Lymphopoiesis in the adult: identification of early progenitors in both B cell and thymocyte development

The progression of commitment from hematopoietic stem cell to progenitors with specific lineage potentials remains largely undefined, in spite of decades of investigation. This dissertation reports the discovery of a novel hematopoietic progenitor population from adult mouse bone marrow defined as Lin\Neg Sca1\Pos Thy1.1\Neg] This population generates both lymphoid and myeloid cell types, but produces lymphoid forms more frequently in vitro and in transplant assays. It also lacks the ability to rescue lethally irradiated mice that is characteristic of stem cells, but as such, it represents a broad intermediary population in lineage commitment. Immunofluorescent staining and multiparameter flow cytometry supported by functional assays for lineage commitment were employed to separate and enrich the progenitors within this compartment responsible for the differing lineage potentials. cKit, the AA4.1 antigen, and L-selectin were found to distinguish functional subsets within this population, and several progenitors were successfully identified. These included a cKit[\Low AA4.1\ progenitor committed to the B cell lineage, but at a CD45R<super>Neg

LYMPHOPOIESIS IN THE ADULT: IDENTIFICATION OF EARLY PROGENITORS IN BOTH B CELL AND THYMOCYTE DEVELOPMENT by S. Scott Perry A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy ill Experimental Pathology Department of Pathology The University of Utah August 2002 Copyright © S. Scott Perry 2002 All Rights Reserved THE UNIVERSITY OF UTAH GRADUATE SCHOOL SUPERVISORY COMMITTEE APPROVAL of a dissertation submitted by S. Scott Perry This dissertation has been read by each merrlber of the following supervisory committee and by majority vote has been found to be satisfactory. " ~i {"" J1Adl~ l /' Wolfram E. Samlowski I / THE UNIVERSITY OF UTAH GRADUATE SCHOOL FINAL READING APPROVAL To the Graduate Council of the University of Utah: I have read the dissertation of s. Scott Perry in its final form and have found that (1) its fOimat, citations, and bibliographic style are consistent and acceptable; (2) its illustrative materials including figures, tables, and charts are in place; and (3) the final manuscript is satisfactory to the supervisory committee and is ready for submission to The Graduate School. Date Gerald J. Spang Chair: Supervisory Committee Approved for the Major Department Chair Approved for the Graduate Council ~-=-J $.~--. David S. Chapma Dean of The Graduate School ABSTRACT The progression of commitment from hematopoietic stem cell to progenitors with specific lineage potentials remains largely undefined, in spite of decades of investigation. This dissertation reports the discovery of a novel hematopoietic progenitor population from adult mouse bone n1arrow defined as LinNeg Scal Pos Thy 1. 1 Neg. This population generates both lymphoid and myeloid cell types, but produces lymphoid forms more frequently in vitro and in transplant assays. It also lacks the ability to rescue lethally irradiated mice that is characteristic of stem cells, but as such, it represents a broad intermediary population in lineage commitment. Immunofluorescent staining and multiparameter flow cytometry supported by functional assays for lineage commitment were employed to separate and enrich the progenitors within this compartment responsible for the differing lineage potentials. cKit, the AA4.1 antigen, and L-selectin were found to distinguish functional subsets within this population, and several progenitors were successfully identified. These included a cKitLOW AA4.1 Pos progenitor committed to the B cell lineage, but at a CD45RNeg stage early in lymphopoiesis. A cKitBright L-se1ectinPos population was found to contain striking pro-thymocyte characteristics, including thymic repopulation kinetics comparable to whole thymocytes and the generation of recurring waves of T cell engrafiment, suggesting it represents a final stage of T lineage development in the marrow prior to thymic colonization. A very primitive hematopoietic population negative for L-selectin but bright for cKit expression was found to have pluripotent lineage potential and the ability to extend the lives of lethally conditioned mice, but not rescue them. These progenitors mark unique intermediates in hematopoietic commitment not previously described, and should prove useful in further illuminating interconnections within the hematopoietic hierarchy. v To Seiko Your wisdom inspired me to attempt this degree. Your persistence motivated me, your love sustained me, and your patience saw me achieve it. Thank you. And to Kenny, Tommy, and Sarah who made graduate school all worthwhile. T ABLE OF CONTENTS ABSTRACT .......................................................... iv LIST OF FIGURES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. IX LIST OF TABLES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Xl ACKNOWLEDGMENTS ............................................... xii CHAPTER 1. INTRODUCTION ............................................... 1 Hematopoietic stem cells: an overview. . . . . . . . . . . . . . . . . . . . . . . .. 2 Isolation of hematopoietic stem and progenitor cells .............. , 3 Conventional concepts of the hematopoietic hierarchy .............. 8 Merging of myeloid and lymphoid lineages ....................... 10 Lineage predisposition vs commitment. ......................... 13 Scope of study ............................................. 14 Significance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 2. RAPID, B LYMPHOID-RESTRICTED ENGRAFTMENT MEDIATED BY A PRIMITIVE BONE MARROW SUBPOPULATION .............. 21 Abstract .................................................. 22 Introduction ............................................... 22 Materials and methods ....................................... 23 Results ................................................... 24 Discussion ................................................ 27 References ................................................ 28 3. PHENOTYPIC DISTINCTION AND FUNCTIONAL CHARACTERIZATION OF PRO-B CELLS IN ADULT MOUSE BONE MARROW ................................ 30 Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1 Introduction ............................................... 31 Materials and methods ....................................... 31 Results ................................................... 32 Discussion ................................................ 37 References ................................................ 39 4. ENRICHMENT OF LONG-TERM THYMIC PROGENITORS FROM ADlTLT MOUSE BONE MARROW .......................... 41 Abstract .................................................. 42 Introduction ............................................... 43 Results ................................................... 45 Discussion ................................................ 75 Experimental procedures ..................................... 79 References ................................................ 84 5. L-SELECTIN SUBSETS OF SCA1POS MOUSE BONE MARROW: SWIMMING UPSTREAM FROM THE COMMON LYMPHOID PROGENITOR .................................................. 88 Abstract .................................................. 89 Introduction ............................................... 90 Results ................................................... 92 Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 Experimental procedures ..................................... 130 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 135 6. SUMMARY .................................................... 138 Discovery of a primitive hematopoietic compartment. ............. 139 In vitro assessment of progenitor potentials ...................... 140 Identification of a committed B cell progenitor .................... 141 T-lineage potential within the Thy 1. I-Negative compartment. ....... 144 Bone marrow-derived pro-thymocytes .......................... 145 Pro-thymocytes and multi potent progenitors ..................... 147 The L-selectin-Negative popUlation is enriched for multiline age potential. .................................... 149 Pro-thymocyte potential within the L-selectin-Positive population. .. 151 Denouement ............................................... 154 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 157 viii LIST OF FIGURES Figures Pages 1.1 Lineage depletion of whole bone marrow. . . . . . . . . . . . . . . . . . . . . . .. 5 1.2 Separation of the LinNeg Sca1pos population according to Thy 1.1 and cKit expression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 7 2.1 Four-color analyses of mouse bone marrow cells .................. 24 2.2 Recovery of blood lineages after transplantation of Thy 1.1 Neg progenitors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 24 2.3 Flow cytometric analysis of PBMC recovery after Thy 1.1 Neg transplant. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 25 2.4 Flow cytometric analysis of cellular recovery in the bone marrow after Thy 1.1 Neg transplant. . . . . . . . . . . . . . . . . . . . . . .. 25 2.5 Recovery of peripheral blood platelet and PBMC counts after Thy 1.1 Neg transplant. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 26 2.6 Evaluation of peripheral blood following Thy 1.1 Neg transplant. . . . .. 27 3.1 Flow cytometric collection of ScalPos sUbpopulations. . . . . . . . . . . .. 33 3.2 Analysis of hematopoietic markers expressed on Thy 1.1 Neg progenitors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 34 3.3 In vitro analysis of Thyl.1 Neg cKitLow AA4.1 subsets ............. 35 3.4 Cloning efficiencies and differentiation potential of cKit AA4.1 subsets of the Thy 1.1 Neg cell population ............ 36 3.5 Analysis ofT cell progenitor frequency in the Thy 1.1 Neg cKitLow population. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 37 3.6 RT-PCR analysis of early B lineage genes expressed by Thy 1.1 Neg cKitLow AA4.1 subsets ............................. 37 3.7 Model of developmental relationships between lymphoid progenitors of the Thy 1.1 Neg compartment. . . . . . . . . . . . . . . . . . . . .. 38 4.1 The Thy 1.1 Neg population and its engraftment kinetics relative to Thy 1.1 Low HSC. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 51 4.2 Engraftment kinetics of Thyl.1 Neg progenitors transplant according to resulting lineage combinations ....................... 57 4.3 Assessment of progenitor frequencies among cKit fractions of the Thy1.1 Neg population .................................. 62 4.4 Engraftment kinetics of Thy 1.1 Neg cKitBright progenitors. . . . . . . . . .. 65 5.1 Limiting dilution analysis of the Thy1.1 Neg cKitBright population ..... 95 5.2 L-selectin separation of the Thy 1.1 Neg population ................. 98 5.3 In vitro potential of Thy 1.1 Neg cKitBright L-selectin subsets ......... 101 5.4 Engraftment following transplant with L-selectin subsets of the Thy 1.1 Neg cKitBright population .......................... 109 5.5 Relationship of L-selectin subsets with other hematopoietic progenitor compartments ......................... 123 6.1 Relationship of cKit and L-selectin expression among Thy 1.1 Neg progenitor populations. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 155 x LIST OF TABLES Tables Pages 2.1 Primary and secondary colony-forming potential of Thy 1.1 Low and Thy 1.1 Neg subsets. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 3.1 Cloning efficiency and lineage potential of Thy 1.1 Low and Thy 1.1 Neg subsets. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 33 3.2 Cloning efficiency and lineage potential of Thy 1.1 Neg subsets defined by cKit and AA4.1. ........................... 35 4.1 Engraftment posttransplant with selected progenitor subsets. . . . . . .. 48 4.2 Frequencies of lineage combinations and thymocyte engraftment resulting from cKitBright Thy 1.1 Neg progenitor transplant. .......... 69 4.3 Peripheral lymphocytes resulting from transplant of Thy 1.1 Neg cKitBright progenitors. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 73 5.1 In vitro analysis of Thy 1.1 Neg cKitBright population subsets. . . . . . .. 104 5.2 Engraftment frequencies following transplant of L-selectin subsets .... 112 ACKNOWLEDGEMENTS The work reported in this dissertation could not have been accomplished without the continual assistance of a great many people. Foremost among these is Dr. Jerry Spangrude, who provided not only the physical resources needed to conduct these experiments, but also a great deal of the training and interpretation necessary to make them meaningful. I am additionally greatly indebted to L. Jeanne Pierce for an array of technical assistance, especially in the daunting task of sorting highly purified hematopoietic progenitors, as well as for manuscript editing, and countless other areas of support. Likewise, A. Elena Searles conducted the initial in vivo assessment of the Thy 1.1 Neg population described in Chapter 2 (The Journal of Immunology, 2000, 165: 67-74). Other technical assistance was kindly offered by Melissa Marx and Chris White, without whom many assay days would have been much longer and more tedious. Doctors William B. Slayton and Meriluz P. Mojica played crucial roles in many capacities. They generously shared resources while planning experiments and freely dispensing background information, in addition to providing a substantial amount of the technical instruction and moral support necessary to complete this degree. Furthermore, Dr. Mojica conducted the majority of in vitro assays needed to characterize the AA4.1 Pos B cell progenitor described in Chapter 3 (The Journal of Immunology, 2001, 166: 3042- 3051). It is not possible to specifically enumerate all of the contributions by so many members of the Spangrude Laboratory, but support from Suzanne Pohlmann, Ann Cline, and Anne Wiesmann was especially appreciated. Assistance in this effort did not end with the Spangrude Lab. In particular, the instruments and resources of the Huntsman Cancer Institute Flow Cytometry Facility were essential for the advancements made in this work, and their expert maintenance and administration by Dr. Wayne Green and Hawk Oh proved indispensable. I am also indebted to Dr. Green for considerable instruction in both the practice and theory of flow cytometry. Doctors John Weis and Wolfram Samlowski deserve recognition for their roles in developing my understanding of immunology as they first served as rotation advisors and then as members of my supervisory committee. I am also grateful for the insight and guidance of Dr. Ray Daynes, and to Dr. Kojo Elenitoba-Johnson for both his assistance on my supervisory committee and his instruction on the histological identification of hematopoietic cell types. In addition to these mentors, Dr. Greg A. Prince, Miriam Darnel, and the rest of the staff at Virion Systems, Inc. were instrumental in the beginning of my interest in immunology, and I am grateful for the Virion Systems Scholarship that allowed me to conduct introductory research with them. Dr. Janet M. Shaw