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Characterization of Thy-1 expression on human hematopoietic cells Craig, William 1993

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We accept this thesis as conforming to the required standardCharacterization of Thy-1 expression on human hematopoietic cellsbyWilliam CraigB. Sc., University of British Columbia, 1989A Thesis submitted in partial fulfillment of the requirements for the degree ofMaster of ScienceinThe Faculty of Graduate Studies(Department of Genetics)THE UNIVERSITY OF BRITISH COLUMBIAMarch 1993© William Craig, 1993.In 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.(SignaturDepartment o- c.Ane .;:c..0 The University of British ColumbiaVancouver, CanadaDate^/ eBDE-6 (2/88)1: ABSTRACTHematopoietic stem cells have extensive proliferative capacity and the ability toproduce daughter cells of all hematopoietic lineages. However, their characterization iscomplicated by their low frequency and the heterogeneous cellular composition of thetissues in which they are found. The ability to obtain pure populations of stem cellswould be useful not only for basic studies of stem cell biology, but additionally forclinical procedures involving gene therapy or tumour cell purging. Numerous techniquesand reagents have been developed towards this goal; however, it has been very difficultto obtain a biologically homogeneous population of human hematopoietic stem cells.With the initial purpose of producing new reagents for human hematopoietic stem cellpurification, I helped to generate a panel of monoclonal antibodies by immunizing micewith various human hematopoietic cell lines. Several monoclonal antibodies wereisolated which reacted with subpopulations of the few percent of human bone marrowcells that express CD34, a molecule that is selectively expressed on hematopoieticprecursor cells including those required for long-term reconstitution of hematopoiesis invivo. The property of one of these monoclonal antibodies, referred to as 5E10, that wasraised following immunization with a human erythroleukemia cell line, HEL, was chosenfor more detailed studies. 5E10 was subsequently shown to react with Thy-1, aphosphatidylinositol-anchored cell surface protein belonging to the immunoglobulinsupergene family. This was established by isolation and sequencing of a cDNA clonedby immunoselection of COS cells transfected with a cDNA library derived from a 5E10+cell line. 5E10 staining of hematopoietic cells from human fetal liver, cord blood, adultperipheral blood, and bone marrow samples revealed that Thy-1 expression is restrictedto approximately 1-4% of fetal liver, cord blood and bone marrow cells. These consist ofa small subset of lymphoid cells and approximately 25% of all CD34+ cells. Thy-1+CD34+ cells were further characterized as CD3810/CD45R0+/CD45RAICD7110/c-kitlo and rhodamine-123dull. When CD34+ cells were sorted on the basis of Thy-1expression, the majority of clonogenic hematopoietic progenitor cells were recovered inthe Thy-1- CD344- fraction, whereas the majority of cells giving rise to clonogenicprogenitors after 5-8 weeks of culture on irradiated marrow adherent feeder layers (i.e.,long-term culture initiating cells or LTC-IC) were recovered in the Thy-l+CD34+fraction. In addition to being expressed on a subset of CD34+ cells, Thy-1 was found ona small (<1%) number of CD3+CD4+ CD34- lymphocytes in bone marrow, cord blood,and peripheral blood. The demonstration of a restricted expression of Thy-1 on primitivehuman hematopoietic cells confirms and extends recent observations of others indicatingthat Thy-1 expression can be used to obtain enriched populations of very primitivehematopoietic cells. While the function of Thy-1 is unknown, its differential expressionon early stages of hematopoietic cell development suggests that it may have a specificrole in very early and/or quiescent cells.ivTable of ContentspageABSTRACT^ iiList of Tables viiList of Figures^ viiiList of abbreviations ixAcknowledgments^ xi1. INTRODUCTION 11.1: The hematopoietic system^ 11.1.1: A complex developmental system^11.1.2: Stem cell properties^ 11.1.3: Methodology for distinguishing/measuring cells at^3different stages of development1.2: Stem cell purification^ 61.2.1: Murine stem cell purification^ 61.2.2: Human stem cell purification 71.3: Experimental design and objectives^ 14page2.MATERIALS AND METHODS^ 162.1 Cells^ 162.1.1 Primary tissue^ 162.1.2 Cell lines 162.2 Flow cytometry^ 172.2.1 Antibodies 172.2.2 Indirect staining^ 192.3 Generation of 5E10 202.4 Cell culture^ 212.4.1 Hematopoietic progenitor assays^212.4.2 Long-term cultures^ 212.4.3 Short-term liquid suspension cultures^222.4.4 Activation of peripheral blood T cells 232.5 Irrununoprecipitation studies^ 233: RESULTS^ 263.1: Generation of 5E10 and initial characterization of the^26reactivity of 5E10 with an antigen on the surface of varioushematopoietic cells3.2: 5E10 immunoprecipitates a 25-351(D protein, and is specific^26for human Thy-13.3 Functional analysis of CD34+ cells from bone marrow^31differing in Thy-lexpressionvi3.4 Phenotypic analysis of Thy- 1+CD34+ subpopulations in^38bone marrow, cord blood, and fetal liver3.5 Phenotypic analysis of Thy-l÷+CD34- cells in bone marrow^46and peripheral blood3.6 Effect of monoclonal antibody 5E10 on cells in culture^464. DISCUSSION^ 515. REFERENCES 56viiList of TablespageTable I^Antibodies used in these experiments^18Table II^LTC-IC and clonogenic cell assays of CD71dull^32CD34+ human bone marrow cells sorted on the basisof Thy-1 expressionTable III Serum-free cultures of Thy-1+7Thy-1- CD71dull^33CD34+ human bone marrow cellsTable IV Clonogenic assays of CD34+ human bone marrow^36cells sorted on the basis of Thy-1 expression.Table V Long-term cultures of CD34+ human bone marrow^37cells sorted on the basis of Thy-1 expression.Table VI Thy-1 expression on human bone marrow, peripheral^43blood, cord blood, and fetal liver cellsviiiList of FigurespageFigure 1^Light scatter and fluorescence gates used for sorting^27bone marrow cellsFigure 2^5E10 staining of human bone marrow cells^29Figure 3^5E10 immunoprecipitates a 25-35 kD protein 30Figure 4^5E10 versus CD45RA staining of human cord blood,^40fetal liver, and bone marrow cellsFigure 5^5E10 staining of CD34+ subpopulations in human^42cord blood, fetal liver, and bone marrowFigure 6^5E10 vs rhodamine staining of human bone marrow^45cellsFigure 7^Lineage marker expression on 5E10++ CD34- human^48bone marrow cellsFigure 8^5E10 staining of phytohemagglutinin and IL-2^49stimulated human peripheral blood cellsixList of abbreviationsA-^avidinb biotinBFU-E^burst-forming unit-erythroidcDNA^copy deoxyribonucleic acidCFU colony-forming unitCFU-E^colony-forming unit-erythroidCFU-G^colony-forming unit-granulocyteCFU-GEMM^colony-forming unit-granulocyte/erythroid/macrophage/megakaryocyteCFU-GM^colony-forming unit-granulocyte/macrophageCFU-M^colony-forming unit-macrophageCML chronic myelogenous leukemiaDMEM^Dulbeco's modified eagle mediumELISA^enzyme linked immunosorbent assayf frequencyFc^constant fragment of immunoglobulinFCS fetal calf serumFITC^fluorescein isothiocyanateFSC forward light scatterG-CSF^granulocyte-colony-stimulating factorGM-CSF^granulocyte/macrophage colony-stimulating factorHAT hypoxanthine, aminopterin, and thyrnidineHFN^Hank's HEPES buffered salt solution containing 2% fetalcalf serum and 0.1% NaN3hi^highHPP-CFC^high proliferative potential colony-forming cellIg^immunoglobulinIL interleukinkD^lcilodaltonlo lowLTC-IC^long-term culture-initiating cellM-CSF^macrophage-colony-stimulating factormAb monoclonal antibody (ies)MGF^mast cell growth factor (also known as steel factor, stem cellfactor, or kit ligand)mRNA^messenger ribonucleic acidND not determinedNHS^normal human serumNSS normal sheep serumPBS^phosphate buffered salinePHA phytohemagglutininPI^propidium iodidePO peroxidaseRPE^R-phycoerythrinSA- streptavidinSAM-FITC^sheep anti-mouse fluorescein isothiocyanateSDS-PAGE^sodium dodecylsulfate-polyacrylamide gel electrophoresisSSC^900 light scatter or side light scatterTNP trinitrophenolxiAcknowledgmentsI would like to thankDr. Peter Lansdorp, my supervisor, for his many helpful suggestions and contributions tomy work while at the Terry Fox Lab, and for the direction he provided to my project.Dr. Connie Eaves, my co-supervisor, for helpful suggestions on experiments to perform,and input on the preparation of my Masters Thesis.Dr. Robert Kay, Dr Fumio Takei, and Dr Wilf Jefferies, members of my advisorycommittee, for helpful input in the preparation of my Masters Thesis, and critical readingof this document.Cam Smith, Gail Thornbury, Colleen McAloney, Visia Dragowska, and Sara Abrahamfor expert technical assistance.Dr Krystal's lab for aliquots of erythropoietinThe Medical Research Council of Canada for financial support11: INTRODUCTION1.1:  The hematopoietic system 1.1.1: A complex developmental systemHematopoiesis is a complex process in which a hierarchy of cells at various stagesof differentiation along each of multiple distinct lineages are produced throughout adultlife. These include the lymphoid lineages which lead to the production of mature B, T,and natural killer cells, and the myeloid lineages leading to the production oferythrocytes, monocytes/macrophages, eosinophilic granulocytes, basophilicgranulocytes, neutrophilic granulocytes, mast cells, and megalcaryocytes. Thehematopoietic hierarchy is composed of a continuous spectrum of cell types that can bearbitrarily subdivided into three compartments: stem cells, committed progenitors, andterminally maturing cells (the differentiation compartment).' The differentiationcompartment consists of immature but morphologically recognizable cells, characterizedby limited or no proliferative potential. Progenitor cells, the immediate precursors ofcells in the differentiation compartment and the progeny of stem cells, are characterizedby an apparent loss of totipotentiality although some progenitors have extensiveproliferative potential and have not yet become restricted to a single lineage.1.1.2: Stem cell propertiesPluripotent hematopoietic stem cells are operationally defined by their ability togenerate many generations of mature cells of all of the hematopoietic lineages and it isbelieved that this is achieved through regulation of self-renewal and differentiativedivisions.1 Early experiments examining stem cell biology involved transplantinggenetically anemic W mutant mice with normal marrow cells that contained radiation-induced chromosomal markers. Chromosomal analysis of hematopoietic tissuesindicated that cells derived from a single cell could repopulate both the myeloid andlymphoid lineages.2 More recent studies have used retrovirally marked cells todemonstrate regenerated clones including both lymphoid and myeloid cells.3,4 When a2cell is infected, the retrovirus integrates into the genome