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UBC Theses and Dissertations

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

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STUDIES OP THE INTERACTIONS BETWEEN STROMAL CELLS AND B LYMPHOID PROGENITORS by F r a n c o i s M. Lemoine  M.D.,  U n i v e r s i t y o f 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 o f P a t h o l o g y )  We accept t h i s t h e s i s as conforming to the r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA September, 1988 ©Francois M. Lemoine, 1988  In  presenting  degree at the  this  thesis  in partial fulfilment of  of  department  this thesis for scholarly or  requirements  for  by  his  or  her  I further agree that permission for  purposes  permission.  Department of  Pathology  The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3  DE-6C3/81)  advanced  representatives.  September  14,  1988  extensive  may be granted by the head of It  is  understood  that  publication of this thesis for financial gain shall not be allowed without  Date  an  University of British Columbia, I agree that the Library shall make it  freely available for reference and study. copying  the  copying  my or  my written  ii  ABSTRACT  The o v e r a l l g o a l of the work, d e s c r i b e d i n t h i s t h e s i s was to  investigate  the m o l e c u l a r mechanisms t h a t r e g u l a t e 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  m o l e c u l a r b i o l o g i c a l t e c h n i q u e s have a l l o w e d a number of s t a g e s of p r e - B differentiation  cell  to be d e f i n e d but l i t t l e i s known about mechanisms c o n t r o l l i n g  their proliferation. S t u d i e s of pre-B c e l l p r o d u c t i o n i n a n i m a l models and i n l o n g - t e r m cultures  t h a t support pre-B c e l l p r o l i f e r a t i o n have suggested t h a t  c e l l s p l a y a key r o l e i n t h i s r e g a r d .  stromal  As a f i r s t s t e p 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 s u p p o r t i v e murine s t r o m a 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 . isolated,  c l o n e d and c h a r a c t e r i z e d .  murine l e u k e m i a v i r u s t r a n s f o r m a n t s used i n c o - c u l t u r e experiments  cell  A number of pre-B c e l l l i n e s were a l s o  From t h e s e , spontaneous and A b e l s o n were d e r i v e d .  to demonstrate  These c e l l l i n e s were then  that stromal  c o n s t i t u t i v e l y s e c r e t e a pre-B s t i m u l a t i n g f a c t o r .  cells  C h a r a c t e r i z a t i o n of  p r e - 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 r o m a l c e l l l i n e showed i t to be a 10 Kd m o l e c u l e s e n s i t i v e  (M2-10B4)  to f r e e z i n g and d i f f e r e n t  c l o n e d h e m o p o i e t i c growth f a c t o r d e s c r i b e d to d a t e .  the  from any  The p o s s i b i l i t y t h a t  e x t r a c e l l u l a r m a t r i x components might be i n v o l v e d i n s t r o m a l 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 t h a t  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 t h o u g h f i b r o n e c t i n by itself  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 r o m a l  cell  s t i m u l a t i o n o f pre-B c e l l g r o w t h . Both o f these mechanisms were found to be a f f e c t e d  i n malignantly  transformed pre-B c e l l p o p u l a t i o n s i r r e s p e c t i v e of the mode of  transformation.  iii 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 c e l l populations.  iv  TABLE OF CONTENTS Page ABSTRACT LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS ACKNOWLEDGEMENTS  CHAPTER I  i i v i i viii x xi  INTRODUCTION  1  1)  1 2 4 5  2)  3)  4)  Hemopoiesis: G e n e r a l Concepts A) Concept o f P l u r i p o t e n t Stem C e l l s B) Concept o f Committed P r o g e n i t o r s C) R e g u l a t i o n o f Hemopoietic Stem C e l l s and Myelopoiesis D) M y e l o p o i e t i c Growth F a c t o r s and Hemopoiesis E) Summary' O r i g i n and Development o f B C e l l s A) R e l a t i o n s h i p Between B Lymphoid P r e c u r s o r s and Other Hemopoietic L i n e a g e s B) Ontogeny o f Lymphopoiesis C) Development o f B C e l l s i n the A d u l t (a) I g Gene Rearrangement and E x p r e s s i o n (b) Changes i n S u r f a c e A n t i g e n s ( c ) Other D i f f e r e n t i a t i o n Markers  8 11 12 12 14 17 18 23 26  R e g u l a t i o n o f B C e l l Development A) Systems f o r Study (a) Short-Term C u l t u r e Systems (b) I L - 3 Dependent B-Lineage C e l l L i n e s ( c ) Long-Term B Lymphoid C u l t u r e s (Lymphoid LTC) (d) Animal Models (i) CBA/N Mice ( i i ) SCID Mice ( i i i ) Motheaten Mice B) Mechanisms (a) Growth F a c t o r s (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  36 36 37 38 39 39 42  Thesis Objectives  43  References  29 30 30 31 32  46  V  CHAPTER I I  MATERIALS AND METHODS  59  1) 2) 3) 4) 5) 6) 7) 8)  59 59 60 60 61 61 63 64  Established C e l l Lines Animals Growth F a c t o r s Antibodies Probes P r o t e i n s and P e p t i d e s I s o l a t i o n and C l o n i n g of Stromal C e l l L i n e s I s o l a t i o n , C l o n i n g and Maintenance of Pre-B C e l l Lines 9) A-MuLV T r a n s f o r m a t i o n of Pre-B C e l l s 10) Immunofluorescence A n a l y s e s 11) H i s t o c h e m i s t r y 12) V i r u s Assays 13) Assays f o r T u m o r i g e n i c i t y 14) C e l l P r o l i f e r a t i o n Assays 15) C e l l Attachment Assays 16) Pre-B C e l l Colony Assays 17) M y e l o i d Colony Assays 18) A n a l y s i s of DNA Rearrangements 19) P r e p a r a t i o n of CM 20) G e l Permeation Chromatography References  CHAPTER I I I PARTIAL CHARACTERIZATION OF A NOVEL STROMAL CELLDERIVED PRE-B CELL GROWTH FACTOR ACTIVE ON NORMAL AND IMMORTALIZED PRE-B CELLS 1) 2)  Introduction 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 S u p p o r t i v e Stromal C e l l L i n e s 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 C) I n V i t r o Growth of H9 and A8 C e l l s D) E v i d e n c e f o r a S o l u b l e Pre-B C e l l Stimulating 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 Stromal C e l l L i n e s F) Response of Feeder-Independent H9 C e l l s to D e f i n e d Growth F a c t o r s 3) Discussion References  65 66 67 67 68 68 69 70 70 71 72 73 74  77  77 78 80 82 87 90 92 92 97  vi CHAPTER IV  AUTOCRINE PRODUCTION OF PRE-B CELL STIMULATING ACTIVITY BY A VARIETY OF TRANSFORMED PRE-B CELL LINES 1) 2)  Introduction Results A) H9 C e l l s Produce a Pre-B S t i m u l a t i n g Activity B) P r o d u c t i o n o f 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 C) C h a r a c t e r i z a t i o n o f the A u t o c r i n e A c t i v i t y Produced by Transformed Pre-B C e l l s 3) Discussion References  CHAPTER V  CHAPTER V I  99 99 100 100 102 109 109 114  ROLE OF FIBRONECTIN IN REGULATING PRE-B CELL PROLIFERATION  116  1) 2)  Introduction Results A) D i f f e r e n t i a l attachment o f normal and malignant pre-B c e l l l i n e s t o FN B) E f f e c t o f FN on the p r o l i f e r a t i o n o f normal and t r a n s f o r m e d pre-B c e l l s i n the presence of s t r o m a l CM C) D i f f e r e n t i a l e f f e c t o f FN-R a n t i b o d i e s on the p r o l i f e r a t i o n o f normal and t r a n s f o r m e d pre-B c e l l s 3) Discussion References  116 117117  SUMMARY AND CONCLUSIONS  129  1)  129  R e g u l a t i o n o f Pre-B C e l l P r o l i f e r a t i o n by Stromal C e l l s A) F a c t o r s B) C e l l - C e l l I n t e r a c t i o n s 2) A l t e r e d Mechanisms i n Transformed Pre-B C e l l s 3) Proposed Model References  120 124 124 128  130 131 132 133 135  vii  LIST OF TABLES 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 o f the monoclonal a n t i b o d i e s used i n t h i s s t u d y .  62  TABLE 4  Comparison of the a b i l i t y o f v a r i o u s i r r a d i a t e d (80 Gy) f e e d e r c e l l l i n e s to s u p p o r t the growth of c u l t u r e d pre-B c e l l s .  79  TABLE 5  Immunological and h i s t o c h e m i c a l c h a r a c t e r i z a t i o n o f the s t r o m a l c e l l l i n e s used i n t h i s s t u d y .  81  TABLE 6  D e m o n s t r a t i o n o f a s o l u b l e f a c t o r d e r i v e d from M2-10B4 c e l l s s t i m u l a t i n g i m m o r t a l i z e d pre-B c e l l l i n e s and normal pre-B c e l l s .  88  TABLE 7  Lack, o f r e s p o n s i v e n e s s of H9 c e l l s t o d e f i n e d growth f a c t o r s .  93  TABLE 8  D e m o n s t r a t i o n 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 .  103  TABLE 9  Transformed phenotype o f c l o n a l lymphoid c e l l l i n e s .  104  TABLE 10  Phenotype d a t a o f normal and t r a n s f o r m e d lymphoid clones.  105  TABLE 11  E f f e c t o f FN on normal pre-B c e l l p r o l i f e r a t i o n induced by s t r o m a l pre-B s t i m u l a t i n g a c t i v i t y (M2-10B4 CM).  122  TABLE 12  E f f e c t o f FN on t r a n s f o r m e d pre-B c e l l p r o l i f e r a t i o n induced by s t r o m a l pre-B s t i m u l a t i n g a c t i v i t y (M2-10B4 CM).  123  viii  LIST OF FIGURES Page FIGURE 1  Schematic r e p r e s e n t a t i o n o f hemopoiesis.  3  FIGURE 2  O r g a n i z a t i o n o f immunoglobulin genes.  19  FIGURE 3  S u r f a c e a n t i g e n e x p r e s s i o n and I g gene rearrangement d u r i n g murine B - c e l l development.  22  FIGURE 4  D e s c r i p t i o n o f the d i f f e r e n t types o f long-term hemopoietic c u l t u r e s .  