................................. 15 2.1. Peripheral Blood Mononuclear Cell Isolation ......................... 15 2.2. Cord Blood Mononuclear Cell isolation ................................. 15 2.3. Natural Killer Cell isolation ................................................... 16 3. Cell Culture .................................................................................... 16 4. Phenotypic Analysis ........................................................................ 18 4.1. Gating Strategy .................................................................... 18 4.1.1. Effects of Immunosuppressants on NK Cell Viability............ 18 4.1.2. Effects of Immunosuppressants on NK Cell Activation ......... 19 5. Proliferation Assay ......................................................................... 20 6. CD107a Degranulation Assay ......................................................... 20 7. RNA isolation ................................................................................. 20 8. Reverse Transcription ..................................................................... 21 9. Real Time PCR ................................................................................ 21 10. Statistical Analysis ...................................................................... 21 Results .............................................................................................................. 23 1. Effects of Immunosuppressants on NK Cell Viability ....................... 23 2. Effects of Immunosuppressants on NK Cell Activation .................... 24 3. Effects of Immunosuppressants on NK Cell Proliferation ................. 26 4. Effects of Immunosuppressants on NK Cell Cytotoxicity ................. 28 5. Relative Quantification of IMPDH1, IMPDH2 and NFATc Expression in PB and CB NK Cells by Quantitative Real Time PCR ................................. 31 Discussion ........................................................................................................ 33 Table of Contents IX Conclusion ........................................................................................................ 39 Future Perspectives ........................................................................................... 41 References ........................................................................................................ 43 Abbreviations XVI Fc Fragment crystallisable G-CSF Granulocyte-colony-stimulating factor GDP Guanosine diphosphate GMP Guanosine monophosphate GTP Guanosine triphosphate GvH Graft versus host GvHD Graft versus host disease GvL Graft versus leukaemia h Hours HLA Human leucocyte antigen HPC Haematopoietic progenitor cell HSC Haematopoietic stem cell HSCT Haematopoietic stem cell transplantation i.e. Id est (the same as “that is”) IFN   Interferon IL Interleukin IMP Inosine monophosphate IMPDH Inosine-5’-monophosphate dehydrogenase KIR Killer immunoglobulin-like receptor KLRG1 Killer cell lectin-like receptor subfamily G member 1 LAK Lymphokine-activated killer LAMP Lysosomal-associated membrane glycoprotein LFA Lymphocyte function-associated antigen LN Lymph node MET Methotrexate MHC Major Histocompatibility Complex mHSC Mobilised haematopoietic stem cell mL Millilitre MMF Mycophenolate mofetil MPA Mycophenolic acid mPB Mobilised peripheral blood mRNA Messenger ribonucleic acid NCR Natural cytotoxicity receptor NFAT Nuclear factor of activated T cells ng Nanogram NK Natural killer Abbreviations XVII NKp NK cell related protein NKPs NK cell precursors NKR NK-cell receptor OD Optical density PB Peripheral Blood PBL Peripheral blood lymphocytes PBMCs Peripheral blood mononuclear cells PBS Phosphate-buffered saline PBSC Peripheral blood stem cell PCR Polymerase chain reaction Pen-Strep Penicillin-streptomycin PILR Paired Ig-like receptor PMA Phorbol 12-myristate 13-acetate PRPP Phosphoribosyl pyrophosphate RNA Ribonucleic acid RPMI Roswell Park Memorial Institute SC Stem cell SD Standard deviation TCR T cell receptor TNC Total nucleated cell TNF Tumour necrosis factor TOP 1 Topoisomerase 1 TRM Treatment-related mortality UBC Ubiquitin C UK United Kingdom VCAM Vascular cell adhesion protein Summary XIX Summary Haematopoietic stem cell transplantation (HSCT) is used to treat haematological malignancies and bone marrow (BM) failures. For many years BM was the only source of haematopoietic stem cells (SCs) for transplantation, but now SCs from mobilised peripheral blood (mPB) and cord blood (CB) are increasingly used. Cord blood transplantation (CBT) presents several advantages compared to bone marrow transplantation (BMT) including a greater tolerance to histocompatibility leukocyte antigen (HLA) disparity, a lower risk of graft versus host disease (GvHD) and a preserved graft versus leukaemia (GvL) effect. However, GvHD remains a major cause of morbidity and mortality after HSCT. Cyclosporine A (CsA) and methotrexate (MET) were the standard immunosuppressive drugs used as prophylaxis treatment for GvHD in the United Kingdom (UK), but over the past twenty years new immunosuppressive drugs were developed in order to improve the efficacy and decrease the side effects of CsA and MET, such as mycophenolate mofetil (MMF), anti-T lymphocyte globulin (ATG), Campath and Rapamycin (RAPA). CsA and MMF have been successfully used alone or in combination to prevent GvHD. The effects of those two immunosuppressive drugs have been studied on T cells, but little is known about their effects on natural killer (NK) cells. NK cells provide the beneficial GvL effect without causing GvHD, contributing to reduced relapse rates. Moreover, NK cells reconstitute early after CBT providing GvL and the first line of defence against infectious organisms. Several studies have