providing a method of uniquelymarking individual stem cells and their progeny. Typically, irradiated recipient animalshave been transplanted with retrovirally-marked syngeneic fetal liver or bone marrowcells, and various hematopoietic tissues have then been examined for the evidence ofuniquely marked clonal populations. For this, DNA isolated from representative tissuesis digested with a restriction enzyme that cuts only once within the provirus therebyreleasing a fragment unique to the integration site, and these are then probed with aretroviral sequence on a Southern blot. Integration of the provirus at unique sites thusresults in the ultimate appearance of different bands, each corresponding to a differentsized fragment. Tissue samples that produce the same sized bands on the Southern blotmay be assumed to have contained cells derived from the same clone. Clones detectedby this approach have been observed to include cells of single and multiple lineages, andin some instances produced progeny present in both myeloid and lymphoid tissues.Successful transplantation of single clones generated in primary recipients to multiplesecondary recipients has also been reported.3,4,5 In other experiments, retrovirallymarked cells have been allowed to proliferate in long-term cultures (LTC), and thensibling clones detected following their transplantation into several mice. In severalinstances, reconstitution of both the marrow and thymus by the same retrovirally markedclone was observed.6 These results indicate that normal hematopoietic stem cells notonly have the potential to contribute to both myeloid and lymphoid lineages, but can alsodisplay extensive proliferative potential and the ability to self-renew theirpluripotentiality.Additional proof of stem cell multipotentiality is provided by humanhematological disorders in which lymphoid and myeloid cells have been shown to bederived from a single malignant clone.7,8 In one such study, Philadelphia chromosomepositive B cell lines derived from a female chronic myeloid leukemia (CML) patientheterozygous for glucose-6-phosphate dehydrogenase expressed the same isoform as the3CML clone.9 Another study described a clonal disorder involving erythrocytes,granulocytes, platelets, macrophages, B and T lymphocytes.8 An alternative techniquefor examining clonality is to analyze differences in the methylation state of certain X-linked genes such as hypoxanthine phosphoribosyltransferase or phosphoglycerate kinasewhich contain a restriction fragment length polymorphism. Recipients of bone marrowfrom women heterozygous for one or more of these X-linked restriction fragment lengthpolymorphisms can be examined for clonal hematopoiesis as the probability of adetectable contribution from more than four clones all with the same inactive Xchromosome is very low. In one such study reported, the majority of patients wererepopulated by multiple clones, however, two patients appeared each to be repopulatedby a single clone.101.1.3: Methodology for distinguishing/measuring cells at different stages ofdevelopmentThe study of primitive hematopoietic cells and early differentiation events iscomplicated by the heterogeneity of cell types found within the hematopoietic tissues andtheir lack of obvious spatial organization. This has necessitated the development oftechniques to separate, assay and thereby characterize primitive cells. Terminallymaturing cells can be identified morphologically by light microscopic examination afterstaining with dyes such as May-Grunwal&Giemsal 1 or Wright's stain.12 In contrast,functionally distinct progenitor cells (and stem cells) all appear morphologically as blastcells without specific features obvious by light microscopy to indicate the lineage towhich they may already belong.1The clonogenic progenitor assay measures primarily the number of committedprogenitors (colony forming units-CFU, or clonogenic cells) present in a sample. Cellsare plated in a semisolid (e.g. methylcellulose) medium and cultures are grown at 37°Cin a humidified environment for up to four weeks, during which time single and multi-lineage colonies develop. Colonies containing multiple lineages or arising after longer4periods in culture are considered to be derived from more primitive progenitors thanmore rapidly maturing lineage committed clonogenic cells. 13A variety of myeloid and erythroid colonies can be produced in standardclonogenic assays. Time course studies indicate that erythroid colony developmentfollows a characteristic pattern in which colonies of increasing size appear and disappear.The first erythroid colonies to mature are composed of small, tight clusters of 8-50 ormore erythroblasts. The cell of origin of such colonies has been defined as the colony-forming unit-erythroid or CFU-E. Much larger, later appearing colonies have beendefined as burst-forming units-erythroid or BFU-E to reflect the delay that occurs duringtheir formation before they can be specifically recognized. BFU-E can be furthersubdivided according to the time when their clonal progeny begin to mature and by theirultimate size (as indicated by the number of clusters they are composed of when mature).The progenitors of smaller, earlier appearing bursts have been defined as mature BFU-Ewhile their precursors, the primitive BFU-E, form larger, later appearing bursts.14Granulocyte colonies (from CFU-G) are composed of neutrophilic granulocytes.15Macrophage colonies (from CFU-M) typically contain fewer cells than granulocyticcolonies and are readily distinguished as the macrophages produced tend to be muchlarger cells. CFU-GM generate colonies containing both granulocytes and macrophages.CFU-Meg generate colonies of >2 megakaryocytes.14 Mixed colonies (generallyreferred to as being derived from CFU-GEMM) are derived from less restrictedprogenitors and mature later in culture.15 High proliferative potential colony-formingcells (HPP-CFC) are also a primitive myeloid progenitor cell type identified by their verylarge proliferative capacity. They produce late appearing, large, densely packed colonieslarger than 1 mrn in size, and are composed primarily of macrophages although they maycontain some granulocytes.16Long-term culture-initiating cells or LTC-IC are defined as cells that give rise toclonogenic myeloid progenitors (BFU-E + CFU-GM + CFU-GEMM) detectable after 55weeks of culture on a competent feeder layer.17 Long-term cultures (LTC) initiated withunseparated marrow cells provide a useful model for investigating hematopoiesis as thecultures reproduce many of the features of stromal cell-mediated regulation in vivo.18Transplantable hematopoietic stem cells and LTC-IC are believed to be closely related ifnot identical populations,19 and, in fact, cells that have been maintained in long-termculture for 10 days have been used successfully to transplant patients who haveundergone myeloablative therapy. 20 Additionally, human LTC-IC and murine long-termrepopulating cells have similar unique phenotypes and growth kinetics in long-termculture.17,19,21Most of the clonogenic cells initially present in a cell suspension will havedisappeared by death or differentiation after 5 to 8 weeks in long-term culture, therefore,clonogenic cells present at this time should be the progeny of LTC-IC in the originalsuspension.22 The number of clonogenic cells present after five to eight weeks islinearly related to the number of LTC-IC present at day 0, with each LTC-IC producingan average of 4 clonogenic cells but capable of producing up to 30.23 Week eight LTC-IC are present in normal adult human marrow at half the frequency of week five LTC-ICand are more resistant to cycle active drugs such as 4-hydroperoxycyclophosphamideand, therefore, probably represent a more slowly cycling population.22In addition to these assays, liquid cultures have been developed that allow theoutput of clonogenic cells for extended periods in the absence of stromal feeders24allowing the effects of individual growth factors or combinations thereof to be analyzed.Different growth factor combinations can be included to support the proliferation anddifferentiation of cells along a particular pathway (e.g. the erythroid lineage).25 Anadvantage of liquid suspension cultures is the ability to easily harvest cells at multipletime points and restain them for analysis or resorting on the fluorescence activated cellsorter. As most clonogenic cells in adult marrow are thought to be cycling at a higher6frequency than LTC-IC (C. J. Eaves, personal communication), more committed cellswould be expected to become activated earlier.1.2: Stem cell purification In order to study stem cells, one requires a population that is highly enriched forprimitive multipotential cells but depleted of committed progenitors as the latter wouldrapidly proliferate and dilute out other cells. Much of our knowledge on stem cells hasbeen derived from murine studies and, indeed, many of the techniques used in suchstudies have been incorporated in human protocols.1.2.1: Murine stem cell purificationPopulations of adult murine bone marrow cells highly enriched for "stem" cellshave been isolated for experimentation for over twenty years. Early techniques relied ondifferences in physical properties between primitive cells and mature end cells. Usingsuccessive rounds of equilibrium density centrifugation it was possible to obtainpopulations enriched up to 30-fold for CFU-spleen, a primitive multipotential progenitorthat seeds to the spleen producing a macroscopically visible colony 8-14 days aftertransplantation.26 Later techniques incorporated wheat germ agglutinin labelling andcell sorting to obtain up to 80-fold enriclunents.27 Cell labelling and sorting techniqueshave continued to evolve and most protocols now include monoclonal antibodies (mAb)specific for certain cell surface proteins for positive selection of primitive hematopoieticcells or depletion of mature cells.13 Antibodies specific for Sca-128 and Thy-1,29proteins expressed on a minority of bone marrow cells including some lymphoid cellsand cells capable of reconstituting a lethally irradiated recipient, are frequently used.Other procedures have included fluorescent dyes such as rhodamine-12330 or Hoechst3334213 that label cycling cells more strongly, or cell cycle specific drugs such as 5fluorouracil 13 to enrich for primitive cells that are quiescent in adult marrow.71.2.2: Human stem cell purificationMany of the techniques and reagents used in murine stem cell purification havebeen applied to human stem cell purification including density separation (i.e. Ficoll orPercoll separation) and flow cytometry reagents like rhodamine-12331 and antibodiesspecific for surface proteins such as Thy-1.36CD34 Antibodies specific for CD34 have been the primary tool used for isolatingenriched populations of primitive human cells. CD34 is a highly glycosylatedtransmembrane protein of 107-120 kD 