Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Studies of the interactions between stromal cells and B lymphoid progenitors Lemoine, François Michel 1988

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1988_A1 L45.pdf [ 7.23MB ]
Metadata
JSON: 831-1.0098069.json
JSON-LD: 831-1.0098069-ld.json
RDF/XML (Pretty): 831-1.0098069-rdf.xml
RDF/JSON: 831-1.0098069-rdf.json
Turtle: 831-1.0098069-turtle.txt
N-Triples: 831-1.0098069-rdf-ntriples.txt
Original Record: 831-1.0098069-source.json
Full Text
831-1.0098069-fulltext.txt
Citation
831-1.0098069.ris

Full Text

STUDIES OP THE INTERACTIONS BETWEEN STROMAL CELLS AND B LYMPHOID PROGENITORS by Francois M. Lemoine M.D., U n i v e r s i t y of P a r i s , 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Pathology) We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1988 ©Francois M. Lemoine, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of P a t h o l o g y The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date September 14, 1988 DE-6C3/81) i i ABSTRACT The o v e r a l l goa l of the work, descr ibed i n t h i s t h e s i s was to i n v e s t i g a t e the molecu lar mechanisms that r egu la te normal pre-B c e l l p r o l i f e r a t i o n and how these may be a l t e r e d i n transformed pre-B c e l l s . Monoclonal a n t i b o d i e s and molecular b i o l o g i c a l techniques have a l lowed a number of stages of pre-B c e l l d i f f e r e n t i a t i o n to be de f ined but l i t t l e i s known about mechanisms c o n t r o l l i n g t h e i r p r o l i f e r a t i o n . S tudies of pre-B c e l l p roduc t ion i n animal models and i n long- term c u l t u r e s that support pre-B c e l l p r o l i f e r a t i o n have suggested that s t roma l c e l l s p lay a key r o l e i n t h i s regard . As a f i r s t s tep to i n v e s t i g a t e the mechanisms i n v o l v e d , a number of pre-B c e l l suppor t ive murine s t romal c e l l l i n e s were i s o l a t e d and c h a r a c t e r i z e d . A number of pre-B c e l l l i n e s were a l s o i s o l a t e d , c loned and c h a r a c t e r i z e d . From these, spontaneous and Abelson murine leukemia v i r u s transformants were d e r i v e d . These c e l l l i n e s were then used i n c o - c u l t u r e experiments to demonstrate that s t romal c e l l s c o n s t i t u t i v e l y secre te a pre-B s t i m u l a t i n g f a c t o r . C h a r a c t e r i z a t i o n of the pre-B c e l l s t i m u l a t i n g a c t i v i t y produced by one s t romal c e l l l i n e (M2-10B4) showed i t to be a 10 Kd molecule s e n s i t i v e to f r e e z i n g and d i f f e r e n t from any c loned hemopoiet ic growth f a c t o r de scr ibed to date . The p o s s i b i l i t y that e x t r a c e l l u l a r mat r ix components might be i n v o l v e d i n s t romal c e l l - m e d i a t e d c o n t r o l of pre-B c e l l growth was a l s o i n v e s t i g a t e d . I t was found that pre-B c e l l s a t t a c h s p e c i f i c a l l y to f i b r o n e c t i n and that a l though f i b r o n e c t i n by i t s e l f cannot support pre-B c e l l p r o l i f e r a t i o n , i t c o n t r i b u t e s to s t romal c e l l s t i m u l a t i o n of pre-B c e l l growth. Both of these mechanisms were found to be a f f e c t e d i n m a l i g n a n t l y transformed pre-B c e l l popu la t ions i r r e s p e c t i v e of the mode of t r a n s f o r m a t i o n . i i i Transformed pre-B cells were found to have acquired the ability to secrete a novel 3 Kd autocrine factor that is also capable of stimulating normal pre-B cells. In addition transformed pre-B cells showed a greatly decreased ability to adhere to fibronectin and had become insensitive to the synergistic stimulating effect of fibronectin. It will be of interest to determine in the future whether these findings have a counterpart in human malignant pre-B cell populations. TABLE OF CONTENTS i v Page ABSTRACT i i LIST OF TABLES v i i LIST OF FIGURES v i i i LIST OF ABBREVIATIONS x ACKNOWLEDGEMENTS x i CHAPTER I INTRODUCTION 1 1) Hemopoiesis: General Concepts 1 A) Concept of P l u r i p o t e n t Stem C e l l s 2 B) Concept of Committed Progenitors 4 C) Regulation of Hemopoietic Stem C e l l s and 5 Myelopoiesis D) Myel o p o i e t i c Growth Factors and Hemopoiesis 8 E) Summary' 11 2) O r i g i n and Development of B C e l l s 12 A) R e l a t i o n s h i p Between B Lymphoid Precursors 12 and Other Hemopoietic Lineages B) Ontogeny of Lymphopoiesis 14 C) Development of B C e l l s i n the Adult 17 (a) Ig Gene Rearrangement and Expression 18 (b) Changes i n Surface Antigens 23 (c) Other D i f f e r e n t i a t i o n Markers 26 3) Regulation of B C e l l Development 29 A) Systems f o r Study 30 (a) Short-Term Culture Systems 30 (b) IL-3 Dependent B-Lineage C e l l Lines 31 (c) Long-Term B Lymphoid Cultures 32 (Lymphoid LTC) (d) Animal Models 36 ( i ) CBA/N Mice 36 ( i i ) SCID Mice 37 ( i i i ) Motheaten Mice 38 B) Mechanisms 39 (a) Growth Factors 39 (b) D i r e c t C e l l u l a r I n t e r a c t i o n s 42 4) Thesis Objectives 43 References 46 V CHAPTER I I MATERIALS AND METHODS 59 1) E s t a b l i s h e d C e l l Lines 59 2) Animals 59 3) Growth Factors 60 4) Antibodies 60 5) Probes 61 6) P r o t e i n s and Peptides 61 7) I s o l a t i o n and Cloning of Stromal C e l l Lines 63 8) I s o l a t i o n , Cloning and Maintenance of Pre-B 64 C e l l Lines 9) A-MuLV Transformation of Pre-B C e l l s 65 10) Immunofluorescence Analyses 66 11) Histochemistry 67 12) V i r u s Assays 67 13) Assays f o r Tumorigenicity 68 14) C e l l P r o l i f e r a t i o n Assays 68 15) C e l l Attachment Assays 69 16) Pre-B C e l l Colony Assays 70 17) Myeloid Colony Assays 70 18) A n a l y s i s of DNA Rearrangements 71 19) Preparation of CM 72 20) Gel Permeation Chromatography 73 References 74 CHAPTER I I I PARTIAL CHARACTERIZATION OF A NOVEL STROMAL CELL- 77 DERIVED PRE-B CELL GROWTH FACTOR ACTIVE ON NORMAL AND IMMORTALIZED PRE-B CELLS 1) I n t r o d u c t i o n 77 2) Results A) C h a r a c t e r i z a t i o n of Pre-B C e l l Supportive 78 Stromal C e l l Lines B) I s o l a t i o n and C h a r a c t e r i z a t i o n of Pre-B 80 C e l l Lines C) In V i t r o Growth of H9 and A8 C e l l s 82 D) Evidence f o r a Soluble Pre-B C e l l 87 S t i m u l a t i n g Factor E) S p e c i f i c i t y of the A c t i v i t y Produced by 90 Stromal C e l l Lines F) Response of Feeder-Independent H9 C e l l s to 92 Defined Growth Factors 3) Discus s i o n 92 References 97 v i CHAPTER IV AUTOCRINE PRODUCTION OF PRE-B CELL STIMULATING 99 ACTIVITY BY A VARIETY OF TRANSFORMED PRE-B CELL LINES 1) I n t r o d u c t i o n 99 2) Results 100 A) H9 C e l l s Produce a Pre-B S t i m u l a t i n g 100 A c t i v i t y B) Production of a Pre-B C e l l S t i m u l a t i n g 102 A c t i v i t y by A-MuLV Transformed Pre-B C e l l Lines C) C h a r a c t e r i z a t i o n of the Autocrine A c t i v i t y 109 Produced by Transformed Pre-B C e l l s 3) Discus s i o n 109 References 114 CHAPTER V ROLE OF FIBRONECTIN IN REGULATING PRE-B CELL 116 PROLIFERATION 1) I n t r o d u c t i o n 116 2) Results 117-A) D i f f e r e n t i a l attachment of normal and 117 malignant pre-B c e l l l i n e s to FN B) E f f e c t of FN on the p r o l i f e r a t i o n of normal 120 and transformed pre-B c e l l s i n the presence of stromal CM C) D i f f e r e n t i a l e f f e c t of FN-R anti b o d i e s on the 124 p r o l i f e r a t i o n of normal and transformed pre-B c e l l s 3) Discus s i o n 124 References 128 CHAPTER VI SUMMARY AND CONCLUSIONS 129 1) Regulation of Pre-B C e l l P r o l i f e r a t i o n by 129 Stromal C e l l s A) Factors 130 B) C e l l - C e l l I n t e r a c t i o n s 131 2) A l t e r e d Mechanisms i n Transformed Pre-B C e l l s 132 3) Proposed Model 133 References 135 LIST OF TABLES v i i Page TABLE 1 Hemopoietic growth f a c t o r s . 9-10 TABLE 2 B c e l l growth f a c t o r s . 40 TABLE 3 L i s t and d e s c r i p t i o n of the monoclonal an t i b o d i e s 62 used i n t h i s study. TABLE 4 Comparison of the a b i l i t y of various i r r a d i a t e d (80 Gy) 79 feeder c e l l l i n e s to support the growth of c u l t u r e d pre-B c e l l s . TABLE 5 Immunological and histoc h e m i c a l c h a r a c t e r i z a t i o n of the 81 stromal c e l l l i n e s used i n t h i s study. TABLE 6 Demonstration of a s o l u b l e f a c t o r derived from M2-10B4 88 c e l l s s t i m u l a t i n g immortalized pre-B c e l l l i n e s and normal pre-B c e l l s . TABLE 7 Lack, of responsiveness of H9 c e l l s to defined 93 growth f a c t o r s . TABLE 8 Demonstration that H9 autocrine a c t i v i t y s t i m u l a t e s 103 normal pre-B c e l l s . TABLE 9 Transformed phenotype of c l o n a l lymphoid c e l l l i n e s . 104 TABLE 10 Phenotype data of normal and transformed lymphoid 105 clones. TABLE 11 E f f e c t of FN on normal pre-B c e l l p r o l i f e r a t i o n 122 induced by stromal pre-B s t i m u l a t i n g a c t i v i t y (M2-10B4 CM). TABLE 12 E f f e c t of FN on transformed pre-B c e l l p r o l i f e r a t i o n 123 induced by stromal pre-B s t i m u l a t i n g a c t i v i t y (M2-10B4 CM). v i i i LIST OF FIGURES Page FIGURE 1 Schematic representation of hemopoiesis. 3 FIGURE 2 Organization of immunoglobulin genes. 19 FIGURE 3 Surface antigen expression and I g gene rearrangement 22 during murine B - c e l l development. FIGURE 4 D e s c r i p t i o n of the d i f f e r e n t types of long-term 33 hemopoietic c u l t u r e s . FIGURE 5 Panel a. Southern b l o t a n a l y s i s of Ig H chain gene 83 rearrangement i n A8 and H9 c e l l s f o l l o w i n g DNA d i g e s t i o n with EcoRI and h y b r i d i z a t i o n to a Jfj probe. Panel b. Southern b l o t a n a l y s i s of the T c e l l receptor g chain gene i n A8 and H9 c e l l s f o l l o w i n g DNA d i g e s t i o n with Hind I I I and h y b r i d i z a t i o n to a Tg cDNA probe. FIGURE 6 P r o l i f e r a t i o n of H9 c e l l s (panel a) and A8 c e l l s 84 (panel b) seeded at d i f f e r e n t c e l l concentrations under d i f f e r e n t c o n d i t i o n s . FIGURE 7 P r o l i f e r a t i o n of 3,000 H9 c e l l s / m l (panel a) and 86 3 x 10^ A8 c e l l s / m l (panel b) when plated on i r r a d i a t e d M2-10B4 c e l l s at d i f f e r e n t M2-10B4 c e l l c oncentrations. FIGURE 8 Panel a. P r o l i f e r a t i o n of H9 c e l l s (3000 c e l l s / m l ) 89 seeded i n the presence of various concentrations of M2-10B4 CM harvested at 1, 2, 3 or 5 days a f t e r adding f r e s h medium to the M2-10B4 c e l l s . Panel b: P r o l i f e r a t i o n of H9 c e l l s (3000 c e l l s / m l ) seeded i n the presence of various concentrations of M2-10B4 Day 1 CM a f t e r heating to 56°C f o r 1 hour; and a f t e r f r e e z i n g and thawing once. FIGURE 9 A r e p r e s e n t a t i v e Sephadex G100 p r o f i l e of the H9 91 s t i m u l a t i n g a c t i v i t y present i n a 1 ml sample of 8X concentrated serum-free M2-10B4 CM. FIGURE 10 H9 s t i m u l a t i n g a c t i v i t y i n H9 CM or M2-10B4 CM. 101 FIGURE 11 Southern b l o t a n a l y s i s of Ig H chain gene 106 rearrangements i n normal pre-B c e l l clones and d e r i v a t i v e A-MuLV transformants. FIGURE 12 H9 c e l l s t i m u l a t i n g a c t i v i t y present i n media conditioned by normal and transformed pre-B c e l l l i n e s . 108 I X FIGURE 13 A r e p r e s e n t a t i v e Sephadex G50 p r o f i l e of the pre-B 110 s t i m u l a t i n g a c t i v i t y present i n a 1 ml sample of 50X concentrated serum-free H9 CM. FIGURE 14 Attachment of normal pre-B c e l l s to FN. 118 FIGURE 15 E f f e c t of s y n t h e t i c peptides on the attachment 119 of normal pre-B c e l l s (Bp) to FN. FIGURE 16 Attachment of transformed pre-B c e l l s to FN. 121 FIGURE 17 Panel a. E f f e c t of FN-R and VN-R ant i b o d i e s on the 125 p r o l i f e r a t i o n of normal pre-B c e l l s . Panel b. Role of FN-R and VN-R ant i b o d i e s on the p r o l i f e r a t i o n of transformed pre-B c e l l s . LIST OF ABBREVIATIONS A-MuLV Abelson murine leukemia virus ATCC American Type Culture Collection BSA Bovine serum albumin C Constant cu Cytoplasmic u CFU-GEMM Colony-forming unit-granulocytic, erythroid, megakaryocytic and macrophagic CFU-S Colony-forming unit-spleen CM Conditioned medium CML Chronic myeloid leukemia CR Complement receptor CSF Colony-stimulating factor D Diversity ECM Extracellular matrix EGF Epidermal growth factor Epo Erythropoietin FACS Fluorescent-activated cell sorter FCS Fetal calf serum FFU Focus-forming unit FITC Fluorescein isothiocyanate FN Fibronectin G6PD Glucose-6-phosphate dehydrogenase G-CSF Granulocyte colony-stimulating factor GM-CSF Granulocyte-macrophage colony-stimulating factor H Heavy HGF Hybridoma growth factor I FN Interferon Ig Immunoglobulin IL Interleukin J Joining L Light LCM Leukocyte conditioned medium LM Laminin LPS Lipopolysaccharide LTC Long-term culture M-CSF Macrophage colony-stimulating factor ME Mercaptoethanol MHC Major histocompatibility complex MLR Mixed lymphocyte reaction MLS Minor lymphocyte stimulating antigens Mo-MuLV Moloney murine leukemia virus PBS Phosphate buffer saline PWM-SCCM Pokeweed mitogen-spleen cell conditioned medium RGD Arg-gly-asp SCID Severe combined immune deficient disease slg Surface immunoglobulin TdT Terminal deoxynucleotidyl transferase TGF Transforming growth factor V Variable VN Vitronectin x i ACKNOWLEDGMENTS I wish to express my s i n c e r e g r a t i t u d e : To my s u p e r v i s o r D r . C . J . Eaves f o r her c r i t i c a l sugges t ions , and constant guidance throughout t h i s p r o j e c t , To Dr s . S. Dedhar, R . K . Humphries, G. K r y s t a l f o r h e l p f u l d i s c u s s i o n s and a c t i v e c o l l a b o r a t i o n , To members of my a d v i s o r y committee, Dr s . D. Brunet te and F. Take i f o r t h e i r i n t e r e s t and f o r r e v i e w i n g t h i s t h e s i s , To W. Dragowska and G. Lima for expert t e c h n i c a l a s s i s t a n c e , To M. Coulombe f o r s e c r e t a r i a l a s s i s t a n c e , To La Ligue N a t i o n a l e Franca i se Contre l e Cancer, the Fondat ion pour l a Recherche M e d i c a l e , the P h i l i p p e Foundation and the A s s o c i a t i o n pour l a Recherche Contre l e Cancer fo r t h e i r f i n a n c i a l support . To a l l the s t a f f i n the Terry Fox Laboratory f o r s h a r i n g e x c i t i n g d i s c u s s i o n s and p r o v i d i n g such a s t i m u l a t i n g environment. And f i n a l l y , to my w i f e Freder ique and my sons Romain and X a v i e r f o r t h e i r p a t i e n t support . 1 C H A P T E R I INTRODUCTION 1) HEMOPOIESIS; GENERAL CONCEPTS Blood c e l l s are produced i n the bone marrow and i n the lymphoid organs throughout a d u l t l i f e . Because most of these c e l l s have a shor t l i f e and cannot renew themselves, they must be c o n t i n u o u s l y rep laced so that t h e i r numbers are ma inta ined . The process by which t h i s i s achieved i s known as hemopoies i s . The most p r i m i t i v e hemopoiet ic c e l l i s p l u r i p o t e n t and has the c a p a c i t y to p r o l i f e r a t e , commit and d i f f e r e n t i a t e through d i f f e r e n t h i e r a r c h i c a l steps to e v e n t u a l l y produce a l l of the formed elements of the b l o o d . These f u n c t i o n a l c e l l s , namely the e r y t h r o c y t e s , p l a t e l e t s , lymphocytes and c e l l s of the granulocyte-monocyte s e r i e s (monocytes, n e u t r o p h i l s , e o s i n o p h i l s and b a s o p h i l s ) are necessary f o r oxygen t r a n s p o r t , hemostasis and r e s i s t a n c e to i n f e c t i o n . The hemopoiet ic system has been separated a r b i t r a r i l y i n t o four compartments a ccord ing to the decreas ing p r o l i f e r a t i v e and s e l f - r e n e w a l c a p a c i t i e s of the c e l l s w i t h i n them: ( i ) stem c e l l s , ( i i ) p r o g e n i t o r s of more r e s t r i c t e d p r o l i f e r a t i v e and d i f f e r e n t i a t i v e c a p a c i t y , ( i i i ) m o r p h o l o g i c a l l y r e c o g n i z a b l e , precursors l i m i t e d to a s i n g l e pathway and a few t e r m i n a l d i v i s i o n s , and ( i v ) the f i n a l mature blood c e l l s . The l a s t two compartments comprise most of the c e l l s i n the system. In c o n t r a s t , the two most p r i m i t i v e compartments are much s m a l l e r . As a r e s u l t , these c e l l types are extremely 2 rare even i n the bone marrow ( l e s s than one i n one thousand nucleated c e l l s ) . T h i s , together w i t h the f a c t that they are not morphologically r e c o g n i z a b l e , e x p l a i n s why hemopoietic progenitors can only be studied by i n d i r e c t (developmental) methods, i . e through the generation of t h e i r r e c o g n i z a b l e , mature progeny. An o u t l i n e of the h i e r a r c h i c a l o r g a n i z a t i o n of the hemopoietic system i s presented i n Figure 1. A) Concept of P l u r i p o t e n t Stem C e l l s In 1961 T i l l and McCulloch (1) described the f i r s t assay f o r a hemopoietic c e l l with stem c e l l p r o p e r t i e s . These c e l l s have the c a p a c i t y to form macroscopic c o l o n i e s c o n t a i n i n g various combinations of e r y t h r o i d , g r a n u l o c y t i c , megakaryocytic and u n d i f f e r e n t i a t e d c e l l s i n the spleens of l e t h a l l y i r r a d i a t e d syngeneic mice and are r e f e r r e d to as CFU-S (colony forming u n i t - s p l e e n ) . Subsequent st u d i e s l a i d the b asis of the concept of a hemopoietic stem c e l l compartment c o n s i s t i n g of p l u r i p o t e n t c e l l s w i t h enormous p r o l i f e r a t i v e c a p a c i t y i n c l u d i n g a capacity f o r generating daughter c e l l s w i t h s i m i l a r l y u n r e s t r i c t e d d i f f e r e n t i a t i o n p o t e n t i a l and high p r o l i f e r a t i v e c a p a c i t y (2). A d d i t i o n a l s t u d i e s with both chromosomally ( 3 ) , or more r e c e n t l y r e t r o v i r a l l y (4,5) marked marrow c e l l s have confirmed that these p r o p e r t i e s are features of a small subset of otherwise poorly understood c e l l s i n the marrow. However, i t i s a l s o known that c e l l s defined as CFU-S represent a heterogeneous population with respect to t h e i r s e l f - r e n e w a l and d i f f e r e n t i a t i o n c a p a c i t i e s . The f i r s t spleen c o l o n i e s that are v i s i b l e (seen 5-7 days a f t e r t r a n s p l a n t a t i o n ) are derived from c e l l s that are n e i t h e r p l u r i p o t e n t i a l nor s e l f - m a i n t a i n i n g ( 6 ) , i n contrast to spleen c o l o n i e s v i s i b l e at l a t e r times (12-14 days a f t e r t r a n s p l a n t a t i o n ) . Figure 1. Schematic repre senta t ion of hemopoiesis . OJ 4 In humans, a comparable i n vivo CFU-S assay cannot, obviously be performed. However, stu d i e s of c l o n a l d i s o r d e r s of hemopoiesis such as chronic myelogenous leukemia (CML), where a l l of the c e l l s i n the n e o p l a s t i c clone c a r r y a s p e c i f i c chromosomal marker ( l i k e the P h i l a d e l p h i a (Ph*-) chromosome i n CML) have a l s o demonstrated the existence of a p l u r i p o t e n t hemopoietic stem c e l l population i n man analogous to that i d e n t i f i e d i n the mouse system (7,8). P l u r i p o t e n t hemopoietic c e l l s of both mouse and human o r i g i n can a l s o be detected i n v i t r o by t h e i r a b i l i t y to generate c o l o n i e s c o n t a i n i n g mature c e l l s of m u l t i p l e myeloid lineages (9,10). Those that i n i t i a t e colony growth r a p i d l y and show l i m i t e d s e l f - r e n e w a l capacity are r e f e r r e d to as CFU-GEMM. Others show delayed i n i t i a t i o n of colony growth and are c h a r a c t e r i z e d by a higher s e l f - r e n e w a l c a p a c i t y . As a r e s u l t , they give r i s e to c o l o n i e s which at an e a r l y stage are composed e n t i r e l y of b l a s t s and the c e l l of o r i g i n has been se p a r a t e l y designated as a CFU-blast or S - c e l l (11). Both CFU-GEMM and CFU-blast ( S - c e l l ) progenitors can generate c e l l s detectable i n the mouse as CFU-S. However, t h e i r a b i l i t y to generate lymphoid c e l l s i s not yet c l e a r . B) Concept of Committed Progenitors Committed, or l i n e a g e - r e s t r i c t e d progenitors are considered to be more d i f f e r e n t i a t e d than t h e i r p l u r i p o t e n t precursors because t h e i r d i f f e r e n t i a t i v e p o t e n t i a l has been determined. Current evidence suggests that committed progenitors may possess an extensive p r o l i f e r a t i v e c a p a c i t y but cannot undergo s e l f - r e n e w a l . Committed progenitors are i d e n t i f i e d i n d i r e c t l y by " i n v i t r o " clonogenic assays i n a s e m i - s o l i d c u l t u r e medium co n t a i n i n g appropriate n u t r i e n t s j serum components and a source of crude or s p e c i f i c growth f a c t o r s . 5 D i f f e r e n t committed p r o g e n i t o r s are i d e n t i f i e d by the v a r i o u s types of c o l o n i e s they produce. These are d i s t i n g u i s h e d i n terms of t h e i r u l t i m a t e s i z e , the time r e q u i r e d f o r mature c e l l s to appear, and the f i n a l c e l l u l a r compos i t ion of the c o l o n y . In g e n e r a l , more p r i m i t i v e p r o g e n i t o r s g i v e r i s e to l a r g e r c o l o n i e s which r e q u i r e 2 to 3 weeks to complete t h e i r growth i n c u l t u r e . Dur ing t h i s time many c e l l d i v i s i o n s occur p r i o r to the appearance of t e r m i n a l l y d i f f e r e n t i a t e d n o n - d i v i d i n g blood c e l l progeny. The more mature p r o g e n i t o r s generate c o l o n i e s of mature c e l l s w i t h i n a week i n v i t r o and these are c o r r e s p o n d i n g l y s m a l l e r . In both the human and murine systems, committed p r o g e n i t o r s f o r each of the myeloid pathways and i n some cases f o r d i f f e r e n t s tages of development a long a pathway can now be measured by the use of i n v i t r o co lony assays and appropr i a t e s c o r i n g c r i t e r i a (12-14) . The names used to des ignate each of these are g iven i n F igure 1. I n t e r e s t i n g l y , a l though d i f f e r e n t s t u d i e s have demonstrated a common c e l l o r i g i n f o r lymphoid and myelo id c e l l s (see be low) , there are not as yet analogous " i n v i t r o " co lony assays to i d e n t i f y e a r l y committed B or T lymphocyte p r o g e n i t o r s . C) R e g u l a t i o n of Hemopoietic Stem C e l l s and M y e l o p o i e s i s S e v e r a l l i n e s of evidence suggest that the p r o l i f e r a t i o n and hence the d i f f e r e n t i a t i o n of hemopoiet ic c e l l s i s regu la ted at l e a s t i n part by v a r i o u s growth f a c t o r s as w e l l as by d i r e c t i n t e r a c t i o n s w i t h the f i x e d " s t r o m a l " ' elements of the bone marrow (15 ,16 ) . However, how l i n e a g e r e s t r i c t i o n i s determined i s not known. U l t r a s t r u c t u r a l s t u d i e s of the bone marrow have d i s c l o s e d a number of i n t i m a t e r e l a t i o n s h i p s between hemopoiet ic c e l l s and the v a r i o u s m o r p h o l o g i c a l l y de f ined s t romal popula t ions (17) . Stromal c e l l s of 6 mesenchymal o r i g i n i n c l u d e e n d o t h e l i a l c e l l s , f i b r o b l a s t s / a d v e n t i t i a l c e l l s , fat-accumulating c e l l s or adipocytes, and o s t e o b l a s t s (18). These c e l l s p a r t i c i p a t e i n the formation of a complex e x t r a c e l l u l a r matrix (ECM) of f i b r o u s and non-fibrous proteins i n c l u d i n g d i f f e r e n t types of c o l l a g e n (19), l a m i n i n (LM) (20), f i b r o n e c t i n (FN) (21), v i t r o n e c t i n (VN) (22), proteoglycans (23) and more r e c e n t l y haemonectin (24). Evidence from both i n viv o and/or i n v i t r o experiments demonstrates that stromal c e l l s and ECM p r o t e i n s i n t e r a c t d i r e c t l y w i t h p r i m i t i v e hemopoietic c e l l s and play a r o l e i n the r e g u l a t i o n of t h e i r growth. I t has been shown that radio-induced damage or mechanical d i s r u p t i o n of the stroma can r e s u l t i n l o s s of hemopoietic f u n c t i o n (25-27). The a b i l i t y of hemopoietic c e l l s to home s p e c i f i c a l l y to the bone marrow microenvironment has been shown by the t r a n s p l a n t a t i o n of bone marrow fragments under the kidney capsule of semi-syngeneic animals. This leads to the formation of bone t i s s u e which then becomes populated by hemopoietic c e l l s of r e c i p i e n t o r i g i n (28). Animal models such as mice bearing a l t e r a t i o n s at the S t e e l locus (29) have a l s o provided evidence f o r stromal mediated r e g u l a t i o n of hemopoietic stem c e l l p r o l i f e r a t i o n . Mutant mice of the S l / S l ^ genotype have a d e f e c t i v e hemopoietic system which i s due to a non-transplantable defect of the hemopoietic microenvironment. S l / S l ^ hemopoietic stem c e l l s are i n t r i n s i c a l l y normal (29); and cure of the anemia that c h a r a c t e r i z e s S l / S l ^ mice r e q u i r e s engraftment of an i n t a c t hematopoietic stroma (30). Long-term marrow c u l t u r e s o r i g i n a l l y described by Dexter (31) reproduce i n many respects the features of hemopoietic stem c e l l r e g u l a t i o n seen i n v i v o . This system has been p a r t i c u l a r l y u s e f u l f o r i n v e s t i g a t i o n s of the i n t e r a c t i o n s that can occur between stromal c e l l s and myelopoietic progenitors (32). This long-term marrow c u l t u r e (LTC) system has been analyzed by 7 changing e i t h e r the stromal c e l l or the hemopoietic c e l l components (33). A l s o , as discussed i n greater d e t a i l below, m o d i f i c a t i o n of the c u l t u r e c o n d i t i o n s allows the s e l e c t i v e long-term stromal cell-dependent propagation of B-lineage r e s t r i c t e d c e l l s (34). This l a t t e r system has been very u s e f u l to study the r e g u l a t i o n of B-lymphopoietic c e l l p r o l i f e r a t i o n . ECM i s an important component of the hemopoietic microenvironment and i s thought to mediate many of i t s r e g u l a t o r y a c t i o n s on the growth of hemopoietic c e l l s (35,36). The a v a i l a b i l i t y of p u r i f i e d ECM molecules has been u s e f u l to study how these molecules i n t e r a c t s p e c i f i c a l l y with hemopoietic c e l l s . For example, i t has r e c e n t l y been shown that hemopoietic c e l l s and p a r t i c u l a r l y B lymphoid progenitors can bind to FN (37,38). Haemonectin, seems to be a s p e c i f i c attachment molecule f o r c e l l s of the g r a n u l o c y t i c l i n e a g e (24). Collagen, appears to be c r u c i a l f o r the establishment of a stroma supportive of hemopoiesis i n LTC as shown by stu d i e s i n which the d e p o s i t i o n of c o l l a g e n was i n h i b i t e d i n the presence of a r e l a t i v e l y s p e c i f i c i n h i b i t o r l i k e C i s-4-hydroxyproline (39,40). LM binds to type IV co l l a g e n (41), and seems to regulat e the types of macromolecules that pass across the basement membrane (42). Thus d i f f e r e n t ECM pro t e i n s may have d i f f e r e n t r e g u l a t o r y f u n c t i o n s . Some may serve p r i m a r i l y a mechanical r o l e to allow a clos e a s s o c i a t i o n between hemopoietic c e l l s and stromal c e l l s to be maintained. As a consequence the hemopoietic c e l l s would be maintained i n c l o s e p r oximity to stromal c e l l derived growth f a c t o r s . Other ECM components may c o n t r o l the re l e a s e of growth f a c t o r s synthesized e i t h e r l o c a l l y or elsewhere and present them or r e t a i n them i n a b i o l o g i c a l l y a c t i v e form f o r p r e s e n t a t i o n to hemopoietic p r o g e n i t o r s . Such a r o l e has been r e c e n t l y documented f o r heparan sulphate; This glycosaminoglycan i s the component of the marrow ECM produced 8 i n LTC to which granulocyte-macrophage colony s t i m u l a t i n g f a c t o r (GM-CSF) i s found to be s e l e c t i v e l y bound (36,43). D) M y e l o p o i e t i c Growth Factors and Hemopoiesis The hemopoietic growth f a c t o r s (HGF) are a l l g l y c o p r o t e i n s that exert r e g u l a t o r y a c t i v i t i e s on hemopoietic c e l l s . A l a r g e number have now been p u r i f i e d , and cloned, and i n some cases t h e i r r e s p e c t i v e receptors p a r t i a l l y c h a r a c t e r i z e d (44). This has allowed the b i o l o g i c a l a c t i v i t i e s of unique, defined r e g u l a t o r y molecules to be studied separately both i n v i t r o and i n v i v o . These molecules have, however, turned out to be extremely complex i n t h e i r range of e f f e c t s on d i f f e r e n t target c e l l types and t h e i r mechanisms of a c t i o n at the c e l l u l a r and s u b c e l l u l a r l e v e l are not yet e l u c i d a t e d . HGF's that s t i m u l a t e myeloid progenitors can be o p e r a t i o n a l l y separated i n t o two c a t e g o r i e s : f i r s t , the colony s t i m u l a t i n g f a c t o r s (CSF's) that s t i m u l a t e d i r e c t l y the p r o l i f e r a t i o n and the d i f f e r e n t i a t i o n of hemopoietic p r o g e n i t o r s ; and second, s y n e r g i s t i c f a c t o r s which alone appear devoid of i n t r i n s i c myeloid c o l o n y - s t i m u l a t i n g a c t i v i t y but can enhance the e f f e c t s of a CSF. The best c h a r a c t e r i z e d of these molecules, some of t h e i r p r o p e r t i e s and b e t t e r known e f f e c t s on myeloid c e l l s are summarized i n Table 1. As a l s o shown i n Table 1, most of the HGF's are produced by e i t h e r a c t i v a t e d stromal c e l l s or a c t i v a t e d T c e l l s . Only Epo, which i s produced by the kidney, i s considered as a c l a s s i c a l hormone. I t i s important to point out that amongst the d i f f e r e n t CSF's a h i e r a r c h i c a l spectrum of a c t i v i t y and a complex set of i n t e r a c t i o n s appear to e x i s t . Growth f a c t o r s able to act on more p r i m i t i v e c e l l s (e.g. I L - 3 , and GM-CSF) can down-regulate receptors f o r growth f a c t o r s of more r e s t r i c t e d Table 1. Hemopoietic Growth F a c t o r s 3 A l t e r n a t i v e Name(s) C e l l u l a r O r i g i n (no rmal) M o l e c u l a r Weight C e l l s Responsive to F a c t o r Alone S y n e r g i s t i c A c t i v i t y GM-CSF (Granulocyte-macrophage c o l o n y - s t i m u l a t i n g f a c t o r ) CSF-2 MGI A c t i v a t e d T lymphocytes F i b r o b l a s t s E n d o t h e l i a l c e l l s 23 ,000 CFU-GM CFU-mix BFU-E G r a n u l o c y t e s Macrophages + CSF-1 s t i m u l a t e s p l u r i p o t e n t stem c e l l s G—CSF ( G r a n u l o c y t e c o l o n y -s t i m u l a t i n g f a c t o r ) A c t i v a t e d T lymphocytes Mac rophages F i b r o b l a s t s 25,000 S u b - p o p u l a t i o n of CFU-GM (CFU-G) G r a n u l o c y t e s M u l t i - C S F ( M u l t i p o t e n t i a l c o l o n y - s t i m u l a t i n g f a c t o r ) IL-3 BPA PSF A c t i v a t e d T lymphocytes 23-28,000 Stem c e l l s CFU-GEMM BFU-E CFU-GM CFU-MK Mast C e l l s + CSF-1 Epo M-CSF (Macrophage colony-s t i m u l a t i n g f a c t o r ) CSF-1 A c t i v a t e d mac rophages F i b r o b l a s t s 70,000 S u b — p o p u l a t i o n of CFU-GM Macrophages Epo ( E r y t h r o p o i e t i n ) Kidney p e r i -t u b u l a r c e l l : 39,000 CFU-E Mature BFU-E EDF ( E o s i n o p h i l i c d i f f e r e n t i a t i o n f a c t o r ) IL-5 TRF BCGF I I T c e l l s 45-60,000 CFU-Eos B c e l l s a T h e i n f o r m a t i o n shown i s r e p r e s e n t a t i v e , not a l l i n c l u s i v e . The lymphoid c e l l s t i m u l a t i n g a c t i v i t y of many of these f a c t o r s i s summarized i n Table 2. Table 1. Hemopoietic Growth F a c t o r s 3 - Continued Name A l t e r n a t i v e Name(s) C e l l u l a r O r i g i n (No rraal) F a c t o r Mo 1ecula r Weight C e l l s Responsive to F a c t o r Alone Syne r g i s t i c A c t i v i t y I n t e r l e u k i n - 1 (IL-1) LAF Hemopo i e t i n-1 A c t i v a t e d monocytes E n d o t h e l i a l c e l l s a f t e r exposure to endotoxin or TNF-a 13-19,000 T and B lymphocytes Hepatocytes S t i m u l a t e s f i b r o b l a s t s & e n d o t h e l i a l c e l l s to produce G-CSF & GM-CSF + CSF-1 or IL-3 s t i m u l a t e s stem c e l l s I n t e r l e u k i n - 4 (IL-4) BSF-1 BCGF-I A c t i v a t e d T lymphocytes 16-21,000 B c e l l s + Epo, G-CSF, GM-CSF or IL-3 s t i m u l a t e s hemopoietic p r o g e n i t o r s I n t e r l e u k i n - 6 (IL-6 ) BSF-2 HGF IFN-02 F i b r o b l a s t s T lymphocytes Monocytes 26,000 B c e l l s Hepatocytes + IL-3 s t i m u l a t e s p l u r i p o t e n t c e l l s TC-1 a c t i v i t y TC-1 c e l l l i n e 200,000 + CSF-1, I L - 3 , or GM-CSF s t i m u l a t e s h e m o p o i e t i c p r o g e n i t o r s a T h e i n f o r m a t i o n shown i s r e p r e s e n t a t i v e , not a l l i n c l u s i v e , i s summarized i n Table 2. The lymphoid c e l l s t i m u l a t i n g a c t i v i t y of many of these f a c t o r R e f e r e n c e s 44-60. 11 l i n e a g e a c t i o n (e.g. G-CSF, M-CSF, Epo) (61) and may a l s o synergize t h e i r a c t i v i t y (59). I n t e r e s t i n g l y , many of the s y n e r g i s t i c f a c t o r s a l s o have e f f e c t s on lymphoid c e l l s (see below). Regulation of myelopoiesis a l s o i n v o l v e s i n h i b i t o r y molecules. Various i n h i b i t o r s of hemopoiesis i n c l u d i n g l a c t o f e r r i n , t r a n s f e r r i n , a c i d i c i s o f e r r i t i n s , E-type prostaglandins, i n t e r f e r o n (IFN), c a c h e c t i n (TNF-a), lymphotoxin (TFN-0), chalones, have been reported (62-65). More r e c e n t l y , transforming growth f a c t o r - 3 (TGF-P), o r i g i n a l l y known f o r i t s a b i l i t y to confer anchorage-independent growth on non-malignant f i b r o b l a s t s (66), has been shown to be a potent i n h i b i t o r of hemopoietic c e l l p r o l i f e r a t i o n (67). TGF-P i s a h i g h l y conserved homodimer of 25,000 daltons produced by a v a r i e t y of c e l l s . Two c l o s e l y r e l a t e d forms: TGF-gi and TGF-f32> that bind to three d i s t i n c t forms of receptors, e x i s t (68). TGF-P^ i n h i b i t s the c y c l i n g of the most p r i m i t i v e myeloid progenitor c e l l types i n both mouse and human marrow (69) . I n t e r e s t i n g l y , TGF-3 i s a l s o a potent i n h i b i t o r of B lymphopoiesis (70) . This f a c t o r a l s o increases the production of ECM FN and type I c o l l a g e n by f i b r o b l a s t s and e p i t h e l i a l c e l l s (71,72). E) Summary < Hemopoiesis may be viewed as a c e l l u l a r pyramid i n which very few stem c e l l s g i v e r i s e to mature blood c e l l s . The r e g u l a t i o n of hemopoiesis i n v o l v e s a set of complex i n t e r a c t i o n s between hemopoietic c e l l s , stromal c e l l s and t h e i r products. The l a t t e r i n c l u d e ECM p r o t e i n s as w e l l as various s t i m u l a t o r y and i n h i b i t o r y molecules. 