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Antileukemic activities of human bone marrow and blood cells after culture in interleukins-2, -7 and… Wong, Elaine Karol 1994

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ANTILEUKEMIC ACTIVITIES OF HUMAN BONE MARROWAND BLOOD CELLS AFTER CULTURE ININTERLEUKINS-2, -7 AND -12byELAINE KAROL WONGBSc., University of British Columbia, 1992A THESIS SUBMITFED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTERS OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Department of Pathology)We accept this thesis as conformingto the re uire ‘St dardTHE UMVERSITY OF BRITISH COLUMBIAApril 1994Elaine Karol Wong, 1994In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of P€L*ioIogThe University of British ColumbiaVancouver, CanadaDate AyvL2Ht4jJ994DE-6 (2/88)11ABSTRACTAcute myeloid leukemia (AML) can be treated by chemotherapy alone, allogeneicbone marrow transplantation (BMT) or autologous BMT. There is a risk of diseaserecurrence with all these methods, the lowest being associated with allogeneic BMT due toan allogeneic antileukemic effect. Despite its benefits, the use of autologous BMT islimited by high relapse rates due to the persistence of leukemic stem cells in the autologousbone marrow (BM) and/or residual disease in the patient. Thus, it would be desirable todevelop methods to decrease relapse rates associated with autologous BMT to a levelsimilar to those of allogeneic BMT. Since individuals who receive BM from a twin aresubject to a high risk of relapse, an active immune component that contributes to theelimination of residual disease must be present in the allogeneic BM graft. This project isbased on the hypothesis that this can also be achieved by appropriate cytokine manipulationof the antiproliferative activity of autologous effector cells. The natural killer (NK) cellpopulation is the first cell subpopulation to reconstitute after BMT and it is, therefore, anobvious candidate for such manipulations.BMT involving interleukin (IL)-2-activated autologous BM is associated withdelayed neutrophil and platelet recovery which can be hastened by the addition of peripheralblood stem cells with the BM. Recent studies with IL-7 and IL-12 suggest that they mightmimic or enhance the cytotoxic and antiproliferative activities of IL-2. The effects of thesecytokines on bone marrow cells (BMC) and peripheral blood mononuclear cells (PBMC)were compared in a model system in order to assess potential alternate strategies for the exvivo purging of leukemic cells from human BMC and PBMC.The cytotoxie activity of BMC and PBMC was measured with 51-Cr release assays.The cytotoxic activity exerted by BMC stimulated with optimal doses of IL-2 was less than111that of PBMC. While neither IL-7 nor IL- 12 induced cytotoxic activity in BMC, activitywas detected in PBMC stimulated with these cytokines. The cytotoxic activity induced byoptimal doses of IL-2 in BMC was not enhanced by IL-7 whereas enhancement wasobserved with IL-12. In contrast, IL-2-induced cytotoxic activity in PBMC was increasedby both IL-7 and IL-12 when suboptimal doses of IL-2 were used.BMC and PBMC were cocultured with K562neor cells and their ability to inhibitleukemic cell survival was measured using an assay to detect clonogenic K562 cells. IL-2-stimulated BMC and PBMC inhibited leukemic cell growth in a manner dependent on theinitial number of leukemic cells in the coculture. IL-2-stimulated BMC and PBMC werethe most efficient in eliminating leukemic cells from the coculture compared to thosestimulated with IL-7 or IL-12. IL-7 did not enhance IL-2-induced inhibitory activity onleukemic cell growth in BMC and PBMC while IL-12 did. There was no difference inBMC-mediated inhibitory activity on leukemic cell growth upon cytokine stimulation whentested after 2, 4, 6, and 8 days of culture. IL-7 did not hasten the development of maximalIL-2-induced inhibitory activity relative to IL-2 alone while IL-12 did. Overall, PBMCmediated inhibitory activity on leukemic cell survival in cocultures with K562-neo’ cellswas significantly greater and faster than that observed in BMC cocultures when equalnumbers of leukemic cells were initially present.These findings suggest that the use of cytokine combinations may have potential inthe clinical setting. The information regarding the relative capacities of BMC and PBMC interms of their antileukemic activities obtained in these experiments will aid in refining the invitro culture system for these cells such that their antileukemic activity may be optimized.ivTABLE OF CONTENTSABSTRACTTABLE OF CONTENTS ivLIST OF TABLES viiLIST OF FIGURES viiiLIST OF ABBREVIATIONS ixACKNOWLEDGMENTS xCHAPTER I INTRODUCTION(1) Normal Immunity1.1. Immunecells 11.2. Cytokines and immune responses 21.3. Immune surveillance 3(2) Cells Involved in Non-specific Immune Responses2.1, Natural killer (NK) cells 42.1.1. Surface antigens of NK cells 42.1.2. Cytotoxic activity of NK cells 62.1.2.1. NKcellactivity 62.1.2.2. ADCC 62.1.2.3. Apoptosis 82.1.3. Proliferative and cytotoxic activities of NK cells 92.1.4. Role of NK cells in normal physiology 102.1.4.1. Antiviralactivity 112.1.4.2. Elimination of neoplastic cells 122.1.4.3. Regulation of hematopoiesis 122.1.5. Clinical applications of NK cells 132.2. Cytokine-induced killer (CIK) cells 142.2.1. ClKcellactivity 152.2.2. Proliferative and cytotoxic activities of CIK cells 162.2.3. Clinical applications of CIK cells 16(3) Cytokines Involved in regulating the Non-specific Cytotoxicand Antiproliferative Activities of Immune Cells3.1. Interleukin-2 (IL-2) 183.1.1. IL-2 receptor (IL-2R) 193.1.2. IL-2 as a biologic response modifier 193.1.3. Proliferative and cytotoxic effects of IL-2 203.1.4. Clinical applications of IL-2 203.1.4.1. Cytokine therapy with IL-2 alone or 21in combination3.1.4.2. Immunotherapy with IL-2 plus CIK 22cells3.1.4.3. Tumour-targeted immunotherapy 223.1.5. Problems associated with IL-2 utilization 233.1.5.1. Exacerbation of malignancy 233.1.5.2. IL-2toxicity 23V3.2. Interleukin-7 (IL-7) 243.2.1. IL-7 receptor (1L-7R) 253.2.20 Proliferative effects of IL-7 263.2.2.1. Effects on T lymphocytes 263.2.2.2. Effects on B lymphocytes 273.2.2.3. Effects on NK cells 273.2.3. Cytotoxic effects of IL-7 283.2.3.1. Effects on T lymphocytes 283.2.3.2. EffectsonNKcells 283.2.3.3. Effects on monocytes/macrophages 293.2.4. Clinical applications of IL-7 293.3. Interleukin-12 (IL-12) 303.3.1. IL-l2receptor(IL-12R) 323.3.2. Proliferative effects of IL-12 323.3.2.1. Effects on T lymphocytes 333.3.2.2. EffectsonNKcells 333.3.2.3. Synergism between IL-2 and IL-12 343.3.3. Cytotoxic effects of IL-12 353.3.4. IL-2-induced IFN-y production 363.3.5, Clinical applications of IL-12 37(4) The Use of Bone Marrow Transplantation in the Treatment ofAcute Leukemia4.1. Cancer 384.1.1. Acuteleukemia 384.1.2. Therapeutic immunomodulation 394.2. Bone marrow transplantation (BMT) 394.2.1. Bone marrow (BM) microenvironment 394.2.2. BMT 404.2.3. TreatmentofAML 414.2.3.1. AllogeneicBMT 424.2.3.2. AutologousBMT 434.2.4. Problems associated with BMT 444,2.5. TherapyinBMT 454.2.5.1. Chemotherapy 464.2.5.2. Depletion of T lymphocytes 464.2.5.3. Cytokine therapy 474.2.5.4. Adoptive immunotherapy 494.2.5.5. Induction of GVHD 514.2.5.6. GVLeffect 514.2.5.7. BMpurging 524.2.6. Future trends in BMT 53(5) Thesis Objectives 45CHAPTER II MATERIALS AND METHODS(1) Cell Preparation1.1. Normal bone marrow (BM) 571.2. Normal peripheral blood (PB) 57vi(2) Tumour Cell Lines 58(3) Cytokines 59(4) Gene Transfer of K562 Target Cells 59(5) Cytotoxicity Assay 60(6) Antiproliferation Assay 62(7) Statistical Analysis 65CHAPTER III RESULTS(1) Cytokine-induced Cytotoxic Activity in BMC and PBMC1.1. Cytotoxic activity of BMC after cytokine stimulation 661.2. Cytotoxic activity of PBMC after cytokine stimulation 67(2) Kinetics of Leukemic Cell Proliferation 69(3) BMC- and PBMC-mediated Inhibition of 70Leukemic Cell Survival3.1. BMC-mediated inhibition of leukemic cell survival in the 71presence of increasing target cell load3.2. Kinetics of BMC-mediated inhibition of leukemic cell 73survival3.3. PBMC-mediated inhibition of leukemic cells survival in the 77presence of increasing target cell load3.4. Kinetics of PBMC-mediated inhibition of leukemic cell 79survivalCHAPTER IV DISCUSSION 83REFERENCES 100viiLIST OF TABLESpageTABLE 1. Percentage Expression of Cell Surface Antigens on NK Cells 5and Other Cytolytic EffectorsTABLE 2. Functional Properties of NK Cells and Other Cytolytic Effectors 6TABLE 3. Effect of Various Cytokines on NK and Cytokine-induced Killer 10(CIK) Cell FunctionsTABLE 4. Predominant Functions of NK Cells in Normal Physiology 11TABLE 5. Precursor and Effector Phenotypes of Cells Mediating CIK Cell 15ActivityTABLE 6. Predominant Signs and Symptoms of IL-2 Toxicity 24TABLE 7. Cell Types and Tissues that Express IL-7 25TABLE 8. Cell Types that Respond to IL-7 26TABLE 9. Predominant in vitro Activities of IL-12 31TABLE 10. Allogeneic versus Autologous BMT 42TABLE 11. Complications Associated with BMT 45TABLE 12. Possible Clinical Applications of Cytokines in BMT Therapy 48TABLE 13. Cytotoxic Activity of BMC After Stimulation with Optimal 67Doses of IL-2, IL-7 and IL-12TABLE 14. Cytotoxic Activity of PBMC After Stimulation with Optimal 68Doses of IL-2, IL-7 and IL-12TABLE 15. Cytotoxic Activity of PBMC After Stimulation with Increasing 69Doses of IL-2 and Optimal Doses of IL-7 and -12viiiLIST OF FIGURESpageFIGURE 1, Sensitivity of K562 and Daudi Cells to NK and CIK Cell 62Killing in a 4-hour 51Cr Release AssayFIGURE 2. K562neor Cell Formation in Methylcellulose Containing 64G418 After Coculture with BMCFIGURE 3. Kinetics of K562-neo’ Proliferation 70FIGURE 4. Inhibition of Leukemic Cell Survival by Cytokine-stimulated 72BMC Cocultured with Increasing Numbers of K562-neo’CellsFIGURE 5. Inhibition of Leukemic Cell Survival by Cytokine-stimulated 74BMC Cocultured with 0.5% K562neor CellsFIGURE 6. Inhibition of Leukemic Cell Survival by Cytokine-stimulated 76BMC Cocultured with 1% K562neor CellsFIGURE 7. Inhibition of Leukemic Cell Survival by Cytokine-stimulated 78PBMC Cocultured with Increasing Numbers of K562-neo’CellsFIGURE 8. Inhibition of Leukemic Cell Survival by Cytokine-stimulated 80PBMC Cocultured with 1% K562neor CellsFIGURE 9. Inhibition of Leukemic Cell Survival by Cytokine-stimulated 82PBMC Cocultured with 4% K562neor CellsFIGURE 10. Proposed Scheme for the Differential Kinetics Involved in NK 98Cell Activation in Bone Marrow and Peripheral BloodixABBREVIATIONSADCC antibody-dependent cellular cytotoxicityAML acute myeloid leukemiaBM bone marrowBMC bone marrow cellBMT bone marrow transplantationBRM biologic response modifierCAM cellular adhesion moleculeCIK cytokine-induced killerCLMF cytotoxic lymphocyte maturation factorconA concanavalin ACr chromiumCTh cytotoxic T lymphocyteDFS disease-free survivalE:T effector:targetFCS fetal calf serumFH ficoll-hypaqueGM-CSF granulocyte-macrophage colony-stimulating factorGVHD graft-versus-host diseaseGVL graft-versus-leukemiaHC hydrocortisoneICAM intercellular adhesion abbremoleculeIFN interferonIL interleukinkbp kilo base pairsLAK lymphokine-activated killerLFA lymphocyte function associated antigenLGL large granular lymphocyteLTBMC long-term bone marrow cultureMC methylcelluloseMHC major histocompatibility complexMRD minimal residual diseaseneo neomycinNK natural killerNKSF natural killer stimulatory factorPB peripheral bloodPBL peripheral blood lymphocytePBMC peripheral blood mononuclear cellPBS phosphate-buffered salinePBSC peripheral blood stem cellPHA phytohemagglutininr resistantR receptorSEM standard error of the meanTBI total body irradiationTcR T cell receptorTNF tumour necrosis factorU unitxACKNOWLEDGEMENTSWhen I first embarked on my journey as a graduate student, I was prepared for thehard work and long hours to come. What I had neglected to account for were theassociated frustrations and lapses of paranoia. Then again, I was also not prepared for thesatisfaction I gained from performing an experiment well or the challenge and excitementthat built up in me with every bit of consistent data.However, no matter how much time and energy I would have exerted, this projectwould not have been possible without the invaluable assistance, support andencouragement of various people I have known and worked with during the course of mywork.I would like to thank Dr. Connie Eaves and Dr. Hans Klingemann, mysupervisors, for their guidance, helpful advice and review of the thesis. Despite theirdemanding schedules, they have always had time to help and listen to me. I am especiallygrateful to Dr. Klingemann for his never-ending support. His continual encouragementand enthusiasm have always been instrumental in building my confidence to tackle newchallenges from the very first time I set foot in his lab as a summer student.I would also like to thank Dr. John O’Kusky, Chairman of my advisory committee,and Dr. Graeme Dougherty and Dr. Hermann Ziltener, my advisory committee members,for their continual interest in my work as well as their critical review of the thesis.Lastly, I would like to extend a most special thanks to a very important group ofpeople - my family and friends. I would like to thank my parents, sister, brother and dogfor their encouragement and patience, especially at those trying times when things did notwork as well as hoped. I am very grateful to my friends for their 24-hour support and forunderstanding all the times we’ve cried and laughed together.Without all of you, this would have been a long, lonely journey. Thank you!1CHAPTER IINTRODUCTION1. NORMAL IMMUNITY1.1. IMMUNE CELLSThe vertebrate immune response to virally-infected and transformed cells ismediated by several phenotypically and functionally distinct sets of lymphocyte effectors[1] which arise from hematopoietic stem cells. These stem cells were first identified by Tilland McCulloch in 1961 and their pluripotential nature allows them to reproduce themselvesand generate all blood cell types [2].There are four basic cell types whose functions are associated with antitumour cellimmunity [3-4]. T lymphocytes, involved in cell-mediated immunity [4], recognize anddestroy tumour cells that express complementary tumour-associated antigens on theirmembranes presented in the context of major histocompatibility (MHC) proteins [3-5]. Blymphocytes mediate humoral immunity through the secretion of antibodies [3-4]. Both Tand B lymphocytes have virtually no spontaneous cytotoxic or other forms of activity [5].Monocytes/macrophages are accessory cells that phagocytose, process and presentantigens to T lymphocytes such that an immune response may be initiated. They participatein the antibody-dependent cellular cytotoxicity (ADCC)-mediated destruction of tumourcells [3]. Unlike T and B lymphocytes, monocytes/macrophages lack antigen specificity.Lastly, natural killer (NK) cells, which constitute a portion of the large granularlymphocyte (LGL) population, can destroy a wide range of tumour cells through NK cell2activity, induction of target cell apoptosis and ADCC. NK cells lack most of thecharacteristic cell surface markers of mature T and B lymphocytes and can lyse a variety oftarget cells without the need for prior sensitization or restriction by MHC proteins.Stimulation of NK cells with a variety of cytokines leads to the generation of cytokineinduced killer (CIK) cells which are able to lyse a broad range of target cells, includingthose resistant to NK cell lysis, in an MHC-nonrestricted manner.1.2. CYTOKINES AND IMMUNE RESPONSESOriginally, the term “lymphokine” was used in reference to factors that modulate thegrowth or mobility of a variety of leukocytes [6-7]. To date, many such molecules havebeen identified as products of both lymphocytic and non-lymphocytic cells [7-11] and, inrecognition of the contribution of nonlymphocytic cells to lymphokine production, Cohenet. al. proposed the term “cytokine” to describe these mediators [7].Cytokines are pleiotropic and redundant since they have multiple biologicalactivities in different target cells [6, 8-9, 11-12] and more than one cytokine can mediate thesame or similar function [8, 11-12]. Pleiotropy and redundancy allow cytokines to form acomplex regulatory network in the immune system [8-9]. The “cytokine cascade” thatarises from one cytokine’s influence on the expression of another cytokine’s generepresents features of cytokine action in the growth, differentiation and function of cells [9,12].Cytokines regulate immunity, inflammation and tissue repair [7, 12]. Morespecifically with respect to immunity, cytokines regulate lymphocyte growth,differentiation and function [8-10]. The specificity of each cytokine’s action is determined3by target cell expression of receptors for the cytokine [13]. The proliferation of antigen-reactive lymphocytes is mediated through the interaction of cytokine growth factors withtheir receptors on the surfaces of activated lymphoid cells; this event is a central feature ofthe immune response [14].1.3. IMMUNE SURVEILLANCEMany tumours arising de novo do not express tumour-specific antigens and thuscannot be recognized as foreign; therefore, they are not eliminated by the immune system[15], There are several immune mechanisms which contribute to the destruction and/orarrest of tumours: (i) the generation of cytotoxic T lymphocytes (CTh) which recognize anddestroy tumour cells bearing specific tumour antigens [16-18], (ii) the non-specific lysis oftumour cells by NK cells [4, 16-18, 19-28], CIK cells [4, 16] and activated monocytes[16, 18], (iii) the production of cytostatic or cytotoxic cytokines (e.g., interferon [IFNI -y,tumour necrosis factor [TNF]-cx) [16-18], and (iv) ADCC [16, 18].42. CELLS INVOLVED IN NON-SPECIFIC IMMUNE RESPONSES2.1. NATURAL KILLER (NK) CELLSNK cells, discovered in 1974 [5], are nonphagocytic cells found in a wide range ofvertebrates, including humans, dogs, mice and fish, and NK-like effectors have beendescribed in animals as primitive as starfish and earthworms [5, 19, 29]. The NK celllineage is poorly understood since these cells share phenotypic markers with both lymphoidand myeloid cells [5, 19, 30-32]. They are LGL [5, 18-19, 24, 26, 29, 32-50] and arebelieved to arise from BM progenitor cells [19, 21, 29, 31, 42, 48, 51-60]. NK cellsdifferentiate in the BM, after which they are released into the peripheral blood (PB) fromwhere a small number migrates to the spleen [19, 59], liver [19, 29], lung [19, 29, 59] andintestine [19, 29]. NK cells comprise 10-15% of human peripheral blood lymphocytes(PBL) [19, 29, 34, 39, 42-43, 47, 50] and up to 25% of murine splenic lymphocytes [29].2. 1. 