32 found on 1-5% of low density mononuclearhuman marrow cells. This fraction includes all LTC-IC and clonogenic cells,22 as wellas cells capable of enhancing hematopoietic recovery in recipients who have undergonemyeloablative therapy.32,33 Anti-CD34 Ab were originally raised against KGla cells,established as an autonomously growing cell line from a patient with acute leukemia,later found to express high levels of CD34.34 CD34 is expressed at relatively low levelson primitive normal human hematopoietic cells, estimated as 50,000 molecules perce11,35 with expression decreasing as cells mature.32A variety of other reagents have been used in conjunction with anti-CD34 Ab forstem cell enrichment. Most protocols have relied on eliminating cells expressing markersof more mature cells. Baum et al36 used a combination of positive selection for CD34and Thy-1, and negative selection against cells expressing markers of mature cells(lineage negative).36 Other markers used in negative selection include CD3337,38 andHLA-DR.22 CD33 (a marker of immature monomyelocytic cells in the marrow),38 andHLA-DR (a major histocompatability class II antigen)22 are present on clonogenic cellsand more mature cells but are absent from LTC-IC.A variety of additional reagents can be used to subdivide CD34+ cells includingrhodamine-12331 and Ab specific for CD71,39 CD38,4° c_kit,41,42 and the highmolecular weight isoform of the leukocyte common antigen, CD45RA.25,438Thy-1 Thy-l-specific Ab have been used for the purification of rat44 and murine45,46hematopoietic stem cells, and has recently been demonstrated on pluripotent humanhematopoietic cells in fetal bone marrow.36 Thy-1 is a phophotidylinositol anchoredmembrane protein originally characterized as a brain and thymic molecule in the mouse.It has since been described in a variety of other tissues and species,47 however, tissuedistribution varies widely between species suggesting that Thy-1 function may havebecome altered during evolution.48 Although rat, human, and murine Thy-1 cDNA'scontain a putative transmembrane domain, this is absent from mature surface Thy-149,50 and is probably being removed during Thy-1 attachment to its glycophospholipidtail.51 In the rat, thymic and brain Thy-1 are differentially glycosylated suggesting theymay interact with different molecules and may, therefore, perform different functions.52Thy-1 expression is highest on neural tissue and this is relatively invariable betweenspecies.53 Thy-1 is a member of the immunoglobulin (Ig) supergene family, a group ofproteins with similar structure that have evolved from a common ancestral gene. Thy-1is most homologous with the Ig variable domain.54 Thy-1 has been hypothesized to bethe ancestral precursor of the other members of the immunoglobulin supergene family55,56 with Thy-1 homologues being found in organisms as far back as tunicates andmollusks.57 While its function is unknown, Thy-1 has been hypothesized to be involvedin cellular recognition,55 adherence,58 and T cell activation.59, 60, 61About 3-6% of murine bone marrow cells are Thy-1+ including primitivemultipotential cells (Thy-110) and T lymphocytes (Thy-1111), while approximately 45% ofrat bone marrow cells are Thy-1+,62 including stem cells, early progenitors, and B and Tcells.63 Most previous reports have claimed that Thy-1 was absent from human bonemarrow and peripheral blood cells48 although as mentioned above Thy-1 expression hasrecently been reported on human fetal bone marrow cells.369Most Thy-1 studies have examined Thy-1 function in T cells, and have suggestedthat Thy-1 has a role in lymphocyte proliferation. Transgenic mice constructed withThy-1 linked to an Ig enhancer developed an inheritable lymphoproliferative disorderdue to inappropriate Thy-1 expression.61 Some anti-Thy-1 Ab are capable ofstimulating T cell proliferation and Ca2+ mobilization with the addition of a secondarycross-linking Ab and a co-stimulator such as 413-phorbo1-12-myristate-13-actetate.64,59Other anti-Thy-1 Ab can stimulate lymphocyte proliferation, Ca2+ mobilization, andinterleukin 2 (IL-2) production without additional stimulators.59 The requirement for asecondary cross-linking Ab suggests that T cell activation via Thy-1 requires theformation of large surface aggregates. This hypothesis is further strengthened by the factthat IgM Ab are particularly potent mitogens and that the requirement for a secondary Abcan be replaced by a second anti-Thy-1 Ab directed against a different epitope.64The T cell receptor appears to be involved in Thy-1 mediated activation. Human(Jurkat) T cell lines transfected with murine Thy-1 but carrying a defective CD3/T cellreceptor complex can be stimulated by mitogen and anti-Thy-1 Ab to release Ca2+ butnot to produce IL-2, whereas normal Jurkat lines transfected with murine Thy-1 wereable to produce IL-2 when stimulated with mitogen and anti-Thy-1 Ab.65 Similarly, Bcells transfected with Thy-1 could be stimulated to mobilize Ca2+, but failed toproliferate or release IL-2.66 Thy-1 mediated T cell activation leads to rapidphosphorylation of the chain of the T cell receptor and 53 and 62 kl) cytoplasmicproteins. In the absence of the T cell receptor, the 62 kD protein is still phosphorylated,suggesting that Thy-1 probably interacts with other proteins in addition to components ofthe T cell receptor. 67Thy-1 is also expressed on cells in a variety of tissues outside of thehematopoietic system, however, such expression varies between species. For example,Thy-1 is expressed on both murine and human cells in the cerebrum, cerebellum, brainstem, connective tissue, and on skin fibroblasts. In the mouse, splenic T cells, thymic10cells, and cells in the kidney urethelium, visceral smooth muscle, and blood vesselsmooth muscle are Thy-1+. In man, Thy-1 is expressed on cells of the kidney tubules,blood vessel endothelium, and connective tissue. Within the hematopoietic system, themajority of murine peripheral T cells and thymocytes are Thy-1+,45 in addition toepidermal dendritic cells68 and primitive multipotential progenitors and long-termrepopulating cells.69 Previous reports of Thy-1 expression on human hematopoieticcells confined Thy-1 expression to primitive multipotential cells in fetal bone marrow36and a small subset of adult thymocytes.48 Transgenic mice constructed with the murineor human Thy-1 gene showed murine and human patterns of expression respectively,suggesting that cis regulatory sequences and consequently, Thy-1 function, may havebecome altered during evolution.70Thy-1 studies on muscle, mammary, brain, and hematopoietic cells havesuggested that Thy-1 may have a role in cell-cell interactions. Studies on rat mammaryepithelial cells suggest that Thy-1 has a role in mammary development as, in the rat,histological staining localized Thy-1 to the contact points between cells, and highconcentrations of anti-Thy-1 Ab could inhibit dome formation.71 During ratmyogenesis, mononucleate myeloblasts are Thy-1+, but become Thy-1- as they fuse intomultinucleate myeloblasts.72 Human studies suggest that Thy-1 may function inrecognition of a ligand on other myeloblasts allowing cell contact and fusion.73 Duringmurine neural development, Thy-1 is absent from growing axons and fetal neural cellswhile adult cells are Thy-1+.74 Neural cell lines transfected with Thy-1 could interactwith astrocytes (Thy-1-) in vitro to limit neurite extension. Thy-1 may directly initiate aneurite growth-inhibitory signal across the membrane of neural cells, possibly by amechanism similar to T cell activation as suggested by studies in the rat.75Thy-1 may function in murine T cell binding to thymic epithelial cells,58possibly through sulfated glycans.76 Sulfated glycans such as pentosan sulfate, dextransulfate, and fucoidan strongly inhibited Thy-1 dependent binding of lymphopoietic cells11to a murine thymic epithelial cell line MTE. Thy-1 specific Ab had a similar effect.76Additionally, these MTE cells were found to bind soluble Thy-1 and this bindinginhibited T cell binding. Inhibition of Thy-l-dependent binding by sulfated glycans anda lack of detectable Thy-1 on MTE cells suggests that Thy-1 binds to a ligand onepithelial cells that is not Thy-1 itself. Thy-1 expression is highest on cortical (mostprimitive) murine thymocytes and decreases significantly when murine thymocytesmigrate to the periphery, and Thy-1- mutants have a reduced ability to bind to culturedepithelial stromal cells further suggesting a role for Thy-1 in murine thymic lympho-epithelial cell binding.58Other surface proteins analyzed in this studyCD71 CD71 (the transferrin receptor) is a high molecular weight (1901(D) cell surfacedimer that forms part of the iron transport cycle into cells. Iron is bound extracellularlyby transferrin which is then bound by CD71, transported into the cell, and released.77Transferrin and CD71 are then recycled to the surface. Actively cycling cells and cellsproducing iron containing proteins such as hemoglobin have a higher iron requirement,and express more CD71.78,79 Rat stem cells are transferrin receptor negative.80Human studies indicate that CD7110 CD34+ bone marrow cells are highly enriched forLTC-IC.39CD45The leukocyte common antigen (CD45) is a highly glycosylated81 cell surfaceprotein found only on hematopoietic cells.82,83,84 All hematopoietic cells, exceptplatelets and more mature erythrocytes, are CD45+.85,86 The alternate splicing of threeexons (4, 5, and 6 or A, B, and C) can produce up to eight possible isoforms,81,82,84 allof which have been detected at the mRNA leve1.82 Additional heterogeneity exists dueto multiple potential 0 and N linked glycosylation sites.83,87 Monoclonal Ab specificfor CD45 are classified as either anti-CD45 (react with determinants present on all12isoforms) or anti-CD45R (specific for restricted determinants).88 Anti-CD45R Ab arefurther defined by the isoform they recognize; RA, RB, RC, or RO, specific for isoformscontaining sequences encoded by exons A, B, C, or none of these respectively.80,86Electrophoresis produces bands of 220, 205, 190, and 1801(D. Anti-RO Ab recognize the180kD low molecular weight isoform, anti-RB the 190, 205, and 220 kD isoforms, andanti-RA the 220 and 205 kD high molecular weight isoforms.88 Previous studies havereported that the majority of LTC-IC are CD45R0+ CD45RA-.25,43c-kitThe c-kit proto-oncogene (c-kit receptor) is a transmembrane protein that hastyrosine kinase activity and homology to the platelet-derived growth factor/ colonystimulating factor-1 receptor fatnily.89 The c-kit ligand (also called mast cell growthfactor [MGR kit ligand, Steel factor, or stem cell factor) can exist either as atransmembrane or as a secreted protein, and may be involved with cell-cell interactionsin its membrane bound form.9° Functional studies have shown that MGF has powerfulsynergistic effects with other growth factors91 particularly on multipotential progenitorswhen included with IL-3, IL-6, or IL-192 although it is not essential in the maintenanceof LTC-IC in long-term culture.93 Murine studies indicate that the most primitive cells(cells with marrow repopulating ability) are c-kit+,92, 94 but human studies are lessconclusive. While studies have shown that multipotential CFU-GEMM are recovered inthe c-kit+ CD34+ fraction, LTC-IC were not assayed in these reports.41,42 Erythroidprogenitors express the most c-kit amongst CD34+ cells, 95 while brain cells have thehighest c-kit expression in the body.96CD_ 3. 