shown that NK cells are beneficial for clinical outcome. To determine the effects of MPA, CsA and the combination of the drugs on peripheral blood (PB) and CB NK cells an approach based on flow cytometry and quantitative real time PCR was used. MPA and MPA+CsA induced cell death of CB NK cells, but not of PB NK cells. The immunosuppressants did not have a significant effect on the expression of the activation markers CD69 and NKp44 by NK cells, but significantly reduced CB NK cell proliferation. MPA reduced perforin production by PB NK cells, while CsA and MPA+CsA drastically decreased PB and CB NK cell degranulation. This study showed that all drugs tested had significant effects on NK cells that could jeopardise their beneficial effects after HSCT. We demonstrated that CB NK cells were more sensitive to the drugs than PB NK cells. However, this difference of sensibility could not be explained by significant differences in the expression of the drug targets, IMPDH1, IMPDH2 and NFATc between PB and CB NK cells. These data suggest that the immunosuppressive drug dosage could be adjusted for CBT or maybe alternatives to MMF Summary XX and CsA could be used in order to prevent GvHD, without jeopardising NK cell functionality. Resumo XXI Resumo O transplante de células estaminais hematopoiéticas é utlizado para tratar doenças hematológicas malignas e falhas da medula óssea. Durante muitos anos a medula óssea foi a única fonte de células estaminais hematopoiéticas utilizadas em transplantes, mas agora células estaminais mobilizadas do sangue periférico e células estaminais do cordão umbilical são cada vez mais utilizadas. Algumas das vantagens do transplante do cordão umbilical em comparação com o transplante de medula óssea incluem uma maior tolerância a incompatibilidades do antígeno leucocitário humano, uma menor incidência da doença do transplante contra o hospedeiro e obtenção do efeito imunológico do enxerto contra leucemia. No entanto, a doença do transplante contra o hospedeiro continua sendo a maior causa de morbilidade e mortalidade após o transplante de células estaminais hematopoiéticas. Cisclosporina A e metotrexato eram os fármacos imunossupressores padrão utilizados no tratamento profilático da doença do enxerto contra o hospedeiro no Reino Unido, mas ao longo dos últimos vinte anos novos fármacos imunossupressores têm sido desenvolvidos com o objectivo de melhorar a eficácia e minimizar os efeitos secundários desses fármacos. Alguns exemplos dos fármacos de nova geração desenvolvidos são o micofenolato de mofetila, globulina anti-linfócito T, campath e rapamicina. Ciclosporina A e micofenolato de mofetila têm sido eficazmente utilizados individualmente ou em combinação para prevenir a doença do enxerto contra o hospedeiro. Os efeitos desses dois fármacos imunossupressores têm sido estudados em linfócitos T mas sabe-se muito pouco sobre os seus efeitos nas células natural killer (NK). As células NK exercem o efeito imunológico benéfico do enxerto contra leucemia sem provocar a doença do enxerto contra o hospedeiro, contribuindo assim para a redução da reincidência da doença. Para além do mais, as células NK reconstituem num curto período de tempo após o transplante do cordão umbilical, proporcionando não só a primeira linha de defesa contra agentes infecciosos, mas também o efeito imunológico do enxerto contra leucemia. Vários estudos têm demostrado que as células NK têm um efeito benéfico no resultado clínico dos pacientes. Para determinar os efeitos do ácido micofenólico, ciclosporina A e da combinação dos fármacos nas células NK do sangue periférico e do cordão umbilical, foi utilizada uma metodologia baseada em citometria de fluxo e reações quantitativa de polimerase em cadeia detectada em tempo real. O micofenolato de mofetil e a sua combinação com ciclosporina A induziu a morte celular das células NK do cordão umbilical, mas não das células NK do sangue periférico. Os imunossupressores não tiveram um efeito significativo na expressão dos marcadores de ativação CD69 e NKp44 pelas células NK, mas reduziram significativamente a proliferação das células NK do cordão umbilical. O micofenolato de mofetila também Resumo XXII reduziu a produção de perforina pelas células NK do sangue periférico, enquanto que a ciclosporina A e a combinação dos fármacos reduziu drasticamente a degranulação das células NK de ambas as fontes. Este estudo mostrou que todos os fármacos testados apresentam efeitos significativos nas células NK que podem prejudicar o seu efeito benéfico após o transplante de células estaminais hematopoiéticas. Demonstramos ainda que as células NK do cordão umbilical são mais susceptíveis aos imunossupressores do que as células NK do sangue periférico. No entanto, esta diferença de susceptibilidade não pode ser explicada com base em diferenças significativas dos alvos moleculares dos fármacos, como IMPDH1, IMPDH2 e NFATc, entre células NK do sangue periférico e do cordão umbilical. Estes resultados sugerem que a dosagem dos fármacos imunossupressores pode ser ajustada para o transplante do cordão umbilical ou então alternativas ao micofenolato de mofetila e ciclosporina A devem ser pensadas de forma a prevenir a doença do enxerto contra o hospedeiro sem prejudicar a funcionalidade das células NK. Introduction 1 Introduction 1. Human Natural Killer Cells Natural killer (NK) cells are large, granular, bone marrow (BM)-derived cells that constitute approximately 10-15% of human peripheral blood lymphocytes (PBL), however, this proportion can vary with age [1]. They are an essential element of the innate immune system, mediating early defence through cellular cytotoxicity against infectious organisms or transformed cells without previous sensitisation and through production of a wide