33  FIGURE 5  P a n e l a. Southern b l o t a n a l y s i s o f I g H c h a i n 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 w i t h EcoRI and h y b r i d i z a t i o n t o a Jfj probe. P a n e l b. Southern b l o t a n a l y s i s o f the T c e l l r e c e p t o r g c h a i n 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 w i t h Hind I I I and h y b r i d i z a t i o n t o a Tg cDNA probe.  83  FIGURE 6  P r o l i f e r a t i o n o f H9 c e l l s ( p a n e l a) and A8 c e l l s ( p a n e l b) seeded a t d i f f e r e n t c e l l c o n c e n t r a t i o n s under d i f f e r e n t c o n d i t i o n s .  84  FIGURE 7  P r o l i f e r a t i o n o f 3,000 H9 c e l l s / m l ( p a n e l a) and 3 x 10^ A8 c e l l s / m l ( p a n e l b) when p l a t e d on i r r a d i a t e d M2-10B4 c e l l s a t d i f f e r e n t M2-10B4 c e l l concentrations.  86  FIGURE 8  P a n e l a. P r o l i f e r a t i o n o f H9 c e l l s (3000 c e l l s / m l ) seeded i n the presence o f v a r i o u s c o n c e n t r a t i o n s o f M2-10B4 CM h a r v e s t e d a t 1, 2, 3 o r 5 days a f t e r adding f r e s h medium t o the M2-10B4 c e l l s . P a n e l b: P r o l i f e r a t i o n o f H9 c e l l s (3000 c e l l s / m l ) seeded i n the presence o f v a r i o u s c o n c e n t r a t i o n s o f M2-10B4 Day 1 CM a f t e r h e a t i n g t o 56°C f o r 1 hour; and a f t e r f r e e z i n g and thawing once.  89  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 o f 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 o f 8X c o n c e n t r a t e d serum-free M2-10B4 CM.  91  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 o r M2-10B4 CM.  101  FIGURE 11  Southern b l o t a n a l y s i s o f I g H c h a i n gene rearrangements i n normal pre-B c e l l c l o n e s and d e r i v a t i v e A-MuLV t r a n s f o r m a n t s .  106  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 c o n d i t i o n e d by normal and transformed pre-B c e l l  lines.  108  IX  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 o f the pre-B s t i m u l a t i n g a c t i v i t y p r e s e n t i n a 1 ml sample o f 50X c o n c e n t r a t e d serum-free H9 CM.  110  FIGURE 14  Attachment of normal pre-B c e l l s to FN.  118  FIGURE 15  E f f e c t o f s y n t h e t i c p e p t i d e s on the attachment of normal pre-B c e l l s (Bp) to FN.  119  FIGURE 16  Attachment o f transformed pre-B c e l l s to FN.  121  FIGURE 17  P a n e l a. E f f e c t o f FN-R and VN-R a n t i b o d i e s on the p r o l i f e r a t i o n o f normal pre-B c e l l s . P a n e l b. R o l e o f FN-R and VN-R a n t i b o d i e s on the p r o l i f e r a t i o n o f transformed pre-B c e l l s .  125  LIST OF ABBREVIATIONS  A-MuLV ATCC BSA C cu CFU-GEMM CFU-S CM CML CR CSF D ECM EGF Epo FACS FCS FFU FITC FN G6PD G-CSF GM-CSF H HGF I FN Ig IL J L LCM LM LPS LTC M-CSF ME MHC MLR MLS Mo-MuLV PBS PWM-SCCM RGD SCID slg TdT TGF V VN  Abelson murine leukemia virus American Type Culture Collection Bovine serum albumin Constant Cytoplasmic u Colony-forming unit-granulocytic, erythroid, megakaryocytic and macrophagic Colony-forming unit-spleen Conditioned medium Chronic myeloid leukemia Complement receptor Colony-stimulating factor Diversity Extracellular matrix Epidermal growth factor Erythropoietin Fluorescent-activated cell sorter Fetal calf serum Focus-forming unit Fluorescein isothiocyanate Fibronectin Glucose-6-phosphate dehydrogenase Granulocyte colony-stimulating factor Granulocyte-macrophage colony-stimulating factor Heavy Hybridoma growth factor Interferon Immunoglobulin Interleukin Joining Light Leukocyte conditioned medium Laminin Lipopolysaccharide Long-term culture Macrophage colony-stimulating factor Mercaptoethanol Major histocompatibility complex Mixed lymphocyte reaction Minor lymphocyte stimulating antigens Moloney murine leukemia virus Phosphate buffer saline Pokeweed mitogen-spleen cell conditioned medium Arg-gly-asp Severe combined immune deficient disease Surface immunoglobulin Terminal deoxynucleotidyl transferase Transforming growth factor Variable Vitronectin  xi  ACKNOWLEDGMENTS  I w i s h to e x p r e s s my s i n c e r e  gratitude:  To my s u p e r v i s o r D r . C . J . Eaves f o r h e r c r i t i c a l s u g g e s t i o n s , and c o n s t a n t guidance throughout t h i s p r o j e c t , To D r 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 active collaboration, To members o f my a d v i s o r y committee, D r s . D. B r u n e t t e and F . T a k e 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 f o r e x p e r t To M. Coulombe f o r s e c r e t a r i a l  technical assistance,  assistance,  To L a L i g u e N a t i o n a l e F r a n c a i s e C o n t r e l e C a n c e r , the F o n d a t i o n pour l a Recherche M e d i c a l e , the P h i l i p p e F o u n d a t i o n and the A s s o c i a t i o n pour l a Recherche C o n t r e l e Cancer f o r t h e i r f i n a n c i a l s u p p o r t . To a l l the s t a f f i n the T e r r y Fox L a b o r a t o r y 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 e n v i r o n m e n t . And f i n a l l y , to my w i f e F r e d e r i q u e and my sons Romain and X a v i e r f o r t h e i r patient support.  1  CHAPTER  I  INTRODUCTION  1)  HEMOPOIESIS; GENERAL CONCEPTS  B l o o d 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 s h o r t l i f e and  cannot renew themselves,  they must be c o n t i n u o u s l y r e p l a c e d so t h a t  numbers are m a i n t a i n e d .  The p r o c e s s by which t h i s i s a c h i e v e d i s known as  hemopoiesis. capacity  The most p r i m i t i v e h e m o p o i e t i c c e l l i s p l u r i p o t e n t and has  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  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 ,  lymphocytes and c e l l s of the g r a n u l o c y t e - m o n o c y t e s e r i e s 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 )  the  through d i f f e r e n t  h i e r a r c h i c a l s t e p s to e v e n t u a l l y produce a l l of the formed elements blood.  their  of  the  platelets, (monocytes,  are n e c e s s a r y f o r oxygen  transport,  h e m o s t a s i s and r e s i s t a n c e to i n f e c t i o n . The h e m o p o i e t i c system has been s e p a r a t e d a r b i t r a r i l y i n t o f o u r compartments capacities  a c c o r d i n g to the d e c r e a s i n g  of the c e l l s w i t h i n them:  (i)  p r o l i f e r a t i v e and stem c e l l s ,  ( i i ) progenitors  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 , recognizable,  precursors  d i v i s i o n s , and ( i v )  o f more  ( i i i ) morphologically  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  the f i n a l mature b l o o d c e l l s .  comprise most of the c e l l s i n the system. compartments  self-renewal  are much s m a l l e r .  The l a s t two  In contrast,  As a r e s u l t ,  these c e l l  compartments  the two most p r i m i t i v e types are  extremely  2  r a r e even i n the bone marrow ( l e s s than one T h i s , together e x p l a i n s why  i n one  thousand n u c l e a t e d  w i t h the f a c t t h a t they are not m o r p h o l o g i c a l l y  hemopoietic progenitors  of t h e i r  recognizable,  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  h e m o p o i e t i c system i s p r e s e n t e d i n F i g u r e  A)  recognizable,  can o n l y be s t u d i e d by i n d i r e c t  ( d e v e l o p m e n t a l ) methods, i . e through the g e n e r a t i o n mature progeny.  cells).  the  1.  Concept of P l u r i p o t e n t Stem C e l l s  I n 1961  T i l l and M c C u l l o c h (1) d e s c r i b e d  h e m o p o i e t i c c e l l w i t h stem c e l l p r o p e r t i e s .  the f i r s t assay f o r a These c e l l s have the c a p a c i t y  to  form m a c r o s c o p i c c o l o n i e s c o n t a i n i n g v a r i o u s c o m b i n a t i o n s of e r y t h r o i d , g r a n u l o c y t i c , m e g a k a r y o c y t i c 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 s p l e e n s of l e t h a l l y i r r a d i a t e d s y n g e n e i c mice and forming u n i t - s p l e e n ) .  are r e f e r r e d to as CFU-S ( c o l o n y  Subsequent s t u d i e s l a i d the b a s i s of the concept of a  h e m o p o i e t i c 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  with  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 c a p a c i t y f o r g e n e r a t i n g 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 p r o l i f e r a t i v e capacity (2).  daughter  high  A d d i t i o n a l s t u d i e s w i t h 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 c o n f i r m e d  that  these p r o p e r t i e s are f e a t u r e s of a s m a l l subset of o t h e r w i s e p o o r l y u n d e r s t o o d c e l l s i n the marrow. represent  However, i t i s a l s o known that c e l l s d e f i n e d as CFU-S  a heterogeneous p o p u l a t i o n w i t h r e s p e c t  differentiation capacities. 5-7  The  to t h e i r s e l f - r e n e w a l  f i r s t spleen c o l o n i e s that are v i s i b l e  days a f t e r t r a n s p l a n t a t i o n ) are d e r i v e d from c e l l s t h a t are  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 c o n t r a s t to s p l e e n 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 ) .  neither colonies  and (seen  OJ  F i g u r e 1.  Schematic r e p r e s e n t a t i o n o f  hemopoiesis.  