then fostered an interest in research and directed me toward the Ph.D. program at the University of Utah. It is most likely that without her patient and guiding hand, I would not have begun the path leading to this degree. I would be remiss if I failed to mention the generous financial support offered to me during the course of my studies. The George Weber Immunology Research xiii Foundation has graciously nlade it possible for me to share my research at several scientific meetings, and the Hematology Training Grant and Lymphoma Foundation have provided a graduate student stipend at varying times during nly research. Finally, my deepest thanks is reserved for my wife, Seiko, for her constant support throughout my education, and for her pragmatism and counsel that has helped me maintain an even keel on the course to completing this degree. xiv CHAPTERl INTRODUCTION 2 Hematopoietic stem cells: an overview Hematopoietic stem cells (HSC) are capable of reconstituting all of the cell lineages forming circulating blood, including the cellular immune system (Spangrude, et aI., 1988). HSC progeny are constantly in demand, as aged, damaged, deleterious, and nonfunctional corpuscles are perpetually being eliminated by various means. For example, circulating neutrophils in mice and humans have an estimated life span of less than 4 days (Hestdal, et aI., 1991), and erythrocytes are replaced every 30 days (van Iperen, et aI., 2000). Lymphoid lineages are among the longer lived hematopoietic cells, with memory Band T cells persisting possibly for the life of an individual (Tough & Sprent, 1995). However, the process of generating lymphocytes that are effective at eradicating pathogens while permissive to self tissues is very costly. Among T cells, this education process can eliminate as many as 99% of candidate thymocytes (Owen & Jenkinson, 1992). As a result of this attrition, maintaining homeostatic hematopoiesis demands a constant and vibrant production of progenitors from HSC for each blood cell lineage, as well as the ability to indefinitely renew the stem cell compartment itself. Transplants of lethally irradiated mice with cells from varying normal tissues showed that bone marrow contained the vast majority of stem cells responsible for maintaining hematopoiesis in adults (Ford, et aI., 1956); however, specific identification of this population remained a daunting task. Early efforts at probing bone marrow for this fraction focused on physical separation processes such as density centrifugation and counter current elutriation (Abramson, et aI., 1977; Miyama-Inaba, et aI., 1987). It was also found that treating mice with drugs toxic to rapidly cycling cells, such as 3 hydroxyurea and (more effectively) 5-fluorouracil, was successful at enriching HSC activity in marrow samples collected from these animals (Bruce & Meeker, 1967; Blackett & Adams, 1972; Bohne, et aI., 1970). Such experiments indicated that HSC are low in physical density relative to other bone marrow fractions and rarely enter the cell cycle. Isolation of hematopoietic stem and progenitor cells It has only been since the introduction of flow cytometric analysis coupled with immunofluorescent staining that specific identification of HSC and intermediates in the dynamic hematopoietic hierarchy has been possible. This process involves labeling antigen specifically expressed by a target cell population with antibodies conjugated to a fluorochrome. Once labeled, cells can be detected and collected from a milieu of similar cells according to the fluorescent properties of the conjugated fluorochrome. Multiple antibody clones, each specific to a separate antigen and conjugated to varying fluoro­chron1es can be simultaneously applied in this method to fractionate progressively sn1aller compartments of a target population. Metabolically specific fluorescent probes can fur­ther enhance this process, as can antibodies conjugated to magnetic particles. This latter technique enables the use of magnetic fields in addition to light properties for separation of specific marrow subsets. The culmination of these methods has enabled the isolation of HSC from both mice (Spangrude, et aI., 1988) and humans (Baum, et aI., 1992). The man1malian HSC was first isolated from mouse bone marrow, where it represents slightly less than 1 in 50,000 nucleated marrow cells (Morrison, et aI., 1995). Selecting a population as rare as this first requires a considerable enrichment before 4 fluorescence activated cell sorting can be effective. This is accomplished by first incubating the marrow with a cocktail of rat monoclonal antibodies optimized for binding an assortment of mouse antigens expressed by mature cells and progenitors committed to specific lineages (Figure 1.1). Next, this mixture of cells, either labeled or not labeled with rat anti-mouse antibodies, is reacted with magnetic beads coupled to sheep immunoglobulin specific to rat antibody. A brief incubation results in attachment of the cells labeled with rat anti-mouse antibodies to the magnetic beads via the sheep immunoglobulin. When a magnetic field is applied perpendicular to gravity, the bead­conjugated, lineage-positive cells are forced to one side of the sample tube. This allows for simple collection of the supernatant with the lineage-negative (LinNeg ) cells it contains. One application of immunoglobulin-coupled magnetic beads in this manner is sufficient to remove 80-900/0 of lineage specific cells from a marrow sample. Two applications are typically conducted, which leaves between 1-50/0 of the original cells. This high degree of enrichment for LinNeg cells enables the use of fluorochrome­conjugated antibodies against specific primitive markers in concert with fluorescent activated cell sorting to subset this popUlation into progressively more primitive compartments. Current technology can separate a cell sample based on more than 11 fluorescent parameters (De Rosa, et aI., 2001; De Rosa & Roederer, 2001); nevertheless, the isolation of HSC from LinNeg marrow typically requires no more than four of these. Commonly, one parameter is used to select viable cells based on exclusion of any of a number of fluorescent dyes. The remaining three parameters are used to select for expression of the primitive hematopoietic markers stem cell antigen-I, cKit, and Thy 1.1. anti-mouse Ab specific for lineage markers: Collect mouse bone marrow • CD2 • CD3 Apply magnetic field • B220 • CDS ·CD8 • CD19 • Ly-6G • Mac 1 • TERl19 Wash ~ Incubate )~ 'L I with sheep ~l anti-rat Ab f~ conjugated to magnetic beads IJnNeg IJone Marrow ..(. :ollect Supernatant FrOlll 1 to 5(/ti of Whole Bone Marro\\' Figure 1.1 Ijneage depletion of whole bone marrow 5 6 Although none of these markers is absolutely specific for mouse stem cells, expression of all three together is a characteristic of stem and progenitor cells within LinNeg bone marrow. Stem cell antigen-1 (Sca1) is expressed by slightly less than 10% of the LinNeg compartment. Sca1 is a glycosyl phosphatidalinositol-linked extracellular membrane protein of undetermined function, but its expression correlates with those cells capable of reconstituting hematopoiesis in vivo. Conversely, the roughly 90% of LinNeg cells that lack the Sca1 antigen are essentially devoid of in vivo hematopoietic potential (Spangrude and Brooks, 1993; van de Rijn, et ai., 1989; Hammmelburger, et ai., 1987). cKit is the receptor for the cytokine steel factor, and is a tyrosine kinase essential for many aspects of development. Homozygous null mutations of this gene are lethal at early stages of embryogenesis, while hypomorphic mutants exhibit macrocytic anemia. cKit is expressed to some degree on the majority of LinNeg cells that also express Sca1 (Sca1 POS); however, HSC have been shown by in vivo reconstitution assays to reside in the brightest cKit fraction of this ccmpartment (cKitBright; Besmmer, 1991; Rawls, et ai., 2001). The Sca1 Pos cKitBright subset includes a subset of cells that are positive for Thy 1 (Figure 1.2). This antigen is expressed by a broad range of mammalian species, including humans (Baum, et ai., 1992), but like Sca 1, its function is unknown. Thy 1 was first described as an early marker in thymocyte development, and is expressed abundantly by T cells; however, its expression in the stem cell compartment is more subdued. In mice homozygous for the Thy 1.1 allele of this gene, all HSC express this marker at low levels LinNeg Seal Pos Po lation Neg Low Thy!.! Expression )Ita Figure 1.2 Separation of the LinNcg Seal Pus population according to Thy1.l and cKit expression 7 (Thy 1.1 Low), but in other mouse strains, Thy 1 is expressed more randomly among primitive hematopoietic progenitors and expression does not completely correlate with HSC function (Spangrude & Brooks, 1992). Coordination of these three parameters, Sca 1 Pos, cKitBright, and Thy 1.1 Low, with 8 cell viability measured in a fourth, marks a specific compartment of LinNeg mouse bone marrow enriched with HSC to the point that as few as 100 cells can rescue a mouse from lethal irradiation. HSC frequency can be even further enriched by exploiting their metabolically quiescent state using fluorescent probes that target mitochondrial activity. Rhodamine 123 is one such dye that has been used effectively to separate a more primitive subset within the LinNeg Scal Pos cKitBright Thy l. 1 Low (Thy!. 1 LOW) population. Rhodamine 123 accumulates in actively respiring mitochondria; consequently, collecting the fraction of Thy 1.1 Low cells that retain the lowest levels of rhodamine 123 substantially enriches for HSC activity. As few as 25 of these RhoLow cells have been shown to effectively reconstitute all hematopoietic lineages when transplanted in competition with whole bone marrow grafts into lethally irradiated hosts (Spangrude & Johnson, 1990; Spangrude, et aI., 1995). Conventional concepts of the hematopoietic hierarchy Both rhodamine separation of HSC and their enrichment in the marrow of mice treated with 5-fluorouracil indicate that HSC are metabolically quiescent and rarely enter the cell cycle. These characteristics contrast starkly with the dramatic tum-over of cells during normal hematopoiesis. Furthermore, mature blood cells replicate only under special circumstances, such as in the context of lymphocyte activation. These observations suggest that the bulk of proliferation necessary to maintain homeostatic hematopoiesis is conducted by the vast pool of immature progenitors harbored in hematopoietic organs. This conclusion is supported by the observation that lymphopoiesis is reconstituted much nl0re rapidly by grafts of whole marrow than by parallel grafts containing an equivalent number of purified HSC (Szilvassy, et aI., 1996; Li, et aI., 1995). 9 Accordingly, current theories in this area propose progenitors generated by the HSC that are actively proliferating but decrease in lineage potential as they become more removed in cell divisions from the stem cell. It has also been proposed that potential lineage fates are restricted within a few divisions of the HSC to produce progenitors capable of repopulating either lymphoid lineages or myelo-erythroid lineages, but not both. This concept is supported by the fact that some gene families are expressed across one or the other of these branches, which would most simply be explained by a common point of control in a likewise common progenitor. For example, a common lymphoid progenitor has long been suspected based on the unusual practice of somatic recombination used to fornl the diverse repertoire of antigen receptors in Band T cells. This activity depends on the recombinase genes RAG-l and RAG-2 expressed in the progenitors of these lymphoid lineages, but not normally expressed in myelo-erythroid cell types (Huang & Muegge, 2001). This position has been further substantiated by the discovery in recent years of a 10 primitive hematopoietic population in adult murine bone marrow that can, from a single cell, generate both B and natural killer (NK) cells in culture, as well as T cells following intra-thymic transplant. This population was isolated using immunomagnetic, immunofluorescent, and flow cytometric techniques similar to those described above for HSC collection. Like stem cells, this population is LinNeg and Scal Pos, but expresses low as apposed to bright levels of cKit. Additionally ~ it is differentiated from HSC by expressing the interleukin-7 (IL 7) cytokine receptor alpha chain (lL 7RPos; Kondo, et ai., 1997). IL 7 is a growth factor critical to murine lymphopoiesis, and would therefore be predicted to be expressed on a progenitor common to lymphoid lineages. Conversely, IL 7 does not critically impact the development of myeloid lineages, and as expression of this marker suggests, this common lymphoid progenitor (CLP) population does not produce granulocytic lineages in either culture or transplant models (Huang & Muegge, 2001). Likewise, similar investigation of murine fetal liver has yielded the isolation of a progenitor capable of regenerating T and NK cells, but not myeloid cell types (Douagi, et ai., 2002), further supporting a common origin for lymphoid lineages. Merging of myeloid and lymphoid lineages In spite of this compelling evidence for an early separation in hematopoiesis between lymphoid and myeloid developlnent, many other lines of investigation suggest that this difference is not developmentally rigid. For example, extensive testing of hematopoietic progenitors from fetal liver has failed to produce a progenitor that can generate both T and B cells in the absence of myeloid progeny. These experiments were 11 conducted by co-culturing fetal liver cells with deoxyguanosine-treated fetal thymic lobes in an environment permissive to both lymphoid and myeloid development. Deoxyguanosine treatment is toxic to dividing cells and effectively conditions thymic lobes for colonization by nascent progenitors. Specifically, a deoxyguanosine-treated fetal thymic lobe was added to each well in a microtiter tissue culture plate with nutrient media containing growth factors permissive to both myeloid and lymphoid development, after which a single fetal liver progenitor cell was added to each well. Following an allotted time for development, the colonies that developed in each well were assayed by immunofluorescent staining and flow cytometric analysis for cells co-expressing a donor­specific CD45 isoform with antigens specific to B cells, T cells, or myeloid lineages. Assays of hundreds of such experimental tissue culture colonies resulted in the observation of single fetal liver progenitors producing B cells, thymocytes, granulocytes, and all combinations of these lineages, with the exception of restricted B cell and thymocyte development from the same progenitor (Kawamoto, et ai., 2000). The lack of clonal BIT readout from this system, in contrast to the studies which defined the adult CLP, may be due to the unnatural conditions present in tissue culture, as well as to differences between adult and fetal hematopoiesis. Nevertheless, both the lack of BIT progeny as well as the presence of BlMyeloid and T/Myeloid readouts indicate that the lymphoid and n1yeloid branches of hematopoiesis are not as rigidly separated as might have been once supposed. This conclusion is supported by the observation in both adult and fetal hematopoiesis of single, non-stem cell progenitors that can generate both B cells and 12 macrophages (Cumano, et aI., 1992; Kee, et aI., 1994; Montecino-Rodriguez, et aI., 2001). The position that lymphoid and myeloid lineage lines are often crossed by bone marrow progenitors is further supported by studies where the low-affinity IL2 receptor beta chain was transgenically expressed in IL 7RPoS CLPs. Since IL2 is a cytokine involved in T cell maturation and activation, it is surprising to note that the presence of IL2R-beta at the CLP stage promoted expression of myeloid-associated growth factor receptors. Furthermore, myeloid progeny including macrophages, subsequently developed from these otherwise "lymphoid-committed" progenitors (Kondo, et aI., 2000). Finally, yet further evidence of the interconnectedness between myeloid and lymphoid development during hematopoiesis comes from cultured progenitors lacking a functional Pax-5 allele. Pax-5 is an important gene in B cell development with several functions. Notably, it cooperates with c-Myb to activate transcription at the RAG-2 locus (Kishi, et ai., 2002). In the absence of Pax-5, lymphoid progenitors can progress in B cell development until somatic recombination of both alleles of the immunoglobulin heavy chain locus are DHJH rearranged. Several B cell-specific genes are also expressed at this point, including CD19, VpreB, and Lambda5. Nevertheless, without a functional Pax-5 allele, somatic recombination cannot be completed, and B-cell development arrests. As long as exogenous IL 7 is supplied, such progenitors remain in this arested state; however, if IL 7 is withdrawn, these progenitors proceed to differentiate into an array of non-B cell lineages, including progeny as diverse as T cells and osteoclasts, according to the growth factors and other environmental stimuli supplied (Rolink, et aI., 2000). 