variety of chemokines and cytokines that influence other cellular compartments of the immune system [1, 2]. NK cell receptors are germline-encoded, but unlike B and T cells, they do not undergo somatic recombination [1]. It is the balance of signals from activating and inhibitory receptors that determines the outcome of NK cell function [3]. Every healthy cell expresses major histocompatibility complex (MHC) class I molecules that are recognised by specific inhibitory NK cell receptors. This interaction allows NK cells to recognise self from non-self preventing autoimmunity [3, 4]. Infections and tumour transformation lead to a loss of MHC class I expression, triggering NK cell activation, as proposed by the “missing-self” hypothesis [5]. NK cells use several mechanisms to kill their cellular targets, including calcium (Ca2+) dependent exocytosis of perforin and granzyme cytotoxic proteins, Fas/FasL mediated apoptosis, membrane bound or secreted cytokines such as tumor necrosis factor- (TNF-[6] and antibody dependent cellular cytotoxicity (ADCC) [7]. Besides target recognition, NK cell effector functions can be triggered by cytokine stimulation. Infected or activated dendritic cells (DCs) and macrophages produce cytokines such as interferon- (IFN-, interleukin (IL)-12, IL-15 and IL18 that stimulate NK cells to produce other cytokines including IFN-and TNF-. In addition, several chemokines are also secreted [8, 9]Regulatory pathways that control NK cell cytokine production are particularly relevant during early phases of inflammatory response [8]. Introduction 2 1.1. NK Cell Subsets Two distinct NK cell subsets in humans can be characterised according to their relative expression of CD56 and to major functional differences: cytolytic activity, cytokine production and homing capabilities [10, 11]. The majority (90%) of human NK cells are CD56dim, meaning they have low surface density expression of CD56. This population is primarily CD16+, killer cell immunoglobulin-like receptor (KIR)+ and display low cytokine production, but potent cytotoxicity. CD56dim cells predominate in peripheral blood (PB) and inflamed tissues. In contrast, a minority of human NK cells (10%) are CD56bright, have low or absent CD16 and KIR expression, have low cytolytic activity, but release high levels of cytokines. They predominate in lymph nodes (LN) (Figure 1) [10, 11]. (a) (b) Figure 1. Schematic of human NK cell subsets exhibiting differential receptor profiles and functions. (a) CD56bright NK cells produce high levels of cytokines and exhibit high lymphokine-activated killer (LAK) activity but low natural cytotoxicity and antibodydependent cellular activity. (b) By contrast, CD56dim NK cells produce low levels of cytokines but are potent mediators of ADCC, LAK and natural cytotoxicity. CD56dim have a more granular morphology and express high-level of KIRs when compared with CD56bright cells. However, CD56bright NK cells display a higher expression of CD94/NKG2A and Lselectin. From Cooper et al., 2001 [11]. 1.2. NK Cell Development As all cells of the haematopoietic system human NK cells derive from CD34+ haematopoietic stem cells (HSCs). It is believed that the BM microenvironment is needed for NK cell full maturation [12]. BM ablation studies in mice provided the first evidence of its importance for NK cell development in vivo [13, 14]. Introduction 3 Mature NK cells do not express CD34 and are CD3-CD56+ [15] although additional antigens are required to distinguish between functionally mature NK cells and immature intermediates. Human NK cell development can be divided into different stages. Briefly, NK cell progenitors in the BM interact with stromal growth factors and generate an intermediate NK cell precursor (CD34+IL-2/IL-15R+CD56-). Becoming responsive to IL-15, this precursor is able to develop into a functional CD56+ NK cell [12]. Freud et al. identified and characterised 4 distinct stages of NK cell differentiation, using CD34+ haematopoietic progenitor cells (HPCs) from secondary lymphoid tissues (SLT), as follows: stage 1 CD34(+)CD117(-)CD94(-); stage 2, CD34(+)CD117(+)-CD94(-); stage 3, CD34(-)CD117(+)CD94(-); and stage 4, CD34(-)CD117(+/-)CD94(+). CD56 expression progressively increases as NK cell differentiation progresses from one stage to another to reach a level similar to PB CD56bright NK cells at stage 4 of differentiation. They also showed that the commitment to NK cell lineage occurs at stage 3, prior to NK cell functional maturity, which occurs from stage 4 [16]. When generated in vitro, NK cells were CD56bright not CD56dim. This finding can be explained by the requirement of other soluble factors or cell-cell interactions that are not present in vitro. Parrish-Novak et al. showed that IL-21 in combination with IL-15 is involved in the development of CD56+CD16+ NK cells from BM HPCs in vitro [17]. However, it is not clear whether these cells are phenotypically and functionally similar to PB CD56dim NK cells. Several studies support the hypothesis that CD56dim population might represent the terminally differentiated stage of NK cell development [16, 18, 19]. 1.3. NK Cell Recognition and NK Cell Receptors Similarly to T cells, human NK cell recognition occurs through cell surface receptors. While T cells possess T cell receptors (TCRs), NK cells display an array of molecules; e.g. immunoglobulin-like and C-type lectin receptors. Normal and healthy cells expressing MHC class I molecules will be specifically recognised by inhibitory NK cell receptors that will prevent NK cell activation while the interaction with transformed or non-self cells that have lost or exhibit altered MHC class I molecules expression will result in NK cell activation. Even though this is the main mechanism of NK cell recognition, MHC class I molecules are not always required for protection from NK cell lysis, and inhibition Introduction 10 Figure 2. De novo and salvage pathways of purine biosynthesis. MPA inhibits IMPDH that catalyses the conversion of inosine monophosphate (IMP) into guanosine monophosphate (GMP). AMP, adenosine monophosphate; ATP, adenosine triphosphate; dADP, deoxyadenosine diphosphate; dATP, deoxyadenosine triphosphate; GTP, guanosine triphosphate; GMP, guanosine monophosphate; dGDP, deoxyguanosine diphosphate; dGTP, deoxyguanosine triphosphate; DNA, deoxyribonucleic acid; RNA ribonucleic acid. Adapted from Allison et al. 2005. 