8CD38 is a 45kD surface protein that has been described as an activation antigenbecause of its expression on activated lymphoid cells. It is also present on thymocytes,natural killer cells, early B lymphocytes and plasma cells, monomyeloid cells, earlyerythroid cells, and about 99% of CD34+ cells. CD38- CD34+ cells are highly enriched13for blast colony-forming cells, a primitive cell type assayable in methylcellulose culturesthat is capable of generating mixed colonies or additional blast cell colonies uponreplating.4°Rhodamine -123 Uptake Rhodamine-123 is a vital dye that binds to mitochondria, and cells staining lessbrightly with rhodamine-123 have been assumed to be quiescent. If rhodamine-123stained samples are incubated at 37°C, the rhodamine-123bright population remainsrhodamine-123bright, while the rhodamine-123dull population is less brightly stainedthan comparable cells left at 4°C suggesting that rhodamine-123 may be pumped out.Recent papers have reported that rhodamine-123 efflux is correlated with the expressionof the multidrug efflux pump P-glycoprotein and have suggested that P-glycoprotein maybe responsible for this efflux.97 Murine30 and human studies31 indicate that cells withmarrow repopulating ability and LTC-IC, respectively, are found in the rhodamine-123du11 fraction.141.3:Experimental design and objectives The study of human hematopoietic stem cells has been complicated by aninability to isolate pure populations of these cells. A variety of techniques that have beenuseful include density separation, immunoaffinity columns, and flow cytometry. Anti-CD34 Ab have been the primary tool used in such studies, however; CD34+ cells are stillheterogeneous both functionally and in terms of the markers expressed on CD34+ cells.Some of these have allowed subpopulations of CD34+ cells to be obtained that are highlyenriched for very primitive cells (i.e., LTC-IC). Antibodies specific for Thy-1 haveproven useful for the purification of murine hematopoietic stem cells and more recentlyfor studies with human fetal bone marrow.My original objective was to isolate and characterize new antibodies that mightimprove our ability to purify the most primitive human hematopoietic cells from CD34+populations. Early on one such antibody, 5E10, appeared of interest on the basis of itsexpression pattern on CD34+ human bone marrow cells. The goal of my project wastherefore initially to correlate expression of the antigen recognized by 5E10 with that ofother markers commonly used in human stem cell purification. A second objective wasthen to correlate data from multi-parameter flow cytometric studies using 5E10 inconjunction with other markers to assess the function of 5E10-positive cells. Thestrategy I planned was to sort cells with defined phenotypes and then assay them forLTC-IC and clonogenic cell content to determine whether 5E10 staining allowed eitheror both of these cells to be enriched. Cord blood and fetal liver samples were alsoincluded to determine whether 5E10 expression is altered during ontogeny.A third objective was to characterize the antigen identified by 5E10. For this Iproposed to use inununoprecipitation studies to begin isolation and characterization ofthe antigen. During the course of this work 5E10 was shown to react with Thy-1 by acDNA immunoselection cloning strategy carried out by Dr. R. Kay. Therefore it has15now become possible to interpret all of the staining and cell isolation data in terms ofThy-1 expression.162: MATERIALS AND METHODS 2.1: Cells2.1.1: Primary tissueHeparinized bone marrow samples were obtained from informed and consentingindividuals donating for allogeneic transplantation. In some experiments, bone marrowcells retrieved from the vertebral bodies of organ donors were used (kindly provided byDr. M. Strong, Northwest Tissue Center, Seattle, WA). Cord blood samples wereobtained from the umbilical cord at the time of birth from full term pregnancies (kindlyprovided by Dr. J. O'Toole and his colleagues at the Royal Columbian Hospital,Coquitlam, BC). Peripheral blood samples were donated by healthy consentingvolunteers. Fetal liver cells were obtained from elective, therapeutic abortions, in the12th through 20th week of gestation. Low density mononuclear cells (<1.077g/cm3 )were isolated by density separation using Ficoll-Paque (Pharmacia LKB, Uppsala,Sweden). Interphase cells were removed and washed twice in Hank's HEPES BufferedSalt Solution containing 2% fetal calf serum (FCS) and 0.1% sodium azide (HFN) beforebeing resuspended at 107 cells/ml in HFN + 5% normal human serum (NHS) to block Fcreceptors. Viable cells were counted on a hemocytometer after samples were stainedwith trypan blue to exclude dead cells. For some experiments, red blood cells were lysedby mixing samples with a ten-fold excess of ammonium chloride lysing solution,incubating on ice for 15 minutes, and washing twice with HFN.2.1.2: Cell linesHEL, K562, KG1a, DHL4, CCRF-CEM, MOLT4, HUT78, and Sp2/0Ag14 (amurine myeloma line) cells were all obtained from the American Type CultureCollection. All cell lines and hybridomas were grown in Dulbeco's Modified EagleMedium supplemented with 10% fetal calf serum and 5x105M 2-mercaptoethanol(DMEM10).172.2: Flow cytometry2.2.1: Antibodies Monoclonal antibody (mAb) specificities and concentrations are listed in Table I.Monoclonal IgGi Ab specific for CD34 (8G12), CD71 (OKT9), CD38 (THB7),CD45RA (8D2), and Thy-1 (5E10) were purified from hybridoma culture supernatantsusing protein G (Pharmacia, Uppsala, Sweden). THB7 and OKT9 were obtained fromthe American Type Culture Collection. 8D298 and 8G1232 were mAb generated in ourlab. Unlabelled inAb were used at the concentrations indicated in Table I. 8G12 labeledwith cyanine 5 was previously described,25 and used at 1014/ml. OKT9, THB7, 8D2,and 8G12 were labeled with fluorescein isothyocyanate (FTTC, product No. f-7250,Sigma, St. Louis, MO), and were used at 1, 2, 2, and 101.1g/m1 respectively. 5E10 waslabeled with either fluorescein (5 and 6-carboxyfluorescein succinimydyl ester,Molecular Probes, Eugene, OR) or biotin (64(6((biotinoyl)amino)hexanoyDamino)hexanoic acid, succinimydyl ester, Molecular Probes, Eugene. OR) and used at 8i.tg/m1and 414/m1 respectively. Ascitic fluid containing 17F11, an anti-c-kit IgM, was obtainedfrom AMAC Monoclonal Antibodies (Westbrook, ME) and was used at a dilution of1:1000. Biotinylated goat anti-mouse immunoglobulin (catalog number 115-065-062,Jackson Immunoresearch Laboratories, Westgrove, PA) and FTTC conjugated sheep anti-mouse Ig (SAM-FITC, Jackson Immunoresearch Laboratories, Westgrove, PA) werediluted 200x and 100x respectively in HFN + 5% normal sheep serum (NSS) + 5%normal human serum.Anti-Leu-4 (anti-CD3), anti-Leu-2a (anti-CD8), anti-Leu-3a (anti-CD4), anti-Leu-19-PE (anti-CD56), and anti-Leu-12-F1TC (anti-CD9) were obtained from BectonDickinson (San Jose, CA), and used 1:5. M110 (anti-CD13), M138 (anti-CD14), M058(anti-CD16), M09 (IgM control), and M112 (anti CD9) ascites were part of the myeloidpanel from the Fourth International Leukocyte Typing Workshop. Ascites fluid was usedat a final concentration of 1:1000.18Table IAntibody Specificity Concentration1.14/m1)8G12 CD34 10OKT9 CD71 1THB7 CD38 28D2 CD45RA 25E10 Thy-1 45E10-b I, 45E10-F „ 817F11 c-kit 1:1000 ascites*anti-Leu-4 CD3 1:5***anti-Leu-2a CD8 1:5***anti-Leu-3a CD4 1:5***anti-Leu-19PE CD56 1:5***anti-Leu-12-F1TC CD9 1:5**M110 CD13 1:1000 ascitesM138 CD14 1:1000 ascitesM058 CD16 1:1000 ascitesM112 CD9 1:1000 ascitesanti-CD32 CD32 *1:5M09 irrelevant IgManti-TNP trinitrophenolanti-PO peroxidaseTable I Antibodies used in these experiments. Irrelevant isotype matchedcontrol antibodies (M09, anti-PO, and anti-TNP) were used at the sameconcentrations as test antibodies. * denotes a commercially produced antibodypreparation. ** The ratio indicates the final concentration at which theantibody was used.19Bispecific tetrameric Ab complexes were prepared for 8D2, 5E10, or irrelevantisotype matched controls as previously described.99 Briefly, 1D3 (anti-R-phycoerythrin,RPE) was cross-linked to the desired mAb by TFL-P9 rat anti-mouse immunoglobulinF(ab)2 fragments, and mixed with an excess of RPE.2.2.2: Indirect staining techniquesAll staining procedures were performed at a final cell concentration of 107cells/ml. For most experiments, indirect staining of cells was used. In all experiments,parallel samples were stained with irrelevant IgGi mAb specific for either trinitrophenol(TNP) or peroxidase (PO) at the concentrations used for relevant mAb. Cells wereincubated with unlabelled primary mAb for 30 minutes at 4°C, followed by two washeswith HFN. Samples were resuspended in biotin anti-mouse Ig (1:200 dilution in HFN +5% NHS + 5% NSS), incubated for 30 minutes at 4°C, washed twice in HFN, andresuspended in a half volume of an irrelevant mAb (10014/m1 of monoclonal anti-PO,IgGi isotype, in HFN) to block any residual biotin anti-mouse Ig. Samples wereincubated 10 minutes at 4°C. Directly conjugated mAb, and streptavidin-R-phycoerythrin (SA-RPE, 1:500 dilution) or avidin-fluorescein isothiocyanate (A-FITC,Becton Dickinson, San Jose, CA) at 314/ml, were then added and cells incubated for anadditional 30 minutes at 4°C, washed twice in HFN, and resuspended in HFN +propidium iodide (PI, cat no p-5264, Sigma) at 2gg/ml. Cells stained for c-kitexpression underwent two rounds of amplification to increase the c-kit signal. After c-kitstaining, samples were incubated with a biotinylated anti-RPE mAb (11.1g/m1) followedby SA-RPE (1:500).100In samples stained with SAM-FITC, cells were incubated with unlabelled rnAb inthe first step, followed by SAM-FITC (1:100 in HFN + 5% NHS + 5% NSS) in thesecond, and then blocked with an irrelevant mAb for 10'. Samples were then labeledwith biotinylated mAb (e.g. 5E10-b), followed by SA-RPE or A-FTTC, and directlyconjugated mAb. If no biotinylated mAb were included in the staining, then directly20labeled Ab were added after the blocking step. Samples were resuspended in PI beforeanalysis on the flow cytometer. Analysis and/or cell sorting was performed on aFACStar+ (Becton Dickinson, San Jose, CA) equipped with a 5W Argon and 30mWHelium Neon Laser.2.3: Generation of 5E10Female Balb/c mice were injected with 107 human erythroleukemia (HEL) cellsin Freund's complete adjuvant. Mice were boosted with 