13 Lineage predisposition vs commitment Collectively, these observations indicate that the hematopoietic hierarchy is more complex than indicated by the branched tree diagrams typically used to describe it. Furthermore, they suggest that environmental stimuli such as growth factor signalling can provide a substantial instructive role during hematopoietic development, and are not entirely limited to permissively fostering a cell fate intrinsically determined within the progenitor. Nevertheless, it should also be noted that control experiments using B cell progenitors containing functional Pax-5 alleles remain restricted to the B cell lineage, and that CLPs not transgenically modified with IL2R-beta do not diverge into myeloid lineages. Therefore, in spite of the instructive roles that environn1ental stimuli might play, the genetic programming of hematopoietic progenitors as they diverge from the stem cell leaves them predisposed to assume certain cell fates over others. This might better be viewed as an iris controlling lineage decision that is wide open for the first few divisions as progenitors progress from the HSC, but, perhaps under the influence of environmental stimuli, gradually restricts to limit the window of lineage possibilities that may be normally assumed. This pattern of predisposition to certain lineages over others can also be seen in populations considerably more primitive than either the CLP or B cell progenitors mentioned here. For example, the HSC compartment defined by the Thy 1.1 Low selection described above is capable of reconstitution all hematopoietic lineages; nevertheless, it repopulates myeloid and erythroid compartments much more rapidly than lymphoid lines (Nib ley and Spangrude, 1998~ Sylvassy, et aI., 1996). Rhodamine separation has 14 indicated that this bias is due to the inclusion of rapidly cycling muitipotent progenitors that do not self-renew over extended periods of time (Nibley, et aI., 1997). This tendency towards myeloid development is not observed, however, if the HSC compartment is selected based simply on LinNeg , Sca 1 Pos, and cKitPOS criteria. Instead, this selection produces a population with engraftment kinetics where lymphoid reconstitution is not delayed, but the HSC activity is somewhat more dilute (Okada, et ai., 1993). The rapid reconstitution of lymphoid lineages suggests that selection based on cKit includes primitive progenitors with a predisposition for lymphoid development, as well as those progenitors with nlyeloid bias selected with the additional criteria of Thy 1.1 expression. Scope o/study Direct examination of the LinNeg Scal Pos population by immunofluorescent staining and flow cytometry for both Thy 1.1 and cKit expression showed that many cKitPOS progenitors were negative for Thy 1. 1 expression (Thyl.l Neg; Figure 1.2), indicating that this might be the fraction responsible for the rapid lymphoid engraftment properties of the LinNeg Sca1 Pos cKitPOS population. This study applies the separation techniques described above to test the hypothesis that the cKitPOS Thy 1.1 Neg fraction of LinNcg Sca 1 Pos adult mouse bone marrow contained the progenitors responsible for the observed acceleration in lymphoid engraftment relative to the Thy 1.1 Low -selected compartment. The data contained herein describe both the engraftment potential of this Thy 1.1 Neg population and its in vitro expansion potential under the influence of various 15 growth factors. The isolation of specific lymphoid progenitor populations included within the Thy 1.1 Neg population is also discussed. Findings from these experiments include the discovery of a B lymphocyte-committed progenitor more primitive than has yet been described in adult mouse bone marrow, as well as convincing evidence for a bone marrow pro-thymocyte and a multipotent progenitor that can extend the life of lethally irradiated mice, but not rescue them. Additionally, these data track rapidly engrafting lymphoid progenitors and show that within the LinNeg compartment, such activity is confined to the Thyl.l Neg population. Moreover, these data indicate discrepancies between in vitro and in vivo potentials of the progenitors examined, supporting the conclusion that environmental stimuli can provide an instructive role in hematopoietic development. Significance Lymphocytes are among the most critical players in eradicating viral infections. Cytomegalovirus and Epstein-Barr virus are normally benign infections in healthy individuals, but become life-threatening pathogens to patients undergoing conditioning and transplant to cure hematopoietic malignancies (Yoshikawa, 2001; Hsieh, et aI., 1999; Wingard, 1999). This may be due in part to the fact that lymphocytes, and especially T cells, are notoriously slow to recover following such treatment (Smith & Thomson, 1999). Enriching grafts for rapidly engrafting, robust lymphoid progenitors may be one mechanism to forestall the complications of infections posttransplant until a mature hematopoietic repertoire can emerge to address them. The studies undertaken in this 16 work describe both the earliest B cell progenitor yet reported, as well as show conclusive enrichment of pro-thymocyte activity from adult mouse bone marrow. As HSC were first isolated in mice before their discovery in humans, it is hoped that the advances described here may provide guidance in selecting homologous human populations. These findings may also assist in elucidation of the erroneous biochemistry that generates hematopoietic malignancies. These studies include a description of combined lymphoid and myeloid potential in some progenitors that may provide a reference of normal tissue to aid in the investigation of malignancies with multilineage characteristics. The B cell and pro-thymocyte populations described here may likewise provide similar references for investigating errors of proliferation and differentiation in other lymphoid leukemias. Beyond these medical considerations, this study clearly renders greater resolution to the classical concept of the hematopoietic hierarchy. Furthermore, these data imply some degree of lineage plasticity among normal progenitors in response to crisis hematopoietic conditions, and show the existence of several intermediates both down- and up- stream from the classically defined common lymphoid progenitor. References Abramson, S., Miller, R.G., and Phillips, R.A. (1977). The identification in adult bone marrow of pluripotent and restricted stem cells of the myeloid and lymphoid systems. J. Exp. Med. 145, 1567-1579. Baum, C.M., Weissman, LL., Tsukamoto, A.S., Buckle, A.M., and Peault, B. (1992) Isolation of a candidate human hematopoietic stem-cell population. Proc. Natl. Acad. Sci. USA 89, 2804-2808. 17 Besmer, P. (1991). The kit ligand encoded at the murine Steel locus: a plieotropic growth and differentiation factor. Blackett, N.M. and Adams, K. (1972). Cell proliferation and the action of cytotoxic agents on haemopoietic tissue. Br. J. Haematol. 23, 751-758. Bohne, F., Haas, R.1., Fliedner, T.M., and Fache, 1. (1970). 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Primitive stem cells alone mediate rapid marrow recovery and multilineage engraftment after transplantation. Bone Marrow Transplant 21, 345-354. 19 Okada, S., Nagayoshi, K., Nakauchi, H., Nishikawa, S., MMiura, Y., and Suda, T. (1993). Sequential analysis of hematopoietic reconstitution achieved by transplantation of hematopoietic stem cells. Blood 81, 1720-1725. Owen, J.1., and Jenkinson, E.1. (1992). Apoptosis and T-cell repertoire selection in the thymus. Ann. NY Accad. Sci. 663,305-310. Rawls, J.F., Mellgren, E.M., and Johnson, S.L. (2001). How the zebrafish gets its stripes. Dev. BioI. 240, 301-314. Rolink, A.G., Schaniel, C., Busslinger, M., Nutt, S.L., and Melchers, F. (2000). Fidelity and infidelity in commitment to B-Iymphocyte lineage development. Imnlunol. Rev. 175, 104-111. Smith, F.O. and Thomson, B. (1999). T-cell recovery following marrow transplant: experience with delayed lymphocyte infusions to accelerate immune recovery or treat infectious problems. Pediatr. Transplant 3, Suppl. 1 :59-64. Spangrude, G.J., and Brooks, D.M. (1992). Phenotypic analysis of mouse hematopoietic stem cells shows a Thy-I-negative subset. Blood 80,1957-1964. Spangrude, G.1., and Brooks, D.M. (1993). Mouse strain variability in the expression of the hematopoietic stem cell antigen Ly-6AJE by bone marrow cells. Blood 82, 3327- 3332. Spangrude, G.1., Brooks, D.M., and Tumas, D.B. (1995). Long-term repopUlation of irradiated mice with limiting numbers of purified hematopoietic stem cells: in vivo expansioin of stem cell phenotype but not function. Blood 85, 1006-1016. Spangrude, G.1., and Johnson, G.R. (1990). Resting and activated subsets of mouse multipotent hematopoietic stem cells. Proc. Nat!. Acad. Sci. USA 87, 7433-7437. Spangrude, G.J., Heimfeld, S., Weissman, LL. (1988). Purification and characterization of mouse hematopoietic stem cells. Science 241, 58-62. Sylvassy, S.1., Weller, K.P., Chen, B., Juttner, C.A., Tsukamoto, A., and Hoffman, R. (1996). Partially differentiated ex vivo expanded cells accelerate hematologic recovery in myeloablated mice transplanted with highly enriched long-term repopulating stem cells. Blood 88, 3642-3653. Tough, D.F., and Sprent, J. (1995). Lifespan of lymphocytes. Immunol. Res. 14, 1-12. 20 van de Rijn, M., Heimfeld, S., Spangrude, G.1., and Weissman, LL. (1989). Mouse hematopoietic stem-cell antigen Sca-l is a member of the Ly-6 antigen family. Proc. Natl. Acad. Sci. USA 86, 4634-4638. van Iperen, C.E., van de Wiel, A. de Bruin, M., and Marx, J.1. (2000). Total hip replacement surgery does not influence RBC survival. Transfusion 40,1235-1238. Wingard, J.R. (1999). Opportunistic infections after blood and marrow transplantation. Transpl. Infect. Dis. 1, 3-20. Yoshikawa, T. (2001). Human herpesvirus 6 infection in transplantation. Nagoya 1. Med. Sci. 64, 11-18. CHAPTER 2 RAPID, B LYMPHOID-RESTRICTED ENGRAFTMENT MEDIA TED BY A PRIMITIVE BONE MARROW SUBPOPULATION (The Journal of Immunology, 2000, 165: 67-74) 22 Rapid, B Lymphoid-Restricted Engraftment Mediated by a Primitive Bone Marrow Subpopulation1 A. Elena Searles,* Suzanne J. Pohlmann,t L. Jeanne Pierce,* S. Scott Perry,t William B. Slayton,:/: Mariluz P. Mojica,§ and Gerald J. Spangrude2*t Utilizing multiparameter Dow cytometry, we have defined a subset of bone marrow celIs containing lymphoid-restricted dUfer­entiation potential after i.v. transplantation. Bone marrow celIs characterized by expression of the Sea-I and c-/cit Ags and lacking Ags of dUferentiating lineages were segregated into subsets based on allele-specific Thy-l.l Ag expression. Although hematopOietic stem celIs were recovered in the Thy-l.llo .. subset as previously described, the Thy-I.I nel! subset consisted of progenitor cells that preferentiaUy reconstituted the B lymphocyte lineage after i.v. transplantation. Recipients of Thy_I.IDel! cells did not survive beyond 30 days, presumably due to the failure of erythroid and platelet lineages to recover after transplants. Thy_l.loeg cells predominantly reconstituted the bone marrow and peripheral blood of lethally irradiated recipients with B lineage ceUs within 2 weeks, although a low frequency of myeloid lineage celIs was also detected. In contrast, myeloid progenitors outnumbered lym­phoid progenitors when the Thy_1.lneR population was assayed in culture. When Thy_1.1IOw stem cells were rigorously excluded from the Thy-1.1nel! subset, reconstitution of T lymphocytes was rarely observed in peripheral blood after i.v. transplantatiou. Competitive repopulation studies showed that the B lymphoid reconstitutiou derived from Thy_1.1nes cells was not sustained over a 20-wk period. Therefore, the Thy.I.lDeIJ population defined in these studies includes transplantable, uon-self-renewiug B lym­phocyte progenitor ceUs. The Journal of Immunology, 2000, 165: 67-74. I n recent years, investigators studying hematopoietic stem cell (HSC)3 engraftment have shifted their focus toward defining the behavior of these cells shortly after transplant. Although the long-term reconstituting activity of HSCs in murine transplant models has been well documented, there is an increasing interest in better understanding the activity of stem and progenitor cell pop­ulations at early times after infusion of a graft. The transition in interest from long- to short-term engraftment in HSC transplanta­tion is partially the result of the need to promote rapid engraftment of functional blood cells in the first few weeks after clinical bone marrow (BM) transplantation. This has led investigators to begin to identify the BM eel! populations that contribute to and facilitate early engraftment and to investigate the intrinsic and extrinsic fac­tors that influence the kinetics of early engraftment after BM trans­plantation (1-4). Departments of ·Oncological Sciences. tpathology. tPediatrics. and }Hwnan Genet­ics. University of UIllh, Salt Lake City, UT 84132 Received for publication November 12. 