4  I n humans, a comparable i n v i v o CFU-S assay cannot, o b v i o u s l y be performed.  However, s t u d i e s o f c l o n a l d i s o r d e r s o f hemopoiesis such as  c h r o n i c myelogenous l e u k e m i a (CML), where a l l o f the c e l l s i n t h e n e o p l a s t i c c l o n e 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 e x i s t e n c e o f a p l u r i p o t e n t h e m o p o i e t i c stem c e l l p o p u l a t i o n  i n man analogous to t h a t 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 h e m o p o i e t i c c e l l s o f 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 g e n e r a t e c o l o n i e s c o n t a i n i n g mature  c e l l s o f m u l t i p l e m y e l o i d l i n e a g e s (9,10).  Those that i n i t i a t e c o l o n y  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 c a p a c i t y a r e r e f e r r e d t o as CFU-GEMM. Others show d e l a y e d i n i t i a t i o n o f colony growth and a r e c h a r a c t e r i z e d by a higher  self-renewal capacity.  As a r e s u l t , they g i v e r i s e t o c o l o n i e s which  at an e a r l y s t a g e a r e composed e n t i r e l y o f b l a s t s and t h e c e l l o f o r i g i n has been s e p a r a t e l y d e s i g n a t e d  as a CFU-blast o r S - c e l l ( 1 1 ) .  CFU-blast ( S - c e l l ) progenitors CFU-S.  B)  Both CFU-GEMM and  can g e n e r a t e c e l l s d e t e c t a b l e  i n the mouse as  However, t h e i r a b i l i t y t o g e n e r a t e lymphoid c e l l s i s n o t y e t c l e a r .  Concept o f Committed  Progenitors  Committed, o r l i n e a g e - r e s t r i c t e d p r o g e n i t o r s a r e c o n s i d e r e d 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 p r e c u r s o r s p o t e n t i a l has been determined. progenitors  clonogenic  differentiative  C u r r e n t e v i d e n c e suggests t h a t committed  may possess an e x t e n s i v e  self-renewal.  because t h e i r  t o be more  Committed p r o g e n i t o r s  p r o l i f e r a t i v e c a p a c i t y but cannot undergo a r e 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 "  a s s a y s i n a s e m i - s o l i d c u l t u r e medium c o n t a i n i n g  appropriate  n u t r i e n t s j serum components and a source o f crude o r 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 a r e i d e n t i f i e d by the v a r i o u s colonies size,  they produce.  types o f  These a r e d i s t i n g u i s h e d i n terms o f t h e i r u l t i m a t e  the time r e q u i r e d f o r mature c e l l s  c o m p o s i t i o n o f the c o l o n y .  In general,  to appear, and the f i n a l  cellular  more p r i m i t i v e p r o g e n i t o r s  give  rise  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 culture.  D u r i n g t h i s time many c e l l d i v i s i o n s o c c u r 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 progenitors  I n both the human and murine s y s t e m s , committed  f o r each o f the m y e l o i d pathways and i n some cases f o r d i f f e r e n t  s t a g e s o f development a l o n g a pathway can now be measured v i t r o c o l o n y assays and a p p r o p r i a t e  s t u d i e s have demonstrated  myeloid c e l l s  The names used  Interestingly,  although  a common c e l l o r i g i n f o r lymphoid and  (see b e l o w ) , t h e r e a r e not as y e t analogous " i n v i t r o " c o l o n y  a s s a y s to i d e n t i f y e a r l y committed B o r T lymphocyte  C)  by the use o f i n  scoring c r i t e r i a (12-14).  to d e s i g n a t e each o f these a r e g i v e n i n F i g u r e 1. different  The more mature  g e n e r a t e c o l o n i e s o f 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 . progenitors  n o n - d i v i d i n g blood c e l l progeny.  progenitors.  R e g u l a t i o n o f 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 o f e v i d e n c e 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 o f h e m o p o i e t i c c e l l s i s r e g u l a t e d a t l e a s t i n p a r t by v a r i o u s growth f a c t o r s elements  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 " '  o f the bone marrow ( 1 5 , 1 6 ) .  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 o f the bone marrow have d i s c l o s e d a number o f i n t i m a t e r e l a t i o n s h i p s between h e m o p o i e t i c c e l l s and the v a r i o u s morphologically defined stromal populations  (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 f a t - a c c u m u l a t i n g c e l l s o r a d i p o c y t e s , and o s t e o b l a s t s ( 1 8 ) .  cells,  These c e l l s  p a r t i c i p a t e i n the f o r m a t i o n of a complex e x t r a c e l l u l a r m a t r i x (ECM)  of  f i b r o u s and n o n - f i b r o u s p r o t e i n s 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 ( 1 9 ) , l a m i n i n (LM) ( 2 0 ) , f i b r o n e c t i n (FN) ( 2 1 ) , v i t r o n e c t i n (VN) ( 2 2 ) , p r o t e o g l y c a n s (23) and more r e c e n t l y haemonectin ( 2 4 ) . E v i d e n c e from both i n v i v o and/or i n v i t r o experiments demonstrates  that  s t r o m a l 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 h e m o p o i e t i c c e l l s and p l a y 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  t h a t r a d i o - i n d u c e d damage or m e c h a n i c a l 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 o f h e m o p o i e t i c f u n c t i o n (25-27).  The a b i l i t y of h e m o p o i e t i c 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 k i d n e y c a p s u l e of s e m i syngeneic animals.  T h i s l e a d s to the f o r m a t i o n of bone t i s s u e which then  becomes p o p u l a t e d by h e m o p o i e t i c c e l l s of r e c i p i e n t o r i g i n ( 2 8 ) .  Animal  models such as mice b e a r i n g a l t e r a t i o n s a t the S t e e l l o c u s (29) have a l s o p r o v i d e d e v i d e n c e f o r s t r o m a l mediated r e g u l a t i o n of h e m o p o i e t i c stem proliferation.  cell  Mutant mice of the S l / S l ^ genotype have a d e f e c t i v e  h e m o p o i e t i c system which i s due to a n o n - t r a n s p l a n t a b l e d e f e c t o f the h e m o p o i e t i c microenvironment.  Sl/Sl^  h e m o p o i e t i c stem c e l l s a r e  i n t r i n s i c a l l y normal ( 2 9 ) ; and cure of the anemia t h a t c h a r a c t e r i z e s  Sl/Sl^  mice r e q u i r e s engraftment of an i n t a c t h e m a t o p o i e t i c stroma ( 3 0 ) . Long-term marrow c u l t u r e s o r i g i n a l l y d e s c r i b e d by Dexter (31) reproduce i n many r e s p e c t s the f e a t u r e s of h e m o p o i e t i c stem c e l l r e g u l a t i o n seen i n vivo.  T h i s 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 t h a t can o c c u r between s t r o m a l c e l l s and m y e l o p o i e t i c p r o g e n i t o r s (32).  T h i s l o n g - t e r m marrow c u l t u r e (LTC) system has been a n a l y z e d by  7  changing e i t h e r the s t r o m a l c e l l o r the h e m o p o i e t i c c e l l components ( 3 3 ) . A l s o , as d i s c u s s e d i n g r e a t e r d e t a i l below, m o d i f i c a t i o n o f the c u l t u r e c o n d i t i o n s a l l o w s the s e l e c t i v e long-term s t r o m a l c e l l - d e p e n d e n t p r o p a g a t i o n of  B-lineage r e s t r i c t e d c e l l s (34).  T h i s l a t t e r system has been v e r y u s e f u l  to s t u d y the r e g u l a t i o n o f B - l y m p h o p o i e t i c c e l l  proliferation.  ECM i s an i m p o r t a n t component o f the h e m o p o i e t i c microenvironment  and i s  thought t o mediate many o f i t s r e g u l a t o r y a c t i o n s on the growth o f h e m o p o i e t i c c e l l s (35,36).  The a v a i l a b i l i t y o f p u r i f i e d ECM m o l e c u l e s has been u s e f u l t o  s t u d y how these m o l e c u l e s i n t e r a c t s p e c i f i c a l l y w i t h h e m o p o i e t i c c e l l s .  For  example, i t has r e c e n t l y been shown that h e m o p o i e t i c c e l l s and p a r t i c u l a r l y B lymphoid p r o g e n i t o r s can b i n d to FN (37,38).  Haemonectin, seems t o be a  s p e c i f i c attachment m o l e c u l e f o r c e l l s o f the g r a n u l o c y t i c l i n e a g e ( 2 4 ) . C o l l a g e n , appears of  t o be c r u c i a l f o r the e s t a b l i s h m e n t o f a stroma s u p p o r t i v e  hemopoiesis i n LTC as shown by s t u d i e s i n which the d e p o s i t i o n o f c o l l a g e n  was  i n h i b i t e d i n t h e presence o f 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  C i s - 4 - h y d r o x y p r o l i n e (39,40).  LM b i n d s to type IV c o l l a g e n ( 4 1 ) , and seems t o  r e g u l a t e the types o f macromolecules (42).  like  t h a t pass a c r o s s the basement membrane  Thus d i f f e r e n t ECM p r o 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  functions.  Some may s e r v e p r i m a r i l y a mechanical r o l e t o a l l o w a c l o s e a s s o c i a t i o n between h e m o p o i e t i c c e l l s and s t r o m a l c e l l s t o be m a i n t a i n e d .  As a  consequence t h e h e m o p o i e t i c c e l l s would be m a i n t a i n e d i n c l o s e p r o x i m i t y t o s t r o m a l c e l l d e r i v e d growth f a c t o r s .  