107 cells four weeks later, andfour days before the fusion was performed. Spleens were disaggregated to form a singlecell suspension, and cells fused at a ratio of 5 spleen cells to 1 Sp2/0Ag14 cell.Hybridomas were plated in DMEM10 + HAT (hypoxanthine, aminopterin, andthymidine) +50U/m1 IL-6 (R & D Systems, Minneapolis, MN). After 7 days, mediumwas removed and fresh DMEM10 + HT + 10U/m1M-6 was added. Hybridomas werescreened on day 13 or 14 as previously described.101 Briefly, 60 well Terasaki plateswere coated with either low density peripheral blood leukocytes or HEL cells. Plateswere fixed with 0.1% paraformaldehyde in PBS (phosphate buffered saline), coated with0.1% gelatin in PBS to block non-specific binding, and stored until needed. Plates wereincubated with undiluted hybridoma supernatants, followed by peroxidase-conjugatedsheep anti-mouse secondary Ab (Dimension Laboratories, Mississauga, Ont), and finallywith the substrate solution containing 0-phenylenediamine dihydrochloride (Sigma, StLouis, MO). Hybridomas producing Ab that reacted with HEL cells but not peripheralblood leukocytes were then tested by flow cytometry on low density bone marrowmononuclear cells for reactivity with CD34+ cells. Hybridomas were cloned by platingcells in semisolid methylcellulose and plucking colonies. Clones were selected byELISA, and reactivity with CD34+ cells confirmed by flow cytometry.212.4: Cell culture2.4.1: Hematopoietic progenitor assaysSorted bone marrow cells were plated in semisolid methylcellulose culturescontaining 3U/m1 human urinary erythropoietin (>1000U/mg), 10% agar-stimulatedhuman leukocyte conditioned medium and 2Ong/m1 of the granulocyte/macrophagecolony-stimulating factor/ interleukin 3 (GM-CSF/IL-3) fusion protein102 and 20ng/m1of human mast cell growth factor103 (MGF, a c-kit ligand) which were both kindlyprovided by Dr. D.E. Williams (Immunex, Seattle, WA). Cultures were grown for 28days at 37°C in a humidified atmosphere containing 5% CO2. Erythropoietic (BFU-E,CFU-E) and granulopoietic (CFU-GM) colonies were scored on day 14, and highproliferative potential colony forming cells (HPP-CFC) on day 28. Control cultures wereinitiated with 30,000 PI- cells, while sorted fractions were plated at severalconcentrations to account for progenitor enrichments. Progenitor recovery wascalculated by multiplying the calculated enrichment by the percentage of cells in thefraction sorted.2.4.2: Long-term culturesBone marrow cells were suspended in 2.5m1 of long-term culture media (alphamedium supplemented with 40mg/1 inositol, 10mg/1 folic acid, 400mg/1 glutamine,12.5% horse serum, 12.5% FCS, and 10-6 M hydrocortisone sodium succinate) andadded to 35-mm Coming tissue culture dishes (Coming Glassworks, Corning, NY) withpre-established, irradiated, normal marrow feeders. To prepare stromal feeders, long-term cultures were initiated 2-6 weeks before the experiment, and maintained as normallong-term cultures. One to 7 days before the experiment, nonadhererent cells wereremoved, and adherent layers harvested by trypsinization. Trypsinized adherent cellswere irradiated with 15 gray of 250 KVp x-rays, and plated at 3 x 105 cells per dish (3 x104 cells/cm2).2222PI- bone marrow cells were plated at 5 x 105 cells/LTC, while sorted sampleswere typically plated at 2,000 to 10,000 cells/LTC. Cultures were initiated and grownfor 4 days at 37°C, then transferred to 33°C. Half of the non-adherent cells wereremoved at weekly intervals and an equal volume of fresh LTC media was added. Atweeks 5 or 8, LTC were harvested by removing all non-adherent cells and trypsinizingadherent layers, cells were washed, and adherent and non-adherent cells mixed andaliquoted for progenitor assays as described earlier. LTC-IC were assumed to produce anaverage of four clonogenic cells each.182.4.3: Short -term liquid suspension cultures Media was prepared fresh for each experiment, and sterilized by 0.21.1m filtration.Iscove's modified Dulbecco's medium (IMDM powder, product no. 17633, Sigma) wasstored at -20°C in 0.177g aliquots. Single aliquots were dissolved in 9.8 ml of doubledistilled water plus 0.2m1 of 7% (weight/volume) NaHCO3. 2.3 ml of a bovine serumalbumin/insulin/transferrin/penicillin/streptomycin mixture, 50g1 of 10mM 2-mercaptoethanol and 80111 of low density lipoproteins (5mg/ml, Product no. L-2139,Sigma) were added to 7.57 ml of the Iscove's medium. The bovine serumalbumin/insulin/transferrin/penicillin/streptomycin mixture was prepared as follows. Asolution of 20% (weight/volume) of bovine serum albumin (Fraction V, cat. no. A4503,Sigma) in double distilled water was de-ionized using 60g of AG-501-X8(D) resin (Bio-Rad Labs, Richmond, CA), filtered and mixed with an equal volume of 2x IMDM (plusbicarbonate) to give a 10% (weight/volume) stock solution that was filter sterilized andstored at -20°C in aliquots. Insulin (cat. no. 40205, Collaborative Research, Bedford,MA) was aliquoted at 1 mg/ml and stored at -20°C. Iron-saturated human transferrin(cat. no. 82-343, ICN ImmunoBiological, Costa Mesa, CA) was aliquoted at 20mg/m1 inLMDM and stored at -20°C. Penicillin (104U/m1) and Streptomycin (5mg/m1) stocksolution was also aliquoted and stored at -20°C. The bovine serumalbumin/insulin/transferrin/penicillin/streptomycin mixture was prepared by adding 1 ml23of penicillin/streptomycin stock solution, 1 ml of insulin stock solution, and 1 ml oftransferrin stock solution to 20m1 of the 10% bovine serum albumin solution. Thismixture was stored at -20°C in 2.3 ml aliquots. Tissue culture supernatants of transfectedCOS cells containing human IL-3 at 214/m1 and human erythropoietin at 150U/m1 wereprovided by colleagues in our laboratory. The final concentrations of growth factorsused in serum-free cultures were: 2Ong,/m1 IL-3, lOng/m1 IL-6, 5Ong/m1MGF, 20 ng/mlof a GM-CSF/IL-3 fusion protein, and 3U/mlerythropoietin.25 Cells were plated at5,000 to 10,000 sorted cells/ml, and harvested on days 5-11 for counting, and restainingfor analysis and/or resorting on the fluorescence activated cell sorter.2.4.4: Activation of peripheral blood T cells Low density peripheral blood cells from a healthy volunteer were cultured at 105cells/ml in Iscove's medium with 10% FCS and containing a 1:200 dilution of a stocksolution of phytohemagglutinin M (PHA, Cat. No. 670-0576AD Gibco BRL,Gaithersberg, MD) as well as IL-2 (Cetus, Emeryville, CA) at 480U/ml. Cultures wereharvested on day 7 and stained for Thy-1 and CD3 expression.2.5: Immunoprecipitation studiesDHL4 (5E10+) and CCRF-CEM (5E10') cells were harvested, washed twice inHanks buffered salt solution, and 107 cells lysed at a final concentration of 2 x 107 cellsper ml of 0.5% NP40, 50mM HEPES, 100mM NaF, 100 mM NaPPO4, 2mM Na3VO4,2mM EDTA + 214/m1 leupeptin, 5mM phenylmethylsulphonylfluoride (Sigma, St Louis,MO) and 100 Kallikrein inhibitor units /m1 aprotinin, pH7.5 in distilled water. Lysedsamples were then centrifuged for 10 minutes at maximum speed in an Eppendorfmicrofuge to remove cell debris including nuclei and cytoskeletal components, and thesupernatants transferred to new tubes. Cell lysates were precleared by two successiverounds of incubation with anti-TNP bound protein-A Sepharose beads (Sigma, St Louis,MO). Anti-TNP (an irrelevant IgG 1 control mAb) was added at 5gg/m1 to protein-ASepharose beads and incubated for 30 minutes at 4°C. 150m1 of this Ab/bead mixture24was added to cell lysates, samples mixed at 4°C on a rotating platform (Nutator) for 1hour, and beads pelleted by microfuging at maximum speed for 1 minute. Supernatantswere transferred to new tubes, and the preclearing step was repeated. Forimmunoprecipitation, unlabelled 5E10 or an isotype matched control Ab (anti-TNP) wereadded at a final concentration of 514/m1, and samples mixed on a Nutator for 30 minutesat 4°C. 200m1 of protein-A Sepharose beads were then added to each tube, and samplesmixed on a Nutator for 1 hour at 4°C. The beads were then pelleted and washed threetimes in low salt buffer (0.2% NP40, 2mM EDTA, 10mM Tris-C1, pH7.5, 150mMNaC1), twice in high salt buffer (0.2% NP40, 2mM EDTA, 10mM Tris-C1, pH7.5,500mM NaC1), and once in 10mM Tris-C1, pH7.5. Proteins were then separated bySDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis) as follows.Four times concentrated sample buffer (30% glycerol, 25% 2-mercaptoethanol, 9.2%SDS, 0.8% bromophenol blue) and distilled water were added to samples (final volumeof 160g1). Samples and low range prestained protein standards (cat. no. 6040SA, BRL,Gaithersberg, MD) were boiled for 2 minutes, then loaded onto a 20cm 15%polyacrylamide gel and run for 16 hours at 100 Volts on a BioRad Protean II gelapparatus. Bands were then electroblotted to an Immobilon membrane (Millipore,Mississauga, Ont.) for 1.5 hours on a BioRad transblot. The membrane was removed andblocked by incubating for 2 hours at room temperature in blocking solution (2% bovineserum albumin, 0.05% NaN3, 0.05% Tween in PBS), followed by three 5 minute rinsesin wash solution (0.1% bovine serum albumin, 0.05% Tween in PBS). The membranewas transferred to wash solution containing 0.514/m1 of biotinylated 5E10 and afterincubation for 30 minutes at room temperature the membrane was rinsed three times inwash solution. The membrane was then incubated for 1 hour in wash solution containingdiluted (1:20,000) streptavidin conjugated horseradish peroxidase (Bio-Can, Mississauga,Ont), rinsed four times in wash solution, and once in PBS. Enhanced chemiluminescencesubstrate solution (Amersharn, Oakville, Ontario, Canada) was added for 1 minute25according to manufacturers instructions before the membrane was removed, wrapped inresonite, and exposed for 2 minutes to Kodak Xomat AR film.263: RESULTS 3.1: Generation of 5E10 and initial characterization of the reactivity of 5E10 withan antigen on the surface of various hematopoietic cells Hybridomas generated from the spleen of a mouse immunized with IIEL werescreened by ELISA on peripheral blood and HEL cells. Hybridomas producingantibodies specific for HEL were further examined by flow cytometry on low densitybone marrow mononuclear cells. Bone marrow cells were labeled with hybridomasupernatants and 8G12-FITC (anti-CD34) to examine reactivity with CD34+ cells. 