1999. Accepted for publication April 12.2000. The costs of publication of this anicle were defrayed in part by the payment of page cbarges. TIris anicle must therefore be bereby marked advem.semem in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. lTIris work was supported by grants from the National Institutes of Health (ROI HL56857 and P50 DK492 19). The Flow Cytometry and Irradiation core facilities of the Huntsman Cancer Institute. supported by National Cancer Institute Cancer Center Support Grant P30 CA42014. were utilized for these studies. Address correspondence and reprint requests to Dr. Gerald Spangrude. 50 North Medical Drive. Room 5C334S0M, Salt Lake City. UT 84132. E-mail address: [e-mail] 3 Abbreviations used in this paper: HSC. hematopoietic stem cell; BM. bone marrow; CLP. common lymphoid progenitor: Lin. lineage; PI. propidium iodide; APC, allo­pbycocyanin; Lin""', BM cells depleted of cells expressing any of a panel of lineage­specific Ags; Thy·LI .. •. Sca-l+c-kit'"Lin""'1by-l.I oc , BM cells: Thy_l.l low • Sea­I'" c-Icit ~Lin""lI'Jby_I.I'oW BM cells; CFU-GM. colonies conlaining only granulocytes and macrophages; CFU·Mix, colonies containing both RBC and nucleated cells; CFU·preB. colonies grown under conditions supportive only for B lymphocyte dif­ferentiation; CFU-S. colony-fonning unit in spleen. Copyright © 2000 by The American Association of Immunologists Studies comparing the kinetics of hematopoietic recovery after transplantation of enriched HSCs, as compared with unfractionated BM containing the same number of HSCs, have revealed a marked difference in reconstitutive capabilities of these two populations early after transplantation. In an analysis of the engraftment kinet­ics of whole BM containing 1000 Sca-l "-Thy_l.llowH_2KW gb cells transplanted into irradiated recipients, Szilvassy et al. (5) showed that the unfractionated cells produced a gradual but steady increase in leukocytes through day 30. However, the transplantation of 1000 isolated Sca-I"-Thy-l.llowH-2KW gh cells potentiated a sharp and transient increase in leukocyte counts 15 days after transplantation. Leukocyte counts then drastically diminished and eventually reached a normal range around day 30. These studies suggested that regulatory interactions between HSC and other BM cells are lost after cellular enrichment before transplantation, a conclusion that is also supported in studies of allogeneic trans­plants (6). Similar studies using a primitive subset of HSC selected based on low retention of the mitochondrial probe rhodamine-123 also demonstrated an early, temporary spike in PBMC counts after transplantation (7). However, analysis of the cells comprising this recovery revealed that they were mainly monocytes and neutro­phils and further showed that recovery of the B lymphoid lineage was not observed until about 30 days posttransplant. These results demonstrated that the HSC population within the BM is not capable of rapidly providing early lymphoid reconstitution. Transplantation of purified populations of actively cycling rhodamine-123nigh multi­potent progenitor ceHs or of the more lineage-restricted Sca-\ neg progenitor cells revealed that neither possessed the capacity to sig­nificantly regenerate leukocytes (7-9). Thus, lymphocyte progen­itor activity observed early after transplantation must be derived from an as yet uncharacterized progenitor cel! population. Interestingly, Okada et al. (10) reported the early recovery (14-21 days) of myeloid and B lymphocytes in the blood after transplantation of highly enriched HSC that were selected based on 0022·1767/00/$02.00 68 expression of the c-kit molecule. This observation suggested that a cell population with different functional activities was isolated by this selection protocol compared with previous studies that had isolated HSC based on low levels of Thy- I.l expression (11). We repon here a series of experiments aimed at testing this hypothesis and show that the addition of c-kit expression to the criteria used in previous studies to isolate HSC allows distinction of a commit­ted progenitor population that predominantly generates B lineage cells within 2 wk of i. v. transplantation. By phenotype and func­tion, these progenitors represent a cell population closely related to the common lymphoid progenitor (CLP) recently identified by Kondo et al. (12). However, the lack of selection for the IL-7R in the present studies resulted in significant myeloid lineage potential as well as lymphoid potential in the isolated progenitor population. Interestingly, the use of different fluorochrome conjugates of the c-kit Ab led to variable results, suggesting that fluorochrome con­jugates used to isolate particular cell populations can influence the in vivo reconstitutive capabilities of cells to which they bind. Materials and Methods Mice BM donor animals were 4- to 8-wk-old B6-Thy-I.I-Ly-S.1 or B6.PL (Thy­I.!. Ly-S.2) congenic mice. C57BU6 (Thy-I.::!. Ly-5.2) or B6.SJL (Thy­I.::!, Ly-S.I) mice between 8 and 10 wk old served as transplant recipients. Animals were bred and maintained at the Animal Resource Facility at the University of Utah (Salt Lake City, UT) or were purchased from The Jack­son Laboratory (Bar Harbor. ME). All animals were maintained on auto­c1aved, acidified water (pH 2.5) and autoclaved chow. Harvest and preparation of 8M cells for cell ,wning BM was harvested from both femora and tibiae of donor mice. The bones were crushed with a mortar and pestle in HBSS containing 5% FCS and JO mM HEPES buffer (HBSSI5). After filtering the cells through nylon mesh (pore size. 85 p.m; Small Pans. Miami Lakes, FL). cells were washed once with HBSS/5 and centrifuged (1:;;00 rpm at 4°C) for S min. The resus­pended pellet was treated with ammonium chloride-potassium solution for 2-3 min to lyse RBCs and then was washed again with HBSS/5. To remove mature blood cells from the BM and thereby enrich for rare stem and progenitor cell populations. BM cells were reacted with a lineage (Lin) cocktail containing optimized concentrations of eight mAbs. These mAbs recognize Ags associated with differentiated blood cells and include CD2 (RM-2.2), CD3 (KT3-l.I), CDS (53-7.3), CD8 (53-6.7). Mac-I (MlnO). TER-119 (an erythroid Ag), Gr-l (RB6-8C5), and B220 (RA3-6B2). Cells were incubated with Lin cocktail at a cell density of 5 X 107 ceUslm! for 20 min. After the incubation period, cells were washed with HBSSI5 and subsequently incubated with sheep anti-rat [g-coupled magnetic beads (Dy­nal, Oslo. Norway). The incubation was perfonned in a volume of I mI at a bead-to-cell ratio of I: I with intermittent mixing over a 20-min period. HBSS/5 was then added to bring the volume to 8 ml before magnetic depletion. This depletion process was repeated. and the remaining cells were then incubated with Ly-6NE (Sca-1. clone 07) mAb conjugated to the fluorochrome PE (PharMingen. San Diego. CA) for 20 min on ice. After a wash, cells were resuspended in HBSSI5 containing 10 p.glm! propidium iodide (PI; Molecular Probes. Eugene. OR) and filtered through nylon mesh. Flow cytometry Cells were sorted for Sca-I"PIDeg cells using a FACS-Vantage instrument (Becton Dickinson. San Jose. CAl in enrich mode. using a threshold on PE staining as a trigger. Sorted cells were recovered by centrifugation and stained with anti-Thy-I.l (clone 19XE5. conjugated in our laboratory to Oregon Green. Molecular Probes). PE-Sca-!. and one of two different anti­c- kit reagents. These were either a biotin conjugate (clone ACK4 (13), kindly provided by SA. Nisrukawa (Kyoto University. Kyoto. Japan) and biotinylated in our laboratory using standard techniques) or an aUophyco­cyanin (APC) conjugate (clone 2B8; PharMingen). The biotin-c-kit reagent was detected with streptavidin-RED6I3 (Life Technologies, Gaithersburg, MD). After staining, the cells were washed and resuspended in HBSS/S containing either To-Pro-2 (Molecular Probes) or PI for dead cell exclu­sion. Cell populations were then sorted using forward scatter triggering in normal recovery mode for Sca-l + c-kit + cells, which were further segre­gated into Thy-l.I Deg and Thy_l.l luw subsets (Fig. I). Hereafter, the terms 23 TRANSPLANTABLE B LYMPHOCYTE PROGENITORS "Thy-I.! neg" and "Thy_l.lIOW .. are used to indicate Linucg populations that are coselected for the Sca-I ... c-kit'" phenotype in addition to the presence or absence of Thy-l.1. as shown in Fig. I. Two different c-kit reagents were utilized during the course of these studies (Fig. I). Although the APC-c-kit conjugate gave superior staining because of a higher signal. cell sorting experiments showed that the degree of engraftment was minimal when Thy-!.I Del populations isolated using the APC-c-kit reagent were transplanted by the i.v. route. In contrast. the use of the biotin-c-kit conjugate for sons resulted in reproducible recovery of transplantable lymphocyte progenitors in the Thy-I.I oei population. Be­cause in vitro culture assays did not demonstrate a difference in clorung efficiency or differentiation potential between cell popUlations isolated us­ing the different Ab conjugates (data not shown), the engmftment defect of Thy-I.I Deg cells isolated using the APC reagent may be due to interference of in vivo homing by the bulky APC protein. Interestingly. Thy_I.I!OW HSC preparations isolated using the two c-kit reagents were equally functional after in vivo transplants. suggesting that the inhibitory effect of the APC­c- kit conjugate was specific for the Thy-I.I neg progenitor cell population. Transplantation of sorted cell populations All recipient mice were exposed to 13 cGy of radiation from a 137 Cs source (Mark I gamma irradiator; J. L. Shepherd & Associates. Glendale, CA) delivered in two equal doses sepanlted by a 3-h delay. Several hours later. mice were anesthetized using methoxyflurane and were administered pu­rified cells in 0.2 mI HBSSI5 by retroorbital injection. Transplant recipients differed from donors at the Ly-5 locus. For competitive repopulation stud­ies, transplants included 10> syngeneic BM cells in addition to purified congenic cells. Recipient animals were maintained on oral neomycin sul­fate (BiosoI. :; mglm!; Upjohn. Kalamazoo, MI) for:; wk after irradiation and transplantation. Analysis of transplant recipiems Engrafrment was evaluated by immunofluorescent staining for the Ly-5 allelic Ag on PBMC. BM. and spleen cells at times ranging from 7 to 140 days after the transplants. For PBMC analysis. ISO p.1 of blood was col­lected from the retroorbital sinus into a tube containing :!O p.1 citrate dex­trose anticoagulant (Fonnula A; Baxter Health Care. Mundelein. IL) using heparinized capillary tubes (Fisher Scientific, Pittsburgh. PAl. A portion of each blood sample was removed to obtain complete blood counts using an automated hematology analyzer (Serono System 9010; Serono Diagnos­tics. Allentown. PAl. The remainder of the blood was added to a volume of 2% Dextran T500 (phannacia Biotech. Piscataway. NJ) in PBS. This mixture was incubated for 30 min in a 37°C water bath to allow for sed­imentation of RBCs. After incubation. the upper layer of PBMC was re­moved from each sample and centrifuged for 5 min at 1200 rpm. The pellet was resuspended in ammonium chloride-potassium solution to lyse resid­ual RBCs and, after a wash in HBSS/S. each sample was aliquoted into three wells of a 96 .. well microtiter plate. The plate was then centrifuged for 2 min and decanted. and immunofluorescent staining was perfonned by reacting cells at a density of S X 107 cellslm! with satur&ing solutions of mAb to define and phenotype donor-derived cells based on the allelic dif­ference at the Ly-5 locus. T lymphocyte. B lymphocyte. and myeloid lin­eages of donor and recipient origin were measured using fluorescein­Ly- S.I staining in combination with CD4 and CD8 (T lineage). B220 (B lineage). or Mac-l and Gr .. 1 (myeloid lineages) mAbs as PE conjugates. Animal groups were set up in duplicate so that no animal was bled more frequently than once per week. BM and spleen tissues were isolated and single-cell suspensions were made as previously described (7). After RBC lysiS, cell counts were de­termined and cells were reacted with mAb to define and phenotype cells of donor and recipient origin as described above. Colony-forming assays CFU content of freshly isolated Thy- U 10w HSC and Thy-l.l neg progeni­tors or of BM tissue after transplantation of these cells was determined by methylcellulose colony assays. Primary CFU activity was evaluated by plating 5 X 104 normal BM cells or I X 102 Thy_1.l 10w HSC or Thy .. l.l neg progenitors per 35-mm culture plate. Methylcellulose medium consisted of (l-MEM (Life Technologies), 1.2% methylcellulose (Shinetsu, Tokyo. Ja­pan). 30% FCS (Life Technologies). I % deionized BSA (Sigma. St. Louis. MO). and 0.1 mM 2-ME (Mallinckrodt Chemical. Chesterfield, MO). My­eloerythroid cultures (CFU .. GM and CFU-Mix) were supplemented with the recombinant cytokines IL-3 (10 nglm!; Peprotech, Rocky Hill. NJ). IL-6 (20 nglml; Peprotech), G-CSF (10 nglml; Amgen, Thousand Oaks, CA). and erythropoietin (5 UlmI; Ortho Pharmaceuticals. Raritan, NJ). For analysis of secondary CFU activity, lethally irradiated transplant recipients were euthanized 13 days after transplant of 103 cells. and BM was isolated. The Journal of Immunology Thy-l.1 Thy-l.1 FIGURE 1. Four·color analyses of mouse BM cells immunomagneti­cally depleted of mature cells based on expression of CD2. CD3. CD5. COg. CDI9. B220. Gr·l. and TER-II9 and gated to include only Sca-l + cells. These Sea· I + Lin""g cells are displayed with respect to c·kit and Thy- 1.1 expression. Viability probes used were PI in combination with APC and To·Pro-2 in combination with RED613. Thy·l.l""g and Thy_l.llow cell populations were isolated using gates corresponding to the upper left and upper right quadrants. respectively, of plot A. Secondary CFU activity was stimulated with IL-3. in the form of :;0% WEHI-3B conditioned medium. plus recombinant human erythropOietin (5 U/rnl; Ortho Pharmaceuticals). Cells were plated at two densities (5 X 104/ml and 5 X IOS/ml) in 35-mm culture plates. Ali cultures were incu­bated at 37°C under 5% CO2, and colonies were enumerated 7-10 days later. CFU·Mix were identified as red colonies by visual inspection or after benzidine staining of hemoglobin. Cultures selective for growth of B lym­phocyte progenitors (CFU-preB) contained steel factor (100 ng/rnl. a kind gjft from Kinn Pharmaceuticals. Tokyo. Japan). Flt3 ligand (75 nglml. kindly provided by lmmunex. Seattle. WA). and IL·7 (10 ngJrnl; Pepro­tech). Colonies were counted after 8 days. Results Definition of two subsets of c-kit+ hematopoietic progenitor cells Previous studies have established that the Sca-l Ag is expressed by virtually all HSC in the BM of adult B6 mice (8, 9). Furthermore, HSC in adult mouse bone marrow express the Thy-I Ag in an allele-specific manner, such that in mouse strains expressing Thy- 1.1 but not Thy-1.2, HSC are entirely contained within the Thy­llow subset of BM cells (14). The tyrosine kinase receptor c-kit is a third