12 2) ORIGIN AND DEVELOPMENT OF B CELLS A) R e l a t i o n s h i p Between B Lymphoid Precursors and Other Hemopoietic  Lineages The sequence of d i f f e r e n t i a t i o n events that leads to the generation of c e l l s r e s t r i c t e d to the B and T lineages i s s t i l l poorly understood. When CFU-S assays were f i r s t described, no evidence of lymphoid c e l l s were found i n the spleen c o l o n i e s produced ( 1 ) . However, chromosomal markers s t u d i e s subsequently enabled the presence of a common stem c e l l of both lymphoid and myeloid c e l l s to be demonstrated (73). Nevertheless, i n one such study, Abramson et a l (74) found a chimerism i n T c e l l s only, i n myeloid c e l l s o nly, or i n B c e l l s plus T c e l l s and myeloid c e l l s . Therefore, the exi s t e n c e of d i f f e r e n t populations of stem c e l l s , i n c l u d i n g those g i v i n g r i s e to non-lymphoid c e l l s only, and those r e s t r i c t e d to T-lineage d i f f e r e n t i a t i o n , as w e l l as t o t a l l y u n r e s t r i c t e d stem c e l l s , was suggested. Very r e c e n t l y , chromosomally marked b l a s t colony c e l l s , generated i n v i t r o i n the presence of a cloned supportive adherent c e l l l i n e , were transplanted i n t o l e t h a l l y i r r a d i a t e d r e c i p i e n t s and shown to produce both myeloid and lymphoid progeny i n the marrow, spleen, mesenteric lymph node and thymus (75). Although these methods have served to e s t a b l i s h the existence of stem c e l l s w i t h both lymphoid and myeloid p o t e n t i a l , k a r y o t y p i c a l l y v i s i b l e but n o n - l e t h a l l e s i o n s cannot be generated at high frequency. Therefore, the random s i t e s of r e t r o v i r a l i n s e r t i o n i n t o hemopoietic c e l l DNA, have proved very u s e f u l f o r i d e n t i f y i n g c l o n a l hemopoietic populations generated i n mice given marrow t r a n s p l a n t s , and hence f o r lineage mapping. Such s t u d i e s (5,76) have shown one stem c e l l can repopulate the spleen, lymph nodes, thymus and 13 bone marrow. These r e s u l t s i n d i c a t e unequivocally the existence of hemopoietic stem c e l l s with both lymphoid and myeloid p o t e n t i a l . In humans, evidence f o r the r e l a t i o n s h i p between lymphoid and hemopoietic c e l l s came from the a n a l y s i s of glucose-6-phosphate dehydrogenase (G6PD) heterozygote women. E l e c t r o p h o r e t i c a l l y d i s t i n c t a l l e l i c forms of G6PD are encoded on the X chromosome, and i n d i v i d u a l somatic c e l l s of heterozygous women express only one of the two a l l e l e s , as a r e s u l t of l y o n i z a t i o n e a r l y i n embryogenesis. Therefore, hematologic d i s o r d e r s that o r i g i n a t e i n a s i n g l e c e l l express only a s i n g l e enzyme phenotype whereas most t i s s u e s o r i g i n a t e from m u l t i p l e c e l l s and therefore show a mixed G6PD population i n female heterozygotes. By a n a l y z i n g the type of G6PD isoenzyme expressed i n d i f f e r e n t c e l l s from a woman with acquired i d i o p a t h i c s i d e r o b l a s t i c anemia, P r c h a l et a l (77) showed that both B and T lymphocytes and myeloid c e l l s came from a common stem c e l l . S i m i l a r l y , G6PD a n a l y s i s and cytogenetic i d e n t i f i c a t i o n of the Phi-chromosome i n CML have shown that the same isoenzyme patt e r n and a l s o the chromosome Ph* are expressed i n both myeloid and B lymphoid c e l l s . These data i n d i c a t e that CML i s a c l o n a l disease r e s u l t i n g from the transformation of a p l u r i p o t e n t stem c e l l common to these pathways (7,78,79). Attempts to determine whether T c e l l s are a l s o involved i n CML, have y i e l d e d l e s s c o n c l u s i v e data and t h i s i s s t i l l a matter of controversy (80). However, rare r e p o r t s i n d i c a t i n g that the T lineage may be involved i n CML (81,82), provide f u r t h e r support f o r the existence of a common p l u r i p o t e n t stem c e l l i n humans a l s o . 14 B) Ontogeny of Lymphopoiesis The B c e l l l ineage receives i t s name from i t s o r i g i n i n the bursa of F a b r i c i u s i n b i r d s and the bone marrow i n mammals. Because B c e l l s and a l l other blood c e l l types o r i g i n a t e from a common p l u r i p o t e n t stem c e l l , t h e i r ontogeny shares some s i m i l a r i t i e s . The embryonic phase of hemopoiesis can be summarized as f o l l o w s . P l u r i p o t e n t hemopoietic stem c e l l s are thought to der i v e from mesenchymal c e l l s l ocated i n the region of the p r i m i t i v e kidneys e a r l y i n embryonic l i f e (29). On migrating i n t o the y o l k sac blood i s l a n d s some of these c e l l s begin to d i f f e r e n t i a t e along the e r y t h r o i d and myeloid pathways. D i f f e r e n t i a t i o n of T and B c e l l s begins l a t e r a f t e r the primary lymphoid organs have developed and i t i s thought that these may provide unique i n d u c t i v e microenvironments e s s e n t i a l f o r B and T c e l l development. In b i r d s , the ontogeny of B c e l l s i s h i g h l y dependent on a s i n g l e t i s s u e , the bursa of F a b r i c i u s . The bursa i s a unique organ, that a r i s e s at day 5 of embryonic l i f e as a d o r s a l d i v e r t i c u l u m of the cloaca. S u r g i c a l bursectomy during e a r l y embryonic development induces severe agammaglobulinemia and r e s u l t s i n the f a i l u r e to mount an immune response to any immunizing antigen (83). Stem c e l l s c o l o n i z e the bursa from the general c i r c u l a t i o n between days 8 to 14 of in c u b a t i o n . They migrate through the b u r s a l mesenchyme where a few may a l s o give r i s e to granulocytes and e r y t h r o i d c e l l s . When they reach the b u r s a l e p i t h e l i u m , they d i v i d e and d i f f e r e n t i a t e to form i n t r a e p i t h e l i a l f o l l i c l e s (84,85). Each f o l l i c l e gives r i s e to a population of c e l l s that express immunoglobulin M (IgM) on t h e i r surfaces. Ninety percent of b u r s a l c e l l s are B lymphocytes by day 20 of embryonic development ( c h i c k s hatch a f t e r the 20th day of i n c u b a t i o n ) . B u r s a l development invol v e s two phases: the intraembryonic phase, which includes the c o l o n i z a t i o n and growth of about 10^ 15 B c e l l s i n the b u r s a l f o l l i c l e s and the post-hatching pe r i o d , which i n c l u d e s the c o n t i n u i n g expansion of the b u r s a l f o l l i c l e s and the seeding of b u r s a l B c e l l s to the periphery. By 4 weeks of age a s u f f i c i e n t number of c e l l s has migrated out of the bursa as postbursal B lineage stem c e l l s , to ensure establishment of the mature chicken B c e l l immune system i n the periphery (86). At t h i s stage, B c e l l s can no longer be generated from a more p r i m i t i v e multipotent stem c e l l because the bursa has i n v o l u t e d and can no longer provide the microenvironment f o r t h i s e a r l y step to occur. In mammals B c e l l s are produced i n the same hemopoietic t i s s u e along w i t h a l l of the other types of blood c e l l s except T c e l l s . Thus, B lymphoid ontogeny i s not r e s t r i c t e d to a s i n g l e t i s s u e and a mammalian t i s s u e e q uivalent of the bursa of F a b r i c i u s of b i r d s has not been demonstrable. I n i t i a l l y various gut-associated lymphoid t i s s u e s , i . e . the appendix, the t o n s i l s and the Peyer's patches, were proposed as p o s s i b l e mammalian b u r s a l e q u i v a l e n t s because t h e i r s u r g i c a l removal induced defects i n antibody formation (29). A c t u a l l y , these t i s s u e s e x h i b i t many features t y p i c a l of secondary lymphoid t i s s u e s . The y o l k sac of the murine embryo was a l s o considered as a candidate equivalent of the bursa i n mammals; however, n e i t h e r I g - s y n t h e s i z i n g , nor l i p o p o l y s a c c h a r i d e (LPS)-responsive B l i n e a g e c e l l s , nor even immediate precursors of B c e l l s have been detected i n the y o l k sac (87). Nevertheless, CFU-S can be detected the murine y o l k sac and hemopoietic stem c e l l s capable of lymphoid d i f f e r e n t i a t i o n are a l s o present w i t h i n the blood i s l a n d s of t h i s extraembryonic t i s s u e i n 8--10 mouse day embryos (88). I t has therefore been suggested that hemopoietic stem c e l l s a r i s e de novo only i n t h i s extraembryonic t i s s u e and subsequently migrate v i a the bloodstream to c o l o n i z e the various lymphoid and hemopoietic organs of the embryo such as the f e t a l l i v e r and bone marrow (88,89). 16 The l i v e r i s an a c t i v e blood-forming t i s s u e during embryonic development before the bone marrow takes over t h i s f u n c t i o n . Hemopoietic a c t i v i t y i n the murine f e t a l l i v e r begins around the 10th day of embryonic l i f e and l a s t s u n t i l a few days a f t e r b i r t h . The f i r s t i d e n t i f i a b l e c e l l s of the B lymphocyte li n e a g e expressing the B220 surface antigen appear i n the f e t a l l i v e r on day 12 of g e s t a t i o n (88). At t h i s stage no such c e l l s can be detected i n e i t h e r the y o l k sac or the embryonic c i r c u l a t i o n . C e l l s c o n t a i n i n g cytoplasmic u chains ( c u + ) are detectable i n 10-12 day mouse embryos. The f i r s t surface immunoglobulin ( s l g ) - b e a r i n g B c e l l s emerge l a t e r , between 16 and 17 days of g e s t a t i o n (88,90,91). Between 13 and 16 days of g e s t a t i o n , there i s a dramatic increase i n the number of committed B - c e l l precursors i n f e t a l l i v e r . These give r i s e to LPS-responsive B - c e l l s (87). A l l of these data i n d i c a t e that an important stage of the development of the humoral immune system i n mammals occurs w i t h i n the f e t a l l i v e r . The production of B - c e l l s l a t e r s h i f t s to the bone marrow when i t becomes the primary blood-forming t i s s u e . Thus, long bones from 15-day-old mouse fetuses can generate B c e l l s i n t h e i r marrow c a v i t i e s a f t e r s e v e r a l days i n c u l t u r e (87). A f t e r b i r t h , the bone marrow continues to be the major s i t e of B - c e l l formation. The production of small lymphocytes i n the marrow of mice has been estimated to be approximately 10^ c e l l s per day (92). While s m a l l lymphocytes 1 represent one quarter of the nucleated c e l l s , the bone marrow a l s o contains precursor c e l l s at various stages of maturity. Some of the newly-formed small lymphocytes leave the bone marrow and migrate to the spleen. 17 C) Development of B C e l l s i n the A d u l t The most important f u n c t i o n of B c e l l s as e f f e c t o r s of the humoral immune system i s to s y n t h e s i z e a n t i b o d i e s ( I g ) . B lymphoid p r e c u r s o r s , t h a t , themselves , o r i g i n a t e from p l u r i p o t e n t stem c e l l s d i f f e r e n t i a t e p r o g r e s s i v e l y i n t o pre-pre-B c e l l s , pre-B c e l l s , immature B c e l l s , mature B c e l l s , and a c t i v a t e d B c e l l s which f i n a l l y become I g - s e c r e t i n g plasma c e l l s . T h i s process i s c h a r a c t e r i z e d by a cascade of molecular events . I t i s convenient to cons ider the process of murine B - c e l l development and d i f f e r e n t i a t i o n i n three phases. The e a r l i e s t phase of B - c e l l development i s ant igen- independent . I t begins w i t h the commitment of p l u r i p o t e n t hemopoiet ic stem c e l l elements to the B - c e l l pathway and the i n i t i a t i o n of Ig gene rearrangement. Cytop la smic u p r o t e i n may then be de tec ted , which i s i n t u r n fo l lowed by the expre s s ion of c e l l sur face I g . Th i s serves as the an t igen s p e c i f i c receptor and represent s the d e f i n i n g c h a r a c t e r i s t i c of the mature B c e l l ( 8 8 , 9 1 , 9 3 ) . Mature B - c e l l s capable of responding to ant igen enter the c i r c u l a t i o n and then migrate to the p e r i p h e r a l lymphoid t i s s u e s where they are a c t i v a t e d by contact w i t h an t igen i n concert w i t h secondary f a c t o r s . T h i s a c t i v a t i o n process c o n s t i t u t e s the second phase of B c e l l development and d i f f e r e n t i a t i o n . F i n a l l y , s p e c i f i c a l l y a c t i v a t e d B c e l l s enter S-phase, p r o l i f e r a t e and begin to produce and secre te l a rge amounts of Ig i n t o the lymphat ic and v a s c u l a r spaces . In a d d i t i o n , some may undergo c l a s s s w i t c h i n g and some may r e t u r n to a quiescent s t a t e as "memory" B c e l l s . 18 (a) Ig Gene Rearrangement and Expression. The molecular events that underlie the commitment of pluripotent stem cells to the lymphoid pathway remain poorly understood because of the difficulties in obtaining pure stem cell populations and the lack of antigenic and molecular markers to facilitate the identification of the first B-restricted stem cells. Nevertheless, substantial progress has been made in delineating the process of Ig gene rearrangement, the earliest known step in the differentiation of pre-pre-B cells. The structural organization of the murine Ig genes is shown diagrammatically in Figure 2. Ig molecules are constructed from three sets of unlinked genes. These encode the various heavy (H) chains, and the K and X light (L) chains. Each of these is, in turn, made up of multiple germline DNA segments which are somatically assembled during the differentiation of B-lineage cells. Ig genes include variable (V) region genes and constant (C) region genes. The variable region of the H chain gene is encoded by 3 germline DNA segments; a V(H) (which encodes the bulk of the gene) which is linked to a D (diversity) segment, which is in turn linked to a J(H) (joining) segment (94,95). There are 4 J(H) segments which l ie approximately 7 kb upstream from the exons that encode the first H chain C region gene expressed during development, the Cy gene. At a distance of from 1 to 80 kb upstream from the J(H) segments l ie approximately 12 D segments. At an unknown distance upstream from the D segments l ie approximately 200 to 1000 V(H) gene segments. D and V segments can be divided into families based on their nucleic acid sequence homology (96). In mice, the K gene, which produces 90% of the serum L chains, is organized in a similar manner as the H chain genes, but it does not have any D segments. The murine X locus is organized in a VH(200-1000) v H x v H 2 v H 1 DH(12) D X D 2 JH(4) Enhancer Constant regions(8) C/x C 5 C-yg C<yi C-y2b ^72a Ce l l l l ' Heavy chain gene 5'-VK (200) VKX VKN VJ a-o-o-J* (5) Enhancer I c K H i l l • M •3' Kappa gene 51 V\2 JX2 C\2 J\4 C\4 V\1 J\3 C\ 3 J\1 C\1 O-i—• • • 3' Lambda gene Figure 2. Organ iza t ion of immunoglobulin genes. 2 0 somewhat d i f f e r e n t fashion with only 2 genes, each of which i s followed downstream by 2 J\-C\ u n i t s . The a v a i l a b i l i t y of tumor c e l l l i n e s and p a r t i c u l a r l y of Abelson murine leukemia v i r u s (A-MuLV) transformed pre-B c e l l l i n e s r e presenting the v a r i o u s stages of pre-B development have been u s e f u l f o r a n a l y s i s of the various ways i n which V(H), D, and J(H) elements may be rearranged (97,98). Recombination between the V region gene elements i s apparently mediated by conserved recombination r e c o g n i t i o n sequences. These c o n s i s t of a palindromic heptamer and a c h a r a c t e r i s t i c nonamer, separated by a spacer of 12 or 23 base p a i r s that d i r e c t s the recombinational machinery (99,100). This process leads to d e l e t i o n from the chromosome of the sequences between the i n v o l v e d V(H) and DJ(H) elements. However, random i n s e r t i o n of small numbers of n u c l e o t i d e s by an enzyme c a l l e d terminal deoxynucleotidyl transferase (TdT) may a l s o occur (101,102). A l t et a l (95) have proposed an ordered mechanism of V(H)DJ(H) gene c o n s t r u c t i o n during pre-B c e l l d i f f e r e n t i a t i o n i n which the f i r s t event i s the j o i n i n g of a D segment to a J(H) segment. This occurs on both chromosomes. Subsequently, the complete V(H)DJ(H) v a r i a b l e region i s assembled v i a the appendage of a V(H) segment to the p r e - e x i s t i n g DJ(H) complex. The complexity of the combinatorial assortment of the d i f f e r e n t component gene segments account i n part f o r the d i v e r s i t y of the s p e c i f i c i t i e s of the population of a n t i b o d i e s ( i . e . Ig) which c o n s t i t u t e the immune r e p e r t o i r e . I t has been c a l c u l a t e d that the combination of one H and one L chain per antigen-combinating s i t e gives the system approximately 10^ p o s s i b l e antigen-combining s i t e s (103). I t i s important to point out that incompletely assembled V region gene segments may be expressed, and that other aberrant and non-productive rearrangements a l s o occur at a r e l a t i v e high frequency (95,104). Such 21 rearrangements, that are t r a n s c r i b e d but do not encode f u n c t i o n a l I g , seem to be involved i n the r e g u l a t i o n of Ig V region gene assembly (95). Once V region genes are completely assembled, t r a n s c r i p t i o n i s i n i t i a t e d from a promoter which l i e s upstream of each germline V region gene (95). The primary t r a n s c r i p t extends downstream through the exons which encode the f i r s t C region gene expressed during development, the Cy gene. Fo l l o w i n g appropriate RNA s p l i c i n g events, the V(H)DJ(H) sequences are fused to the C(H) coding sequences to form the complete H chain mRNA. H chain V region genes are assembled e a r l i e r during pre-B c e l l d i f f e r e n t i a t i o n than are the genes f o r the L chains. However, a s i m i l a r process i s involved. Complete messenger f o r Ig H chains c a r r i e s information f o r both a membrane-bound and a secreted form of the y p r o t e i n . I t has been observed that i n a c t i v e s y n t h e s i s of I g molecules, only one of a p a i r of chromosomes i s i n v o l v e d . This process i s c a l l e d a l l e l i c e x c l u s i o n (98,105). Studies of the assembly and expression of Ig genes during the a n t i g e n -independent stages of pre-B c e l l d i f f e r e n t i a t i o n has revealed a c o n s i s t e n t sequence of events that are now used to define d i f f e r e n t stages of pre-B c e l l development (Figure 3). A " n u l l " pre-B c e l l which has DJ(H) rearrangements on both chromosomes and no L chain gene rearrangements i s the f i r s t stage that can be i d e n t i f i e d . Such a very e a r l y pre-B c e l l , by making a productive V(H)DJ(H) rearrangement, then becomes a c y + pre-B c e l l . A f r a c t i o n of these pre-B c e l l s then undergo rearrangement and expression of e i t h e r t h e i r K or X L chain genes i n order to assemble complete IgM subunits as a n t i g e n - s p e c i f i c receptors on the c e l l surface (106,107). These sIgM + B c e l l s c o n s t i t u t e the most immature B c e l l s . With f u r t h e r maturation B c e l l s begin to express surface IgD molecules and other surface antigens (see below). At t h i s stage, mature B c e l l s are capable of binding and responding to s p e c i f i c antigens. BONE MARROW Peripheral Blood Stem Cell Proliferating Precursors Non-dividing Small Lymphocytes Mature B Lymphocytes Oo°o°o Pre—Pre—B cells Pre—B cells B Lymphocytes low IIIII Thy-I TdT B 220 ThB BP-I/6C3 O S/x HC gene G/G LC gene G/G II II II II II II II I II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II I II II II II II II II II II I II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II G/G G/G G/G G/G R/G G/G R/R G/G R/R R/R R/R G/G-G/R G/R G/R and/or or or R/R R/R R/R Figure 3. Surface ant igen express ion and Ig gene rearrangement d u r i n g murine B - c e l l development. ro 23 During the course of the response to antigen, another kind of gene rearrangement termed H chain class switching occurs. This phenomenon allows a single clone of B cells to generate progeny which maintain the same antigen binding specificity linked to different C(H) effector functions like those of IgG or IgA (108,109,110). (b) Changes in Surface Antigens. For a long time, surface markers that could be used for detecting and isolating B cell precursors were not known and the only unique marker for distinguishing pre-B cells was the presence of cu. However, detection of cu requires fixation of the cells and could therefore not be used as a basis for isolating viable pre-B cells. Nevertheless, measurements of pre-B population size and cell kinetic parameters could be performed and these showed that actively proliferating large pre-B cells give rise to small pre-B cells, which then, without further division, differentiate into B lymphocytes bearing surface u H chains (sy) in whole IgM molecules (111). With the introduction of monoclonal antibody (MoAb) technology, several groups began to make monoclonal antibodies that recognize antigens present on pre-B cells. Only the best characterized antigens expressed during the earlier stages of murine B-lineage development will be reviewed here. Figure 3 summarizes surface antigen expression and Ig gene rearrangement during murine B-cell development. B220 is a large (220 kD) surface glycoprotein detected by several monoclonal antibodies (112,113) and has been useful to detect various stages of pre-B cell development. B220 is related to the T200 antigen. T200 is also called Ly-5, a protein family init ial ly described on T cells but now known to be expressed by most hemopoietic cells (114,115). Immunofluorescent studies 24 performed with anti-B220 MoAb's have, however, shown that the expression of B220 i s B - l i n e a g e - s p e c i f i c . Indeed, the f i r s t immunofluorescent s t a i n i n g of d i f f e r e n t lymphoid organs with anti-B220 showed that about 30% of bone marrow c e l l s , 50% of spleen c e l l s , 40% of mesenteric lymph nodes and l e s s than 2% of thymocytes were p o s i t i v e (116). Experiments using the f l u o r e s c e n t - a c t i v a t e d c e l l s o r t e r (FACS) to analyze and separate doubly immunofluorescent c e l l s l a b e l l e d w i t h B220 Mo-Abs and a n t i - y or a n t i - K a n t i b o d i e s have subsequently demonstrated that B220 i s expressed by a l l B lymphocytes, as w e l l as by both s m a l l and l a r g e pre-B c e l l s i n mouse bone marrow (116). Some f i n d i n g s i n d i c a t e that mouse bone marrow a l s o contains a population of B220 + c e l l s that do not contain u chains (107,111) suggests that B220 expression may precede y chain s y n t h e s i s . Very r e c e n t l y , Park and Osmond (111) have reported the e x i s t e n c e of three d i s c r e t e cy~ large pre-B c e l l populations. The e a r l i e s t c e l l s are TdT+ B220~ cy~. The next c e l l s are TdT+ B220+ cy", and the t h i r d p o pulation i s TdT~ B220+ cy~. The ThB antigen was i n i t i a l l y described on mouse myeloma c e l l s , but has a l s o been detected on B c e l l s and approximately 50% of thymocytes, but not on p e r i p h e r a l T c e l l s (116,117). Immunofluorescence s t a i n i n g with anti-ThB MoAb has shown that 15-20% of bone marrow c e l l s are ThB + and that n e a r l y a l l of these are small lymphocytes (116). S o r t i n g and r e s t a i n i n g w i t h e i t h e r B220 or s l g MoAb demonstrated that a l l ThB + c e l l s are a l s o B220+ and one h a l f of them are s l g ~ , s i n c e the other h a l f are s l g + c e l l s (116). These data have allowed the d e f i n i t i o n of a population of small B220 + ThB+ s l g - c e l l s that represent the most mature pre-B c e l l stage. These mature without f u r t h e r d i v i s i o n i n t o s l g + B c e l l s expressing both y and L chains. Thy-1 was one of the f i r s t mouse lymphocyte d i f f e r e n t i a t i o n antigens to be discovered (118,119). I t was i n i t i a l l y described as a marker of thymocytes 25 and thymus-dependent c e l l s i n the spleen (118). L a t e r , i t was shown that low amounts of Thy-1 are detectable on p l u r i p o t e n t stem c e l l s (120). This has allowed the d i f f e r e n t i a t i o n of Thy-l +(l° w) stem c e l l s i n t o pre-pre-B c e l l s to be i n v e s t i g a t e d . Using a c o c k t a i l of monoclonal an t i b o d i e s d i r e c t e d against B220, Thy-1, and d i f f e r e n t mature T c e l l and myeloid antigens to s o r t normal bone marrow c e l l subpopulations with the capacity to e i t h e r repopulate i r r a d i a t e d mice or to generate B-lymphoid long-term c u l t u r e s (see below), two p h e n o t y p i c a l l y d i s t i n c t B c e l l precursors were i s o l a t e d (121). One (Thy-1~, B220 -) can i n i t i a t e pre-B c e l l c u l t u r e s i n which B220 + c e l l s are r a p i d l y produced but only f o r ~1 weeks. The second ( T h y - l + ( l o w ) , B 2 2 0 - ) can i n i t i a t e long-term B220+ pre-B c e l l c u l t u r e s and can a l s o r e c o n s t i t u t e hemopoiesis i n l e t h a l l y i r r a d i a t e d congenic mice. These r e s u l t s i n d i c a t e that t h i s second population (-0.1% of bone marrow) contains p l u r i p o t e n t hemopoietic stem c e l l s . BP-1 was r e c e n t l y described by Cooper et a l and i s another surface marker of the e a r l y pre-B c e l l d i f f e r e n t i a t i o n (122). By FACS a n a l y s i s , anti-BP-1 MoAb s t a i n s approximately 10% of nucleated c e l l s i n the bone marrow of which 90% are pre-B and newly formed B c e l l s . The s t a i n i n g i n t e n s i t y of BP-1 i s r e l a t i v e l y low on normal pre-B c e l l s . Transformed pre-B c e l l s e x h i b i t an higher expression of BP-1 which decreases, however, as a f u n c t i o n of the maturational stage of the transformed c e l l (122). BP-1 i s a homodimeric g l y c o p r o t e i n of 140 kD. I n t e r e s t i n g l y , BP-1 i s immunoprecipitated by a Mo-AB (6C3) p r e v i o u s l y described by P i l l e m e r et a l as a tumor-associated marker of A-MuLV-induced pre-B lymphoma c e l l s e x h i b i t i n g high tumorigenic p o t e n t i a l (123). Recently, i t has a l s o been shown that stromal c e l l l i n e s that support pre--B c e l l p r o l i f e r a t i o n i n v i t r o , i . e i n B lymphoid LTC's, a l s o express the 6C3 epitope. Comparative s t u d i e s between BP-1 and 6C3 MoAb i n d i c a t e that both i d e n t i f y the same antigen but a d i f f e r e n t epitope. The 26 p o s s i b i l i t y that BP-1/6C3 may play a r o l e i n stromal cell-dependent pre-B c e l l p r o l i f e r a t i o n as w e l l as the p r o l i f e r a t i o n of n e o p l a s t i c pre-B c e l l s has t h e r e f o r e been the subject of some s p e c u l a t i o n (124). (c) Other D i f f e r e n t i a t i o n Markers. Soon a f t e r they are formed i n the bone marrow, s l g + B c e l l s undergo a s e r i e s of changes i n t h e i r c e l l s u rface components and i n t h e i r f u n c t i o n a l c a p a b i l i t i e s as they mature i n t o r e s t i n g B c e l l s (88,91,93). At the same time they enter the c i r c u l a t i o n and migrate to s p e c i f i c regions i n the spleen, lymph nodes and other secondary lymphoid t i s s u e s . F o l l o w i n g a c t i v a t i o n by a n t i g e n i c s t i m u l a t i o n they p r o l i f e r a t e and d i f f e r e n t i a t e i n t o Ig s e c r e t i n g c e l l s or plasma c e l l s . This f i n a l a n t i g e n -dependent phase of B c e l l d i f f e r e n t i a t i o n requires the p a r t i c i p a t i o n of helper T c e l l s and accessory c e l l s of the monocyte-macrophage l i n e a g e . A number of markers of d i f f e r e n t i a t i o n are used to d i s t i n g u i s h B c e l l subpopulations w i t h d i f f e r e n t f u n c t i o n a l c a p a b i l i t i e s and to c h a r a c t e r i z e d i s c r e t e steps i n the development of plasma c e l l s from antigen-stimulated B c e l l s . The appearance of slgD on the m a j o r i t y of sIgM + c e l l s has s t i m u l a t e d a great deal of i n t e r e s t with regard to the r o l e of these Ig's i n B c e l l f u n c t i o n . During f e t a l development and the 2 f i r s t weeks of p o s t - n a t a l l i f e , i t was shown that & f i r s t appears on sIgM+ c e l l s that bear high d e n s i t i e s of slgM. However, while the amount of slgM on i n d i v i d u a l B c e l l s d iminishes, the amount of slgD increases. Thus, the r a t i o of y/S on the t o t a l B c e l l p o p ulation changes from a very high number f o r f e t a l mice to approximately one i n a d u l t mice (125). Even i n the a d u l t , two major groups of p e r i p h e r a l IgM + B c e l l s can be d i s t i n g u i s h e d : those with l i t t l e or no slgD and a heterogeneous d i s t r i b u t i o n of slgM (sIgM3+ s l g D - ) , and those with low-to-intermediate amounts of slgM and intermediate-to-high amounts of slgD (sIgM + sIgD3+) (125). 27 During the progressive d i f f e r e n t i a t i o n of a c t i v a t e d B c e l l s i n t o plasma c e l l s , slgD expression i s l o s t before slgM. The r o l e of coexpressed slgM and slgD i s not yet c l e a r . I t appears that t h e i r antigen-binding s p e c i f i c i t i e s on i n d i v i d u a l B c e l l s are i d e n t i c a l (126). However, they may convey d i f f e r e n t s i g n a l s to the c e l l i n response to e i t h e r thymic-dependent (TD) or c l a s s 1 thymic-independent (TI) antigens (127). Ia molecules represent an important surface component of B c e l l s because they serve as r e c o g n i t i o n elements i n the i n t e r a c t i o n of B and T c e l l s w i t h antigen presenting c e l l s . These g e n e t i c a l l y polymorphic g l y c o p r o t e i n s are encoded by genes w i t h i n the midportion of the murine major h i s t o c o m p a t i b i l i t y complex (MHC) and are therefore a l t e r n a t i v e l y r e f e r r e d to as MHC c l a s s I I molecules. They are expressed on subpopulations of macrophages, some a c t i v a t e d T c e l l s and most B c e l l s (91). However, immature IgM + B c e l l s i n f e t a l l i v e r , bone marrow, and spleen of neonatal mice express l i t t l e or no det e c t a b l e I a (128,129). During B c e l l maturation, I a determinants are acquired i n p a r a l l e l with s l g by newly formed B c e l l s i n the marrow (91,130). Two other antigens, the Fc receptor (FcR) f o r the Fc p o r t i o n of Ig and the mouse B lymphocyte antigen (MBLA) have been detected on a l l s l g + c e l l s of f e t a l and adult mice ,but not pre-B c e l l s (131,132). While MBLA i s unique to B c e l l s , FcR i s a l s o found on phagocytic c e l l s where i t s plays an important r o l e i n the i n t e r n a l i z a t i o n of antibody-coated p a r t i c l e s such as opsonized b a c t e r i a (133). The f u n c t i o n a l r o l e of MBLA or FcR on B c e l l s i s unknown. However, there i s evidence that the B c e l l FcR may act to regula t e the magnitude of the B c e l l response to a given antigen (134). In c o n t r a s t to both FcR and MLBA, the complement receptor (CR) molecule and minor lymphocyte s t i m u l a t i n g (Mis) antigens are not expressed on a l l murine B c e l l s . The CR i s found on macrophages, granulocytes and 28 approximately 50% of s p l e n i c B c e l l s . The absence of CR + B c e l l s i n the bone marrow suggests that t h i s receptor marks a la t e - d e v e l o p i n g B c e l l subpopulation (91). Further support f o r t h i s was obtained from the demonstration that when adult B c e l l s were separated i n t o CR + and CR~ populations, expression of CR was found to begin a f t e r IgM + c e l l s had acquired IgD. However, B c e l l s cease to express CR a f t e r they are a c t i v a t e d (91). The Mis determinants on the surface of lymphocytes are encoded by a s i n g l e gene or gene complex d i s t i n c t from the MHC. These determinants are res p o n s i b l e f o r p r o l i f e r a t i v e responses i n mixed lymphocyte r e a c t i o n s (MLR) when lymphocytes from H-2 i d e n t i c a l , Mis d i s t i n c t s t r a i n s are c u l t u r e d together. The c e l l s which s t i m u l a t e an Mis-determined MLR are mature and not immature B c e l l s (135,136). Both CR and Mis markers have been u s e f u l i n d i s t i n g u i s h i n g subpopulations of B lymphocytes. Three sIgD + B c e l l populations have been i d e n t i f i e d . The e a r l i e s t appearing are IgM + ( h i g h ) , F c + , MBLA+, I a + , IgD +. The next two are IgM+ (low-intermediate), Fc+, MBLA+, Ia+, IgD+, CR + and IgM+ (low), F c + , MBLA+, Ia+, IgD+, CR+, M l s + (91). A number of a d d i t i o n a l markers have been i d e n t i f i e d on the surface of B c e l l s . These are Lyb-3, Lyb-5 and Lyb-7. By comparing B c e l l f u n c t i o n s i n normal and immuno-defective CBA/N mice with s p e c i f i c a n t i s e r a developed against Lyb determinants, i t has been p o s s i b l e to f u r t h e r d e l i n e a t e a number of mature B c e l l subpopulations. CBA/N mice ca r r y an X l i n k e d gene, x i d , that causes a defect i n c e r t a i n B c e l l f unctions (137,138). The l a t e s t - a p p e a r i n g B c e l l s are Lyb-3 +, Lyb-5 + and Lyb-7 + and thus can be c h a r a c t e r i z e d as IgM + (low), F c + , MBLA+, Ia+, IgD+, CR+, Mls+, Lyb-3+, 5+, 7+ (91). F u n c t i o n a l s t u d i e s have shown that Lyb-3 +, 5 +, 7 + B c e l l s i n a d d i t i o n to being r e s p o n s i b l e f o r the immune response to thymus-independent type 2 antigens, are 29 c r i t i c a l f o r B c e l l responses to low doses of thymus-dependent antigens and are a l s o important f o r the IgM to IgG switch during a primary immune response. The absence of Lyb-3 +, 5 + and 7 + determinants on the surface of the B c e l l s of ad u l t x i d mice suggests that the immune defects i n these animals may be e n t i r e l y due to the l a c k of t h i s subpopulation (91). This i s supported by the f i n d i n g that both T c e l l s and accessory c e l l s are normal i n x i d mice (88). Moreover, the r e l a t i v e l y good secondary IgG response induced by thymus-dependent antigens i n CBA/N mice suggests that the c e l l s that are a c t i v a t e d to become memory B c e l l s a f t e r a n t i g e n i c s t i m u l a t i o n are d i s t i n c t , at l e a s t i n par t , from the Lyb-3 +, 5 +, 7 + B c e l l population (88,91,93). 