5E10labeled a minority of bone marrow cells including approximately 25% of CD34bri ghtcells and a small population of CD34- cells (Figures 2A and C). It was difficult todiscriminate between 5E10 and control stained samples without gating on CD34+ cells(Figures 2A and B) as only a few percent of bone marrow cells are 5E10+ (1-4%) andthe determinant recognized by 5E10 is apparently expressed at low levels on humanhematopoietic progenitors.3.2: 5E10 immunoprecipitates a 25-35kD protein, and is specific for human Thy - 1 Several human hematopoietic cell lines were examined for 5E10 staining. CCRF-CEM, MOLT4 and HUT78 are T cell lines, DHL4 a B cell lymphoma line, and K562,HEL, and KG 1 a are all leukemic lines with various characteristics of early hematopoieticblast cells. HEL, DHL4, and HUT78 were brightly stained by 5E10, MOLT4 onlyweakly, and K562 and CCRF-CEM not detectably. Only a few percent of KGla cellswere 5E10+, and staining of these cells was weak (data not shown). From these results, itwas decided to use DHL4 (brightest staining) and CCIZF-CEM (lowest backgroundstaining) cell lysates for immunoprecipitation studies with 5E10.DMA. (Figure 3, lanes 1 and 2) and CCRF-CEM (Figure 3, lanes 3 and 4) cellswere lysed and immunoprecipitated with either 5E10 (Figure 3, lanes 1 and 3) or anisotype matched irrelevant control Ab (Figure 3, lanes 2 and 4). Immune complexeswere then run on a 15% gel, transferred to an Immobilon membrane, and00,00 200 400 600 800 1000FSC0 200 480 680 800 1000FSC27280 400 600 800 IOWSSCFigure 1. Light scatter and fluorescence gates used for sorting human CD34+ bonemarrow cells. Bone marrow cells were gated to exclude dead cells and cells with highside scatter (SSC, A), and include cells with low SSC and low to medium forward lightscatter (FSC, B) and low side scatter and high CD34 expression (C). The same gateswere used for cord blood and fetal liver samples.28Figure 2. 5E10 staining of human bone marrow cells.  Low density bone marrowmononuclear cells were labeled with 5E10 or isotype matched control Ab (anti-TNP oranti-PO) together with Ab against CD34. 5E10 stains a small population of CD34- cells(boxed area, A) and 20-30% of CD34+ cells. Indirect staining results in the brighteststaining but also the highest background. Note that 5E10 appears to react preferentiallywith CD341"i ght cells.5E10/bGamiSA-RPE aTNP/bGam/SA-RPE5E10/bGam/SA-RPE aTNP/bGam/SA-RPE5E1 0-F aPO-Feg)^CS". "...eD1**.cEn105E10b/SA-RPE aP0b/SA-RPE29301^2 3 445-30-19-16-Figure 3. 5E10 immunoprecipitates a 25-3510 protein.  DHL4 (5E10+) and CCRF-CEM (5E10-) cells were lysed, and subjected to immunoprecipitation with either 5E10 oran irrelevant isotype matched control antibody anti-trinitrophenol (T'NP) and Westernblotted with 5E10. Lane 1: DHL4 cells subjected to immunoprecipitation with 5E10;lane 2: DHL4 cells subjected to immunoprecipitation with anti-TNP; lane 3: CCRF-CEM cells subjected to immunoprecipitation with 5E10; lane 4: CCRF-CEM cellssubjected to immunoprecipitation with anti-TNP. The very strong band around 25-35kDwas specific for DHL4 cells, and was not precipitated by the control antibody.31blotted with 5E10. Several weak bands were seen in all lanes, but a single broad band at25-351(D was specific for the 5E10 immunoprecipitated DHL4 lysates (Figure 3, lane 1).In separate studies, the gene for the protein recognized by 5E10 was cloned froma HEL cell expression library by Dr. Rob Kay and the sequence data obtained for itindicates that the gene is identical to human Thy-1 (Craig,W.H.; Kay, R.; Cutler, R.C.and Lansdorp, P.M. Expression of Thy-1 on human hematopoietic progenitor cells. InPress, J. Exp. Med.).3.3: Functional analysis of CD34+ cells from bone marrow differing in Thy-1 expression.To functionally characterize Thy-1+ CD34+ cells, normal adult bone marrowcells were sorted on the basis of CD34 and Thy-1 expression as well as CD71 expressionand plated in serum-free cultures, long-term cultures, and clonogenic progenitor assays.For the initial experiments, organ donor bone marrow cells were thawed and stained withmAb specific for Thy-1 (5E10), CD34, and CD71. Low side scatter CD71dull CD34+cells were sorted into Thy-1+ and Thy-1- fractions, and cultured for 10 days in serum-free cultures with (epo mix: IL-6, IL-3, MGF, and erythropoietin) or without growthfactors. Parallel LTC and clonogenic cell assays were performed. The majority ofclonogenic cells and of week 5 and 8 LTC-IC were recovered in the Thy-1+ fraction ofthe CD71dull CD34+ population (Table II). Serum-free cultures were harvested on days5, 7, and 10, counted, and restained for analysis on the fluorescence activated cell sorter.Thy- i+ can dull CD34+ cells survived better in culture without added growth factors,and took longer to become activated than the Thy-1- fraction (Table III). When sampleswere restained on day 5 and analyzed on the fluorescence activated cell sorter, cells fromcultures initiated with Thy-1- CD71dull CD34+ cells remained Thy-1, while a smallproportion of cells in cultures initiated with Thy-1+ cmidull CD34+ cells maintainedtheir Thy-1+ cmidull CD34+ phenotype (Table III). Thy-1+ cells decreased in relationTable HCFU week 5 LTC-IC week 8 LTC-ICfractionsorted%sortedf per2x106enrich-ment%recoveryf per2x106enrich-ment%recoveryf per2x106enrich-ment%recoveryungated 100 10,900 1 100 1680 1 100 540 1 100Thy-1-cD71dullcD34+0.49 62,500 5.7 3 25,500 15 7.5 6,000 11 5.4Thyl+CD71dullCD34+1.99 180,000 16.5 32 48,000 29 57 39,000 72 143Table II LTC-IC and clonogenic cell assays of CD71dull-CD34+ human bone marrow cells sorted on thebasis of Thy-1 expression. Organ donor bone marrow CD71dull CD34+cells were sorted on the basis ofThy-1 expression and assayed for long-term culture initiating cells (LTC-IC) and clonogenic cells (CFU).Enrichments were calculated by dividing the frequency (f) of CFU or LTC-IC in purified fractions by thefrequency for ungated viable cells. Recoveries were calculated by multiplying the enrichment by thepercentage of cells sorted.33Table IIISample day 0 day 5 day 7 day 10 increase noThy-1- 10,000 3.7x104 4.1x105 7.2x106 720x 4,125Thy-1+ 10,000 2.7x104 8.1x104 1.2x106 120x 6,700Table III Serum-free cultures of Thy-1±/Thy_  i_-__cmidull  cD34__+ human bone marrowcells. Organ donor bone marrow CD71dulICD34+ cells were sorted on the basis of Thy-1 expression and placed in serum-free cultures with or without added growth factors, asdescribed in the Materials and Methods section. Sorted cells came from the sameexperiment as those in Table II. Growth factor (0)-containing cultures were harvestedon days 5, 7, and 10, and cultures lacking growth factors on day 10. Cells were countedon a hemocytometer and stained with Ab specific for CD34, CD71, and Thy-1 foranalysis on the fluorescence activated cell sorter.34to input cell number and as proportion of total cells during culture. A higher proportionof the cells in the Thy-1+ cultures were CD71dull CD34+ (4.3% of total cells, or 1160cells) than in the Thy-1- cultures (0.7% of total cells or 259 cells respectively) on day 5.Thy-1+ cultures also produced more CD34+ cells after 10 days (71% versus 16% of totalcells or 1.94 x104 versus 0.6 x 104 cells respectively from 104 input cells). These resultssuggest that Thy-1+ CD34+ cells are probably the precursors of Thy-1- CD34+ cells.The above experiment was repeated with cells from the same marrow using amore restrictive gate on CD71 and Thy-1 expression. Cells were sorted and plated inlong-term cultures, methylcellulose progenitor assays, and serum-free cultures with (epomix or myeloid mix: GM/M-3 fusion protein, IL-6, G-CSF [granulocyte-colony-stimulating factor], M-CSF [macrophage-colony-stimulating factor], MGF) or withoutadded growth factors. Serum-free cultures were analyzed on days 6, 9, and 10. Resultswere consistent with the first experiment except that recoveries of LTC-IC were muchpoorer, probably due to the very restrictive sort gates. Thy-1- cultures were virtually allCD71+ CD34+ by day 9, and survived poorly without added growth factors (10%survival for Thy-1- cells versus 30% for Thy-1+ cells). A proportion of the cells in theThy-1+ cultures appeared to remained quiescent (Thy-1+ cwidull cD34+) for 9 days.These cells were resorted and plated in serum-free cultures with the same growth factorcombinations in which they had been plated originally. Cell production continued foranother 12 days at which time cultures were then terminated.Initial LTC-IC and clonogenic progenitor assays indicated that Thy-1+ CD34+cells were highly enriched for week 5 and 8 LTC-IC. To further examine this, fournormal adult bone marrow samples were stained and sorted on the basis of CD34 andThy-1 expression. Sort windows were set to give a clear separation of Thy-1+ andThy-1- cells, therefore, some Thy-11° cells were excluded and this resulted in poorrecoveries for some experiments (Tables IV and V). In three of four experiments,myeloid and erythroid clonogenic cells were depleted in the Thy-1+ fraction while35Table IV Clonogenic assays of CD34± human bone marrow cells sorted on the basis of Thy-1 expression. Bone marrow cells were labeled with antibodies specific forCD34 and Thy-1, and CD34+ cells were sorted into Thy-1+ and Thy-1- populations.Sorted cells were plated in clonogenic progenitor assays. Myeloid (CFU-GM ) anderythroid (BFU-E and CFU-GEMM) colonies were scored on day 14, and highproliferative potential colonies (HPP-CFC) on day 28. Enrichments were calculated bydividing the frequency of colonies in purified fractions by the frequency (f) ofcolonies in cultures initiated with flow sorted viable, ungated cells. Recoveries werecalculated by multiplying the enrichment by the percent sorted.Table IVBFU-E + CFU-GEMM CFU-GM HPP-CFCExp.No.Fractionsorted%sortedf per2x106enrich-ment%recoveryf per2x106enrich-ment%recoveryf per2x106enrich-ment%recovery1 ungated 100 11,500 1 100 8,740 1 100 366 1 100Thy-1-CD34+2.1 215,000 19 39 272,500 31,^65 2,500 6.8 14Thy1+CD34+ 0.56 115,000 10 5.6 325,000 37 21 27,500 75 422 ungated 100 16,600 1 100 9,600 1 100 2,000 1 100Thy-1-CD34+2.6 398,000 24 62 110,000 11.5 30 0 0 0Thy-+CD34+ 0.39 10,000 0.6 0.2 88,000 9.2 3.6 60,000 30 123 ungated 100 5,160 1 100 3,740 1 100 360 1 100Thy-1CD34+ 1.5 195,000 38 53 175,000 47 71 0 0 0Thy1+CD34+ 0.48 190,000 37 18 422,500 113 54 57,500 160 764 ungated 100 3,060 1 100 3,000 1 100 720 1 100Thy-1-CD34+1.4 92,000 30 42 160,000 53 74 10,000 13.8 19Thy1+CD34+ 0.15 90,000 29 4.4 95,000 32 4.8 60,000 83 12.5mean +1- SEMungated 100 9,080+/-5,3401 100 6,720+/-2,9301 100 862+1-6371 100Thy- 1"CD34+1.9+/-0.5225,000+/-110,00028+/-749+/-9180,000+/-59,00035.6+/-1660+/-17.63,125+/-4,1005.2+/-5.78.3+1-8.4Thy1+CD34+ 0.4+/-0.15101,000+/-64,00019+/-14.57+/-6.67.1+/-6.647.8+/-3920.9+/-20.351,250+/-13,75087+/-4735.6+1-26.337Table Vweek 5 LTC-IC week 8 LTC-ICExp. Fraction sorted % f per enrich- % f per enrich- %No. sorted 2x106 ment recovery 2x106 ment recovery1 ungated 100 775 1 100 214 1 100Thy-1-CD34+ 2.1 5,250 6.7 0.14 375 1.8 3.8Thy-1+CD34+ 0.56 159,000 204 114 44,800 209 1172 ungated 100 1,660 1 100 638 1 100Thy-1-CD34+ 2.6 9,150 5.5 14 2,550 4 10Thy-1+CD34+ 0.39 80,500 48 19 45,900 72 283 ungated 100 ND ND ND ND ND NDThy-1-CD34+ 1.5 ND ND ND ND ND NDThy-1+CD34+ 0.48 ND ND ND ND ND ND4 ungated 100 174 1 100 68 1 100Thy-1-CD34+ 1.4 3,000 17 24 900 13 18Thy-1+CD34+ 0.15 112,000 644 97 43,800 644 97mean +/- SEMungated 100 870 1 100 306 1 100+/- +1-610 242Thy-1-CD34+ 1.9 5,800 9.7 12.7 1,275 6.3 10.6+/- +/- +/- +/- +/- +/- +1-0.5 2,540 5.2 9.8 927 4.8 5.8Thy-1+CD34+ 0.4 117,200 