4.1.2. Effects on B and T Lymphocytes There are two IMPDH isoforms: IMPDH1 and IMPDH2. While most of the cells express IMPDH1, IMPDH2 is almost exclusively expressed by B and T lymphocytes. Since MMF is 5 times more active on IMPDH2, it selectively affects the proliferation of activated lymphocytes, which is almost entirely dependent on the de novo pathway for purine synthesis [80]. Several papers have reported MMF/MPA as an inductor of apoptosis in T lymphocytes [81-83]. Human T lymphocytic and monocytic cell lines treated with MPA showed an increase in apoptotic cells [84]. Moreover, MMF supresses cytotoxic T lymphocyte generation [72]. Eugui et al. described MPA as an inhibitor of antibodies formation by activated B cells [71]. Introduction 11 4.1.3. Effects on NK Cells Recently, Ohata et al. described MPA as an inhibitor of PB NK cell proliferation and cytotoxic function. They suggested that the inclusion of MMF in the GvHD prophylaxis treatment may not be advantageous [85]. Eissens et al. demonstrated that MPA impairs human PB NK cell cytotoxicity due to differential effects on NK cell phenotype. The most important effects observed were the inhibition of the proliferation of the CD56brightCD16+/- subset and the inhibition of IFN- production, suggesting that MPA might have a negative effect on NK cell mediated GvL responses in vivo after HSCT [86]. 4.1.4. Effects on Dendritic Cells DCs are the most important antigen presenting cells, especially during primary immune responses. Mehling et al. showed that MMF impairs the maturation and function of murine dendritic cells due to the downregulation of co-stimulatory molecules, CD40 and CD86, required for the maturation process. Similarly, studies on human monocyte-derived DCs showed that MPA suppresses their maturation and allostimulatory functions, highlighting a beneficial role for transplantation through tolerance induction [87]. 4.1.5. Effects on Adhesion Molecules An in vitro study conducted by Blaheta et al. showed that MMF suppresses in a dose-dependent manner the glycosylation and expression of several adhesion molecules such as vascular cell adhesion protein (VCAM)-1, E-selectin and Pselectin on T lymphocytes [88]. Furthermore, it was shown that MMF impairs T cell transendothelial migration [88]. In vivo, MMF decreased leucocytic infiltration into rat kidney allografts, reducing the incidence of acute rejection [89]. 4.2. Cyclosporine A The cyclic oligopeptide, CsA, was primarily described as derived from the fungus Tolypocladium inflatum, but several other microorganisms produce it. CsA presents a variety of biological activities, including immunosuppressive, antiinflammatory, antifungal and antiparasitic properties [90]. Introduction 12 Borel et al. were the first to show in 1976 that CsA delays the rejection of skin transplants and prevents/treats GvHD in mice [91]. Since approved for clinical use as immunosuppressant in 1983, CsA has been used in preventing allograft rejection and treating certain autoimmune diseases [90]. 4.2.1. Mechanism of Action Within the cell, CsA binds to cyclophilin and the complex CsA-cyclophilin inhibits calcineurin, a Ca2+-dependent serine threonine phosphatase. This consequently inhibits the nuclear factor of activated T cells (NFAT) dephosphorylation, preventing its translocation into the nucleus. The repression of NFAT translocation into the nucleus prevents the transcription of early genes, such as IL-2, IL-4, IFN-, TNF, but also FasL and CD40L. Thus, CsA exerts its main immunosuppressive effect by inhibiting T cell activation [92]. 4.2.2. Pharmacokinetic CsA pharmacokinetic is highly variable in humans according to the type of organ transplanted, the co-administration with other drugs that can interact with CsA, the disease state and age of the patient. Its metabolisation occurs in the liver and the half-life of the drug is estimated as being between 6.4-8.7 hours [92]. 4.2.3. Toxic Effects In addition to its immunologic effects, CsA presents several side effects, partly due to calcineurin inhibition in non-lymphatic tissues. Among the most prominent side effects are nephrotoxicity and hypertension [92]. However, coadministration of CsA with other immunosuppressive drugs has shown a beneficial synergistic effect with a decrease of its toxic effects [90]. 