3) REGULATION OF B CELL DEVELOPMENT Just as our knowledge and a b i l i t y to study B c e l l populations preceded the c h a r a c t e r i z a t i o n of pre-B c e l l s , so to has information about the r e g u l a t i o n of B c e l l a c t i v a t i o n and p r o l i f e r a t i o n been accumulated much f a s t e r than f o r pre-B c e l l s . A number of molecular r e g u l a t o r s of B c e l l d i f f e r e n t i a t i o n produced by T c e l l s and macrophages have been p u r i f i e d and i n some instances MoAb to these lymphokines have a l s o been obtained (51). The a v a i l a b i l i t y of the^se molecules i n pure form has been i n v a l u a b l e i n a l l o w i n g t h e i r i n d i v i d u a l r o l e s i n the r e g u l a t i o n of the antigen-dependent phase of B c e l l development to be defined. The mechanisms that regulate the commitment of p l u r i p o t e n t stem c e l l s to the B c e l l pathway and that regulate the s i z e s of the populations of the d i f f e r e n t subsequent stages of pre-B c e l l development are s t i l l l a r g e l y unknown. However, r e c e n t l y s e v e r a l l i n e s of evidence obtained from both animal models and LTC's (see below) have suggested that stromal c e l l s r e g u l a t e 30 the earliest phases of B lymphopoiesis possibly via direct contact with developing pre-B cells and by the secretion of specific growth factors (106,124,139-142). In the following section, the main systems for studying pre-B and B cells are briefly described. Current knowledge of how these cell populations are regulated is then summarized. A) Systems for Study (a) Short-Term Culture Systems. The hemolytic plaque assay originally described by Jerne et al detects single mature Ig-producing cells on the basis of Ig binding to surrounding antigen-carrying erythrocytes which then fix complement either directly or indirectly (by the addition of an antiglobulin antibody) and yield a plaque (143). The short-term culture system init ial ly described by Mishell and Dutton for detecting splenic precursors (B cells) of Ig-producing cells has also been useful for analyzing B cell proliferation (cell count, DNA precursor uptake) and Ig synthesis and secretion (144). The use of a combination of these two techniques has allowed the biological activities of various lymphokines to be evaluated. For example, by adding a purified (or recombinant) lymphokine to a liquid culture containing splenic B cells, previously activated with anti-Ig or LPS, it is possible to determine and to quantitate whether the molecule acts as a proliferative and/or a differentiation factor. Colony-forming assays for B-cells (CFU-B) is another method for quantitating B-lineage cells (88,145). Short-term cultures in soft agar enable membrane Ig-bearing B cells to form colonies in the presence of mitogens that are native to laboratory grade agar with the further addition to 31 the c u l t u r e s of e i t h e r SRBC, LPS or adherent l a y e r s of p e r i t o n e a l exudate macrophages (146). Further c h a r a c t e r i z a t i o n of the c e l l s that can be cloned i n s e m i s o l i d agar has shown that the CFU-B assay can detect B-lineage c e l l s at a v a r i e t y of maturation stages i n c l u d i n g those that are IgD + and IgD~ stages and I a + and I a - (88). However, t h i s c l o n a l p r o l i f e r a t i o n assay does not detect e a r l y B c e l l progenitors as s e m i s o l i d assays f o r myeloid committed progenitors do. Recently, a clonogenic assay f o r B c e l l precursors has been described (147). Although, pre-B c e l l s that do not express membrane IgM are not capable of forming agar c o l o n i e s under CFU-B c u l t u r e c o n d i t i o n s , they are capable to form c o l o n i e s i n agar i f they are allowed to d i f f e r e n t i a t e to s l g + B c e l l s e i t h e r p r i o r to s o f t agar c u l t u r e or a f t e r c u l t u r e i n the presence of feeder l a y e r s of f e t a l l i v e r - a d h e r e n t c e l l s (147,148). Thus, Paige described a double-layer agar assay i n which B c e l l precursors already present i n 12-day f e t a l l i v e r can generate c o l o n i e s which contain a n t i b o d y - s e c r e t i n g c e l l s w i t h i n 8 days. Colony growth requires s o l u b l e mediators provided by p l a s t i c -adherent f e t a l l i v e r c e l l s growing under the agar l a y e r . The d e t e c t i o n of antibody s e c r e t i o n i s achieved a f t e r t r a n s f e r of the uppermost agar l a y e r to a gl a s s s l i d e , by a m o d i f i c a t i o n of the Jerne plaque assay. The c o l o n i e s obtained produce both K and X L chains (150). This assay system detects c e l l s that d i f f e r from mature CFU-B (148) and points out that B c e l l precursor growth i s dependent on adherent accessory c e l l s . (b) IL-3 Dependent B-Lineage C e l l L i n e s . Long-term c u l t u r e s of normal mouse B lymphocytes were f i r s t described by Howard et a l . They showed that s p l e n i c B c e l l s could be maintained f o r 10 months i n media conditioned by Con-A-stimulated spleen c e l l s (149). P a l a c i o s et a l subsequently reported the 32 establishment and maintenance of pre-B cell lines from marrow and spleen cells in IL-3 containing media (151,152). They also reported that "the CC11"M6-Ab, thought to be against the IL-3 receptor (153), could be used to distinguish two major populations of marrow B cell precursors: one which was CC11+ and also IL-3-responsive, the other being CC11~ and unresponsive to IL-3 (154). While such factor-dependent pre-B and B cell lines have been useful for evaluating factors promoting early and late stages of lymphopoiesis, they do not allow direct analysis of the mechanism by which stromal cells influence B cell precursors. (c) Long-Term B Lymphoid Cultures (Lymphoid LTC). In 1977, Dexter et al (31) described a marrow culture system that allowed the continuous production of cells of multiple hemopoietic cell lineages, including granulocytes, macrophages, and precursors of myeloid and erythroid cells, for many weeks. However, no identifiable cells of the lymphoid series are produced in these cultures, although transplantation experiments have shown that very primitive precursors of both B- and T-cell lineages are present (155,156). In 1982, Whitlock and Witte described a modification of this LTC method that resulted in the sustained production of B lymphocytes and their precursors and in the concomitant loss of myeloid cells and of CFU-S (33,157). Figure 4 shows diagrammatically the similarities and differences between the methodologies used for the two types of LTC systems. A common feature of both types of cultures is the organization of the cells in two layers: an adherent layer containing stromal cells and some hemopoietic cells, and a non-adherent layer composed of the more mature hemopoietic cells. In both systems, the continued production of non-adherent cells (myeloid cells in Dexter cultures and lymphoid cells in Witte cultures) is dependent on the cells in the 33 Myeloid LTC 8 1 0 6 b o n e marrow cells/ml ••••I • • • UtW 3 3 ° 1 2 . 5 % HS 1 2 . 5 % FCS 1 0 0 / i M 2 - mercaptoethanol culture medium (a) 1 0 ~ 6 M hydrocortisone Lymphoid LTC 3 7 ° 5 % FCS 5 0 iiM 2 - mercaptoethanol culture medium (RPMI 1 6 4 0 ) Figure 4. D e s c r i p t i o n of the d i f f e r e n t types of long-term hemopoietic c u l t u r e s . 34 adherent l a y e r and f a c t o r s they produce. The d i f f e r e n t c o n d i t i o n s obtained i n the two types of LTC r e s u l t i n a d i f f e r e n t morphology of the "stromal" c e l l s seen i n the adherent l a y e r . In myeloid LTC's, the stromal c e l l s i n c l u d e e n d o t h e l i a l - l i k e c e l l s , d e n d r i t i c - r e t i c u l a r c e l l s , l i p i d - f i l l e d adipocytes and phagocytic mononuclear c e l l s (158). In lymphoid LTC's the adherent c e l l p o p ulation does not appear to contain l i p i d - c o n t a i n i n g adipocytes and i s made of l a r g e , adherent, mononuclear c e l l s with numerous pleomorphic cytoplasmic processes. Although c o n d i t i o n s i n myeloid LTC's, i n p a r t i c u l a r the use of c o r t i c o s t e r o i d s which are t o x i c f o r B c e l l s , are c l e a r l y not s u i t a b l e f o r supporting the p r o l i f e r a t i o n of B lymphoid c e l l s , e s t a b l i s h e d myeloid LTC can be converted to productive lymphoid LTC's by simply s w i t c h i n g the c u l t u r e s to lymphoid LTC maintenance c o n d i t i o n s (34,159). However, the opposite experiment does not work. I f lymphoid LTC's are switched back to Dexter c o n d i t i o n s , only low l e v e l s of t r a n s i e n t myelopoiesis i s observed (34). These f i n d i n g s show that there are c e l l s i n myeloid LTC's that can support B lymphopoiesis and suggested that the f a i l u r e of lymphopoiesis i n myeloid LTC's i s not due to an inadequate "stroma". These f i n d i n g s a l s o suggest that lymphoid LTC can be i n i t i a t e d by a committed B-lymphocyte stem c e l l or i t s p l u r i p o t e n t parent. The establishment of lymphoid LTC's i s h i g h l y reproducible w i t h s e l e c t e d batches of f e t a l c a l f serum (FCS). Superior r e s u l t s have been claimed f o r marrow from c e r t a i n ages and s t r a i n s of mice (106,157). The c u l t u r e s t y p i c a l l y progress through d i s t i n c t i v e phases. During the two f i r s t weeks, wh i l e the adherent l a y e r i s forming, the number of non-adherent c e l l s d e c l i n e s . During the next few weeks, there i s a " c r i s i s " phase when few non-adherent c e l l s can be detected i n the c u l t u r e s . Then, patches of small non-adherent c e l l s reappear i n the adherent c e l l l a y e r and these subsequently 35 expand i n number. Most of these c e l l s resemble small lymphocytes, although some appear to be b l a s t s . The f i r s t evidence that B c e l l s are among the non-adherent c e l l s i n these c u l t u r e s came from b i o s y n t h e t i c l a b e l l i n g experiments. These shoved a la r g e amount of H and L chain synthesis (33). Autoradiography of ^H-thymidine l a b e l l e d c e l l s i n d i c a t e d that a large p r o p o r t i o n of the c e l l s were a c t i v e l y d i v i d i n g (106). Phenotyping s t u d i e s i n d i c a t e d that the c u l t u r e s i n the f i r s t three months contained 10-20% membrane Ig-bearing c e l l s . However, at 15-20 weeks, subpopulations of e i t h e r pre-B or B c e l l s tend to dominate the c u l t u r e s , and the patter n of H and L chains synthesized suggests that the lymphoid c e l l s have become p a u c i c l o n a l (33). 10-30% of the c e l l s express a low l e v e l of B220. These synthesize few i f any H chains but produce high amounts of TdT (106). A n a l y s i s of rearrangements at the Ig H chain locus has shown that 10-30% of the DNA contained H chain sequences i n an unrearranged, germline s t a t e . These data i n d i c a t e that the n u l l c e l l lymphoid population present represent very e a r l y B-lineage c e l l s . Thus, lymphoid LTC's allow the maintenance, p r o l i f e r a t i o n and d i f f e r e n t i a t i o n of very e a r l y B c e l l precursors to Ig-bearing B c e l l s i n the presence of a stromal c e l l l a y e r . The i n t r o d u c t i o n of t h i s c u l t u r e system has provided a powerful t o o l to study the r e g u l a t i o n of pre-B c e l l lymphopoiesis. At l e a s t three d i f f e r e n t aspects of B c e l l development have been i n v e s t i g a t e d . The f i r s t has been an a n a l y s i s of the molecular changes that accompany pre-B c e l l transformation. Such c e l l s , i n p a r t i c u l a r l a r g e c y + pre-B c e l l s , are s e n s i t i v e targets f o r A-MuLV (160), and A-MuLV transformed c e l l l i n e s r e p r e s e n t i n g d i f f e r e n t stages of e a r l y B c e l l ontogeny can be e a s i l y obtained by i n f e c t i n g lymphoid LTC c e l l s (161). A n a l y s i s of I g gene l o c i i n these c e l l s a f t e r continued maintenance has shown that some A-MuLV transformed subclones can undergo extensive Ig gene rearrangement s h o r t l y a f t e r v i r a l 36 infection (97,161). It has also been observed that the proliferation of such transformants can become feeder-independent after 1-3 months in culture (162). This has led to the suggestion that A-MuLV induces or regulates normal mechanisms within the cell so that it acquires the capacity for autonomous growth. A second application of lymphoid LTC's has been to study B lymphoid ontogeny in fetal liver cultures (163). Fetal liver cells are, themselves, incapable of forming an adherent layer which can support long-term lymphopoiesis, but this deficiency can be overcome by using pre-established adherent layers from bone marrow. The fetal liver lymphoid LTC's thus obtained are identical in appearance to those initiated with marrow but retain an earlier presumptive B cell precursor population which do not undergo further differentiation in vitro. This could be due to a developmental block imposed by the culture conditions or to an intrinsic defect in the B lineage cells derived from fetal liver (163). Finally, lymphoid LTC's have been used as a basis for analyzing the role of the stromal cells in supporting B lymphoid progenitors. (d) Animal Models. A number of animals bearing genetically determined immunodeficiencies have been useful for studies of the regulation of lymphopoiesis. Reconstitution experiments in which isolated cell populations are injected into such immunodeficient animals have been used to analyze the cell type capable of complementing the defect and thereby to provide an "assay" for the stage of cell whose production is affected. (i) CBA/N Mice. CBA/N mice possess a defective gene on the X chromosome that leads to a series of B-lymphocyte abnormalities, including the absence of a particular B cell subpopulation and unresponsiveness to TNP-Ficoll and other 37 thymus-independent type 2 antigens (91). However, T c e l l s , accessory c e l l s and stromal c e l l s are normal i n these x i d mice (88,138). Such CBA/N mice have been used by Kurland (164) i n order to demonstrate the presence of a B - c e l l precursor subpopulation i n lymphoid LTC's. He showed that i n j e c t i o n of c e l l s from lymphoid LTC's ( e s t a b l i s h e d from CBA/N mice) i n t o s u b l e t h a l l y i r r a d i a t e d (CBA/NxBALB/c) r e c i p i e n t s r e s u l t e d i n s u c c e s s f u l r e c o n s t i t u t i o n of the B-lymphocyte compartment of the i n j e c t e d mice. ( i i ) SCID Mice. Another animal model i s that bearing a g e n e t i c a l l y determined severe combined immune d e f i c i e n c y disease (SCID) (165,166). This mouse s t r a i n i s homozygous f o r an autosomal mutation which r e s u l t s i n the almost complete absence of normal B and T lymphocytes and serum I g . Bone marrow of SCID mice have no detectable pre-B or B c e l l s . However, other hemopoietic c e l l s , i n c l u d i n g CFU-S, n a t u r a l k i l l e r c e l l s , myeloid c e l l s , and antigen-presenting c e l l s appear to be unaffected by the defect (165-167). SCID marrow c e l l s are not able to r e c o n s t i t u t e the lymphoid systems of i r r a d i a t e d normal hosts (168). In c o n t r a s t , normal bone marrow or even c e l l s from normal lymphoid LTC's are able to f u l l y c o r r e c t the defect i n SCID mice (169). This i n d i c a t e s that the d e f i c i e n c y i n SCID i s i n t r i n s i c to a lymphoid progenitor population and i s not due to a d e f e c t i v e microenvironment provided by non-transplantable stromal c e l l s (169). SCID marrow can be used to s u c c e s s f u l l y i n i t i a t e lymphoid LTC's (170). This shows that SCID mice have progenitors of B c e l l s that are able to p r o l i f e r a t e i n v i t r o , although they do not give r i s e to c u + c e l l s . Southern b l o t a n a l y s i s of the c e l l s produced i n these SCID LTC's demonstrated that aberrant or nonproductive H gene rearrangements are unusually common (170). This may account f o r the absence of pre-B and B c e l l s i n SCID mice. 38 Recently, SCID mice have a l s o been used to study B c e l l development i n f e t a l l i v e r LTC's (171). Normal lymphoid f e t a l l i v e r LTC's are composed predominantly of pre-pre-B lymphocytes which have not undergone Ig gene rearrangement and do not contain sIgM + c e l l s . Nevertheless, t r a n s p l a n t a t i o n of f e t a l l i v e r LTC c e l l s to SCID mice was found to r e c o n s t i t u t e s p l e n i c B c e l l s and serum IgM. However, T lymphocyte r e c o n s t i t u t i o n was not observed and serum IgG l e v e l s were very low i n d i c a t i n g impaired c l a s s s w i t c h i n g i n the lymphocytes produced. When thymocytes were c o - i n j e c t e d with these f e t a l l i v e r LTC c e l l s , the B lymphocytes became able to undergo c l a s s s w i t c h i n g and responded normally to T-dependent antigens (171). Thus, the use of LTC and SCID r e c i p i e n t s has allowed the separation of B lymphocyte development i n t o 3 stages: e a r l y d i f f e r e n t i a t i o n i n v i t r o , progression to IgM s e c r e t i o n i n v i v o , and l a t e d i f f e r e n t i a t i o n dependent upon mature T lymphocytes i n v i v o . ( i i i ) Motheaten Mice. Mice homozygous f o r the autosomal r e c e s s i v e s i n g l e gene a l l e l i c mutation, motheaten (me/me) or v i a b l e motheaten (me/me v), are s e v e r e l y d e f i c i e n t i n B and T lymphocytes as w e l l as NK c e l l s and develop autoimmune disease c h a r a c t e r i z e d by hyperimmunoglobulinemia, expression of m u l t i p l e autoantibodies and d e p o s i t i o n of immune complexes i n numerous t i s s u e s (172,173). Because of the severe immunologic disturbances i n these mice, they di e at an e a r l y age. Several s t u d i e s have shown that the development of prothymocytes, pre-B c e l l s , and TdT + lymphoid precursors i n the bone marrow of motheaten mice i s d e f e c t i v e because of a l a c k of an appropriate microenvironment f o r the generation of TdT + bone marrow c e l l s (174,175). By comparing lymphoid LTC's from motheaten and normal mice, i t has been r e c e n t l y reported that the stromal l a y e r of motheaten c u l t u r e s i s overgrown by a macrophage-like c e l l population and me/mev c e l l s suppress the growth of normal progenitors i n lymphoid LTC's (176). Thus, motheaten (me/me) or v i a b l e 39 motheaten (me/mev) mice represent an interesting model in which the severe defective lymphopoiesis may involve defective stromal cell function. B) MECHANISMS As for myelopoiesis, lymphopoiesis appears to be regulated by both growth factors and direct interactions with regulatory cells. (a) Growth Factors. Growth factors affecting B-lineage cells can be divided into two categories: those involved in the development of mature B cells from early progenitors, and those directing humoral responses to specific antigens. In Table 2 is summarized a number of effects that various well characterized (cloned) hemopoietic growth factors have on B cell maturation. After antigenic stimulation, B cells respond to humoral factors produced by helper T cells and also antigen-presenting cells. For example, IL-4, IL-5, and IL-6 act on B-cells to promote their activation, proliferation and differentiation. As previously discussed (see Table 1), these molecules can also influence the proliferation of variety of myeloid cell types. Other lymphokines, e.g. IL-1, IL-2, and IFNy may also modulate B cell functions. Recently, it has been shown that in the mouse two distinct subsets of helper T cel l lines, Thl and Th2 may produce different lymphokines (177). Thl cells produce IFNy and IL-2, and Th2 T cells produce IL-4, IL-5 and possibly IL-6. Because these different lymphokines appear to modulate Ig-class switching of B cells differentially, this mechanism may be controlled at the cellular level through the interaction of activated B cells with particular T helper subsets (177). Table 2. B C e l l Growth Factors Name E f f e c t s on B C e l l s I n t e r l e u k i n - 1 (IL-1) Induces: p r o l i f e r a t i o n w i t h a n t i - I g + IL-4 p r o l i f e r a t i o n & d i f f e r e n t i a t i o n of Ag-stimulated B c e l l s maturation of pre-B c e l l s I n t e r l e u k i n - 2 (IL-2) Induces: p r o l i f e r a t i o n with a n t i - I g + LPS p r o l i f e r a t i o n & d i f f e r e n t i a t i o n of Ag-stimulated B c e l l s d i f f e r e n t i a t i o n w i t h a n t i - I g + IL-4 I n t e r l e u k i n - 3 (IL-3) Supports growth of pre-B c e l l l i n e s I n t e r l e u k i n - 4 (IL-4) Induces p r o l i f e r a t i o n with a n t i - I g T I a on r e s t i n g B c e l l s t FcR expression t IgG^ IgE s e c r e t i o n by LPS a c t i v a t e d B c e l l s •*• IgG2a» x g G 2 b ' J S M & x g G 3 s e c r e t i o n by LPS-activated B c e l l s I n t e r l e u k i n - 5 (IL-5) t p r o l i f e r a t i o n of a c t i v a t e d normal B c e l l s & BCL-1 c e l l s t IgM & IgG s e c r e t i o n by a c t i v a t e d normal B c e l l s & BCL-1 c e l l s T IgA s e c r e t i o n by LPS-stimulated B c e l l s T IL-2 receptor expression I n t e r l e u k i n - 6 (IL-6) Induces growth of B c e l l hybridomas & plasmacytomas I n t e r f e r o n Y ( I F N - Y ) Induces: Ig s e c r e t i o n w i t h IL-2 Ig s e c r e t i o n by r e s t i n g B c e l l s t I g G 2 a , I I g G 2 b Gi G 3 by LPS stimulated B c e l l s 4- IL-4 a c t i v i t y From Reference 51. 41 Factors that regulate e a r l y stages of B c e l l development have f o r the most part not been w e l l c h a r a c t e r i z e d . As a s t a r t i n g p o i n t , s e v e r a l groups ( i n c l u d i n g our own - see Chapter I I I ) have i s o l a t e d stromal c e l l l i n e s that support pre-B c e l l growth and/or d i f f e r e n t i a t i o n (60,124,139,140,142). Dorshkind et a l (142) r e c e n t l y described the c h a r a c t e r i s t i c s of a l i n e (S17) derived from the adherent l a y e r of Dexter LTC which can support B c e l l d i f f e r e n t i a t i o n . Whitlock et a l (124) have s i m i l a r l y i s o l a t e d cloned stromal c e l l l i n e s from LTC's which support the d i f f e r e n t i a t i o n of e a r l y B progenitors i n t o B220+ and c y + c e l l s but without f u r t h e r d i f f e r e n t i a t i o n i n t o slgM c e l l s . Another l i n e (ALC) i s o l a t e d from lymphoid LTC's that supports the i n v i t r o growth of e a r l y lymphoid and myeloid c e l l s has been described by Hunt et a l (140), and Quesenberry and co-workers have described an adherent marrow c e l l l i n e (TC-1) that produces a m u l t i l i n e a g e s y n e r g i s t i c a c t i v i t y which a l s o supports pre-B c e l l growth (60). Hunt et a l (140) a l s o showed that ALC c e l l s produce a f a c t o r that s t i m u l a t e s the p r o l i f e r a t i o n of pre-B c e l l s . Using a c l o n a l pre-B c e l l l i n e they showed that the s t i m u l a t o r y f a c t o r i s o l a t e d was a 30 kD molecule. A pre-B s t i m u l a t i n g f a c t o r of 25 kD has a l s o been r e c e n t l y d e scribed, p u r i f i e d and cloned by Namen (178,179). The TC-1 a c t i v i t y reported by Quesenberry appears to be a high molecular weight g l y c o p r o t e i n (60). Landreth (142) p u r i f i e d two f a c t o r s from the conditioned medium of S17 c e l l s , a 50-60 kD molecule and a 10 kD molecule. Both induce B220 and cy p r o t e i n expression i n pre-B c e l l s that have not yet begun to express these markers. I n t e r e s t i n g l y , none of the d i f f e r e n t pre-B c e l l s t i m u l a t i n g stromal c e l l s i s o l a t e d to date have been found to secrete detectable IL-3 and none of the d i f f e r e n t pre-B c e l l growth f a c t o r s or pre-B d i f f e r e n t i a t i o n f a c t o r s resemble any known hemopoietic growth f a c t o r s . I t thus appears that the r e g u l a t i o n of the e a r l y events of B c e l l development may be complex i n v o l v i n g s e v e r a l new 42 factors produced by stromal cells* The role or in vivo significance of IL-3 as a pre-B stimulating factor remains unclear. As in myelopoiesis, TGF-0 has an inhibitory effect on B lymphoid cells. Kincade (70) has shown that TGF-f3i as well as TGF-P2 inhibits the acquisition by pre-B cells of K expression. TFG-0 was also found to inhibit the increase in K chain expression observed after exposure of mature B cells to LPS. Thus, TGF-0 may function during specific stages of B cell differentiation by inhibiting the initiation of, or increased transcription of, Ig genes. It may, therefore, be a potent inhibitor of the transition of pre-B cells to mature, functional B cells. (b) Direct Cellular Interactions. Very l i t t le is known about the role or mechanisms by which stromal cells and B cell progenitors might interact directly. Bernard! (38) have recently shown that precursor B lymphoid cell lines blocked at specific stages of differentiation, but not peripheral blood lymphocytes, adhere specifically to FN and not to LM or collagen type I. In addition, they also found that these lymphoid cell lines can adhere to two different sites on the FN molecule. FN possesses both a cell-binding domain containing the arg-gly-asp (RGD) sequence and a high-affinity binding site for heparin. Very early B progenitors adhere preferentially to the RGD ce l l -binding site and more differentiated B cells may tend to adhere to the heparin site. These findings suggest that different FN receptors (FN-R) with different specificity may be expressed in the course of B cell maturation and that these multiple receptors may play a role in normal lymphoid precursor cel l adhesion to the ECM (38). Another interesting study relevant to this topic was recently reported by Kierney et al (141). They showed that B lymphocyte precursors in diffusion chambers placed on top of adherent stromal 43 c e l l s d i d not d i f f e r e n t i a t e suggest ing that op t ima l pre-B c e l l s t i m u l a t i o n r e q u i r e s s i g n a l s from s t romal c e l l s that are not achieved without c e l l c o n t a c t . 4) THESIS OBJECTIVES Dur ing the l a s t ten year s , numerous advances have been made i n the development of new technolog ie s s p e c i f i c a l l y a p p l i c a b l e to i n v e s t i g a t i n g the r e g u l a t i o n of B c e l l development. The genera t ion of MoAb aga ins t B c e l l l i n e s has a l lowed the i d e n t i f i c a t i o n of s e v e r a l s p e c i f i c markers of B c e l l p r o g e n i t o r s . Molecu l a r a n a l y s i s of Ig gene s t r u c t u r e and Ig gene rearrangement i n i s o l a t e d c lones has l e d to an understanding of the succe s s ive g e n e t i c m o d i f i c a t i o n s that lead to the expres s ion of cy and s l g . The d i s c o v e r y of a system f o r suppor t ing the long-term p r o l i f e r a t i o n of normal pre-B c e l l s has a l s o been i n s t r u m e n t a l . These c u l t u r e s represent an i n v i t r o model of B c e l l development and have a l lowed mechanisms of s t roma l mediated c o n t r o l of pre-B c e l l p r o l i f e r a t i o n and d i f f e r e n t i a t i o n to begin to be d e f i n e d . However, a number of major ques t ions s t i l l remain unso lved . FACS a n a l y s i s has shown that p l u r i p o t e n t and B-committed stem c e l l s express d i f f e r e n t phenotypes (121), and a comparison between myeloid and lymphoid LTC' s has suggested that the commitment of p l u r i p o t e n t stem c e l l s i n t o B-stem c e l l s i s i r r e v e r s i b l e (34). S t i l l a mystery i s the mechanism that determines how a p l u r i p o t e n t stem c e l l becomes committed to the lymphoid pathway and the extent to which t h i s may be subject to e x t r i n s i c i n f l u e n c e s . 44 A second ques t ion concerns the d e f i n i t i o n of how feedback, c o n t r o l of B c e l l p r o d u c t i o n i s maintained i n v i v o . I t has been shown that B - l i n e a g e stem c e l l s g i v e r i s e i n a s tep-wise f a sh ion a s soc i a ted w i t h p r o l i f e r a t i o n , to mature f u n c t i o n a l s m a l l lymphocytes . Somehow, the p roduc t ion and death of p e r i p h e r a l blood lymphocytes i s kept constant throughout a d u l t l i f e . T h i s suggests the e x i s t e n c e of r e g u l a t o r y mechanisms c o n t r o l l i n g the balance between these two parameters. Although i t has been shown very r e c e n t l y tha t s t roma l c e l l s can secre te s e v e r a l pre-B growth f a c t o r s , the a c t u a l mechanism by which these f a c t o r s exert t h e i r e f f e c t s on pre-B c e l l s has not yet been e l u c i d a t e d . Indeed, i t i s not yet c l e a r how many d i f f e r e n t pre-B f a c t o r s may e x i s t , nor have t h e i r normal r o l e s i n v i v o or i n transformed lymphoid c e l l popu la t ions been e s t a b l i s h e d . The major g o a l of the work undertaken for t h i s t h e s i s was to d e f i n e at a c e l l u l a r and molecular l e v e l mechanisms that r egu la te normal and transformed pre-B lymphoid c e l l s by s t romal c e l l s . When t h i s p r o j e c t was s t a r t e d three years ago, very l i t t l e was known about the nature of the i n t e r a c t i o n s that occur between s t romal c e l l s and pre-B c e l l s (106) . The s p e c i f i c o b j e c t i v e s of the work were there fore as f o l l o w s : to determine whether s t romal c e l l s s e c r e t e s o l u b l e pre-B c e l l s t i m u l a t i n g f a c t o r ( s ) ; to determine whether a d i r e c t i n t e r a c t i o n between s t romal c e l l s and pre-B c e l l s p lays a r o l e i n s t r o m a l -mediated support of pre-B c e l l growth; and f i n a l l y to determine whether e ' i ther of these mechanisms are a l t e r e d i n transformed pre-B c e l l popu la t ions that have become malignant and capable of autonomous growth i n v i t r o . In order to f u l f i l l the o b j e c t i v e s c i t e d above, a n a l y s i s of mechanisms o p e r a t i v e i n lymphoid LTC's appeared to be the most u s e f u l s t a r t i n g p o i n t . However, the f ac t that such LTC's are composed of both an heterogeneous adherent c e l l l a y e r and of a non-adherent lymphoid l a y e r which i n c l u d e s c e l l s 45 at d i f f e r e n t stages of B c e l l development, suggested that the i s o l a t i o n of c loned r e g u l a t o r y ( s t romal ) and responder (pre-B) c e l l c o n s t i t u e n t p o p u l a t i o n s would f a c i l i t a t e the s t u d i e s to be i n i t i a t e d . Thus, the f i r s t o b j e c t i v e was to generate and c h a r a c t e r i z e such l i n e s and then to de f ine how they might be used i n a c o - c u l t u r e s t r a t e g y to study how normal and transformed pre-B c e l l growth i s r e g u l a t e d . The f i r s t ques t ion to be asked was whether s t romal c e l l s s t i m u l a t e pre-B c e l l s through the s e c r e t i o n of a s o l u b l e f a c t o r ( s ) or whether d i r e c t c e l l contact i s r e q u i r e d . To address t h i s ques t ion r e q u i r e d the use of an exper imenta l system where i t would be p o s s i b l e to measure the s t i m u l a t o r y r o l e ( i f any) of s t romal c e l l s on pre-B c e l l p r o l i f e r a t i o n i n the absence of any d i r e c t contact between these two c e l l types . The next q u e s t i o n was to determine whether any ECM component and/or m e t a b o l i c a l l y i n a c t i v e ( i . e . f i x e d w i t h g l u t a r a l d e h y d e ) s t romal c e l l s could alone or s y n e r g i s t i c a l l y promote the p r o l i f e r a t i o n of pre-B c e l l s . The f i n a l o b j e c t i v e was to determine whether the malignant t r a n s f o r m a t i o n of pre-B c e l l s a l t e r s t h e i r i n t e r a c t i o n s w i t h s t romal c e l l s and the nature or ba s i s of any changes found. Although many transformed pre-B c e l l l i n e s were known to be a v a i l a b l e , the c e l l s from which they had been der ived were n o t , thus making d i f f i c u l t any a s s o c i a t i o n s of unique fea tures they might e x h i b i t w i t h t h e i r transformed s t a t e . To overcome t h i s c r i t i c i s m an attempt was t h e r e f o r e made to i s o l a t e and c lone normal pre-B c e l l l i n e s and then d e r i v e c loned transformants from these c e l l s u s i n g A-MuLV. In t h i s way, normal and transformed parent-progeny l i n e s could be used s imul taneous ly to e x p l o r e the p o s s i b i l i t y that they i n t e r a c t e d d i f f e r e n t l y w i t h s t romal c e l l s . The r e s u l t s of a l l of these s t u d i e s are presented i n Chapters I I I , IV and V. 46 REFERENCES 1. T i l l JE, McCulloch EA. A d i r e c t measurement of the r a d i a t i o n s e n s i t i v i t y of normal bone marrow c e l l s . R a d i a t i o n Research 14:213, 1961. 2. T i l l JE, McCulloch EA. Hemopoietic stem c e l l d i f f e r e n t i a t i o n . B i ochimica et Biophysica Acta 605:431, 1980. 3. Becker AJ, McCulloch EA, T i l l JE. C y t o l o g i c a l demonstration of the c l o n a l nature of spleen c o l o n i e s derived from transplanted mouse marrow c e l l s . Nature 197:452, 1963. 