1. Surface Antigens of NK CellsNK cells share surface antigens with both lymphoid and myeloid cells [5, 19, 30-32] and, to date, no single surface receptor or target ligand is able to unambiguouslyidentify all human NK cells. Robertson et. at. have shown that NK cells do not possessclonotypic, antigen-specific receptors [61-62]. However, several antigens have been usedas NK cell markers for clinical and basic research purposes. These include CD56 (Leu19)[19, 24, 29, 31-32, 34, 37, 39, 47, 52-53, 56, 62-63] and CD16 (FcyRIII for IgG) [5, 17,19, 24, 29, 31-32, 34, 37, 39, 47, 52, 56, 62-63]. CD56 itself does not participatedirectly in the NK cell killing of most target cells, but it mediates the homotypic adhesion of5NK cells to CD56 tumour cells [19]. Human NK cells are best phenotypically defmed asCD3CD56 lymphocytes [19, 24, 34, 37, 53, 64].NK cell target specificity is regulated by changes in the expression of cellularadhesion molecules (CAM) [19]. A number of these molecules play a significant role inpromoting NK and target cell interactions: (i) 2 integrins, such as lymphocyte functionassociated antigen (LFA)-1 (CD11aJCD18), Mac-i (CD1 lb/CD 18) and P150/95(CD11c/CD18), (ii) CD2 (LFA-2, E-rosette receptor), and (iii) CD56 [19, 29, 47, 63],LFA- 1 on NK cells interacts with intercellular cell adhesion molecule (ICAM)- 1 andICAM-2 to promote target cell cytolysis [19, 29, 47]. Virtually all NK cells express CD2which interacts with LFA-3 (CD58) as an accessory/adhesion molecule in the NK celleffector system [19, 29]. Some of the cell surface antigens found on cytolytic immunecells are shown in Table 1.TABLE 1. Percentage Expression of Cell Surface Antigens on NK Cellsand Other Cytolytic Effectors(Adapted from Robertson MJ & Ritz 3 [19])Surface Antigens NK Cells T Cells MonocytesCD2 70-90 >95 <5CD3 0 >95 0CD8 30-40 30-40 <5CD11b 80-90 10-15 >90CD15 <5 <6 <5CD16 80-90 <5 <5CD56 >95 <5 <5Less than 5% of normal T lymphocytes expresses both CD56 and CD3; theseCD56CD3 T lymphocytes are capable of MHC-unrestricted cytotoxicity [19, 32, 52, 65-668]. Culture of these CD3+ T lymphocytes leads to the induction of CD56 expression inparallel with acquisition of NK activity [19].2.1.2. Cytotoxic Activity of NK CellsNK cells possess functional properties, summarized in Table 2, which areimportant to the immune system. Three of these - (i) MHC-unrestricted cytotoxic activity(i.e., NK cell activity), (ii) ADCC and (iii) cytokine secretion - are discussed below.TABLE 2, Functional Properties of NK Cells and Other CytolyticEffectors(Adapted from Robertson MJ & Ritz 3 [19])Functional Attribute NK Cells T Cells MonocytesNK cell activity yes no* noADCC yes no yesPhagocytosis no no yesImmunologic memory no yes noProliferative capability yes yes no* except for a small (i.e.<5%) number of T lymphocytes which express CD562.1.2.1. NK Cell ActivityNK cell activity is functionally defined as the ability to lyse transformed and virally-infected cells without prior sensitization or restriction by MHC antigens. It was firstdescribed in the 1970s when it was observed that freshly-isolated lymphocytes fromunimmunized hosts could lyse allogeneic tumour cell lines [19]. NK cell activity is7different from the cytolytic activity of T lymphocytes since it (i) occurs rapidly after targetcell exposure without prior immunization, (ii) does not require expression of self-MHCantigens by target cells, and (iii) does not involve clonally distributed specificity [19, 61].NK cells of normal individuals have spontaneous activity which can be rapidlyaugmented. Resting NK cells constitutively express a high level of the intermediate IL-2receptors (R) and a low number of high-affinity IL-2R [19, 52, 62, 69-70]. NK cells arehighly responsive to IL-2 [30-31, 62, 65, 68, 71-72] and, to a lesser degree, IL-7 [62, 65,71] and IL-12 [30, 62, 65, 71]. IL-2 stimulation alone induces in vivo and in vitro NK cellproliferation, leading to the generation of CIK cell activity and enhanced expression ofhigh-affinity IL-2R [73]. Additionally, IL-2 acts as a chemoattractant for already activatedNK cells.NK cell proliferation in response to optimal concentrations of IL-2 is at least 10-fold greater than those in response to optimal concentrations of IL-7 and IL- 12 [62]. Thedifferences in the induction pattern of cytokine (e.g., granulocyte-macrophage-colonystimulating factor [GM-CSF], IFN-y, TNF-a) secretion [56] and receptor (e.g., IL-2R,TNFR) expression in vivo mediated by NK cells in response to IL-2, IL-7 or IL- 12stimulation indicate that the roles of IL-2, IL-7 and IL- 12 in the regulation of NK cellactivities are different [65].The development of cytotoxic activity in culture is coincidental to the acquisition ofLGL morphology [52, 74]. The mechanism of NK cell killing is dependent on divalentcalcium and magnesium ions and involves (i) target cell adhesion, (ii) effector cellrecognition/signal transduction, (iii) triggering of the NK cytolytic apparatus, (iv) deliveryof the lethal signal to the target cell, and (v) effector cell detachment, recycling and targetcell cytolysis [5, 24, 29, 32-33, 41, 52].82.1.2.2. ADCCNK cells can mediate ADCC whereby antibody-bound target cells are lysed byeffector cells bearing CD 16, which binds to the Fc portion of the IgG molecule [19, 25,29, 31, 62, 68, 75]. Effector cells which express the FcyRIII (CD16) and are capable ofbinding to antibody-coated cells include NK cells [19, 29, 39], neutrophils [39] andactivated monocytes and macrophages [19]. Perturbation of CD16 can induce calciummobilization and activate antigen expression, cytokine production and cytolytic activity inNK cells [62].Although the receptors for NK activity and ADCC are distinct, these cytolyticpathways involve common terminal effector mechanisms [19]. Resting NK cells havepreformed cytolytic granules containing performs, serine esterases and chondroitin sulfateproteoglycans which are discharged upon exposure to target cells [5, 19, 24, 76].Performs insert into the target cell membrane as monomers and polymerize to formtransmembrane pores that permit osmotic lysis of the target cell [19]. Serine proteases areinvolved in the activation of performs and other lytic effector molecules whileproteoglycans bind granular components to protect effector cells from their own lyticfactors [19].2,1.2.3. ApoptosisNK cells can mediate apoptosis, programmed cell death characterized by DNAfragmentation. Upon target cell stimulation, NK cells secrete soluble toxins (i.e., NK cellcytotoxic/cytostatic factors) which activate endogenous target cell endonucleases that9degrade target cell genomic DNA within minutes of cellular contact [33, 77]. NK cellcytotoxic/cytostatic factors include lymphotoxin, TNF-a [17, 24, 32, 45, 47, 59, 78-81],IFN-x [32, 47, 59, 79], IFN-y [17, 24, 32, 44-45, 59, 78, 80, 82-85] and IL-i [19, 24,32, 41, 45, 82, 85]. These cytotoxic/cytostatic factors modulate (i) the presence andcytotoxicity of T lymphocytes in primary and secondary antitumour responses and (ii) therelease of additional lymphokines, such as macrophage migration and inhibitory factors andleukocyte migration, adherence and inhibitory factors, from T lymphocytes followingtumour cell contact [33].2.1.3. Proliferative and Cytotoxic Activities of NK CellsSeveral cytokines have been shown to affect NK cell proliferation and/or cytotoxicactivity. These are summarized in Table 3. Two major regulators are IL-2 and WN- Themajority of NK cells in the PB constitutively express IL-2R and can proliferate and displayenhanced cytolytic activity in response to IL-2 alone [5, 19, 66, 69, 87-89]. Activation ofNK cell cytolytic activity is mediated by the intermediate affinity IL-2R while NK cellproliferation requires the expression of high-affinity IL-2R [19]. IL-2 induces theexpression of some NK cell CAM, which partially mediate the induction of cytotoxicity forNK cell-resistant targets, and stimulate NK cell expression of mRNA for some serineproteases [19].NK cells can proliferate in response to IFN-y [66, 86] Several groups, including asTrinchieri et. al. [90], have shown that IFN-y and IFN-y-inducers rapidly augment the invivo and in vitro cytotoxic activity of NK cells [26, 9 1-93]. IL-2 can increases NK cellactivity independently of IFN-y while IFN-y increases the expression of IL-2R on NKcells, thus enhancing NK cell responsiveness to IL-2 [92].10TABLE 3. Effect of Various Cytokines on NK and Cytokine-inducedKiller (CIK) Cell Functions(Adapted from Robertson MJ & Ritz J [19])Cytokine NK Cells CIK Cells______________Cytotoxicity Proliferation Cytotoxicity ProliferationIL-i augments no effect augments augmentsIL-2 augments augmentsIL-3 no effect no effect no effect unknownIL-4 no effect no effect variable variableIL-7 augments unknown no effect unknownIL-12 augments no effect unknown no effectIFN-& augments no effect augments inhibitsIFN-y augments no effect augments no effectTNF-a augments unknown augments unknown2.1.4. Role of NK Cells in Normal PhysiologyNK cells synthesize several cytokines that are involved in the modulation ofhematopoiesis, immune response and the regulation of their own activities. The ability ofNK cells to release cytokines broadens their action in nonadaptive resistance by enablingthem to recruit other relevant effector cells. Since NK cells do not require presensitizationto manifest their cytotoxic potential they play a role in natural host defense againstinfectious diseases, immunoresistance to neoplastic growth and the immunoregulation ofhematopoietic growth. However, NK cells have also been shown to participate in severaldisease states and defective NK activity is frequently associated with disturbances of thehematopoietic system and immunodeficiency [20, 22, 24, 29, 94-95]. The major functionsof NK cells are summarized in Table 4.11TABLE 4. Predominant Functions of NK Cells in Normal Physiology(Adapted from O’Shea J & Ortaldo R [47])1. Control of tumour cell growth2. Control of microbial infectionsa. Viral infectionsb. Parasites (intracellular and extracellular)c. Fungid. Bacteria3. Immunoregulationa. Production of cytokinesb. Control of hematopoietic SC growth and differentiationc. Involvement in allograft rejection (mice)4. Disease statesa. Involvement in graft-versus-host disease (mice)b. contribute to some forms of aplastic anemia, neutropeniac. Potentiate autoimmune and neurological diseased. contribute to development of some forms of diabetese. Involved in various gastrointestinal diseases2.1,4,1, Antiviral ActivityNK cells are an important immune defense mechanism against viral infections.Cells exhibiting NK phenotype and activity localize in the lungs and gastrointestinal tract,both important portals of viral entry, as well as the liver and spleen, sites of viralaccumulation [29, 68]. NK cells selectively lyse virally-infected target cells while sparinguninfected cells [5, 19].NK cells proliferate at early times after viral infection while T cells proliferate atlater times [40]. NK cell proliferation correlates with the production of virally-induced IFNwhile IL-2 production is associated with T cell proliferation [40]. The expression of viralantigens or other surface structures by infected cells appears to render them more sensitive12to NK cytolysis [19]. IFN secreted by accessory cells or NK cells themselves in responseto viral infections potentiates their cytolytic activity [19].2.1.4.2. Elimination of Neoplastic CellsThe current theory of immune surveillance postulates that immune effector cells canrecognize and destroy spontaneously arising tumour cells. Historically, this activity wasattributed to CTL [19]. However, there is considerable experimental evidence to suggestthat the destruction of autologous tumours is predominantly mediated by NK cells [4, 19,24-26, 27-28]. It has been proposed that NK cells regulate tumour metastases; a majorphysiologic reservoir of NK activity is the PB, a route by which metastases typicallydisseminate. Unstimulated NK cells can readily lyse cultured leukemic cells and tumourcell lines isolated from patients with lymphoid and myeloid leukemia in an MHCunrestricted manner [19].2.1,4.3. Regulation of HematopoiesisThe role of NK cells in the regulation of hematopoiesis is controversial. They arebelieved to be the primary cells responsible for hybrid resistance in mice [5, 19, 29]. Theysecrete a variety of soluble factors, such as IFN-y [5, 65, 17, 29, 32, 45, 56, 78, 80, 82-84, 96], GM-CSF [19, 21, 45, 56, 65, 80] and IL-3 [19, 65] which have eitherstimulatory or inhibitory activity and affect the in vitro growth of normal humanhematopoietic progenitors [19, 29].132.1.5. Clinical Applications of NK CellsNK cells are important in BMT since they regulate stem cell engraftment andhematopoiesis and their ability to generate lymphokines may essential for successfulmarrow graft take and the reappearance of a functioning immune system [82]. NK cellsrecover quickly after BMT and are among the earliest cells to regenerate after BMT inpatients not receiving post-transplant immunosuppression; they represent the majority ofPBL during the first 4 to 6 weeks post-transplant [19, 48, 55, 58-59, 82, 96-99]. Thus,NK cell activity is fully restored much earlier than that of most other classical lymphocytes,including T and B lymphocytes.Dokhelar et. al. demonstrated that after 6 weeks, there are no distinguishabledifferences between the activities of NK cells in BM recipients and those of normalindividuals [48]. NK cell function is resistant to irradiation so both donor and recipientcells may contribute to net NK activity in the early post-transplant period [19, 54, 59, 82-83, 99]. It has been shown that lymphocytes with NK cell surface phenotype and activitycan be generated from BM precursor cells in vitro [19]. Activated NK effector cells arepresent in individuals who have undergone autologous and allogeneic BMT but they areabsent in those receiving chemotherapy alone [19, 94, 100-103]. They are hypothesized tobe the predominant mediators of antileukemic responses in individuals who haveundergone BMT and they can inhibit the growth of lymphocyte clones derived from the PBof these patients [19].NK cells are important immunoregulatory cells since they can provide accessoryfunction for a variety of immune responses that are affected by IL-2 or IFN [5]. Inaddition to their direct antitumour effects, NK cells are able to react with a wide range offoreign materials by producing soluble factors that can induce antiviral resistance and14cytostasis of tumour cells [5]. The ability of NK cells to produce WN-y [17, 29, 32, 45,78, 80, 82-84, 96] and IL-2 [5, 32, 41, 45, 59, 82-84] provides a mechanism for positiveself-regulation.2.2. CYTOKINE-INDUCED KILLER (CIK) CELLSThe stimulation of unfractionated PBMC with IL-2 for 5 to 7 days results in thegeneration of what have been classically known as “lymphokine-activated killer (LAK)cellst’. In this study, because BMC, as well as PBMC, and IL-7 and IL-12, in addition toIL-2, were used, it is more appropriate to adopt a name which will encompass thesebroader conditions. Thus, the tenn “cytokine-induced kifler (CIK) cells” will be used inthis thesis.CIK cells, originally described in the early 1980s by Rosenberg et. al. represent aunique system of cytotoxic cells distinct from classical CTL and NK cells due todifferences in their kinetics of activation, target cell specificity and phenotype of precursorand effector cells [52, 56, 70, 76, 87, 104-1101. The predominant CIK cell in PB displaysa CD3CD56CD16 phenotype [66]. Morphologically, CIK cells and NK cells areidentical. CIK cells possess cytoplasmic granules containing various enzymes and lyticproteins [7610 CIK cells are generated from several different cell types that conthbute to theoverall cytotoxicity; LGL contribute the major portion of CIK cell activity [32, 52, 57, 66,69, 70-71, 111-113]. However, populations naturally devoid of NK activity have beenshown to give rise to CIK cell activity [72, 104].152.2.1. CIK Cell ActivityThe CIK cell phenomenon is defined as the ability of PBL or splenocytes to kill NKcell-resistant tumour cell lines or fresh tumour cells in an MHC-unresthcted manner afterIL-2 stimulation. It is neither generated from a unique precursor nor mediated by a uniqueeffector. This IL-2-induced cytotoxic reaction is mediated by a heterogeneous mixture ofcells bearing phenotypic surface markers characteristic of both T and NK cells; of these,cells bearing the CD56 NK cell marker play a predominant role [32, 70-7 1, 76, 107, 111-112, 115]. Table 5 compares the precursor and effector cells involved in CIK cell activity.T and NK cells generate cytotoxic activity against fresh tumour cells at differentrates. Cytotoxic activity has been detected in LGL populations with as little as 24 hours ofstimulation with optimal doses of IL-2 while PBL and T cell populations require at least 48hours under the same conditions [66, 116-117]. This may be because (i) CIK cells areactivated more rapidly than T cells or (ii) intercellular interactions occur in the LGLpopulation which may optimize the development of CIK cell activity [661.TABLE 5. Precursor and Effector Phenotypes of Cells Mediating CIKCell Activity(Adapted from Hiserodt JC [52])Precursor Cells Effector Cellsmainly LGL mainly LGLmainly CD56 mainly CD56mainly CD16 variable expression of CD16mainly CD3 CD5- mainly CD3CD5mostly LLME* sensitive mostly L-LME sensitive* L-LME is a lysosomotrophic amine that removes granular lymphocytes162.2.2. Proliferative and Cvtotoxic Activities of CIK CellsAs shown in Table 3, the proliferation of CIK cells and the generation of CIK cellactivity are influenced by various cytokines, In enriched NK populations, IL-2 [71, 86,104, 111], IL-7 [71, 111], IFN-a [71, 104, 1111 or IFN-y [71, 86, 104, 111-112] alonecan induce CIK cell activity. Low concentrations of IL-2 increase cellular cytotoxicity byinducing secondary cytokine production and several cytokine combinations have been citedto enhance CIK cell activation. A combination of IL-2 and TNF costimulate CIK cellactivity and it has been proposed that TNF released by CIK cells following IL-2 stimulationmay act in an autocrine fashion to induce CIK cell activity [17]. It has also been shownthat CIK cells spontaneously secrete cytokines, such as IFN-y and TNF-oc, which haveadditional antileukemic activities of their own [103].2.2.3. Clinical Applications of CIK CellsEndogenously activated CIK cells are found in patients in complete remissionfollowing allogeneic or autologous BMT, but not after conventional chemotherapy [19-20,94, 100-103]. This suggests a potential role for CIK cells in the elimination of residualleukemic cells responsible for the relapse occurring in a majority of patients treated withchemotherapy alone [19, 52, 94, 100-103]. Although the contribution of CIK cells toantitumour responses is not clearly established, preclinical models have shown that thenumber and cytolytic activity of adoptively-transferred CIK cells correlate with tumourregression [61, 76, 118-120]. Several groups, including Lotzova et. al. have demonstratedthat CIK cells are capable of inhibiting the clonogenic growth of cells isolated from patientswith AML [22] and the ability of these cells to mediate tumour rejection is dependent onsimultaneous systemic IL-2 administration [19, 121]. CIK cells obtained from both normal17donors and those with AML have similar in vitro lytic activity on fresh allogeneic andautologous leukemic blasts [94, 96].The success of IL-2-CIK cell therapy relies on the sensitivity of leukemic cells toCIK cell lysis and the inducibility of high CIK cell activity in the patient [94]. DefectiveCIK cell generation or a decreased susceptibility of blasts to autologous CIK cell effectshas been documented in many acute leukemia patients [20, 22, 24, 29, 94-95] while themajority of patients in complete remission have good CIK cell activity [20, 22, 80, 105,119]. Long et. al. have proposed using CIK cells in the elimination of malignant cells inharvested BM as part of the therapy for autologous BMT in the treatment of widespreadneoplastic disease [122]. Animal studies performed by the same group have demonstratedthat the treatment of normal BM with CIK cells does not significantly reduce the ability ofthe BM to fully reconstitute the animal [52, 96, 122]. Thus, the use of CIK cells incombination with current methods of BM purging may be effective in completelyeradicating tumours.183. CYTOKINES INVOLVED IN REGULATING THE NON-SPECIFICCYTOTOXIC AND ANTIPROLIFERATIVE ACTIVITIES OFIMMUNE CELLS3.1. INTERLEUKIN-2IL-2 was isolated in 1976 by Morgan, Ruscetti and Gallo [123-125] as ahydrophobic 15 lcD glycoprotein produced by activated helper T lymphocytes [17, 24, 70,123, 126-129], It was