4.2.4. Effects on Lymphocytes and other Cell Types In 1976, Borel and colleagues [91] thought that CsA was specific of lymphocytes, but over the years CsA was shown to have effects on a wide range Introduction 13 of cells. In Table 3 are summarised several studies reporting the effects of CsA on various cell types. Table 3. Summary of studies reporting the effects of CsA on T cells, NK cells and other cell types. inhibition; induction;  no difference --- not applicable/not studied; AICD activation-induced cell death. T lymphocytes / cytotoxic T lymphocytes NK cells B cells / B cell line Mast cells Basophils Degranulation [93] --- --- [94] [94, 95] Proliferation [91] [85, 96]; (trend) [86] --- --- --- Cytolytic function [93] [96]; [97]; [85, 86]; --- --- --- Cytokine production IFNandTNF TGF- IFN- IFN- --- --- --- Apoptosis [99]; [100] [96] [100] --- --- AICD [101, 102] --- [103] --- --- 5. Aim MMF and CsA have been successfully used worldwide alone or in combination for GvHD prophylaxis [104-106]. Although the effects of these immunosuppressive drugs have been extensively studied in T cells, little is known about their effects on NK cells. The aim of the present in vitro study was to gain a better understanding of the effects of MPA and/or CsA on PB and CB NK cell phenotype and function. This study intended to determine if CB NK cells are more sensitive to the immunosuppressants tested than PB NK cells. NK cells have been described as effectors of the GvL effect without causing GvHD. Thus, a fully functional NK cell population after HSCT is important to obtain a more favourable outcome. Hence we intended to identify the immunosuppressive regimen that preserves better NK cell functionality. Materials and Methods 15 Materials and Methods 6. Collection of Blood Samples PB samples from healthy volunteers were collected in sterile heparinised tubes. CB samples were obtained from the Anthony Nolan Cord Blood Bank, Nottingham, United Kingdom. Samples were collected, using routine banking procedures, into a CB donation bag containing a Citrate-Phosphate-Dextrose anticoagulant buffer. Only fresh CB units (up to 48h after collection) were used. All samples were collected under written informed consent. 7. Cell Isolation 7.1. Peripheral Blood Mononuclear Cell Isolation The blood was diluted 1:1 in phosphate buffered saline (PBS) 1X (Lonza, Basel). Lympholyte-H® (Cederlane, Gateshead) density gradient was used to isolate PBMCs. Cells were washed and counted using trypan blue® (Sigma-Aldrich, Dorset) to assess cell viability. Thereafter, cells were either cultured in RPMI-1640 (Lonza) supplemented with 10% foetal bovine serum (FBS) (Lonza), 1% penicillinstreptomycin™ (Pen-Strep) (Lonza) and 0.1% -mercaptoethanol (Gibco Invitrogen, Paisley) or resuspended in PBS 1X supplemented with 0.5% bovine serum albumin (BSA) and 2 mM EDTA (Gibco Invitrogen) for NK cell isolation. 7.2. Cord Blood Mononuclear Cell isolation The blood was diluted 1:1 in RPMI-1640 supplemented with 10% FBS, 1% Pen-Strep, 33% trisodium citrate® (AnalaR, Poole) and 0.05 µM -mercaptoethanol and allowed to equilibrate at room temperature for 45 minutes. Ficoll-Paque™ Plus (GE Healthcare, Chalfont St Giles) density gradient was used to isolate CB mononuclear cells (CBMCs). CBMCs were treated with lysing buffer 1X (BD Pharmingen, Oxford) at room temperature for 2 minutes to lyse red blood cells, washed and counted with Türk solution (methylene blue and 1% acetic acid (v/v)) to discriminate red blood cells and trypan blue to assess cell viability of white Materials and Methods 16 blood cells. CBMCs were either cultured in RPMI-1640 supplemented with 10% FBS, 1% Pen-Strep and 50 µM -mercaptoethanol or resuspended in PBS 1X supplemented with 0.5% BSA and 2 mM EDTA for NK cell isolation. 7.3. Natural Killer Cell isolation CB samples were treated with the human granulocyte depletion cocktail (StemCell Technologies, Grenoble) before CBMCs isolation to deplete granulocytes that could block the column during the CD3-CD56+ enrichment step. CD3-CD56+ populations were obtained by negative selection using the NK cell isolation kit (Miltenyi Biotec, Bergisch Gladbach) according to the manufacturer instructions; however, some modifications were applied to improve the quality of isolation. After labelling with the NK cell microbead cocktail, cells were washed with 25 mL of buffer rather than 1-2 mL of buffer per 107 cells, centrifuged at 300xg for 5 minutes and then resuspended in 5 mL of buffer instead of 500 µL (up to 108 cells). The magnetic separation was performed using LS columns (Miltenyi Biotec) up to 1x109 total cells per column. The labelled cell suspension was applied to the column and washing steps were performed twice with 7 mL of PBS 1X supplemented with 0.5% BSA and 2 mM EDTA. The enriched NK cell fraction (negative fraction) was centrifuged; cells were resuspended in RPMI-1640 and counted with trypan blue. 8. Cell Culture PBMCs and CBMCs were cultured in flat-bottom 96 well plates in RPMI-1640 supplemented with 10% FBS, 1% Pen-Strep, 50 µM -mercaptoethanol without IL stimulation in order to test the effects of immunosuppressants on resting NK cells. However, after 24 hours of culture, most CB NK cells were dead (Figure 3), while PB NK cells could withstand without stimulation for a longer time period. For that reason the protocol had to be optimised to address the needs of CB NK cells and 1000 IU/mL of IL-2 was added to the cultures. Immunosuppressive drugs were added at the beginning of the culture. Materials and Methods 17 Figure 3. Comparison between resting PB and resting CB NK cell population at 24h after cell isolation. FACS plots from a representative donor showing staining for CD3 and CD56 of resting PBMCs compared to resting CBMCs in the absence of immunosuppressive drugs at 24h after cell isolation. PB and CB isolated NK cells were cultured in round-bottom 96 well plates in RPMI-1640 supplemented with 10% FBS, 1% Pen-Strep, 50 µM -mercaptoethanol and IL-2 at 1000 IU/mL for 5 days. Immunosuppressive drugs were added at day 5. Cells were maintained in an incubator (Sanyo) at 37ºC, in a humidified atmosphere with 5% CO2. The various concentrations of immunosuppressants used in different experiments are listed in Table 4. Table 4. List of immunosuppressants and their corresponding concentration used in different experiments. Immunosuppressants Viability Activation Granzyme B and perforin production CD107a degranulation assay Proliferation assay MPA (M) 2.5, 5, 7.5 and 10 5 and 10 CsA (ng/mL) 5, 50, 500 and 5000 50 and 5000 MPA (M) + CsA (ng/mL) 2.5+5, 5+50, 7.5+500 and 10+5000 5+50 and 10+5000 Materials and Methods 18 9. Phenotypic Analysis The following anti-human monoclonal antibodies were supplied by BD Pharmingen: CD3-FITC (HIT3), CD3-PE (HIT3), CD3-PerCP (HIT3), CD16-FITC (NKp15), CD56-APC (B159), CD56-PE (B159), CD69-PerCP (L78), CD107a (H4A3), granzyme B-FITC (GB11) and perforin-PE (G9). 