4. W i l l i a m s DA, Lemischka IR, Nathan DG, M u l l i g a n RC. I n t r o d u c t i o n of new genetic m a t e r i a l i n t o p l u r i p o t e n t haematopoietic stem c e l l s of the mouse. Nature 310:476, 1984. 5. K e l l e r G, Paige C, G i l b o a E, Wagner EF. Expression of a f o r e i g n gene i n myeloid and lymphoid c e l l s derived from multipotent haematopoietic precursors. Nature 318:149, 1985. 6. Magli MC, Iscove NN, Odartchenko N. Transient nature of e a r l y haemopoietic spleen c o l o n i e s . Nature 295:527, 1982. 7. Fialkow PJ, Jacobson RJ, Papayannopoulou T. Chronic myelocytic leukemia: c l o n a l o r i g i n i n a stem c e l l common to the granulocyte, e r y t h r o c y t e , p l a t e l e t and monocyte/macrophage. Am J Med 63: 125, 1977. 8. Fialkow PJ. C l o n a l and stem c e l l o r i g i n of blood c e l l neoplasms. In : "Contemporary Hematology/Oncology, Volume I " , (ed. J Lobuc), Plenum Press, New York, pp 1, 1980. 9. Humphries RK, Eaves AC, Eaves CJ. C h a r a c t e r i z a t i o n of a p r i m i t i v e e r y t h r o p o i e t i c progenitor found i n mouse marrow before and a f t e r s e v e r a l weeks i n c u l t u r e . Blood 54:746, 1979. 10. Fauser AA, Messner HA. I d e n t i f i c a t i o n of megakaryocytes, macrophages, and e o s i n o p h i l s i n c o l o n i e s of human bone marrow c o n t a i n i n g n e u t r o p h i l i c granulocytes and e r y t h r o b l a s t s . Blood 53:1023, 1979. 11. Ogawa M, Pharr PN, Suda T. S t o c h a s t i c nature of stem c e l l f u n c t i o n s i n c u l t u r e . I n : "Progress i n C l i n i c a l and B i o l o g i c a l Research, V o l 184, Hematopoietic Stem C e l l Physiology", (eds. EP C r o n k i t e , N Dainiak, RP McCaffrey, J Palek, PJ Quesenberry P J ) , Alan R L i s s , Inc, New York, pp 11, 1985. 12. Johnson GR, Metcalf D. Pure and mixed e r y t h r o i d colony formation i n v i t r o s t i m u l a t e d by spleen conditioned medium with no detectable e r y t h r o p o i e t i n . Proc N a t l Acad S c i USA 74:3879, 1977. 13. Gregory CJ, Eaves AC. Three stages of e r y t h r o p o i e t i c progenitor c e l l d i f f e r e n t i a t i o n d i s t i n g u i s h e d by a number of p h y s i c a l and b i o l o g i c a l p r o p e r t i e s . Blood 51:527, 1978. 47 14. McLeod DL, Shreeve M, Axelrad AA. Induction of megakaryocyte c o l o n i e s w i t h p l a t e l e t formation i n v i t r o . Nature 261:492, 1976. 15. Wolf NS, T r e n t i n J J . Hemopoietic colony s t u d i e s V. E f f e c t of hemopoietic organ stroma on d i f f e r e n t i a t i o n of p l u r i p o t e n t stem c e l l s . J Exp Med 127:205, 1967. 16. T r e n t i n J J . Influence of hematopoietic organ stroma (Hematopoietic i n d u c t i v e microenvironments on stem c e l l d i f f e r e n t i a t i o n ) . In: "Regulation of Hematopoiesis", (ed. Gordon AS), Appleton-Century-Crofts, New York, pp 161, 1970. 17. Lichtman MA. The r e l a t i o n s h i p of stromal c e l l s to hemopoietic c e l l s i n marrow. In: "Long-Term Bone Marrow C u l t u r e " , (eds. DG Wright, JS Greenberger), Alan R L i s s , Inc, New York, pp 13, 1984. 18. A l l e n TD, Dexter TM. The e s s e n t i a l c e l l s of the hemopoietic microenvironment. Exp Hematol 12:517, 1984. 19. Reddi AH, Gay R, Gay S, M i l l e r EJ. T r a n s i t i o n s i n c o l l a g e n types during matrix-induced c a r t i l a g e , bone, and bone marrow formation. Proc N a t l Acad S c i USA 74:5589, 1977. 20. Von der Mark K, Kuhl U. Laminin and i t s receptor. Biochimica et Biophysica Acta 823:147, 1985. 21. Yamada KM, Hayashi M, Hirano H, Akiyama SK. F i b r o n e c t i n and c e l l s u rface i n t e r a c t i o n s . In: "The r o l e of E x t r a c e l l u l a r Matrix i n Development", Alan R L i s s , Inc, New York, pp 89, 1984. 22. Hayman EG, E n g v a l l E, A'Hearn E, Barnes D, Pierschbacher M, R u o s l a h t i E. C e l l attachment on r e p l i c a s of SDS-polyacrylamide g e l s reveals two adhesive plasma p r o t e i n s . J C e l l B i o l 95:20, 1982. 23. A l b e r t s B, Bray D, Lewis J , Raff M, Roberts K, Watson JD. In: "Molecular Bi o l o g y of the C e l l " , Garland P u b l i s h i n g Inc, New York, pp 703, 1983. 24. Campbell AD, Long MW, Wicha MS. Haemonectin, a bone marrow adhesion p r o t e i n s p e c i f i c f o r c e l l s of granulocyte l i n e a g e . Nature 329:744, 1987. 25. Knospe WH, Blom J , Crosby WH. Regeneration of l o c a l l y i r r a d i a t e d bone marrow I . Dose dependent, long-term changes i n the r a t , w i t h p a r t i c u l a r emphasis upon va s c u l a r and stromal r e a c t i o n . Blood 28:398, 1966. 26. Knospe WH, Blom J , Crosby WH. Regeneration of l o c a l l y i r r a d i a t e d bone marrow I I . Induction of regeneration i n permanently a p l a s t i c medullary c a v i t i e s . Blood 31:400, 1968. 27. Patt HM, Maloney MA. Bone marrow regeneration a f t e r l o c a l i n j u r y : A review. Exp Hematol 3:135, 1975. 28. F r i e d e n s t e i n AJ, Petrakova KV, Kurolesova AI, Frolova GP. Heterotopic t r a n s p l a n t s of bone marrow. A n a l y s i s of precursor c e l l s f o r osteogenic and hematopoietic t i s s u e s . T r a n s p l a n t a t i o n 6:230, 1968. 48 29. Metcalf D, Moore MAS. Haemopoietic c e l l s . In: " F r o n t i e r s of B i o l o g y , Volume 24", North-Holland P u b l i s h i n g Company, Amsterdam, 1971. 30. B e r n s t e i n SE. Tissue t r a n s p l a n t a t i o n as an a n a l y t i c therapeutic t o o l i n the treatment of h e r e d i t a r y anemias. Amer J Surg 119:448, 1970. 31. Dexter TM, A l l e n TD, L a j t h a LG. Conditions c o n t r o l l i n g the p r o l i f e r a t i o n of haemopoietic stem c e l l s i n v i t r o . J C e l l P h y s i o l 91:335, 1977. 32. Bentley SA. A cl o s e range c e l l : c e l l i n t e r a c t i o n required f o r stem c e l l maintenance i n continuous bone marrow c u l t u r e . Exp Hematol 9:305, 1981. 33. Whitlock CA, Witte ON. Longterm c u l t u r e of B lymphocytes and t h e i r precursors from murine bone marrow. Proc N a t l Acad S c i USA 79:3608, 1982. 34. Dorshkind K. In v i t r o d i f f e r e n t i a t i o n of B lymphocytes from p r i m i t i v e hemopoietic precursors present i n long-term bone marrow c u l t u r e s . J Immunol 136:422, 1986. 35. Coulombel L, V u i l l e t MH, Leroy C, Tchernia G. Lineage- and stage-s p e c i f i c adhesion of human hematopoietic progenitor c e l l s to e x t r a c e l l u l a r matrices from marrow f i b r o b l a s t s . Blood 71:329, 1988. 36. Roberts R, Gallagher J , Spooncer E, A l l e n TD, Bloomfield F, Dexter TM. Heparan sulphate bound growth f a c t o r s : A mechanism f o r stromal c e l l mediated haemopoiesis. Nature 332:376, 1988. 37. G i a n c o t t i FG, Comoglio PM, Tarone G. Fibronectin-plasma membrane i n t e r a c t i o n i n the adhesion of hemopoietic c e l l s . J C e l l B i o l 103:429, 1986. 38. Bernardi P, P a t e l VP, Lodish HF. Lymphoid precursor c e l l s adhere to two d i f f e r e n t s i t e s on f i b r o n e c t i n . J C e l l B i o l 105:489, 1987. 39. Kao WW-Y, Prockop DJ. Can p r o l i n e analogues be used to prevent f i b r o b l a s t s from overgrowing c u l t u r e s of e p i t h e l i a l c e l l s ? B i r t h D e f e c t s : 0 r i g i n a l A r t i c l e S e r i e s , XVI(Number 2):53, 1980. 40. Zuckerman KS, Rhodes RK, Goodrum DD, P a t e l VR, Sparks B, Wells J , Wicha MS, Mayo LA. I n h i b i t i o n of c o l l a g e n d e p o s i t i o n i n the e x t r a c e l l u l a r matrix prevents the establishment of a stroma supportive of hematopoiesis i n long-term murine bone marrow c u l t u r e s . J C l i n Invest 75:970, 1985. 41. Terranova VP, Rohrbach DH, Martin GR. Role of la m i n i n i n the attachment of PAM 212 ( e p i t h e l i a l ) c e l l s to basement membrane c o l l a g e n . C e l l 22:719, 1980. 42. Vrako R. Basal lamina s c a f f o l d anatomy and s i g n i f i c a n c e f o r maintenance of o r d e r l y t i s s u e s t r u c t u r e . Am J Pathol 77:314, 1974. 43. Gordon MY, R i l e y GP, Watt SM, Greaves MF. Compartmentalization of a haemopoietic growth f a c t o r (GM-CSF) by glycosaminoglycans i n the bone marrow microenvironment. Nature 326:403, 1987. 49 44. S i e f f CA. Hematopoietic growth f a c t o r s . J C l i n Invest 79:1549, 1987. 45. Whetton AD, Dexter TM. Haemopoietic growth f a c t o r s . TIBS 11:207, 1986. 46. Metcalf D. The granulocyte-macrophage colony s t i m u l a t i n g f a c t o r s . C e l l 43:5, 1985. 47. Metcalf D. The molecular biology and functions of the granulocyte-macrophage c o l o n y - s t i m u l a t i n g f a c t o r s . Blood 67:257, 1986. 48. Sanderson CJ, Warren DJ, S t r a t h M. I d e n t i f i c a t i o n of a lymphokine that s t i m u l a t e s e o s i n o p h i l d i f f e r e n t i a t i o n i n v i t r o . I t s r e l a t i o n s h i p to i n t e r l e u k i n 3, and f u n c t i o n a l p r o p e r t i e s of e o s i n o p h i l s produced i n c u l t u r e s . J Exp Med 162:60, 1985. 49. Lopez AF, Gegley CG, Williamson DJ, Warren DJ, Vadas MA, Sanderson CJ. Murine e o s i n o p h i l d i f f e r e n t i a t i o n f a c t o r - an e o s i n o p h i l s p e c i f i c colony s t i m u l a t i n g f a c t o r with a c t i v i t y f o r human c e l l s . J Exp Med 163:1085, 1986. 50. Warren DJ, Moore MAS. Synergism among i n t e r l e u k i n 1, i n t e r l e u k i n 3, and i n t e r l e u k i n 5 i n the production of e o s i n o p h i l s from p r i m i t i v e hemopoietic stem c e l l s . J Immunol 140:94, 1988. 51. O'Garra A. Umland S, De France T, C h r i s t i a n s e n J . " B - c e l l f a c t o r s " are p l e i o t r o p i c . Immunol Today 9:45, 1988. 52. Z u c a l i JR, D i n a r e l l o CA, Obion DJ, Gross MA, Anderson L, Weiner RS. I n t e r l e u k i n 1 s t i m u l a t e s f i b r o b l a s t s to produce granulocyte-macrophage c o l o n y - s t i m u l a t i n g a c t i v i t y and prostaglandin E2- J C l i n Invest 77:1857, 1986. 53. Le J , V i l c e k J . Tumor nec r o s i s f a c t o r and i n t e r l e u k i n - 1 : C y t o k i n e s w i t h m u l t i p l e overlapping b i o l o g i c a l a c t i v i t i e s . Lab Invest 56:234, 1987. 54. Peschel C, Paul WE, Ohara J , Green I . E f f e c t s of B C e l l s t i m u l a t o r y f a c t o r - l / I n t e r l e u k i n 4 on hematopoietic progenitor c e l l s . Blood 70:254, 1987. 55. Rennick D, Yang G, Muller-Sieburg C, Smith C, A r a i N, Takabe Y, Gemmel L. I n t e r l e u k i n 4 ( B - c e l l s t i m u l a t o r y f a c t o r - 1 ) can enhance or antagonize the factor-dependent growth of hemopoietic progenitor c e l l s . Proc N a t l Acad S c i USA 84:6889, 1987. 56. Wong GG, C l a r k SC. M u l t i p l e a c t i o n s of i n t e r l e u k i n 6 w i t h i n a cytokine network. Immunol Today 9:137, 1988. 57. Gauldie J , Richards C, Harnish D, Lansdorp P, Baumann H. I n t e r f e r o n 3 2 / B - c e l l f a c t o r 2 shares i d e n t i t y with monocyte-derived hepatocyte-s t i m u l a t i n g f a c t o r and regulates the major acute phase p r o t e i n response i n l i v e r c e l l s . Proc N a t l Acad Sci USA 84:7251, 1987. 58. Ikebuchi K, Wong GG, C l a r k SC, I h l e JN, H i r a i Y, Ogawa M. I n t e r l e u k i n 6 enhancement of i n t e r l e u k i n 3-dependent p r o l i f e r a t i o n of m u l t i p o t e n t i a l hemopoietic progenitors. Proc N a t l Acad S c i USA 84:9035, 1987. 50 59. Quesenberry PJ. S y n g e r g i s t i c hematopoietic growth f a c t o r s . Int J C e l l Cloning 4:3, 1986. 60. Quesenberry P, Song Z, McGrath E, McNiece I , Shadduck R, Waheed A, Baber G, Kleeman E, Kai s e r D. M u l t i l i n e a g e s y n e r g i s t i c a c t i v i t y produced by a murine adherent marrow c e l l l i n e . Blood 69:827, 1987. 61. Walker F, N i c o l a NA, Metcalf D, Burgess AW. H i e r a r c h i c a l down-modulation of hemopoietic growth f a c t o r receptors. C e l l 43:269, 1985. 62. Lord BI, Wright EG. I n t e r a c t i o n s of i n h i b i t o r s and s t i m u l a t o r s i n the r e g u l a t i o n of CFU-S p r o l i f e r a t i o n . Leukemia Res 6:541, 1982. 63. Broxmeyer HE, G e n t i l e P, Bognacki J , Ralph P. L a c t o f e r r i n , t r a n s f e r r i n and a c i d i c i s o f e r r i t i n s : Regulatory molecules with p o t e n t i a l t h e r a p e u t i c value i n leukemia. Blood C e l l s 9:83, 1983. 64. Murphy M, Pe r u s s i a B, T r i n c h i e r i G. E f f e c t s of recombinant tumor n e c r o s i s f a c t o r , lymphotoxin, and immune i n t e r f e r o n on p r o l i f e r a t i o n and d i f f e r e n t i a t i o n of enriched hematopoietic precursor c e l l s . Exp Hematol 16:131, 1988. 65. Pelus LM, Ottmann 0G, Nocka KH. S y n e r g i s t i c i n h i b i t i o n of human marrow granulocyte-macrophage progenitor c e l l s by prostaglandin E and recombinant interferon-a,-|3, and -y and an e f f e c t mediated by tumor ne c r o s i s f a c t o r . J Immunol 140:479, 1988. 66. Sporn MB, Roberts AB, Wakefield LM, Assoian RK. Transforming growth f a c t o r - 0 : B i o l o g i c a l f u n c t i o n and chemical s t r u c t u r e . Science 233:532, 1986. 67. Ohta M, Greenberger JS, A n k l e s a r i a P, Bassols A, Massague J . Two forms of transforming growth f a c t o r - 0 d i s t i n g u i s h e d by m u l t i p o t e n t i a l haemopoietic progenitor c e l l s . Nature 329:539, 1987. 68. C h e i f e t z S, Weatherbee JA, Tsang MLS, Anderson JK, Mole JE, Lucas R, Massague J . The transforming growth f a c t o r - g system, a complex p a t t e r n of c r o s s - r e a c t i v e l i g a n d s and receptors. C e l l 48:409, 1987. 69. Eaves AC, Eaves CJ. Maintenance and p r o l i f e r a t i o n c o n t r o l of p r i m i t i v e hemopoietic progenitors i n long-term c u l t u r e s of human marrow c e l l s . Blood C e l l s ( i n p r e s s ) . 70. Lee G, E l l i n g s w o r t h LR, G i l l i s S, Wall R, Kincade PW. |3 transforming growth f a c t o r s are p o t e n t i a l r e g u l a t o r s of B lymphopoiesis; J Exp Med 166:1290, 1987. 71. Ignotz RA, Massague J . C e l l adhesion p r o t e i n receptors as targ e t s f o r transforming growth f a c t o r - P a c t i o n . C e l l 51:189, 1987. 72. Penttinen RP, Kobayashi S, Bornstein P. Transforming growth f a c t o r 0 increases mRNA f o r matrix proteins both i n the presence and i n the absence of changes i n mRNA s t a b i l i t y . Proc N a t l Acad S c i USA 85:1105, 1988. 51 73. Wu AM, T i l l JE, Siminovitch L, McCulloch EA. C y t o l o g i c a l evidence f o r a r e l a t i o n s h i p between normal hematopoietic colony-forming c e l l s and c e l l s of the lymphoid system. J Exp Med 127:455, 1968. 74. Abramson S, M i l l e r RG, P h i l l i p s RA. I d e n t i f i c a t i o n of p l u r i p o t e n t and r e s t r i c t e d stem c e l l s of the myeloid and lymphoid systems. J Exp Med 145:1567, 1972. 75. Leung LC, Johnson GR. In v i t r o maintenance of hemopoietic stem c e l l s w i t h lymphoid and myeloid repopulating a b i l i t y by a cloned murine adherent bone marrow c e l l l i n e . Exp Hematol 15:989, 1987. 76. Lemischka IR, Raulet DH, Mu l l i g a n RC. Developmental p o t e n t i a l and dynamic behavior of hematopoietic stem c e l l s . C e l l 45:917, 1986. 77. P r c h a l JT, Throckmorton DW, C a r r o l AJ, Fuson EW, Gams RA, P r c h a l JF. A common progenitor f o r human myeloid and lymphoid c e l l s . Nature 274: 90, 1978. 78. N i t t a M, Kato Y, S t r i f e A, Wachter M, F r i e d J , Perez A, Jhanwar S, Duigou-Osterndof R, Chagante RSK, Clarkson B. Incidence of involvement of the B and T lymphocyte lineages i n chronic myelogeneous leukemia. Blood 66: 1053. 79. Martin PJ, N a j f e l d V, Hansen JA, Penfold GK, Jacobson RJ, Fialkow PJ. Involvement of the B-lymphoid system i n chronic myelogeneous leukaemia. Nature 287: 49, 1980. 80. Bartram CR, Raghavachar A, Anger B, S t a i n C, Bettelheim P. T lymphocytes l a c k rearrangement of the bcr gene i n P h i l a d e l p h i a chromosome-positive chronic myelocytic leukemia. Blood 69:1682, 1987. 81. Hernandez P, Carnot J , Cruz C. Chronic myeloid leukemia b l a s t c r i s i s w i t h T c e l l f e a t u r e s . Br J Haematol 51:175, 1982. 82. Chan LC, Furley AJ, Ford AM, Yardumian DA, Greaves MF. C l o n a l rearrangement and expression of the T c e l l receptor ft gene and involvement of the breakpoint c l u s t e r region i n b l a s t c r i s i s of CGL. Blood 67:533, 1986. 83. Cooper MD, Cain WA, Van A l t e n PJ, Good RA. Development and f u n c t i o n of the immunoglobulin-producing system: I . E f f e c t of bursectomy at d i f f e r e n t stages of development on germinal centers, plasma c e l l s , immunoglobulins and antibody production. Int Arch A l l e r g y Appl Immunol 35:242, 1969. 84. Le Douarin NM, Houssaint E, Jotereau FV, Belo M. O r i g i n of haemopoietic stem c e l l s i n the embryonic bursa of F a b r i c i u s and bone marrow studi e d through i n t e r s p e c i f i c chimaeras. Proc N a t l Acad S c i USA 72:2701, 1975. 85. Lydyard PM, G r o s s i CE, Cooper MD. Ontogeny of B c e l l s i n the chicken: I . Sequential development of c l o n a l d i v e r s i t y i n the bursa. J Exp Med 144:79, 1976. 86. W e i l l J-C, Reynaud C-A. The chicken B c e l l compartment. Science 238:1094, 1987. 52 87. Owen JJT, Raff MC, Cooper MD. Studies on the generation of B lymphocytes i n the mouse embryo. Eur J Immunol 5:468, 1975. 88. Kincade PW. Formation of B lymphocytes i n f e t a l and adult l i f e . Adv Immunol 31:177, 1981. 89. Moore MAS, Owen JJT. Stem c e l l migration i n developing myeloid and lymphoid systems. Lancet 2:658, 1967. 90. Owen JJT, Cooper MD, Raff MC. In v i t r o generation of B lymphocytes i n mouse f e t a l l i v e r : A mammalian "bursa equivalent". Nature 249:361, 1974. 91. Scher I . B-lymphocyte ontogeny. CRC C r i t Rev Immunol 3:287, 1981. 92. Rosse C. Perspectives of lymphocyte production and c e l l u l a r t r a f f i c i n the bone marrow. I n : "Handbook of Cancer Immunology, Volume 6", (ed. H. Waters), Garland STPM Press, New York, pp 250, 1981. 93. Cooper MD, Kearney J , Scher I . B lymphocytes. In: "Fundamental Immunology", (ed. WE P a u l ) , Raven Press, New York, pp 43, 1984. 94. Tonegawa S. Somatic generation of antibody d i v e r s i t y . Nature 302:575, 1983. 95. A l t FW, B l a c k w e l l TK, DePinho RA, Reth MG, Yancopoulos GD. Regulation of genome rearrangement events during lymphocyte d i f f e r e n t i a t i o n . Immunol Rev 89:5, 1986. 96. Brodeur P, R i b l e t t R. The immunoglobulin heavy chain v a r i a b l e region (Ig-H-V) i n the mouse. I . 100 IgH-V genes comprise 7 f a m i l i e s of homologous genes. Eur J Immunol 14:922, 1984. 97. A l t F, Rosenberg N, Lewis S, Thomas E, Baltimore D. Organization and r e o r g a n i z a t i o n of immunoglobulin genes i n A-MuLV-transformed c e l l s : rearrangement of heavy but not l i g h t chain genes. C e l l 27:381, 1981. 98. A l t FW, Yancopoulos GD, B l a c k w e l l TK, Wood C, Thomas E, Boss M, Coffman R, Rosenberg N, Tonegawa S, Baltimore D. Ordered rearrangement of immunoglobulin heavy chain v a r i a b l e region segments. EMB0 J . 3:1209, 1984. 99. E a r l y P, Huang H> Davis M, Calame K, Hood L. An immunoglobulin heavy chain v a r i a b l e region gene i s generated from three segments of DNA: Vp_, D and J H . C e l l 19:981, 1980. 100. Hood L, Kronenberg M, Hu n k a p i l l e r T. T c e l l antigen receptor and the immunoglobulin supergene family C e l l 40:225, 1985. 101. Baltimore D. Is terminal deoxynucleotidyl transferase a somatic mutagen i n lymphocytes? Nature 248:409, 1974. 102. Landau NR, Schatz DG, Rosa M, Baltimore D. Increased frequency of N-region i n s e r t i o n i n a murine p r e - B - c e l l l i n e i n f e c t e d w i t h a t e r m i n a l d e o x y n u c l e o t i d y l t ransferase r e t r o v i r a l expression vector. Molec C e l l B i o l 7:3237, 1987. 53 103. Golub ES. Somatic mutation: D i v e r s i t y and r e g u l a t i o n of the immune r e p e r t o i r e . C e l l 48:723, 1987. 104. Yancopoulos GD, A l t FW. Regulation of the assembly and expression of v a r i a b l e - r e g i o n genes. Ann Rev Immunol 4:339, 1986. 105. A l t PW. E x c l u s i v e immunoglobulin genes. Nature 312:502, 1984. 106. Whitlock C, Denis K, Robertson D, Witte 0. In v i t r o a n a l y s i s of murine B - c e l l development. Ann Rev Immunol 3:213, 1985. 107. Coffman RL, Weissman IL . Immunoglobulin gene rearrangement during pre-B c e l l d i f f e r e n t i a t i o n . J Mol C e l l Immunol 1:31, 1983. 108. Marcu K, Cooper M. New views of the immunoglobulin heavy-chain s w i t c h . Nature 306:243, 1982. 109. Shimizu A, Honjo T. Ig c l a s s s w i t c h i n g . C e l l 36:801, 1984. 110; Burrows PD, Beck-Engeser GB, Wabl MR. Immunoglobulin heavy-chain c l a s s s w i t c h i n g i n a pre-B c e l l l i n e i s accompanied by DNA rearrangement. Nature 306:243, 1983. 111. Park Y-H, Osmond DG. Phenotype and p r o l i f e r a t i o n of e a r l y B lymphocyte precursor c e l l s i n mouse bone marrow. J Exp Med 165:444, 1987. 112. Coffman RL, Weismann IL. B220: a B c e l l - s p e c i f i c member of the T200 g l y c o p r o t e i n f a m i l y . Nature 289:681, 1981. 113. Kincade PW, Lee G, Watanabe T, Sun L, Scheid MP. Antigens d i s p l a y e d on murine B lymphocyte precursors. J Immunol 127:2262, 1981. 114. Trowbridge IS. I n t e r s p e c i e s spleen-myeloma hybrid producing monoclonal a n t i b o d i e s against mouse lymphocyte surface g l y c o p r o t e i n , T200. J Exp Med 148:313, 1978. 115. Siadak AW, Nowinski RC. I d e n t i f i c a t i o n of Ly-5 and T200 antigens on i d e n t i c a l c e l l surface p r o t e i n s . J Immunol 125:1400, 1980. 116. Coffman RL. Surface antigen expression and immunoglobulin gene rearrangement during mouse pre-B c e l l development. Immunological Rev 69:5, 1982. 117. Eckhardt LA, Herzenberg LA. Monoclonal antibody to ThB detect c l o s e l i n k a g e of Ly-6 and a gene r e g u l a t i n g ThB expression. Immunogenetics 11:275, 1980. 118. R e i f AE, A l l e n JMV. The AKR thymic antigen and i t s d i s t r i b u t i o n i n leukemias and nervous t i s s u e s . J Exp Med 120:413, 1964. 119. Ledbetter JA, Herzenberg LA. Xenogeneic monoclonal a n t i b o d i e s to mouse lymphoid d i f f e r e n t i a t i o n antigens. Immunol Rev 47:63, 1979. 120. Basch RS, Berman JW. Thy-1 determinants are present on many murine hematopoietic c e l l s other than T c e l l s . Eur J Immunol 12:359, 1982. 54 121. M u l l e r - S i e b u r g CE, Whitlock CA, Weissman IL. I s o l a t i o n of two e a r l y B lymphocyte progenitors from mouse marrow: a committed pre-pre-B c e l l and a clonogenic Thy-l-*-0 hematopoietic stem c e l l . C e l l 44:653, 1986. 122. Cooper MD, Mulvaney D, Coutinho A, Cazenave P-A, A novel c e l l surface molecule on e a r l y B-lineage c e l l s . Nature 321:616, 1986. 123. P i l l e m e r E, Whitlock C, Weissman IL . Transformation-associated p r o t e i n s i n murine B - c e l l lymphomas that are d i s t i n c t from Abelson v i r u s gene products. Proc N a t l Acad S c i USA 81:4434, 1984. 124. Whitlock CA, Tidmarsh GF, Muller-Sieburg C, Weissman IL. Bone marrow stromal c e l l l i n e s with lymphopoietic a c t i v i t y express high l e v e l s of a pre-B ne o p l a s i a - a s s o c i a t e d molecule. C e l l 48:1009, 1987. 125. Scher I , Berning AK, K e s s l e r S, Finkelman FD. Development of B lymphocytes i n the mouse; studies of the frequency and d i s t r i b u t i o n of surface IgM and IgD i n normal and immunodefective CBA/N F^ mice. J Immunol 125:1686, 1980. 126. Goding JW, Layton JE. Antigen-induced co-capping of IgM and I g D - l i k e receptors on murine B c e l l s . J Exp Med 144:852, 1976. 127. Kettman JR, Cambier JC, Uhr JW, L i g l e r F, V i t t e t a ES. The r o l e of receptor IgM and IgD i n determining t r i g g e r i n g and i n d u c t i o n of tolerance i n murine B c e l l s . Immunol Rev 43:69, 1979. 128. Gelfand MC, E l f e n b e i n GJ, Frank MM, Paul WE. Ontogeny of B lymphocytes. I I . R e l a t i v e rates of appearance of lymphocytes bearing surface immunoglobulin and complement receptors. J Exp Med 139:1125, 1974. 129. Hammerling U, Chin AF, Abbot J , Scheid MP. The ontogeny of murine B lymphocytes. I . Induction of phenotypic conversion of I a - to I a + lymphocytes. J Immunol 115:1425, 1975. 130. Kearny JF, Cooper MD, K l e i n J , Abney ER, Parkerhouse RME, Lawton AR. Ontogeny of I a and IgD on IgM-bearing B lymphocytes i n mice. J Exp Med 146:297, 1977. 131. Bianco C, P a t r i c k R, Nussenzweig V. A population of lymphocytes bearing a membrane receptor f o r antigen-antibody-complement complexes. I ; Separation and c h a r a c t e r i z a t i o n . J Exp Med 132:702, 1970. 132. Scher I . The CBA/N mouse s t r a i n : An experimental model i l l u s t r a t i n g the i n f l u e n c e of the X-chromosome on immunity. Adv Immunol 33:1, 1981. 133. V i t t e t a ES, Brooks K, Isakson P, Layton J , Pure E, Yuan D. B lymphocyte receptors. In: "Fundamental Immunology", (ed. WE P a u l ) , Raven Press, New York, pp 221, 1984. 134. Berman MA, Weigle W0. B-lymphocyte a c t i v a t i o n by the Fc region of IgG. J Exp Med 146:241, 1977. 55 135. F e s t e n s t e i n H. Immunogenetic and b i o l o g i c a l aspects of i n v i t r o lymphocyte a l l o t r a n s f o r m a t i o n (MLR) i n the mouse. Transplant Rev 15:62, 1973. 136. F e s t e n s t e i n H. Pe r t i n e n t features of M locus determinants i n c l u d i n g r e v i s e d nomenclature and s t r a i n d i s t r i b u t i o n . T r a n s p l a n t a t i o n 18:555, 1974. 137. Huber BR, Gershon RD, Cantor H. I d e n t i f i c a t i o n of a B c e l l surface s t r u c t u r e involved i n antigen-dependent t r i g g e r i n g : Absence of t h i s s t r u c t u r e on B c e l l s from CBA/N mutant mice. J Exp Med 145:10, 1977. 138. Scher I , Steinberg AD, Berning AK, Paul WE. X - l i n k e d B-lymphocyte immune defect i n CBA/N mice. I I . Studies of the mechanisms u n d e r l y i n g the immune defect. J Exp Med 142: 637, 1975. 139. Lemoine FM, Humphries RK, Abraham SDM, K r y s t a l G, Eaves CJ. P a r t i a l c h a r a c t e r i z a t i o n of a novel stromal c e l l - d e r i v e d pre-B c e l l growth f a c t o r a c t i v e on normal and immortalized pre-B c e l l s . J Exp Hematol 16:718, 1988. 140. Hunt P, Robertson D, Weiss D, Rennick D, Lee F, Witte ON. A s i n g l e bone marrow-derived stromal c e l l type supports i n v i t r o growth of e a r l y lymphoid and myeloid c e l l s . C e l l 48:997, 1987. 141. Kierney PC, Dorshkind K. B lymphocyte precursors and myeloid progenitors s u r v i v e i n d i f f u s i o n chamber c u l t u r e s but B c e l l d i f f e r e n t i a t i o n r equires c l o s e a s s o c i a t i o n with stromal c e l l s . Blood 70:1418, 1987. 142. Landreth KS, Dorshkind K. Pre-B c e l l generation p o t e n t i a t e d by s o l u b l e f a c t o r s from a bone marrow stromal c e l l l i n e . J Immunol 140:845, 1988. 143. Jerne NK, Nordin AA. Plaque formation i n agar by s i n g l e antibody-producing c e l l s . Science 140:405, 1963. 144. M i s h e l l RI, Dutton RW. Immunization of d i s s o c i a t e d spleen c e l l c u l t u r e s from normal mice. J Exp Med 126:423, 1967. 145. Metcalf D, Nossal GJV, Warner NL, M i l l e r JFAP, Mandel TE. Growth of B lymphocyte c o l o n i e s i n v i t r o . J Exp Med 142:1534, 1975. 146. Kincade PW, Ralph P, Moore MAS. Growth of B-lymphocyte clones i n s e m i s o l i d c u l t u r e s i s mitogen dependent. J Exp Med 143:1265, 1976. 147. Paige CJ. Surface immunoglobulin-negative B - c e l l precursors detected by formation of an t i b o d y - s e c r e t i n g c o l o n i e s i n agar. Nature 302:711, 1983. 148. Paige CJ, G i s l e r RH, McKearn JP, Iscove NN. D i f f e r e n t i a t i o n of murine B c e l l precursors i n agar c u l t u r e . Frequency, surface marker a n a l y s i s and requirements f o r growth of clonable pre-B c e l l s . Eur J Immunol 14:979, 1984. 56 149. Sauter H, Paige CJ. Detection of normal B - c e l l precursors that g i v e r i s e to c o l o n i e s producing both K and X l i g h t immunoglobulin chains. Proc N a t l Acad S c i USA 84:4989, 1987. 150. Howard M, K e s s l e r S, Chused T, Paul WE. Long term c u l t u r e of normal mouse B lymhocytes. Proc N a t l Acad S c i USA 78:5788, 1981. 151. P a l a c i o s R, Henson G, Steinmetz M, McKearn JP. I n t e r l e u k i n - 3 supports growth of mouse pre-B c e l l clones i n v i t r o . Nature 309:126, 1984. 152. P a l a c i o s R, Steinmetz M. IL3-dependent mouse clones that express B-220 surface antigen, contain I g genes i n germ-line c o n f i g u r a t i o n , and generate B lymphocytes i n v i v o . C e l l 41:727, 1985. 153. P a l a c i o s R, Neri T, Brochaus M. Monoclonal a n t i b o d i e s s p e c i f i c f o r i n t e r l e u k i n 3 - s e n s i t i v e murine c e l l s . J Exp Med 163:369, 1986. 154. P a l a c i o s R, Leu T. CC11: a monoclonal antibody s p e c i f i c f o r I n t e r l e u k i n 3 - s e n s i t i v e mouse c e l l s defines two major populations of B c e l l precursors i n the bone marrow. Immunol Rev 93:125, 1986. 155. Schrader JW, Schrader S. In v i t r o s t u d i e s on lymphocyte d i f f e r e n t i a t i o n . I . Long-term i n v i t r o c u l t u r e of c e l l s g i v i n g r i s e to f u n c t i o n a l lymphocytes i n i r r a d i a t e d mice. J Exp Med 148:823, 1978. 156. Dorshkind K, P h i l l i p s RA. C h a r a c t e r i z a t i o n of e a r l y B lymphocyte precursors present i n long-term bone marrow c u l t u r e s . J Immunol 131:2240, 1983. 157. Whitlock CA, Robertson D, Witte ON. Murine B c e l l lymphopoiesis i n long term c u l t u r e . J Immunol Meth 67:353, 1984. 158. Dorshkind K, Witte ON. Long-term murine hemopoietic c u l t u r e s as model systems f o r a n a l y s i s of B lymphocyte d i f f e r e n t i a t i o n . Curr Topics M i c r o b i o l Immunol 135:23, 1987. 159. Denis KA, Witte ON. In v i t r o development of B lymphocytes from long-term c u t l u r e d precursor c e l l s . Proc N a t l Acad S c i USA 83:441, 1986. 160. Siden EJ, Baltimore D, C l a r k D, Rosenberg NE. Immunoglobulin s y n t h e s i s by lymphoid c e l l s transformed i n v i t r o by Abelson murine leukemia v i r u s . C e l l 1979:16, 389. 161. Whitlock CA, Z i e g l e r SF, Treiman L J , S t a f f o r d J l , Witte ON. . D i f f e r e n t i a t i o n of cloned populations of immature B c e l l s a f t e r transformation w i t h Abelson murine leukemia v i r u s . C e l l 32:903, 1983. 162. Whitlock CA, Z i e g l e r SF, Witte ON. Progression of the transformed phenotype i n c l o n a l l i n e s of Abelson v i r u s - i n f e c t e d lymphocytes. Molec C e l l B i o l 3:596, 1983. 163. Denis KA, Treiman L J , St C l a i r e J l , Witte ON. Long-term c u l t u r e s of murine f e t a l l i v e r r e t a i n very e a r l y B lymphoid phenotype. J Exp Med 160:1087, 1984. 57 164. Kurland J I , Z i e g l e r SF, Witte ON. Long-term c u t l u r e d B lymphocytes and t h e i r precursors r e c o n s t i t u t e the B-lymphocyte lineage i n v i t r o . Proc N a t l Acad S c i USA 81:7554, 1984. 165. Bosma GC, Custer RP, Bosma MJ. A severe combined immunodeficiency mutation i n the mouse. Nature 301:527, 1983. 166. Custer RP, Bosma GC, Bosma MJ. Severe combined immunodeficiency (SCID) i n the mouse: pathology, r e c o n s t i t u t i o n , neoplasms. Am J P a t h o l 120:464, 1985. 167. Dorshkind K, P o l l a c k SB, Bosma MJ, P h i l l i p s RA. N a t u r a l k i l l e r c e l l s are present i n mice with severe combined immunodeficiency ( s c i d ) . J Immunol 134:3798, 1985. 168. O'toole M, Bosma MJ. F u n c t i o n a l status of c e l l s from lymphoid and myeloid t i s s u e s i n mice with severe combined immunodeficiency disease. J Immunol 132:1804, 1984. 169. Fulop GM, P h i l l i p s RA. F u l l r e c o n s t i t u t i o n of the immune d e f i c i e n c y i n s c i d mice with normal stem c e l l s r equires low-dose i r r a d i a t i o n of the r e c i p i e n t s . J Immunol 136:4438, 1986. 170. Witte PL, Burrows PD, Kincade PW, Cooper MD. C h a r a c t e r i z a t i o n of B lymphocyte lineage progenitor c e l l s from mice with severe combined immune d e f i c i e n c y disease (SCID) made p o s s i b l e by long term c u l t u r e . J Immunol 138:2698, 1987. 171. Denis KA, Dorskhind K, Witte ON. Regulated progression of B lymphocyte d i f f e r e n t i a t i o n from c u l t u r e d f e t a l l i v e r . J Exp Med 166:391, 1987. 172. Schultz LD. P l e i o t r o p i c mutations causing abnormalities i n the murine immune system and the s k i n . Curr P r o b l Dermatol 17:236, 1987. 173. Kincade PW. Experimental models f o r understanding B lymphocyte formation. Adv Immunol 41:181, 1987. 174. Greiner DL, Goldschneider I , Komschlies KL, Medlock ES, Bollum F J , Schultz L. Defective lymphopoiesis i n bone marrow of motheaten (me/me) and v i a b l e motheaten (mev/mev) mutant mice. I . A n a l y s i s of development of prothymocytes. E a r l y B lineage c e l l s , and terminal d e o x y n u c l e o t i d y l t r a n s f e r a s e - p o s i t i v e c e l l s . J Exp Med 164:1129, 1986. 175. Medlock ES, Goldschneider I , Greiner DL, Schultz L. D e f e c t i v e lymphopoiesis i n the bone marrow of motheaten (me/me) and v i a b l e motheathen (me v/me v) mutant mice. I I . D e s c r i p t i o n of a microenvironmental defect f o r the generation of terminal d e o x y n u c l e o t i d y l t r a n s f e r a s e - p o s i t i v e bone marrow c e l l s i n v i t r o . J Immunol 138:3590, 1987. 176. Hayashi S-I, Witte PL, Shultz LD, Kincade PW. Lymphohemopoiesis i n c u l t u r e i s prevented by i n t e r a c t i o n with adherent bone marrow c e l l s from mutant v i a b l e motheaten mice. J Immunol 140:2139, 1988. 58 177; Coffman RL, Seymour BWP, Lebman DA, H i r a k i DD, C h r i s t i a n s e n JA, Shrader B, Cherwinski HM, Savelkoul HFJ, Finkelman FD, Bond MW, Mosmann TR. The r o l e of helper T c e l l products i n mouse B c e l l d i f f e r e n t i a t i o n ad i s o t y p e r e g u l a t i o n . Immunol Rev 102:190, 1988. 178. Namen AE, Schmierer AE, March CJ, O v e r e l l RW, Park LS, Urdal DL, . Mochizuki DY. B c e l l precursor growth-promoting a c t i v i t y . J Exp Med 167:988, 1988. 179. Namen AE, Lupton S, H j e r r i l d K, Wignall J , Mochizuki DY, Schmierer A, Mosley B, March CJ, Urdal DL, G i l l i s S, Cosman D, Goodwin RG. S t i m u l a t i o n of B - c e l l progenitors by cloned murine i n t e r l e u k i n - 7 . Nature 333:571, 1988. 