purified from a heterogeneous mixture of immunomodulatorysubstances collectively termed ‘T cell growth factorst [10, 70, 123, 126, 128, 130-133].The IL-2 gene is located on chromosome 4q26-28 [125, 128]. Since its discovery, it hasbeen shown to be involved in the growth, differentiation, activation and function of avariety of immune cells, including B lymphocytes, monocytes and NK cells [126, 130].3.1,1. IL-2 Receptor (IL-2R)IL-2-responsive immune cells are activated by JL-2 following its interaction withIL-2R on the cell surface. The IL-2R contains at least two subunits, a and f3, and exists inthree isoforms distinguished by their affinities for IL-2: high-affinity (a/f3 heterodimer),intermediate-affinity (3 chain, p75) and low-affinity (a chain, p55) [17, 19, 70, 88-89,120, 123-124, 126, 128-129, 132-137]. A third chain, the ychain was cloned in 1992 bySugamura et. al. [136] and is believed to participate in the formation of high- andintermediate-affinity IL-2R [136, 138] and successful receptor-mediated internalization ofIL-2 [136, 138]. It also interacts with downstream signaling molecules and IL-2, thus,influencing IL-2R affinity [138].19Resting lymphocytes express few high-affinity IL-2R and show little or noresponse to low concentrations of IL-2 in vitro [70, 128]. Using polymerase chain reaction[65] and flow cytometric analysis [111], Naume et. al. [139] demonstrated the constitutiveexpression of high-affinity IL-2R mRNA in a small fraction of NK cells isolated fromhealthy donors [70]. Exposure to IL-2 [65, 80, 96, 111, 140], IL-7 [65, 111] and IL-12[65, 111] dramatically increases the expression of high-affinity IL-2R on NK cells. It isbelieved that these IL-2R are involved in NK cell proliferation, but not to the initialgeneration of CIK cell activity [70, 139].3.1.2. IL-2 as a Biologic Response ModifierBiologic response modifiers (BRM) comprise a group of factors which exert theireffects indirectly to mediate tumour regression by augmenting the host’s ability to mountspecific and nonspecific immune responses against invading neoplasms [70]. IL-2 is a trueBRM since all of the physiological effects it exerts appear to be the indirect result of otherimmunomodulatory substances synthesized and released by the differentiation andproliferation of activated lymphocytes.IL-2 induces the release of hematopoietic growth factors, including GM-CSF [70,141, 142] and IL-3 [141-142] from T lymphocytes and NK cells. It also induces therelease of secondary cytokines, such as IFN-y [6, 10, 23, 47, 65-66, 74, 78-80, 84, 96,123-124, 126, 128-129, 135, 143-147] and TNF-x [10, 65, 78-80, 96, 123, 129, 134-135, 139, 142, 145, 147], both of which have direct anti-leukemic effects, from Tlymphocytes, NK cells and monocytes. In order to enhance the cellular responsiveness tothese cytokines, JL-2 upregulates the transcriptional rate and expression of these cytokinereceptors on lymphocytes [13, 47, 65, 141,148-149] as well as some CAM [148].203.1.3. Proliferative and Cytotoxic Effects of IL-2IL-2 induces the in vitro differentiation of T lymphocytes from hematopoieticprogenitor clones generated by IL-3 [8]. It is necessary for the expansion of most Tlymphocytes [3, 10, 13, 66, 70, 78, 126, 129, 148, 150-152] and is capable of activatingand maintaining their proliferative and cytolytic responses [70, 123, 153].Additionally, IL-2 alone is sufficient and necessary for the expansion of NK cells[13, 31, 40, 55, 61, 66, 68, 70, 72, 74, 86, 119, 121, 150-15 1, 154-155] and can activateand maintain their proliferative and cytolytic responses [3, 25, 70, 78, 92, 120, 128-129,146-147, 149, 152-153, 156-157]. It promotes the differentiation of NK cells into CIKcells [10, 47, 74, 79, 87, 106, 120, 123, 126, 128-129, 140, 145-148, 153]. PBL orsplenocytes cultured for 3-5 days with 100-1000 U/ml IL-2 develop significant CIK cellactivity [2, 45, 52, 70, 110, 116].3.1.4. Clinical Applications of IL-2In describing the clinical biology and mechanisms of action of systemicallyadministered IL-2, Voss and Sondel propose the idea that all of the components necessaryfor an antitumour response are present in a tumour-bearing individual [70]. Nevertheless,the challenge remains in activating these effector elements and targeting them to the tumourtissue. IL-2 has shown great potential in activating antitumour activity in both in vitro andin vivo systems. The antitumour effects of high-dose IL-2 may be mediated by the (i)direct tumour cytolysis by CIK cells induced in vivo [140], (ii) induction of cytokinesecretion [74, 140, 146] and/or (iii) stimulation of proliferation and cytotoxic function ofCTL, NK cells or macrophages [140].21In vitro studies of the phenotype and functional immunological activity oflymphocytes obtained during rebound lymphocytosis have shown that the systemicadministration of low doses of IL-2 is accompanied by an increase in both the percentageand absolute number of CD56 NK cells [70, 154]. High doses of IL-2 increase thepercentage of both circulating NK cells and CD3 T lymphocytes [70, 154]. The enhancedCIK cell activity observed in vitro in freshly obtained PBL following IL-2 therapy does notresult only from an increase in the number of CD56+ NK cells, but also from a heightenedresponsiveness of these cells to subsequent IL-2 exposure in terms of proliferation andcytolysis due to in vivo activation [70].3.1.4.1. Cytokine Therapy with IL-2 Alone or in CombinationSystemically administered IL-2 augments cytolytic activity and causes theregression of certain malignant tumours in animals and humans [17, 121, 158-160].However, when given in doses sufficient to cause cellular activation in vivo, IL-2 alsoinduces significant systemic toxicity in these individuals [158]. While murine studies haveshown that low doses of IL-2 have little in vivo effect due to its rapid compartmentalequilibrium and clearance [70], those using high doses of IL-2, whose level wasmaintained by continuous infusion or repeated injections, demonstrated significantantitumour responses in vivo [70, 140].IL-2 preserves the antileukemic effect of lymphocytes [159] by activating andtriggering the lytic activity of cytotoxic cells in response to tumour cell presence [119,161]. Foa et. a!. have shown that the administration of IL-2 to patients with AML who arein relapse may lead to the disappearance of the leukemic population [119]. Subsequent invitro studies performed by this group showed that IL-2 hindered the in vitro proliferative22capacity of these leukemic blasts, suggesting that IL-2 may block their in vivo growth[153]. Charak et. al. [161] have shown that the preactivation of BM with IL-2 does notchange the progenitor cell activity nor does it impair the engrafting potential of the marrowgraft [79]. Additionally, several cytokines other than IL-2, such as TNF-cz, IFN-”y, IL-7and IL-12, are capable of inducing demonstrable antitumour activity and the possibility ofutilizing combination cytokine therapy is currently being explored.3.1.4.2. Immunotherapy with IL-2 plus CIK CellsAntitumour responses are dependent on three main factors: (i) the extent of thetumour burden, (ii) the dose of IL-2 administered, and (iii) the degree of lymphocyteactivation achieved [70]. Murine studies have shown that the adoptive transfer of ex vivoIL-2-activated syngeneic CIK cells, together with concomitantly administered IL-2, canproduce greater antitumour responses than the administration of IL-2 alone [52, 70, 80,106, 140, 153] and this has led to much interest in this area.3.1.4.3. Tumour-targeted ImmunotherapyThe systemic administration of IL-2 induces the expansion of NK cells, many ofwhich bear the FcyRIII (CD 16) for IgG and are, thus, capable of mediating ADCC [70].The addition of tumour-specific monoclonal antibodies to current IL-2 regimens may confera degree of specificity to these nonspecific immune effectors and enhance the specifictargeting of FcyRIII-bearing NK cells to sites of tumour growth. In this way, by avoidingnonspecific immune-mediated damage to normal tissues, fewer side effects may bemanifested [70].233. 1.5. Problems Associated with IL-2 Utilization3.1.5.1. Exacerbation of MalignancyIt is of concern as to whether or not IL-2 can exacerbate the growth and progressionof some malignancies by acting as a growth factor or through immunomodulation [145].Some malignant cells possess IL-2R and can proliferate in response to IL-2 stimulation[145]. However, IL-2 has only rarely been shown to induce such a stimulatory effect onacute leukemia cells [83, 96, 121, 126, 150, 153].3.1.5.2. IL-2 ToxicityThe systemic administration of IL-2 is capable of inducing immune-relatedantitumour responses in a minority of patients yet it induces some degree of toxicity in amajority of patients [96, 123, 129, 134, 160]. IL-2 utilization has been shown to inducecardiovascular, renal, pulmonary, gastrointestinal toxicity [52, 70, 80, 96, 123, 129, 145,162]. Additionally, autoimmune responses and neurologic and hematologic malignancieshave been associated with IL-2 utilization [52, 70, 80, 96, 123, 129, 145, 162]. Theseconditions are summarized in Table 6.24TABLE 6. Predominant Signs and Symptoms of IL-2 Toxicity(Adapted from Balkwil FR [81])FeverChillsAnemiaHypotensionErythematous rashFatigueThrombocytopeniaWeight gain increasingDiarrhea frequencyHeadacheNauseaMyagliaConfusionDisorientationDyspneaMyocardial infarction3.2. INTERLEUKIN-7IL-7 is a 25 kD glycoprotein which was identified, purified and cloned by Namenet. al. from a murine BM stromal cell line based on its ability to stimulate and support thegrowth of murine pre-B cells [8, 97, 130, 139, 148, 163-186]. It has also been known asmurine pre-B cell growth factor [163] and lymphopoietin-1 [148, 163, 167, 171, 174,178, 182]. Human IL-7 was cloned from a human hepatoma cell line, SK-HEP-1 usingmurine cDNA as the probe [174]. The IL-7 gene has six exons distributed over 33 kbpfound on chromosome 8q12-13 [130, 174, 178, 187].IL-7 is produced by BM and thymic stromal cells [9, 163, 174-175, 177, 188], asindicated by Table 7. In humans, the highest levels of IL-7 mRNA are found in the spleen[165-167, 174, 178, 183, 185]. While immature IL-7-responsive cells are found inprimitive hematopoietic tissues they are rare in adult hematopoietic tissues, such as the BM[189].25TABLE 7. Cell Types and Tissues that Express IL-7(Adapted from Appasamy PM [174])IL-7 Producer Protein mRNABone marrow stromal cells + +Spleen ?* +Fetal thymus + +Adult thymus +Cultured thymic stromal cells + +Kidney +Fetal liver +* ? indicates that the presence of the IL-7 protein has not yet been documented3.2.1. IL-7 Receptor (IL-7R)The IL-7R is a member of the hematopoietin receptor family [174, 178]. Itpossesses both high- and low-affinity binding sites [174] and is expressed on pre-B cells,some T cell lines and BM-derived macrophages but is absent on mature B lineage lines[174. 178, 183]. IL-7R expression on NK cells has not yet been defined [163]. Many celltypes have been observed to be IL-7-responsive. These are presented in Table 8.26TABLE 8. Cell Types that Respond to IL-7(Adapted from Appasamy PM [174])Cell Target Effect(s)B lineagepro-B cells Proliferationpre-B cells ProliferationT lineageBulk thymocytes (fetal, adult) ProliferationCD3CD4CD8 Proliferation, differentiation?CD4CD8- ProliferationCD4CD8 ProliferationBulk peripheral T lymphocytes Proliferation only with other mitogensCytotoxic T lymphocytes Proliferation, cytotoxic activity, CTL generationLAK cells of the T lineage Generation of LAK, proliferation, cytotoxic activityCommon B, Tprogenitor? Proliferation?, differentiation?NK lineageLAK cells of the NK lineage Generation of LAK, proliferation, cytotoxic activityMyeloid lineageMonocytes/macrophages Tumouricidal activity3.2.2. Proliferative Effects of IL-73.2.2.1. Effects on T LymphocytesIL-7 plays a pivotal role in the development of fetal thymocytes although it does notaffect the growth or differentiation of prethymic and inirathymic T lymphocyte progenitorclones [8, 130, 135, 139, 148, 165-166, 168-169, 172, 174, 183-184, 190-191]. It aloneis mitogenic towards resting CD4 and CD8 thymocytes that express either a/b or g/dTcR [130, 135, 139, 148, 165-167, 169, 174, 177-178, 180-182, 184-186, 188-190,19 1-195] and comitogenic towards peripheral T lymphocytes activated by concanavalin A27(conA) and phytohemagglutinin (PHA) [167, 174, 183, 192-194, 196] or antibodies toCD3 [174, 179-180, 194]. IL-7 augments IL-2R expression and IL-2 production [148,163, 165, 167, 174, 176, 178, 184, 191, 194, 196-197], as well as the expression ofICAM-1 on CD4 and CD8 thymocytes [6, 148, 163].3.2.2.2. Effects on B LymphocytesIL-7 is a hematopoietic growth factor for early B lineage lines. It induces theexpansion of immature B lymphocytes (i.e., pro-B and pre-B lymphocytes) [139, 148,163, 165-170, 174-175, 177-182, 184-185, 188, 190, 192, 196, 1981. It can support thein vitro growth of pre-B lymphocytes in the absence of stromal elements and can sustainthe proliferation of normal B lymphocyte precursors for several weeks in the presence ofBM-derived stromal cells [189, 199]. Henney [159] has reported that cultures of Blymphocytes maintained in IL-7 showed no evidence of either pro-B or pre-B lymphocytedifferentiation; thus, the role of IL-7 on B lymphocytes is solely proliferative [175].Mature B lymphocytes and plasma cells lack IL-7R and are, therefore, unresponsive to IL7 [163, 167, 174, 178, 189, 198].3.2.2.3. Effects on NK CellsIL-7-induced proliferation of NK cells from populations of resting PBMC occursindependently of IL-2 [139, 163]. Murine models have demonstrated that lymphoid cellscultured with IL-7 show enhanced recovery of viable lymphoid cells possibly due (i) tocontinued proliferation of these cells or (ii) to IL-7 acting as a “survival factor” that28promotes the increased recovery of lymphoid cells in culture in the absence of high levelsof proliferation [2].3.2.3. Cvtotoxic Effects of IL-73.2.3,1. Effects on T LymphocytesIL-7 generates cytotoxic T lymphocytes with CIK cell activity from thymocytes[97, 164, 169, 174, 176-178, 181, 184, 186, 190, 192-193, 200-202]. The generation ofantitumour CTL by IL-7 is as effective as IL-2 with respect to cytolysis and IL-7-generatedCTh are more specific than those generated by IL-2 [174, 176, 200]. Studies conductedby Pavietic et. a!. suggest that cells preactivated in vivo by IL-2 therapy may be moreresponsive to IL-7 in terms of CIK cell induction [203].3.2.3.2, Effects on NK CellsIL-7 induces CIK cell activity in lymphoid cells from secondary lymphoid tissues(e.g., lymph nodes, spleen) but not from primary lymphoid tissues (e.g., BM, thymus)independently of IL-2 [130, 178]. Murine studies conducted by Pavletic et. a!. showedthat the ability of IL-7 to induce the generation of CIK cell activity in populations of restingPBL appears to be mediated by a population of cells distinct from those stimulated by IL-2[159]; this may also hold true in the human system.High doses of IL-7 generate low CIK cell activity, independently of IL-2 [97, 111,139, 148, 163, 166, 169, 174, 177-178, 181, 184, 190, 192-193, 201, 203], from29PBMC. Although IL-7-induced CIK cell activity is about 10-fold lower in potencycompared to IL-2-induced CIK cell activity [139, 193, 201], its specific cytolytic activity isequal to that seen with IL-2 [176, 193]. The kinetics of IL-7-induced CIK cell activity areslower than those of IL-2; IL-2 requires 3 to 4 days [130] while IL-7 requires 6 to 8 days[130, 174, 178] to induce maximal cytolytic activity. Masuda et. at. suggest that IL-7 ismore effective in maintaining CIK cell cytotoxic activity over a longer period of time withrespect to IL-2 [178], Additionally, IL-7 has been shown to potently increase TNF-ocmRNA [65, 139], and to a lesser extent, IFN-’y mRNA [65, 193] in CD56CD3 NK cellsafter 24 hours in culture.3.2.3.3. Effects on Monocvtes/MacrophagesIL-7 IL-icc, IL-113, IL-6 and TNF-oc secretion by, and tumouricidal activity of,monocytes to enhance their tumouricidal activity [97, 169, 174, 177, 184-185, 190].3.2.4. Clinical Applications of IL-7IL-7 holds promise in the field of immunotherapy since it induces the proliferationand activation of cellular effector functions [163]. It also has direct effects on maturelymphoid cells which are engaged in immune responses. Cytokine therapy with IL-7 afterBMT has been considered because IL-7 may generate antitumour effects through amechanism different from that of IL-2 [97]. In addition, IL-7 may serve as alymphopoietin by acting on early hematopoietic elements; thus, it can provide a means forexpanding the immune repertoire from immature lineages in order to facilitate immunerecovery after BMT [163, 174]. The murine studies of Faltynek et. at. have shown that IL-307 accelerates the regeneration of lymphoid lineages after chemotherapy, suggesting that itmay be therapeutically useful as a lymphocyte-reconstituting agent for congenital oracquired lymphocytopenia [174, 185, 190].Systemic IL-7 administration exerts antitumour activity in animal models withouttoxicity [97, 177]. Tushinski et. al. [189] and Aoki et. a!. [177], among others, havedemonstrated that local IL-7 production by gene transfer into tumour cells can result in thedestruction of an otherwise progressively growing tumour [174, 177, 184, 192, 201].Additionally, the adoptive transfer of syngeneic lymphoid cells after in vitro incubationwith IL-7 induces tumour regression in tumour-bearing hosts; cells incubated with IL-7induced equal or greater tumour regression than those incubated with IL-2 [97].3.3. INTERLEUKIN- 12IL-12 was identified and purified from the conditioned medium of an EBVtransformed human B lymphoblastoid cell line by Gubler et. al. and Wolf et. a!. [16, 61,73, 111, 204-22 1]. It has also been known as cytotoxic lymphocyte maturation factor(CLMF) [14, 16, 51, 61, 73, 111, 198, 204-206, 208, 210-212, 214-216, 219, 222-229]and natural killer cell stimulatory factor (NKSF) [14, 16, 51, 61, 73, 111, 158, 204-206,208, 210-212, 214-2 16, 219, 22 1-228, 230]. IL-12 is a 70 kD heterodimer composed ofcovalently linked glycosylated 35 and 40 kD subunits encoded by separate genes, both ofwhich are required for biological activity [14, 16, 51, 57, 61, 73, 158, 204-208, 210-228,23 1-233]. The p35 chain shares homology with IL-6 while the p4.0 chain shares homologywith the extracellular domain of the IL-6R; thus, IL-12 appears to be composed of areceptor-like subunit linked to a cytokine-like ligand [16, 214-215, 217-218, 222, 224-225, 227-228, 231].31PBMC constitutively produce low levels of the heterodimer [204-205, 209, 232]and the 40 kD chain [205, 208, 218]. Biologically-active levels of IL-12 are produced byaccessory and antigen-presenting cells, including monocytes/macrophages [204-205, 218,224, 227, 231-232, 234-235] and B lymphocytes [204-205, 218, 224, 227, 230, 232,234] in response to bacteria, bacterial products or parasites.