7-aminoactinomycin D (7-AAD), annexin-V-FITC, mouse IgG1 κ isotype control FITC (MOPC-21), mouse IgG2b κ isotype control PE (G9) were also supplied by BD Pharmingen. NKp44-APC (P44-8) was purchased from Biolegend (London). For surface labelling, cells were incubated with antibodies in the dark at 4°C for 10 minutes, washed and resuspended in either cold PBS 1X with 2% FBS or Annexin-V binding buffer (BD Pharmingen) 1X. Annexin-V and 7AAD were used to assess NK cell viability, while CD69 and NKp44 were the receptors chosen to study NK cell activation. Perforin and granzyme B expression was assessed by intracellular staining after cell membrane permeabilisation with the Cytofix/CytopermTM kit from BD Pharmingen. Briefly, a 10% mouse serum blocking step preceded CD3, CD56 and CD16 surface labelling at 4°C for 10 minutes in the dark to avoid nonspecific binding. Cells were fixed and permeabilised with Cytofix/CytopermTM at 4°C for 20 minutes in the dark. After a second blocking step, cells were incubated either with the target or the isotype control antibodies in the dark at 4°C for 1 hour, washed twice with Perm/WashTM buffer 1X and then resuspended in PBS 1X with 0.1% FBS. Cells were acquired using a FACSCalibur flow cytometer (BD Biosciences) and the data were analysed using FlowJo software (Tree Star, Oshland). 9.1. Gating Strategy 9.1.1. Effects of Immunosuppressants on NK Cell Viability The lymphocyte population and the smaller population representing dying cells were gated based on forward and side scatter. Subsequent analyses were done on CD3-CD56+ cells. Cell death was determined by the expression of annexin-V and 7AAD (Figure 4). Materials and Methods 19 Figure 4. Gating strategy used for the viability study of NK cells. The example represents the analysis of one PBMCs sample. The same gating strategy was applied to CBMCs samples. 9.1.2. Effects of Immunosuppressants on NK Cell Activation The lymphocyte population was gated based on forward and side scatter. CD69 and NKp44 mean fluorescence intensity (MFI) was determined on NK cells by gating on CD3-CD56+ cells (Figure 5). Figure 5. Gating strategy used to analyse the expression of activation markers on NK cells. The example represents the analysis of one PBMCs sample. The same gating strategy was applied to CBMCs samples. Results 26 Figure 9. Influence of immunosuppressants on the expression of activation markers by isolated NK cells. MFI of CD69 and NKp44 activation markers on IL-2 (1000 IU/mL) activated PB (N=5) and CB (N=6) NK cells at day 7 of culture. MPA (2.5, 5, 7.5 and 10 µM) and/or CsA (5, 50, 500, 5000 ng/mL) were added at day 5 of culture. 3. Effects of Immunosuppressants on NK Cell Proliferation To study the effects of MPA, CsA and MPA+CsA on NK cell proliferation, freshly isolated PB or CB NK cells were labelled with CFSE and cultured up to 5 days with 1000 IU/mL of IL-2 in the presence or absence of increasing concentration of MPA and/or CsA. The CFSE levels were determined by flow cytometry at day 2 and 5 of culture. All NK cells proliferated at day 2 compared to day 0 when stimulated with 1000 IU/mL of IL-2. At day 2 the drugs did not affect the proliferation of PB or CB NK cells compared to untreated cells. At day 5 PB NK cells were divided into 2 groups as they showed differences in proliferation in the presence of the drugs. Two out of six samples tested (group 1) showed reduced proliferation in the presence of the drugs. MPAand MPA+CsA-treated NK cells showed a similar inhibition of proliferation compared to untreated NK cells independently of the dose tested, while CsA decreased NK cell proliferation in a dose dependent manner. In group 2, none of the drugs inhibited NK cell proliferation (Figure 10). Results 27 In contrast, the immunosuppressive drugs inhibited NK cell proliferation of all CB NK cell samples. However, CsA (p<0.05) showed to be less potent than MPA (p<0.001) and MPA+CsA (p<0.001) (Figure 10). Regarding proliferation, CB NK cells showed to be more sensitive to the immunosuppressants than PB NK cells. Figure 10. Influence of immunosuppressants on NK cell proliferation. CFSE division profile at day 2 and day 5 of IL-2 (1000 IU/mL) stimulated untreated (dashed line, open histograms) compared to drugs-treated (heavy line, open histograms) NK cells. The CFSE content at day 0 is represented by a heavy line, closed histograms. As at day 5 PB NK cells from different individuals showed distinct profiles, they were divided in group 1 (N=2), in which the immunosuppressive drugs inhibited NK cell proliferation and group 2 (N=4), in which the immunosuppressive drugs did not inhibit NK cell proliferation. Regarding CB NK cells, the figure shows a set of histograms from a representative sample. NK cells from 4 individual CB samples showed similar results. Results 28 4. Effects of Immunosuppressants on NK Cell Cytotoxicity NK cells are important effectors of the innate immune system that mediate cytotoxicity against infectious organisms and tumour cells without prior sensitisation by exocytosis of perforin and granzyme B. Perforin creates pores in the cell membrane of the target cell while granzyme B, a serine protease, enters the cell via those pores and induces apoptosis. To investigate the effects of immunosuppressants on NK cell killing machinery, NK cells were activated for 5 days with 1000 IU/mL of IL-2 and the drugs were added at day 5 of culture. At day 7 of culture granzyme B and