Other ECM components may c o n t r o l t h e  r e l e a s e o f growth f a c t o r s s y n t h e s i z e d e i t h e r l o c a l l y o r elsewhere and p r e s e n t them o r 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 t o h e m o p o i e t i c 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;  T h i s g l y c o s a m i n o g l y c a n i s the component o f the marrow ECM produced  8  i n LTC  to which 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 (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 F a c t o r s and  The  h e m o p o i e t i c growth f a c t o r s (HGF)  Hemopoiesis  are a l l g l y c o p r o t e i n s  r e g u l a t o r y a c t i v i t i e s on h e m o p o i e t i c c e l l s . p u r i f i e d , and  c l o n e d , and  c h a r a c t e r i z e d (44). defined vivo.  that  A l a r g e number have now  i n some cases t h e i r r e s p e c t i v e r e c e p t o r s  T h i s has a l l o w e d  exert been  partially  the b i o l o g i c a l a c t i v i t i e s of u n i q u e ,  r e g u l a t o r y m o l e c u l e s to be s t u d i e d s e p a r a t e l y both i n v i t r o and These m o l e c u l e s have, however, turned  out  to be e x t r e m e l y 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 t a r g e t c e l l types and 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 y e t  HGF's t h a t s t i m u l a t e m y e l o i d p r o g e n i t o r s i n t o two  categories: f i r s t ,  t h e i r mechanisms of elucidated.  can be o p e r a t i o n a l l y s e p a r a t e d  the colony s t i m u l a t i n g f a c t o r s (CSF's) t h a t  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 p r o g e n i t o r s ; and  in  the d i f f e r e n t i a t i o n of h e m o p o i e t i c  second, s y n e r g i s t i c f a c t o r s which a l o n e appear d e v o i d  of  i n t r i n s i c m y e l o i d 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 m o l e c u l e s , some of t h e i r p r o p e r t i e s  b e t t e r known e f f e c t s on m y e l o i d c e l l s are summarized i n T a b l e 1.  As  also  shown i n T a b l e 1, most of the HGF's are produced by e i t h e r a c t i v a t e d c e l l s or a c t i v a t e d T c e l l s . considered  Only Epo,  stromal  which i s produced by the k i d n e y ,  is  as a c l a s s i c a l hormone.  I t i s i m p o r t a n t to p o i n t 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 exist.  and  a complex set of i n t e r a c t i o n s appear to  Growth f a c t o r s a b l e to a c t on more p r i m i t i v e c e l l s (e.g. I L - 3 ,  GM-CSF) can down-regulate r e c e p t o r s  and  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.  CSF-2 MGI  G—CSF ( G r a n u l o c y t e c o l o n y stimulating factor)  Growth  Factors  3  Molecular Weight  C e l l s Responsive to F a c t o r Alone  Activated T lymphocytes Fibroblasts Endothelial cells  23 ,000  CFU-GM CFU-mix BFU-E Granulocytes Macrophages  Activated T lymphocytes Mac r o p h a g e s Fibroblasts  25,000  Sub-population of CFU-GM (CFU-G) Granulocytes  Alternative Name(s)  GM-CSF ( G r a n u l o c y t e - m a c r o p h a g e colony-stimulating factor)  Hemopoietic  Cellular Origin (no r m a l )  + CSF-1 s t i m u l a t e s p l u r i p o t e n t stem cells  + CSF-1 Epo  Multi-CSF (Multipotential colony-stimulating factor)  IL-3 BPA PSF  Activated 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  M-CSF ( M a c r o p h a g e c o l o n y stimulating factor)  CSF-1  Activated mac r o p h a g e s Fibroblasts  70,000  Sub—population of CFU-GM Macrophages  Kidney p e r i tubular cell:  39,000  CFU-E Mature  T  45-60,000  Epo  (Erythropoietin)  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 factor)  a  The information shown f a c t o r s i s summarized  IL-5 TRF BCGF I I  i s representative, i n T a b l e 2.  cells  not a l l i n c l u s i v e .  The l y m p h o i d  Synergistic Activity  BFU-E  CFU-Eos B cells  cell  stimulating  activity  o f many o f t h e s e  Table  Name  Alternative Name(s)  LAF  Interleukin-1 (IL-1)  Hemopo i e t i n - 1  1.  Hemopoietic  Growth F a c t o r s  Cellular Origin (No rraal)  3  - Continued  C e l l s Responsive to F a c t o r A l o n e  Factor Mo 1 e c u l a r Weight  Activated monocytes Endothelial cells a f t e r exposure t o e n d o t o x i n o r TNF-a  13-19,000  T and B l y m p h o c y t e s Hepatocytes Stimulates fibroblasts & endothelial cells to p r o d u c e G-CSF & GM-CSF  + CSF-1 o r I L - 3 s t i m u l a t e s stem cells  + Epo, G-CSF, GM-CSF or I L - 3 s t i m u l a t e s hemopoietic progenitors  Interleukin-4 (IL-4)  BSF-1 BCGF-I  Activated T lymphocytes  16-21,000  B  Interleukin-6 (IL-6 )  BSF-2 HGF IFN-02  Fibroblasts T lymphocytes Monocytes  26,000  B cells Hepatocytes  TC-1  a  activity  TC-1  cell  line  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 , n o t a l l i n c l u s i v e , i s s u m m a r i z e d i n T a b l e 2. References  44-60.  Syne r g i s t i c Activity  cells  200,000  The  lymphoid  + IL-3 s t i m u l a t e s pluripotent cells  + C S F - 1 , I L - 3 , o r GM-CSF stimulates hemopoietic progenitors  cell  stimulating activity  o f many o f t h e s e  factor  11  l i n e a g e a c t i o n ( e . g . G-CSF, M-CSF, Epo) a c t i v i t y (59).  (61) and may  also synergize their  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). R e g u l a t i o n of m y e l o p o i e s i s a l s o i n v o l v e s i n h i b i t o r y m o l e c u l e s . i n h i b i t o r s of hemopoiesis  including lactoferrin, transferrin,  Various  acidic  i s o f e r r i t i n s , E-type p r o s t a g l a n d i n s , i n t e r f e r o n ( I F N ) , c a c h e c t i n (TNF-a), l y m p h o t o x i n (TFN-0), c h a l o n e s , have been r e p o r t e d (62-65). t r a n s f o r m i n g growth  More r e c e n t l y ,  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  c o n f e r anchorage-independent  growth on non-malignant  to  f i b r o b l a s t s ( 6 6 ) , has  been shown to be a potent i n h i b i t o r of h e m o p o i e t i c c e l l p r o l i f e r a t i o n ( 6 7 ) . TGF-P i s a h i g h l y conserved homodimer of 25,000 d a l t o n s produced of c e l l s .  Two  c l o s e l y r e l a t e d forms:  by a v a r i e t y  TGF-gi and TGF-f3 > t h a t b i n d to t h r e e 2  d i s t i n c t forms of r e c e p t o r s , e x i s t ( 6 8 ) .  TGF-P^ i n h i b i t s the c y c l i n g o f the  most p r i m i t i v e m y e l o i d p r o g e n i t o r 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 l y m p h o p o i e s i s  (70) .  T h i s f a c t o r a l s o i n c r e a s e s the p r o d u c t i o n of ECM  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  E)  FN and type I c o l l a g e n  (71,72).  Summary  Hemopoiesis  <  may  be viewed as a c e l l u l a r pyramid i n which v e r y few stem  c e l l s g i v e r i s e to mature b l o o d 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 s e t o f complex i n t e r a c t i o n s between h e m o p o i e t i c c e l l s , s t r o m a l c e l l s t h e i r products.  The l a t t e r i n c l u d e ECM  s t i m u l a t o r y and i n h i b i t o r y m o l e c u l e s .  p r o t e i n s as w e l l as v a r i o u s  and  12  2)  ORIGIN AND DEVELOPMENT OF B CELLS  A)  R e l a t i o n s h i p Between B Lymphoid P r e c u r s o r s and Other  Hemopoietic  Lineages  The sequence o f d i f f e r e n t i a t i o n events t h a t l e a d s t o the g e n e r a t i o n o f c e l l s r e s t r i c t e d to the B and T l i n e a g e s i s s t i l l p o o r l y u n d e r s t o o d .  When  CFU-S a s s a y s were f i r s t d e s c r i b e d , no e v i d e n c e o f lymphoid c e l l s were found i n the s p l e e n c o l o n i e s produced  (1).  However, chromosomal markers  studies  s u b s e q u e n t l y enabled the presence o f a common stem c e l l o f both lymphoid and m y e l o i d c e l l s t o be demonstrated  (73).  N e v e r t h e l e s s , i n one such s t u d y ,  Abramson e t a l (74) found a chimerism i n T c e l l s o n l y , i n m y e l o i d c e l l s o n l y , or  i n B c e l l s p l u s T c e l l s and m y e l o i d c e l l s .  T h e r e f o r e , the e x i s t e n c e o f  d i f f e r e n t p o p u l a t i o n s o f 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 t o nonlymphoid c e l l s o n l y , and those r e s t r i c t e d t o T - l i n e a g e 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. chromosomally  Very  recently,  marked b l a s t c o l o n y c e l l s , generated i n v i t r o i n t h e presence o f  a c l o n e d s u p p o r t i v e adherent c e l l l i n e , were t r a n s p l a n t e d i n t o  lethally  i r r a d i a t e d r e c i p i e n t s and shown t o produce both m y e l o i d and lymphoid  progeny  i n t h e marrow, s p l e e n , m e s e n t e r i c lymph node and thymus ( 7 5 ) . A l t h o u g h these methods have s e r v e d t o e s t a b l i s h t h e e x i s t e n c e o f stem c e l l s w i t h both lymphoid and m y e l o i d 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 a t h i g h frequency.  Therefore, the  random s i t e s o f r e t r o v i r a l i n s e r t i o n i n t o h e m o p o i e t i c c e l l DNA, have proved v e r y 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 h e m o p o i e t i c p o p u l a t i o n s g e n e r a t e d i n mice g i v e n marrow t r a n s p l a n t s , and hence f o r l i n e a g e mapping.  Such s t u d i e s (5,76)  have shown one stem c e l l can r e p o p u l a t e the s p l e e n , lymph nodes, thymus and  13  bone marrow.  