Ag characteristic of mouse HSC (13). However, the B lym­phoid lineage has been reported to engraft within 2 weeks after transplantation of BM progenitors selected on the basis of dual expression of Sca-l and c-kit (10). In contrast, BM progenitors selected on the basis of Sca-l and Thy-l.l expression require 4- wk to regenerate B lymphocytes in the peripheral blood (7). To ad­dress this discrepancy, we evaluated the coexpression of Thy-l.l and c-kit among Sea-l TLinne !! BM cells. As shown in Fig. 1, c-ki('-Sca·l-Linneg BM cells were subdivided into two popula­tions of approximately equal frequency by Thy-l.l staining. Fur· thermore, cells expressing Thy-I. I appeared relatively homoge­neous for a high level of c-kit expression, whereas those not expressing Thy-l.l included both c-kifoW and c-kirugh subsets (Fig. lB). Although equivalent staining patterns and subset fre­quencies were observed with two different anti-c-kit reagents, an APC-conjugated reagent gave brighter staining and superior reso­lution of the different levels of c-kit expression compared with a biotin-conjugated reagent that was detected with streptavidin­RED613 (compare Fig. 1, A and B). However, cells selected as C-kiI +Thy·l.l neg using the APC conjugate consistently failed to engraft in irradiated recipient animals, whereas the same popula­tion selected using the biotin-avidin combination resulted in en­graftment after transplantation. Interestingly, the engraftment de­fect observed when Thy- Ll nc .. populations were selected using APC·c-kit was not observed when Thy_l.l low c_kil'igh HSC were e ID- ,e.. . )( (.) :s Dl a.. E al-e )( (.) III a: al~ e )( -(I) G.I '-ii CIS c: 25 A 20 15 10 5 0 0 12 6 4 2 0 1.5 1.0 0.5 0.0 0 10 20 I e Thy-1.1 neg o Thy-1.1 Iow 10 20 30 40 50 Days Post-Transplant 24 69 60 60 60 FIGURE 2. Recovery of PBMC (A), RBCs (B). and platelets (C) after transplantation of Thy-I.I neg progenitors (e) or Thy_I.IIOW HSC (0) into lethally irradiated recipiem mice. The cell populations were enriched as shown in Fig. I. selecting for c-kit+ Sca-! ~ cells from lineage-depleted BM and separating these into two subsets based on expression of the Thy-I.1 Ag. The two cell populations were transplanted at a dose of 5000 cells per mouse, and cell counts were performed using a Serono hematology counter. No individual mouse was bled more frequently than once weekly. Data from three separate experiments were pooled and are depicted as mean counts :!: SEM. In some cases. error bars were too small to plot. isolated in the same sorts and transplanted into irradiated recipients (data not shown). Consequently, in this paper we repon only the transplant experiments performed using the avidin-biotin selection protocol. Thy· I. ]""8 and Thy_l.llow cell populations differentially reconstitute blood lineages in irradiated animals After transplantation of 5000 BM c-ki("Sca-l""Linncg cells, se­lected as either Thy-l.l neg or Thy_l.llow, into lethally irradiated recipient animals, we evaluated the recovery of peripheral blood cellularity over time (Fig. 2). In both transplant groups, PBMC counts were below the level of detection «0.2 X 106/mI) until 12 days after transplantation. Beginning at day 14, recipients of the Thy- L1 neg subset exhibited highly variable PBMC counts, which in some cases exceeded the values observed in samples obtained from normal animals by severalfold (Fig. 2A). The variability in PBMC counts was to some extent due to the automated hematol­ogy counter detecting clumps of activated platelets or circulating normoblasts in the PBMC window, because flow cytometric anal­ysis of the same samples showed few PBMe. In other cases, flow 70 ..0. .,.----,.------, A ... g B co .. Donor Ly-5 Allele --+- FIGURE 3. F!ow cytometric analysis of PBMC recovery 14 days after transplantation of 5000 Thy_l.l 1oW HSC or 5000 Thy-I.! IlCg progenitor cells. A and B, Analysis of a periphenu blood sample from :l recipient of Thy-I.! n"~ cells. C and D. Analysis of a peripheral blood sample from a recipient of Thy_l.llow cells. In each case. the graft -derived cells are de­tected by Ly-5 staining and are evaluated as lymphoid lineage by 8220 staining (A and C) or as myeloid by Mac-lIGr-J staining (B and D). In each case. data representing the entire peripheral blood sample was collected by flow cytometry, so the density of dots is proportional to the PBMC count. cytometry confinued high PBMC counts, and phenotypic analysis showed that the majority of circulating PBMC were of the B lym­phoid lineage based on a B220+ Mac-lIGr-lneg phenotype (Fig. 3, A and B). In contrast to recipients of Thy-I.l neg cells, animals transplanted with Thy_l.llOW cells exhibited a gradual and very consistent increase in PBMC counts beginning at day 12 and reaching near-nonnallevels by day 30 (Fig. 2A). Phenotypic anal­ysis demonstrated that the majority of cells appearing in the pe­ripheral bLood early after transplantation were of the myeLoid lin­eage (Fig. 3, C and D). In one representative experiment, groups of four animals transplanted with either Thy-l.l neg or Thy_l.llow cells were evaluated for the frequency of B lymphoid and myeloid cells in peripheral blood 14 days after transplantation. In recipients of Thy-Uncg grafts. donor-derived cells in the peripheral blood consisted of (mean ::!: SO) 87.1 ::!: 3.5% B lineage cells and 12.8 :!:: 3.5% myeloid cells. In contrast, recipients of Thy_l.llow HSC grafts included 16.3 ::!: 6.8% B Lineage cells and 83.3 :!:: 6.9% myeloid cells among donor-derived peripheral blood cells. In the particular experiment from which the representative phenotypic analysis shown in Fig. 3 was derived, the average PBMC count at 14 days was 2-fold higher in recipients of Thy_l.lnc!! cells com­pared with recipients of Thy_l.llow cells (Fig. 24). In absolute numbers, the circulating B lineage cells ranged from 1.3 to 6.7 X 106 ceIlslml in recipients of Thy-l.I ncg grafts and from 0.1 to 0.4 X 106 ceIlslml in recipients of Thy_l.llow grafts at day 14. - 25 TRANSPLANTABLE B LYMPHOCYTE PROGENITORS All recipients of Thy_1.110W HSC grafts survived lethal irradia­tion, whereas 31 of 35 recipients of Thy-l.l neg cells died of he­matopoietic failure between days 25 and 30. The four surviving mice were observed in a single experiment in which contamination of the Thy-l.l neg cell preparation with Thy_l.llow HSC was ap­parent on reanalysis of the sorted cells; these animals are excluded from the data shown in Fig. 2. The contamination of Thy_l.llow cells in the Thy-l.l'leg population likely accounts for the survival of the animals, because even a 5% contamination among 5000 total cells will result in a sufficient number (250) of Thy_l.l10W HSC to mediate engraftment (11). Therefore. the use of an optimized anti­Thy- l.l reagent and rigorous flow cytometric separation with re­analysis of the two isolated cell populations was critical in sepa­rating progenitor cells from HSC for these experiments. The absence of hematopoietic recovery in the erythroid and platelet lineages in recipients of Thy-l.l m'l! grafts is shown in Fig. 2, Band C. Suppression of both lineages was apparent in the first week after radiation conditioning, regardless of whether Thy- 1.1 neg or Thy_l.llow cells were transplanted. The earliest recovery of erythropoiesis after highly enriched HSC grafts has been re­ported to occur at about day 12 (5, 7), and the results shown in Fig. 2B are consistent with this finding. Irradiated recipients of Thy­l. llow HSC exhibited prompt erythroid recovery that was main­tained throughout the 60-day course of study with a mild degree of anemia. In contrast, animals transplanted with Thy-l.l neg cells ex­hibited no erythroid recovery. and by 22 days, RBC counts were 3.33 :!:: 0.95 X 109/ml (mean::!: SO; n 7) compared with the nonnal value of 9.79 :!:: 0.7 X 109/ml (mean::!: SO; n = 22). Platelet counts reached a deep nadir in both experimental groups II days posttransplant, but by 14 days, recipients of Thy_l.llow HSC showed evidence of platelet engraftment and platelet counts were within the normal range (1.013 ::!: 0.339 X 109/ml; mean :!:: SO. n 22) by 26 days posttransplant. Recipients of Thy-l.l neg cells exhibited no evidence of platelet engraftment before death (Fig.2C). Differential BM engraftment by subsets of cells defined by Thy-i.] expression To test whether preferential engraftment of the B lymphocyte lin­eage by Thy-l.t neg cells was due to the reconstitution of the BM with B lineage progenitors, we evaluated recovery of BM 13 days after transplantation of 103 purified cells. Although equivalent numbers of cells (5.5 X 106 cellslfemur) were recovered from transplant recipients of either Thy_l.l1ow or Thy-l.l neg cells, the phenotypic and functional composition of the BM was dramati­cally different. As shown in Fig. 4, the frequencies of the lymphoid and myeloid components of BM obtained from recipients of Thy- 1.1 1ow HSC closely resembled those observed in BM obtained from normal, untreated mice. In contrast, the balance between lym­phoid and myeloid differentiation in the BM samples from recip­ients of Thy-l.l neg progenitors was heavily skewed toward the FIGURE 4. Flow cytometric analysis of cellular recovery in the BM after trans­plantation of 5000 Thy-I, llow HSC or 5000 Thy-l.l IIC, progenitor cells. BM cells collected 13 days posttransplant were evaluated for expression of the 8 lineage Ag B220 correlated with the myeloid Ags Mac-l and Gr-!. Bone marrow cells de­rived from a normal animal were pro­cessed in parallel as a controL 34% Normal 8M 29% Thy_1.110W BM 72"t ... : . Thy-1.1 n419 BM : "C - '0 .s::. Co - E >- -' 2 - The Journal of Immunology Table I. Primary and secondnry colony1orming pOlential of Thy-l.llo", and Thy-I.}""/( subsets Colony Type Primary colony­fonning activity per 104 cells" CFU-GMb CFU-Mix CFU-preB Secondary colony-fonning activity per 106 BM cellsc CFU-GM'/ CFU-Mix NormalBM 9.7 0.9 1.5 ::!: 0.3 0.75 0.25 5.600::: 380 690::: 90 880::: 220 564::: 96 4.5::: 1..2 7.3 L3 1.450::: 340 50::: 20 525::: 130 ::: 1.7' oj ., Normal SM. Thy. 1.I iO_. and Thy·UU"' cells were freshly obtained from mice and cultured in methylcellulose containing cytokines selective for myeloerythroid or lymphoid progenitor cell growth. Colony growth was scored after 7-10 days and is expressed as the mean :': SEM of the number of colonies per 104 celts plated (n 4-12 methylcellulose cultures). h CFU.GM and CFU·Mix colonies were stimulated with steel factor. lL-3. IL-6. G-CSF. and erythropoietin. CFU-Mix colonies were defined by visible red coloring or by benzidine staining of hemoglobin. CFU-preB colonies were sumulated with steel factor. FIG-ligand. and IL-7. , BM harvested 13 days after transpla.mation of 10J Thy-I.I low or Thy-I. I n<~ cells into lethally irradiated recipients was cultured to determine colony-forming potential. Colony growth was scored after 7-10 days and is expressed as the mean :': SEM of the number of ,olonies per 100 celts plated in each of four methylcellulose cultures. d CFU.GM and CFU-Mix colonies were stimulated using 20% WEHI·3B super­natant :l5 a source of lL·3 and erythropoietin. , Microcoionies; much smaller than typical CFU·GM, f No colonies detected in four cultures seeded with 5.6 x lOs cells each, lymphoid lineage, conSisting largely of lymphoid cells and includ­ing very few cells expressing myeloid Ags (Fig. 4). Consistent with this finding, spleens harvested from the recipient mice re­vealed 3.0 ::!: \.4 splenic colony-forming units (CFU-S) per 100 Thy_l.llow cells transplanted but fewer than 0.2 CFU-S per 100 Thy-i.l neg cells. Primary and secondary colony-fvnning potential vf Thy-l.r,·g and Thy- 1. llow cells To evaluate the colony-forming potential of Thy-I.l n .. g and Thy­l. l loW cells, methylcellulose assays were performed. The results of primary cultures of normal BM cells and each Thy-l.l subset are shown in Table L Both subsets included a high frequency of my­eloid progenitors compared with unfractionated BM. However, Thy- Ll low HSC contained .. Hold more CFU-GM and 14-fold more CFU-Mix relative to Thy-l.l Deg progenitor cells. The fre­quency of CFU-preB was approximately equal in the two subsets and was enriched 700- to lOOO-fold compared with normal BM (Table i). To evaluate the recovery of progenitors for the hematopoietic lineages after transplantation, secondary colony-fOrming assays were preformed. BM cells were isolated 13 days after transplants of 103 cells of each Thy-I.I subset and were cultured in colony assays. As shown in Table I. BM obtained from recipienls of Thy­l. llow cells contained progenitor cells capable of growing in con­ditions supporting either myeloid or lymphoid differentiation. The frequency of colonies containing granulocytes and macrophages in the absence of erythroid development (CFU -GM) was slInilar to that observed in cultures of normal BM cells. and myeloid colonies containing erythroid differentiation (CFU -Mix) were also observed at a low frequency (4.5 CFU-Mix per 106 cells compared with 1.5 CFU-Mix per [Q4 cells in primary colony-forming assays). Colo­nies capable of growth under conditions supporting only B lym- 20 Weeks Post-Transplant 26 71 FIGURE 5. Recovery of peripheral blood platelet (A) and PBMC (8) counts after transplantation of 10] TI1Y-1.1 neg or Thy·l.I low HSC in the presence of a competlIlg dose of 105 nonna! BM cells. phoid progenitors (CFU-preB \ were observed at a IO-fold lower frequency in cultures initlated from BM of Thy_l.llow transplant recipients compared with normal BM. In contrast the frequency of CFU-GM in cultures initiated from BM obtained from Thy-l.l neg transplant recipients was IOO-fold lower than that in control 8M cultures. and no erythroid or lymphoid colonies were detected. CollectivelY, the results shown in Figs. 2-4 and Table I suggest that although the Thy-l.1 neg subset includes both myeloid and lymphOId progenitors. the cells capable of hOming to the BM after i.v. transplantation are predominantly committed to differentiation along the lymphoid lineages and fail to regenerate significant num­bers of additional progenitor cells. T lineage progenitors are rare among Thy-l.J"~g cells and self-renewal is limited The failure to observe CFU-preB in BM of mice transplanted with Thy-i.l neg cells suggests that these cells reconstitute the B lineage progenitor compartment in the BM with non-self-renewing pro­genitors after transplantation. To further evaluate the potential of Thy-l.l nell cells to contribute to the lymphoid lineages, a compet­itive repopulation experiment was performed. Because Thy-i.1 neg cells were incapable of providing protection from letha! doses of radiation. cotransplantation of L05 normal BM cells of Ly-5.2 Of­igin along with L03 cells of either the Thy-I.l neg or Thy-l.1 low subsets (Ly-5.l) was performed into lethally irradiated Ly-5.2 re­cipient mice. The contribution of each Thy-I. I-defined population to peripheral biood lineages was then evaluated over 20 wk. using the Ly-5 allele to distinguish progeny of tht: BM cells from those of the Thy-1.1 subsets. The differential contribution of the Thy- t.1 neg and Thy_l.llow