perforin production was accessed by intracellular staining. It is interesting to note that CB NK cells produced far less perforin than PB NK cells. Furthermore, the drugs affected differentially PB and CB NK cells. The percentage of cells producing perforin was analysed and the perforin content/cell (MFI) was measured. MPA significantly decreased the percentage of perforin producing cells in a dose dependent manner, as well as the quantity of perforin produced by PB NK cells. Although CsA increased significantly the percentage of perforin producing cells, it did not significantly affect the global perforin production. Interestingly, although MPA+CsA significantly decreased the percentage of perforin producing PB NK cells, MPA+CsA only showed a trend to decrease the perforin production. In contrast, the combination of drugs seemed to have the opposite effect on CB NK cells, significantly increasing the percentage of perforin producing cells (Figure 11). The percentage of granzyme B producing cells, as well as the quantity of granzyme B produced by these cells, was very similar between PB and CB NK cells. The immunosuppressants did not show major effect on granzyme B production. Although CsA showed a trend towards the decrease of granzyme B production, only the highest concentration of the combination of drugs showed a significant decrease of granzyme B production by CB NK cells (Figure 12). Results 29 Figure 11. Influence of immunosuppressants on perforin expression by NK cells. Percentage of perforin-producing PB (N=4) and CB (N=4) NK cells (top graphs) and MFI of perforin produced by isolated PB (N=4) and CB (N=4) NK cells (bottom graphs) activated with 1000 IU/mL of IL-2 in the presence of MPA (5 and 10 µM), CsA (50 and 5000 ng/mL) and MPA+CsA (5 µM + 50 ng/mL and 10 µM + 5000 ng/mL). The drugs were added at day 5 of culture and the cells were stained at day 7 of culture. *p<0.05 and **p<0.01 compared to untreated cells. Figure 12. Influence of immunosuppressants on granzyme B expression by NK cells. Percentage of granzyme B-producing PB (N=4) and CB (N=4) NK cells (top graphs) and MFI of granzyme B produced by isolated PB (N=4) and CB (N=4) NK cells (bottom graphs) activated with 1000 IU/mL of IL-2 in the presence of MPA (5 and 10 µM), CsA (50 and 5000 ng/mL) and MPA+CsA (5 µM + 50 ng/mL and 10 µM + 5000 ng/mL). The drugs were added at day 5 of culture and the cells were stained at day 7 of culture. *p<0.05. Results 30 The previous data elucidated how the immunosuppressants affect the intracellular content of perforin and granzyme B. However, the cells are functional only if they are able to degranulate following stimulation. Therefore, the cells were activated with 1000 IU/mL of IL-2 for five days and the drugs were added at day 5 of culture. At day 7 of culture cells were stained for CD107a after stimulation with 100 ng/mL of PMA and 1 µg/mL of ionomycin for 2 hours. CD107a, also known as lysosomal-associated membrane protein-1 (LAMP-1), is a marker of degranulation for cytotoxic lymphocytes such as CD8+ T cells and NK cells. Figure 13. Influence of immunosuppressants on NK cell degranulation. Degranulation of PMA and ionomycin treated NK cells in the presence of immunosuppressive drugs. Flow cytometry histograms showing one representative example of CD107a surface expression on PB (N=3) and CB (N=4) untreated NK cells compared to MPA- (5 and 10 µM), CsA- (50 and 5000 ng/mL) and MPA+CsA- (5 µM+50 ng/mL and 10 µM+5000 ng/mL) treated NK cells at day 7 of culture after 2 hours of stimulation with 100 ng/mL of PMA and 1 µg/mL of ionomycin. Isotype controls are shown as closed histograms. Results 31 Figure 13 shows one representative example of PB and CB NK cell degranulation in the presence or absence of the drugs. In this experiment, although a great variability in the amount of degranulation was observed between samples, all the samples showed the same degranulation profile in the presence of the drugs. These preliminary data showed that CsA-treated PB and CB NK cells exhibited a drastic reduction of degranulation compared to untreated cells. MPA+CsA inhibited degranulation almost totally. Surprisingly, MPA slightly increased PB NK cell degranulation, but slightly decreased CB NK cell degranulation. 5. Relative Quantification of IMPDH1, IMPDH2 and NFATc Expression in PB and CB NK Cells by Quantitative Real Time PCR To understand the difference of sensitivity to the immunosuppressants between PB and CB NK cells, a relative quantification of the drug targets; IMPDH1, IMPDH2 and NFATc expression by resting and activated PB and CB NK cells was performed by quantitative real time PCR. No significant changes were observed in the expression of IMPDH1, IMPDH2 and NFATc between PB and CB resting or activated NK cells. However, activated CB NK cells showed a trend towards a reduced expression of IMPDH2 and NFATc compared to PB NK cells (Figure 14). Figure 14. Relative expression of IMPDH1, IMPDH2 and NFATc mRNA levels by resting and activated NK cells. mRNA levels analysed by RT-PCR in freshly isolated PB (N=5) and CB (N=5) NK cells (left) and isolated PB (N=4) and CB (N=5) NK cells activated with IL-2 (1000 IU/mL) for 5 days (right). Discussion 33 Discussion CB has been increasingly used as a source of SCs for transplantation as an alternative to BM and mPB due to a decreased GvHD incidence, off-the-shelf availability and a greater tolerance to HLA disparity. NK cells reconstitute early after CBT, providing GvL effect, without causing GvHD. These cells have already proven to be beneficial for clinical outcome [65, 66]. Therefore, it is important to study the effects of immunosuppressive drugs on NK cells in order to select a prophylaxis regimen with less impact on NK cell functionality. Although the effects