59 C H A P T E R I I MATERIALS AND METHODS 1) ESTABLISHED CELL LINES B6SUtA c e l l s (1) were maintained i n RPMI 1640 supplemented with 20% FCS and medium conditioned by pokeweed mitogen stimulated mouse spleen c e l l s (PWM-SCCM) at a f i n a l concentration of 5% ( 2 ) . 32D clone 3 c e l l s ( 1 ) , were s i m i l a r l y maintained i n RPMI 1640 supplemented with 20% FCS, but 15% WEHI 3B conditioned medium (CM) ins t e a d of PWM-SCCM was used as a source of IL-3. NIH 3T3 f i b r o b l a s t s were grown i n DMEM supplemented with 10% FCS. 2) ANIMALS Balb/cJ - +/+ and Balb/cJ - nu/nu mice were o r i g i n a l l y purchased e i t h e r from Jackson Laboratories (Bar Harbour,ME) or from Simonsen l a b o r a t o r i e s ( G i l r o y , CA) and then bred and maintained i n our f a c i l i t y . (C57B1/6J x C3H/HeJ) ? 1 hybrid mice and (WB/Re x C57B1/6J) F1 - +/+ mice were bred and maintained i n our f a c i l i t y , s t a r t i n g from p a r e n t a l stocks o r i g i n a l l y purchased from Jackson L a b o r a t o r i e s . 60 3) GROWTH FACTORS Recombinant human IL-1|3, recombinant murine IL-3 and recombinant murine GM-CSF were k i n d l y provided as h i g h l y p u r i f i e d reagents a f t e r expression i n E. c o l i by Biogen (Boston, MA). Highly p u r i f i e d forms of recombinant human IL-2 ( l o t 103A), recombinant human G-CSF, and recombinant human EGF were purchased from Amgen (Thousand Oaks, CA). P a r t i a l l y p u r i f i e d n a t u r a l GM-CSF and recombinant murine IL-4 were purchased from Genzyme (Boston, MA). Highly p u r i f i e d recombinant hybridoma growth f a c t o r (which i s the same as IFN-32 (3)) was a g i f t from Dr. L.A. Aarden (Amsterdam, The Netherlands). H i g h l y p u r i f i e d recombinant human I F N - Y was obtained from Dr. J . Gutterman (M.D. Anderson H o s p i t a l and Tumor I n s t i t u t e , Houston, TX). P u r i f i e d r a t TGF-a was purchased from Bachem Inc. (Torrance, CA) and p u r i f i e d porcine TGF-P^ was purchased from R. and D. Systems, Inc. (Minneapolis, MN). 4) ANTIBODIES Rabbit a n t i - l a m i n i n , a n t i - t y p e I c o l l a g e n and a n t i - t y p e IV c o l l a g e n a n t i b o d i e s were obtained from Dr. H. Furthmayr (Yale U n i v e r s i t y , New Haven, CT) and had been generated by immunization of r a b b i t s with the corresponding antigen p u r i f i e d from the ECM (4,5). Rabbit anti-FN receptor (FN-R) and anti-VN receptor (VN-R) a n t i b o d i e s were k i n d l y provided by Dr.M. Pierschbacher (La J o l l a Cancer Research Foundation, La J o l l a , CA). They had been generated by immunization of r a b b i t s w i t h the corresponding receptor p r e v i o u s l y a f f i n i t y - p u r i f i e d from human placenta as described (6,7). Immune sera were then p u r i f i e d by a f f i n i t y chromatography on receptor Sepharose columns. 61 Rabbit anti-human f a c t o r V I I I , which c r o s s - r e a c t s w i t h mouse f a c t o r V I I I ( 8 ) , was obtained from Dako Corporation (Santa Barbara, CA). A l i s t and d e s c r i p t i o n of the s p e c i f i c i t y and source of the r a t and mouse MoAb's used i s given i n Table 3. F l u o r e s c e i n i s o t h i o c y a n a t e (FITC) l a b e l l e d goat a n t i - r a b b i t IgG and FITC l a b e l l e d F(ab')2 goat anti-mouse IgG were purchased from Cappel L a b o r a t o r i e s ( C o c h r a n v i l l e , PA). Monoclonal mouse a n t i - r a t K antibody (from ATCC) was l a b e l l e d w i t h FITC and k i n d l y provided by Dr. P. Lansdorp (Terry Fox Laboratory, Vancouver, BC). FITC l a b e l e d goat anti-mouse IgM (y chain s p e c i f i c ) was obtained from Cedarlane Laboratories (Hornby, ONT). 5) PROBES To detect I g H chain gene rearrangement a 0.770 kb Xbal-EcoRI i n s e r t corresponding to a region 3' of J g ^ and 5' of Cu was i s o l a t e d from the p J H 12/23 plasmid (from F. A l t , Columbia U n i v e r s i t y , New York, NY - 20). For T i p gene rearrangements a plasmid 86T5 c o n t a i n i n g a 0.650 kb EcoRI cDNA fragment (from M. Davis, Stanford U n i v e r s i t y , Stanford, CA - 21) encompassing v a r i a b l e , j o i n i n g and constant regions of the T i p gene was used. To detect v - a b l sequences i n c l o n a l c e l l l i n e s , a plasmid pABlsub9 (from D.Baltimore, Massachusetts I n s t i t u t e of Technology, Cambridge, MA -22) was used. 6) PROTEINS AND PEPTIDES FN and VN were k i n d l y provided by Dr. M.D. Pierschbacher and were p u r i f i e d from human plasma. P u r i f i e d c o l l a g e n type I (Sigma, St. Lo u i s , M0) 62 Table 3. L i s t and d e s c r i p t i o n of the monoclonal a n t i b o d i e s used i n t h i s study. S p e c i f i c Antibodies Used f o r A n a l y s i s Reagent O r i g i n S p e c i f i c i t y Reference RA3-3A 1/6.1 (B220) YE1/30 (Thy 1) YE1/21 (T200) YE1/9 Ml/69 YE4/20 ThB YE6/26 Ml/70 (Mac-1) 10-4-22 BP-1 34-5-8S 15-5-5S a MK-D6 14-4-4S Anti-mouse IgM (u chain s p e c i f i c ) TdT Rat Rat Rat Rat Rat Rat Rat Rat Rat Mouse Mouse Mouse Mouse Mouse Mouse Goat pre-B & B lymphocytes thymocytes & p r i m i t i v e hemopoietic c e l l s hemopoietic c e l l s (except e r y t h r o i d c e l l s ) t r a n s f e r r i n receptor hemopoietic c e l l s (except p e r i p h e r a l T lymphocytes) IgM a l l o t y p e thymocytes and B lymphocytes Mo-MuLV gp70 monocytes and granulocytes IgD of a,c,d, or f a l l o t y p e e a r l y pre-B, newly formed B lymphocytes H2-Dd H2-Dk I-A d I-E k/C k l a t e pre-B lymphocyte 9 10 10 11 12 13 14 13 12 15 16 17 18 19 18 Rabbit T lymphocytes e a r l y pre-B lymphocytes a T h i s antibody c r o s s - r e a c t s with H2-K.d and the f haplotype. 63 was a g i f t from Dr. S.R. Dedhar. LM was purchased from C o l l a b o r a t i v e Research (Lexington, MA). The s y n t h e t i c peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) was obtained from La J o l l a Cancer Research Foundation (La J o l l a , CA). The s y n t h e t i c peptide Gly-Arg-Ala-Asp-Ser-Pro (GRADSP) was purchased from the Department of Biochemistry, U n i v e r s i t y of V i c t o r i a ( V i c t o r i a , BC). 7) ISOLATION AND CLONING OF STROMAL CELL LINES Marrow stromal l i n e s (e.g. M2-10B4 and Ml-Bl) were i s o l a t e d from primary c u l t u r e s of marrow c e l l s from (C57Bl/6JxC3H/HeJ) F^ hyb r i d mice. These were i n i t i a l l y e s t a b l i s h e d and maintained according to standard procedures f o r ac h i e v i n g long-term myelopoiesis (23, 24) except that hydrocortisone ( S o l u c o r t e f , Upjohn Co.) was omitted from the growth medium and the c u l t u r e s were kept at 37°C. Adherent l a y e r c e l l s were t r y p s i n i z e d and subcultured i n RPMI 1640 plus 15% f e t a l c a l f serum (FCS) when the c u l t u r e s were 5 weeks o l d and showed l i t t l e r e s i d u a l evidence of myelopoiesis. A f t e r 1 or 2 f u r t h e r passages (1 and 2 months l a t e r ) , clones of f i b r o b l a s t - l i k e c e l l s were i s o l a t e d from low d e n s i t y c u l t u r e s (seeded at 300 c e l l s per 60 mm dish) using c l o n i n g w e l l s . Clones were then expanded, recloned and propagated by weekly s u b c u l t u r i n g at 5 x 10^ c e l l s per 25 cm 2 f l a s k i n RPMI 1640 plus 15% FCS. Primary c u l t u r e s of spleen f i b r o b l a s t s were e s t a b l i s h e d by a l l o w i n g s m a l l minced fragments of spleen to adhere to p l a s t i c c u l t u r e dishes or f l a s k s i n a sm a l l volume of RPMI 1640 plus 15% FCS. When s i g n i f i c a n t outgrowth of f i b r o b l a s t s had occurred (2-4 weeks l a t e r ) , the c e l l s were t r y p s i n i z e d , subcultured, cloned and maintained as f o r marrow c e l l l i n e s . S5-2 i s such a l i n e i s o l a t e d from a (WB/Re x C57B1/6J) F^ - +/+ mouse. 64 8) ISOLATION, CLONING AND MAINTENANCE OF PRE-B CELL LINES Lymphoid LTC's were e s t a b l i s h e d from the marrow of 3-4 week-old B a l b / c J or (C57B1/6J x C3H/HeJ) F1 hybrid mice and maintained i n RPMI 1640 plus 5% FCS and 50 uM 2-mercaptoethanol (ME) as described by Whitlock and Witte (25), w i t h the m o d i f i c a t i o n that marrow c e l l s were often seeded onto p r e - e s t a b l i s h e d syngeneic adherent marrow c e l l l a y e r s , as we found t h i s improved the r e l i a b i l i t y of o b t a i n i n g productive c u l t u r e s . In order to t r y and e s t a b l i s h feeder-dependent pre-B c e l l l i n e s , non-adherent c e l l s from w e l l e s t a b l i s h e d lymphoid LTC's were p e r i o d i c a l l y t r a n s f e r r e d onto various i r r a d i a t e d (80 Gy) stromal c e l l l i n e s , expanded i n the same medium used to e s t a b l i s h lymphoid LTC's and then cloned by l i m i t i n g d i l u t i o n as described below. Cloned c e l l l i n e s were then expanded and maintained on i r r a d i a t e d M2-10B4 c e l l s l i k e standard LTC's. Bn and Bp represent 2 normal c l o n a l pre-B c e l l l i n e s obtained from a C57B1/6J x C3H/HeJ c u l t u r e i n t h i s way. In one of these secondary lymphoid LTC's o r i g i n a l l y of Balb/cJ o r i g i n , a subpopulation of non-adherent c e l l s evolved 6 months l a t e r that could be maintained i n continuous suspension c u l t u r e i n the absence of stromal feeder c e l l s . Both "feeder-dependent" and "feeder-independent" cloned l i n e s were then i s o l a t e d by p l a t i n g c e l l s from these two types of c u l t u r e at l i m i t i n g d i l u t i o n (0.3 c e l l s per w e l l ) i n 96 w e l l (flat-bottomed) m i c r o t i t r e p l a t e s seeded the previous day with 6000 M2-10B4 c e l l s that had been i r r a d i a t e d w i t h 80 Gy 250 Kvp X-rays. C e l l s f o r c l o n i n g were f i r s t sorted w i t h a FACS 440 (Bectori Dickinson, Sunnyvale, CA) to exclude stromal c e l l s , d e b r i s and dead c e l l s by appropriate g a t i n g of c e l l s by s i z e , forward s c a t t e r and propidium i o d i d e - p o s i t i v i t y . A f t e r 10 days, p o s i t i v e w e l l s were i d e n t i f i e d and the 65 clones i n them a m p l i f i e d and then tested f o r t h e i r a b i l i t y to grow d i r e c t l y on p l a s t i c (feeder-independent clones, e.g. H9 c e l l s ) or only on p r e - e s t a b l i s h e d feeders (feeder-dependent clones, e.g. A8 c e l l s ) . This c l o n i n g and screening procedure was repeated at l e a s t once f o r each cloned l i n e subsequently used i n these s t u d i e s . I n i t i a l l y a l l l i n e s were maintained i n RPMI 1640 plus 5% FCS and 50 mM 2-ME. However, l a t e r s t u d i e s revealed that A8 c e l l s grew o p t i m a l l y i n 15% FCS (see R e s u l t s ) . Thereafter, the growth medium used f o r maintenance of t h i s l i n e and a l l experiments with A8 c e l l s was RPMI 1640 plus 15% FCS and 50 mM 2-ME unless otherwise i n d i c a t e d . L + VII.A8 c e l l l i n e i s another, p h e n o t y p i c a l l y normal, pre-B c e l l l i n e d e rived from a lymphoid LTC e s t a b l i s h e d from a f r a c t i o n a t e d Balb/cJ marrow population seeded onto a M2-10B4 feeder l a y e r . 9) A-MULV TRANSFORMATION OF PRE-B CELLS C l o n a l i s o l a t e s from normal lymphoid LTC's were exposed to A-MuLV by in c u b a t i n g 10^ c e l l s at room temperature i n 0.5 ml of f r e s h RPMI c o n t a i n i n g 5% FCS, 50 uM ME and A-MuLV (pl60 - o r i g i n a l l y obtained from Dr. 0. Witte) at a f i n a l v i r u s concentration of 5 x 10^ focus forming u n i t s / m l . A f t e r 3 hours, c e l l s were spun once f o r 10 minutes at 1200 rpm, resuspended i n f r e s h medium and then pla t e d onto a pr e - e s t a b l i s h e d i r r a d i a t e d M2-10B4 feeder l a y e r (4 x IO 4 M2-10B4 c e l l s / w e l l ) i n 24 w e l l (flat-bottomed) Linbro 7603305 p l a t e s at a conc e n t r a t i o n of 5 x 10^ c e l l s / m l . Thus, ABn and ABp c e l l l i n e s are the A-MuLV transformants of Bn and Bp pre-B c e l l s r e s p e c t i v e l y . As a c o n t r o l , a d d i t i o n a l a l i q u o t s from the same o r i g i n a l normal clones were incubated under the same c o n d i t i o n s with a stock of Moloney helper v i r u s (Mo-MuLV) or medium alone. These c e l l s were maintained using the same p r o t o c o l as f o r standard 66 lymphoid LTC's f o r the f i r s t 3 months a f t e r i n f e c t i o n . At t h i s time, the c e l l s that had been i n f e c t e d w i t h A-MuLV became capable of autonomous growth and were t r a n s f e r r e d to new w e l l s and maintained t h e r e a f t e r as suspension c u l t u r e s . 10) IMMUNOFLUORESCENCE ANALYSES For the s t a i n i n g of stromal c e l l s , these were c u l t u r e d f o r 48 hours on s t e r i l e c o v e r s l i p s , a i r d r i e d , f i x e d w i t h methanol f o r 5 minutes, washed with phosphate buffered s a l i n e (PBS) c o n t a i n i n g 2% FCS, s t a i n e d w i t h the appropriate f i r s t ( r a b b i t or r a t ) antibodies followed by FITC conjugated goat a n t i - r a b b i t (or r a t ) Ig antibody. Controls included s t a i n i n g w i t h non-immune r a b b i t serum as a f i r s t reagent or w i t h FITC conjugated antibody alone. For s t a i n i n g of B c e l l clones f o r cu, c y t o s p i n preparations were f i x e d w i t h methanol and s t a i n e d d i r e c t l y with FITC l a b e l e d goat anti-mouse IgM (y chain s p e c i f i c ) using normal bone marrow, as a p o s i t i v e c o n t r o l and MLB-2 c e l l s (a Moloney v i r u s transformed T - c e l l l i n e - 26) as a negative c o n t r o l . To s t a i n f o r TdT, c e l l s were s i m i l a r l y s t a i n e d but r a b b i t anti-TdT antibody (Gibco/BRL, Gaithersburg, MD) was used as the f i r s t reagent and FITC l a b e l e d goat a n t i - r a b b i t Ig was used as the second antibody. A l l immunofluorescent s l i d e preparations were examined under a Zeiss episcope and at l e a s t 100 c e l l s were evaluated. To detect various surface markers, f r e s h c e l l s were s t a i n e d using the appropriate i n d i r e c t procedure, i n c l u d i n g a b r i e f exposure to propidium i o d i d e (2 ug/ml) and then analyzed with a FACS 440. As p o s i t i v e c o n t r o l s , c e l l suspensions from normal Balb/cJ thymus, spleen and bone marrow were a l s o 67 st a i n e d and analyzed. As negative c o n t r o l s , c e l l s were l a b e l e d with the second FITC l a b e l e d reagent only, or with an i r r e l e v a n t mouse antibody as the f i r s t reagent. To estimate the proportion of p o s i t i v e c e l l s , the channel number where the negative c o n t r o l and the te s t sample curves crossed each other was f i r s t determined. The number of negative c e l l s to the r i g h t of t h i s channel (higher fluorescence values) was subtracted from the number of c e l l s i n the te s t sample that f e l l to the r i g h t of the crossover p o i n t . The percentage of p o s i t i v e c e l l s r e l a t i v e to negative c o n t r o l antibody was determined on 10^ c e l l s . 11) HISTOCHEMISTRY Stromal c e l l s were prepared as described f o r immunofluorescence s t a i n i n g , a i r d r i e d , and then f i x e d and stained f o r a c i d phosphatase, c h l o r o a c e t a t e esterase, alpha-naphthyl-acetate esterase, peroxidase, and a l k a l i n e phosphatase using standard techniques. 12) VIRUS ASSAYS Fre s h l y or r a p i d l y frozen c e l l l i n e supernatants were tested f o r v i r u s e s s e n t i a l l y as o r i g i n a l l y described by Scher and S i e g l e r (27). B r i e f l y , e a r l y passage NIH 3T3 c e l l s were seeded i n 60 mm p l a s t i c Falcon dishes (10^/dish) i n medium with 10 ug/ml of polybrene. The next day the medium was replaced w i t h 0.5 ml of v i r a l stock or a l i q u o t s of te s t supernatants and incubated at 37°C f o r 1 hour with o c c a s i o n a l rocking of the dishes. Medium was changed again a f t e r 5 days and transformed f o c i scored on the 10th day using an i n v e r t e d microscope. 68 13) ASSAYS FOR TUMORIGENICITY 10 5-10 6 t e s t c e l l s i n 100 u l were i n j e c t e d i n t r a d e r m a l l y i n t o untreated syngeneic, Balb/cJ-nu/nu mice and/or i n t o syngeneic-+/+ mice that had been immunocompromised by treatment with 7 Gy of 270 Kvp X-rays and a g r a f t of 3.6 x 10^ syngeneic marrow c e l l s a week p r e v i o u s l y . Tumor development was monitored f o r up to 6 months. 14) CELL PROLIFERATION ASSAYS Pre-B c e l l growth and population doubling times were determined e i t h e r d i r e c t l y from v i a b l e c e l l counts ( N i g r o s i n dye e x c l u s i o n ) or from measurements of ^rl-thymidine i n c o r p o r a t i o n , u s u a l l y a f t e r 3-4 days of c u l t u r e , i n f l a t bottom w e l l s i n 96 w e l l Costar m i c r o t i t r e p l a t e s (Costar 3596, Cambridge, MA). P r e l i m i n a r y experiments e s t a b l i s h e d that maximum ^H-thymidine i n c o r p o r a t i o n was seen at t h i s time. To measure -^-thymidine i n c o r p o r a t i o n , 1 y C i of 3H~thymidine (20 mCi/mmole, Amersham, O a k v i l l e , ONT) i n 20 y l of growth medium was added to each 100 y l of c u l t u r e and the c e l l s were then incubated f o r an a d d i t i o n a l 4 hours. C e l l u l a r DNA was harvested onto g l a s s f i b e r f i l t e r paper and the amount of ^H-thymidine i n c o r p o r a t i o n determined by s c i n t i l l a t i o n counting. In experiments where the e f f e c t of a 0.35% agarose (Sigma, St. L o u i s , M0) i n t e r l a y e r was examined (Chapter I I I ) , 0.7 ml pre-B c e l l c u l t u r e s were f i r s t incubated i n 24 w e l l (flat-bottomed) Linbro 7603305 p l a t e s . Cultures were i n i t i a t e d by p l a t i n g 2.1 x 10 4 A8 c e l l s or 2100 H9 c e l l s e i t h e r d i r e c t l y i n the w e l l s or onto a confluent 80 Gy i r r a d i a t e d M2-10B4 feeder l a y e r (4 x 10^ M2-10B4 c e l l s / w e l l ) , i n each case with or without an intermediate 69 0.2 ml i n t e r l a y e r of 0.35% agarose i n the same medium. A f t e r the 4 hour i n c u b a t i o n w i t h -^-thymidine, the c e l l s on top of the agarose i n each w e l l were resuspended without d i s r u p t i n g the agarose i n t e r l a y e r , and then t r a n s f e r r e d i n d i v i d u a l l y to the w e l l s of a 96 fl a t - b o t t o m w e l l m i c r o t i t r e p l a t e p r i o r to ha r v e s t i n g the DNA. For some of the co- c u l t u r e experiments described i n Chapter IV, 3 x 10 6 i r r a d i a t e d H9 c e l l s / m l were mixed i n RPMI 1640 c o n t a i n i n g 5% FCS, 50 yM 2-ME and 0.35% Noble agar. 0.2 ml of t h i s mixture were then p l a t e d i n 24 w e l l (flat-bottomed) Linbro 7603305 p l a t e s ( i . e . 6 x 10 5 H9 c e l l s / w e l l ) . A second 0.2 ml agar i n t e r l a y e r without c e l l s was then poured on top of the l a y e r c o n t a i n i n g the H9 c e l l s and then f i n a l l y overlayed with 0.7 ml of medium c o n t a i n i n g 2 x 10^ c e l l s / m l . A f t e r 3 days of c u l t u r e , 3 H -thymidine uptake i n t o the c e l l s i n the upper, l i q u i d l a y e r was measured. 15) CELL ATTACHMENT ASSAYS Mi c r o w e l l p l a t e s (96 w e l l , Costar 3596, Cambridge, MA) were coated with the designated p r o t e i n s by incubating the p r o t e i n s o l u t i o n s at various concentrations i n the p l a t e s overnight at 4°C. Unbound p r o t e i n s were removed from the p l a t e s by washing with PBS. The p l a t e s were then incubated w i t h RPMI 1640 c o n t a i n i n g bovine serum albumin (BSA; 2.5 mg/ml; Sigma) f o r 2 hours at 37°C. A f t e r washing twice i n PBS, pre-B c e l l s were resuspended i n RPMI 1640 c o n t a i n i n g BSA (2.5 mg/ml) at 5 x 10^ c e l l s / m l . One hundred u l of each c e l l suspension was added to the protein-coated w e l l s , and the p l a t e s were incubated at 37°C f o r 1 hr. A f t e r washing gently with PBS, attached c e l l s were f i x e d w i t h 3% paraformaldhyde (100 u l ) f o r 30 minutes at room temperature and then s t a i n e d with 0.5% t o l u i d i n e blue i n 3.7% paraformaldhyde (100 u l ) 70 overnight. The number of attached c e l l s was counted under an i n v e r t e d microscope. 16) PRE-B CELL COLONY ASSAYS A8 or H9 c e l l s were plated i n 35 mm t i s s u e c u l t u r e dishes at a f i n a l c o n c e n t r a t i o n of 300 c e l l s per ml i n 1.1 ml volumes of 0.8% m e t h y l c e l l u l o s e c u l t u r e medium (2) co n t a i n i n g 5% FCS and 50 uM 2-ME. In some cases, the dishes had been seeded the previous day with 2 x 10^ i r r a d i a t e d (80 Gy) M2-10B4 c e l l s , i . e . s u f f i c i e n t c e l l s to give a confluent monolayer. The m e t h y l c e l l u l o s e medium co n t a i n i n g A8 or H9 c e l l s was then p l a t e d e i t h e r d i r e c t l y on top of the feeder l a y e r , or on top of an intermediate 0.5 ml l a y e r of the same medium (without m e t h y l c e l l u l o s e ) but s o l i d i f i e d w i t h 0.35% agarose. A f t e r 7 days incubation at 37°C, col o n i e s c o n t a i n i n g >10 c e l l s were counted d i r e c t l y i n the dishes using an i n v e r t e d microscope. 17) MYELOID COLONY ASSAYS Fre s h l y suspended (C57B1/6J x C3H/HeHJ) F^ bone marrow c e l l s were p l a t e d at a f i n a l concentration of 3 x 10^ c e l l s i n 1.1 ml of a c u l t u r e medium c o n t a i n i n g 0.8% m e t h y l c e l l u l o s e , 30% FCS, 1% deionized BSA, 100 uM 2-ME, 3 u n i t s of Epo (28) per ml, and e i t h e r 10% M2-10B4 CM (or S5-2CM) or 10% H9 CM, or 1% PWM-SCCM i n alpha medium (2). Cultures were then incubated f o r 14 days at 37°C and co l o n i e s c o n t a i n i n g more than 20 granulocytes, macrophages, e r y t h r o b l a s t s , megakaryocytes and mast c e l l s s i n g l y or i n var y i n g combinations scored d i r e c t l y i n the dishes. Under these c o n d i t i o n s , c u l t u r e s c o n t a i n i n g 71 only Epo as an added s p e c i f i c HGF y i e l d e d <10 such c o l o n i e s per c u l t u r e and these were small pure e r y t h r o i d or small macrophage c o l o n i e s . 18) ANALYSIS OF DNA REARRANGEMENTS High molecular weight DNA was i s o l a t e d by proteinase K d i g e s t i o n of c e l l s f ollowed by phenol and chloroform e x t r a c t i o n (29). DNA was digested w i t h EcoRI f o r v-a b l sequences and f o r Ig heavy chain gene a n a l y s i s , and w i t h Hind I I I f o r T c e l l receptor 0 chain (Tig) gene a n a l y s i s . The digested fragments were separated by e l e c t r o p h o r e s i s f o r 16 hours at 2 volts/cm i n a 1% agarose g e l and t r a n s f e r r e d e i t h e r to n i t r o c e l l u l o s e papers ( S c h l e i c h e r and S c h u e l l , Keene, NY) using standard methods (29) or to nylon f i l t e r s (Zeta-probe, Biorad) by the a l k a l i n e t r a n s f e r technique (30). N i t r o c e l l u l o s e f i l t e r s were pr e h y b r i d i z e d , h y b r i d i z e d , washed using standard techniques (29). For nylon f i l t e r s , p r e h y b r i d i z a t i o n was f o r 4 hours at 60°C i n 0.9% M NaCl, 10% formamide (BRL), 1% SDS, 2 mM EDTA, 2% non-fat d r i e d m i l k and 0,5 mg/ml denaturated salmon sperm DNA. H y b r i d i z a t i o n b u f f e r included the same components except f o r formamide (20%). The probes used to detect e i t h e r v - a b l sequences i n c l o n a l c e l l l i n e s or T i g gene rearrangements were ^ 2 P - l a b e l l e d by n i c k t r a n s l a t i o n (Nick T r a n s l a t i o n K i t , Bethesda Research Laboratory, Gaithersburg, MD). The probe used f o r Ig heavy chain gene rearrangement, was ^ 2 P - l a b e l l e d by the hexamer primer method (Pharmacia, o l i g o l a b e l l i n g k i t ) (31). A f t e r h y b r i d i z a t i o n f o r 18-24 hours, f i l t e r s were washed 3 times i n 0.1% SDS, 0.1% SSC and 0.1% sodium pyrophosphate at 60°C f o r 30 minutes with the exception that an a d d i t i o n a l washing procedure under more s t r i n g e n t c o n d i t i o n s ( i . e . 0.1% SSC and 0.1% SDS at 68°C) was used f o r the J H probe i n order to 72 e l i m i n a t e the h y b r i d i z a t i o n of r e p e t i t i v e sequences (32). Autoradiography was performed at -70°C f o r 1-4 days using Kodak. XAR-5 f i l m and an i n t e n s i f y i n g screen. 19) PREPARATION OF CM M2-10B4 or S5-2 c e l l s were seeded i n 75 cm 2 Corning f l a s k s c o n t a i n i n g 10 ml of RPMI 1640 supplemented with 15% FCS. When the c e l l s were s t i l l subconfluent and i n a l o g growth phase, the o l d medium was removed and the c e l l s r i n s e d once with serum-free RPMI 1640. Then f r e s h RMPI 1640 supplemented e i t h e r w i t h 0.2% FCS only, or with 50 uM 2-ME only, was added. CM was harvested a f t e r 24 hours and stored at 4°C p r i o r to t e s t i n g f o r pre-B s t i m u l a t i n g a c t i v i t y , unless otherwise s p e c i f i e d . H9 and A-MuLV transformed pre-B c e l l clones i n l o g phase were washed twice w i t h serum-free RPMI 1640 supplemented only with 50 uM 2-ME. They were then resuspended i n 10 ml of the same f r e s h medium at a concentration of 5 x 10^ c e l l s / m l and placed i n 75 cm 2 f l a s k s f o r 24-36 hours. CM was ce n t r i f u g e d to remove c e l l s and then f i l t e r e d through a 0.22 y f i l t e r (Nalgene). CM was heated to 56°C f o r 1 hour to i n a c t i v a t e any v i r u s present. CM was stored at 4°C p r i o r to t e s t i n g f o r pre-B s t i m u l a t i n g a c t i v i t y , unless otherwise s p e c i f i e d . Medium conditioned by human p e r i p h e r a l blood leukocytes incubated i n agar (agar-LCM, 33) or a c t i v a t e d i n suspension c u l t u r e by PHA (PHA-LCM, 34), and PWM-SCCM were prepared i n our la b o r a t o r y using standard procedures ( 2 ) . 73 20) GEL PERMEATION CHROMATOGRAPHY Fre s h l y harvested M2-10B4 CM was f i l t e r e d through a 0.22 y f i l t e r (Nalgene), l y o p h i l i z e d overnight and then resuspended i n s t e r i l e double d i s t i l l e d water to obtain a 10-fold concentration. One ml of the concentrated sample was run i n t o a 1.5 cm x 48 cm ( i . e . 84.5 ml bed volume) s i z e e x c l u s i o n column (Sephadex G100, Bio-Rad, Mississauga, ONT) and el u t e d w i t h PBS (pH + 7.4) at a flow r a t e of 5.4 ml/h. 0.9 ml f r a c t i o n s were c o l l e c t e d at 4°C and tested f o r b i o l o g i c a l a c t i v i t y . The column was c a l i b r a t e d w i t h : 1) Dextran Blue (void volume), 2) Ovalbumin (MW = 45,000), 3) Cytochrome C (MV = 12,384), 4) Phenol red (MW = 354). Fr e s h l y harvested H9 CM was l y o p h i l i z e d overnight and then resuspended i n s t e r i l e double d i s t i l l e d water to obtain a 50 - f o l d concentration. One ml of the concentrated sample was run i n t o a 1.5 cm x 55 cm ( i . e . 97 ml bed volume) s i z e e x c l u s i o n column (Sephadex G50, Bio-Rad, Mississauga, ONT) e q u i l i b r a t e d and e l u t e d w i t h PBS (pH 7.4) at a flow r a t e of 5 ml/h. F r a c t i o n s (0.83 ml) were c o l l e c t e d at 4°C and tested f o r b i o l o g i c a l a c t i v i t y . The column was c a l i b r a t e d w i t h : 1) Dextran blue (void volume), 2) Ovalbumin (MW = 45,000), 3) Cytochrome C (MW = 12,384), 4) Vitamin B12 (MW = 1,355), 5) Phenol red (MW = 354). 74 REFERENCES 1. Greenberger JS, Eckner RJ, Sakakeeny M, Marks P, Reid D, Nabel G, Hapel A, I h l e JN, Humphries RK. I n t e r l e u k i n 3-dependent hematopoietic progenitor c e l l l i n e s . Federation Proceedings 42:2762, 1983. 2. Eaves CJ, K r y s t a l G, Eaves AC. E r y t h r o p o i e t i c c e l l s . In: " B i b l i o t h e c a Haematologica, No 48 - Current Methodology i n Experimental Hematology", (ed. SJ Baum), S. Krager, Basel, pp 81, 1984. 3. Aarden LA, de Groot ER, Schaap OL, Lansdorp PM. Production of hybridoma growth f a c t o r by human monocytes. Eur J Immunol 17: 1411, 1987. 4. Madri JA, Dryer B, P i t l i c k F, Furthmayr H. The collagenous components of the subendothelium: c o r r e l a t i o n of s t r u c t u r e and f u n c t i o n . Lab Invest 43:303, 1980. 5. Madri JA, Williams SK, Wyatt T, Mezzio C. C a p i l l a r y e n d o t h e l i a l c e l l c u l t u r e s : phenotypic modulation by matrix components. J C e l l B i o l 97:153, 1983. 6. Hayman EG, E n g v a l l E, A'Hearn E, Barnes D, Pierschbacher MD, R u o s l a h t i E. C e l l attachment on r e p l i c a s of SDS polyacrylamide g e l s r e v e l s two adhesive plasma p r o t e i n s . J C e l l B i o l 95:20, 1982. 7. P y t e l a R, Pierschbacher MD, Ruoshlahti E. I d e n t i f i c a t i o n and i s o l a t i o n of a 140 kd c e l l surface g l y c o p r o t e i n w i t h p r o p e r t i e s expected of a f i b r o n e c t i n receptor. C e l l 40:191, 1985. 8. Zuckerman KS, Wicha MS. E x t r a c e l l u l a r matrix production by the adherent c e l l s of long-term murine bone marrow c u l t u r e s . Blood 61:540, 1983. 9. Coffman RL, Weismann IL. B220: a B c e l l - s p e c i f i c member of the T200 g l y c o p r o t e i n f a m i l y . Nature 289:681, 1981. 10. Takei F. A novel d i f f e r e n t i a t i o n antigen on p r o l i f e r a t i n g murine thymocytes i d e n t i f i e d by a r a t monoclonal antibody. J Immunol 132:766, 1984. 11. Takei F. Two surface antigens expressed on p r o l i f e r a t i n g mouse T lymphocytes defined by r a t monoclonal a n t i b o d i e s . J Immunol 130:2734, 1983. 12. Springer T, G a l f r e G, Secher DS, M i l s t e i n C. Monoclonal xenogeneic a n t i b o d i e s to murine c e l l surface antigens: i d e n t i f i c a t i o n of novel leukocyte d i f f e r e n t i a t i o n antigens. Eur J Immunol 8:539, 1978. 13. Takei F. Murine T lymphoma c e l l s express a novel membrane-associated antigen w i t h unique fea t u r e s . J Immunol 139:649, 1987. 14. Eckhardt LA, Herzenberg LA. Monoclonal antibody to ThB detect c l o s e l i n k a g e of Ly-6 and a gene r e g u l a t i n g ThB expression. Immunogenetics 11:275, 1980. 75 15. Oi VT, Jones PP, Goding JW, Herzenberg LA, Herzenberg LA. P r o p e r t i e s of monoclonal an t i b o d i e s to mouse Ig a l l o t y p e s , H-2, and I a antigens. Curr Top M i c r o b i o l Immunol 81:115, 1978. 16. Cooper MD, Mulvaney D, Coutinho A, Cazenave P-A. A novel c e l l s u rface molecule on e a r l y B-lineage c e l l s . Nature 321:616, 1986. 17. Ozato K, Mayer NM, Sachs DH. Monoclonal a n t i b o d i e s to mouse major h i s t o c o m p a t i b i l i t y complex antigens. T r a n s p l a n t a t i o n 34:113, 1982. 18. Ozato K, Mayer NM, Sachs DH. Hybridoma c e l l l i n e s s e c r e t i n g monoclonal a n t i b o d i e s to mouse H-2 and I a antigens. J Immunol 124:533, 1980. 19. Kappler JW, Skidmore B, White J , Marrack P. A n t i g e n - i n d u c i b l e , H - 2 - r e s t r i c t e d , i n t e r l e u k i n - 2 - p r o d u c i n g T c e l l hybridomas. J Exp Med 53:1198, 1981. 20. A l t FW, Yancopoulos GD, B l a c k w e l l TK, Wood C, Thomas E, Boss M, Coffman R, Rosenberg N, Tonegawa S, Baltimore D. Ordered rearrangement of immunoglobulin heavy chain v a r i a b l e region segments. EMBO J 3:1209, 1984. 21. Hedrick SM, N i e l s e n EA, Kavaler J , Cohen DI, Davis MM. Sequence r e l a t i o n s h i p s between p u t a t i v e T - c e l l receptor polypeptides and immunoglobulins. Nature 308:153, 1984. 22. Wang JY, Baltimore D. C e l l u l a r RNA homologous to the Abelson murine leukemia v i r u s transforming gene expression and r e l a t i o n s h i p to the v i r a l sequence. Mol C e l l B i o l 3:773, 1983. 23. Greenberger JS. S e n s i t i v i t y of corticosteroid-dependent i n s u l i n -r e s i s t a n t l i p o g e n e s i s i n marrow preadipocytes of obese-diabetic (db/db) mice. Nature 275:752, 1978. 24. Eaves C, Coulombel L, Eaves A. A n a l y s i s of hemopoiesis i n long-term human marrow c u l t u r e s . In: "Haemopoietic Stem C e l l s " . (eds. Sv-Aa Killmann, EP Cronki t e , CN M u l l e r - B e r a t ) , Munksgaard, Copenhagen, pp 287, 1983. 25. Whitlock CA, Witte ON. Long-term c u l t u r e of B lymphocytes and t h e i r precursors from murine bone marrow. Proc N a t l Acad S c i USA 79:3608, 1982. 26. Chan P-Y, Takei F. Expression of a T c e l l r e c e p t o r - l i k e molecule on normal and malignant T c e l l s detected by r a t monoclonal a n t i b o d i e s to nonclonotypic determinants. J Immunol 136:1346, 1986. 27. Scher CD, S i e g l e r R. D i r e c t transformation of 3T3 c e l l s by Abelson murine leukemia v i r u s . Nature 253:729, 1975. 28. K r y s t a l G, Pankratz HRC, Farber WM, Smart JE. P u r i f i c a t i o n of human e r y t h r o p o i e t i n to homogeneity by a r a p i d f i v e step procedure. Blood 67:71, 1986. 76 29. M a n i a t i s T, F r i t s c h EF, Sambrook J . Molecular c l o n i n g . I n : "A Laboratory Manual", Cold Spring Harbor Laboratory, New York, 1982. 30. Reed KC, Mann DA. Rapid t r a n s f e r of DNA from agarose g e l s to nylon membranes. N u c l e i c Acids Res 13:7207, 1985. 31. Feinberg AP, V o g e l s t e i n B. A technique f o r r a d i o l a b e l i n g DNA r e s t r i c t i o n endonuclease fragments to high s p e c i f i c a c t i v i t y . Anal Biochem 132:6, 1983. 32. A l t F, Rosenberg N, Lewis S, Thomas E, Baltimore D. Organization and r e o r g a n i z a t i o n of immunoglobulin genes i n A-MuLV-transformed c e l l s : rearrangement of heavy but not l i g h t chain genes. C e l l 27:381, 1981. 33. Eaves CJ, Eaves AC. E r y t h r o p o i e t i n (Ep) dose response curves f o r three c l a s s e s of e r y t h r o i d progenitors i n normal human marrow and i n p a t i e n t s wit h polycythemia vera. Blood 52:1196, 1978. 34. Cashman J , Henkelman D, Humphries K, Eaves C, Eaves A. I n d i v i d u a l BFU-E on polycythemia vera produce both e r y t h r o p o i e t i n dependent and independent progeny. Blood 61:876, 1983. 