[L-12 induces a wide range of immunoregulatory functions on different lymphoidsubpopulations. It enhances cellular proliferation, cytotoxicity and cytokine production byT and NK cells [14, 16, 205, 208-2 10, 227, 234] and may regulate T and NK cells duringlocalized immune responses and inflammation [205]. IL-l2 upregulates and prolongs theexpression of several CAM, such as CD2 [61, 236], CD11a [61], CD56 [61, 216, 236],ICAM-1 [61, 216, 236] and LFA-1 [61, 2361, as well as several cytokine receptors,including IL-2Roc, IL-2Rf, and the 75 kD TNFR, on purified NK cells [16, 61, 111,216,237]. Together, these effects allow IL-12 to enhance immune function by inducing theproduction of essential cytokines and facilitating accessory cell functions in PBL cultures.The predominant in vitro functions of IL- 12 are summarized in Table 9.TABLE 9. Predominant in vitro Activities of IL-12(Adapted from Quesniaux VFJ [191])1. Stimulation of CD4 and CD8 T lymphocyte and PHA-activatedlymphoblast proliferation2. Activation of NK-mediated cytotoxicity3. Synergizes with IL-2 to activated cytotoxic lymphocytes andgenerate CIK cells4. Induction of IFN-y production by resting and activated PBL, Tlymphocytes and NK cells by increasing IFN-y mRNA transcriptionand stability323.3,1. IL-12 Receptor (IL-12R)The IL-12R is a 100 kD membrane protein expressed at low levels on restingPBMC [51, 217-218, 224, 231]. High levels of IL-12R are found on activated CD56NK cells [16, 51, 73, 204, 216, 231], and CD4 and CD8 T lymphocytes [16, 51, 73,204-206, 231]. Stimulation of PBMC leads to an increased cellular expression of IL-12R,which reaches maximal levels after 6 to 8 days of culture [51]. Radiolabeled ]L-12 bindingassays conducted by Desai et. a!. [51] showed that PHA-activated lymphoblasts express1000 to 9000 IL-12 binding sites per cell [218]. Steady-state binding analysis performedby the same group determined that the Kd of the IL-12R to be approximately 100 to 600pM [51]. Thus, only a small fraction of IL-12R needs to be occupied in order to trigger abiologic response. Desai et. a!. [511 also demonstrated a close correlation between thekinetics of IL- 12R expression and the kinetics of both the proliferative response and theacquisition and decay of biologic responsiveness to IL- 12 in PHA- or IL-2-activatedPBMC [51]. The expression of IL-12R on activated PBMC does not decrease in thepresence of anti-IL-2 or anti-IL-2R antibodies; therefore, the upregulation of the IL-i 2Roccurs independently of IL-2 [51].3.3.2. Proliferative Effects of IL-12IL- 12 potently induces the proliferation of activated T lymphocytes and NK cells.The concentration of IL-12 required to stimulate the half-maximum proliferation ofactivated T and NK cells is 1-8 pM (i.e., 6-8-fold lesser than the amount of IL-2 required);however, the maximum proliferation elicited by IL-12 is only 40-50% of the maximumstimulated by IL-2 [16].333.3.2.1. Effects on T LymphocytesIL-12 causes the proliferation of PHA- or IL-2-activated lymphoblastsindependently of IL-2 but has little effect on unstimulated PBL [14, 73, 205, 210, 214,216-217, 222]. It directly stimulates the proliferation of activated T lymphocytes [16, 61,73, 205-206, 208, 210-213, 215-216, 219, 222, 224, 228, 234]. Results obtained byBertagnolli et. a!. suggest that low concentrations of IL-2 in combination with IL- 12 actthrough independent pathways to induce T cell proliferation [206]. IL-12 also facilitatesthe differentiation of CTh precursors into lytically-active CTL [227] through the alterationof MHC antigen expression on stimulator cells [216]. IL-i 2-facilitated allogeneic CTLresponses are blocked by antibodies to IL-2, indicating that IL-2 produced in situ, either inresponse to allogeneic cells or to IL-12, is involved [16, 216].3,3.2,2, Effects on NK CellsIL-12 directly stimulates proliferation of NK cells [16, 61, 73, 205, 208, 210, 213,216, 219, 222, 224, 234]. NK cells constitute the predominant lymphocyte subpopulationthat proliferates in PBMC cultures in response to IL-12 [14, 16, iii, 221]. Stimulation ofPBMC with IL-2, mitogens or antibodies to CD3 for at least 4 days results in an increase inthe cellular expression of functional IL-12R, leading to an increase in IL-12-inducedproliferation of IL-2-activated NK cells [61, 206].343.3.2.3. Synergism Between IL-2 and IL-12IL- 12 either enhances or antagonizes IL-2-induced responses. It antagonizes high-dose IL-2-induced proliferation of NK cells [16, 205-206, 219] but enhances and prolongsthe proliferation of resting PBMC which have been activated by suboptimal doses of IL-2[14, 16, 51, 111, 203, 206, 210, 213, 219, 221-222]. The finding by Gately et. at. thatthis enhanced proliferation is partially inhibited by antibodies to IL-12 suggests that IL-2induces the in situ production of IL-12 [14]. This group has also suggested thatenhancement may result from an IL-12-induced alteration in the responding PBMCpopulation that enables them to proliferate in response to lower concentrations of IL-2 [14].It is believed that low concentrations of IL-2 in combination with IL- 12 may act throughindependent pathways to induce NK cell proliferation; each of these cytokines delivers apartial signal that is augmented in combination [61, 206].The mechanism by which IL-2 and IL-12 in combination cause synergisticproliferation of NK cells may involve (i) IL-12-induced prolongation of IL-2R expression[16, 111], (ii) IL-2-induced upregulation of IL-12R expression [14, 16, 111, 206], and(lii) alteration of the signal transduction process so that fewer occupied IL-2R are requiredto trigger the proliferative response [14]. Any, or all, of these actions can enhance theresponse of these cells to both cytokines [16, 1111. The ability of IL- 12 to prolong IL-2-induced expression of IL-2R supports the idea that enhanced proliferation in the presenceof IL-12 is partially due to enhanced responsiveness to IL-2 [14]. IL-12 is also believed toprolong the half-life of IFN-y mRNA [218].353.3.3. Cytotoxic Effects of IL-12IL-12 augments all known cytolytic activities mediated by NK cells. Extremely lowconcentrations of IL-12 are able to potently stimulate NK cell cytotoxic functions with rapidkinetics [208-209, 217]; IL-12-induced morphological signs of NK cell activation areevident with as little as 4 hours of stimulation [209-210, 215-216]. Activated NK cellsexpress high-affinity IL-12R; thus, in addition to its indirect effects mediated by othercytokines produced in response to IL-2, IL-12 can directly affect the function of CD56NKICIK cells [61, 205, 209-210, 215-216, 219-220, 237-238]. IL-12 may, at leastpartially, exert its effects on NK cells by upregulating CAM expression by NK cells [61,73, 111, 210, 218, 237]. It causes minimal proliferation of resting NK cells so its effectson the functional state of lytic effector cells appear to be independent of its ability tostimulate proliferation [216, 218, 239].IL-12 upregulates the cytotoxic activity of NK cells against tumour-derived celllines in short-term cultures at molar concentrations of 100-1000-fold lower than thoserequired for IL-2 [61, 73, 158, 209, 214, 222, 226-227] or IFN-o [61, 209], However,the maximum lytic activity elicited by IL-12 is about 50% that of IL-2 in purified NK cells[16]. The diminished response seen in IL- 12-stimulated long-term cultures compared tothat seen in IL-2 cultures may be partially due to the lower ability of IL-12 to cause theproliferation of NK cells with respect to IL-2 [7].IL-12 alone is unable to activate CIK cells in the presence of hydrocortisone (HC),but in the absence of HC, it synergizes with IL-2 in activating CIK cells [51, 111, 216,231]. Glucocorticoids minimize endogenous cytokine production. The ability of IL-12 toinduce CIK cell activity in the absence, but not in the presence, of HC could reflect arequirement for the in situ production of one or more cytokines, such as IFN-y, TNF-ct,36IL-i, IL-4, IL-6 and/or IL-7, which play a role in IL-12-induced activation of CIK cells[16, 65, 216].3,3.4. IL-12-induced IFN-y ProductionIL-12 potently induces 1FN-y production from resting and activated T and NK cells[14, 16, 51, 61, 65, 73, 111, 204-208, 210-224, 226-227, 230-233, 235-236, 240]. Itsynergizes with low concentrations of other IFN-y inducers, such as IL-2 [205, 224, 232],ligands for TcR-CD3 [16, 205, 208, 224, 232], allogeneic antigens [16, 205, 208, 232],PHA [16, 21 1-212] and phorbol diesters [16, 205, 208, 211-212, 232] in inducing IFN-ygene expression. Chan et. al. demonstrated that this synergistic effect occurs at both thetranscriptional and post-transcriptional levels [211].The synergistic effect IL- 12 has with other IFN-y-inducing factors results mainlyfrom the ability of the cytokine combination to increase IFN-y mRNA in individualproducer cells, rather than through the recruitment of new IFN-y-producing cells [211-212], and/or from increasing the transcriptional rate of IFN-y mRNA [2111. The activationof the IFN-y gene by IL-2, IL-12 and phorbol diesters is independent of de novo proteinsynthesis and this suggests that IL-12 acts directly on ]FN-y-producing cells [211]. TheIFN-y produced directly and indirectly conthbutes to the augmentation of the proliferativeresponse [14]. IL-12 synergizes with IFN-y to enhance IL-12 production by monocytesand macrophages in a positive feedback loop [224].373.3.5. Clinical Applications of IL-12IL- 12 exerts a number of immunoenhancing effects on the activities of NK and CIKcells and T lymphocytes and its biological activities suggest that it may have therapeuticutility as an antitumour agent when used alone or in combination with other cytokines. Itsuse has been considered in antitumour therapies based on its potential in expanding and/ormaintaining CIK cell functions in adoptive immunotherapy [16]. The systemicadministration of IL-12 into normal mice is associated an increase in NK cell and CTLfunction [227, 233], as well as with little or no toxicity [233]. Additionally, iransfection ofpoorly immunogenic tumours with the IL-12 gene results in their conversion into onesstrongly immunogenic; these converted tumours are then able to elicit a systemic immuneresponse that will lead to the destruction of both nontransfected and transfected tumourcells [16, 227]. The ability of IL- 12 to facilitate nonspecific CIK cell and specific CTLresponses indicates that it may be useful as a therapeutic agent against some tumours andinfectious diseases [216].384. THE USE OF BONE MARROW TRANSPLANTATION IN THETREATMENT OF ACUTE LEUKEMIA4.1. CANCERMost deaths from cancer are due to the growth of metastases that are resistant toconventional treatment. The lack of successful strategies in the treatment of cancer arisesfrom limits placed by the location of the tumour on the dose of therapeutic agent that can bedelivered to it and the heterogeneity of malignant neoplasms [241]. Metastatic cells aregenetically unstable and diverse subpopulations of these cells are able to evade hostresponses that occur during progressive tumour growth and the establishment of metastaticlesions [241].Despite considerable progress in clinical oncology, successful curative treatment formost patients with established tumours remains an elusive goal. Although BRM mediatesome antitumour effects directly, they primarily exert their effects indirectly by mediatingtumour regression by augmenting the host’s ability to mount both specific and nonspecificimmune responses against invading neoplasms [242]. Because of this, they have beenactively explored for their potential role in the treatment of leukemia.4.1.1. Acute LeukemiaAcute leukemias arise from the clonal expansion of a single transformedundifferentiated hematopoietic cell as well as from mutations which may occur during thisprocess [114]. Impaired regulation of the genes responsible for the normal growth anddifferentiation of hematopoietic cells initiates molecular events that can lead to the39transformation and proliferation of these cells, resulting in a state which is clinicallyrecognized as leukemia [101].4.1 , 2. Therapeutic ImmunomodulationNonspecific effectors of immune surveillance include NK cells, circulatingmonocytes, tissue macrophages and certain CTL. These cells form the first line of hostdefense against neoplasms by mediating the MHC-unrestricted recognition of selectedtumour cell lines. Direct tumour cell lysis is mediated by either intercellular contact or therelease of diffusible cytoactive molecules, such as chemotactic factors, growth peptides,proteases, TNF, IFN-y and interleukins, which can potentially inhibit tumour growth. Inimmunologically normal individuals, it is possible to eliminate spontaneously-arisingneoplasms by a combination of nonspecific and specific immune effector mechanisms. Bytreating patients with cytokine-induced effector cells and pharmacological doses of agentsnormally involved in regulating antitumour effects in vivo, it may be possible to restimulatethe apparently quiescent immune effector mechanisms that originally allowed tumourdevelopment to now mediate tumour regression.4.2. BONE MARROW TRANSPLANTATION (BMT4.2.1. Bone Marrow (BM) MicroenvironmentHematopoietic stem cells are pluripotent and have extensive renewal ability; theseproperties allow them to repopulate BM after ablation [243]. Stromal cells, made up ofmacrophages, fibroblasts and adipocytes [166, 185, 243], support myeloid and lymphoid40proliferation and/or differentiation through direct cell-cell interaction and/or the release ofcytokines and growth factors [166, 189, 243], which either have stimulatory or inhibitoryeffects on the proliferation and differentiation of stem cells during regenerative and steady-state conditions. Molecules in the extracellular matrix bind and present regulatorymolecules to stem cells and their more differentiated progeny [243].4.2.2. BMTOriginally, therapy for leukemia has been approached with the assumption thathematopoietic malignancies could be cured by administering megadoses of chemotherapyand radiation. Unfortunately, the therapeutic window is limited by toxicity to other organsystems [244]. Improved disease control, such as higher doses of total body irradiation(TBI), frequently results in higher toxicity without an increase in overall disease-freesurvival (DFS) [244], BMT is performed with the goals of facilitating hematopoietic andimmunologic reconstruction following marrow grafting and decreasing the period duringwhich the patient is at risk of developing bleeding and infections during the period oftransplant-related immunocompromise. This could be achieved through either theadministration of cytokines following BMT to enhance the proliferation, differentiation andfunction of various cells of the immunohematopoietic system or the activation of progenitorcells prior to grafting [100].BMT for malignant lymphohematopoietic disease is an established therapeuticoption for an increasing number of patients. Residual disease following remission can beeradicated in a proportion of patients by high-dose chemoradiotherapy along withallogeneic or autologous BMT [151]. Allogeneic and autologous BMT for hematologicmalignancy are associated with a lower risk of relapse and improved long-term DFS than41treatment with cytotoxic chemotherapy alone [103]. BMT is most effective when used forpatients with early well-controlled diseases, rather than as a rescue procedure for therapy-resistant malignancies [244-2451. Patients with AML transplanted in first remission showa higher probability of DFS than those transplanted in early relapse or second remission[244].Patients who obtain complete remission with conservative induction therapy havethree options for future treatment: (i) chemotherapy alone, (ii) allogeneic BMT with anHLA-compatible donor or (iii) autologous BMT with his own BM. In AML, long-termDFS with chemotherapy alone is about 20%; disease recurrence occurs due to minimalresidual disease in the patient [96, 103, 102, 151]. Prospects of longer DFS are associatedwith allogeneic and autologous BMT, namely 80% and 50% respectively [96, 245]; thisimprovement over chemotherapy alone may result from an antileukemic effect largelymediated by MHC-unrestricted CIK cells which are absent after treatment withchemotherapy alone [102].4.2.3. Treatment of AMLIn Canada, 650 new cases of AML are diagnosed each year and less than 25% ofthese patients under the age of 55 maintain durable clinical remission with conventionalchemotherapy [96]. A major aim in AML is to prevent relapse and achievement of this goalrests on finding ways to fully eradicate disease. Part of the antileukemic effect of BMTderives from the regenerating BM to constitute an antileukemic effect that is most evidentafter allogeneic BMT. Table 10 summarizes some of the major differences betweenallogeneic and autologous BMT.42TABLE 10. Allogeneic versus Autologous BMT(Adapted from Goldstone AH [246])Parameter Allogeneic Autologous_____________________BMT BMTProblems with matching yes noGraft versus tumour effect yes noAge limitation up to 50 years up to 60 yearsGraft-versus-host disease yes usually noVenoocciusive complications yes yes but littleInterstitial pneumonitis yes yes but littlePotential marrow contamination no yes4.2.3.1. Allogeneic BMTAllogeneic BMT is associated with a high probability (i.e., 70% to 80%) of long-term DFS [247-248], and is, thus, the choice in maintaining remission in AML. However,an age restriction of 55 years and the need for an HLA-matched donor limit the number ofpatients who are able to access this treatment [96, 247]. The high probability of attaininglong-term DFS in individuals undergoing allogeneic BMT may be related to thedevelopment of graft-versus-host disease (GVHD); it has been observed since 1979 thatindividuals who developed GVHD post-BMT frequently experienced lower relapse rates[79, 244, 247]. Disease control by allogeneic BMT is postulated to be mediated by thepreparative regimen and interactions between graft-derived cells of the immune surveillancesystem and residual malignant cells of the host [96, 244].434.2.3.2. Autologous BMTAutologous BMT is becoming increasingly popular as a procedure that allows forthe administration of more aggressive chemotherapy to patients with advanced malignancieswhile avoiding GVHD and other complications associated with allogeneic BMT [100, 122,246, 248-249]. It is favoured over allogeneic BMT because it negates the need for amatched donor, there is no age limit, and the procedure-related risk is diminished due to theabsence of the rejection phenomenon and interactions of immunocompetent donor T cellsagainst the host [96, 100, 248]. Syngeneic BMT shows that the myeloablative (e.g.,cyclophosphamide, ThI) treatment itself is well-tolerated; this demonstrates that autologousBMT can be applied to a greater percentage of AML patients with acceptable toxicity [96,247].However, the recurrence of leukemia is more common after autologous BMT thanafter allogeneic BMT for AML in first remission [17, 154, 245]. Despite the use of high-dose chemoradiotherapy in the treatment of acute leukemia, relapse continues to be themajor cause of death in patients undergoing autologous BMT [78, 97, 245]. Thereinfusion and regrowth of tumour cells with the autologous marrow graft and/or thepersistence of minimal residual disease (MRD) in the patient can give rise to thereappearance of disease due to the absence of an allogeneic antileukemic effect [17, 78-79,96, 245-247, 250].In autologous BMT, the patienttsBM is harvested prior to intensive chemotherapy,treated in vitro and then readministered; this allows for the administration of high levels ofcytotoxic drugs and/or irradiation that would otherwise be limited by host BM toxicity andfailure [249]. Patients should be harvested during remission when the tumour burden has44been reduced by aggressive chemoradiotherapy to decrease the potential for reexposure ofthe patient to residual neoplastic cells in the BM [249].Although the BM may still contain a small number of tumour cells, methods haverecently been developed for the in vitro purging of harvested BM to remove contaminatingmalignant cells. An increase in DFS after autologous BMT can be achieved by enhancingthe antitumour activity of the BM by improving systemic therapy and purging.Postremission treatment may help decrease the number of leukemic cells in the autograftand in the patient at the time of BMT and removal of the least possible volume of BMminimizes the number of leukemic cells in the graft [247].4.2.4. Problems Associated with BMTComplications seen after BMT are related to the intensity of the conditioningregimen, BM suppression and related immunodeficiency [162]. Major problems associatedwith BMT include (i) acute and chronic GVHD, (ii) leukemic relapse, (iii) graft failure inpatients given HLA-nonidentical or T lymphocyte-depleted grafts, (iv) infections, and (v)toxicity from conditioning programs [251]. Some of the predominant problems associatedwith BMT are listed in Table 11.45TABLE 11. Complications Associated with BMT (In increasing severity)(Adapted from Deeg HJ [162])GastroenteritisOral mucositisVenoocciusive diseaseLeaky capillariesHemolysisAcute GVHDGraft failureInfectionsImmunodeficiencyInterstitial pneumonitisMarrow dysfunctionSecondary malignancyChronic GVHDChronic lung diseaseAutoimmune disordersEndocrine dysfunctionCataractsDental problemsRadiation nephritis4.2.5. Therapy in BMTImprovements in the therapeutic regimens that currently exist for the treatment ofacute leukemias stem from the utilization of high-dose therapy with autologous BMT earlierin the disease and from the addition of BRM or alternative agents, either during, orimmediately after, recovery from BMT so that such immunomodulation may control thedisease [246]. It is desirable to develop an optimal regimen to control MRD in patientswith malignant disease in order to decrease the rate of relapse. This may be achieved byeradicating as many clonogenic tumour cells as possible before BMT and by improving theintensity of the immune-mediated interactions of the donor immune cells against residualhost tumour cells that escape pre-transplant chemoradiotherapy [100].464.2.5.1. ChemotherapyChemotherapy commonly involves cyclophosphamide with TBI [251]; however,this is insufficient to eradicate leukemia in all cases and is accompanied by a high incidenceof graft failure in BMT patients [247]. Immunosuppressive drugs, such as cyclosporineand methotrexate, have been used alone or in combination in the prevention of GVHI) aftergrafting [251]. However, clinical evidence supports the idea that all approaches involvingchemotherapy with or without TBI have reached the limits of nonhematopoietic toxicity andit appears impossible to achieve any further improvements in these approaches [2511.4.2.5.2. Depletion of T LymphocytesAcute GVHD is induced by donor immunocompetent T lymphocytes. Thepretranspiant depletion of donor immunocompetent T lymphocytes by immunologic ormechanical methods may reduce or prevent GVHD [82, 98, 100-101, 244, 250-252]. Bydepleting BM of T lymphocytes, the most differentiated lymphocytes causing GVHD areeliminated. Thus the immune system returns to an early prenatal state in which newlyformed T lymphocytes can accept the host antigenic environment as “self” and becometolerant to it [251]. Clinical studies described by Slavin and Nagler have shown that aslong as graft rejection is absent, BM depleted of T lymphocytes will engraft normally[100].Although the incidence of acute GVHD decreases with the removal of Tlymphocytes, that of graft rejection and leukemic relapse increase [19, 83, 98, 159, 162,247-248, 250-252, 253], possibly due to the loss donor T lymphocytes which eitherdirectly or indirectly mediate an antileukemic effect [98, 252]. Drobyski et. a!. have shown47that the depletion of T lymphocytes does not remove CIK cell precursors [72, 110, 252,254]. Although donor NK cells activated in vitro can mediate graft-versus-leukemia (GVL)activity, Mackinnon et. a!. have proposed the possibility that the failure of helper T cells tofully activate NK cells in vivo following BMT with T lymphocyte-depleted BM leads to aloss of GVL activity, resulting in an increase in leukemic relapse [98].4.2.5.3. Cytokine TherapyTherapy with recombinant cytokines after autologous BMT is actively beingexplored as a way to prevent relapse. Cytokine-based tumour therapy involves locally-produced cytokines to take advantage of their immunoenhancing properties while avoidingsystemic toxicity [16]. As reviewed by Slavin and Nagler, cytokines can increase theefficacy of tumour eradication prior to BMT by improving host tolerance to higher andmore frequent doses of cytotoxic agents [100]. The eradication or control of MRD may beaccomplished through cytokine-mediated immunotherapy or cell-mediated cytokineactivated immunotherapy, both of which induce antigen-nonspecific or immune-specificeffector mechanisms against residual tumour cells [100]. Cytokines, either alone or incombination, have been studied in terms of their ability to enhance the immune system afterBMT; some of these are presented in Table 12.48TABLE 12. Possible Clinical Applications of Cytokines in BMT Therapy(Adapted from Slavin S & Nagler A [100])CytokineIFNsBiologic ActivityAntiviral activityPotential ApplicationInfectious diseasesCytokine-induced amplification of natural host defense mechanisms against cancermediated by MHC-unrestricted NK cells and a variety of cytokines produced by activatedNK cells which have direct or indirect antitumour activity enable the elimination orprevention of residual tumour cells from escaping high-dose chemoradiotherapy given withBMT [100]. They may induce the rapid repopulation of donor hematopoietic cells whichwill inhibit residual host tumour cells from escaping chemoradiotherapy in BMT bycompeting with tumour cells for available BM niches [100]. Additionally, cytokines maybe involved in increasing and synchronizing the proliferation and differentiation of cancercells that are able to respond to a given factor, thus rendering these cells more susceptible tothe actions of cytotoxic agents or high-dose chemoradiotherapy used in BMT [100].IL-i Radioprotection Cancer radiotherapyIL-2 Antitumour cytotoxicity, Cancer immunotherapyimmune reconstitution,improved activity againstinfectious agents?IL-i, IL-2, GM-CSF Hematopoiesis BMTG-CSF, M-CSF, IL-6 Immunologic reconstitution Chemoradiotherapy ofcancerIL-i, IL-4, IL-5, IL-6, IL-7 Improved activity against Immunodeficiencyinfections agents, antitumour Cancer immunotherapy?cytotoxicity?Erythropoietin Erythropoiesis AnemiaIFN, IFN-cc, IFN-y, TNF Antitumour activity Cancer therapy49Immunotherapy by IL-2 with or without ex vivo-generated CIK cells, if utilizedearly after autologous BMT [97], has been proposed as a possible means by which relapserates may be decreased since it induces the regression of some hematologic malignancies.IL-2 infusion induces the proliferation and activation of circulating CD3CD 16- Tlymphocytes and CD3CD16 CIK cells, as well as the precursor activity of CIK cells inPB [61, 83, 100, 103, 123, 126, 154]. However, systemic IL-2 administration isassociated with significant toxicity [97]. Because of its short half-life (reported to rangefrom 7 minutes to 7 hours) [70, 123, 143], administration of high doses is required toachieve the desired physiologic responses [16].Pancytopenia secondary to the intense preparative regimen threatens successfulBMT; fortunately, the availability of hematopoietic growth factors, such as GM-CSF, JL-3and IL-6, can shorten the period of neutropenia by enhancing the proliferation ofprogenitors involved in the engraftment process [244].Since patient toxicity due to cytokines occurs in a concentration-dependent manner,synergistic drug interactions are being investigated in hopes of decreasing the concentrationof each individual cytokine to keep the therapeutic benefit high while diminishing theseverity and incidence of toxic side effects [143]. Combination therapy with certaincytokines may be synergistic and permit therapy with lower and less toxic side effects.4.2.5.4. Adoptive ImmunotherapyAdoptive immunotherapy is being explored as a means by which residual leukemiamay be eradicated after BMT [154]. The transfer of donor immunocompetent cells with theallogeneic BM contributes to the antileukemic effect of allogeneic BMT [154, 252]. IL-250has been utilized in adoptive immunotherapy and may contribute to tumour regression[140, 153, 163]. The generation of antitumour cells with activity in vitro and therapeuticefficacy in vivo is seen with CIK cells, tumour-infiltrating lymphocytes and CTL [164].Slavin et. a!. have reported that IL-2 activation does not impair BM reconstitution postBMT [100]. As reported by Parmiani, tumour-bearing animals can be cured by adoptiveimmunotherapy with tumour-specific T lymphocytes; they reject their own immunogenicneoplasms and develop long-lasting immunity resulting from the long-term persistence ofdonor immune T cells or from immunization caused by tumour destruction [115].Endogenously generated NK cells circulate after allogeneic or autologous BMT butnot after chemotherapy [19, 94, 100-103]. NK cell function recovers early after BMT [19,48, 58-59, 82] and IL-2-responsive CIK precursor cells can be detected in the PB as earlyas 18 days after autologous BMT [97]. As reported by Hogan, significant levels of IL-2are spontaneously released after BMT; this is associated with an increase in NK cellfunction [59, 101-102, 147] and the release of cytotoxic cytokines such as TNF-cx [56, 69,102] and IFN-y [30, 56, 102].The CIK cell phenomenon has been proposed as a new immunotherapeuticapproach to human cancer since CIK cells can lyse fresh non-cultured cells as well astumour cell lines, of both lymphoid and myeloid origins, while showing minimal toxicitytoward normal cells [105, 154, 225, 249]. Thus, the endogenous generation of CIK cellsafter allogeneic and autologous BMT contributes to the reduced relapse following BMT[94, 103]. Fierro et. a!. [105] as well as Foa [252] have reported that it is possible to elicitCIK cell activity in PBL from patients in complete clinicohematological remission [20, 22,80, 105, 119]. Additionally, CIK cells spontaneously secrete other cytokines that haveadditional antileukemic activity [154]. Thus, MRD may be eliminated or suppressed by51stimulating the antileukemic potential of CIK cells once the patient achieves remission [69,151].4.2.5.5. Induction of GVHDGVHD which develops after allogeneic BMT may be controlled post-BMT throughthe use of antibodies specific for either the effector cells of GVHD or the cell products thatare involved in the induction of GVHD [162]. Post-transplant therapy, such as theinduction of GVHD after autologous BMT, as a therapeutic maneuver has been investigatedas a means of decreasing relapse rate. It is hoped that this GVHD syndrome can provide aclinical antitumour effect similar to that following allogeneic BMT in patients undergoingautologous BMT [79, 245, 247].4.2.5.6. GVL EffectA GVL effect is frequently observed with allogeneic BMT. This antileukemic effectis hypothesized to be mediated predominantly by the NK and CIK cell populations [79, 96]and Slavin et. a!. [100] have reported that a GVL effect can be induced with IL-2 or IFN-cc[247] treatment during the postgraft period. Strategies in which immune effectormechanisms may be activated after autologous BMT such that a GVL effect may beachieved are currently being investigated [78, 100].524.2.5.7. BM PurgingIn autologous BMT, the risk of the reinfusion of tumour cells in the BM may bedecreased by purging the autologous BM by immunological [100, 122, 154, 161, 245-246, 249], mechanical [100, 246] or pharmacological [100, 122, 161, 245-246, 248-2491means, as well as long-term BM cultures (LTBMC) [78, 243, 245]. Long et. al. haveproposed treatment with CIK cells prior to BMT as a method by which leukemic cellpurging may be achieved [249]. The freeze-thawing of autologous BM grafts or themodulation of immune mechanisms operative in autologous BMT could potentially beharnessed in the eradication of tumours from BM grafts [245]. Monoclonal antibodies,conjugated to radioactive isotopes or immunotoxins, designed to interact specifically withleukemic cells may be a means by which leukemic cells may be destroyed [247].In LTBMC, stromal cells promote self-renewal without the need to add exogenousgrowth factors. Although normal hematopoiesis is promoted, the survival and growth ofleukemic cells are inhibited in over 50% of all cases [243, 247, 255]. Thus, leukemic cellsdie preferentially in LTBMC possibly due to (i) the absence of factors in cultures, (ii) highneeds for factors produced in cultures compared to normal cells, or (iii) the selective killingby cells or cell products [243]. Additionally, leukemic cells may have intrinsically poorerself-renewal probability or be defective in their ability to attach to BM stromal cells or theextracellular matrix and, therefore, have inadequate access to necessary growth factors[243].534.2.6. Future Trends in BMTPeripheral blood stem cells (PBSC) represent a pooi of committed progenitor cellswhich can give rise to early and rapid recovery of PB counts. They are capable ofrepopulating ablated patients; their number maximizes after chemotherapy when intensiveleukophoresis yields a sufficient number to ensure hematopoietic regeneration [243, 247].PBSC provide a purer source of stem cells and are less likely to be contaminated withmalignant cells compared to BM [243, 247].BMT is associated with delayed neutrophil and platelet recovery. PBSC have beenused in autologous BMT in the treatment of AML; hematopoietic recovery is reached earlierwith the rapid reappearance of neutrophils and platelets [96, 247]. Although rapidengraftment occurs after the infusion of PBSC, it may be unstable [243] and, becausePBSC may not be able to maintain long-term engraftment alone, BM is used as a stem cellsource to supplement PBSC [96].545. THESIS OBJECTIVESThere are three main ways by which AML can be treated: chemotherapy alone,allogeneic BMT and autologous BMT. However, there is an associated risk of diseaserecurrence with all these methods; the relative relapse rates are approximately as follows:80% for chemotherapy alone, 20% for allogeneic BMT and 50% for autologous BMT. Theobservation that individuals who receive BM from a twin are still subject to a 60% chanceof disease recurrence indicates that the provision of “clean” BM alone to the patient isinsufficient in eliminating disease and that there must be some active immune componentinvolved in the eradication of residual disease.The use of autologous BMT in patients with acute leukemia is an ideal treatment dueto the lack of age restrictions and the requirement for an HLA-matched donor.Additionally, the immunological problems incurred to varying degrees after allogeneicBMT are completely avoided. Nevertheless, the use of autologous BMT is severely limitedby high relapse rates due to (i) the persistence of leukemic stem cells in the autologous BMand (ii) residual disease in the patient. Either one of these problems is sufficient to causedisease recurrence due to the lack of an allogeneic antileukemic effect.It would be desirable to develop a means by which autologous BM could be utilizedwith a relapse rate that is decreased to a level similar to that of allogeneic BM. An activeimmune component must be provided with the BM graft in order to eliminate MRD and thiscould be achieved by appropriate cytokine manipulation of autologous effector cells toenhance their antiproliferative activity. The NK cell population is an obvious candidate forsuch a manipulation; it is the first cell subpopulation to reconstitute after BMT.55A clinical protocol in which transplantation of IL-2-activated BM alone to the patienthas been developed by the Klingemann and Eaves Laboratories at the Terry Fox Laboratoryfor Hematology/Oncology and used in collaboration with the BMT Program of BritishColumbia as part of a Phase I clinical trial. This treatment has been used in nine patients sofar [96], and although the results appear promising, one major drawback has been delayedneutrophil and platelet recovery (median of 49 days).Phase II of this project involves the transplantation of IL-2-activated PBSC to thepatient. To date, five patients have received this treatment and it has been observed thatthere is an improvement in the time required for platelet and neutrophil recovery; neutrophilrecovery has occurred, on average, 12 days post-BMT. However, as reported by Testaand Dexter [243] and by our group [96], the rapid engraftment associated with thetransplantation of PBSC may be unstable and, thus, may not be able to maintain long-termengraftment alone. Therefore, it appears that BM may be required as a supplementarysource of stem cells in PBSC transplants.The objective of my thesis was to study the modulatory effects of IL-2, IL-7 andIL- 12 on the cytotoxic and antiproliferative activities of BMC and PBMC. These particularcytokines have been shown to mimic or enhance the cytotoxic and antiproliferative activitiesof IL-2 and act directly on NK cells, which are hypothesized to be the predominantmediator of antileukemic effects after BMT. My first aim was to study the differentialeffects of IL-2, IL-7 and IL- 12 on BMC and PBMC in terms of inducing direct cytotoxicityagainst tumour target cells using a release assay against K562 and Daudi target cells,which are classical tumour cell lines used to measure NK and CIK cell activities,respectively.56Secondly, I was interested in the ability of these cytokines to induceantiproliferative activity in terms of direct cytotoxicity as well as indirectly throughcytostatic effects, against neomycin-resistant K562 (K562neor) tumour cells using anassay developed in our laboratory to detect the survival of clonogenic K562 cells. Byusing this clonogenic assay, I measured the ability of BMC and PBMC to inhibit thesurvival of K562neor tumour target cells after a fixed coculture period in the presence ofincreasing initial target cell contaminations. Using the same system, I investigated thekinetics involved in the cytokine stimulation of BMC and PBMC by measuring the survivalof K562-neo’ tumour cells at various time points after coculture with BMC and PBMCeffector cells.By carrying out these experiments, it was my aim to refine the in vitro culturesystem for BMC and PBMC and to assess the relative abilities of these two cell populationsin terms of their cytotoxic and antiproliferative activities after cytokine stimulation. Theinformation obtained from these experiments will aid in refining the ex vivo culture systemfor BMC and PBMC such that their antileukemic effects may be optimized.57CHAPTER IIMATERIALS AND METHODS1. CELL PREPARATION1.1. Normal Bone Marrow (BM’The BM samples used in these studies were acquired from aspirate harvestsobtained with informed consent from normal BM donors for autologous or allogeneic BMTafter approval of the University of British Columbia Clinical Screening Committee forResearch Involving Human Subjects. Cells were collected in TC199 medium with 100U/mi preservative-free heparin and kept on ice.Light-density BMC were isolated by density-gradient centrifugation on FicollHypaque (FH) (Pharmacia, Piscataway, NJ) at a density of 1.077. The BM sample wasdiluted at a 1:3 ratio with phosphate buffered saline (PBS) (StemCell Technologies,Vancouver, B.C.) at room temperature and then overlayed onto FH at a 3:2 ratio in a 50 mlFalcon tube (Becton-Dickinson Canada, Ontario). The mixture was centrifuged at 600g for45 minutes at room temperature. The interface layer was removed and washed twice withRPMI 1640 medium (StemCell Technologies) supplemented with 2 mM L-glutamine, 1mM sodium pyruvate, 0.1 mM nonessential amino acids, 50 U/mi penicillin, 25 mM hepesand 5% heat-inactivated fetal calf serum (FCS) (Hycione, Logan, UT) at 200g for 10minutes. This medium will be referred to as RPMI/5%FCS hereafter. Nucleated cellcounts were performed using a hematocytometer in the presence of 3% acetic acid.581.2. Normal Peripheral Blood (PBPB cells were obtained by venipuncture after informed consent from normal donorsor from normal platelet donors undergoing platelet/leukapheresis at the Cell Separator Unitof the Vancouver General Hospital. Cells were originally collected in 13x 100 mmVacutainer tubes (Becton-Dickinson Canada) containing 100 U of heparin and kept at roomtemperature,Light-density PBMC were isolated by density-gradient centrifugation on FH at adensity of 1.077 (Pharmacia). The PB sample was diluted at a 1:3 ratio with PBS at roomtemperature and then overlayed onto FH at a 3:2 ratio in a 50 ml Falcon tube. The mixturewas then centrifuged at 600g for 45 minutes at room temperature. The interface layer wasremoved and washed twice with RPMJJ5%FCS at 200g for 10 minutes. Nucleated cellcounts were performed using a hematocytometer in the presence of 3% acetic acid.2. TUMOUR CELL LINESK562 is an NK-sensitive human myelogenous leukemia derived from a patient withCML in blast crisis. Daudi is an NK-resistant human B cell tumour line derived from apatient with Burkitt lymphoma. Both cell lines were obtained from the American TypeCulture Collection (ATCC, Rockville, MD) and maintained in continuous culture in RPMI1640 medium (StemCell Technologies) supplemented with 2 mM glutamine, 1 mM sodiumpyruvate, 0.1 mM nonessential amino acids, 50 U/ml penicillin, 25 mM hepes and 10%heat-inactivated FCS, hereafter referred to as RPMJJ1O%FCS, at 37°C in a humidifiedatmosphere of 5% CO2 in air.593. CYTOKINESRecombinant human IL-2 was obtained from Hoffmann-LaRoche Limited (Bern,Switzerland). It had a specific activity of lx i07 U/mg. Recombinant human ]L-7, clonedand expressed in E. coli, was provided as a gift by Immunex Research and DevelopmentCorporation (Seattle, WA). It had a specific activity of 2x107 U/mg. Recombinant humanIL-12, cloned and expressed in CR0, was provided as a gift from the Genetics InstituteIncorporated (Cambridge, MA). It had a specific activity of 3.57x106U/mg.4. GENE TRANSFER OF K562 TARGET CELLSThe neor gene, coding for resistance to neomycin, was transfected into K562 cellsby electroporation. 