subsets to the platelet lineage was again c\eariy apparent in these experiments. despite the cotransplanted BM (Fig. 5A). Compared with recipients of Thy- \.l low cells, recipients of Thy_l.ln";; cells exhibited consistently lower platelet counts but equivalent PBMC counts throughout the 20-wk study period (Fig. 5B). This is con­sistent with the data shown in Fig. 2. indicating that Thy_I.I!OW but not Thy-l.l neg cells contribute to reconstitution of the platelet lin­eage. Therefore. the slow platelet recovery observed in recipients of Thy-l.l neg cells in the competitive transplant assay (Fig. SA) is due solely to the cotransplanted syngeneic BM, whereas the more rapid platelet recovery seen after transplantation of Thy -\. r low cells represents tht: aggregate contributions of both the cotrans­planted BM and the Thy- i .llow HSC. The relative contributions of 27 72 TRANSPLANTABLE B LYMPHOCYTE PROGENITORS 100 100 100 ~ A B FIGURE 6. After troillSplantation of IOJ Thy­U Del or Thy-Li low HSC in lhe presence of a com­peting dose of 105 normal BM cells. engraftment of nucleated cells derived from each population was evaluated in lhe peripheral blood using Ly-5 staining in combination with Ags associated with T lymphoid (A). B lymphoid (8). and myeLoid (C) lineage::;, Ab­solute numbers of cells were calculated using PBMC counts performed at each timepoinL The limit of de­tection was defined as 0.5% positive cells at a PBMC count of 106 ceLlsirnl. which' corresponds to 5 x !O3 donor cells/rnl (hatched area in A). Donar-derived T cells were not detected above this level in six of seven recipients of Thy-I.! neg cells. See text for detmls. e 11)-'0 ;! ~ ~ aI / ~ II) ;!10 )( G) OJ ('II ~ lC ~ 1 :10~ l- t -! CcII r I 1I -a- Thy-nag ! :::i 1-0.1 0.01 purified cells vs cotransplanted BM to the platelet lineage were not directly assessed in these experiments because of the lack of Ly-5 expression by platelets. Contributions to the T lymphoid. B lymphoid, and myeloid lin­eages by Thy-I.! neg and Thy-I. i low cells are shown in Fig. 6. In six of seven mice transplanted with Thy-l.l neg cells, T lympho­cvtes were not observed above the threshold of detection (0.5% of PBMC, or 5 X 103 cells/ml). One mouse of seven showed T ceil engraftment, which reached a total of 2 x 105 congenic T cells/ml by week 20. This animal also demonstrated about :2 X 105 con­genic myelocytes and B220'" cells/ml in the peripheral blood. We have excluded this animal from the data shown in Fig. 6 because we cannot exclude the possibility of engraftment by a low level of contaminating Thy_l.llow HSC contained in the Thy-!.l neg cell preparation. Engraftment in this animal may also have been due to the presence of a minor subpopulation of Thy -1.1 "eg cells capable of T cell development. In contrast to the absence of T cell engrafunent in the majority of recipients of Thy_l.lueg cells, all animals transplanted with Thy­I. l lnw HSC demonstrated robust, long-term T cell engraftment (Fig. 6A). Animals in both transplant groups exhibited equivalent engraftment of the B lymphoid lineage at 4 wk posttransplant (Fig. 68). ALthough the absolute number of B220'" cells in both trans­plant groups increased over the next 4 wk, the recipients of Thy­l. llow HSC contained about 20-fold more of these cells per ml of blood at 8 wk compared with recipients of Thy-l.l neg progenitors. Between 8 and 20 wk posttransplant. the number of B lineage cells continued to increase in Thy-l.llow recipients but declined in re­cipients of Thy-l.l neg cells. Myeloid engraftment was high at all times posttransplant of Thy-I.t low HSC but was represented at 50- to 100-fold lower levels in recipients of Thy-I.I neg progenitors (Fig.6C). Discussion Although a variety of studies have documented commitment to the 8 lymphoid lineage and have traced lineage relationShips based on expression patterns of cell surt'ace molecules (15, 16), relatively few studies have shown activity of comnutted B lineage progen­iLors in transplant experiments (17, 18). Due to the high degree of proliferative potential inherent in uncommitted hematopoietic stem cells, clonal transplant assays can be utilized to demonstrate mul­ulineage engraftment in vi vo from singie cells (19 -21). in con­trast, lineage-specific progenitor cells Lack extensive proliferative potential (22). Because of this, lineage-restricted reconstituting ac­tivity of hematopoietic progenitor cells has been difficult to dem­onstrate in transplant models. A critical considera[ion in experiments such as those reported in this study is that putative progenitor popuiations must be isolated ! :J t ! 'I --- Thy-low I lEI J I 0.11 0.1 ! 20 4 20 20 Weeks Post-Transplant in the absence of HSC to demonstrate lineage restriction in non­clonal models. The high proliferative potential of HSC will kad to long-term reconstitution even when a low level ot' stem cell con­tamination is present. If early commitment events lead to a re­stricted representation of lineages in a clone derived from a HSC the results can be erroneously interpreted as engraftment of a lin­eage- restricted progenitor. Immunodeficient mouse strains such as severe combined immunodeficiency mutants or recombinase knockouts have been exploited as animal models permissive for lymphoid engraftment in transplant experiments (12, 23, 24). However, the use of immunodeficient mouse strains as transplant recipients may preferentially select for lymphoid engrafunent be­cause the nonlymphoid lineages in these animals are normal. This may lead to underestimates of the myeloid and erythroid potentials of transplanted cell populations. In support of this concept. immu­nodeficient patients who are treated with allogeneic bone marrow transplants often demonstrate mixed chimerism that results in do­nor- derived lymphoid but not myeloid reconstitution (25, 26). These considerations must be taken into account in transplant stud­ies using early hematopoIetic progenitors. The studies reported in this paper document a transplantable progenitor with restricted lineage potential. The cell population described in these studies has a pattern of surface Ag expression that is very similar to that of HSC, with the exception being a difference in expression of Thy-l.l. Elsewhere we have shown that mouse HSC invariably express Thy-l.l (14), and it is this lack of HSC contamination among Thy-I.I neg cells that results in the abil­ity to completely separate the lymphoid progenitor population from multipotent HSC. This separation will not work in mouse strains expressing the Thy-1.2 alleie because about 50% of the HSC in these strains lack Thy-l expression. Contamination of Thy_l.lneg cell preparations by Thy-l.l 10"" HSC was noted by sort reanalysis and by functional studies in some of our experiments, underscoring the importance of carefully controiled cell sorting techlllques in hematopoietic progenitor separations. The Thy-l.l neg progenitor cell population we have character­ized largely overlaps with a CLP cell subset previously isolated on the basis of expression of IL-7R (12). In those studies, CLP were selected by a Linne!! phenotype in combination with low levels of c-kiz and Sca-l expression as well as IL-7R expressIon. The ma­jority of these cells, which comprised 0.02% of 8M, lacked ex­pression of Thy-I.!. The selection described herein utilizes selec­tion for Thy-l.I. Sca-l, and c-kit expression among Lin"~g cells but does not include discrimination based on IL-7R and thus se­lects a larger population of cells (0.1 % of total BM). It is likely that the lack of selection for IL-7R in the present studies resulted in the recovery of myeloid progenitors in addition to lymphoid progen­itors because IL-7R expression is linuted to the lymphoid lineage. The Journal of Immunology Interestingly, the presence of a significant number of myeloid pro­genitors among Thy-I.l neg cells (Table 1) did not result in mea­surable engraftment of platelets, RBCs, or myelomonocytic cells in transplant experiments (Figs. 2, 4, and 6). This may be due to a relative lack of proliferation potential in early myelopoiesis com­pared with the significant expansion that occurs at the pro-B stage of development. Alternatively, the myeloid progenitors present in the Thy- 1.1 neg subset may lack the ability to home to the BM and establish myelopoiesis (Fig. 4). These studies support the concept of an early separation between erythroid and platelet lineages from other myeloid progenitors because these lineages were not ob­served to recover after transplantation of Thy_Llneg cells (Fig. 2) and because erythroid colonies were rarely observed in cultures (Table 1). Evidence of a separation of erythroid and platelet pro­genitors from other myeloid lineages early in hematopoietic de­velopment has recently been demonstrated in cell enrichment stud­ies (27). Kondo et al. (12) utilized clonal analysis to demonstrate that clones generated from single CLP included both T and B lineage potentiaL However, T lineage potential was evaluated by direct intrathymic injection of individual clones. The low frequency of T cell potential in the Thy-I.I neg subset isolated in our studies may indicate a defect in thymic homing because our i. v. transplant as­say requires both homing, intrathymic proliferation and export of a sufficient number of mature T lymphocytes for detection in the peripheral blood. However, the relevant BM progenitor for T cells is also required to perform these functions to maintain the periph­eral pool of T lymphocytes. Therefore, our studies suggest that the cell population that normally seeds the thymus may be a minor subpopulation of the Thy-I.1 neg BM subset, despite the ability of freshly isolated cells or clones derived from those cells to differ­entiate along the T lineage when injected into the thymus (12). ntis interpretation is supported by a recent study that shows that B lineage progenitors isolated from Pax5 knockout animals can be reprogrammed to differentiate into multiple hematopoietic lin­eages, induding the T cell lineage (28). It is possible that the in vitro culture utilized by Kondo et a!. (12) induced such a lineage reprogramming process in cells that are normally committed to the B lymphoid lineage. Alternatively, segregation of the Thy_Uneg population using additional Ags may reveal a small subset with T lineage potential that was transplanted at a frequency too low to be detected in the present studies. The robust reconstitution of BM and peripheral blood B lineage cells but not other hematopoietic lineages by Thy-!.I neg cells sug­gests lhat this cell population predominantly includes progenitors committed to the B lineage. In separate studies, we have used cell separation techniques and in vitro cultures to show that over 50% of the cells contained in the Thy-l.l neg subset are committed my­eloid progenitors that can be separated from the lymphoid progen­itors based on levels of c-kil expression.4 Interestingly, myeloid engraftment was minimal in the studies reported in this paper (Figs. 3, 4, and 6), despite the presence of many myeloid progen­itors in the transplanted Thy-l.l neg population (Table I). This sug­gests that the homing or proliferation of the myeloid progenitor cells contained within the Thy-I.l neg subset is insufficient to result in significant engraftment. Alternatively, the progeny of the my­eloid progenitors may be rapidly sequestered in tissues or may have too short of a lifespan to be detected at the time we evaluated engraftment posttransplant. We assume that the minor degree of myeloid engraftment observed in the present studies (Figs. 3, 4, • M. P. Mojica, S. S. Perry. A. E. Searles. K. S. 1. Elenitoba·Johnson. L. J. Pierce. A. Wiesmann. W. B. Slayton. and O. J. Spangrude. 2000. Expression of AA4.1 defines the onset of Pax5 transcription in mouse pro-B cells. Submitted for publication. 28 73 and 6) was due to the myeloid progenitors contained within the Thy_l.Ineg cell population. Bauman et aL (29) reported effects of specific Ab detection sys­tems on hematopoietic engraftment analogous to those observed in our studies. In those studies, it was shown that anti-class I MHC Abs detected using an anti-rat secondary reagent inhibited CFU-S activity, whereas anti-class I MHC Abs conjugated to biotin did not have this inhibitory effect. The group concluded that the re­duction in CFU-S numbers was due to the phagocytosis of cells opsonized with anti-rat Abs. This did not occur in the case of staining with the avidin-fluorochrome conjugate, possibly because of the presence of fewer Fc regions or because of masking of Fc determinants by the biotin-avidin complexes, which prevented rec­ognition and subsequent phagocytosis of these cells by macro­phages. In our studies, a similar mechanism may be causing the preferential removal of cells bound to APC-conjugated Abs. Such a response may be stimulated by the large size and foreign nature of APC (derived from algae), which could activate macrophages to phagocytose Thy-U neg cells opsonized with APC-labeled Abs in a manner similar to that described by Bauman et al. (29). However, the lack of an inhibitory effect of the APC-conjugated c-kil reagent on transplantation of Thy_l.1 low cells, which express very high levels of c-kit (Fig. I), makes this interpretation difficult to justify. Additional experiments will be required to resolve this question. The clear clinical value of the Thy 1.1 neg cell population in me­diating early, albeit transient, PBMC recovery in the posttransplant setting makes it important for us to define the mechanisms that regulate engraftment of this cell population and its ability to fa­cilitate lymphoid reconstitution. In addition, as transplant products are increasingly subjected to in vitro manipulations before infusion to engineer specific balances of graft-vs-Ieukemia effects with he­matopoietic engraftment (30, 31), it will be increasingly important to clarify biologic effects of Ab staining on subsequent transplan­tation potential. References I. Kaufman. C. L.. Y. L. Colsoo. S. M. Wren. S. Watkins. R. L. Simmons. and S. T. lids tad. 1994. Phenolypic characterization of a novel bone marrow-derived cell that facilitates engrafiment of allogeneic bone marrow stem cells. Blood 84:2436. 2. Papayannopoulou, T.. C. Craddock. B. Nakamoto. O. V. Priestley. and N. S. Wolf. 1995. 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T. lIdslad. 1998. Effect of Fl. 1'3 ligand and granulocyte colony-stimulating faclor on expansion and mobilization of facilitating cells and hematopoietic stem cells in mice: ki­netics and repopulating potentiaL Blood 92:3177. 7. Nibley. W. E .. and O. J. Spangrude. 1998. Primitive stem cells alone mediate rapid marrow recovery and multilineage engraftment after Iransplanlation. Bone Marrow TrlVlsplant. 21:345. 8. Uchida. N .• and !. L. Weissman. 1992. Searching for hematopoietic stem cells: evidence thaI Thy·l.I'oLin - Sca-I· cells are the only stem cells in C57BUKa· Thy-I.! bone marrow. J. Exp. Med. 175:175. 9. Spangrude. G. 1.. and D. M. Brooks. 1993. Mouse Slralo variabilily in the ex· pression of the hematopoietic stem cell antigen Ly-6AIE by bone marrow cells. Blood 82:3327. 