77 C H A P T E R I I I PARTIAL CHARACTERIZATION OF A NOVEL STROMAL CELL-DERIVED PRE-B CELL GROWTH FACTOR ACTIVE ON NORMAL AND IMMORTALIZED PRE-B CELLS 1) INTRODUCTION As reviewed i n Chapter I , a n t i g e n - r e s t r i c t e d mature B lymphocytes a r i s e from more p r i m i t i v e precursors i n the marrow that possess a broader range of p r o l i f e r a t i v e and d i f f e r e n t i a t i v e p o t e n t i a l i t i e s . These i n c l u d e a population of p l u r i p o t e n t hemopoietic stem c e l l s which p e r s i s t throughout ad u l t l i f e (1,2) and which are capable of generating various myeloid daughter c e l l types as w e l l as T and B c e l l s . The extent to which B lymphocyte numbers are normally maintained by p l u r i p o t e n t hemopoietic stem c e l l s or d e r i v a t i v e l i n e a g e - r e s t r i c t e d but p r i m i t i v e pre-B c e l l precursors i s not known. D e l i n e a t i o n of the sequence of Ig H and L chain gene rearrangement and expression i n developing B c e l l s has allowed the d e f i n i t i o n of s e v e r a l stages of d i f f e r e n t i a t i o n along t h i s pathway ( 3 ) . Of i n t e r e s t would be the p a r a l l e l i d e n t i f i c a t i o n of f a c t o r s that regulate the s i z e of these various pre-B c e l l p opulations. The l o c a l i z a t i o n of B c e l l development w i t h i n s p e c i f i c regions of hemopoietic and lymphoid t i s s u e s suggests that i n v i v o the sources of such f a c t o r s may include f i x e d elements of non-hemopoietic o r i g i n . Recently, an i n v i t r o system that supports the p r o l i f e r a t i o n and d i f f e r e n t i a t i o n of very e a r l y B c e l l precursors f o r s e v e r a l months has been described ( 4 ) . A key featu r e of these pre-B lymphoid LTC's, l i k e the myeloid LTC's from which they were 78 adapted (5) and from which they can be derived by a l t e r i n g the growth medium used ( 6 ) , i s the presence of an adherent l a y e r of accessory c e l l s . Further a n a l y s i s of the mechanisms involved i n the r e g u l a t i o n of e a r l y pre-B c e l l p r o l i f e r a t i o n should be f a c i l i t a t e d by the a v a i l a b i l i t y of a v a r i e t y of cloned c e l l l i n e s representing the d i f f e r e n t components of the long-term pre-B lymphoid c u l t u r e system. Recently, a number of such l i n e s have been described (7-12). During the course of experiments i n our l a b o r a t o r y i n v o l v i n g the r o u t i n e establishment and maintenance of pre-B c e l l LTC's from mouse marrow, a spontaneously immortalized l i n e arose that was i n i t i a l l y feeder-dependent but over time generated feeder-independent v a r i a n t s . A d e s c r i p t i o n of the c h a r a c t e r i s t i c s of r e p r e s e n t a t i v e clones of each of these phenotypes as w e l l as s e v e r a l cloned stromal c e l l l i n e s that supported t h e i r growth i s described below together with t h e i r use i n s t u d i e s of the mechanism of stromal support of pre-B c e l l s . 2) RESULTS A) C h a r a c t e r i z a t i o n of Pre-B C e l l Supportive Stromal C e l l Lines A number of adherent f i b r o b l a s t - l i k e c e l l l i n e s were i s o l a t e d from marrow and spleen c e l l c u l t u r e s and cloned as described i n the M a t e r i a l s and Methods. Table 4 shows the growth supporting c a p a c i t y of 3 of these when tested as feeders f o r nonadherent c e l l s from marrow c u l t u r e s o r i g i n a l l y e s t a b l i s h e d as described by Whitlock and Witte (4). In the p a r t i c u l a r experiment presented i n Table 4, a s i m i l a r growth supporting r o l e of 3T3 c e l l s i s a l s o demonstrated. A d d i t i o n a l experiments of t h i s type showed that 2 x 10^ feeder c e l l s per cm 2 was s u f f i c i e n t to give optimal s t i m u l a t i o n of such nonadherent, 79 Table 4. Comparison of the a b i l i t y of various i r r a d i a t e d (80 Gy) feeder c e l l l i n e s to support the growth of c u l t u r e d pre-B c e l l s . No. of C e l l s A f t e r 2 Weeks (x 10 5) Feeder C e l l a Non-Adherent T o t a l C o n t r o l 0.8 0.8 M2-10B4 53 107 Ml-Bl 20 66 S5-2 91 94 3T3 54 58 a T r y p s i n i z e d feeder c e l l s were i r r a d i a t e d i n suspension with 80 Gy to prevent t h e i r subsequent growth and were then plat e d i n d u p l i c a t e at 2 x 10^ c e l l s per 35 mm t i s s u e c u l t u r e d i s h . Twenty-four hours l a t e r 10^ nonadherent c e l l s from an e s t a b l i s h e d longterm lymphoid marrow c u l t u r e were added to each d i s h . A f t e r 2 weeks, both nonadherent c e l l s and adherent c e l l s were suspended and the number of v i a b l e c e l l s counted. Nonadherent lymphoid c e l l s c u l t u r e d i n dishes without feeders, i . e . d i r e c t l y on p l a s t i c , served as c o n t r o l s . 80 c u l t u r e d p r e - B - c e l l s whether or not the feeder population had been p r e v i o u s l y i r r a d i a t e d w i t h 80 Gy (data not shown, see a l s o Figure 7 below). A l l 4 of the adherent c e l l l i n e s tested i n Table 4 showed a s i m i l a r f i b r o b l a s t - l i k e morphology, and were c o n s i s t e n t l y found to be p o s i t i v e f o r LM and i n some cases f o r c o l l a g e n IV a l s o (Table 5 ) , suggestive of e n d o t h e l i a l r a t h e r than f i b r o b l a s t c e l l d i f f e r e n t i a t i o n (13). On the other hand, no evidence of Factor V I I I associated antigen, a common marker of mature e n d o t h e l i a l c e l l s , was detected. Some c e l l s i n a l l l i n e s could be converted i n t o o i l red 0 - p o s i t i v e l i p i d laden adipocytes f o l l o w i n g a d d i t i o n of hydrocortisone to the c u l t u r e medium as described by others (7,14). None of the stromal l i n e s were p o s i t i v e f o r any of the hemopoietic c e l l markers tested (Table 5). B) I s o l a t i o n and C h a r a c t e r i z a t i o n of Pre-B C e l l Lines A cloned l i n e of feeder-dependent and feeder-independent pre-B c e l l s , designated as A8 and H9 c e l l s r e s p e c t i v e l y , were obtained as described i n the M a t e r i a l s and Methods. H9 c e l l s have remained s t a b l y feeder-independent f o r more than 2 years, but feeder-independent v a r i a n t s were found to a r i s e continuously from A8 c e l l s at a frequency of approximately 10~^. Thus to maintain A8 c e l l s as a homogeneous feeder-dependent population t h i s l i n e was recloned every 2-3 months. Both A8 and H9 c e l l s resemble lymphoblasts morphologically. A d d i t i o n a l c h a r a c t e r i z a t i o n s t u d i e s showed that no A8 or H9 c e l l s are p o s i t i v e f o r surface (IgM or IgD) or cytoplasmic (y chain) I g , B220 antigen, TdT, or any of the myeloid or T c e l l markers tested. The l a t t e r i n c l u d e Thy-1, T200, Ml/69, ThB, and Mac-1. However, >90% of c e l l s i n both l i n e s s t r o n g l y express BP-1, Table 5. Immunological and histo c h e m i c a l c h a r a c t e r i z a t i o n of the stromal c e l l l i n e s used i n t h i s study. M2-10B4 Ml-Bl S5-2 3T3 Laminin + + + + Collagen Type IV + + - +/-Collagen Type I - -Factor V I I I - -Ml/70 ( M a c - l ) a - -YE1/21 (T200) a - -A c i d Phosphatase - - +++ Chloroacetate esterase + + - -« naphthyl-acetate esterase - -Myeloperoxidase - - ND^ ND A l k a l i n e Phosphatase - -aMonoclonal r a t anti-mouse a n t i b o d i e s . Ml/70 (Mac-1) i s s p e c i f i c f o r monocytes and granulocytes, YE1/21 (T200) reacts g e n e r a l l y with hemopoietic c e l l s , except e r y t h r o i d c e l l s . bND = not done. 82 an antigen c h a r a c t e r i s t i c of pre-B c e l l s (15). A8 and H9 c e l l s a l s o express r e a d i l y d e t e c t a b l e l e v e l s of Class I MHC antigens and the t r a n s f e r r i n receptor. A n a l y s i s of A8 and H9 c e l l DNA revealed that both a l l e l e s of J H, region appear to be i d e n t i c a l l y rearranged i n both l i n e s (Figure 5a). In c o n t r a s t , a n a l y s i s w i t h a Tg probe revealed no evidence of rearrangement of t h i s gene (Figure 5b). These data are consi s t e n t with a pre-B c e l l o r i g i n of both A8 and H9 c e l l s , and suggest that they represent subclones o r i g i n a l l y d erived from the same immortalized c e l l . C) In V i t r o Growth of H9 and A8 C e l l s We f i r s t analyzed the FCS requirement of A8 and H9 c e l l s and found that optimal growth of A8 c e l l s r equires 15% FCS, but that growth of H9 c e l l s i s not improved with FCS concentrations above 5% (data not shown). Therefore, these c o n d i t i o n s were used i n a l l subsequent experiments. However, even when the FCS concentration i s optimized, c e l l p r o l i f e r a t i o n i n both cases i s h i g h l y c e l l concentration-dependent, as i l l u s t r a t e d i n Figure 6a. At concentrations above*10^ c e l l s / m l , H9 c e l l p r o l i f e r a t i o n appears to be completely feeder and 2-ME independent. However, at concentrations below 10^ c e l l s / m l , H9 c e l l p r o l i f e r a t i o n i s enhanced by c o - c u l t i v a t i o n on s u i t a b l e feeders and by supplementation of the medium with 2-ME. This can be seen by the increased slope of the curve f o r H9 c e l l s alone by comparison to H9 c e l l s c u l t u r e d on M2-10B4 c e l l s . At concentrations below 3,000 H9 c e l l s / m l p r o l i f e r a t i o n ceases. Thus H9 c e l l s show some feeder and 2-ME dependence, but only at c e l l c oncentrations much lower than those t y p i c a l l y used f o r l i n e maintenance (>10^ c e l l s / m l ) . This e x p l a i n s the i n i t i a l l y p a r a d o x i c a l f i n d i n g that 83 1 2 3 4 5 23 9.6- 23 6J6-U — 6 . 2 Kb | | 4 3 -• 4.3 2.3 23- 2 2 - Kb Kb -—9.4 Kb — 3.1 Kb Figure 5. Panel a. Southern b l o t a n a l y s i s of Ig H chain gene rearrangement i n A8 and H9 c e l l s f o l l o w i n g DNA d i g e s t i o n with EcoRI and h y b r i d i z a t i o n to a J H probe. Lane 1, Balb/cJ spleen; lane 2, A20 B c e l l s (from ATCC, R o c k v i l l e , MD); lane 3, M2-10B4 c e l l s ; lane 4, H9 c e l l s ; and lane 5, A8 c e l l s . An arrow marks the expected p o s i t i o n of the 6.2 Kb germ l i n e fragment. Rearrangement i s evident i n the A20 B c e l l l i n e ( p o s i t i v e c o n t r o l ) and i n both H9 and A8 c e l l s . Panel b. Southern b l o t a n a l y s i s of the T c e l l receptor 3 chain gene i n A8 and H9 c e l l s f o l l o w i n g DNA d i g e s t i o n with Hind I I I and h y b r i d i z a t i o n to a Tp cDNA probe. Lane 1, Balb/cJ spleen; lane 2, A20 B c e l l s ; lane 3, A-Kl T c e l l s (16); lane 4, Balb/cJ thymus; lane 5, M2-10B4 c e l l s ; lane 6, H9 c e l l s ; and lane 7, A8 c e l l s . Arrows mark the expected p o s i t i o n s of the 9.4 and 3.1 Kb germ l i n e fragments. Rearrangement i s evident i n A-Kl T c e l l s and B a l b / c J thymus ( p o s i t i v e c o n t r o l s ) . 0 150 1500 1 5 0 0 0 150000 0 150 1500 15000 150000 N u m b e r ol FI-H9 c e l l s p e r ml N u m b e r ° ' F D " A 8 c e l l s P e r m l Panel a Panel b Figure 6. P r o l i f e r a t i o n of H9 c e l l s (panel a) and A8 c e l l s (panel b) seeded at d i f f e r e n t c e l l concentrations under d i f f e r e n t conditions. on pre-established i r r a d i a t e d M2-10B4 stromal c e l l s (at 6 x 10 4 c e l l s / m l or 2 x 10 4 c e l l s / c m 2 ) ; on p l a s t i c ; - - - on p l a s t i c i n the absence of 2-ME. I I I I I background due to M2-10B4 c e l l s alone (6 x 10 4 c e l l s / m l or 2 x 10 4 c e l l s / c m 2 ) . 85 feeder-independent c e l l s could not be cloned i n the absence of a feeder. In c o n t r a s t , A8 c e l l s do not p r o l i f e r a t e at a l l (or even remain v i a b l e ) on p l a s t i c at concentrations below 3 x 10 4 c e l l s / m l (Figure 6b). Above 6 x 10 4 c e l l s / m l , some A8 c e l l p r o l i f e r a t i o n can be detected but t h i s i s s h o r t - l i v e d and occurs only i n the presence of 50 yM 2-ME. Under optimal c o n d i t i o n s the population doubling times of H9 and A8 c e l l s a l s o d i f f e r and are approximately 15 and 43 hours, r e s p e c t i v e l y . Figure 7 shows a more d e t a i l e d a n a l y s i s of the dose-dependence of the s t i m u l a t o r y e f f e c t of i r r a d i a t e d M2-10B4 c e l l s on H9 and A8 c e l l s . The c o n c e n t r a t i o n of H9 c e l l s used i n t h i s p a r t i c u l a r experiment was 3,000 c e l l s / m l , i . e . s u f f i c i e n t to give s u b s t a n t i a l counts under optimal c o n d i t i o n s , but low enough to allow the response to M2-10B4 feeders to a l s o be revealed. I t can be seen that optimal s t i m u l a t i o n of 3,000 H9 c e l l s / m l i s achieved with a feeder c e l l concentration of 3,000 M2-10B4 c e l l s / m l ( i . e . 10 4 c e l l s / cm^) (Figure 7a). At M2-10B4 concentrations above 10^ c e l l s / m l , i n h i b i t i o n of H9 c e l l p r o l i f e r a t i o n i s apparent. I t should a l s be noted that h e a v i l y i r r a d i a t e d M2-10B4 c e l l s alone demonstrate s i g n i f i c a n t •^H-thymidine i n c o r p o r a t i o n up to concentrations of 3 x 10 4 c e l l s / m l . Figure 7b shows the r e s u l t s f o r A8 c e l l s i n an experiment of s i m i l a r design. However, i n t h i s case A8 c e l l s were c u l t u r e d at a higher c o n c e n t r a t i o n (3 x 10 4 c e l l s / m l ) to give a comparable feeder-dependent p r o l i f e r a t i v e response ( i . e . to achieve the production i n the same period of time - 4 days - o f approximately the same number of c e l l s ) . The data f o r A8 c e l l s c u l t u r e d under these c o n d i t i o n s can be superimposed on the data f o r H9 c e l l s shown i n Figure 7a but with a 20-fold s h i f t ( i n c r e ase) i n the r e l a t i v e M2-10B4 c e l l requirement of A8 c e l l s . E a o ~i 1 r 0 150 1500 15000 1500C0 Number ol M2-10B4 cells per ml ~i r 0 150 1500 15000 150000 Number of M2-10B4 cells per ml Panel a Panel b Figure 7. P r o l i f e r a t i o n of 3,000 B9 c e l l s / m l (panel a) and 3 x 10 4 A8 c e l l s / m l (panel b) when plated on i r r a d i a t e d M2-10B4 c e l l s at d i f f e r e n t M2-10B4 c e l l concentrations. The s o l i d l i n e shows the r e s u l t s for cu l t u r e s containing H9 or A8 c e l l s . The dashed l i n e shows the background due to the i r r a d i a t e d M2-10B4 feeder c e l l s only. 87 D) Evidence f o r a Soluble Pre-B C e l l S t i m u l a t i n g Factor To determine whether the stromal c e l l s t i m u l a t i n g a c t i v i t y might be a t t r i b u t a b l e to the release of a so l u b l e growth f a c t o r , the e f f e c t of sep a r a t i n g the target A8 or H9 c e l l s from M2-10B4 c e l l s by an agarose i n t e r l a y e r was examined. In these experiments A8 or H9 c e l l s were seeded at low c e l l d e n s i t y e i t h e r i n m e t h y l c e l l u l o s e f o r assessment of growth by colony formation, or i n l i q u i d medium f o r assessment of growth by ^H-thymidine i n c o r p o r a t i o n . Results of a t y p i c a l experiment are shown i n Table 6. The presence of an agarose i n t e r l a y e r d i d not a l t e r the number of H9 c o l o n i e s obtained but markedly reduced A8 c e l l p l a t i n g e f f i c i e n c y . The a b i l i t y of M2-10B4 c e l l s to s t i m u l a t e H9 and A8 c e l l p r o l i f e r a t i o n i n l i q u i d c u l t u r e s separated by an agarose i n t e r l a y e r was a l s o r e a d i l y demonstrated. As f u r t h e r shown i n Table 6, M2-10B4 c e l l s were a l s o able to s t i m u l a t e normal pre-B c e l l s (obtained from a r e c e n t l y e s t a b l i s h e d long-term pre-B lymphoid c u l t u r e ) when separated by an agarose i n t e r l a y e r . S i m i l a r r e s u l t s were obtained when non-i r r a d i a t e d M2-10B4 feeders were used (data not shown). To f u r t h e r c h a r a c t e r i z e the a c t i v i t y produced by M2-10B4 c e l l s , CM were assayed f o r t h e i r a b i l i t y to s t i m u l a t e A8 or H9 c e l l p r o l i f e r a t i o n i n low de n s i t y c u l t u r e s . P r e l i m i n a r y experiments e s t a b l i s h e d that a p r o l i f e r a t i v e response of A8 c e l l s , and a l s o of normal pre-B c e l l s , could not be r e a d i l y detected w i t h M2-10B4 CM. However, H9 c e l l s gave a c l e a r dose-dependent response (Figure 8a). CM harvested a f t e r 1-2 days were c o n s i s t e n t l y found to con t a i n the most H9 c e l l - s t i m u l a t i n g a c t i v i t y . A f t e r 3 days s t i m u l a t o r y f a c t o r s could no longer be detected. S i m i l a r r e s u l t s have been obtained w i t h S5-2 CM. As shown i n Figure 8b, the H9 c e l l - s t i m u l a t i n g a c t i v i t y produced by M2-10B4 c e l l s i s h i g h l y l a b i l e to f r e e z i n g and thawing, but i s completely Table 6. Demonstration of a sol u b l e f a c t o r derived from M2-10B4 c e l l s s t i m u l a t i n g immortalized pre-B c e l l l i n e s and normal pre-B c e l l s . Colony Assay 3 C e l l P r o l i f e r a t i o n Assay 0 Conditions H9 A8 H9 A8 Normal Pre-B C e l l s on p l a s t i c 0 0 on agarose 0 0 on i r r a d i a t e d 47,43 43,33 M2-10B4 on i r r a d i a t e d 48,47 22,9 M2-10B4 + agarose 129 + 16 238 + 33 387 + 39 71 +14 76 + 21 134 + 13 1,788 + 108 c 11,561 + 810 c 16,511 + l , 7 5 0 d 1,179 + 100 2,504 + 93 3,468 + 187 a R e s u l t s represent d u p l i c a t e measurements of colony numbers (>10 c e l l s / c o l o n y ) per 1.1 ml me t h y l c e l l u l o s e c u l t u r e from 2 representative experiments. C e l l s were plate d at 300 c e l l s per 1.1 ml c u l t u r e and colonies were scored on Day 7. D R e s u l t s represent the mean cpm + SEM from t r i p l i c a t e assays of 2 re p r e s e n t a t i v e l i q u i d c u l t u r e experiments. In one, H9 c e l l s were cultured at a concentration of 3,000 c e l l s / m l (or 2100 c e l l s / w e l l ) and A8 c e l l s were cultured at a concentration o f 3 x 10 4 c e l l s / m l ( or 2.1 x 1 0 4 c e l l s / w e l l ) . In the other, B220-negative pre-B c e l l s from the non-adherent f r a c t i o n of a 4-month old long-term lymphoid marrow c e l l c u l t u r e were obtained by FACS and cu l t u r e d at a concentration of 2 x 10 4 c e l l s / m l . »^Level of ^H-thymidine i n c o r p o r a t i o n i n t o i r r a d i a t e d M2-10B4 c e l l s alone, which was 874 (+ 121) ( c ) , and 498 + 46 (d), i s included i n these r e s u l t s . Panel a Panel b Figure 8. Panel a. P r o l i f e r a t i o n of H9 c e l l s (3000 c e l l s / m l ) seeded i n the presence of various concentrations of M2-10B4 CM harvested at 1 • , 2 A, 3 • or 5 O days a f t e r adding f r e s h medium to the M2-10B4 c e l l s . |||| H9 c e l l s (3000 c e l l s / m l ) seeded with medium alone. Panel b: P r o l i f e r a t i o n of H9 c e l l s (3000 c e l l s / m l ) seeded i n the presence of various concentrations of M2-10B4 Day 1 CM a f t e r heating to 56°C f o r 1 hour O ; and a f t e r f r e e z i n g and thawing once B . The dash l i n e represents a p o s i t i v e c o n t r o l which i s s i m i l a r to DI curve from panel A. |||| H9 c e l l s (3000 c e l l s / m l ) seeded with medium alone. 90 r e s i s t a n t to heating at 56°C f o r 1 hour. Figure 9 shows a r e p r e s e n t a t i v e p r o f i l e of the H9 c e l l - s t i m u l a t i n g a c t i v i t y found i n serum-free M2-10B4 CM a f t e r chromatography on Sephadex G100. I t can be seen that a l l of the a c t i v i t y e l u t e s from the column i n a s i n g l e peak, with an apparent molecular weight of approximately 10,000. E) S p e c i f i c i t y of the A c t i v i t y Produced by Stromal C e l l Lines To determine whether the a c t i v i t y i n M2-10B4 CM could be a t t r i b u t e d to a known growth f a c t o r , the a b i l i t y of t h i s CM to s t i m u l a t e the p r o l i f e r a t i o n of B6SUtA (17) and 32D clone 3 (17) c e l l s was tested by Dr. G. K r y s t a l using the same ^H-thymidine i n c o r p o r a t i o n p r o t o c o l described f o r H9 c e l l s , except that the target responder c e l l s were harvested a f t e r 24 hours ra t h e r than a f t e r 3-4 days. B6SUtA c e l l s which respond to IL-3 (17), GM-CSF and IL-4 (Dr. G. K r y s t a l , personal communcation), and 32D clone 3 c e l l s which are e x c l u s i v e l y IL-3 responsive (17), were n e i t h e r stimulated nor i n h i b i t e d by M2-10B4 CM (data not shown). This CM was a l s o tested by Dr. F. Lee (DNAX Research I n s t i t u t e of Molecular and C e l l B i o l ogy, Palo A l t o , CA) and not found to co n t a i n detectable IL-4 a c t i v i t y . M2-10B4 CM (Day 1) when tested f o r i t s a b i l i t y to s t i m u l a t e myeloid or e r y t h r o i d colony formation by 3 x 10 4 normal mouse marrow c e l l s p l a t e d i n standard m e t h y l c e l l u l o s e c u l t u r e s c o n t a i n i n g 3 un i t s / m l of Epo (18) supported the growth of only a few co l o n i e s c o n t a i n i n g <20 macrophages. However, M2-10B4 CM harvested a f t e r longer periods s t i m u l a t e d l a r g e r numbers of col o n i e s c o n t a i n i n g up to 500 macrophages (only) suggesting that M2-10B4 c e l l s may produce some M-CSF. 91 Phenol red 60 Fract ion number Figure 9. A re p r e s e n t a t i v e Sephadex G100 p r o f i l e of the H9 s t i m u l a t i n g a c t i v i t y present i n a 1 ml sample of 8X concentrated serum-free M2-10B4 CM. Each point represents the mean of 3 r e p l i c a t e s + SEM of the a c t i v i t y present i n f r a c t i o n s assayed at a f i n a l concentration ol 5%. Arrows i n d i c a t e the e l u t i o n p o s i t i o n s of dextran blue (V0), ovalbumin (OVA), cytochrome C (Cyt. C) and phenol red. 92 F) Response of Feeder-Independent H9 C e l l s to Defined Growth Factors Table 7 shows the r e s u l t s of a r e p r e s e n t a t i v e experiment i n which H9 c e l l s were tested f o r t h e i r p r o l i f e r a t i v e response to a wide v a r i e t y of crude CM or defined growth f a c t o r s , many of which are known to o p t i m a l l y s t i m u l a t e or i n h i b i t various hemopoietic and/or lymphoid c e l l types at the intermediate concentrations used. None of these stimulated H9 c e l l s . 3) DISCUSSION We have described here the i s o l a t i o n , c l o n i n g and c h a r a c t e r i z a t i o n of two c l o s e l y r e l a t e d murine pre-B c e l l l i n e s o r i g i n a l l y derived from the nonadherent f r a c t i o n of a long-term lymphoid marrow c u l t u r e . One of these (A8) i s of i n t e r e s t because i t i s h i g h l y dependent on mesenchymal c e l l s of the f i b r o b l a s t - e n d o t h e l i a l - a d i p o c y t e lineage f o r continued s u r v i v a l and growth. Such a property i s a feature of f r e s h l y i s o l a t e d pre-B c e l l s (21) and of t h e i r progeny generated over the f i r s t s e v e r a l weeks i n r e c e n t l y e s t a b l i s h e d l o n g -term lymphoid marrow c u l t u r e s ( 4 ) . Extensive c h a r a c t e r i z a t i o n of A8 c e l l s has shown that they l a c k a l l B - c e l l phenotypic markers examined ( i . e . B220, surface and cytoplasmic I g , ThB, and TdT), w i t h the exception of BP-1 which i s expressed at r e l a t i v e l y high l e v e l s on a l l A8 c e l l s . I t i s perhaps noteworthy that a s i m i l a r high expression of BP-1 has been p r e v i o u s l y a s s o c i a t e d w i t h the transformation of murine B-lineage c e l l s i n contrast to normal B-lineage c e l l s which are only weakly BP-1 p o s i t i v e (15, and data not shown). I n t e r e s t i n g l y , more recent s t u d i e s have demonstrated that A8 c e l l s are tumorigenic i n syngeneic nude mice although they do not release transforming v i r u s a c t i v e i n a focus forming assay on 3T3 c e l l s , and do not express the Gp70 antigen of 93 Table 7. Lack of responsiveness of H9 c e l l s to defined growth f a c t o r s . H9 c e l l s (300 c / w e l l ) a Growth Factors' 3 cpm C o n c e n t r a t i o n 0 p o s i t i v e c o n t r o l ^ 28729 7905 + + 875 303 H9 c e l l s + M2-10B4 c e l l s M2-10B4 CM 25% (1-50%) negative c o n t r o l e 818 ± 125 medium alone r h IL-1 0 978 + 145 120 U/ml (12 - 1200 U/ml) r h IL-2 1370 + 117 10 U/ml (10 - 100 U/ml) r m IL-3 583 + 152 5 U/ml (0.5 - 50 U/ml) r m IL-4 965 + 300 100 U/ml (10 - 1000 U/ml) r m GM-CSF 931 + 114 25 U/ml (2.5 - 250 U/ml) r h G-CSF 1030 + 98 100 U/ml (10 - 1000 U/ml) r h IFNy 894 + 489 100 U/ml (10 - 1000 U/ml) r h HGF 1044 + 58 100 U/ml (1 - 1000 U/ml) TGF p x 76 + 16 5 ng/ml (0.5 - 50 ng/ml) TGF a 569 + 110 50 ng/ml (5 - 500 ng/ml) EGF 807 + 92 0.5 ng/ml (0.5 - 5 ng/ml) EMT-6 CMf 653 + . 47 5% ( 1 % - 10%) : PWM - SCCM 649 + 108 0.1% (0.1% - 10%) Agar-LCM 1155 + 61 1% (1 - 10%) PHA-LCM 820 + 93 5% (1 - 10%) a R e s u l t s represent the mean of t r i p l i c a t e s and are expressed i n cpm + SEM b I L - l , 3 and GM-CSF were obtained as p u r i f i e d f a c t o r s from Biogen. IL-2, G-CSF and EGF were purchased from Amgen (Thousand Oaks, CA); n a t u r a l GM-CSF and IL-4 were purchased from Genzyme (Boston, MA); HGF, which i s the same as i n t e r f e r o n 0 2 a n d IL-6 was obtained from Dr. L.A. Aarden (19); i n t e r f e r o n y (IFNy) was obtained from Dr. J . Gutterman (Houston, TX); porcine TGF-P^ was purchased from R. & D. Systems, Inc. (Minneapolis, MN); and r a t TGF-a was purchased from Bachem Inc. (Torrance, CA); r = recombinant, h = human, m = murine. c ( ) concentration range tested. ^Feeder alone 335 + 13. e C e l l s c u l t u r e d i n medium alone on p l a s t i c . ^EMT-6 CM contains M-CSF a c t i v i t y and was prepared as described p r e v i o u s l y (20). 94 murine Moloney v i r u s (Chapter I V ) . The pre-B phenotype of A8 c e l l s was confirmed by Southern a n a l y s i s which i n d i c a t e d that both J J J a l l e l e s were rearranged. An unproductive rearrangement of both J J J a l l e l e s could e x p l a i n the observed f a i l u r e of these c e l l s to generate more mature phenotypes (22). The second cloned l i n e of pre-B c e l l s (H9) has very s i m i l a r f eatures to A8 c e l l s and was derived from the same o r i g i n a l c u l t u r e . Both H9 and A8 c e l l s appear to have i d e n t i c a l gene rearrangements and therefore probably d e r i v e from the same o r i g i n a l l y immortalized pre-B c e l l . The H9 c e l l l i n e appears to d i f f e r only with regard to i t s much lower (>20-fold) requirement f o r a stromal c e l l feeder l a y e r and a higher s e n s i t i v i t y to a pre-B s t i m u l a t o r y f a c t o r produced by such c e l l s . To f a c i l i t a t e a n a l y s i s of the pre-B c e l l s t i m u l a t o r y mechanism mediated by stromal c e l l s , we have a l s o i s o l a t e d and c h a r a c t e r i z e d a number of adherent c e l l l i n e s of marrow and spleen o r i g i n that are able to support pre-B c e l l growth. A l l three such l i n e s described i n t h i s study share a common phenotype which, l i k e 3T3 c e l l s , appears to be intermediate between f i b r o b l a s t s and e n d o t h e l i a l c e l l s . P h e n o t y p i c a l l y s i m i l a r stromal c e l l s have a l s o r e c e n t l y been described by others (7-12). In s p i t e of some phenotypic homogeneity amongst d i f f e r e n t stromal c e l l l i n e s , evidence f o r v a r i a t i o n s of f u n c t i o n a l p o t e n t i a l has been reported (23-25), and l i n e s d i f f e r i n g i n t h e i r a b i l i t y to promote myelopoiesis and/or lymphopoiesis have been i d e n t i f i e d ( 9 ) . However, such b i o a c t i v i t y measurements must be i n t e r p r e t e d with caution s i n c e both s t i m u l a t o r y and i n h i b i t o r y a c t i v i t i e s may be produced by the same c e l l s and the r e l a t i v e amounts and hence b i o l o g i c a l importance of these may a l s o vary under d i f f e r e n t c o n d i t i o n s . Nevertheless, d i f f e r e n c e s i n the c o n s t i t u t i v e or i n d u c i b l e production of various hemopoietic growth f a c t o r s such as GM-CSF and 95 G-CSF by d i f f e r e n t stromal l i n e s has a l s o been documented (11, and R.K. Humphries, personal communication). The s i g n i f i c a n c e of these l a t t e r d i f f e r e n c e s to the mechanism of pre-B c e l l s t i m u l a t i o n i s not yet c l e a r . We have shown that at l e a s t two of our stromal c e l l l i n e s produce a f a c t o r that can s t i m u l a t e normal pre-B c e l l s , as w e l l as both A8 and H9 c e l l s . Time course s t u d i e s showed that the most potent stromal c e l l CM was obtained when serum-free medium c o n t a i n i n g 2-ME was harvested a f t e r 1 day; t h e r e a f t e r l e v e l s d e c l i n e d . The a c t i v i t y i s r e s i s t a n t to heating to 56°C but unusually s e n s i t i v e to f r e e z i n g and thawing. A d d i t i o n a l experiments showed that our stromal c e l l l i n e s do not make dete c t a b l e I L - 3 , GM-CSF or IL-4. Moreover, since H9 c e l l s are not responsive to any of these growth f a c t o r s , nor to IL-1, IL-2, and IFN02> the pre-B c e l l s t i m u l a t i n g a c t i v i t y must be due to a d i f f e r e n t molecule. Although pre-B c e l l s t i m u l a t i o n by IL-3 has been reported (26), others have not found pre-B c e l l s derived from long-term lymphoid marrow c u l t u r e to be IL-3 responsive (12). This appears to be the case f o r A8 and H9 c e l l s a l s o . Recently two other groups (12,27) have reported a novel a c t i v i t y produced by d i f f e r e n t stromal c e l l l i n e s and a c t i v e on B-lineage c e l l s . However, the apparent molecular weights of the pre-B c e l l s t i m u l a t i n g f a c t o r s i d e n t i f i e d here (-10,000) and by Hunt et a l ! (12) (~35,000) are q u i t e d i f f e r e n t . Further information on both f a c t o r s w i l l be required to e s t a b l i s h whether these are d i s t i n c t gene products. The a v a i l a b i l i t y of v a r i a n t subclones of immortalized pre-B c e l l s that show marked and co n s i s t e n t d i f f e r e n c e s i n t h e i r dependence on mesenchymal stromal c e l l s and i n t h e i r s e n s i t i v i t y to a s o l u b l e f a c t o r produced by such stromal c e l l s , should prove u s e f u l i n the f u r t h e r c h a r a c t e r i z a t i o n of pre-B c e l l growth f a c t o r s and t h e i r mechanism of a c t i o n . P r e l i m i n a r y data i n d i c a t e 96 that these l i n e s w i l l a l s o be u s e f u l f o r studies of changes that accompany pre-B c e l l transformation. 97 REFERENCES WU AM, Siminovitch L, T i l l JE, McCulloch EA. Evidence f o r a r e l a t i o n s h i p between mouse hemopoietic stem c e l l s and c e l l s forming c o l o n i e s i n c u l t u r e . Proc N a t l Acad S c i USA 59:1209, 1968. Dick. JE, Magli MC, Huszar D, P h i l l i p s RA, B e r n s t e i n A. I n t r o d u c t i o n of a s e l e c t a b l e gene i n t o p r i m i t i v e stem c e l l s capable of long term r e c o n s t i t u t i o n of the hemopoietic system of w/wv mice. C e l l 42:71, 1985. A l t FW, B l a c k w e l l TK, DePinho RA, Reth MG, Yancopoulos GD. Regulation of genome rearrangement events during lymphocyte d i f f e r e n t i a t i o n . Immunol Rev 89:5, 1986. Whitlock CA, Witte ON. Long-term c u l t u r e of B lymphocytes and t h e i r precursors from murine bone marrow. Proc N a t l Acad S c i USA 79:3608, 1982. Dexter TM, A l l e n TD, L a j t h a LG. Conditions c o n t r o l l i n g the p r o l i f e r a t i o n of hemopoietic stem c e l l s i n v i t r o . J C e l l P h y s i o l 91:355, 1977. Dorshkind K. In v i t r o d i f f e r e n t i a t i o n of B lymphocytes from p r i m i t i v e hemopoietic precursors present i n long-term bone marrow c u l t u r e s . J Immunol 136:422, 1986. Kodama H-A, Hagiwara H, Sudo H, Arnagai Y, Yokota T, A r a i N, Kitamura Y. MC3T3-G2/PA6 preadipocytes support i n v i t r o p r o l i f e r a t i o n of hemopoietic stem c e l l s through a mechanism d i f f e r e n t from that of i n t e r l e u k i n 3. J C e l l P h y s i o l 129:20, 1986. Whitlock CA, Robertson D, Witte ON. Murine B c e l l lymphopoiesis i n long term c u l t u r e . J Immunol Meth 67:353, 1984. C o l l i n s LS, Dorshkind K. A stromal c e l l l i n e from myeloid long-term bone marrow c u l t u r e s can support myelopoiesis and B lymphopoiesis. J Immunol 138:1082, 1987. Quesenberry P, Song Z, McGrath E, McNiece I , Shadduck R, Waheed A, Baber G, Kleeman E, K a i s e r D. M u l t i l i n e a g e s y n e r g i s t i c a c t i v i t y produced by a murine adherent marrow c e l l l i n e . Blood 69:827, 1987. Rennick D, Yang G, Gemmell L, Lee F. Co n t r o l of hemopoiesis by a bone marrow stromal c e l l clone: l i p o p o l y s a c c h a r i d e - and i n t e r l e u k i n -1 - i n d u c i b l e production of c o l o n y - s t i m u l a t i n g f a c t o r s . Blood 69:682, 1987. Hunt P, Robertson D, Weiss D, Rennick D, Lee F, Witte ON. A s i n g l e bone marrow-derived stromal c e l l type supports i n v i t r o growth of e a r l y lymphoid and myeloid c e l l s . C e l l 48:997, 1987. Zuckerman KS, Wicha MS. E x t r a c e l l u l a r matrix production by the adherent c e l l s of long-term murine bone marrow c u l t u r e s . Blood 61:540, 1983. 98 14. Greenberger JS. S e n s i t i v i t y of corticosteroid-dependent i n s u l i n -r e s i s t a n t l i p o g e n e s i s i n marrow preadipocytes of obese-diabetic (db/db) mice. Nature 275:752, 1978. 15. Cooper MD, Mulvaney D, Coutinho A, Cazenave P-A. A novel c e l l surface molecule on e a r l y B-lineage c e l l s . Nature 321:616, 1986. 16. Takei F. Murine T lymphoma c e l l s express a novel membrane-associated antigen with unique fea t u r e s . J Immunol 139:649, 1987. 17. Greenberger JS, Eckner RJ, Sakakeeny M, Marks P, Reid D, Nabel G, Hapel A, I h l e JN, Humphries RK. I n t e r l e u k i n 3-dependent hematopoietic progenitor c e l l l i n e s . Federation Proceedings 42:2762, 1983. 18. Eaves CJ, K r y s t a l G, Eaves AC. E r y t h r o p o i e t i c c e l l s . I n: " B i b l i o t h e c a Haematologica, No 48 - Current Methodology i n Experimental Hematology", (ed. SJ Baum ), S. Krager, B a s e l , pp 81, 1984. 19. Aarden LA, de Groot ER, Schaap OL, Lansdorp PM. Production of hybridoma growth f a c t o r by human monocytes. Eur J Immunol 17:1411, 1987. 20. Gregory CJ, Eaves AC. In v i t r o s t u d i e s of e r y t h r o p o i e t i c progenitor c e l l d i f f e r e n t i a t i o n . In: " D i f f e r e n t i a t i o n of Normal and N e o p l a s t i c Hematopoietic C e l l s " , (eds. B Clarkson, PA Marks, JE T i l l ) , Cold Spring Harbor Laboratory, New York, pp 179, 1978. 21. Muller-Sieburg CE, Whitlock CA, Weissman IL. I s o l a t i o n of two e a r l y B lymphocyte progenitors from mouse marrow: a committed pre-pre-B c e l l and a clonogenic Thy-1-'-0 hematopoietic stem c e l l . C e l l 44:653, 1986. 22. A l t F, Rosenberg N, Lewis S, Thomas E, Baltimore D. Organization and r e o r g a n i z a t i o n of immunoglobulin genes i n A-MuLV-transformed c e l l s : rearrangement of heavy but not l i g h t chain genes. C e l l 27:381, 1981. 