5x107 leukemic cells were suspended in 0.8 ml of RPMJJ5%FCS andincubated on ice for 10 minutes with 30 ig of the pMC1-Neo plasmid (provided by Dr. K.Humphries, Terry Fox Laboratory for Hematology/Oncology, Vancouver, B.C.). Thecells were exposed to a single voltage pulse (125 hF, 0.4 KV) at room temperature andallowed to remain in buffer for 10 minutes. The cells were then transferred into 25 cm2 50ml tissue culture flasks (Falcon, Lincoln Park, NJ) and incubated at 37°C in a humidifiedatmosphere of 5% CO2 in air for two days. Transfected cells were transferred to 35 mmpetri dishes (Greiner, Nurtingen, Germany) and selected in 0.8% Iscove’s methylcellulose(MC) medium (StemCell Technologies) supplemented with 30% FCS, i0 M 2-mercaptoethanol and 1 mM L-glutamine supplemented with 0.8 mg/mi (active weight)G418 (Geneticin, 0418 sulfate, Gibco-BRL, Grand Island, NY). 0418 was prepared bydissolving the drug in PBS at room temperature. After 2 weeks, neor clones wereidentified and isolated from the MC and maintained in RPMII1O%FCS containing 0.8mg/mi G418.605. CYTOTOXICITY ASSAYBMC and PBMC were cultured in human myeloid long-term culture mediumcontaining 12.5% FCS 12.5% horse serum, io4 M 2-mercaptoethanol, 2 mM Lglutamine, 0.2 mM i-inositol and 20 mM folic acid (StemCell Technologies) supplementedwith 106Mhydrocortisone sodium succinate (Sigma Chemical Company, St. Louis, MO)at a concentration of 1x106cells/ml in 35 mm tissue culture dishes (Coming, NY) in a finalvolume of 2 ml at 37°C in a humidified atmosphere of 5% CO2 in air. Cultures containedno cytokine, 10 to 500 U/mi IL-2, 250 U/mi IL-7, 4 U/mi IL-12 or a combination of IL-2and TL-7 or IL-12.After 6 days, the cultures were harvested by trypsin treatment. The cultures wereaspirated from the dishes which were then rinsed with Hank’s salt (StemCellTechnologies). 0.5 ml of trypsin (0.25% trypsin in citrate saline, StemCell Technologies)was added to each dish which was then incubated for 2 minutes at 37°C in a humidifiedatmosphere of 5% CO2 in air. The dishes were scraped with a rubber policeman andrinsed twice with PBS supplemented with 2% FCS (PBS/2%FCS). The cells werewashed twice with RPMIJ5%FCS at 200g for 10 minutes and then resuspended in freshRPMJJ1O%FCS, Nucleated cell counts were performed using a hematocytometer in thepresence of 3% acetic acid.Harvested BMC and PBMC were analyzed for cytotoxic activity against K562 andDaudi target cells in a 51Cr release assay at effector:target (E:T) ratios of 20:1 and 5:1. NKcell activity was defined as the ability of freshly isolated BMC and PBMC to lyse NK cellsensitive K562 target cells and CIK cell activity was defined as the ability of freshlyisolated BMC and PBMC to lyse NK cell-resistant Daudi target cells in a 4-hour 51Cr61release assay after incubation in medium alone or with IL-2, IL-7 IL-12 alone or incombination. This is represented in Figure 1.K562 and Daudi target cells were labeled with 0.1 tCi 5 (Na5‘Cr04 in normalsaline, Dupont, North Billerica, MA) per 1x106 cells. The cells were incubated for onehour at 37°C in a humidified atmosphere of 5% CO2 in air, washed twice withRPMIJ5%FCS at 200g for 10 minutes and then resuspended in fresh RPMJJ1O%FCS.100 i1 of the BMC or PBMC effector cell suspensions and 1x104 5target cells in 100 tl were added in triplicate to each well in a 96-round-bottomed microtitreplate (Nunc, Denmark) to give a final volume of 200 tl. The plates were centrifuged at200g for 1 minute and incubated for 4 hours at 37°C in a humidified atmosphere of 5%CO2 in air.After incubation, 100 ml of the supernatants were aspirated from each well and theradioactivity released into the supernatants by the lysed target cells was determined using aGamma 5500 gamma scintillation counter (Beckman Instruments, Incorporated, Fullerton,CA). The percentage of specific lysis was defined as follows:experimental release - spontaneous releasex100%maximal release - spontaneous releaseSpontaneous release was measured by culturing target cells in medium alone. Maximalrelease was measured by lysing triplicate wells of target cells with 20% Triton X- 100 (toctylphenoxypolyethoxyethanol) (Sigma Chemical Company). All data shown representthe means of triplicate measurements.62IL-xNK Cells CIK CellsK562 Target Cells Daudi Target CellsFIGURE 1. Sensitivity of K562 and Daudi Cells to NK and CIK CellKilling in a 4-hour 51Cr Release AssayStimulation of.NK cells with IL-x (x =2,7, or 12) results in the generationof CIK cells. While NK cells display cytotoxic activity towards K562target cells, CIK cells exhibit cytotoxic activity towards both K562 andDaudi target cells.6. ANTIPROLIFERATION ASSAYBMC and PBMC were cultured in human myeloid long-term culture mediumsupplemented with 10-6Mhydrocortisone at a concentration of lx 106 cells/ml in a 24-wellflat-bottomed tissue culture plate (Nunc) in a final volume of 0.5 ml. K562-neo’ cells wereadded at 0.125, 0.25, 0.5 and 1% to BMC cultures and at 1, 2, 4, and 8% to PBMCcultures which contained no cytokine, 500 U/ml IL-2, 250 UIml IL-7, 4 U/ml TL-12 or acombination of IL-2 and IL-7 or IL-12. The cocultures were incubated at 370C in ahumidified atmosphere of 5% CO2 in air.The cocultures were harvested after 2, 4, 6, or 8 days by trypsin treatment. Thecultures were aspirated from the wells which were then rinsed with Hank’s salt. 0.5 ml oftrypsin was added to each well and the plate was incubated for 2 minutes at 37°C in a63humidified atmosphere of 5% CO2 in air. The wells were scraped with a rubber policemanand rinsed twice with PBS/2%FCS. The cell mixture was washed twice withRPMJJ5%FCS at 200g for 10 minutes and then resuspended in fresh RPMI/10%FCS.Nucleated cell counts were performed using a hematocytometer in the presence of 3% aceticacid.1x103 cells from the cell suspension were suspended in 0.8% Iscove?s MCcontaining 0.8 mg/mi G418 in a final volume of 1.1 ml. The mixture was plated onto 35mm petri dishes (Greiner) and incubated at 37°C in a humidified atmosphere of 5% CO2 inair. After 8 days, the surviving K562neor colonies were counted. Colony formation isillustrated in Figure 2. Results are presented as the mean ± for K562neor survival relativeto the medium control.64FIGURE 2. K562neor Cell Colony Formation inContaining G418 After Coculture with BMCBMC ± K562neor cells were cocuitured for 6 days and plated into MCcontaining 0.8 mg/mi G418. Subsequent colony formation was countedafter 8 days. (1) BMC alone; (2) K562neor cells alone; (3) BMCcocuitured with 0.125% K562neor cells after a 6-day culture and 8 days inmethylcelluloseMethylcellulose•0I’:4WI__a-. :.0•..• a• •1(.•b..b..00’S2I3657. STATISTICAL ANALYSISStatistically significant differences between cultures incubated in the presence andabsence of cytokines were determined by the student’s T test and the results are expressedas the mean ± standard deviation. A probability of p<O.O5 was considered statisticallysignificant.66CHAPTER IIIRESULTS1. CYTOKINE-INDUCED CYTOTOXIC ACTIVITY IN BMC ANDPBMCDose-response curves were performed with IL-2, IL-7 and ]L-12 alone in order todetermine the appropriate doses of these cytokines to be used in subsequent experiments.Based on the data obtained from 51Cr release assays performed against K562 and Dauditumour target cells cocultured with PBMC for 4 hours, it was determined that 500 U/mi IL-2, 250 U/mi IL-7 and 4 U/mi IL-12 elicited maximal cytotoxic activity from PBMC andthese doses were used in subsequent experiments with both BMC and PBMC.1.1. Cytotoxic Activity of BMC After Cytokine StimulationBMC cultures were stimulated with 500 U/ml IL-2, 250 U/mi TL-7 or 4 U/mi IL-12, alone or in combination, for 6 days and then tested for their cytotoxic activity againstK562 and Daudi target cells at 20:1 and 5:1 E:T ratios. Table 13 summarizes the resultsobtained in these assays. It can be seen that BMC cultured with medium alone did notexhibit significant cytotoxic activity against K562 and Daudi target cells. Culturesstimulated with IL-2 alone exerted cytotoxic activity against both K562 and Daudi cells.Neither IL-7 alone nor IL-12 alone induced significant tumour target cell killing in the BMCcultures. IL-2-induced cytotoxicity against K562 and Daudi cells was not augmented byIL-7. In contrast, IL-12 significantly enhanced IL-2-induced cytotoxicity; K562 cell killingwas increased 1.6-fold while that of Daudi cells was boosted 2.5-fold when compared at20:1 E:T ratios.67Culture % K562 Killing % Daudi KillingCondition 20:1 5:1 20:1 5:1Medium 1.9 ± 0.96 0.7 ± 0.30 1.4 ± 0.72 1.6 ± 1.1IL-2 39.7 ± 5.0 18.5 ± 2.2 33.2 ± 3.4 18.9 ± 5.21L-7 6.1 ± 1.5*** 2.7 ± 0.57*** 7.9 ± 2.4*** 6.3 ± 2.5***IL-12 2.3 ± 0.92*** 2.3 ± l.4*** 4.2 ± 3.6*** 8.5 ± 6.8***TL-2 + IL-7 46.3 ± 4.5 27.2 ± 4.1 46.8 ± 6.9 29.3 ± 5.2IL-2+IL-12 63.8±7.2* 46.8±8.l** 82.5±7.5*** 76.3± 14.4**TABLE 13. Cytotoxic Activity of BMC After Stimulation with OptimalDoses of IL-2, IL-7 and IL-12BMC were stimulated with 500 U/mi IL-2, 250 U/mi IL-7 and 4 U/mi IL-12, alone or in combination, for 6 days. Results are shown as the mean ±SEM for the data obtained. Comparisons of all culture conditions weremade to IL-2 alone. Values of p<O.05 were considered significant (n.s. =not significant); * p<0.05, ** p.<0.Ol, p<0.OOl. The sample sizeranged from n=6- 18.1.2. Cytotoxic activity of PBMC after Cytokine StimulationPBMC cultures were stimulated with 500 U/mi IL-2, 250 U/mi IL-7 or 4 U/ml IL-12, alone or in combination, for 6 days and then tested for their cytotoxic activity againstK562 and Daudi target cells at 20:1 and 5:1 E:T ratios. Table 14 summarizes the resultsobtained in these assays. It can be seen that PBMC cultured with medium alone did notexhibit significant cytotoxic activity against K562 and Daudi target cells. Culturesstimulated with an optimal dose of IL-2 alone at 500 U/mI exerted significant cytotoxicactivity against both K562 and Daudi cells. Both IL-7 and IL-12 alone induced tumourtarget cell killing, although both IL-7- and -12- induced cytotoxic activity was less than thatobtained with 500 U/mi IL-2 alone. Additionally, IL-2-induced K562 and Daudi cellkilling was not augmented by either IL-7 or IL-l2.68TABLE 14. Cytotoxic Activity of PBMC After Stimulation with OptimalDoses of IL-2, IL-7 and IL-12PBMC were stimulated with 500 U/mi IL-2, 250 U/mi IL-7 and 4 U/mi IL-12, alone or in combination, for 6 days. Results are shown as the mean ±SEM for the data obtained. Comparisons of all culture conditions weremade to IL-2 alone. Values of p<O.O5 were considered significant (n.s. =not significant); * p<O.05, ** p.<O.Ol, p<0.OOl, The sample sizeranged from n=13-24,Since neither IL-7 nor IL-12 enhanced the cytotoxic activity induced by optimaldoses of ll..-2, suboptimal doses of IL-2 at 10 U/mi and 50 U/mi were then tested. As seenin Table 15, a dose-dependent increase in the cytotoxicity of PBMC against K562 andDaudi cells was observed as the dose of IL-2 was increased from 10 to 500 U/mi. Similarto that observed with 500 U/mi IL-2, no enhancement of IL-2-induced cytotoxic activity byIL-7 or IL-12 was observed when the dose of IL-2 was decreased to 50 U/mi. The dose ofIL-2 was further decreased to 10 U/mi IL-2 and the cytotoxic activity resulting from IL-2stimulation was enhanced by both IL-7 and IL-12.CultureCondition% K56220:1Killing5:1% Daudi20:1MediumIL-2IL-7IL- 12IL-2 + IL-7IL-2 + IL-12Killing5:18.3 ± 2.370.2 ± 2.947.5± 53**30.2 ± 5.0***66.6 ± 4.562.3 ± 4.63.6 ± 0.7956.5 ± 4.625.515.6 ± 4.2***51.1 ± 5.541.5 ± 5.0*3.5 ± 0.8677.5 ± 4. 142.3 ± 59***27.5 ± 55***7 1.3 ± 4.774.0 ± 4. 11.5 ± 0.6361.5 ± 6.929.3 ± 6.1***13.2 ± 47***58.1 ±5.959.7 ± 5.869500 U/mi IL-2 70.2 ± 2.9 56.3 ± 5.5 77.5 ± 4.1 61.5 ± 6.9IL-2 + 1L-7 66.6 ± 4.5 51.1 ± 5.5 71.3 ± 4.7 58.1 ± 5.9IL-2 + IL-12 62.3 ± 4.6 41.6 ± 5.0 74.0 ± 4.1 59.7 ± 5.850 U/mi IL-2 52.0 ± 9.5 36.3 ± 9.5 66.0 ± 11.1 48.3 ± 11.9IL-2+IL-7 43.5 ±9.0 34.7 ±7.5 66.3±11.1 61.0±9.2IL-2 + IL-12 56.5 ± 6.7 39.3 ± 3.8 76.5 ± 4.1 61.3 ± 2.2CultureCondition% K56220:1Killing5:1% Daudi20:1Killing5:110 U/mi IL-2 32.5 ± 6.9 11.3 ± 2.3 21.3 ± 2.9 10.3 ± 1.91L-2 + JL-7 36.0 ± 8.8 13.5 ± 3.6 43.5 ± 4.4 313 ±79*IL-2 + IL-12 62.0 ± 4.6* 31.0 ± 4.0* 44.8 ± 6.0* 30.0 ± 95*TABLE 15. Cytotoxic Activity of PBMC After Stimulation with IncreasingDoses of IL-2 and Optimal Doses of IL-7 and IL-12PBMC were stimulated with 10-500 U/mi IL-2, 250 U/ml IL-7 and 4 U/mlIL-12, alone or in combination, for 6 days. Results are shown as the mean± SEM for the data obtained. Comparisons of all culture conditions weremade to IL-2 alone. Values of p<O.05 were considered significant (n.s. =not significant); * p<0.05, ** p<0.01, p<O.OO1. The sample size for500 U/mi IL-2 ranged from n=15-22, n=3-5 for 50 U/mi IL-2 and n=3-4for 10 U/mi IL-2.2. KINETICS OF LEUKEMIC CELL PROLIFERATIONDirect tumour target cell killing can be quantitatively assessed with 51Cr releaseassays. However, it is known that target cell killing can be achieved by indirectmechanisms as well. Thus, an assay was developed in which the survival of clonogenicK562neor target cells previously cocultured with either BMC or PBMC, in the presence orabsence of cytokines, could be quantitatively measured.It is important to determine the normal kinetics of K562-neo’ cell proliferation. Inorder to assess this, 1x104 K562neor cells were cultured alone under experimental70conditions previously described for up to 8 days. The cultures were plated in MCcontaining G418, a neomycin analogue, after 2, 4, 6 and 8 days of culture and subsequentcolony formation was observed. By day 8, a significant amount of cell death wasobserved, resulting from the exhaustion of nutrients from the growth medium. Figure 3shows the number of colonies formed per culture of by K562neor cells after variousculture periods.zCC41:DAYS IN CULTUREFIGURE 3. Kinetics of K562-neo’ Cell Proliferationlx K562neor cells were cultured for up to 8 days and plated into MCcontaining 0.8 mg/mi G418. The subsequent colony formation by thesurviving leukemic cells was counted after 8 days in MC.15000010000000 2 4 6713. BMC- AND PBMC-MEDIATED INHIBITION OF LEUKEMIC CELLSURVIVAL3.1 BMC-mediated Inhibition of Leukemic Cell Survival in the Presenceof Increasing Tumour Target Cell LoadBMC were cocultured with increasing numbers (0.125% to 1%) of K562neor cellsin the presence of 500 U/mi IL-2, 250 U/mi IL-7 or 4 U/mi IL-12, alone or in combination,for 4, 6 and 8 days, and the percentage survival of leukemic cells per culture, relative tococultures in medium alone, was determined with a clonogenic assay in which thecocultures were plated in MC containing G418.The leukemic cell survival pattern in the cocultures did not vary significantlybetween 4, 6 and 8 days of culture at any of the K562neor cell contamination ratios; onlythe data obtained for day 6 are shown in Figure 4. It can be seen that IL-2 inhibited K562-neor cell survival per culture in a fashion dependent on the number of ieukemic cellscocultured with the BMC; cell survival increased in the cocultures as the ieukemic cellcontamination increased from 0.125% to 1%. Stimulation of the cocultures with either IL-7 or IL- 12 alone did not significantly affect K562neor cell survival at all leukemic cellcontaminations. Overall, the ability of either of these cytokines in inhibiting leukemic cellgrowth was not better than that of IL-2.IL-7 did not augment the IL-2-induced inhibition of K562neor cell survival in theBMC cocultures; a similar pattern of leukemic cell survival was observed betweencocultures stimulated with IL-2 alone and those stimulated with a combination of IL-2 andIL-7. In contrast, a combination of IL-2 and IL- 12 significantly augmented the inhibitoryactivity induced by IL-2 alone on K562-neo cell survival. However, this synergistic effectis only evident at a high K562neor cell contamination.72120100•8060C40.1.IL-2 IL-7 IL-12 IL-2 ÷ IL-71L-2 ÷ IL-12• 0,125% K562-neor D 0.25% K562-neorQ 0.5% K562-neor 0 1% K562-neorCULTURE CONDITIONFIGURE 4. Inhibition of Leukemic Cell Survival by Cytokine-stimulateclBMC Cocultured with Increasing Numbers of K562neor CellsBMC were cocultured with increasing numbers of K562neor cells for 6days in the presence or absence of 500 U/mi IL-2, 250 U/ml IL-7 or 4 U/miIL-12, alone or in combination. The leukemic cell survival per culture wasmeasured by plating the cocultures into MC containing 0.8 mg/mi G418 andcounting the subsequent colony formation after 8 days. The data shown arethe percentage K562neor cell survival per culture ± SEM after cytokinestimulation relative to cocultures in medium alone in which ieukemic cellsurvival was considered 100%.733.2. Kinetics of BMC-mediated Inhibition of Leukemic Cell SurvivalBMC were cocultured with 0.5% and 1% K562neor cells in the presence of 500U/mi IL-2, 250 U/mi IL-7 or 4 U/mi IL-12, alone or in combination, and the percentagesurvival of leukemic cells per culture, relative to cocultures in medium alone, wasdetermined after 2, 4, 6 and 8 days of coculture with a clonogenic assay in which thecocultures were plated in MC containing 0418.Cocultures of BMC with 0.5% K562-neo’ cells showed a time-dependent, yetstatistically insignificant, decrease in leukemic cell survival when stimulated with IL-2.Cocultures stimulated with either IL-7 or IL- 12 alone yielded relatively high leukemicsurvival which did not vary significantly over the 8-day coculture period. IL-7 did notenhance IL-2-induced antiproliferative activity. However, when IL-2 and IL-12 were usedin combination, leukemic cell survival significantly decreased with respect to culturesstimulated with IL-2 alone. These data are shown in Figure 5.74140120100•80060 T\ T4020 [. . . .IL-2 IL-7 IL-12 IL-2 + IL-71L-2 + IL-12• Day 2 Q Day 4 D