10. Okada, S .• K. Nagayoshi. H. Nakauchi. S. I. Nishikawa. Y. Miura. and T. Suda. 1993. Sequential analysis of hemalopoietic reconstirution achieved by IraIlsplan­tation of hematopoietic stem cells. Blood 81:1720. I!. Spangrude, 0.1.. S. Heimfeld. and I. L. Weissman. 1988. Purification and char· acterization of mouse hematopoietic stem cells. Science 241 :58. 12. Kondo. M .. I. L. Weissman. and K. Akashi. 1997. Identification of clonogenic corumoo lymphoid progemtors in mouse bone marrow. Cell 91:661. 74 13. Ogawa, M., Y. MatsuzaJd. S. Nisbikawa. S. Hayasbi, T. Kunisada. T. Sudo, T. Kina. and H. Nakauchi. 1991. Ellpression and function of c-/cit in hemopoietic progenitor cells. J. Exp. Med. 174:63. 14. Spangrude. G. I .. and D. M. Brooks. 1992. Phenotypic analysis of mousc hema­topoietic stem cells shows a Thy-I-negative subset. Blood 80:1957. 15. Hayakawa. K., Y. S. Li, R. Wasserman. S. Sauder, S. Shinton. and R. R. Hardy. 1997. B lymphocyte developmental lineages. Ann. NY Acad. ScL 815:15. 16. Osmond, D. G., A. Rolink. and F. Melchers. 1998. Murine B lymphopoiesis: towards a unified model. ImmunoL Todc.y 19:65. 17. Kantor. A. B .. A. M. Stall. S. Adams. K. Watanabe. and L. A. Henenberg. 1995. De novo development and self-replenishment of B cells. Int. ImmWlol. 7:55. 18. Rolinlc. A .. D. Haasner. S. Nisbikawa. and F. Melchers. 1993. Changes in fre­quencies of clonable pre B cells during life in different lymphoid organs of mice. Blood 81:2290. 19. Smith. L. G .. I. L. Weissman. and S. Heimfeld. 1991. Clonal analysis of hem a­topoietic stem-cell differentiation in vivo. proc. NatL Acad. Sci. USA 88:2788. 20. Spangrude. G. I .. D. M. Brooks. and D. B. Tumas. 1995. Long-term repopulation of irradiated mice with limiting numbers of purified hematopoietic stem cells: in vivo ellpansion of stem cell phenotype but not function. Blood 85: 1006. 21. Momson. S. J .• and I. L. Weissman. 1994. The long-term repopulating subset of hematopoietic stem cells is deterministic and Isolatable by phenotype. Immunity 1:661. 22. McNiece. l. K .. I. Bertoncello. A. B. Kriegler. and P. 1. Quesenberry. 1990. Colony-forming cells with high proliferative potential (HPP·CFC). Int. 1. Cell Cloning 8: 146. 23. Fulop. G. M., and R. A. Phillips. 1989. Use of scid mice to identifY and quantiwe lymphoid-restricted stem cells in long-term bone marrow cultures. Blood 74: 1537. 29 TRANSPLANTABLE B LYMPHOCYTE PROGENITORS 24. Hackelt. J.. 1r .• G. C. Bosma. M. 1. Bosma. M. Bennett. and V. Kumar. 1986. Transplantable progenitors of natural killer cells are distinct from those of T and B lymphocytes. Proc. NatL Acad. Sci. USA 83:3427. 25. van Leeuwen, J. E~ M. J. van Tol. A. M. Joosten, P. T. Schellekens. R. L van den Bergh. 1. L Waaijer. N. 1. 0Weman-Gruber. C. P. van dcr Weijden-Ragas. M. T. Roos. m1 E. 1. Genilsen. 1994. Relalioosbip between pattrms of CIIgraftment in peripbcral blood and immUDe recoostillllioo after allogeneic booe marrow transpIamalioo for (severe) c0m­bined lmmUDodeficietlC}'. Blood 84:3936. 26. Brady. K. A .• M.l. Cowan, and A. D. Leavitt. 1996. Circulating red cells usually remain of host origin after bone marrow transplantation for severe combined immUDOOeficiency. Transfusion 36:314. 27. Akashi. K .• D. Traver. T. Miyamoto. and I. L. Weissman. 2000. A clonogenic common myeloid progenitor that gives risc to all myeloid lineages. Namre 404: 193. 28. Rolink. A. G .• S. L. Nun. F. Melchers. and M. Busslinger. 1999. Long-term in vivo reconstirution of T-cell development by PallS-deficient B-cell progenitors. Narure 401 :603. 29. Bauman. 1. G. 1 .. A. H. Mulder. and G. J. van den Engh. 1985. Effect of surface antigen labeling on spleen colony formation: comparison of the indirect immu­nofluorescence and the biotin-avidin methods. Exp. HemaIol. 13:760. 30. Bonini. C. G. Ferrari. S. Veneletti. P. Servida. E. Zappone. L. Ruggieri. M. POllZoni. S. Rossini. F. Mavilio. C. Traversari. and C Bordignon. 1997. HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft­versus- leukemia. Science 276:/719. 31. Scho. L. H .. E. P. Alyea. E. Weller. C. Canning. S. Lee. 1. Ritz, J. H. Antin. and R. 1. Soiffer. 1999. Comparative outcomes of T-cell-depleted and non-T-~ell­depleted allogeneic bone marrow transplantation for chronic myelogenous leu­kemia: impact of donor lymphocyte infusion. 1. Clin. Oneol. 17:561. CHAPTER 3 PHENOTYPIC DISTINCTION AND FUNCTIONAL CHARACTERIZATION OF PRO-B CELLS IN ADULT MOUSE BONE MARROW (The Journal of Immunology, 2001, 166: 3042-3051) 31 Phenotypic Distinction and Functional Characterization of Pro-B Cells in Adult Mouse Bone Marrowl Mariluz P. Mojica,* s. Scott Perry/ A. Elena Searles,:J: Kojo S. J. Elenitoba-Johnson/ L. Jeanne Pierce,§ Anne Wiesmann,§ William B. Slayton,lI and Gerald J. Spangrude2t:J:§ A lymphoid-committed progenitor population was isolated from mouse bone marrow based on the cell surface phenotype Thy- 1.1oegSca_lposc_KitIoWLinoeg. These cells were CD43posCD24POs on isolation and proliferated in response to the cytokine combi­nation of steel factor, IL-7, and Flt3ligand. Lymphoid-committed progenitors could be segregated into more primitive and more dilferentiated subsets based on expression of AA4.1. The more dilferentiated subset generated only B lymphoid cells in 92% oftolal colonies assayed. lacked T lineage potential, and expressed Pax5. These studies have therefore defined and isolated a B lymphoid­committed progenitor population at a developmental stage corresponding to the initial expression of CD45R. The Journal of Immunology, 2001, 166: 3042-3051. Combinations of surface proteins expressed by cells at dif­ferent stages of blood cell development have enabled the isolation and subsequent functional characterization of phenotypically defined hemopoietic progenitor cell populations. In the adult mouse. the bone marrow (BM? is the site of hemopoiesis where mature blood cell lineages are generated from self-renewing multipotent hemopoietic stem cells (HSC) (1). These HSC express low levels of Thy-l.l and high levels of stem cell Ag-l (Sca_l pOS ) and lack expression of lineage-associated markers (Lin°eg) (2). Phenotypically defined progenitor cell populations with restricted myeloid and/or lymphoid lineage potentials have recently been described (3-6). Expression of CD45R has been used by a number of investigators to isolate and characterize lymphoid progenitors (7. 8), while stages of B lymphoid development before expression of CD45R are only beginning to be explored (4,9, 10). A goal in enriching for early B lymphoid progenitors is to sep­arate these cells from both HSC and differentiated cells of the B and other blood cell lineages. The latter can be achieved by de­pleting BM of cells expressing CD45R and other lineage-associ­ated markers. Segregating early lymphoid progenitor cells from HSC has been more challenging. Previous studies have shown that although the Thy-l.llowSca-1 posLinoeg (Thy_l.llow) cell popula-tion is markedly enriched for HSC, it is functionally heterogeneous Departments of *Human Genetics. '"Pathology, ~Medicine (Division of Hematology), ~Oncological Sciences. and 'lpediatrics, University of Utah, Salt Lake City. lIT 84132 Received for publication August 28. 2000. Accepted for publication December 18.2000. The costs of publication of this miele were defrayed in pan by the payment of page cbarges. This article must therefore be hereby marked advertisemenr in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I This work was supponed by grants from the National Institutes of Health (ROIHL56857 and P50DK49219). the Lymphoma Foundation. and the Blood and Marrow Transplant Program at the University of Utah. The Flow Cytometry and Irradiation core facilities of the Huntsman Cancer Institute. supponed by National Cancer Institute Cancer Center Suppon Grant P30CA42014. were used for these studies. 2 Address correspondence and reprint requests to Dr. GerJld 1. Spangrude, Depanment of Oncological Sciences. University of Utah, 50 Nonh Medical Drive. Room 5034, Salt Lake City. lIT 84132. E-mail address: [e-mail] 3 Abbreviations used in this paper: BM. bone marrow; HSC. hemopoietic stem cells: STL. Steel factor: Flt3L. Flt3 ligand; Epo. erythropoietin: Lin°es. bone marrow cells depleted of cells expressing any of a panel of lineage-specific Ags: Thy.l.! low. Sea· I +c.Kit+LinDeIThy. 1.1 low 8M cells; Thy· 1.1 neg. Sea· I +c·KitTLinnegThy.l.l°e, BM cells: PI. propidium iodide; SAv. s[feplavidin: Rag. recombinase·activating gene. Copyrigbt © 2001 by The American ASSOCiation of Immunologists (II. 12). This heterogeneity, which is reflected in the ability of the cell population to mediate both short term and long term BM en­graftment, can be dissected using separations based on cell cycle activity and activation state 03, 14). Although experiments de­signed to segregate progenitors for lymphoid lineages have shown that subsets of Thy_l.l1oW HSC engraft in the BM. the kinetics of B cell engrafunent exhibit a 2-wk delay relative to whole BM transplants (15). This suggests that a lymphoid progenitor cell pop­ulation that is present in normal BM is missing from the Thy- 1.lloW HSC population. The c-Kit molecule has also been used extensively as a cell surface marker in the purification of HSCs (16-18). We recently found that the c- KitPOS subset of Sca-l POSLinOe!! BM cells contains two populations of cells which differ with respect to expression of Thy-l.l. The majority of Thy_l.lIOW cells express c-Kit at high levels (C_Ki~gh). while Sca_lPo'Linneg BM cells lacking the Thy- 1.1 Ag (Thy-l.l neg cells) include c_Kirow as well as c-Kirugb sub­sets. Transplant studies demonstrated that the Thy_l.llowc_Kit"igh subset mediated full hemopoietic engraftment of lethally irradiated recipient animals with prominent erythroid. myeloid and platelet reconstitution and delayed lymphoid engraftment ( 19). In contrast, the Thy-L1 neg subset failed to provide erythroid and platelet en­grafunem to transplant recipients, but mediated rapid lymphoid engraftment with a minor degree of myeloid recovery. In the ab­sence of full hemopoietic recovery, transplant recipients of Thy­Uueg cells survive only 25-30 days before death due to hemo­poietic failure. The current studies were initiated to resolve the lymphoid and my­eloid potentials of Thy_l.lneg cells at a clonal leveL Using a number of additional phenotypic markers. we demonstrate three separate pro­genitor populations within the Thy-I.I neg subset of Sca-l poliLinneg BM cells. These include separate committed progenitors for lymphOId and myeloid (predominantly macrophage) lineages as well as a mixed lineage progenitor population. Interestingly, expression of the AA4.1 Ag defines the onset of Pax5 transcription and marks the loss of pro-T cell potential. Thus. these studies have defined a very early stage of mouse pro-B cell development. Materials and Methods Mouse strains B6.PL and AKR mice were obtained from The Jackson Laboratory (Bar Harbor. ME). while C57BUKa, B6-Thy-l.l-Ly·5.1. and B6-Ly-l.l mice 0022·1767/01/$02.00 The Journal of lnununology were bred and maintained at the Animal Resource Center facility of the University of Utah. Mice used were 4-16 wk of age. Cytokines and Abs Steel factor (SlL) and G-CSF were gifts from Gemini Science (San Diego, CA). a subsidiary of Kirin Brewery (Tokyo, Japan). At3 ligand (FIt3L) and IL-6 were kindly provided by Immunex (Seattle. W A). Recombinant hu­man erythropoietin (Epo) was purchased from Ortho (Raritan, NJ). Re­combinant murine IL-3 and IL-7 were purchased from PeproTech (Rocky Hill. NJ). The cytokines were used at the following concentrations: SlL, 100 nglml; G-CSF, 10 nglml; At3L. 75 nglml; IL-6, 20 nglml; Epo. 5 UIml; IL-3. 10 nglml: and lL-7. 10 ngfml. mAbs against CDS (53-6.7). Mad (MlnO). erythrocytes (TERI19), Gr-l (RB6-8C5). CD3 (KT3-1.1). CD5 (53-7.3), CD2 (Rm2.2). mouse Ig (RAM-HB58), CD45R (RA3-6B2). Thy-L1 (I9XE5). c-Kit (ACK-4), early B cell (AA4.l). and IL-7R were purified from media of cultured hybridoma cell lines. while the mAb agaillst CD 19 was purchased from PharMingen (San Diego. CA). mAbs used for cell surface staining of CD45R, Thy-I.l. c-Kit. Gr-\. CD62L. AM. I. and IL-7R were conjugated with biotin. PE, or ATC in our laboratory. Biotinylated Abs were second­arily stained with either PE-streptavidin (PE-SAv; Biomeda. Foster City. CA) or PE-Texas Red-SAv (Red613; Life Technologies, Grand Island. NY). In addition. PE-conjugated mAbs to Sca-I. Mac-I. and CD45R. bi­otinylated Abs to CD24 (M 1169) and CD43 (S7). and allophycocyanin­conjugated c-Kit Ab were purchased from PharMingen. The IL-7R clone used in these studies was a gift from Richard R. Hardy (Institute for Cancer Research. Fox Chase Cancer Center, Philadelphia, PA). Preparation of BM cells and isolation of hemopoietic stem/progenitor cell populations The procedure for the preparation of BM cells for sorting has been previ­ously described (20). Briefly. BM cells were isolated from femurs and tibia of donor mice. and the RBCs were lysed in an ammonium chloride potas­sium solution. The ceils were incubated in a lineage cocktail containing optimized concentrations of Abs to CD2. CD3, CD5. CD8. Mac-I. Gr-l. TERI19. CD45R. and CDI9. The CD45R Ab was not included in the lineage cocktail whenever CD45R expression was evaluated after lineage depletion. Lineage depletion was conducted by two successive incubations of the BM cells in sheep anti-rat Ig-coupled magnetic beads (Dynal. Oslo. Norway). The Lin""8 cells were stained with PE-Sca-I and sorted using the FACSVantage (Becton Dickinson. San Jose. CA) set at enrichment mode and thresholding on PE emissions above background levels. Dead cells were excluded from all analyses and sorts by gating on forward scatter and PI staining. The sorted Lin .... Sca-I pos cells were pelleted and stained with allophycocyanin-c-Kit and ATC-Thy-I.l and resorted into Thy_l.lIOW c­KitPOs and Thy- Ll De3C_ KitPO' subsets. In experiments in which the Thy­Ll ae'c-Kitpo, subset was further fractionated, the appropriate biotin-con­jugated Ab stain was added and visualized using Red613-SAv. AIl cell sorting steps were performed using the F ACS Vantage. and an aliquot of the sorted cell population was always taken for reanalysis. Methy[rellulose assays The sorted cell populations were cultured in methyl cellulose at a plating density of -100 cells/35-mm culture dish. Each milliliter of culture me­dium contained a-MEM (Life Technologies). 1.2% methyl cellulose (Shi­netsu. Tokyo. Japan). 30% FCS (Life Technologies). I % deionized BSA (Sigma, St. Louis. MO). and 0.1 roM 2-ME (Mallinckrodt, Chesterfield. MO) supplemented with the indicated cytokine combinations. Culture dishes were inCUbated at 37°C and infused with 5% CO~. The number of colonies was counted using an inverted microscope after'7 days of culture to detertnine the cloning efficiency of each sorted cell population. Four to six plates were scored for each group, and the results were expressed as percentage of the total cells plated. In addition. individual colonies were plucked between days 6 andl2 of culture and analyzed for both cell surface staining and cell morphology. Two-thirds of the cells harvested from each colony were stained with Abs to CD45R and Gr-l and analyzed by flow cytometry. while cytospins were prepared from the remaining cells. Cyto­spins were stained with May-Grunwald-Giernsa for morphological analysis. Liquid cultures Liquid cultures of sorted cell populations were conducted using a-MEM (Life Technologies) containing 10% FCS. 1 roM MEM sodium pyruvate solution (Life Technologies). 