299 77 44,800 308 80.7+/- +/- +/- +/- +/- +/- +1-0.15 32,250 252 41 860 244 38.1Table V Long-term cultures of CD34± human bone marrow cells sorted on the basis of Thy-1 expression. Sorted cells were placed in long-term cultures and maintainedfor 5-8 weeks, at which time cultures were harvested and clonogenic progenitor assaysperformed. Enrichments were calculated by dividing the frequency (f) of colonies inpurified fractions by the frequency of colonies in cultures initiated with flow sortedviable, ungated cells. Recoveries were calculated by multiplying the enrichment bythe percentage of cells sorted. ND = not determined.38the majority of these colony forming cells were recovered in the Thy-1- fraction (TableIV). High proliferative potential colony-forming cells (HPP-CFC), a more primitiveprogenitor cell that can be assayed in semi-solid medium,24 were highly enriched in theThy-1+ fraction (30-160 fold, Table IV) and were either absent from or only moderatelyenriched in the Thy-1- fraction (Table IV). Similarly, week 5 and 8 LTC-IC were highlyenriched (48-644 and 72-644 fold respectively) in the Thy-1+ fraction (Table V). Theseresults indicate that Thy-1 expression is highest on week 8 LTC-IC, followed by week 5LTC-IC, and HPP-CFC, and is absent from the majority of the most mature clonogeniccells.3.4: Phenotypic analysis of Thy-1+ CD34+  subpopulations in bone marrow, cordblood, and fetal liver. Functional studies indicated that the majority of LTC-IC were recovered in the Thy-1+subpopulation of CD34+ cells. This population was further characterized by multiparameterflow cytometry to correlate Thy-1 expression with additional markers, primarily to investigatethe possibility of subdividing Thy-1+ CD34+ cells using a third reagent. Cord blood and fetalliver samples were included to examine whether Thy-1 expression is altered during ontogeny.Low density mononuclear cells from bone marrow, cord blood, and fetal liver were labeled inthe same experiments with 5E10, 8G12 (anti-CD34) and either rhodamine-123 or a mAbspecific for c-kit, CD38, CD71, or CD45RA. Dead cells were excluded by gating out PI+ cells.Additional gates were set to exclude cells with high side scatter (SSC) and to restrict analysis tocells with medium to high forward scatter (FSC), and high CD34 expression (Figure 1). Profilesfor bone marrow, cord blood, and fetal liver CD34+ cells are shown in Figures 4 and 5 and aresummarized in Table VI. Again, without further gating it was difficult to discriminate 5E10staining from background staining (compare Figures 4A, E, and I with Figures 4B, F and J). Upto 50% of CD34+ cells from fetal liver were Thy-1+ (Figure 4D) and this population wassmaller but clearly present in CD34+ cells from cord blood and bone marrow (Figures 4H andL). Note that CD45RA expression is low on fetal liver cells, high on some cord blood and39Figure 4. 5E10 versus CD45RA staining of human cord blood, fetal liver, and bone marrow cells. Low density mononuclear cells from fetal liver, cord blood, and bone marrow werestained with antibodies specific for CD34, CD45RA, and Thy-1 (5E10) or an isotype matchedcontrol antibody. Gates were set to exclude dead and high side scatter cells, as shown in Figure1, to reduce nonspecific events, and to restrict analysis to CD34+ cells. 5E10 staining wasstrongest on CD45RA- CD341- cells, a population previously shown to be highly enriched forLTC-IC. Note that some fetal liver CD34+ cells express more Thy-1 than any cord blood orbone marrow CD34+ cells.All cells inLS, PI gate CD34+ cells inLS, PI gate7• leo Tes 142 163 i 0"". CO^• Ov s^ ,(34^1 Ta^M•" '” a7 04BONEMARROW 6▪ .1CD45RA-FO 0;00' • I e ' • "112— "^-"mg^:70260 To, ----re2CD45RA-F CD45RA-FFETALLIVERCORDBLOOD7csLIJ400- ..."io^Te2^--rem^Floo -"at --;62CD45RA-F41Figure 5. 5E10 staining of CD34+ subpopulations in human fetal liver, cord blood andbone marrow. Bone marrow, cord blood, and fetal liver cells were labeled withantibodies specific for CD34, Thy-1 (5E10), and a third marker (c-kit, CD38, or CD71).Gates were set to restrict analysis to cells with low side scatter, low to medium forwardscatter, and high CD34 expression as described in Figure 1. The majority of Thy-1+(5E10+) CD34+ cells were CD7110 and CD38-. Thy-ft c-kit+ CD34+ cells expressedlow to intermediate amounts of c-kit.Z17ow >03:Iz33mo * 5E10-F.4 e0 Al. ..1,2. _193. 1045E10-PEr---n7ernrAil_....I-5E10-PE5E10-F.1 .^i2^j.31 4^4 qo^ 123, 1, ...1? ...!93. ...tbr75E10-F5E10-PE5E10-PE_leo 1st 192 03 10461 no ., L• . . n Y.1....w....193, 1 0.,eDY 0 a.,..i5E10-PE.400toil^2j93 lir..^-75E10-PE_100... 1 cA 1. ...192..__I93. •Lew;.1.1uC,043Table VICD34+Sample N Eate LS gate all CD71- CD38" CD45RA- c-kit+bonemarrow2.4 - 25 (2.7) 45 80 - -4.2 4.0 20 (3.2) 15 32 25 -2.5 5.2 41(2.3) 75 75 52 702.5 2.0 26 (1.7) - - - -1.0 1.0 22(3.2) - - - -2.0 4.5 27(4.0) - 87 - -1.8 2.4 21(1.5) - - - -0 0 12(2.5) - - - -2.6 0.6 30 (4.6) - - 40 50peripheraI blood0.20.30.7cordblood5.8 1.2 54 (0.4) 56 45 - -0 0 10(1.0) - - 20 -1.6 0 25(0.4) - - 30 -4.5 3.5 40(1.4) - - 43 40fetal liver 3.0 3.0 18 (1.0) 30 25 20 707.0 7.0 35 (7.4) 50 65 45 601.4 1.3 38(1.3) 65 80 50 30Table VI Thy-1 expression on human bone marrow, peripheral blood, cord blood, andfetal liver cells. Bone marrow, cord blood, fetal liver, and peripheral blood cells werelabeled with antibodies specific for CD34, Thy-1, and a third marker. Gates were set asin Figure 1 to examine Thy-1 expression on: (1) all viable cells, (2) viable cells with lowside scatter and low to medium forward scatter, and (3) viable cells with low side scatterand low to medium forward scatter, and high CD34 expression (value in brackets).Additional gates were set to examine CD34+ subpopulations. Results are expressed asthe percent positive cells within the indicated cell fraction (- = not tested). The percentThy-1+ was determined by subtracting percent positive for control stained samples fromthe percent positive in 5E10 stained samples.44bone marrow cells and that Thy-1 expression appears restricted to CD45RA- CD34+cells in adult bone marrow but includes some CD45RA+ CD34+ cells from cord blood(Figures 4D, H and L).c-kit expression was restricted to a few percent of bone marrow, cord blood andfetal liver cells (not shown) but the majority of CD34+ cells from each source was c-kit+(Figures 5A, D and G). In all instances when CD34 was plotted against c-kit, the c-kitlocells expressed the highest levels of CD34 (data not shown) and Thy-1 expression wasfound to be restricted to cells that express intermediate to low levels of c-kit (Figures 5A,D and G). Thy-1 expression was slightly lower on cord blood than on bone marrow orfetal liver. 5E10-FITC was used in these experiments because detection of c-kitexpression required sensitive indirect staining. Staining with 5E10-FITC resulted insignificantly weaker staining than in comparable stainings in which a biotinylatedprimary or secondary Ab was used (Figure 2). The majority of Thy-l+CD34+ cells wereCD7110 in all tissues and Thy-1 expression decreased with increased CD71 expression(Figures 5B, E and H). Similarly, Thy-1 staining was highest on CD38-CD34+ cells, anddecreased with increased CD38 expression (Figures 5C, F and I). Rhodamine-123staining and Thy-1 expression on bone marrow CD34+ cells was inversely correlated(Figure 6). Thy-1+ CD34+ cells were rhodamine-123du11, with rhodamine-123 stainingincreasing as Thy-1 expression decreased. Resolution between rhodamine-123"11 andrhodamine-123bright cells was improved by incubating rhodamine-123 stained samplesin medium at 37°C to allow dye efflux (compare Figures 6C and D). Data derived fromthree color stainings indicate that Thy-1 has very restricted expression that, within theCD34 compartment, is confined to cells that are phenotypically similar to the mostprimitive hematopoietic cells.&^/60^rill^re2113^ie..'454°C^37°CLUO.6iiiiLai^ i^103^104Rhodamine 123Figure 6. 5E10 vs rhodamine staining of human bone marrow cells.  Cells were labeledwith 8G12 (anti-CD34), rhodamine-123, and either an irrelevant isotype matched controlantibody (plots A and B) or 5E10 (plots C and D). After rhodamine-123 staining at 37°C, samples were split and either left on ice at 4°C (plots A and C), or incubated anadditional 20 minutes at 37°C (plots B and D) to allow dye efflux. Within the CD34gate, Thy-1 expression (5E10 staining) and rhodamine-123 efflux were tightly correlated.463.5: Phenotypic analysis of Thy-1++ CD34- cells in bone marrow and peripheralblood.One bone marrow and one peripheral blood sample were analyzed for the co-expression of Thy-1 and various lymphocyte and myeloid markers. Samples werelabeled with 5E10, 8G12 (anti-CD34), and various lineage markers (Figure 7) and Thy-1++CD34- cells (gate shown in Figure 2A) analyzed for the expression of lineagemarkers. The Thy-1 gate was set high in order to include only cells that were morebrightly stained than control stained samples (Figures 2A and B). It is possible that, as aresult, some weakly stained Thy-1+ cells were excluded, however discriminationbetween specific and non-specific staining is difficult in this region. Thy-l++CD34-cells comprised 0.1-0.3% of low density bone marrow and peripheral blood cellsincluding a small subset of lymphoid cells as judged by their light scatter. As shown inFigure 7, the majority of Thy-1++CD34- cells were CD3+ and CD4+. Interestingly,when peripheral blood leukocytes were stimulated with PHA and IL-2, the proportion ofThy-l+CD3+ cells increased dramatically (Figure 8). Cells observed in cytospinpreparations of Thy-1++ CD34- peripheral blood cells were small dense lymphocytes(results not shown).3.6: Effect of mAb 5E10 on cells in culture.Experiments indicated that the 5E10 Ab has no effect on hematopoietic cells invitro. 5E10 or control Ab were added at 1014/m1 to purified bone marrow cells (CD7110CD45RA10CD34+ ) plated in long-term culture (2000 cells/dish) and progenitor assays(400/dish). No significant difference in colony formation was seen between the cultureswith (22 colonies/dish) or without added 5E10 Ab (25 colonies/dish). Long-termcultures harvested after 5 weeks produced 172 colonies/dish and 141 colonies/dish forcultures with and without added 5E10 respectively. Week 8 harvested long-term culturesproduced 70 colonies/dish with and without added 5E10. From these experiments it was47Figure 7. Lineage marker expression on 5E10++ CD34- human bone marrow cells.Normal bone marrow cells were labeled with antibodies specific for CD34, Thy-1(5E10), and various lineage markers. Histograms on the left of the figure are thefluorescence histograms of gated viable low density mononuclear cells obtained with theindicated marker, whereas histograms in the right of the figure represent lineage markerexpression on cells with an additional 5E10++ CD34- gate (shown in Figure 2a). Notethat Thy-1++ CD34- cells appear to be CD3+CD4+ lymphocytes.48Pr 5E10++ CD34" PI"migGiCD3CD19CD4CD8CD13CD14CD9CD56CD3+ cells492u4Unstimulated•1StimulatedmIgGi-F^5E10-FFigure 8. 