These r e s u l t s i n d i c a t e u n e q u i v o c a l l y the e x i s t e n c e o f  h e m o p o i e t i c stem c e l l s w i t h both lymphoid and m y e l o i d p o t e n t i a l . I n humans, e v i d e n c e f o r the r e l a t i o n s h i p between lymphoid and c e l l s came from the a n a l y s i s of glucose-6-phosphate h e t e r o z y g o t e women.  hemopoietic  dehydrogenase (G6PD)  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 a r e  encoded on the X chromosome, and i n d i v i d u a l somatic c e l l s of h e t e r o z y g o u s women e x p r e s s o n l y one of the two a l l e l e s , as a r e s u l t o f 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 e x p r e s s o n l y 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 t h e r e f o r e show a mixed G6PD p o p u l a t i o n 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 w i t h a c q u i r e d 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 t h a t both B and T lymphocytes and m y e l o i d 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 c y t o g e n e t i c i d e n t i f i c a t i o n o f the Phi-  chromosome i n CML  have shown t h a t the same isoenzyme  p a t t e r n and a l s o the  chromosome Ph* a r e expressed i n both m y e l o i d and B lymphoid c e l l s . i n d i c a t e t h a t CML  These d a t a  i s a c l o n a l d i s e a s e r e s u l t i n g from the t r a n s f o r m a t i o n o f a  p l u r i p o t e n t stem c e l l common to these pathways (7,78,79). determine whether T c e l l s are a l s o i n v o l v e d i n CML,  Attempts  have y i e l d e d  c o n c l u s i v e d a t a and t h i s i s s t i l l a matter of c o n t r o v e r s y ( 8 0 ) . r e p o r t s i n d i c a t i n g t h a t the T l i n e a g e may  be i n v o l v e d i n CML  to  less However, r a r e  (81,82), p r o v i d e  f u r t h e r s u p p o r t f o r the e x i s t e n c e of a common p l u r i p o t e n t stem c e l l i n humans also.  14  B)  Ontogeny o f Lymphopoiesis  The B c e l l l i n e a g e r e c e i v e s  i t s name from i t s o r i g i n i n the b u r s a o f  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  o t h e r b l o o d 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 s h a r e s some s i m i l a r i t i e s . summarized  as f o l l o w s .  The embryonic phase o f hemopoiesis can be  P l u r i p o t e n t h e m o p o i e t i c stem c e l l s a r e thought t o  d e r i v e from mesenchymal c e l l s l o c a t e d i n the r e g i o n o f the p r i m i t i v e k i d n e y s e a r l y i n embryonic l i f e  (29).  On m i g r a t i n g  i n t o the y o l k s a c b l o o d i s l a n d s  some o f these c e l l s b e g i n t o d i f f e r e n t i a t e a l o n g the e r y t h r o i d and m y e l o i d pathways.  D i f f e r e n t i a t i o n o f T and B c e l l s b e g i n s l a t e r a f t e r the p r i m a r y  lymphoid organs have developed and i t i s thought that these may p r o v i d e 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.  I n b i r d s , the ontogeny o f B c e l l s i s h i g h l y dependent the b u r s a o f F a b r i c i u s .  on a s i n g l e t i s s u e ,  The b u r s a i s a unique organ, that a r i s e s a t day 5 o f  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 o f the c l o a c a . d u r i n g e a r l y embryonic development  Surgical  bursectomy  induces s e v e r e agammaglobulinemia  and  r e s u l t s i n the f a i l u r e to mount an immune response t o any immunizing a n t i g e n (83).  Stem c e l l s c o l o n i z e the b u r s a from the g e n e r a l c i r c u l a t i o n between days  8 t o 14 o f i n c u b a t i o n .  They m i g r a t e through the b u r s a l mesenchyme where a few  may a l s o g i v e r i s e t o g r a n u l o c y t e s and e r y t h r o i d c e l l s . bursal epithelium, follicles  (84,85).  When they r e a c h the  they d i v i d e and d i f f e r e n t i a t e t o form i n t r a e p i t h e l i a l Each f o l l i c l e g i v e s r i s e t o a p o p u l a t i o n  e x p r e s s immunoglobulin M (IgM) on t h e i r s u r f a c e s .  B u r s a l development  i n t r a e m b r y o n i c phase, which i n c l u d e s  that  Ninety percent of b u r s a l  c e l l s a r e B lymphocytes by day 20 o f embryonic development the 20th day o f i n c u b a t i o n ) .  of c e l l s  (chicks hatch a f t e r  i n v o l v e s two phases: the  the c o l o n i z a t i o n and growth o f 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 p o s t - h a t c h i n g p e r i o d , which  includes  the c o n t i n u i n g e x p a n s i o n of the b u r s a l f o l l i c l e s and the s e e d i n g of b u r s a l B c e l l s to the p e r i p h e r y .  By 4 weeks of age a s u f f i c i e n t number o f c e l l s  m i g r a t e d out of the b u r s a as p o s t b u r s a l B l i n e a g e stem c e l l s ,  has  to ensure  e s t a b l i s h m e n t of the mature c h i c k e n B c e l l immune system i n the p e r i p h e r y (86).  At t h i s s t a g e , B c e l l s can no l o n g e r be g e n e r a t e d from a more p r i m i t i v e  m u l t i p o t e n t stem c e l l because the b u r s a has i n v o l u t e d and can no l o n g e r p r o v i d e the microenvironment  f o r t h i s e a r l y s t e p to o c c u r .  I n mammals B c e l l s a r e produced  i n the same h e m o p o i e t i c t i s s u e a l o n g w i t h  a l l of the o t h e r types of b l o o d c e l l s except T c e l l s . ontogeny  Thus, B lymphoid  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 u i v a l e n t of the b u r s a 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 v a r i o u s g u t - a s s o c i a t e d lymphoid t i s s u e s , i . e . the appendix,  the  t o n s i l s and the Peyer's p a t c h e s , 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 d e f e c t s i n a n t i b o d y 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 f e a t u r e s 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  c o n s i d e r e d as a c a n d i d a t e e q u i v a l e n t of the b u r s a i n mammals;  also  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 ( L P S ) - r e s p o n s i v e B l i n e a g e c e l l s , nor even immediate sac ( 8 7 ) .  p r e c u r s o r s of B c e l l s have been d e t e c t e d i n the y o l k  N e v e r t h e l e s s , CFU-S can be d e t e c t e d the murine y o l k sac and  h e m o p o i e t i c stem c e l l s c a p a b l e of lymphoid d i f f e r e n t i a t i o n a r e a l s o p r e s e n t w i t h i n the b l o o d i s l a n d s of t h i s e x t r a e m b r y o n i c t i s s u e i n 8--10 mouse day embryos ( 8 8 ) .  I t has t h e r e f o r e been suggested t h a t h e m o p o i e t i c stem c e l l s  a r i s e de novo o n l y i n t h i s e x t r a e m b r y o n i c t i s s u e and s u b s e q u e n t l y m i g r a t e v i a the b l o o d s t r e a m to c o l o n i z e the v a r i o u s lymphoid and h e m o p o i e t i c 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 b l o o d - f o r m i n g  t i s s u e d u r i n g embryonic development  b e f o r e 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 o f the B  lymphocyte l i n e a g e e x p r e s s i n g the B220 s u r f a c e a n t i g e n appear i n the f e t a l l i v e r on day 12 o f g e s t a t i o n ( 8 8 ) .  At t h i s stage no such c e l l s can be  d e t e c t e d i n e i t h e r the y o l k sac o r the embryonic c i r c u l a t i o n .  Cells  c o n t a i n i n g c y t o p l a s m i c u c h a i n s ( c u ) a r e d e t e c t a b l e i n 10-12 day mouse +  embryos.  The f i r s t s u r f a c e 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 o f g e s t a t i o n (88,90,91). g e s t a t i o n , there i s a dramatic precursors i n f e t a l l i v e r .  Between 13 and 16 days o f  i n c r e a s e i n the number o f committed B - c e l l  These g i v e r i s e to L P S - r e s p o n s i v e  A l l o f these d a t a i n d i c a t e that an important  B-cells (87).  stage o f the development o f the  humoral immune system i n mammals occurs w i t h i n the f e t a l  liver.  The p r o d u c t i o n o f 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 p r i m a r y b l o o d - f o r m i n g f e t u s e s can generate c u l t u r e (87).  tissue.  Thus, l o n g bones from 15-day-old  mouse  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  A f t e r b i r t h , the bone marrow c o n t i n u e s to be the major s i t e o f  B - c e l l formation.  The p r o d u c t i o n o f s m a l l lymphocytes i n the marrow o f mice  has been e s t i m a t e d  t o be a p p r o x i m a t e l y  10^ c e l l s per day ( 9 2 ) .  lymphocytes r e p r e s e n t one q u a r t e r o f the n u c l e a t e d c e l l s ,  the bone marrow a l s o  1  c o n t a i n s p r e c u r s o r c e l l s a t v a r i o u s stages of m a t u r i t y .  While s m a l l  Some o f the newly-  formed s m a l l lymphocytes l e a v e the bone marrow and m i g r a t e  to the s p l e e n .  17  C)  Development of B C e l l s i n the A d u l t  The most i m p o r t a n t 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 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 ) . themselves,  of the humoral immune  B lymphoid p r e c u r s o r s ,  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  that, progressively  i n t o p r e - p r e - 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 . p r o c e s s i s c h a r a c t e r i z e d by a cascade of m o l e c u l a r e v e n t s .  