10 roM HEPES (pH 7.3), 100 Ulml penicil­lin, 100 J.Lglml streptomycin. 2 roM glutamine. and 0.1 roM 2-ME (Mallinckrodt) and supplemented with the indicated cytokine combina- 32 3043 lions. Cells were either grown in bulk in 24-well plates or seeded at lim­iting dilution (one cell per well) in 96-well plates with or without stromal cell feeder layers as indicated. Culture plates were incubated at 37°C and infused with 5% CO2, The 2018 stromal cell line (a gift from Kateri Moore) was maintained at 31-33°C. Cells were prepared the day before coculture by seeding 10,000 cells/well in 24-well plates for bulk cultures and 1.000 cells/well in 96-well plates for the limited dilution (clonal) as­says. The presence of a single cell per well in 96-well plates was confirmed whenever possible after overnight culture using an inverted microscope. Positive clones (wells) were scored by day 5 and harvested for analysis between days 6 and 14. For bulk cultures. representative wells were har· vested for analysis between days 5 and 14. lntrathymic T cell development assay Sublethally irradiated B6 (4- to 6-wk-old females) mice were anesthetized and immobilized with rubber bands. The skin over the chest was incised to reveal the sternum, which was cut. The thymus was visualized within the thoracic cavity. and 3 J.Ll of fluid containing the sorted cell population of interest was directly injected into the thymic tissue using a Hamilton sy­ringe (Reno, NV). The ches! was closed using stainless steel surgical clips. The cells to be transplanted were obtained from the B6-Thy-1.I-Ly-5.1 double-congenic strain and were sorted directly into a microfuge tube con­taining a known amount of Hanks' 10% FCS so that eacb I J.LI of fluid contained a known number of cells. Graded doses of cells were injected into groups of animals (10 animals/group) in the presence of an excess of Lin""8 cells obtained from a second B6 congenic strain (B6-Ly-I.I), which served as a carrier and as an internal control to indicate successful mJec­tions. Three or 4 wk later. the recipient B6 mice were sacrificed, and thy­mic tissue was isolated for analysis by flow cytometry to identify thymic lobes containing progeny cells derived from the injected populations. Suc­cessful intrathymic transfers were identified by the presence of Ly-l.l pos cells. and positive thymic lobes were scored for the presence of Ly-5.1 pos cells. Limiting dilution statistics were applied to the resulting data to derive the frequency of repopulating cells in the sorted population. RT-PCR assay Sorted cells were lysed using 500 J.LI of TRIzol (Life Technologies) with 20 J.Lg of glycogen (Roche. Indianapolis. IN) added as a carrier. The TRIzol protocol for RNA isolation prescribed by the manufacturer was followed using half volumes. After isopropanol precipitation. the RN A pellet was washed twice in 70% ethanol and resuspended in 8 J.Ll of diethylpyrocar­bonate- treated water. The RNA samples were incubated with I J.Ll of am­plification grade DNase I (Life Technologies) and I J.Llof lOX DNase I buffer at room temperature for 15 min to eliminate any contaminating DN A. The reaction was stopped with the addition of I J.LI of 25 roM EDT A and heating at noc for 10 min. Water was added to bring the total volume of each reaction to 20 J.LI. Five to 10 J.Ll from each total RNA sample was used for first-strand synthesis using random primers (Life Technologies) and Moloney murine leukemia virus reverse transcriptase (Life Technol­ogies) following the protocol provided by the manufacturer. Semiquantitative PCR was used to compare the expression of genes between sorted cell populations. All primer sequences used in this study have been previously described. To equalize for cDNA input. each sample was first amplified by PCR using GAPDH primers (21). and the amount of input cDNA was adjusted to provide equivalent signals. Subsequent PCR amplifications used the predetennined amount of cDNA with gene-specific primers for sterile Ig heavy chain transcript J.Lo. Rag-2. E2A. Pax-5. and CDI9 (7.22.23). PCR cycle parameters used for GAPDH. J.Lo. and Rag-] were described by Li et al. (22). while those for E2A. Pax-5. and CDI9 were described by Bain et al. (23). Fifteen-microliter aliquots were with­drawn at cycles 24. 27. and 30 (GAPDH) or cycles 27. 30. and 33 (/-Lo' Rag-2, E2A. Pax-5. and CDI9) to assure that amplification was within the linear range. The PCR products were separated by 1% agarose gel elec­tropboresis. Quantitation was perfonned using the MultiAnalyst progr.un (Bio-Rad. Hercules. CAl. lndividual bands were measured and nonnalized using tbe GAPDH signal for each sample. Comparison of gene expression between samples was achieved by comparing the nonnalized value for each sample to the value obtained for CD45RPOs cells. Results The Thy- J. Jn~1J cell population contains three separate progenitor subsets Linneg mouse BM cells were stained with Abs to Sea-I. c-Kit, and Thy-l.l and sorted to recover the Thy_l.1 low and Thy-l.l neg cell populations (Fig. 1). The Thy-l.l neg subset comprised -0.05 :::: 33 3044 DEFINITION OF EARLY B LYMPHOID-COMMITTED PROOalTORS B -!i! U Thy-1.1 o reanalysis i -:x U Thy-1.1 Thy-1.1 FIGURE 1. BM cells isolated from adult mice were lineage depleted and subsequently stained with rnAbs to c· Kit. Sea· I. and Thy-I.I. Dead cells were excluded using propidium iodide staining and forward scalter gating. Sea-l pos cells were selected by gating (A) and analyzed for Thy-I.I expression (8). Thy- !.l""gSca-1 PO'c-KitPO' Lin""g (Thy-I.! a.s) and Thy- 1.1 low Sea.l PO'c-KitPO' Linneg (Thy_U1o, cells were isolated by cell sort­ing as described in Materials and Methods. and aiiquots were taken for reanalysis (C :md D). 0.01 % (mean SD; n 8) of nucleated BM cells, a frequency very similar to that of the Thy_l.llow subset as previously reported (I, 2). Virtually all cells expressing low levels of Thy-I.1 were c-Kif'igh, while the TI1y-l.lnt!g population included cells express­ing both low and high levels of c-Kit. Cells lacking c-Kit expres­sion were not further characterized in these studies. Because of concerns regarding contamination of Thy -1.1 Deg cell preparations with Thy-I. I low HSC, reanalysis of sorted populations was always performed as shown in Fig. 1, and Thy-l.l neg populations con­taining any discemable contamination with cells expressing Thy- 1.1 at a level 5- to lO-fold above background levels were not used for functional studies. Although our previous transplant studies demonstrated an inhibitory influence of allophycocyanin-conju­gated c-Kit Abs on in vivo engraftment of Thy-l.l Deg cells (19). direct comparisons of cloning efficiencies and lineage potentials of Thy-l.1 neg cells isolated using biotin or allophycocyanin conju­gates of anti-c-Kit Abs showed no differences in our in vitro studies. Initial in vivo transplant studies demonstrated that the Thy- 1.1 neg cell population mediates rapid BM engraftment and con-tributes to both lymphoid and myeloid lineages (19). To detennine wbether the Thy- L I neg cell subset consisted of multipotent pro­genitors or separate progenitor cells committed to either lymphoid or myeloid lineages, we conducted clonal progenitor cell assays. The soned cells were cultured in methylcellulose or single-cell liquid cultures supplemented with different cytokine combinations. and individual colonies were analyzed after 6-12 days in culture by both flow cytometry and cell morphology. These analyses showed that the Thy-I.1 neg cell subset contained three types of progenitors (Table I). Stimulation with a mixture of seven cyto­kines, as detailed in Table 1, allowed differentiation of multiple hemopoietic lineages. Under these culture conditions, we observed colonies consisting solely of myeloid lineage cells (macrophages, primitive granulocytes, and erythroid cells), colonies consisting solely of lymphoid lineage cells (CD45R,K>SGr_l neS ), and mixed colonies containing both lineages. Lymphoid lineage colonies rep­resented 33% of the total Thy-l.l neg colonies analyzed, while those consisting of myeloid lineage cells represented 54% (Table I). Mixed lineage colonies, containing CD45RPOs cells as well as myeloid cells, were observed at a frequency of 13%. Similar re­sults were obtained in liquid cultures initiated from single cells, suggesting that the mixed lineage colonies were not the result of sampling error in the methylcellulose assay. When Thy_l.1 low HSC were cultured under the same conditions, pure lymphoid col­onies were not observed, and very few mixed lineage colonies containing CD45RPoS cells were scored (4%, or 2 colonies of 45 examined; Table I). Stimulation of Thy_l.l'oW HSC with lym­phOid- specific cytokines (STL, IL-7, and FIGL) resulted in a marked decrease in cloning efficiency (88 vs 581 colonies/lOoo cells plated). IL-3 has been reported to inhibit early B lymphoid differentia­tion (24, 25). Consistent with this observation, we observed that omission of IL-3 from the cytokine cocktail resulted in a 40-50% decrease in the cloning efficiency of Thy-l.l neg cells. Analysis of the colonies that grew in the absence of IL-3 demonstrated a de­crease in the frequencies of pure myeloid and mixed colonies and a concomitant increase in the proportion, but not the absolute num­ber, of pure lymphoid colonies. Comparison between S7F6EG stimulation with or without IL-3 showed that 33% of 148 44 colonies were lymphoid in the presence of IL-3 (48.S :::t 14 colo­nies/ lOOO), whereas 60% of Sl :::t 29 colonies were lymphoid in the absence of IL-3 (48.6 17 colonies/lOoo; Table I). Thy-l.1 neg cells grown in methyJcellulose cultures supplemented with cyto­kines permissive only for lymphoid differentiation (S7F) exhibited a 6-fold decrease in cloning efficiency compared with the more complex cytokine combination, and all colonies evaluated from Table I. Cloning efficiency and lineage potential o/Thy-l.ll",., and 171y-l.reg subsets" Lineage Content (% of total, n)d Cytokine Colonies/lOOO Cells Cell Population Stimulationb :2: SDc Lymphoid Myeloid Mhed Thy-l.I neg progenitors S7F36EG 148 ±: 44 33%,16 54%.26 13%.6 S7F6EG (no IL-3) 81 :29 60%,1:2 35%,7 5%,1 S7F :25 ±: I 100%,21 0%,0 0%.0 Thy_l.llow HSC S7F36EG 581 ±:71 0%.0 96%.43 4%,:; 88 44 ND ND ND u Linneg 8M cells were sorted to isolate Thy-Ll ", .. Sca-lPO< c_kit""S HSC (Thy_l.llOW) or Thy. LInes Sea-II"'" c·kit""" progenitor cells (Thy·uneg ) as shown in I. b Cultures were initiated in methylcellulose medium containing the indicated cytokines as described in Materials and Methods. Cytokines are abbreviated as follows: S1L: 7. tL-7; F. Flt3L; 3. IL·3; 6. ll..·6; E, Epo; G. G-CSF. C Colonies were counted on days 7-9 of culture. Cultures initiated with Thy-l.t ·°1 cells stimulated with S7F contained 600 celis/pIate; all other cultures were initiated with 120 ceBsiplate. d To evaluate lineage content. individual colonies were isolated from the rnethylcellulose medium and split for flow cytometric and histological analysis as described in Marertals and Methods. Of all colonies isolated from cullure. 70% (Thy-I. I neg) [0 90% (Thy_l.llOW) contained enough cells for analysis. The percentage of the total and number of colonies evaluated (n) are presented. The Journal of Immunology these cultures contained only cells with lymphoid morphology and surface Ag expression. The presence of pure lymphoid colonies after stimulation of Thy-l.I neg cells with the complex cytokine mixture confirms that a committed lymphoid progenitor population is wntained within the Thy-Uneg cell subset, but not in the Thy- 1.110'"' HSC population. These cells grow in response to the cyto­kine combination of S7F and are insensitive to the inhibitory ef­fects of IL-3. A number of early 8 lymphoid markers fractionate the Thy­l. r<'f? cell population into distinct subsets To determine whether additional markers could potentially frac­tionate the Thy-I.l neg cell population into functionally distinct subsets, we isolated Thy-l.l neg cells and evaluated the expression of a number of cell surface markers known to be expressed during the early stages of lymphoid development. Representative FACS plots of the stainmg analyses are shown in Fig. 2. As shown in Fig. 2A, the AA4.1 mAb identifies a subset comprising 30-50% of the Thy-l.l !leg cells that expresses low levels of c-Kit. Expression of IL-7R also separated the Thy- 1.1 neg population into two distinct clusters that correlated with c-Kit staining intensities. Cells that were fL-7Rl'm invariably ex.pressed low leveis of c-Kit. while IL- 7Rnc~ cells were both c_KitiOW and c_Kithigh (Fig. 28). In contrast. CD62UMEL 14 expression was observed on both the c-KitlOW and the c- Kithigh subsets of Thy-I.I neg cells (Fig. 2C). Of the other cell surface Ags tested, CD4 staining revealed only a minor subpopu­lalion of positive cells (l0% of Thy-l.l neg cells, equally distrib­uted between the c_KitlOW and c_KiJ!llSh subsets, Fig. '20), which was similar to the distribution of the Sca-2 Ag (data not shown). CD3 served as a negative control and was expressed by < I % of Thy_Ll"Cg ceI1s (Fig. 2E). Since expression of AA4.1 and IL-7R have previously been associated with early lymphoid progenitors (4, 6, 7, 26), we focused on the c_KitiOW subset of cells for addi­tional phenotypic and functional analysis. A hierarchy of progenitors in the B lymphocyte developmental pathway has been described by Hardy and colleagues, who used ceil surface Ag expression to define specific developmental stages (4,7,27). To better place the Thy_I.l"e"'c_Kirow progenitor pop­uiation in the context of the previous studies, we evaluated CD45R, CD24, and CD43 expression by these cells. Unlike the primitive pro-B cell described by Hardy's stage A, Thy_Llnegc_ Kirow cells largely lack expression of CD4SR and express high levels of CD24 (Fig. 2, F and G). In common with Hardy's stage A of development, TI ;-1.1 neg c- KitloW cells express lower levels of CD43 relative to Thy_l.llow stem cells (Fig. 2N). Hardy and coileagues used expression of AA4. i along with CD4. CD24, and CD43 to identify a CD45Rn "g stage of B cell development pre­ceding the A stage (7), but the isolation protocol used in those studies failed to segregate these cells away from erythroid lineage progenitors (4). Furthennore, functional assessment of B lineage precursors in adult mouse bone marrow established that most early B lineage progenitors ex.press CD24, as detected using either the Ml/69 or 30-Fl Ab 19). To summarize. Thy-l.l''''Sc-KitIOW cells overlap with LinnegTdr" cells as defined by Tudor ;;;t al. (9), but differ from fraction Ao as defined by Hardy and colieagues (7) in that most Thy- 1.1 negc_