23. Roberts RA, Spooncer E, Parkinson EK, Lord BI, A l l e n TD, Dexter TM. M e t a b o l i c a l l y i n a c t i v e 3T3 c e l l s can s u b s t i t u t e f o r marrow stromal c e l l s to promote the p r o l i f e r a t i o n and development of multipotent haemopoietic stem c e l l s . J C e l l P h y s i o l 132:203, 1987. 24. Z i p o r i D, Toledo J , von der Mark K. Phenotypic heterogeneity among stromal c e l l l i n e s from mouse bone marrow d i s c l o s e d i n t h e i r e x t r a c e l l u l a r matrix composition and i n t e r a c t i o n s w i t h normal and leukemic c e l l s . Blood 66:447, 1985. 25. A n k l e s a r i a P, Klassen V, Sakakeeny MA, F i t z G e r a l d TJ, Harrison D, Rybak ME, Greenberger JS. B i o l o g i c a l c h a r a c t e r i z a t i o n of cloned permanent stromal c e l l l i n e s from anemic S l / S l d mice and +/+ l i t t e r m a t e s . Exp Hematol 15:636, 1987. 26. P a l a c i o s R, Henson G, Steinmetz M, McKearn JP. I n t e r l e u k i n - 3 supports growth of mouse p r e - B - c e l l c l o n e s . i n v i t r o . Nature 309:126, 1984. 27. Whitlock CA, Tidmarsh GF, Muller-Sieburg C, Weissman IL. Bone marrow stromal c e l l l i n e s with lymphopoietic a c t i v i t y express high l e v e l s of a pre-B neop l a s i a - a s s o c i a t e d molecule. C e l l 48:1009, 1987. 99 C H A P T E R I V AUTOCRINE PRODUCTION OF PRE-B CELL STIMULATING ACTIVITY BY A VARIETY OF TRANSFORMED PRE-B CELL LINES 1) INTRODUCTION The p r o l i f e r a t i o n and d i f f e r e n t i a t i o n of p r i m i t i v e B-lineage c e l l s appears to be regulated by i n t e r a c t i o n s w i t h mesenchymally derived "stromal" elements ( 1 ) . Although d i r e c t contact between the pre-B c e l l and the stromal c e l l may be involved ( 2 ) , there i s a l s o evidence that supportive mesenchymal c e l l s can release s o l u b l e pre-B c e l l s t i m u l a t i n g f a c t o r s (3-5). Decreased dependence on exogenously provided growth r e g u l a t o r s i s now a w e l l documented featu r e of many malignant c e l l types i n c l u d i n g examples i n the hemopoietic system. In some experimental s i t u a t i o n s , the explanation f o r the autonomous growth c a p a c i t y of the transformed c e l l s can be d i r e c t l y a t t r i b u t e d to the i n i t i a t i o n of s e l f - s t i m u l a t i n g f a c t o r production (6,7). In spontaneously transformed hematopoietic c e l l s the r o l e of autocrine growth f a c t o r s i s l e s s c l e a r . A number of steps i n the s i g n a l transduction pathway c l e a r l y represent p o t e n t i a l targets f o r malignant transformation. Nevertheless, a c t i v a t i o n of aut o c r i n e growth f a c t o r production appears to be a more common event than i n i t i a l l y a n t i c i p a t e d (8-10). Two l i n e s of evidence suggested that autocrine mechanisms might play a r o l e i n the e v o l u t i o n of malignant pre-B c e l l populations. The f i r s t d e r i v e d from s t u d i e s of A-MuLV-induced transformation of pre-B c e l l s i n v i t r o . These 100-demonstrated that A-MuLV i n f e c t e d pre-B c e l l s o f t e n r e q u i r e an i n i t i a l period of 1-2 months before they evolve the cap a c i t y f o r autonomous, i . e . stromal cell-independent, growth (11). The second derived from the st u d i e s described i n Chapter I I I of s e v e r a l spontaneously transformed murine pre-B c e l l l i n e s which were found to e x h i b i t d i f f e r e n t degrees of autonomy i n v i t r o , depending on the concentration of the c e l l s i n the c u l t u r e s used to te s t f o r autonomy. 2) RESULTS A) H9 C e l l s Produce a Pre-B C e l l S t i m u l a t i n g A c t i v i t y CM obtained from the spontaneously transformed H9 pre-B c e l l l i n e was assayed f i r s t f o r i t s a b i l i t y to s t i m u l a t e the p r o l i f e r a t i o n of H9 c e l l s i n low d e n s i t y suspension c u l t u r e s . Figure 10 shows the r e s u l t s of a t y p i c a l t i t r a t i o n of H9 CM by comparison to CM from M2-10B4 c e l l s . I t can be seen that H9 c e l l s produce a r e a d i l y - d e t e c t a b l e s e l f - s t i m u l a t i n g a c t i v i t y , although conside r a b l y l e s s e f f i c i e n t l y than M2-10B4 c e l l s s i n c e the H9 CM i s 5 to 10 f o l d l e s s potent, even though the concentration of H9 c e l l s used to prepare the CM was almost 10 times higher. This d i f f e r e n c e i n the H9 c e l l s t i m u l a t i n g a c t i v i t y of H9 and M2-10B4 CM was a reproducible f i n d i n g . I n t e r e s t i n g l y , although H9 CM proved l e s s s t i m u l a t o r y , i t a l s o appeared to be fr e e of the i n h i b i t o r s r o u t i n e l y detectable i n M2-10B4 CM when t h i s was tested at concentrations above 25%. In order to determine whether H9 c e l l s produced a f a c t o r that could a l s o s t i m u l a t e normal pre-B c e l l s , the l a t t e r were incubated i n the presence or absence of H9 c e l l s separated by an agar i n t e r l a y e r . As a source of pure normal pre-B c e l l s , c l o n a l l y expanded l i n e s from lymphoid LTC's were used i n these experiments. The r e s u l t s of a t y p i c a l experiment are shown 101 12 -Concentration of Conditioned Medium (%) Figure 10. H9 s t i m u l a t i n g a c t i v i t y i n H9 CM. ( ), or M2-10B4 CM ( ). ( I l l ) background 3H-thymidine i n c o r p o r a t i o n by H9 c e l l s at 3,000 c e l l s / m l and c u l t u r e d i n medium alone. 102 i n Table 8. They demonstrate that H9 c e l l s secrete a s o l u b l e f a c t o r that s t i m u l a t e s normal pre-B c e l l s as w e l l as H9 c e l l s , themselves. B) Production of a Pre-B C e l l S t i m u l a t i n g A c t i v i t y by A-MuLV Transformed  Pre-B C e l l Lines To i n v e s t i g a t e whether other transformed pre-B c e l l l i n e s a l s o demonstrate a c t i v a t i o n of autocrine growth f a c t o r production, two ph e n o t y p i c a l l y d i f f e r e n t l i n e s (ABn and ABp) generated independently us i n g A-MuLV as the transforming agent, were s e l e c t e d f o r such s t u d i e s . As shown i n Table 9, both of these, l i k e H9 c e l l s , produced tumors i n immunocompromised mice. In c o n t r a s t , the two ph e n o t y p i c a l l y normal pre-B c e l l clones (Bn and Bp) from which the re s p e c t i v e A-MuLV transformants were de r i v e d , or which were exposed only to Mo-MuLV, were not tumorigenic, as expected. Assessment of the presence of Mo-MuLV Gp70 antigen confirmed the e f f e c t i v e n e s s of i n f e c t i o n s w i t h both A-MuLV and Mo-MuLV stocks and showed that t h i s antigen was absent i n both normal pre-B c e l l clones and i n H9 c e l l s . When supernatants from each of the c e l l l i n e s were assayed f o r focus-forming a c t i v i t y on NIH-3T3 c e l l s , only the two A-MuLV transformants were p o s i t i v e . Southern a n a l y s i s of DNA from a l l of the l i n e s confirmed the presence of v-abl bands e x c l u s i v e l y i n the A-MuLV transformants (data not shown). The r e s u l t s of a d d i t i o n a l s t u d i e s e s t a b l i s h i n g the pre-B c e l l phenotype of the A-MuLV derived transformants are shown i n Table 10. Both A-MuLV l i n e s express B220, T200 and TdT. One l i n e expresses BP-1 (15). This general pattern i s d i f f e r e n t from that t y p i c a l of e i t h e r the normal pre-B c e l l clones or the H9 c e l l l i n e . Ig gene rearrangement a n a l y s i s (Figure 11) showed the same rearrangement of one a l l e l e of Jp; i n the two normal and two d e r i v a t i v e A-MuLV transformed c e l l l i n e s 103 T a b l e 8. Demonstration t h a t H9 a u t o c r i n e a c t i v i t y s t i m u l a t e s normal pre-B c e l l s . C u l t u r e C o n d i t i o n U n d e r l a y e r H9 c e l l s i n T a r g e t c e l l s i n H-thymidine bottom agar l a y e r l i q u i d o v e r l a y i n c o r p o r a t i o n ( cpm) 1 M2-10B4 No Normal pre-B 10,097 + 441 monolayer c e l l s 2 - Yes - 1 4 7 + 1 4 3 - No Normal pre-B 6 7 + 9 c e l l s 4 - Yes Normal pre-B 3,728 + 397 c e l l s A l l c u l t u r e s c o n t a i n e d 2 agar l a y e r s : the lower one with or without H9 c e l l s as i n d i c a t e d , the upper one without any c e l l s to serve as a spacer p r e v e n t i n g any c o n t a c t between the H9 or M2-10B4 c e l l s and the normal pre-B t a r g e t c e l l s which were p r e s e n t i n a l i q u i d s u s p e n s i o n c u l t u r e on top of the upper agar l a y e r . For a d d i t i o n a l d e t a i l s , see M a t e r i a l s and Methods. 104 T a b l e 9. Transformed phenotype of c l o n a l lymphoid c e l l l i n e s . C e l l l i n e s * Tumor i n c i d e n c e Mo-MuLV Gp70 A-MuLV p r o d u c t i o n 0 10^ c e l l s / m o u s e 10^ c e l l s / m o u s e (% p o s i t i v e c e l l s ) (FFU/ml) H9 3/3 2/2 0 0 Bn ( o r i g i n a l l i n e ) ND d 0/2 0 0 Bn + MoMuLV ND 0/2 31 0 Bn + A-MuLV 2/2 2/2 95 4 x 10 3 Bp ( o r i g i n a l l i n e ) ND 0/2 0 0 Bp + MoMuLV ND 0/2 43 0 Bp + A-MuLV 2/2 2/2 98 8 x 10 3 A l l c e l l l i n e s were t e s t e d more than 4weeks a f t e r exposure to v i r u s (Bn, B p - s e r i e s ) or c l o n i n g ,(H9 ) . Gp70 a n t i g e n encoded by Mo-MuLV was d e t e c t e d by u s i n g YE6/26 (12) i n an i n d i r e c t i mmunofluorescent s t a i n i n g procedure and FACS | n a l y s i s . j P o s i t i v e c o n t r o l ( v i r u s s tock s u p e r n a t a n t ) : 10 f o c u s - f o r m i n g u n i t s (FFU/ml). ND = not done. 105 T a b l e 10. P h e n o t y p e d a t a o f n o r m a l a n d t r a n s f o r m e d l y m p h o i d c l o n e s . A n t i b o d i e s u s e d f o r a n a l y s i s C e l l l i n e s (% p o s i t i v e c e l l s ) R e a g e n t O r i g i n S p e c i f i c i t y N o r m a l T r a n s f o r m e d Bn Bp H9 ABn ABp ( s p o n t a n e o u s ) (A-MuLV) (A-MuLV) RA3-3A /6.1 ( B 2 2 0 T Y E 1 / 3 0 ( T h y - 1 ) Y E 1 / 2 1 ( T 2 0 0 ) Y E 1 / 9 BP-1 T d T R a t R a t R a t Hous< A n t i IgM R a b b i t A n t i - / / c h a i n R a b b i t R a b b i t P r e - B , B l y m p h o c y t e s T h y m o c y t e s , p r i m i t i v e h e m o p o i e t i c c e l l s H e m o p o i e t i c c e l l s ( e x c e p t e r y t h r o i d c e l l s ) T r a n s f e r r i n r e c e p t o r E a r l y p r e - B , n e w l y f o r m e d B l y m p h o c y t e s S u r f a c e IgM I n t r a c y t o p l a s m i c / / - c h a i n p r o t e i n T L y m p h o c y t e s e a r l y p r e - B l y m p h o c y t e s 99 98 76 51 0 0 98 97 0 0 61 99 95 0 0 10 15 99 100 95 14 0 10 15 B220 was o b t a i n e d f r o m A m e r i c a n T y p e C u l t u r e C o l l e c t i o n ( R o c k v i l l e , MD). Y E 1 / 3 0 ( 1 3 ) , Y E 1 / 2 1 ( 1 3 ) , Y E 1 / 9 ( 1 4 ) a n t i b o d i e s w e r e k i n d l y p r o v i d e d f r o m D r . F. T a k e i ( T e r r y F o x L a b o r a t o r y , B.C. C a n c e r R e s e a r c h C e n t r e , V a n c o u v e r , B . C . ) . BP-1 ( 1 5 ) was a g i f t f r o m D r . M.D. C o o p e r ( U n i v e r s i t y o f A l a b a m a , B i r m i n g h a m , A L ) . 106 23 1 2 3 4 5 6 7 i I I I I I I 9.6 -6.6 -4.3 -2.3 ~ Kb M | ; •+ M ii M M ) t 6.2 Kb Figure 11. Southern b l o t a n a l y s i s of Ig H chain gene rearrangements i n normal pre-B c e l l clones and d e r i v a t i v e A-MuLV transformants. C e l l u l a r DNA was digested with EcoRI and h y b r i d i z e d to a probe as described i n M a t e r i a l s and Methods. Lane 1, A20 B c e l l s (from the American Type C u l t u r e C o l l e c t i o n , R o c k v i l l e , MD); lane 2, thymus; lane 3, A-Kl T c e l l s ( 1 3 ) ; lane 4, Bn c e l l s ; lane 5, A-MuLV Bn c e l l s ; lane 6, Bp c e l l s ; lane 7, A-MuLV Bp c e l l s . An arrow marks the expected p o s i t i o n of the 6.2 Kb germ l i n e fragment. A l l lanes were loaded w i t h 10 ug of DNA. 107 t e s t e d . However, i n both A-MuLV transformed l i n e s the i n t e n s i t y of the germ l i n e a l l e l e was decreased (lanes 5 and 7), and new bands were apparent suggesting that secondary rearrangements had occurred f o l l o w i n g A-MuLV transformation. A n a l y s i s w i t h a Tp probe revealed no evidence of rearrangements of t h i s gene i n these c e l l s (data not shown). H9 c e l l s that were derived independently from a d i f f e r e n t long-term pre-B c e l l c u l t u r e a l s o show rearrangement of both a l l e l e s of the Jrj gene and no rearrangement of t h e i r Tp genes ( 5 ) . Cytospin preparations showed that a l l c e l l s i n a l l l i n e s e x h i b i t e d a p r i m i t i v e lymphoblast morphology. CM were prepared from c e l l s from A-MuLV i n f e c t e d c u l t u r e s at va r i o u s times a f t e r exposure to A-MuLV, and from c o n t r o l c u l t u r e s according to the p r o t o c o l described i n M a t e r i a l s and Methods. These d i f f e r e n t CM were then tested f o r t h e i r a b i l i t y to s t i m u l a t e H9 c e l l p r o l i f e r a t i o n . As shown i n Figure 12, normal pre-B c e l l s d i d not produce detectable H 9 - s t i m u l a t i n g a c t i v i t y . Weak a c t i v i t y was present i n media conditioned by A-MuLV i n f e c t e d c e l l s that were s t i l l h i g h l y stromal c e l l dependent ( i . e . 4 wks post-A-MuLV i n f e c t i o n ) and t h i s increased (on a per c e l l b a s i s ) when prepared from c e l l s of the same l i n e that were more advanced and capable of autonomous growth i n the absence of a stromal l a y e r ( i . e . 8 wks post A-MuLV). To t e s t whether the H9 c e l l s t i m u l a t i n g a c t i v i t y produced by A-MuLV i n f e c t e d c e l l s had an autocrine e f f e c t , the same A-MuLV c e l l CM were tested f o r t h e i r a b i l i t y to s t i m u l a t e the A-MuLV transformed c e l l s d e rived from the A-MuLV i n f e c t e d c u l t u r e s used to generate the CM. A s i m i l a r increase i n aut o c r i n e f a c t o r concentration was apparent between 4 and 8 weeks post A-MuLV i n f e c t i o n (data riot shown). 108 0 1 10 100 Concentration of Conditioned Medium (%) Figure 12. H9 c e l l s t i m u l a t i n g a c t i v i t y present i n media conditioned by normal and transformed pre-B c e l l l i n e s : ( ) H9 CM ( p o s i t i v e c o n t r o l ; (. . . .) normal (Bp) pre-B c e l l CM; ( ) CM obtained from A-MuLV i n f e c t e d Bp c e l l s obtained 4 weeks a f t e r i n f e c t i o n , and (-•- _^ ) CM obtained from c e l l s from the same A-MuLV i n f e c t e d Bp c u l t u r e s obtained 8 weeks a f t e r i n f e c t i o n . 109 C) C h a r a c t e r i z a t i o n of the Autocrine A c t i v i t y Produced by Transformed  Pre-B C e l l s As a f i r s t step towards the i d e n t i f i c a t i o n of the a c t i v i t y produced by transformed pre-B c e l l s , t h e i r CM's were tested by Dr. G. K r y s t a l using a number of standard bioassays. A d d i t i o n of up to 20% CM from any of the normal or transformed pre-rB c e l l l i n e s , i n c l u d i n g both H9 and A-MuLV transformants, to B6SUtA or 32D clone 23 c e l l c u l t u r e s f a i l e d to s t i m u l a t e d e t e c t a b l e ^H-thymidine uptake i n t o these c e l l s (data not shown). This suggests that n e i t h e r the normal nor the transformed pre-B c e l l s produce IL-3, GM-CSF or IL-4 to which B6SUtA c e l l s ( a l l three) and 32D c e l l s (IL-3 only) respond. Pre-B c e l l CM's at concentrations of up to 10% a l s o f a i l e d to support the growth of any co l o n i e s i n standard m e t h y l c e l l u l o s e assays f o r myeloid progenitors (data not shown). We have p r e v i o u s l y demonstrated that H9 c e l l s do not respond to GM-CSF, G-CSF, IL-1, IL-2, IL-3, IL-4, IL-6 (= I F N - 0 2 ) , I F N - Y , EGF, PWM-SCCM, agar-LCM or PHA-LCM (5). The s e l f - s t i m u l a t i n g a c t i v i t y produced by H9 c e l l s , l i k e that produced by A-MuLV transformants c e l l s i s there f o r e l i k e l y to be d i f f e r e n t from any of these w e l l c h a r a c t e r i z e d growth f a c t o r s . To f u r t h e r c h a r a c t e r i z e the autocrine growth f a c t o r a c t i v e on H9 c e l l s , H9 CM was concentrated and subjected to chromatography on Sephadex G50. Figure 13 shows the p r o f i l e of the H9 s t i m u l a t i n g a c t i v i t y which was re p r o d u c i b l y eluted as a s i n g l e peak with an apparent molecular weight of approximately 3,000. This suggests a d i f f e r e n c e from the pre-B c e l l f a c t o r s produced by M2-10B4 c e l l s (5) or described by others (3,16). However, sequence data may be required before the i n t e r r e l a t i o n s h i p s between these f a c t o r s can be f u l l y e s t a b l i s h e d . 110 Figure 13. A r e p r e s e n t a t i v e Sephadex G50 p r o f i l e of the pre-B s t i m u l a t i n g a c t i v i t y present i n a 1 ml sample of 50X concentrated serum-free H9 CM. Arrows i n d i c a t e the p o s i t i o n at which ovalbumin (Ova), cytochrome C (Cyt C), v i t a m i n B^2 ( v i t B12)> a n (* phenol red were eluted from t h i s column. Each point shows the mean + 1 SEM of 3 r e p l i c a t e s . The a c t i v i t y present i n each f r a c t i o n was assayed at a f i n a l concentration of 10%. I l l 3) DISCUSSION A n a l y s i s of the c o n s t i t u e n t s and f u n c t i o n of d i f f e r e n t c e l l s present i n long-term lymphoid marrow c u l t u r e s has been instrumental i n i d e n t i f y i n g mesenchymal stromal elements as a source of p o s i t i v e growth f a c t o r s e s s e n t i a l f o r the maintenance and p r o l i f e r a t i o n of normal murine pre-B c e l l s (3-5). In c o n t r a s t , some types of transformed pre-B c e l l s are c h a r a c t e r i z e d by an a b i l i t y to grow autonomously i n v i t r o , although a c q u i s i t i o n of t h i s property may not represent the f i r s t change to occur. This has been w e l l documented i n the case of transformed pre-B c e l l s developing a f t e r A-MuLV i n f e c t i o n of mouse marrow c e l l s (11). More r e c e n t l y , a s i m i l a r delay i n the time required f o r A-MuLV induced mast c e l l transformants to become autonomous of exogenous growth f a c t o r requirements has been documented (17,18). In t h i s study, we have shown that both A-MuLV and spontaneous mechanisms of pre-B c e l l t ransformation are associated with a c t i v a t i o n of a utocrine growth f a c t o r production. I n t e r e s t i n g l y , we found that A-MuLV transformed mast c e l l s i s o l a t e d under s i m i l a r c o n d i t i o n s i n v i t r o c o n s i s t e n t l y show the a c t i v a t e d expression of s e v e r a l hemopoietic growth f a c t o r s i n c l u d i n g a f a c t o r a c t i v e on H9 c e l l s as w e l l as IL-3 and GM-CSF, the l a t t e r two being f a c t o r s that are required f o r the production and s u r v i v a l i n v i t r o of normal mast c e l l precursors (19,20). On the other hand, A-MuLV transformed mast c e l l s generated under d i f f e r e n t c o n d i t i o n s have been found to show increased production of a d i f f e r e n t spectrum of growth f a c t o r s (21). In the present experiments, a c t i v a t i o n of myeloid growth f a c t o r s was a l s o not detected. Thus, there i s c l e a r l y heterogeneity i n the growth f a c t o r a c t i v a t i o n p a t t e r n obtained when the same transformation c o n d i t i o n s are a p p l i e d to d i f f e r e n t 112 target c e l l s (B c e l l and mast c e l l precursors) or when d i f f e r e n t c o n d i t i o n s are used to i s o l a t e tranformants of the same lin e a g e . How (or whether) t h i s may r e l a t e to the phenotype of the target c e l l , the i n i t i a l transforming event and the co n d i t i o n s i n which the c e l l subsequently i s stimulated to d i v i d e w i l l r e q u i r e a d d i t i o n a l experiments to r e s o l v e . These s t u d i e s a l s o r a i s e questions about the p o t e n t i a l s i g n i f i c a n c e of abnormal growth f a c t o r production by malignant B lineage c e l l s i n human disease (10,22). The mechanism(s) underlying a c t i v a t i o n of growth f a c t o r genes i n malignant hemopoietic and lymphoid c e l l s are, i n most in s t a n c e s , a complete mystery. Enhancer i n s e r t i o n , as appears to have occurred i n the development of the IL-3 producing WEHI-3B c e l l l i n e , i s one model (23), but t h i s i s u n l i k e l y to e x p l a i n the a c t i v a t i o n of m u l t i p l e growth f a c t o r s as has been seen i n both n a t u r a l l y a r i s i n g human leukemias (22,24) and i n A-MuLV transformed mast c e l l s (19-21). Further i n v e s t i g a t i o n of the mechanisms involved w i l l depend on the a v a i l a b i l i t y of more s p e c i f i c reagents to i d e n t i f y the pre-B c e l l a c t i v i t y i t s e l f and the gene that encodes i t . D i f f e r e n c e s between the a c t i v i t y produced by the one spontaneously transformed pre-B c e l l l i n e s t u d i e d to date and the pre-B c e l l s t i m u l a t i n g a c t i v i t y produced by the M2-10B4 stromal c e l l l i n e (5) suggests that d i f f e r e n t , or d i f f e r e n t l y processed, f a c t o r s may be in v o l v e d . A d d i t i o n a l pre-B c e l l s t i m u l a t i n g f a c t o r s with unique g e l f i l t r a t i o n p r o f i l e s have a l s o been reported r e c e n t l y (3,16). None of the transformed pre-B c e l l s showed as high a l e v e l of c o n s t i t u t i v e pre-B c e l l growth f a c t o r production when compared to M2-10B4 c e l l s , a r e p r e s e n t a t i v e cloned marrow stromal c e l l l i n e . I t i s p o s s i b l e that l e s s f a c t o r may be required i f produced by the responding c e l l i t s e l f , and that some s t i m u l a t i o n might a l s o occur without the n e c e s s i t y of e x t r a c e l l u l a r 113 s e c r e t i o n . Autocrine s t i m u l a t i o n by i n t r a c e l l u l a r growth f a c t o r has been documented i n other transformed c e l l types, i n c l u d i n g examples responding to both PDGF (6) and GM-CSF (7). In a d d i t i o n , the transformed pre-B c e l l s s t u d i e d here may w e l l have undergone other changes c o n t r i b u t i n g to a decreased dependence on exogenous s t i m u l a t i o n under c o n d i t i o n s operative i n v i v o . Nevertheless, i t seems l i k e l y that a c t i v a t i o n of an autocrine growth f a c t o r mechanism may play a r o l e i n A-MuLV transformation of murine pre-B c e l l s , s i n c e f a c t o r production increased concomitant with the a c q u i s i t i o n of autonomous growth p o t e n t i a l i n v i t r o . The f a c t that a s i m i l a r mechanism was a l s o found i n a spontaneously transformed pre-B c e l l l i n e f u r t h e r suggests that autocrine growth f a c t o r production may be a r e l a t i v e l y common event i n the development of transformed pre-B c e l l s . 114 REFERENCES 1. Whitlock C, Denis K, Robertson D, Witte 0. In v i t r o a n a l y s i s of murine B - c e l l development. Ann Rev Immunol 3:213, 1985. 2. Kierney PC, Dorshkind K. B lymphocyte precursors and myeloid progenitors s u r v i v e i n d i f f u s i o n chamber c u l t u r e s but B c e l l d i f f e r e n t i a t i o n r e q u i r e s c l o s e a s s o c i a t i o n with stromal c e l l s . Blood 70:1418, 1987. 3. Hunt P, Robertson D, Weiss D, Rennick D, Lee F, Witte ON. A s i n g l e bone marrow-derived stromal c e l l type supports i n v i t r o growth of e a r l y lymphoid and myeloid c e l l s . C e l l 48:997, 1987. 4. Whitlock CA, Tidmarsh GF, Muller-Sieburg C, Weissman IL. Bone marrow stromal c e l l l i n e s with lymphopoietic a c t i v i t y express high l e v e l s of a pre-B ne o p l a s i a - a s s o c i a t e d molecule. C e l l 48:1009, 1987. 5. Lemoine FM, Humphries RK, Abraham SDM, K r y s t a l G, Eaves CJ. P a r t i a l c h a r a c t e r i z a t i o n of a novel stromal c e l l - d e r i v e d pre-B c e l l growth f a c t o r a c t i v e on normal and immortalized pre-B c e l l s . J Exp Hematol 16:718, 1988. 6. W a t e r f i e l d MD, Scrace GT, W h i t t l e N, Stroobant P, Johnsson A, Wasteson A, Westermark B, Heldin C-H, Huang JS, Deuel TF. P l a t e l e t - d e r i v e d growth f a c t o r i s s t r u c t u r a l l y r e l a t e d to the p u t a t i v e transforming p r o t e i n p 2 8 s l s of simian sarcoma v i r u s . Nature 304:35, 1983. 7. Lang RA, Metcalf D, Gough NM, Dunn AR, Gonda TJ. Expression of a hemopoietic growth f a c t o r cDNA i n a factor-dependent c e l l l i n e r e s u l t s i n autonomous growth and t u m o r i g e n i c i t y . C e l l 43:531, 1985. 8. Young DC, G r i f f i n JD. Autocrine s e c r e t i o n of GM-CSF i n acute m y e l o b l a s t i c leukemia. Blood 68:1178, 1986. 9. Gordon I , Ley SC, Melamed MD, E n g l i s h LS, Hughes-Jones NC. Immortalized B lymphocytes produce B c e l l growth f a c t o r . Nature 310:145, 1984. 10. P i s t o i a V, Ghio R, Roncella S, Cozzolino F, Zupo S, F e r r a r i n i M. Production of c o l o n y - s t i m u l a t i n g a c t i v i t y by normal and n e o p l a s t i c human B lymphocytes. Blood 69:1340, 1987. 11. Whitlock CA, Z i e g l e r SF, Witte ON. Progression of the transformed phenotype i n c l o n a l l i n e s of Abelson v i r u s - i n f e c t e d lymphocytes. Mol C e l l B i o l 3:596, 1983. 12. Takei F. Murine T lymphoma c e l l s express a novel membrane-associated antigen w i t h unique features. J Immunol 139:649, 1987. 13. Takei F. A novel d i f f e r e n t i a t i o n antigen on p r o l i f e r a t i n g murine thymocytes i d e n t i f i e d by a r a t monoclonal antibody. J Immunol 132:766, 1984. 115 14. Takei F. Two surface antigens expressed on p r o l i f e r a t i n g mouse T lymphocytes defined by rat monoclonal a n t i b o d i e s . J Immunol 130:2794, 1983. 15. Cooper MD, Mulvaney D, Coutinho A, Cazenave P-A. A novel c e l l s u rface molecule on e a r l y B-lineage c e l l s . Nature 321:616, 1986. 16. Landreth KS, Dorshkind K. Pre-B c e l l generation p o t e n t i a t e d by s o l u b l e f a c t o r s from a bone marrow stromal c e l l l i n e . J Immunol 140:845, 1988. 17. Wong PMC, Humphries RK, Chen TR, Eaves CJ. Evidence f o r a m u l t i - s t e p pathogenesis i n the generation of tumorigenic c e l l l i n e s from hemopoietic c o l o n i e s exposed to Abelson v i r u s i n v i t r o . Exp Hematol 15:280, 1987. 18. Wong PMC, Chung S-W, Raefsky E, Eaves CJ, Nienhuis AW. B l a s t c o l o n i e s c o n t a i n i n g hemopoietic progenitor c e l l s can give r i s e to Abelson v i r u s (A-MuLV)-transformed c e l l l i n e s . Exp Hematol 16:5, 1988. 19. Chung SW, Wong PMC, Shen-Ong G, R u s c e t t i S, I s h i z a k a T, Eaves CJ. Production of granulocyte-macrophage c o l o n y - s t i m u l a t i n g f a c t o r by Abelson v i r u s - i n d u c e d tumorigenic mast c e l l l i n e s . Blood 68:1074, 1986. 20. Humphries RK, Abraham S, K r y s t a l G, Lansdorp P, Lemoine F, Eaves CJ. A c t i v a t i o n of m u l t i p l e hemopoietic growth f a c t o r genes i n Abelson v i r u s transformed murine myeloid c e l l s . Exp Hematol, i n press. 21. Brown MA, P i e r c e JH, Watson CJ, Falco J , I h l e JN, Paul WE. B c e l l s t i m u l a t o r y f a c t o r - l / I n t e r l e u k i n - 4 mRNA i s expressed by normal and transformed mast c e l l s . C e l l 50:809, 1987. 22. Freeman G, Freedman A, Rabinowe S, S e g i l J , Nadler LM. Expression of IL-6 (BCDF) and IL-4 (BSF-1) genes i n normal a c t i v a t e d and n e o p l a s t i c B c e l l s . Blood 70 (Suppl 1):257a, 1987. 23. Ymer S, Tucker W, Sanderson C, Hapel A, Campbell H, Young I . C o n s t i t u t i v e synthesis of i n t e r l e u k i n - 3 by leukaemia c e l l l i n e WEHI-3B i s due to r e t r o v i r a l i n s e r t i o n near the gene. Nature 317:255, 1985. 24. Oster W, Lindemann A, Horn S, Mertelsmann R, Herrmann F. C o n s t i t u t i v e expression of genes f o r hematopoietic growth f a c t o r s i n acute m y e l o b l a s t i c leukemia. Blood 70 (Suppl 1):266a, 1987. 116 C H A P T E R V ROLE OF FIBRONECTIN IN REGULATING PRE-B CELL PROLIFERATION 1) INTRODUCTION A number of f i n d i n g s suggest that the r e g u l a t i o n of pre-B c e l l p r o l i f e r a t i o n and d i f f e r e n t i a t i o n by stromal c e l l s may i n v o l v e more complex mechanisms than simple growth f a c t o r production, A mechanism i n v o l v i n g d i r e c t c e l l u l a r i n t e r a c t i o n s was suggested by the f a c t that pre-B c e l l d i f f e r e n t i a t i o n r e q u i r e s close a s s o c i a t i o n with stromal c e l l s ( 1 ) . The p r o l i f e r a t i o n a l s o of pre-B c e l l s , as shown i n Chapters I I I (2) and IV ( 3 ) , i s not s t i m u l a t e d as w e l l with stromal c e l l CM or by stromal c e l l s a c t i n g at a d i s t a n c e as when d i r e c t contact i s e s t a b l i s h e d . Although very l i t t l e i s known about the types of i n t e r a c t i o n s that may occur between pre-B c e l l s and stromal c e l l s and t h e i r ECM p r o t e i n s , Bernardi et a l (4) using an i n v i t r o adhesion assay r e c e n t l y showed that precursor lymphoid c e l l l i n e s blocked at s p e c i f i c stages of d i f f e r e n t i a t i o n a ttach s p e c i f i c a l l y to FN. Whether FN plays a r o l e i n f a c i l i t a t i n g stromal cell-mediated support of normal pre-B c e l l p r o l i f e r a t i o n was not i n v e s t i g a t e d . The present s t u d i e s were ther e f o r e i n i t i a t e d to address t h i s question. Because most transformed pre-B c e l l l i n e s become capable of stromal-independent growth i n v i t r o , and a number of c l o s e l y r e l a t e d cloned normal and d e r i v a t i v e transformed pre-B c e l l l i n e s had already been generated i n t h i s study (see Chapters I I I and I V ) , p a r a l l e l experiments 117 were undertaken with both to look f o r changes that might be r e l a t e d to the transformation process. 2) RESULTS A) D i f f e r e n t i a l Attachment of Normal and Malignant Pre-B C e l l Lines to FN The a b i l i t y of normal pre-B c e l l s to adhere to various defined ECM components was tested under serum free c o n d i t i o n s using the cloned, s t r o m a l -dependent l i n e s described i n Table 10. Figure 14 shows that normal pre-B c e l l s a t t a c h to FN i n a dose-dependent manner, and under the c o n d i t i o n s used a plateau i s reached at a FN concentration of 10 yg/ml. In c o n t r a s t , i n the same experiments these normal pre-B c e l l s d i d not adhere to VN, c o l l a g e n type I (COL I ) (data not shown), or LM even at high p r o t e i n concentrations (up to 20 ug/ml). In order to determine which binding s i t e s of the FN molecule i n t e r a c t w i t h pre-B c e l l s , microwells were coated with a f i x e d c oncentration of FN (5 yg/ml) and then pre-B c e l l s tested f o r t h e i r c a p a c i t y to adhere i n the presence of i n c r e a s i n g concentrations of the peptide GRGDSP (a sequence that recognizes the c e l l b inding s i t e of FN). Figure 15 shows that the attachment of pre-B c e l l s to FN can be i n h i b i t e d i n a dose dependent manner by GRGDSP. The GRADSP peptide, which serves as a negative c o n t r o l , has no e f f e c t on the bin d i n g of pre-B c e l l s to FN. In a next s e r i e s of experiments, :an A-MuLV transformed d e r i v a t i v e stromal-independent pre-B c e l l l i n e (Table 10, Chapter IV) and a spontaneously transformed pre-B c e l l l i n e (H9) with s i m i l a r p r o p e r t i e s (see Chapters I I I and 118 Figure 14. Attachment of normal pre-B c e l l s to FN. — - -# : Normal pre-B c e l l s (Bp) added to FN pre-coated w e l l s ^ : Mo-MuLV pre-B c e l l s (MoBp) added to FN pre-coated w e l l s (These c e l l s , l i k e the normal pre-B c e l l s , r e q u i r e the presence of stromal c e l l s f o r growth, see Chapter IV.) . , . O " • : Normal pre-B c e l l s (Bp) added to LM pre-coated w e l l s 25 -Figure 15. E f f e c t of s y n t h e t i c peptides on the attachment of normal pre-B c e l l s (Bp) to FN. GRGDSP: 1 mg = 1.7 y mole GRADSP: 1 mg = 1.42 y mole _ : Normal pre-B c e l l s (Bp) + GRGDSP s y n t h e t i c peptide : Normal pre-B c e l l s (Bp) + GRADSP s y n t h e t i c peptide used as negative c o n t r o l 120 IV) were tested i n the same way. As shown i n Figure 16 even at 20 ug/ml of FN, which i s twice the concentration required f o r maximum attachment of the normal pre-B c e l l s , both transformed pre-B c e l l l i n e s d i d not adhere to FN. B) E f f e c t of FN on the P r o l i f e r a t i o n of Normal and Transformed Pre-B  C e l l s i n the Presence of Stromal CM To t e s t the r o l e of FN i n s t i m u l a t i n g pre-B c e l l p r o l i f e r a t i o n , pre-B c e l l s were put i n t o w e l l s that had been pre-coated with FN and thymidine uptake was then measured 3 days l a t e r . The r e s u l t s of one such experiment are shown i n Table 11. FN by i t s e l f was unable to support the p r o l i f e r a t i o n of normal pre-B c e l l s . However, when M2-10B4 CM was added to FN-coated w e l l s , the p r o l i f e r a t i o n of normal pre-B c e l l s was s l i g h t l y , although s i g n i f i c a n t l y enhanced compared to the e f f e c t of M2-10B4 CM alone (p<0.05, by t - t e s t ) . This suggests that the s t i m u l a t i o n of normal pre-B c e l l s by the pre-B s t i m u l a t i n g f a c t o r known to be present i n M2-10B4 CM can be synergized by FN. Transformed pre-B c e l l s were a l s o tested i n t h i s type of experiment (Table 12). As p r e v i o u s l y shown i n Figure 10 (Chapter I V ) , M2-10B4 has a detectable s t i m u l a t o r y e f f e c t on transformed pre-B c e l l s when these are c u l t u r e d at low c e l l d e n s i t y . However, t h i s was not f u r t h e r s i g n i f i c a n t l y enhanced by FN (p>0.05, by t - t e s t ) . The fa c t that the p r o l i f e r a t i o n of the transformed pre-B c e l l s was the same on p l a s t i c as on FN pre-coated w e l l s a l s o shows that the FN prepa r a t i o n d i d not contain any n o n - s p e c i f i c i n h i b i t o r s . 