of these drugs have been well studied on T cells, their effects on PB and CB NK cells have been scarcely studied. MPA and CsA have been successfully used worldwide, alone or in combination for GvHD prophylaxis. In the present study, we examined if CB NK cell sensitivity to MPA and/or CsA differs from their adult counterparts in terms of viability, activation, proliferation and cytotoxicity. We first studied the effects of MPA and/or CsA on PB and CB NK cell viability. We showed that MPA significantly increase CB NK cell death. From our knowledge, we were the first to show that MPA induces cell death of CB NK cells in therapeutic and supratherapeutic doses at day 7 of culture. As MPA and the combination of the drugs increased cell death by 20%, this might impact on the post-transplant outcome, reducing the GvL effect and increasing the risk of infections. This data indicates that CB NK cells are more sensitive to MPA than their adult counterparts. In accordance with our results, a few papers have reported MPA as an inductor of apoptosis in lymphocytes [81-83]. Human T lymphocytic and monocytic cell lines treated with MPA also showed an increase in apoptosis [84]. On the other hand the role of CsA in inducing or inhibiting apoptosis is controversial [99, 102, 103, 107, 108]. This might be explained by experimental design differences and different cell type tested. In our experiments, CsA had no effects on PB or CB NK cell viability. CsA is a calcineurin inhibitor that blocks NFAT translocation into the nucleus and consequently the transcription of several cytokine genes such as IL-2, IL-4 and IFN-, inhibiting cell activation. We investigated if the drugs affect NK cell activation by comparing the expression of CD69 and NKp44 by drug-treated NK cells with untreated NK cells. We found no significant difference in the expression of both activation markers, which is not in accordance with the results published Discussion 34 by others (Table 6). In addition to the different experimental design regarding the stimulus used for NK cell activation, drugs concentration and time of incubation, there is another major difference that must be underlined. We added the drugs to NK cells activated with IL-2 for 5 days, while in other studies the drugs were added to freshly isolated NK cells. Studies on T cell activation reported that if CsA was given within 1 hour after activation, it resulted in a total inhibition of T cell proliferation and function. On the other hand, if CsA was given more than 6 hours after activation there was little effect on T cell proliferation [109]. These data indicate that the time of drug addition is crucial, at least for CsA to prevent cell activation. Table 6. Studies reporting the effects of MPA and CsA on the expression of CD69 and NKp44 by NK cells. downregulation; no difference. *p<0.05 and **p<0.01. References Source Stimulus Days Drug Concentration Activation marker expression Wang et al. (2007) PB IL-2 (100 IU/mL) + IL-15 (10 ng/mL) 7 CsA 1000 ng/mL NKp44 (p=0.06) Lin et al. (2008) PB/CB IL-15 (10 ng/mL) 18 h CsA 1000 ng/mL PB CD69 (trend) CB  CD69* Eissens et al. (2010) PB IL-2 (100 IU/mL) + IL-15 (10 ng/mL) 5 MPA 500 ng/mL ≈ 1.56 µM NKp44** CsA 1000 ng/mL NKp44 CsA 10-10000 ng/mL CD69 (trend) MPA 10-10000 ng/mL ≈ 0.0312-31.2 µM CD69 (trend) Ohata et al. (2011) PB IL-2 (100 IU/mL) + IL-15 (10 ng/mL) 7 MPA 10 µg/mL ≈ 31.2 µM NKp44* Both Eissens et al. and Ohata et al. showed that MPA donwregulates NKp44 on PB NK cells while we only showed a trend towards the downregulation of NKp44 on these cells. Moreover, the results from Wang et al. and Eissens et al. are not in accordance about the effects of CsA on the expression of NKp44. Wang et al. showed a downregulation of NKp44, while Eissens et al. showed no significant differences in the expression of NKp44. We showed a trend towards the donwregulation of NKp44 by CsA on both PB and CB NK cells. MPA+CsA treated cells followed the same pattern that MPA or CsA treated cells. To our knowledge, the effects of MPA or CsA on NKp44 expression by CB NK cells were not studied by other groups. Nevertheless, CB and PB NK cells showed the same Discussion 35 pattern of NKp44 expression in the presence of drugs. However, it is interesting to note that CB NK cells expressed more NKp44 than PB NK cells. None of the published studies showed a significant difference of CD69 expression when PB NK cells were treated by either MPA or CsA, but they showed a trend towards the downregulation of CD69, which is in accordance with our results. The only study on CB NK cells showed that CsA dowregulates CD69, but we found no significant difference compared with the untreated cells. This might be explained by experimental design differences in particular by the time of drug addition. Comparing our results with what was published in the literature: we can conclude that there is a difference in the expression of activation markers if the immunosuppressive drugs are added to freshly isolated NK cells or if they are added to activated NK cells. Preliminary data obtained in our laboratory showed similar results to the literature when the drugs were added to freshly isolated NK cells. This might be relevant for immunotherapies of immunosuppressed patients using NK cell infusions, however further studies are needed. In contrast with what was published by other groups (Table 7), we showed minor effects of the drugs on PB NK cell proliferation. Only two out of six samples treated with MPA and MPA+CsA showed a reduced proliferation at day 5 of culture. CsA also inhibited PB NK cell proliferation in these two samples in a dose dependent manner. MPA and MPA+CsA inhibited more potently CB NK cell proliferation than CsA. 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