This  I t i s convenient  to c o n s i d e r the p r o c e s s 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 t h r e e phases. The e a r l i e s t  phase of B - c e l l development i s a n t i g e n - i n d e p e n d e n t .  It  b e g i n s w i t h the commitment of p l u r i p o t e n t h e m o p o i e t i c stem c e l l elements the B - c e l l pathway and the i n i t i a t i o n of I g gene rearrangement.  to  Cytoplasmic u  p r o t e i n may then be d e t e c t e d , which i s i n t u r n f o l l o w e d by the e x p r e s s i o n of c e l l surface  Ig.  T h i s s e r v e s as  the a n t i g e n s p e c i f i c r e c e p t o r and  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  represents  (88,91,93).  Mature B - c e l l s capable of r e s p o n d i n g to a n t i g e n e n t e r the c i r c u l a t i o n and then m i g r a t e 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 c o n t a c t w i t h a n t i g e n i n c o n c e r t w i t h secondary f a c t o r s . process c o n s t i t u t e s  This a c t i v a t i o n  the second phase of B c e l l development and  differentiation. Finally,  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 e n t e r S-phase, p r o l i f e r a t e and  b e g i n to produce and s e c r e t e l a r g e amounts of I g i n t o the l y m p h a t i c and vascular spaces.  I n 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 q u i e s c e n t 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 c e l l 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. unlinked genes.  Ig molecules are constructed from three sets of  These encode the various heavy (H) chains, and the K and X  light (L) chains.  Each of these i s , in turn, made up of multiple germline DNA  segments which are somatically assembled during the differentiation of B-lineage cells. region genes.  Ig genes include variable (V) region genes and constant (C)  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) segment (94,95).  (joining)  There are 4 J(H) segments which l i e 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 i e approximately 12 D segments.  At an unknown  distance upstream from the D segments l i e 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  D (12)  V (200-1000) v  x H  v  2 H  v  J (4)  H  H  D  1 H  D  X  H  Enhancer C/x  2  Constant regions(8)  C 5 C-yg C<yi C-y2b ^72a Ce  llll ' Heavy chain gene  VX K  5'-  J* (5)  V (200) V N VJ K  K  a-o-o-  Enhancer I c K  Hill •M  •3'  Kappa gene  V\2 JX2 C\2 J\4 C\4 5  V\1 J\3 C\  3  J\1 C\1  O-i—• •  1  •  Lambda gene  Figure 2.  O r g a n i z a t i o n of immunoglobulin genes.  3'  20  somewhat d i f f e r e n t f a s h i o n w i t h o n l y 2 downstream by 2 J\-C\ The  genes, each of which i s f o l l o w e d  units.  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 A b e l s o n murine  l e u k e m i a v i r u s (A-MuLV) transformed  pre-B c e l l l i n e s r e p r e s e n t i n g the v a r i o u s  s t a g e s 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 v a r i o u s ways i n which V(H), D, and J(H) elements may  be r e a r r a n g e d  (97,98).  Recombination  between the V r e g i o n gene elements i s a p p a r e n t l y 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 p a l i n d r o m i c heptamer  and a c h a r a c t e r i s t i c nonamer, s e p a r a t e d  by a spacer of 12 or 23 base p a i r s  t h a t d i r e c t s the r e c o m b i n a t i o n a l machinery (99,100).  This process  leads  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) DJ(H)  elements.  and  However, random i n s e r t i o n of s m a l l numbers of n u c l e o t i d e s  an enzyme c a l l e d 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 r a n s f e r a s e (TdT) (101,102).  to  A l t et a l (95) have proposed an ordered  may  also  by  occur  mechanism of V(H)DJ(H)  gene c o n s t r u c t i o n d u r i n g 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. chromosomes.  S u b s e q u e n t l y , the complete V(H)DJ(H) v a r i a b l e r e g i o n i s  assembled v i a the appendage o f a V(H) complex.  The  T h i s o c c u r s on both  segment to the p r e - e x i s t i n g DJ(H)  c o m p l e x i t y of the c o m b i n a t o r i a l assortment of the d i f f e r e n t  component gene segments account i n p a r t f o r the d i v e r s i t y of the  specificities  o f the p o p u l a t i o n of a n t i b o d i e s ( i . e . I g ) which c o n s t i t u t e the immune repertoire.  I t has been c a l c u l a t e d that the combination  c h a i n per a n t i g e n - c o m b i n a t i n g antigen-combining  sites  I t i s important segments may  of one H and one  s i t e g i v e s the system a p p r o x i m a t e l y  L  10^ p o s s i b l e  (103).  to p o i n t out t h a t i n c o m p l e t e l y assembled V r e g i o n gene  be e x p r e s s e d ,  and  that o t h e r a b e r r a n t and  rearrangements a l s o occur at a r e l a t i v e h i g h frequency  non-productive (95,104).  Such  21  rearrangements, t h a t a r e 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 i n v o l v e d i n the r e g u l a t i o n of I g V r e g i o n gene assembly ( 9 5 ) . Once V r e g i o n genes are c o m p l e t e l y  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 g e r m l i n e V r e g i o n gene ( 9 5 ) . primary  The  t r a n s c r i p t extends downstream through the exons which encode the  C r e g i o n gene expressed a p p r o p r i a t e RNA  d u r i n g development, the Cy gene.  s p l i c i n g events,  first  Following  the V(H)DJ(H) sequences are fused to the  c o d i n g sequences to form the complete H c h a i n mRNA.  C(H)  H c h a i n V r e g i o n genes  a r e assembled e a r l i e r d u r i n g pre-B c e l l d i f f e r e n t i a t i o n than a r e the genes f o r the L c h a i n s .  However, a s i m i l a r process  i s involved.  Complete messenger f o r  I g H c h a i n s c a r r i e s i n f o r m a t i o n f o r both a membrane-bound and a s e c r e t e d of the y p r o t e i n . molecules,  form  I t has been observed t h a t i n a c t i v e s y n t h e s i s of I g  o n l y one o f a p a i r o f chromosomes i s i n v o l v e d .  T h i s process  is  c a l l e d a l l e l i c e x c l u s i o n (98,105). S t u d i e s of the assembly and e x p r e s s i o n of I g genes d u r i n g the  antigen-  independent s t a g e s of pre-B c e l l d i f f e r e n t i a t i o n has r e v e a l e d a c o n s i s t e n t sequence of events t h a t a r e now development ( F i g u r e 3).  used to d e f i n e d i f f e r e n t s t a g e s o f pre-B  A " n u l l " pre-B c e l l which has DJ(H)  rearrangements on  both chromosomes and no L c h a i n gene rearrangements i s the f i r s t s t a g e can be i d e n t i f i e d .  Such a v e r y e a r l y pre-B c e l l , by making a  V(H)DJ(H) rearrangement, then becomes a c y  +  pre-B c e l l .  cell  that  productive  A f r a c t i o n of  these  pre-B c e l l s then undergo rearrangement and e x p r e s s i o n o f e i t h e r t h e i r K o r X L c h a i n genes i n o r d e r to assemble complete IgM r e c e p t o r s on the c e l l s u r f a c e (106,107). most immature B c e l l s .  s u b u n i t s as a n t i g e n - s p e c i f i c  These s I g M  With f u r t h e r m a t u r a t i o n  s u r f a c e IgD m o l e c u l e s and o t h e r s u r f a c e a n t i g e n s mature B c e l l s a r e capable  of b i n d i n g and  +  B c e l l s c o n s t i t u t e the  B c e l l s begin to (see below).  responding  express  At t h i s  to s p e c i f i c  stage,  antigens.  Peripheral Blood  BONE MARROW Stem Cell  Non-dividing Small Lymphocytes  Proliferating Precursors  Mature B Lymphocytes  o°o°o  O Pre—Pre—B cells  Thy-I low  IIIII  TdT  Pre—B cells  B Lymphocytes  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  B 220 ThB BP-I/6C3  O S/x  HC gene G/G  G/G  G/G  R/G  R/R  R/R  R/R  R/R  LC gene G/G  G/G  G/G  G/G  G/G  G/G-G/R  G/R  G/R  and/or  or  or  R/R  R/R  R/R ro  F i g u r e 3.  Surface a n t i g e n e x p r e s s i o n and I g gene rearrangement d u r i n g murine B - c e l l development.  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 i n i t i a l l y described on T cells but now known to be expressed by most hemopoietic cells (114,115).  Immunofluorescent studies  24  performed w i t h a n t i - B 2 2 0  MoAb's have, however, shown t h a t the e x p r e s s i o n  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 d i f f e r e n t lymphoid organs w i t h a n t i - B 2 2 0  immunofluorescent s t a i n i n g of  showed t h a t about 30% of bone marrow  c e l l s , 50% of s p l e e n c e l l s , 40% of m e s e n t e r i c thymocytes were p o s i t i v e (116).  lymph nodes and  l e s s than 2%  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 demonstrated t h a t B220 i s expressed  subsequently  do not c o n t a i n u c h a i n s (107,111) suggests  t h a t B220 e x p r e s s i o n may  c e l l s a r e TdT+ B220~ cy~.  The  that  precede the  earliest the  third  i n i t i a l l y d e s c r i b e d on mouse myeloma c e l l s , but  (116,117).  Immunofluorescence s t a i n i n g w i t h a n t i - T h B  these a r e s m a l l lymphocytes (116). s l g MoAb demonstrated t h a t a l l T h B  S o r t i n g and +  +  and  on  MoAb  that n e a r l y a l l of  r e s t a i n i n g w i t h e i t h e r B220 o r  c e l l s a r e a l s o B220+ and one h a l f of them  a r e s l g ~ , s i n c e the o t h e r h a l f are s l g  +  c e l l s (116).  the d e f i n i t i o n of a p o p u l a t i o n of s m a l l B 2 2 0 the most mature pre-B c e l l s t a g e .  