5E10 staining of phytohemagglutinin and 11,-2 stimulated human peripheral blood cells. Normal peripheral blood leukocytes were split into two fractions and eitherfrozen (unstimulated, A and B) or cultured with phytohemagglutinin and EL-2(stimulated, C and D). On day 7, frozen samples were thawed, and stimulated culturesharvested. Cells were stained with anti-CD3, and 5E10 (B and D) or an isotype matchedcontrol (A and C). Samples were gated for CD3+ cells, and analyzed for Thy-1expression. No significant populations of 5E1O+CD3- cells were observed.50concluded that, under conditions used, 5E10 did not appear to interfere with theproduction and/or formation of colonies from primitive hematopoietic cells. No effectwas seen when this experiment was performed with cells sorted and plated in liquidsuspension cultures.514: DISCUSSIONCharacterization of Thy-1 expression on human hematopoietic cells wasperformed with a new mAb, 5E10, which immunoprecipitated a 25-35 kD protein asexpected of an anti-Thy-1 Ab48, 104 and was formally demonstrated to be directedagainst Thy-1 by cDNA cloning. 105Thy-l+CD34+ cells are highly enriched for LTC-IC measured at week 5 and 8and for HPP-CFC, but are relatively depleted of most types of clonogenic cells. Resultsfrom the fluorescence activated cell sorter indicated that Thy-1 expression was highest onCD34+ cells that were previously described as being highly enriched for primitivehematopoietic cells (Figure 4,5) in that Thy-1 expression decreased with increasedexpression of CD45RA, CD38, or CD71 expression.25, 40,43 These findings are inagreement with studies of rat' 4 and murine106,107 hematopoietic cells indicating thatrepopulating cells, HPP-CFC, BFU-E, and CFU-GM are Thy-1+, whereas morecommitted progenitors are not.Thy-1 expression appears to be very low on most CD34+ cells, as staining, evenwith very sensitive techniques, was weak. This weak staining is unlikely to reflect lowAb affinity because 5E10 could be used at relatively low concentrations (<514/m1), andcertain cell lines, in addition to the rare population of Thy-1++ CD34" normal lymphoidcells, were very brightly stained even by directly conjugated Ab.Cord blood, bone marrow, and fetal liver profiles obtained with 5E10 and CD34were quite similar except that CD34 and Thy-1 expression appeared higher on some fetalliver cells than on any bone marrow or cord blood cells, and CD34+ cells in most fetalliver samples examined lacked CD45RA or expressed very little (Figures 4D, H, and Lrespectively). It is tempting to speculate that this population of Thy-1++ CD34+ cellsrepresents a more primitive population of cells that is lost during ontogeny. Althoughbackground staining was greater on fetal liver CD34+ cells than on cord blood or bone52marrow CD34+ cells, the increased 5E10 staining is unlikely to be a staining artifact.The higher background may be due to low affinity Fc receptors present on fetal liverblast cells.108 Indeed, when fetal liver cells were stained for Fc receptors (CD 16, CD32)a significant proportion (>30%) of fetal liver but less than 5% of cord blood or bonemarrow CD34+ cells were found to express CD32 and CD16 in agreement with findingsby others.108 However, all 5E10+CD34+ cells lacked Fc receptors (results not shown).The lack of CD45RA expression on the majority of fetal liver CD34+ cells examinedcoincides with expression of CD45R0 (Craig,W.H.; Poppema,S. and Lansdorp, P.M.,manuscript in preparation) and underscores the importance of CD45 isoform expressionin the differentiation of hematopoietic cells.43Mast cell growth factor (MGF), a ligand for the c-kit receptor tyrosine kinase, haspowerful synergistic effects with other early acting growth factors on primitivehematopoietic cells, 103, 25 and murine studies indicate that very primitive hematopoieticcells (including at least some of those with marrow repopulating ability) are c-kit+.92We have found that, on human hematopoietic cells, c-kit is expressed at low levels onpopulations that contain the most primitive cells. Thy-1 expression appears also to berestricted to these most primitive cells and is likewise restricted to c-kitlo cells. Allrhodamine-123du11 CD34+ cells were Thy-1+, with Thy-1 expression being inverselyrelated to rhodamine-123 staining (Figure 6). This is in contrast to the report of Baum etal that Thy-l+CD34+ cells could be subdivided by rhodamine-123 staining.The function of bone marrow and cord blood Thy-1++CD34- lymphoid cells isunknown. The CD3+CD4+ phenotype suggests a mature cell type, and, cells in cytospinpreparations did indeed appear like small lymphocytes. We found that the proportion ofThy-1+ lymphocytes increased dramatically upon stimulation with phytohemagglutininand IL-2 (Figure 8) suggesting that Thy-1 expression could be related to T cellactivation. To our knowledge, this is the first report of Thy-1+ lymphocytes in normalhuman bone marrow, although previous reports have described a population of Thy-1+53thymocytes that were believed to be prothymocytes.48 The only other report of maturehuman Thy-1+ hematopoietic cells described a population of Thy-1+ infiltratingleukocytes in several patients with malignant breast tumours,109 however, thispopulation was not further characterized.Some reports have suggested that IL-3, but not other hematopoietic growthfactors, may up regulate Thy-1 expression on Thy-1- murine hematopoietic progenitorsor stimulate the production of myeloid progenitors from Thy-1- precursor cells110, 111contradicting our results and those of others. 106, 44 Results from our IL-3 containingserum-free cultures showed a consistent decrease in Thy-1+ cells with time under both•erythroid and myeloid promoting conditions. Reports on murine106, 107 and rat44 Thy-1 expression describe an inverse relationship between Thy-1 expression and the stage ofdifferentiation, however, one report suggested that murine cells of the monocyte lineagemay pass through a stage of development in which they express high levels of Thy- 1.46IL-3 stimulated production of Thy-1+ cells from a Thy-1- population may be unique tothe culture conditions used or to murine progenitors, particularly given numerous reportsindicating that Thy-1 is lost during differentiation, rather than gained. i06, 107 ourserum-free cultures contained M-3 in addition to other growth factors and this mayaccount for the differences in results.The function of Thy-1 on hematopoietic stem cells is unknown. Rat stem cellsexpress more Thy-1 than primitive hematopoietic cells of murine62 or human origin, butthe fact that Thy-1 expression is conserved on these cells suggests an important role forThy-1 in early hematopoiesis. As Thy-1 expression appears to be conserved on the mostprimitive cells, it would be interesting to analyze Thy-1 function in hematopoiesis bygene knockout studies in a murine model. The resulting Thy-1- bone marrow cells couldthen be transplanted into syngeneic recipients for further studies. Recently it wasreported that some cells with marrow repopulating ability (presumably stem cells) wereThy-1- in Thy-1.2 strains of mice, whereas all cells with marrow repopulating ability in54Thy-1.1 strains were Thy-1+.112 However, in view of the weak expression of Thy-1 onhematopoietic cells, these findings may reflect unusual binding properties of the Ab used.Thy-1 has been hypothesized to be involved in cellular recognition,55, 65adherence,57 and T cell activation.59, 66, 60, 61 Some Ab against Thy-1 are able tostimulate intracellular Ca2+ release61 and phosphorylation of cytoplasmic proteins,113suggesting Thy-1 may be involved in cell activation. Thy-1 is anchored in the membraneby a phosphatidylinositol anchor and has no direct link to the cytoplasm,47 therefore, if ithas a role in transmembrane signaling, additional associated proteins would be required.Some murine studies have linked Thy-1 to CD45, (a transmembrane protein withtyrosine phosphatase activity) in co-capping, and cross-linking and immunoprecipitationstudies.114, 115 Anti-Thy-1 Ab also co-immunoprecipitate p561ck,116 (a src relatedtyrosine kinase that can associate with CD45) from murine T cells.117 It is interesting tospeculate that Thy-1 and CD45R0 may be functionally associated, given their co-expression on the most primitive CD34+ cells, and the biochemical data physicallylinking CD45 and Thy-1.114, 115, 116Thy-1 may be important in adherence of primitive hematopoietic cells to stroma,possibly providing a growth inhibitory signal as has been suggested for neuronal Thy-1.75 Studies on myogenesis73 and murine thymocytes58 have indicated that Thy-1 mayfunction as a receptor for a ligand on other cells allowing cells to contact and adhere.Thy-1 may have an analogous role on stem cells, allowing them to recognize and bind tostromal determinants. The slightly lower expression of Thy-1 on circulating cord bloodcells may result from a reduced requirement for adherence and is in agreement with thisproposed function of Thy-1. Binding of Thy-1 to a ligand in the stromal microenvironment, possibly a sulfated glycan as has been suggested for thymocyte-epithelialcell bincling,76 may produce a growth inhibitory signal in stem and progenitor cells, ashas been suggested for neuronal cells.75 However, in our studies anti-Thy-1 antibodyaddition to LTC where hematopoiesis is stromal cell dependent had no effect.55Monoclonal Ab against human Thy-1 appear to be attractive tools for stem cellpurification techniques because of the very restricted pattern of Thy-1 expression withinthe human hematopoietic system and the possibility to target such rare cells byimmunoaffinity techniques. Anti-Thy-1 Ab have been used previously for thepurification of primitive murine,45, 46 rat,44 and recently human fetal bone marrow36cells. Our findings indicate that anti-Thy-1 Ab may also be useful for the purification ofstem cells from adult human bone marrow. The 5E10 antibody characterized in thisstudy gave a higher percentage of Thy-1+ CD34+ cells than reported by Baum et a1,36but this probably reflects differences in the Ab used or, alternatively, tissue-specificdifferences. Previous studies have used anti-Thy-1 Ab to purge marrow for autologousbone marrow transplantation in neuroblastoma patients as the majority of malignantneuronal tumours express very high levels of Thy-1. 104, 118 While such techniquesmay spare Thy-1+ bone marrow cells because of their much lower Thy-1 expression,improvements in purging techniques could result in the unwanted elimination of Thy-1+hematopoietic cells, and such techniques should therefore be approached with caution.565: REFERENCES 1. Lansdorp, P.M., and Thomas, T.E. 1991. Selection of human hemopoietic stemcells. In Bone Marrow Processing and Purging. A Practical Guide. ed A.P. 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