Day 6 Day 8CULTURE CONDITIONFIGURE 5. Inhibition of Leukemic Cell Survival by Cytokine-stimulatedBMC Cocultured with 0.5% K562-neo’ CellsBMC were cocuitured with 0.5% K562neor cells for 6 days in the presenceor absence of 500 U/mi IL-2, 250 U/mi IL-7 or 4 U/mi IL-12, alone or incombination. The leukemic cell survival per culture after 2, 4, 6, and 8days of coculture was measured by plating the cocultures into MCcontaining 0.8 mg/mi G418 and counting the subsequent colony formationafter 8 days. The data shown are the percentage K562neor cell survival perculture ± SEM after cytokine stimulation relative to cocuitures in mediumalone in which leukemic cell survival was considered 100%.75The survival of 1% K562-neo” cells cocultured with BMC stimulated with IL-2, -7and -12, either alone or in combination, did not vary significantly over the 8-day cocultureperiod. The minor changes seen in Figure 6 are not statistically significant. Neither IL-7nor IL-12 alone significantly inhibited leukemic cell survival under these conditions. WhileIL-7 did not enhance IL-2-induced inhibition of leukemic cell survival in the BMCcocultures, IL-2 and IL-12 used in combination exhibited significantly more inhibitoryactivity on K562neor cell survival per culture compared to cocultures stimulated with IL-2alone.7614012080rj60’1In 4°’2:IL-2 IL-7 IL-12 IL-2 + IL-71L-2 + IL-12• Day 2 El Day 4 D Day 6 Day 8CULTURE CONDITIONFIGURE 6. Inhibition of Leukemic Cell Survival by Cytokine-stimulatedBMC Cocultured with 1% K562neor CellsBMC were cocukured with 1% K562neor cells for 6 days in the presenceor absence of 500 U/mi IL-2, 250 U/mi IL-7 or 4 U/mi IL-12, alone or incombination. The leukemic celi survival per culture after 2, 4, 6, and 8days of coculture was measured by plating the cocultures into MCcontaining 0.8 mg/mi 0418 and counting the subsequent colony formationafter 8 days. The data shown are the percentage K562neor cell survival perculture ± SEM after cytokine stimulation relative to cocultures in mediumalone in which leukemic cell survival was considered 100%.773.3. PBMC-mediated Inhibition of Leukemic Cell Survival in the Presenceof Increasing Tumour Target Cell LoadPBMC were cocultured with increasing numbers (1% to 8%) of K562-neo’ cells inthe presence of 500 U/ml IL-2, 250 U/ml IL-7 or 4 U/ml IL-12, alone or in combinations,for 6 days, and the percentage survival of leukemic cells per culture, relative to coculturesin medium alone, was determined with a clonogenic assay in which the cocultures wereplated in MC containing G418.As seen in Figure 7, IL-2 inhibited K562neor cell survival per culture in a mannerdependent on the number of leukemic cells cocultured with the PBMC; cell survivalincreased in the cocultures as the leukemic cell contamination increased from 1% to 8%.Stimulation of the cocultures with either IL-7 or IL-12 alone was also accompanied by aleukemic cell survival pattern dependent on the number of K562-neo’ cells contaminatingthe coculture. Overall leukemic cell survival in cocultures stimulated with IL-7 or IL- 12was higher than in those stimulated with IL-2, showing that the ability of both of thesecytokines in inhibiting leukemic cell growth was not better than that of TL-2.IL-7 did not augment the IL-2-induced inhibition of K562-neo’ cell survival in thePBMC cocultures; a similar pattern of leukemic cell survival was observed betweencocultures stimulated with IL-2 alone and those stimulated with a combination of IL-2 andIL-7. In contrast, a combination of IL-2 and IL-12 significantly augmented the inhibitoryactivity induced by IL-2 alone on K562neor cell survival. However, this synergistic effectis only evident at a high K562neor cell contamination.781401201oo.4i[ .iIL-2 IL-7 IL-12 IL-2 + IL-71L-2 + IL-12• 1% K562-neor 0 2% K562-neorQ 4% K562-neor 8% K562-neorCULTURE CONDITIONFIGURE 7. Inhibition of Leukemic Cell Survival by Cytokine-stimulatedPBMC Cocultured with Increasing Numbers of K562-neo’CellsPBMC were cocultured with increasing numbers of K562neor cells for 6days in the presence or absence of 500 U/mi U-2, 250 U/ml IL-7 or 4 U/miIL- 12, alone or in combination. The leukemic cell survival per culture wasmeasured by plating the cocuitures into MC containing 0.8 mg/mi G418 andcounting the subsequent colony formation after 8 days. The data shown arethe percentage K562neor cell survival per culture ± SEM after cytokinestimulation relative to cocultures in medium alone in which leukemic cellsurvival was considered 100%,793.4. Kinetics of PBMC-mediated Inhibition of Leukemic Cell GrowthPBMC were cocultured with 1% and 4% K562neor cells in the presence of 500U/mi IL-2, 250 U/mi IL-7 or 4 U/mi IL-12, alone or in combination, and the percentagesurvival of leukemic cells per culture, relative to cocultures in medium alone, wasdetermined after 2, 4, 6 and 8 days of coculture with a clonogenic assay in which thecocultures were plated in MC containing G418.Cocultures of PBMC with 1% K562-neo’ cells showed a time-dependent decreasein leukemic cell survival when stimulated with IL-2, IL-7 or IL-12 alone, as shown inFigure 8, with IL-2 being the most efficient. Neither IL-7 nor IL-12 enhanced IL-2-induced inhibitory activity on K562neor cells at this relatively low level of leukemic cellcontamination.• 80120100•I_IL-2 IL-7 IL-12 IL-2 + IL-71L-2 + IL-12• Day 2 El Day 4 El Day 6 D Day 8CULTURE CONDITIONFIGURE 8. Inhibition of Leukemic Cell Survival in 8-day CoculturesContaining PBMC with 1% K562neor CellsPBMC were cocultured with 1% K562neor cells for 6 days in the presenceor absence of 500 U/mi IL-2, 250 U/mi IL-7 or 4 U/mi IL- 12, alone or incombination. The leukemic cell survival per culture after 2, 4, 6, and 8days of coculture was measured by plating the cocultures into MCcontaining 0.8 mg/mi G418 and counting the subsequent colony formationafter 8 days. The data shown are the percentage K562neoT cell survival perculture ± SEM after cytokine stimulation relative to cocuitures in mediumalone in which leukemic cell survival was considered 100%,81Since the initial K562neor cell contamination of 1% was low, the inhibitory effectof IL-2-stimulated of PBMC on leukemic survival induced by IL-2 alone nearly completelyeradicated the leukemic cells from the coculture. Therefore, it was difficult to determine theeffect of IL-7 and IL-12 on IL-2-induced inhibition. Therefore, the contamination level ofK562-neo’ cells was increased to 4%. As shown in Figure 9, the leukemic cell survival inall of the cocultures did not vary significantly over the 8-day coculture period. IL-7 did notenhance IL-2-induced inhibitory activity on leukemic cell survival. IL-12, when used incombination with IL-2, exerted a synergistic effect in inhibiting leukemic cell survivalcompared to IL-2 alone. However, this effect was significant only towards the latter halfof the coculture period.82120100806040__I IL..IL-2 IL-7 IL-12 IL-2 + IL-71L-2 ÷ IL-12R Day 2 El Day 4 El Day 6 Day 8CULTURE CONDITIONFIGURE 9. Inhibition of Leukemic Cell Survival in 8-day CoculturesContaining PBMC with 4% K562nr CellsPBMC were cocultured with 4% K562neor cells for 6 days in the presenceor absence of 500 U/mi IL-2, 250 U/mi IL-7 or 4 U/mi IL-12, alone or incombination. The leukemic cell survival per culture after 2, 4, 6, and 8days of cocuiture was measured by plating the cocultures into MCcontaining 0.8 mg/mi G418 and counting the subsequent colony formationafter 8 days. The data shown are the percentage K562neor cell survival perculture ± SEM after cytokine stimulation relative to cocultures in mediumalone in which leukemic cell survival was considered 100%.83CHAPTER IVDISCUSSIONIndividuals with AML may be treated by chemotherapy alone, allogeneic BMT orautologous BMT. Each of these treatments is associated with a risk of disease recurrence;the relative relapse rates are approximately as follows: 80% for chemotherapy alone, 20%for allogeneic BMT and 50% for autologous BMT. Although allogeneic BMT offers a highprobability of DFS, its widespread use is hampered by such problems as GVHD and thelimited availability of HLA-matched donors.The use of autologous BMT avoids the practical and immunological problemsincurred after allogeneic BMT. Despite these benefits, the use of autologous BMT islimited by high relapse rates resulting from the persistence of leukemic stem cells in theautologous BMT and/or residual disease in the patient. Either one of these problems issufficient to cause disease recurrence due to the lack of an allogeneic antileukemic effect.Thus, it would be desirable to develop methods by which the relapse rate associated withautologous BMT could be decreased to a level similar to those characteristic of allogeneicBMT.Transplants involving BM from a twin donor are subject to a high probability ofrelapse. This indicates that an active immune component must be provided with theallogeneic BM graft that contributes to the elimination of residual disease. It ishypothesized that this can also be achieved by appropriate cytokine manipulation of theantiproliferative activity of autologous effector cells. The NK cell population is an obviouscandidate for such manipulations since it is the first cell subpopulation to reconstitute afterBMT and it is believed to be the predominant mediator of antileukemic effects in vivo.84Cytokines play a role in the immunotherapy of leukemia. IL-2, IL-7 and IL-12were used in these experiments since they have been shown to be directly stimulatorytowards NK cells. Additionally, previous studies have demonstrated that IL-7 and IL- 12may mimic or enhance the cytotoxic and antiproliferative effects of IL-2. Clinical protocolsinvolving the in vitro activation of BM with optimal doses of IL-2 for 8 days in order toactivate antileukemic activity in the BM prior to reinfusion into the patient have beendeveloped. Because of delayed neutrophil and platelet recovery associated with thisprocedure, the transplantation of PBSC along with, or instead of, the BM is being exploredas a means by which neutrophil and platelet recovery may be hastened. The aim of thisthesis was to compare the relative antiproliferative capacities of BMC and PBMC towardsleukemic cells after cytokine stimulation. This knowledge can be used to refine the in vitroculture conditions of these cells and to assess the ex vivo purging ability of BM and PB;such data will be applicable in the clinical setting.Because patient toxicity in response to cytokine therapy occurs in a dose-dependentmanner, cytokine combinations are being investigated for possible synergistic effects sothat the severity and incidence of toxic side effects associated with high doses of eachcytokine alone may be diminished. Since systemic administration of high doses of Th-2 isassociated with toxicity, it would be desirable to utilize cytokine combinations which couldelicit similar levels of antileukemic activity using a lower dose of IL-2. It is also hoped thatthe time period required to induce maximal antileukemic activity could be shortened fromthe current 8-day period.A 4-hour 51Cr release assay was utilized as a means for determining the directcytotoxic potentials of BMC and PBMC with and without cytokine stimulation. The killingof K562 target cells reflected NK cell activity while that of Daudi target cells reflected CIKcell activity. Additionally, the antiproliferative activities of BMC and PBMC were85determined by measuring the survival of K562neor cells after their coculture with BMC orPBMC in the presence or absence of cytokine stimulation. An assay was developed in ourlaboratory in which the plating of cocultured effector and target cells into MC containing0418 allowed the detection of clonogenic K562neor cells. These data can be consideredquantitative read-outs for the ability of these effector cells to inhibit leukemic cell survivalsince only those K562neor cells that survive the initial coculture are able to subsequentlyform colonies in the MC.Two aspects of the inhibitory activities of BMC and PBMC on the survival ofK562neor cells in coculture were studied. Firstly, the efficiency of BMC and PBMC ineliminating increasing K562-neo’ cell load in the coculture was determined. Increasinginitial numbers of leukemic cells were added to the BMC and PBMC cultures and theleukemic cell survival per culture was measured after 6-days. Secondly, the kinetics ofcytokine-induced inhibition of leukemic cell survival exerted by BMC and PBMC weredetermined. A fixed number of K562-neo’ cells was added to BMC and PBMC culturesand the leukemic cell survival was measured after 2, 4, 6, and 8 days.Under identical conditions, BMC and PBMC differed significantly in their cytotoxicpotential towards K562 and Daudi target cells. PBMC stimulated with an optimal dose ofIL-2 (i.e., 500 U/ml) displayed 1.8-fold greater cytotoxic activity against K562 target cellsand 2.3-fold greater cytotoxic activity against Daudi target cells at a 20:1 E:T ratio thanBMC. While BMC did not exhibit any cytotoxic activity after stimulation with optimaldoses of either IL-7 or IL-12, PBMC stimulated by IL-7 and IL-12 were cytotoxic towardsK562 and Daudi cells. IL-7 did not enhance the cytotoxic activity induced by optimaldoses of IL-2 in both BMC and PBMC cultures. In contrast, IL- 12 augmented thecytotoxic activity of BMC stimulated with optimal doses of IL-2; K562 cell killingincreased 1.6-fold while Daudi cell killing increased 2.5-fold when compared at a 20:1 E:T86ratio. However, enhancement of IL-2-induced cytotoxic activity was not observed inPBMC cultures until IL-2 was used at a suboptimal dose (i.e., 10 U/ml). Nevertheless, acombination of optimal doses of IL-2 and IL-12 augmented the cytotoxic activity of BMCto a level equal to that seen in PBMC stimulated with optimal doses of IL-2 alone.IL-2 is stimulatory towards NK cells and can activate and maintain their cytolyticand proliferative responses [3, 13, 25, 31, 40, 55, 61, 66, 68, 70, 72, 74, 78, 86, 92,119-121, 128-129, 146-147, 149-157]. Voss et. al. have reported that in vitro stimulationof PBMC for 3 to 5 days with 100 to 1000 U/ml IL-2 results in the development ofsignificant CIK cell activity [2, 45, 52, 70, 110, 1161 and the data presented in this thesisare confirmatory of this finding. Hiserodt [52] and Keever et. al. [256] have shown that 5to 7 days is required for CIK cell activation in BMC. Thus, a 6-day culture period in theseexperiments was considered to be sufficient for the activation of NK cells and thegeneration of maximal cytotoxic activity; this has been confirmed by time-courseexperiments previously done in our laboratory.Optimal doses of IL-2 induced cytotoxic activity towards K562 and Daudi targetcells in both PBMC and BMC. Voss and Sondel [70] report that NK cells isolated fromPB express IL-2fR and are capable of responding to IL-2 [19, 52, 62, 69]. Thus, IL-2can act directly on these cells and induce secondary effects involved in the activation ofCIK cell activity. These effects include the upregulation of cytokine receptor expression[13, 47, 65, 80, 148-149] and the prolongation of secondary cytokine, such as IFN-y [6,10, 23, 47, 65-66, 70, 74, 78-80, 84, 96, 123-124, 128-129, 135, 142-1471 and TNF-x[10, 65, 78-80, 96, 123, 129, 134-135, 139, 142, 145, 147], release. These secondarymechanisms induced by IL-2 contribute to the overall effects attributed to initial IL-2stimulation.87The cytotoxic activity towards K562 and Daudi target cells was significantly higherin PBMC than in BMC. In contrast to PBMC, which contains mature NK cells and CIKcell precursors, the majority of the cells isolated from BM are immature and require amaturation period which may manifest as a delay in the acquisition of cytotoxic activity.Several groups, including our laboratory, have demonstrated that unstimulated BMC havenegligible levels of cytotoxic activity. Thus, the difference in mature NK cell and CIK cellprecursor composition of the PB and BM can be postulated to contribute largely to thecontrasting leukemic cell killing observed in these two settings.The killing of K562 and Daudi target cells has been classically used to measure NKand CIK cell activities. Although these target cells are considered exquisitely sensitive toNK and CIK cell killing, they can be killed by T lymphocytes. The cytotoxic activityexerted by IL-2-stimulated BMC seen in these experiments may arise from the activation ofimmature, and a few mature, IL-2-responsive NK cells. In addition, T lymphocytespresent in the BM could contribute to target cell killing. Keever et. a!. have shown that BMfrom needle aspirates is diluted with PB to a variable extent, ranging from 28% to 96% andabout 10% of BM mononuclear cells obtained after FH treatment may be PBMC [157].Thus, the cytotoxic activity in the BM cultures may arise from the NK and CIK cell activityin the contaminating PB, rather than the BM itself.When BMC and PBMC were cocultured with K562-neo’ cells in the presence ofoptimal doses of IL-2, PBMC were more efficient in eliminating tumour cell load thanBMC. The patterns in leukemic cell inhibition mediated by BMC and PBMC arecomparable, but the initial leukemic cell contamination in the PBMC cultures was at least 4-fold greater than that in the BMC cultures. There was no significant difference in theinhibitory activity exerted by BMC on leukemic cell growth over the 8-day culture periodwhile PBMC attained their maximal inhibitory activity by day 4 at the same initial K562-88neor cell contamination. Because of the high degree of sensitivity of K562 cells to NK andCIK cell killing, the effects observed in these assays can be considered reflective of NKand CIK cell activity.PBMC display higher efficiency and faster kinetics in terms of inhibiting leukemiccell survival than BMC. The presence of more mature effector cells in the PB allows formore rapid leukemic cell elimination, as well as faster induction of secondary effects, suchas cytokine secretion and the upregulation of cytokine receptors. Therefore, anamplification of the antitumour immune response can be more quickly achieved such that aneffective immune response can occur earlier. In contrast, the majority of cells in the BMare immature and must first be activated; hence less efficient and slower induction ofinhibitory activity is observed.IL-2 was the most effective in inducing antileukemic activity from PBMC and BMCwhen optimal doses of the three cytolcines were compared. Many of its effects are achievedindirectly through the induction of secretion of other cytokines. IL-2 induces theproduction of TNF-a, which synergizes with IL-2 to induce LGL differentiation into CIKcell effectors and enhance CIK cell activity [10, 161, 222]. Blay et. at. have demonstratedthat the inhibition of CIK cell activity occurs in parallel to the inhibition TNF binding siteson LGL [69]. TNF-a increases the expression of IL-2R and TNFR on effectorlymphocytes [6, 69, 85, 161], which produce additional TNF-x, IFN-y, IL-i and IL-6[70]. By increasing T cell proliferation, TNF-OL augments IL-2 production [10, 161], thusforming a feedback loop which potentiates the effects of these two cytokines.TNF-a and IL-2 synergize to induce IFN-y production by T lymphocytes and NKcells [85, 161], IFN-y contributes both directly and indirectly towards antineoplasticactivity [10, 257] and can slow down the growth and proliferation of some tumour cells [3,89258]. By increasing their tumour cell expression of TNFR [6, 96], MHC class I proteins[256, 258] and/or tumour-associated antigens [256], IFN-y may predispose tumours toimmune rejection. It escalates the rate of NK cell lysis of tumour target cells by increasingNK cell recycling [24] and stimulates the maturation of pre-NK cells which, in turn,produce additional IFN-y [44]. IFN-y