has  50% of thymocytes, but not  has shown t h a t 15-20% of bone marrow c e l l s a r e T h B  +  The  next c e l l s a r e TdT+ B220+ c y " , and  a l s o been d e t e c t e d on B c e l l s and a p p r o x i m a t e l y  slg  cells  B220+ cy~.  a n t i g e n was  peripheral T cells  +  Very r e c e n t l y , Park and Osmond (111) have r e p o r t e d  e x i s t e n c e of t h r e e d i s c r e t e cy~ l a r g e pre-B c e l l p o p u l a t i o n s .  The ThB  both  Some f i n d i n g s  i n d i c a t e t h a t mouse bone marrow a l s o c o n t a i n s a p o p u l a t i o n of B 2 2 0  p o p u l a t i o n i s TdT~  cells  by a l l B lymphocytes, as w e l l as by  l a r g e pre-B c e l l s i n mouse bone marrow (116).  y chain synthesis.  of  Experiments u s i n g 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 a n a l y z e and s e p a r a t e doubly immunofluorescent  s m a l l and  of  +  These d a t a have a l l o w e d  ThB+ s l g  These mature w i t h o u t  -  c e l l s that  represent  further division  into  B c e l l s e x p r e s s i n g both y and L c h a i n s . Thy-1  was  be d i s c o v e r e d  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 a n t i g e n s (118,119).  I t was  to  i n i t i a l l y d e s c r i b e d as a marker of thymocytes  25  and  thymus-dependent c e l l s i n the s p l e e n (118).  amounts of Thy-1  L a t e r , i t was  shown t h a t low  a r e d e t e c t a b l e on p l u r i p o t e n t stem c e l l s (120).  This  a l l o w e d the d i f f e r e n t i a t i o n of Thy-l (l° ) stem c e l l s i n t o pre-pre-B +  has  cells  w  to  be i n v e s t i g a t e d . U s i n g a c o c k t a i l of monoclonal a n t i b o d i e s d i r e c t e d a g a i n s t B220, Thy-1, and d i f f e r e n t mature T c e l l and m y e l o i d a n t i g e n s to s o r t normal bone marrow c e l l s u b p o p u l a t i o n s w i t h the c a p a c i t y 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 b e l o w ) , 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 p r e c u r s o r s were i s o l a t e d (121). B 2 2 0 ) 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 B 2 2 0 -  produced but o n l y f o r ~1 weeks.  The second ( T h y - l ( +  c e l l s are  +  l o w  One  ),B220 ) -  two  (Thy-1~,  rapidly can  initiate  l o n g - t e r m 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  in  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 t h a t t h i s second p o p u l a t i o n (-0.1% BP-1  was  of bone marrow) c o n t a i n s p l u r i p o t e n t h e m o p o i e t i c  r e c e n t l y d e s c r i b e d by Cooper et a l and i s another s u r f a c e 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). MoAb s t a i n s a p p r o x i m a t e l y  r e l a t i v e l y low on normal pre-B c e l l s . h i g h e r e x p r e s s i o n of BP-1  The s t a i n i n g i n t e n s i t y o f BP-1 Transformed pre-B c e l l s e x h i b i t  c e l l (122).  I n t e r e s t i n g l y , BP-1  BP-1  an  the  by 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 h i g h  tumorigenic  R e c e n t l y , i t has a l s o been shown t h a t s t r o m a l c e l l  t h a t 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 the 6C3 e p i t o p e .  is  i s a homodimeric  i s immunoprecipitated  Mo-AB (6C3) p r e v i o u s l y d e s c r i b e d by P i l l e m e r et a l as a  p o t e n t i a l (123).  anti-BP-1  which d e c r e a s e s , however, as a f u n c t i o n of  m a t u r a t i o n a l s t a g e of the transformed g l y c o p r o t e i n of 140 kD.  By FACS a n a l y s i s ,  10% of n u c l e a t e d c e l l s i n the bone marrow o f which  90% a r e pre-B and newly formed B c e l l s .  express  stem c e l l s .  Comparative s t u d i e s between BP-1  lines  LTC's, a l s o  and 6C3 MoAb  i n d i c a t e t h a t both i d e n t i f y the same a n t i g e n but a d i f f e r e n t e p i t o p e .  The  26  possibility  t h a t BP-1/6C3 may  play a r o l e i n stromal cell-dependent  pre-B  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  cell  has  t h e r e f o r e been the s u b j e c t 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.  bone marrow, s l g components and  +  Soon a f t e r they a r e formed i n the  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  surface  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 e n t e r the c i r c u l a t i o n and m i g r a t e  to  s p e c i f i c r e g i o n s i n the s p l e e n , lymph nodes and o t h e r secondary lymphoid tissues.  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  d i f f e r e n t i a t e i n t o I g 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  and  antigen-  dependent phase of 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 the p a r t i c i p a t i o n of h e l p e r T c e l l s and a c c e s s o r y  c e l l s of the monocyte-macrophage l i n e a g e .  A number o f  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 s u b p o p u l a t i o n s 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  with  to c h a r a c t e r i z e d i s c r e t e s t e p s i n the  development of plasma c e l l s from a n t i g e n - s t i m u l a t e d B c e l l s . The  appearance of s l g D on the m a j o r i t y of s I g M  +  c e l l s has s t i m u l a t e d a  g r e a t d e a l of i n t e r e s t w i t h r e g a r d to the r o l e o f these I g ' s i n B function.  D u r i n g f e t a l development and  cell  the 2 f i r s t weeks of p o s t - n a t a l  life,  i t was  shown t h a t & f i r s t appears on sIgM+ c e l l s t h a t bear h i g h d e n s i t i e s o f  slgM.  However, w h i l e the amount of slgM on i n d i v i d u a l B c e l l s d i m i n i s h e s ,  amount of s l g D i n c r e a s e s .  Thus, the r a t i o of y/S  on the t o t a l B  cell  p o p u l a t i o n changes from a v e r y h i g h number f o r f e t a l mice to a p p r o x i m a t e l y i n a d u l t mice (125).  the  Even i n the a d u l t , two major groups of p e r i p h e r a l I g M  one +  B  c e l l s can be d i s t i n g u i s h e d : those w i t h l i t t l e or no s l g D and a heterogeneous d i s t r i b u t i o n of slgM (sIgM3+ s l g D ) , and -  amounts of slgM and  those w i t h  low-to-intermediate  i n t e r m e d i a t e - t o - h i g h amounts of slgD ( s I g M  +  sIgD3+) ( 1 2 5 ) .  27  D u r i n g the p r o g r e s s i v e d i f f e r e n t i a t i o n o f 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 , s l g D e x p r e s s i o n i s l o s t b e f o r e slgM. not y e t c l e a r .  The r o l e o f coexpressed  slgM and s l g D i s  I t appears t h a t t h e i r a n t i g e n - b i n d i n g 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 identical s i g n a l s t o the c e l l i n response thymic-independent I a molecules  (126).  However, they may convey d i f f e r e n t  t o e i t h e r thymic-dependent (TD) o r c l a s s 1  (TI) antigens  (127).  r e p r e s e n t an important s u r f a c e component o f B c e l l s  because  they s e r v e 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 o f 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  glycoproteins are  encoded by genes w i t h i n the m i d p o r t i o n o f the murine major h i s t o c o m p a t i b i l i t y complex (MHC) and a r e t h e r e f o r e a l t e r n a t i v e l y r e f e r r e d t o as MHC c l a s s I I molecules.  They a r e expressed  on s u b p o p u l a t i o n s o f 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 ( 9 1 ) .  However, immature I g M  +  B cells i n  f e t a l l i v e r , bone marrow, and s p l e e n o f n e o n a t a l mice express l i t t l e o r no d e t e c t a b l e I a (128,129).  During B c e l l maturation, I a determinants a r e  a c q u i r e d i n p a r a l l e l w i t h s l g by newly formed B c e l l s i n the marrow  (91,130).  Two o t h e r a n t i g e n s , the Fc r e c e p t o r (FcR) f o r the Fc p o r t i o n o f I g and the mouse B lymphocyte a n t i g e n (MBLA) have been d e t e c t e d on a l l s l g f e t a l and a d u l t mice ,but not pre-B c e l l s (131,132).  +  c e l l s of  While MBLA i s unique t o  B c e l l s , FcR i s a l s o found on p h a g o c y t i c c e l l s where i t s p l a y s an i m p o r t a n t r o l e i n the i n t e r n a l i z a t i o n o f a n t i b o d y - c o a t e d b a c t e r i a (133).  p a r t i c l e s such as o p s o n i z e d  The f u n c t i o n a l r o l e o f MBLA o r FcR on B c e l l s i s unknown.  However, t h e r e i s evidence  t h a t the B c e l l FcR may a c t to r e g u l a t e the  magnitude o f t h e B c e l l response  t o a g i v e n a n t i g e n (134).  In c o n t r a s t to both FcR and MLBA, the complement r e c e p t o r (CR) m o l e c u l e and minor lymphocyte s t i m u l a t i n g ( M i s ) a n t i g e n s a r e not expressed murine B c e l l s .  The CR i s found on macrophages, g r a n u l o c y t e s and  on a l l  28  approximately  50% o f s p l e n i c B c e l l s .  The absence o f CR  +  B c e l l s i n the bone  marrow s u g g e s t s t h a t t h i s r e c e p t o r marks a l a t e - d e v e l o p i n g B c e l l subpopulation  (91).  