121 20-eg i 16-X E f 12-o 8-4 -[FNj^g/ml Figure 16. Attachment of transformed pre-B c e l l s to FN. : A-MuLV transformed pre-B c e l l s (ABp) added to FN pre-coated w e l l s : H9 (spontaneously transformed pre-B c e l l s ) added to FN pre-coated w e l l s (These c e l l s do not re q u i r e the presence of stromal c e l l s f o r growth when c u l t u r e d at concentrations >2 x 10 4 c e l l s / m l , see Figure 6 and Chapter IV.) : Attachment of normal (Bp) pre-B c e l l s (data from Figure 14) 122 Table 11. E f f e c t of FN on normal pre-B c e l l p r o l i f e r a t i o n induced by stromal pre-B s t i m u l a t i n g a c t i v i t y (M2-10B4 CM). C e l l P r o l i f e r a t i o n (cpm) a Conditions Bp L+VII A8 b (20,000 c e l l s / m l ) (50,000 c e l l s / m l ) P l a s t i c 0 FN M2-10B4 CM FN + M2-10B4 CM 103 + 17 79 + 9 912 + 7 1,174 + 63 179 + 27 169 + 8 1,489 + 50 2,064 + 67 a R e s u l t s represent the mean cpm + SEM from t r i p l i c a t e assays D L + V I I A8 c e l l s are c u + s l g M - pre-B c e l l s derived from a lymphoid LTC. They can be maintained e i t h e r i n the presence of M2-10B4 c e l l s or w i t h 50% M2-10B4 CM alone. In contrast to the usual pre-B assays (e.g. Bp c e l l s ) where -^-thymidine i n c o r p o r a t i o n was measured a f t e r 3 days of c u l t u r e , the L + V I I A8 c e l l assay was terminated a f t e r 1 day of c u l t u r e . °The d i f f e r e n c e i n cpm between c u l t u r e s on p l a s t i c and FN was not s i g n i f i c a n t (p>0.05 by t - t e s t ) . The d i f f e r e n c e i n cpm between c u l t u r e s w i t h M2-10B4 CM and M2-10B4 CM + FN was s i g n i f i c a n t (p<0.05 by t - t e s t ) . 123 Table 12. E f f e c t of FN on transformed pre-B c e l l p r o l i f e r a t i o n induced by stromal pre-B s t i m u l a t i n g a c t i v i t y (M2-10B4 CM). C e l l P r o l i f e r a t i o n (cpm) a Conditions H9 ABp 3,000 c e l l s / m l 20,000 c e l l s / m l 20,000 c e l l s / m l P l a s t i c 5 131 +4 915 + 55 21,920 + 126 FN 118 + 2 1,012 + 32 24,264 + 719 M2-10B4 CM 189 +24 2,106 + 38 24,330 + 108 FN + M2-10B4 CM 235 + 10 2,225 + 62 24,319 + 22 a R e s u l t s represent the mean cpm + SEM from t r i p l i c a t e assays. C e l l p r o l i f e r a t i o n was measured by ^H-thymidine i n c o r p o r a t i o n as described i n Chapter I I . D N e i t h e r the d i f f e r e n c e i n cpm between c u l t u r e s on p l a s t i c and FN nor the d i f f e r e n c e i n cpm between c u l t u r e s with M2-10B4 CM and M2-10B4 CM + FN was s i g n i f i c a n t (p>0.05 by t - t e s t ) . 124 C) D i f f e r e n t i a l E f f e c t of FN-R Antibodies on the P r o l i f e r a t i o n of Normal  and Transformed Pre-B C e l l s The above r e s u l t s suggested that the process of transformation l e a d i n g to a stromal-independent phenotype may be commonly associated with an impairment of the a b i l i t y of normal pre-B c e l l s to bind to FN and hence a reduced a b i l i t y to be s t i m u l a t e d by stromal c e l l s . To t e s t t h i s f u r t h e r , the e f f e c t of a n t i -FN-R a n t i b o d i e s on normal and transformed pre-B c e l l p r o l i f e r a t i o n was i n v e s t i g a t e d . As shown i n Figure 17 anti-FN-R an t i b o d i e s were able to i n h i b i t up to 70% of the thymidine incorporated by normal pre-B c e l l s , w h i l e anti-VN-R a n t i b o d i e s (used as a negative c o n t r o l ) had no e f f e c t . In c o n t r a s t , the p r o l i f e r a t i o n of two d i f f e r e n t transformed pre-B c e l l l i n e s was l e s s i n h i b i t e d by the same FN-R-antibodies, by a f a c t o r of two. 3) DISCUSSION FN, l i k e VN, i s w e l l known f o r i t s strong adhesive c a p a b i l i t i e s and has been g e n e r a l l y assumed to play a r o l e i n c e l l - c e l l i n t e r a c t i o n s . The recent demonstration that FN can r e s t o r e the d e f e c t i v e a l l o - a n t i g e n induced p r o l i f e r a t i o n of lymphocytes from marrow transplant p a t i e n t s l e d to the suggestion that FN may enhance t h e i r binding to accessory c e l l s ( 5 ) . Subsequently the FN-R was demonstrated on the surface of normal p e r i p h e r a l blood lymphocytes (6) and monocytes (7,8). FN may a l s o play a r o l e i n the r e g u l a t i o n of hemopoietic c e l l p r o l i f e r a t i o n . This i s suggested by the recent demonstration that hemopoietic progenitors adhere s p e c i f i c a l l y to FN (9) and by the f a c t that optimal growth of hemopoietic progenitors i n myeloid LTC r e q u i r e s t h e i r d i r e c t i n t e r a c t i o n with stromal c e l l s (10) which l i k e 3T3 c e l l s A N T I B O D Y C O N C E N T R A T I O N ( * j / m l , A N T I B O D Y C O N C E N T R A T I O N (j-Q/ml) Figure 17. Panel a. A Panel a Panel b E f f e c t of FN-R and VN-R antibodies on the p r o l i f e r a t i o n of normal pre-B c e l l s . : Normal pre-B c e l l s (Bp) + FN-R antibodies : Normal pre-B c e l l s (Bp) + VN-R antibodies Panel b. Role of FN-R and VN-R antibodies on the p r o l i f e r a t i o n of transformed pre-B c e l l s . A-MuLV transformed pre-B c e l l s + FN-R antibodies H9 spontaneously transformed pre-B c e l l s + FN-R anti b o d i e s Transformed pre-B c e l l s + VN-R antibodies : Normal pre-B c e l l s (Bp) + FN-R antibodies (see panel a) Antibodies were mixed at in c r e a s i n g concentrations with e i t h e r normal or transformed pre-B c e l l s (Bp and ABp - 2000 c e l l s / w e l l , H9 - 300 c e l l s / w e l l ) which were subsequently p l a t e d on a pre-established M2-10B4 stromal c e l l layer (6000 c e l l s / w e l l ) . Then, thymidine uptake was measured a f t e r 3 days of c u l t u r e as described i n Chapter I I . 126 produce FN (11). In t h i s study, we focussed on the i n t e r a c t i o n of pre-B c e l l s w i t h FN and obtained evidence that FN plays a s i g n i f i c a n t r o l e i n enhancing t h e i r normal a b i l i t y to be stimulated by i n t e r a c t i o n s between stromal c e l l s . Normal pre-B c e l l s were shown to attach s p e c i f i c a l l y to FN, and although n e i t h e r FN alone (Table 11), nor m e t a b o l i c a l l y i n a c t i v e ( f i x e d - ) stromal c e l l s (data not shown) could support pre-B c e l l p r o l i f e r a t i o n , FN was able to enhance the p r o l i f e r a t i v e response of normal pre-B c e l l s to f a c t o r s present i n stromal c e l l CM. In contrast two independently i s o l a t e d transformed pre-B c e l l l i n e s transformed by d i f f e r e n t mechanisms, showed the same impaired a b i l i t y to a t t a c h to FN. In a d d i t i o n , the p r o l i f e r a t i o n of both of these transformed l i n e s had become i n s e n s i t i v e to the s y n e r g i s t i c s t i m u l a t i n g e f f e c t s of FN. This l a t t e r f i n d i n g i n d i c a t e s that pre-B c e l l transformation i s commonly as s o c i a t e d w i t h a change i n the i n t e r a c t i o n s between pre-B c e l l s and stromal c e l l s that are normally mediated by FN. Whether t h i s c o n t r i b u t e s to, or r e s u l t s from, the malignant, stromal-independent phenotype c h a r a c t e r i s t i c of these c e l l s i s not yet c l e a r . Changes i n adhesive p r o p e r t i e s i s common to many transformed c e l l types, although l i t t l e i s known i n t h i s area with regard to hemopoietic c e l l s . Recently, however, a l t e r a t i o n s i n the i n t e r a c t i o n of CML progenitors with marrow stromal c e l l s was reported (12). To date, very l i t t l e has been reported about the r o l e of FN i n B lymphopoiesis. Bernardi et a l has suggested that the attachment of transformed pre-B c e l l l i n e s to FN involves both the FN c e l l b i n d i n g s i t e and the heparin binding s i t e , whereas transformants a r r e s t e d at a more mature stage of B c e l l d i f f e r e n t i a t i o n l a c k the a b i l i t y to a t t a c h to the c e l l - b i n d i n g s i t e ( 4 ) . St. John et a l a l s o r e c e n t l y reported ( i n an a b s t r a c t , 13) that lymphoid c e l l l i n e s adhere to the heparin-binding domain of FN. At f i r s t 127 glance, these r e s u l t s appear to d i f f e r from those described here, where transformation of pre-B c e l l s r e s u l t e d i n a l o s s of t h e i r a b i l i t y to_adhere_to FN. However, i t i s important to note that serum was present i n the assays performed by Bernardi et a l (4) whereas those presented i n Figure 16 were performed i n the absence of serum. Serum contains s e v e r a l ECM components and i t s presence therefore complicates i n t e r p r e t a t i o n of the f i n d i n g s . For example, i t i s p o s s i b l e that the attachment of transformed pre-B c e l l s to the heparin domain of FN might be undetectable under serum-free c o n d i t i o n s . I n t e r e s t i n g l y , a l l three s t u d i e s are c o n s i s t e n t with the idea that transformed pre-B c e l l s do not a t t a c h to the c e l l - b i n d i n g domain of FN, although t h i s does appear to be a property of normal pre-B c e l l s . The present s t u d i e s provide the f i r s t report of a f u n c t i o n a l consequence of FN b i n d i n g to normal pre-B c e l l s . This was seen as an a b i l i t y to enhance the p r o l i f e r a t i o n obtained e i t h e r i n the presence of stromal c e l l s or stromal c e l l CM. Whether FN simply plays a mechanical r o l e by improving the contact between growth f a c t o r producing stromal c e l l s and the pre-B target c e l l s , or whether FN concentrates or s t a b i l i z e s the growth f a c t o r ( s ) they produce remains to be determined. I t i s a l s o p o s s i b l e that FN binding a l t e r s the responsiveness of the pre-B c e l l r ather than p l a y i n g a r o l e p r i m a r i l y by i n c r e a s i n g the concentration of growth f a c t o r the c e l l i s exposed to. Further a n a l y s i s of both normal and transformed pre-B c e l l s should throw f u r t h e r l i g h t on t h i s problem and s i m i l a r l y help to e s t a b l i s h the s i g n i f i c a n c e of the a l t e r e d a b i l i t y of transformed c e l l s to respond to, or bind to, FN. 128 REFERENCES I-. Kierney PC, Dorshkind K. B lymphocyte precursors and myeloid p r o g e n i t o r s — s u r v i v e i n d i f f u s i o n chamber c u l t u r e s but B c e l l d i f f e r e n t i a t i o n r e q u i r e s c l o s e a s s o c i a t i o n with stromal c e l l s . Blood 70:1418, 1987. 2. Lemoine FM, Humphries RK, Abraham SDM, K r y s t a l G, Eaves CJ. P a r t i a l c h a r a c t e r i z a t i o n of a novel stromal c e l l - d e r i v e d pre-B c e l l growth f a c t o r a c t i v e on normal and immortalized pre-B c e l l s . J Exp Hematol 16:718, 1988. 3. Lemoine FM, K r y s t a l G, Humphries RK & Eaves CJ. Autocrine production of pre-B c e l l s t i m u l a t i n g a c t i v i t y by a v a r i e t y of transformed pre-B c e l l l i n e s . Cancer Res, i n press. 4. Bernardi P, P a t e l VP, Lodish HF. Lymphoid precursor c e l l s adhere to two d i f f e r e n t s i t e s on f i b r o n e c t i n . J C e l l B i o l 105:489, 1987. 5. Klingemann H-G, Tsoi M-S, Storb R. F i b r o n e c t i n r e s t o r e s d e f e c t i v e i n v i t r o p r o l i f e r a t i o n of lymphocytes of p a t i e n t s a f t e r marrow g r a f t i n g . T r a n s p l a n t a t i o n 42:412, 1986. 6. Klingemann H-G, Dedhar S, Kohn FR, P h i l l i p s GL. F i b r o n e c t i n increases lymphocyte p r o l i f e r a t i o n by mediating adhesion between immunoreactive c e l l s . J C e l l Biochem (Suppl 12E):174, 1988. 7. Hosein B, Bianco C. Monocyte receptors f o r f i b r o n e c t i n c h a r a c t e r i z e d by a monoclonal antibody that i n t e r f e r e s with receptor a c t i v i t y . J Exp Med 162:152, 1985. 8. Wright SD, Meyer BC. F i b r o n e c t i n receptor of human macrophages recognizes the sequence Arg-Gly-Asp-Ser. J Exp Med 162:762, 1985. 9. G i a n c o t t i FG, Comoglio Pm, Tarone G. Fibronectin-plasma membrane i n t e r a c t i o n i n the adhesion of haemopoietic c e l l s . J C e l l B i o l 103:429, 1086. 10. Dexter TM, Spooncer E, Toksoz D, L a j t h a LG. The r o l e of c e l l s and t h e i r products i n the r e g u l a t i o n of i n v i t r o stem c e l l p r o l i f e r a t i o n and granulocyte development. J Supramolec Structure 13:513, 1980. 11. Z i p o r i D, Toledo J , von der Mark K. Phenotypic heterogeneity among stromal c e l l l i n e s from mouse bone marrow d i s c l o s e d i n t h e i r e x t r a c e l l u l a r matrix composition and i n t e r a c t i o n s w i t h normal and leukemic c e l l s . Blood 66:447, 1985. 12. Gordon MY, Dowding CR, R i l e y GP, Goldman JM, Greaves MF. A l t e r e d adhesive i n t e r a c t i o n s with marrow stroma of haematopoietic progenitor c e l l s i n chronic myeloid leukaemia. Nature 328:342, 1987. 13. St. John J , Applegreen RA, Liao N-S, Cheung HT. Adherence of lymphoid c e l l l i n e s to the carboxy-terminal heparin binding domain of f i b r o n e c t i n . J C e l l B i o l 105:45a, 1987. 129 C H A P T E R V I SUMMARY AND CONCLUSIONS 1) REGULATION OF PRE-B CELL PROLIFERATION BY STROMAL CELLS Both i n v i v o and i n v i t r o f i n d i n g s have suggested that the p r o l i f e r a t i o n of many types of hemopoietic c e l l s i n c l u d i n g pre-B c e l l s i s regulated by t h e i r i n t e r a c t i o n s w i t h marrow stromal c e l l s . I t i s c l e a r that stromal c e l l s can be a c t i v a t e d to secrete a number of w e l l c h a r a c t e r i z e d s o l u b l e growth f a c t o r s that act e i t h e r on myeloid or a c t i v a t e d mature B c e l l s (Tables 1 and 2). The molecular mechanisms by which stromal c e l l s may regulate B c e l l production from t h e i r precursors i n the marrow are now a l s o beginning to be defi n e d . The lymphoid LTC was chosen as a basis f o r i n i t i a t i n g t h i s i n v e s t i g a t i o n of stromal-mediated c o n t r o l of pre-B c e l l s because i t o f f e r e d the p o t e n t i a l of d e l i n e a t i n g mechanisms thought to be relevant i n v i v o and a f f e c t e d by malignant transformation. However, because lymphoid LTC's are composed of both an adherent heterogenous stromal c e l l l a y e r and a non-adherent lymphoid c e l l f r a c t i o n i n c l u d i n g c e l l s at d i f f e r e n t stages of pre-B c e l l d i f f e r e n t i a t i o n , i t seemed that a c r i t i c a l f i r s t step to f u r t h e r a n a l y s i s would be to i s o l a t e c l o n a l populations corresponding to each of the two types of c e l l populations. Stromal c e l l l i n e s were therefore screened f o r t h e i r a b i l i t y to support the growth of pre-B c e l l s derived from the non-adherent f r a c t i o n of lymphoid LTC's and then cloned. Permanent stromal cell-dependent l i n e s of pre-B c e l l s were a l s o i s o l a t e d . From these, cloned l i n e s of 130 spontaneous or A-MuLV induced transformants were derived. The a v a i l a b i l i t y of these l i n e s then made p o s s i b l e a s e r i e s of experiments to analyze the nature of the i n t e r a c t i o n s that occur between pre-B c e l l s and stromal c e l l s , and how they might be a f f e c t e d by d i f f e r e n t mechanisms of pre-B c e l l transformation. A) Factors One of the stromal c e l l l i n e s (M2-10B4), was shown to c o n s t i t u t i v e l y secrete a s o l u b l e f a c t o r able to s t i m u l a t e both normal pre-B c e l l s as w e l l as a spontaneously transformed but s t i l l stromal c e l l - r e s p o n s i v e pre-B c e l l l i n e (H9). Further c h a r a c t e r i z a t i o n of the H9 c e l l s t i m u l a t i n g a c t i v i t y produced by M2-10B4 c e l l s i n d i c a t e d that i t represents a p r e v i o u s l y undescribed HGF w i t h an apparent molecular weight of 10,000 daltons. Very r e c e n t l y s e v e r a l f a c t o r s produced by d i f f e r e n t stromal c e l l l i n e s and a c t i n g on B c e l l precursors have been i d e n t i f i e d i n d i f f e r e n t l a b o r a t o r i e s (1-4). We a l s o found that a pre-B c e l l s t i m u l a t i n g stromal c e l l l i n e derived from the spleen (S5-2, described i n Chapter I I I ) secretes a pre-B s t i m u l a t i n g f a c t o r that appears d i f f e r e n t (-30,000 daltons) from that produced by M2-10B4 c e l l s (data not shown). Thus, i t i s p o s s i b l e that d i f f e r e n t stromal c e l l s secrete d i f f e r e n t s t i m u l a t i n g and/or d i f f e r e n t i a t i n g pre-B f a c t o r s which might act on the same or d i f f e r e n t l e v e l s of B c e l l development. On the other hand, some of these f a c t o r s may represent d i f f e r e n t l y g l y c o s y l a t e d versions of the same gene product. Therefore, the p u r i f i c a t i o n and/or the c l o n i n g of these f a c t o r s w i l l be of great importance to evaluate t h e i r degree of overlap and f u n c t i o n a l s p e c i f i c i t i e s i n v i t r o and i n v i v o . 131 B) C e l l - C e l l I n t e r a c t i o n s When pre-B c e l l s were stimulated by stromal c e l l s through an i n t e r l a y e r of agar, or by stromal c e l l conditioned medium (CM), the extent of p r o l i f e r a t i o n was never as good as when pre-B c e l l s were plat e d d i r e c t l y on stromal c e l l s . This suggests that c e l l contact plays a r o l e i n i n c r e a s i n g the s t i m u l a t i o n of pre-B c e l l growth by stromal c e l l s . Since other pre-B c e l l l i n e s had been shown to bind s p e c i f i c a l l y to FN, and other stromal c e l l s , l i k e 3T3 c e l l s make FN ( 5 ) , FN was considered as a p o s s i b l e binding intermediate between pre-B c e l l s and stromal c e l l s . The stu d i e s performed here showed that FN not only adheres s p e c i f i c a l l y to normal pre-B c e l l s , i t a l s o s t i m u l a t e s t h e i r p r o l i f e r a t i o n i n concert with a stromal pre-B f a c t o r . This increased p r o l i f e r a t i o n could be explained by one or more of a number of p o s s i b i l i t i e s . B i n d i n g could permit a higher l o c a l exposure of pre-B c e l l s to stromal derived growth f a c t o r s by f i x i n g them i n c l o s e r proximity to the producer c e l l s . A l t e r n a t i v e l y , FN might i t s e l f bind and hence concentrate pre-B growth f a c t o r ( s ) and protect them from enzymatic degradation, as r e c e n t l y was suggested f o r heparan s u l f a t e with respect to IL-3 and GM-CSF (6 ) . Further experiments are required to determine whether FN acts alone or j o i n t l y w i t h other ECM components, such as heparan s u l f a t e . In t h i s regard, i t i s i n t e r e s t i n g to note that FN possesses a heparin binding s i t e . I t i s a l s o p o s s i b l e that FN can serve as a second s y n e r g i z i n g s i g n a l , or may cause c e l l s u r face or conformational changes i n pre-B c e l l s that f a c i l i t a t e s t h e i r response to growth f a c t o r s . The a v a i l a b i l i t y of a simple model i n which the s t i m u l a t i n g (stromal c e l l ) and responding (pre-B c e l l ) components can be r e a d i l y obtained as separate homogeneous cloned populations should be u s e f u l f o r f u r t h e r a n a l y s i s of the p r e c i s e r o l e played by FN. Such s t u d i e s may 132 provide important i n s i g h t s not only i n t o the r e g u l a t i n g i n t e r a c t i o n s of growth f a c t o r s and ECM f o r pre-B c e l l s , but a l s o f o r other c e l l types. 2) ALTERED MECHANISMS IN TRANSFORMED PRE-B CELLS Recent observations of autocrine hemopoietic growth f a c t o r production i n human AML (7) and some lymphoid malignancies (8,9), plus the demonstrable a c t i v a t i o n of growth f a c t o r gene expression i n a v a r i e t y of transformed hemopoietic c e l l s (10,11) has focussed i n t e r e s t on the p o s s i b l e relevance of such changes to the a c q u i s i t i o n of autonomous growth p o t e n t i a l by transformed B-lineage c e l l s . The present s t u d i e s have y i e l d e d data that bear on t h i s . Both spontaneous and A-MuLV transformation of pre-B c e l l s were found to lead to the a c q u i s i t i o n of autocrine growth f a c t o r production. S i m i l a r pre-B a u t o c r i n e a c t i v i t y has been a l s o found i n A-MuLV transformed mast c e l l l i n e s i n which m u l t i p l e hemopoietic growth f a c t o r genes were a c t i v a t e d f o l l o w i n g A-MuLV i n f e c t i o n (11). Thus, i t i s p o s s i b l e that expression of v-abl can deregulate the expression of s e v e r a l d i f f e r e n t hemopoietic growth f a c t o r genes according to the type of c e l l s i n i t i a l l y i n f e c t e d . I t i s i n t e r e s t i n g that the autocrine f a c t o r produced by the spontaneous transformed H9 c e l l l i n e can a l s o s t i m u l a t e normal pre-B c e l l s . This suggests a p h y s i o l o g i c r o l e f o r t h i s f a c t o r . One might speculate as to whether normal pre-B c e l l s , which can p r o l i f e r a t e i n v i t r o under some circumstances i n the presence of stromal CM ( 1 ) , may do so because they a l s o are programmed to produce low l e v e l s of t h i s f a c t o r . D i f f e r e n c e s i n the apparent molecular weight between a l l of the described pre-B c e l l s t i m u l a t i n g f a c t o r s and the H9 133 a u t o c r i n e f a c t o r suggest that these are d i f f e r e n t ; however, as mentioned above sequence data w i l l be required before t h i s issue can be c o n c l u s i v e l y r e s o l v e d . The f a c t that the a b i l i t y of normal pre-B c e l l s to adhere to FN i s g r e a t l y decreased a f t e r transformation can be compared to the anchorage-independence observed i n transformed f i b r o b l a s t s (12). In the l a t t e r c e l l s , t ransformation has been shown to cause an a l t e r a t i o n of the FN-R expression. Whether such an a l t e r a t i o n precedes or f o l l o w s the a c q u i s i t i o n of a u t o c r i n e s e c r e t i o n during the oncogenic progression of transformed pre-B c e l l s remains to be answered. In f a c t , t h i s can be viewed as part of the l a r g e r question of whether there i s a r e l a t i o n s h i p between autocrine s e c r e t i o n , a l t e r a t i o n of FN-R and the expression of oncogenes that are known to be involved i n B-lineage malignancies (13-16). The myc fa m i l y of genes, i . e . c-myc, N-myc, L-myc are d i f f e r e n t i a l l y expressed during murine B c e l l development (13) and these as w e l l as c-myb have been found to be e i t h e r overexpressed or a m p l i f i e d i n both A-MuLV and spontaneously transformed B lymphoid c e l l l i n e s (13-15). I t has a l s o been shown that c o n s t i t u t i v e c-myc expression enhanced the anchorage-independent growth of an e s t a b l i s h e d mouse embryo l i n e s t i m u l a t e d w i t h PDGF, EGF and FGF as w e l l as TGF-f3 (17). 3) PROPOSED MODEL The information obtained from the r e s u l t s presented i n t h i s t h e s i s together with the f i n d i n g s of others suggest that the r e g u l a t i o n of normal and transformed pre-B c e l l growth by stromal c e l l s might be described by the f o l l o w i n g model. Stromal c e l l s c o n t r o l the p r o l i f e r a t i o n and the d i f f e r e n t i a t i o n of normal pre-B c e l l precursors by s e c r e t i n g d i f f e r e n t s o l u b l e pre-B f a c t o r s . D i r e c t contact between stromal c e l l s and pre-B c e l l s mediated 134 by FN can increase the response to secreted f a c t o r s and/or promote the p r o l i f e r a t i o n and d i f f e r e n t i a t i o n of pre-B c e l l s . Even normal pre-B c e l l s may have the cap a c i t y f o r producing some autocrine growth f a c t o r s although these are not detectable i n the supernatant of normal pre-B c e l l c u l t u r e s . In pre-B malignancies, one or s e v e r a l growth f a c t o r genes may become a c t i v a t e d so that the c e l l s become independent of stromal c e l l s i n order to p r o l i f e r a t e . This may be d r i v e n by e a r l i e r changes r e s u l t i n g i n the overexpression or a m p l i f i c a t i o n of c-myc which i n turn leads to an a l t e r a t i o n i n FN-R expression and l o s s of the normal a b i l i t y to i n t e r a c t with stromal c e l l s . This would place a strong s e l e c t i v e pressure on autocrine v a r i a n t s . A l t e r n a t i v e l y , a u t o c r i n e mechanisms may be a c t i v a t e d e a r l y on i n the transformation process but be i n s u f f i c i e n t f o r a f u l l y malignant phenotype, f o r example by f a i l i n g to block, stromal induced quiescence or d i f f e r e n t i a t i o n . The goal of t h i s t h e s i s was to obtain f u r t h e r information about mechanisms r e g u l a t i n g normal and transformed pre-B c e l l growth. Although some questions have been answered, many more have been r a i s e d . In p a r t i c u l a r i t i s hoped that the f i n d i n g s w i l l be u s e f u l as a basis f o r improving our knowledge of the mechanisms that are a l t e r e d i n human B-lineage malignancies. The s u c c e s s f u l development f o r human c e l l s of a lymphoid LTC system analogous to that a v a i l a b l e f o r murine c e l l s would be an important f i r s t step i n that d i r e c t i o n . 135 REFERENCES 1. Hunt P, Robertson D, Weiss D, Rennick D, Lee F, Witte ON. A s i n g l e bone marrow-derived stromal c e l l type supports i n v i t r o growth of e a r l y lymphoid and myeloid c e l l s . C e l l 48:997, 1987. 2. Landreth KS, Dorshkind K. Pre-B c e l l generation po t e n t i a t e d by s o l u b l e f a c t o r s from a bone marrow stromal c e l l l i n e . J Immunol 140:845, 1988. 3. Namen AE, Schmierer AE, March CJ, O v e r e l l RW, Park LS, Urdal DL, Mochizuki DY. B c e l l precursor growth-promoting a c t i v i t y . J Exp Med 167:988, 1988. 4. Lemoine FM, Humphries RK, Abraham SDM, K r y s t a l G, Eaves CJ. P a r t i a l c h a r a c t e r i z a t i o n of a novel stromal c e l l - d e r i v e d pre-B c e l l growth f a c t o r a c t i v e on normal and immortalized pre-B c e l l s . J Exp Hematol 16:718, 1988. 5. Z i p o r i D, Toledo J , von der Mark K. Phenotypic heterogeneity among stromal c e l l l i n e s from mouse bone marrow d i s c l o s e d i n t h e i r e x t r a c e l l u l a r matrix composition and i n t e r a c t i o n s with normal and leukemic c e l l s . Blood 66: 447, 1985. 6. Roberts R, Gallagher J , Spooncer E, A l l e n TD, Bloomfield F, Dexter TM. Heparan sulphate bound growth f a c t o r s : A mechanism f o r stromal c e l l mediated haemopoiesis. Nature 332:376, 1988. 7. Young DC, G r i f f i n JD. Autocrine s e c r e t i o n of GM-CSF i n acute m y e l o b l a s t i c leukemia. Blood 68:1178, 1986. 8. Gordon I , Ley SC, Melamed MD, E n g l i s h LS, Hughes-Jones NC. Immortalized B lymphocytes produce B c e l l growth f a c t o r . Nature 310:145, 1984. 9. P i s t o i a V, Ghio R, Roncella S, Cozzolino F, Zupo S, F e r r a r i n i M. Production of c o l o n y - s t i m u l a t i n g a c t i v i t y by normal and n e o p l a s t i c human B lymphocytes. Blood 69:1340, 1987. 10. Freeman G, Freedman A, Rabinowe S, S e g i l J , Nadler LM. Expression of IL-6 (BCDF) and IL-4 (BSF-1) genes i n normal a c t i v a t e d and n e o p l a s t i c B c e l l s . Blood 70 (Suppl 1):257a, 1987. 11. Humphries RK, Abraham S, K r y s t a l G, Lansdorp P, Lemoine F, Eaves CJ. A c t i v a t i o n of m u l t i p l e hemopoietic growth f a c t o r genes i n Abelson v i r u s transformed murine myeloid c e l l s . Exp Hematol, i n press. 12. Shin SI, Freedman UH, R i s s e r R, P o l l a c k R. Tumorigenicity of v i r u s -transformed c e l l s i n nude mice i s c o r r e l a t e d s p e c i f i c a l l y with anchorage independent growth i n v i t r o . Proc N a t l Acad S c i USA 72:4435, 1975. 13. Zimmerman KA, Yancopoulos GD, Collum RG, Smith RK, Kohl NE, Denis KA, Nau MM, Witte ON, Toran-Allerand D, Gee CE, Minna JD, A l t FW. D i f f e r e n t i a l expression of myc family genes during murine development. Nature 319:780, 1986. 136 14. C i t r i Y, Braun J , Baltimore D. Elevated myc expression and c-myc a m p l i f i c a t i o n i n spontaneously o c c u r r i n g B lymphoid c e l l l i n e s . J Exp Med 165:1188, 1987. 15. Bender TP, Kuehl WM. D i f f e r e n t i a l expression of the c-myb proto-oncogene marks the pre-B c e l l / B c e l l j u n c t i o n i n murine B lymphoid tumors. J Immunol 139:3822, 1987. 16. Cheah MSC, Ley TJ, Tronick SR, Robbins KC. f g r proto-oncogene mRNA induced i n B lymphocytes by Epstein-Barr v i r u s i n f e c t i o n . Nature 319:238, 1986. 17. Sorrentino V, Drozdoff V, McKinney MD, Z e i t z L, F l e i s s n e r E. P o t e n t i a t i o n of growth f a c t o r a c t i v i t y by exogenous c-myc expression. Proc N a t l Acad S c i USA 83:8167, 1986. PUBLICATIONS Francois M. Lemoine 1. Lemoine F & Dao C. Diagnosis of purpuras. Rev Med P a r i s 22: 2449-2457, 1981. 2. Metman EH, Danquechin-Dorval E, Lemoine F & Bertrand J . F i b e r endoscopy and treatment of duodenal u l c e r . Rev Med Tours 16: 217-218, 1982. 3. Lemoine F & Dao C. Les purpuras thrombopeniques, l e s thrombopathies, thrombocytemies et thrombocytoses. Encycl Med C h i r P a r i s CP Hematologic 4.6.10: 2399, 1982. 4. Khayat D, Ricome JL, Richard C, Lemoine F, Rimailho A & Auzepy P. I n t o x i c a t i o n aique aux b a r b i t u r i q u e s l e n t s et incompetence cardiaque t r a n s i t o i r e . Nouv Presse Med 45: 3354, 1982. 5. Lemoine F, Benatre A, Metman EH, D e l s o l AL, Lhuintre JP, Danquechin-Dorval E & B r i s o n J . G a s t r i t e granulomateuse r e v e l a t r i c e d'une v a s c u l a r i t y granulomateuse d i g e s t i v e . G a s t r o e n t e r o l C l i n B i o l 7: 546-548, 1983. 6. Richard C, Ricome JL, Lemoine F, Rimailho A, Lantz 0 & Auzepy P. I n t e r e t du C a p t o p r i l dans l e traitement d'une hypertension a r t e r i e l l e systemique et pulmonaire avec a c t i v i t e renine plasmatique augmentee au cours d'une sclerodermic. Rev Med Int 4: 125-129, 1983. 7. Lemoine F, Najman A, Laporte JP, Gorin NC & Duhamel G. Vindesine-Prednisone i n the treatment of b l a s t c r i s i s of chronic myeloid leukemia. Cancer Treat Rep 69: 203-204, 1985. 8. Lemoine F, Najman A, B a i l l o u C, Stachowiak J , Boffa G, Aegerter P, Douay L, Laporte JP, Gorin NC & Duhamel G. A prospective study of the value of bone marrow e r y t h r o i d progenitor c u l t u r e s i n polycythemia. Blood 68: 996-1002, 1986. — 9. V i l l e v a l JL, Cramer P, Lemoine F, Henri A, B e t t a i e b A, Bernaudin F, Beuzard Y, Berger R, F l a n d r i n G, Breton-Gorius J & Vainchenker W. Phenotype of e a r l y e r y t h r o b l a s t i c leukemias. Blood 68: 1167-1174, 1986. 10. Lemoine FM, Humphries RK, Abraham SDM, K r y s t a l G & Eaves CJ. P a r t i a l c h a r a c t e r i z a t i o n of a novel stromal c e l l - d e r i v e d pre-B c e l l growth f a c t o r a c t i v e on normal and immortalized pre-B c e l l s . Exp Hematol 16: 718-726, 1988. 11. Lemoine FM, Humphries RK, Abraham SDM, K r y s t a l G & Eaves CJ. P a r t i a l c h a r a c t e r i z a t i o n of a novel stromal c e l l - d e r i v e d pre-B c e l l growth f a c t o r a c t i v e on normal and immortalized pre-B c e l l s . Exp Hematol 16: 717-726, 1988. ~ 12. Humphries RK, Abraham S, K r y s t a l G, Lansdorp P, Lemoine F & Eaves CJ. A c t i v a t i o n of m u l t i p l e hemopoietic growth f a c t o r genes i n Abelson v i r u s transformed myeloid c e l l s . Exp Hematol ( i n press) 13. Lemoine FM, K r y s t a l G, Humphries RK & Eaves CJ. Autocrine production of pre-B c e l l s t i m u l a t i n g a c t i v i t y by a v a r i e t y of transformed pre-B c e l l l i n e s . Cancer Res ( i n press) REVISED: September 15, 1988 ABSTRACTS Francois M. Lemoine 1. Lemoine F, Ricome JL, Richard C, Khayat D, Rimailho A & Auzepy P. C o l e c t a s i e s aigues sous v e n t i l a t i o n a s s i s t e e : Role de l a Phenoperidine. Congres de Medecine Interne, Geneve, J u i n 1983. 2. Lemoine F, T a b i l i o A, Breton-Gorius J , Najman A & Duhamel G. Leucemie aigue c h i m i o - i n d u i t e survenant au cours d'une leucemie lymphoide chronique: I n t e r e t de l a cytochimie u l t r a s t r u c t u r a l e et des techniques de marquage a l ' a i d e d'anticorps monoclonaux dans l a c a r a c t e r i s a t i o n des c e l l u l e s b l a s t i q u e s . Societe Francaise d'Hematologic, P a r i s , Mars 1984. Nouv Rev Fr Hemat 26: 108, 1984. 3. V i l l e v a l JL, Matamis H, Lemoine F, Bernaudin F, Cramer P, Rochant H, Vainchenker W & Breton-Gorius J . D i f f e r e n t phenotypes of b l a s t s i n erythroleukemia: I d e n t i f i c a t i o n of e a r l y stages of Glycophorin A negative e r y t h r o i d d i f f e r e n t i a t i o n by two new markers. Blood 64 (Suppl 1): 199a, 1984. 4. Lemoine F, Najman A, B a i l l o u C, Stachowiak J , Laporte JP, Gorin NC & Duhamel G. Valeur diagnostique de l a c u l t u r e de progeniteurs e r y t h r o i d e s au cours des p o l y g l o b u l i e s . Congres Francais d'Hematologic, Mai 1985. Nouv Rev Fr Hematol 27: 71, 1985. 5. Lemoine F, Najman A, B a i l l o u C, Stachowiak J , Laporte JP, Gorin NC & Duhamel G. Endogenous e r y t h r o i d c o l o n i e s are a major c r i t e r i o n f o r the c l a s s i f i c a t i o n of polycythemia. Blood 66 (Suppl 1): 178a, 1985. 6. V i l l e v a l JL, Cramer P, Henri A, Lemoine F, Berger R, Breton-Gorius J & Vainchenker W. Phenotypes of e a r l y e r y t h r o b l a s t i c leukemia. Blood 66 (Suppl 1): 184a, 1985. 7. Lemoine FM, Humphries RK & Eaves CJ. I s o l a t i o n of cloned l i n e s of e a r l y pre-B c e l l s that show v a r i a b l e degrees of autonomy but r e t a i n responsiveness to a f a c t o r released by a cloned mesenchymal c e l l l i n e . Blood 68 (Suppl 1): 97a, 1986. 8. Humphries RK, Abraham S, Lemoine F, Lansdorp P & Eaves CJ. A c t i v a t i o n of m u l t i p l e hemopoietic growth f a c t o r genes i n Abelson v i r u s transformed murine myeloid c e l l s . Exp Hematol 15 (Suppl 5): 499, 1987. 9. Lemoine FM, K r y s t a l G, Humphries RK & Eaves CJ. Autocrine s e c r e t i o n of a new pre-B s t i m u l a t i n g f a c t o r by a v a r i e t y of transformed murine pre-B c e l l l i n e s . Blood 70 (Suppl 1): 176a, 1987. 10. Lemoine FM, Dedhar SR & Eaves CJ. D i f f e r e n t i a l attachment of normal and transformed pre-B c e l l s to f i b r o n e c t i n . J C e l l Biochem (Suppl 12B): 112, 1988. 11. Lemoine FM, Dedhar S, Gray V & Eaves CJ. A l t e r a t i o n s i n f i b r o n e c t i n receptors on murine pre-B c e l l s are associated with transformation and the a c q u i s i t i o n of an autocrine phenotype. Blood ( i n press) 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0098069/manifest

Comment

Related Items