enhances the expression of IL-2R on NK cells [41,92, 96, 112, 161] and augments the lytic activity of CIK cells [26, 44, 47, 64, 85, 90-93,96, 161, 256, 259].Additionally, IL-2 induces the differentiation of macrophages into cytotoxic effectorcells [64, 96, 256] and primes them to produce TNF-a and IL-i, both of which amplifyIL-2 secretion and IL-2R expression [2-3, 10, 24, 92, 96, 112, 161]. It can be seen thatIL-2 directly and indirectly stimulates the antileukemic activity of BMC and PBMC byinducing a series of feedback loops with other cytokines such that its effects areaugmented.Murine studies performed by Lynch et. al. [130] demonstrate that the proliferativeeffects of 1L-7 on CD56 NK cells occur independently of IL-2 [97, 111]. They have alsoshown that, although IL-7 supports NK cell proliferation to a lesser extent than IL-2, IL-7-induced proliferation persists longer [130]. It requires TNF-cc as a cofactor for biologicalactivity [65, 111, 139]; Naume et. al, report that endogenously-produced TNF-ot isinvolved in the activation of IL-7-responsive NK cells [111]. IL-7 also induces thesecretion of IL-2 [95, 193], TNF-a [193] and IFN-y [65, 193] from NK cells [5] andupregulates expression of TNFR [65, 139, 193], IL-2Rcz (CD25) [139, 163, 194] and IL2Rf3 [139, 163, 194]. Naume et. al. have shown that IL-7 upregulates the IL-2Rexpression in NK cells to a greater extent than IL-2 [139]. Additionally, IL-2 upregulatesthe expression of several CAM, including CD56 and ICAM-1 [163], involved in NK cellcytolysis.90High doses of IL-7 can generate CIK cell activity independently of IL-2 [97, 111,139, 148, 163] from resting PBMC in a dose-dependent manner after 1 to 3 days ofstimulation [65]. The data presented in this thesis confirm that optimal doses of IL-7 canindeed generate cytotoxic activity from PBMC which, however, was absent in the BMCcultures. Although immature IL-7-responsive cells are found in primitive hematopoietictissues, there are very few in adult hematopoietic tissues, such as the BM [189] andAppasamy [174] and Masuda et. al. [178] report that JL-7 induces CIK cell activity fromsecondary, but not primary lymphoid tissues, such as the BM. Thus, this may largelyaccount for the lack of cytotoxic activity in the BMC cultures.Several groups, including Pavletic et. al. have suggested that the ability of IL-7 toinduce CIK cells in populations of resting PBMC is mediated by a population of cellsdistinct from those stimulated by IL-2 [97]. Alderson et. al. report that the PB of normalindividuals contains a 5-fold greater frequency of IL-2-responsive CIK precursor cellscompared to those responsive to IL-7 [260]. Therefore, the higher frequency of IL-2-responsive CIK cell precursors compared to IL-7-responsive ones may account for thelower level of CIK cell induction seen with IL-7 stimulation compared to that of IL-2.CIK cell activity can be generated after exposure of PBMC to IL-2 for 3 to 5 days[2, 45, 52, 70, 116] while that induced by IL-7 requires 6 to 8 days [130, 174, 178].Thus, it can be seen that maximal IL-7-induced CIK cell activity is not attained until IL-2-induced CIK cell activity is at a maximum and declining. Because the cytotoxic activity ofthe cultures was tested after 6 days of stimulation, IL-7 may not have exerted its full effectson responsive cells at that time; a longer culture period may be required in order to detectpotential IL-7-induced cytotoxic activity. In addition, many of the IL-7-induced effects aremediated through the release of secondary cytokines and the upregulation of surface91receptors and CAM that enhance NK cell and target cell interactions [19, 29, 47].Production of these factors is associated with a lag phase during which protein synthesisoccurs so a 6-day culture may not have been sufficient for the maximal induction of IL-7-mediated antileukemic activity.Naume et. a!. have shown that IL-7 induces high CIK cell cytotoxicity from pureCD56 NK cells but not from bulk PBMC populations [139]. In contrast, the datapresented in this thesis show that cytotoxic activity was present in the PBMC cultures.Being a potent stimulus for T lymphocyte proliferation, it is possible that IL-7 stimulatedthe proliferation of these cells, which then developed cytotoxic activity against the targetcells. Thus, the cytotoxic activity observed in these assays may reflect T lymphocyte-mediated cell lysis, rather than that mediated by NK and CIK cells. It has also beendemonstrated that IL-7 enhances the tumouricidal activity of monocytes and macrophagesby augmenting their secretion of such cytokines as Th-lcx, IL-1, IL-6 and TNF-oc [97,169, 174, 177, 184-185, 190]. These cells and cytokines may contribute to the overallcytotoxic and growth inhibitory effects exerted by BMC and PBMC.Additionally, the results presented in this thesis confirm what several groups haveshown: optimal doses of IL-2 and IL-7 do not synergize in inducing CIK cell activity inunseparated PBMC [163]. The data presented show that synergy between IL-2 and IL-7 isevident in PBMC only when IL-2 is present at suboptimal doses while it is absent in BMC.The contrast between PBMC and BMC cultures could result from the differences in thestarting composition of the effector cell populations. Because IL-7 acts on NK cells,PBMC are more readily activated than BMC since there are more mature NK cells presentin the PB. BM lacks mature NK cells; thus, a maturation period is required which may beseen as a delay in the acquisition of antileukemic activity. It is possible that a longer culture92period may be required for the detection of potential synergy between IL-2 and IL-7 inBMC cultures.IL-7-stimulated BMC did not induce significant inhibitory effect on leukemic cellsurvival, regardless of the initial number of K562neor cells in the coculture. In contrast,IL-7-stimulated PBMC exerted inhibitory activity on leukemic cell survival in a mannerdependent on the initial number of K562neor cells in the coculture. There was nosignificant difference in the kinetics involved in the acquisition of antileukemic activity inIL-7-stimulated BMC and PBMC over the coculture period when the initial leukemic cellcontamination is high. The absence of differences in terms of the acquisition and efficiencyof inhibitory activity on leukemic cell survival in BMC cocultures may relate to the kineticsof IL-7-induced activation of NK and other CIK cell precursors.Unlike the cytotoxicity assays in which the effector cells are activated in cultureprior to testing, these antiproliferation assays involve the coculture of effector and targetcells. Perhaps the entire 6-day period is required for the IL-7-mediated activation of BMCand PBMC and the termination of the coculture on day 6 is too early to see inhibitoryactivity against leukemic cell survival. Once the coculture is plated in the presence ofG4 18, sensitive effector cells are killed; therefore, no further inhibition of leukemic cellgrowth can be attained. Alternatively, since the doubling time of K562-neo’ cells isapproximately 48 hours, the observation that leukemic cell survival appears steady over the8-day coculture period may indicate that the antileukemic activities of BMC and PBMCactually increase so that these effector cells may be able to arrest leukemic cell survival.While IL-12-stimulated BMC did not exhibit any cytotoxic activity towards targetcells, PBMC cultured with IL- 12 did. IL- 12-induced CIK cell activity is predominantlymediated by mature CD56+ lymphocytes. Since the proportion of mature NK cells is muchgreater in PB than in BM, higher cytotoxic activity can be induced in PBMC cultures. In93comparing the level of cytotoxic activity induced by IL-2, IL-7 and IL-12 under identicalconditions, IL- 12 was the least effective. It is possible that this may be due to the lowerability of IL- 12 to cause the proliferation of NK cells with respect to IL-2 and IL-7although several groups have shown that the proliferative and cytotoxic activities of CIKcells induced by IL-12 are independent [23, 71, 111, 218].While the overall cytotoxic activity induced by IL-12 alone was less than thatinduced by IL-2, IL-12 augments all NK cytolytic activities [61] with rapid kinetics at lowconcentrations [208-209, 217]. Morphological signs of NK cell activation can be seen asearly as 4 hours after IL-12 stimulation [209-210, 215-216]. IL-12 upregulates thecytotoxic activity of NK cells towards target cells at molar concentrations 100- to 1000-foldlower than those required for IL-2 [61, 73, 158, 209, 214, 222-223, 226-227]; picomolarconcentrations of IL-12 are as effective as nanomolar concentrations of IL-2 in augmentingthe cytolytic activity of normal PBMC.IL-12 increases the expression of several CAM, such as CD56 [111], CD2 [218,216], CD11a [61], CD54 (ICAM-1) [208, 216], and LFA-1 [16] to enhance the lysis ofNK-resistant targets by NK and CIK cells. It also upregulates the expression of severalreceptors, such as those for IL-2 and TNF [16, 61, 1111, on NK cells to enhance theirresponsiveness to these factors. IL-12 potently induces IFN-y production by resting andactivated T and NK cells [14, 16, 51, 61, 65, 73, 111, 204-208, 210-224, 226-227, 230-233, 235-236, 240]. It synergizes with low concentrations of other IFN-y inducers, suchas IL-2 [205, 224, 232], in inducing IFN-y gene expression at the transcriptional and posttranscriptional levels [16, 61, 111, 205, 209-213, 223-224] and increases IFN-y mRNA inindividual producer cells [211-212].94IL-12 either enhances or antagonizes IL-2-induced responses, depending on theconcentrations of the cytokines and culture conditions used [16, 205-206, 219]. It alonehas no proliferative effect on resting PBMC but it enhances their proliferation whencombined with suboptimal levels of IL-2 [14, 16, 51, 111, 206, 210, 213, 219, 222].This effect is partially inhibited by antibodies to IL-12, suggesting that IL-2 induces the insitu production of IL-12 [14]. In the presence of optimal concentrations of IL-2, IL-12does not affect IL-2-stimulated PBMC proliferation; in fact, several groups have shownthat it causes modest inhibition of IL-2-induced NK cell proliferation [16, 210].Bertagnolli et. a!. have demonstrated that IL-2 and IL- 12 act through independentpathways to induce cellular proliferation through the delivery of partial signals that areaugmented in combination [206]. IL-12 prolongs IL-2R expression while IL-2 upregulatesIL-12R expression; these effects result in enhanced responsiveness to both IL-2 and [L-12[14, 16, 111, 206]. Additionally, IL-12 may alter PBMC to enable them to proliferate inthe presence of lower concentrations of IL-2 or it may enhance IL-2R expression and/oralter the signal transduction process so that a proliferative response may be triggered byfewer occupied IL-2R [14].Unlike IL-2 and IL-7, IL-12 selectively induces the outgrowth and enhances thelytic potential of CD56 NK cells in PBMC cultures independently of IL-2 [14, 16, 1111and IFN-y [210]. Phenotypic analysis conducted by Gately et. a!. has shown that IL- 12directly increases the percentage of CD3CD56+ NK cells [14]. This, again, may accountfor the presence of cytotoxic activity in PBMC cultures which is absent in cultures ofBMC.IL-12-induced CIK cell activation is dependent on the in situ production of othercytokines. Glucocorticoids, such as hydrocortisone (HC), inhibit the secretion of95endogenously-produced cytokines. Thus, the ability of IL-12 to activate CIK cells isimpaired in the presence of HC. However, in the absence of HC, IL- 12 causes dose-dependent CIK cell activation [216]. The loss of cytokines potentially involved inmediating IL-12 effects may account for the inability of IL-12 alone to induce CIK cellactivity in the presence of HC [16, 216]. Since HC was present in the culture medium ofBMC and PBMC, its inhibitory effect on endogenous cytokine production may beaccountable for the lesser antileukemic activity in PBMC and its absence in BMC comparedto IL-2.IL- 12 exhibits costimulatory activities with IL-2 in inducing cytotoxic activity.Although IL-12 alone did not induce any cytotoxic activity in BMC cultures, it enhancedthe leukemic cell killing induced by optimal doses of IL-2. In contrast, it failed to enhancecytotoxic activity in PBMC cultures stimulated with optimal doses of IL-2. Synergy wasnot detected even when the dose of IL-2 was decreased by 10-fold. However, IL- 12combined with suboptimal doses of IL-2 resulted in significant amplification of cytotoxicactivity against K562 and Daudi cells in PBMC. These data confirm the observation thatIL-12 synergizes with ]L-2 only when IL-2 is present in suboptimal doses. However, IL-2and IL-12 synergized in inducing cytotoxic activity in BMC even when both cytokineswere at optimal doses. The original dose-response curves were performed in PBMC todetermine an “optimal” dose; thus, it is possible that what may be an optimal dose forPBMC is actually a suboptimal dose for BMC.The synergy between IL-2 and IL-12 may partially result from the ability of thesetwo cytokines to induce IFN-y production. IL-12-mediated NK cell proliferation involvesendogenously-produced TNF-cc whose production is induced by IL-2. Despite theinability of optimal doses IL- 12 to elicit cytotoxic activity at levels comparable to optimaldoses of IL-2, the activity induced by the synergistic combination of a suboptimal dose of96IL-2 and an optimal optimal IL-12 equals that of an optimal dose of IL-2 alone. Thisdemonstrates that it is possible to achieve equipotent cytotoxic activity with significantlylower doses of IL-2 in the presence of IL- 12. Additionally, stimulation of BMC with acombination of optimal doses of IL-2 and IL-12 resulted in leukemic cell killing that wasequal to that induced by optimal doses of IL-2 in PBMC cultures. The potency of IL- 12 insynergizing with IL-2 is highly evident. It has also been shown that IL-12 has a longerhalf-life compared to IL-2; its accumulation may be a useful fact in future considerations forits use.IL-12-stimulated BMC did not induce significant inhibitory effect on leukemic cellsurvival, regardless of the initial number of leukemic cells in the coculture. In contrast, IL-12-stimulated PBMC inhibited leukemic cell survival in a manner dependent on the initialnumber of contaminating leukemic cells. Looking at the kinetics involved in IL-12-inducedinhibitory activity in BMC or PBMC on leukemic cell survival, it appears that thisinhibitory activity is attained earlier than that induced by IL-2 alone.In summary, the objectives of this thesis - i) to study the differential effects of IL-2,IL-7 and IL- 12 in inducing cytotoxic activity in BMC and PBMC, ii) to study the efficiencyof BMC and PBMC in inhibiting leukemic cell growth in the presence of increasing tumourtarget cell load and iii) to study the kinetics involved in the acquisition of this inhibitoryactivity - were achieved. The data obtained enabled the assessment of the relative abilitiesof BMC and PBMC in terms of their cytotoxic and antiproliferative activities after cytokinestimulation.The cytotoxic activity of PBMC is significantly higher than that of BMC. IL-2, IL7 and IL-12 induced cytotoxic activity in the PBMC cultures against the target cells testedwhereas only IL-2 was able to exert this effect in BMC. While only IL-12 synergizes with97optimal doses of IL-2 in BMC cultures in inducing cytotoxic activity, both IL-7 and IL-12can enhance IL-2-induced cytotoxic activity when PBMC are stimulated with suboptimaldoses of IL-2. The level of cytotoxicity attained in the presence of suboptimal doses of IL-2 and optimal doses of IL-12 equals that induced by optimal levels of IL-2 alone. IL-12also synergizes with optimal levels of IL-2 in BMC to induce cytotoxic activity equal to thatseen in IL-2-stimulated PBMC.PBMC were more efficient in eliminating higher numbers of leukemic cells in thecoculture compared to BMC, possibly due to the faster activation of the NK cells into CIKcell effectors in the PB. While BMC may only be able to push leukemic cell growth into aplateau phase, PBMC may actually eliminate them from the coculture. Since the doublingtime for K562 cells is approximately 48 hours, the observation that the inhibition ofleukemic cell growth in the BMC and PBMC cocultures does not vary significantly over the8-day coculture period, except when stimulated with a combination of IL-2 and IL- 12,suggests that the antileukemic effects of these immune cells come into play early in thecoculture period and are maintained and/or increased over the culture period. Since theleukemic cells are continuously proliferating, these antiproliferative actions can arrest theirgrowth early on. As seen in the data presented, these inhibitory effects are mostpronounced early in the coculture period.Because the BM contains immature NK cell progenitors, there is a time delay in theinduction of NK and CIK cell activities due to the additional requirement for a maturationstage which is not needed in the PB environment. A major consideration in BMT is the invitro culture period required to elicit maximal cytotoxic activity in the BM graft; it would bedesirable to decrease it from its present length of 8 days. This may be possible through theuse of cytokine combinations, as represented in Figure 10.98Maturation timeShorten time with IL-12?FIGURE 10. Proposed Scheme for the Differential Kinetics Involvedin NK Cell Activation in Bone Marrow and PeripheralBlood(x = 2, 7 or 12)Because patient toxicity in response to cytokine therapy occurs in a dose-dependentmanner, cytokine combinations are being investigated for possible synergistic effects inorder to diminish the severity and incidence of toxic side effects associated with high dosesof each cytokine alone. Several groups, using animal models, have demonstrated that thesystemic administration of IL-7 [177, 2031 and IL-12 [1581 is not associated withsignificant toxicity. This may also hold true in the human system. Both IL-7 and IL- 12alone induce CIK cell activation and promote tumour regression; thus, these cytokines mayBMIL-2progenitonitorIL-xPBI-IL-x—1Time saved99have potential use in the immunotherapy of cancer, either alone of in combination with lowdoses of IL-2.PBMC were superior in terms of both cytotoxic and antiproliferative activitiescompared to BMC under the same culture and assay conditions. Therefore, not only doesthe transplantation of PBSC hasten neutrophil and platelet recovery, it has potential inoffering additional antileukemic capacity for the elimination of residual disease in the patientand the BM graft. Although the transplantation of PBSC is unstable [243], it offers theinitial stabilization of the patient and elimination of most of the residual disease. BMCtransplanted along with the PBSC can offer long-term engraftment and additionalantileukemic activity. Additionally, the data presented in this thesis demonstrate that BMCstimulated with a combination of IL-2 and IL- 12 attained levels of cytotoxic activity equalto those induced by PBMC stimulated with optimal doses of IL-2 alone. This suggests thattransplanted BMC have greater potential for the enhancement of antileukemic activity andcan, thus, offer additional antileukemic effects.BMT is an established treatment for acute leukemia. Despite considerable progressin this area, the widespread use of BMT is limited. Much investigation has been performedin the area of autologous BMT in terms of improving the antileukemic effects of thetransplanted cells through the manipulation of NK cells in order to eliminate residualdisease. This project studied the modulatory effects of IL-2, IL-7 and IL-12 on theantileukemic effects of BMC and PBMC. 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