F u r t h e r support f o r t h i s was o b t a i n e d  d e m o n s t r a t i o n t h a t when a d u l t B c e l l s were separated populations, expression IgD.  from the  i n t o CR  o f CR was found t o begin a f t e r I g M  +  +  and CR~  c e l l s had a c q u i r e d  However, B c e l l s cease t o express CR a f t e r they a r e a c t i v a t e d ( 9 1 ) . The  M i s d e t e r m i n a n t s on the s u r f a c e o f lymphocytes a r e encoded by a  s i n g l e gene o r gene complex d i s t i n c t from the MHC. responsible for p r o l i f e r a t i v e  These d e t e r m i n a n t s a r 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 , M i s d i s t i n c t s t r a i n s a r e 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 a r e mature and not  immature B c e l l s (135,136). Both CR and M i s 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 of B lymphocytes.  Three s I g D  e a r l i e s t appearing  are IgM  +  +  B c e l l p o p u l a t i o n s have been i d e n t i f i e d . The  ( h i g h ) , F c , MBLA+, I a , I g D . +  +  IgM+ ( l o w - i n t e r m e d i a t e ) , Fc+, MBLA+, Ia+, IgD+, CR MBLA+, Ia+, IgD+, CR+, M l s  +  subpopulations  +  +  The next two a r e  and IgM+ ( l o w ) , F c , +  (91).  A number o f 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 s u r f a c e o f B cells.  These a r e 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 w i t h s p e c i f i c a n t i s e r a developed a g a i n s t Lyb d e t e r m i n a n t s ,  i t has been p o s s i b l e t o 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 s u b p o p u l a t i o n s .  CBA/N mice c a r r y an X l i n k e d gene, x i d , t h a t  causes a d e f e c t i n c e r t a i n B c e l l f u n c t i o n s (137,138). c e l l s are Lyb-3 , Lyb-5 +  +  and L y b - 7  +  The l a t e s t - a p p e a r i n g B  and thus can be c h a r a c t e r i z e d as I g M  ( l o w ) , F c , MBLA+, Ia+, IgD+, CR+, Mls+, Lyb-3+, 5+, 7+ ( 9 1 ) . +  s t u d i e s have shown t h a t L y b - 3 , 5 , 7 +  +  +  +  Functional  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 t o thymus-independent type 2 a n t i g e n s , a r e  29  c r i t i c a l f o r B c e l l responses t o low doses o f thymus-dependent a n t i g e n s and are  a l s o i m p o r t a n t f o r the IgM t o IgG s w i t c h d u r i n g a p r i m a r y immune response.  The absence o f L y b - 3 , 5 +  +  and 7  +  d e t e r m i n a n t s on the s u r f a c e o f the B c e l l s o f  a d u l t x i d mice s u g g e s t s t h a t the immune d e f e c t s i n these a n i m a l s may be e n t i r e l y due to the l a c k o f t h i s s u b p o p u l a t i o n ( 9 1 ) .  T h i s i s s u p p o r t e d by the  f i n d i n g t h a t both T c e l l s and a c c e s s o r y c e l l s a r e normal i n x i d mice ( 8 8 ) . Moreover,  the r e l a t i v e l y good secondary IgG response induced by thymus-  dependent a n t i g e n s i n CBA/N mice suggests t h a t the c e l l s t h a t a r e a c t i v a t e d t o 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 a r e d i s t i n c t , a t l e a s t i n p a r t , from the L y b - 3 , 5 , 7 +  3)  +  +  B c e l l p o p u l a t i o n (88,91,93).  REGULATION OF B CELL DEVELOPMENT  J u s t as our knowledge and a b i l i t y t o study B c e l l p o p u l a t i o n s preceded the c h a r a c t e r i z a t i o n o f pre-B c e l l s , so t o has i n f o r m a t i o n about the r e g u l a t i o n o f 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 o f m o l e c u l a r r e g u l a t o r s o f 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 i n s t a n c e s MoAb t o these lymphokines have a l s o been o b t a i n e d ( 5 1 ) .  The  a v a i l a b i l i t y o f the^se m o l e c u l e s 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 o f the antigen-dependent  phase o f B  c e l l development t o be d e f i n e d . The mechanisms t h a t r e g u l a t e the commitment o f p l u r i p o t e n t stem c e l l s t o the B c e l l pathway and t h a t r e g u l a t e the s i z e s o f the p o p u l a t i o n s o f the d i f f e r e n t subsequent unknown.  s t a g e s o f pre-B c e l l development a r e s t i l l  largely  However, r e c e n t l y s e v e r a l l i n e s o f e v i d e n c e o b t a i n e d from both  a n i m a l models and LTC's ( s e e below) have suggested that s t r o m a l 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 i n i t i a l l y  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 c e l l s , 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 o f e i t h e r SRBC, LPS o r adherent l a y e r s o f p e r i t o n e a l exudate macrophages (146).  F u r t h e r c h a r a c t e r i z a t i o n o f the c e l l s t h a t can be c l o n e d  i n s e m i s o l i d agar has shown t h a t the CFU-B assay can d e t e c t B - l i n e a g e a v a r i e t y o f m a t u r a t i o n s t a g e s i n c l u d i n g those that a r e I g D and  I a and I a ( 8 8 ) . +  -  +  c e l l s at  and IgD~ s t a g e s  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 m y e l o i d committed  p r o g e n i t o r s do. Recently, (147).  a clonogenic  assay f o r B c e l l p r e c u r s o r s  has been  described  A l t h o u g h , pre-B c e l l s t h a t do not express membrane IgM a r e not c a p a b l e  of f o r m i n g 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 a r e c a p a b l e t o form c o l o n i e s i n agar i f they a r e a l l o w e d  to d i f f e r e n t i a t e to s l g  +  B cells  e i t h e r p r i o r t o s o f t agar c u l t u r e o r a f t e r c u l t u r e i n the presence o f f e e d e r l a y e r s o f 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). double-layer  Thus, P a i g e d e s c r i b e d  agar assay i n which B c e l l p r e c u r s o r s  a l r e a d y p r e s e n t i n 12-day  f e t a l l i v e r can g e n e r a t e c o l o n i e s which c o n t a i n a n t i b o d y - s e c r e t i n g w i t h i n 8 days.  Colony growth r e q u i r e s s o l u b l e mediators p r o v i d e d  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 . antibody  by p l a s t i c -  The d e t e c t i o n o f  produce both K and X L c h a i n s  (150).  The c o l o n i e s  T h i s assay system d e t e c t s  t h a t d i f f e r from mature CFU-B (148) and p o i n t s out t h a t B c e l l growth i s dependent on adherent a c c e s s o r y  (b)  cells  s e c r e t i o n i s a c h i e v e d a f t e r t r a n s f e r o f the uppermost agar l a y e r t o a  g l a s s s l i d e , by a m o d i f i c a t i o n o f the J e r n e plaque assay. obtained  a  precursor  cells.  I L - 3 Dependent B-Lineage C e l l L i n e s .  mouse B lymphocytes were f i r s t d e s c r i b e d  cells  Long-term c u l t u r e s o f normal  by Howard e t a l .  They showed t h a t  s p l e n i c B c e l l s c o u l d be m a i n t a i n e d f o r 10 months i n media c o n d i t i o n e d by C o n - A - s t i m u l a t e d s p l e e n c e l l s (149).  P a l a c i o s e t a l s u b s e q u e n t l y r e p o r t e d 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 - c e l l 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  Lymphoid LTC  Myeloid LTC  8  ••••I • • •  1 0 b o n e marrow cells/ml 6  UtW  ° 1 2 . 5 % HS 1 2 . 5 % FCS 1 0 0 / i M 2 - mercaptoethanol 3  3  3 7 ° 5 % FCS 5 0 iiM 2 - mercaptoethanol culture medium (RPMI 1 6 4 0 )  culture medium (a) 1 0 ~ M hydrocortisone 6  F i g u r e 4.  D e s c r i p t i o n o f the d i f f e r e n t types o f l o n g - t e r m 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  The d i f f e r e n t c o n d i t i o n s o b t a i n e d i n  two types of LTC r e s u l t i n a d i f f e r e n t morphology  seen i n the adherent l a y e r .  of the " s t r o m a l "  I n m y e l o i d LTC's, the s t r o m a l c e l l s  cells  include  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 a d i p o c y t e s and p h a g o c y t i c mononuclear  c e l l s (158).  I n lymphoid LTC's the adherent  cell  p o p u l a t i o n does not appear to c o n t a i n l i p i d - c o n t a i n i n g a d i p o c y t e s and i s made of  l a r g e , a d h e r e n t , mononuclear  c e l l s w i t h numerous pleomorphic c y t o p l a s m i c  processes. A l t h o u g h c o n d i t i o n s i n m y e l o i d 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 a r e t o x i c f o r B c e l l s , a r e c l e a r l y not s u i t a b l e f o r s u p p o r t i n g 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 m y e l o i d LTC  can  be c o n v e r t e d to p r o d u c t i v e lymphoid LTC's by s i m p l y s w i t c h i n g the c u l t u r e s to lymphoid LTC maintenance does not work.  c o n d i t i o n s (34,159). However, the o p p o s i t e experiment  I f lymphoid LTC's a r e s w i t c h e d back to D e x t e r c o n d i t i o n s , o n l y  low l e v e l s of t r a n s i e n t m y e l o p o i e s i s i s observed ( 3 4 ) .  These f i n d i n g s show  t h a t t h e r e a r e c e l l s i n m y e l o i d LTC's that can support B l y m p h o p o i e s i s and suggested t h a t the f a i l u r e of l y m p h o p o i e s i s i n m y e l o i d LTC's i s not due to an i n a d e q u a t e "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 p a r e n t .  The e s t a b l i s h m e n t of lymphoid LTC's i s h i g h l y r e p r o d u c i b l e 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).  S u p e r i o r r e s u l t s have been c l a i m e d 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). t y p i c a l l y p r o g r e s s through d i s t i n c t i v e phases.  The  cultures  D u r i n g the two f i r s t weeks,  w h i l e the adherent l a y e r i s f o r m i n g , the number of non-adherent declines.  cells  D u r i n g the next few weeks, t h e r e i s a " c r i s i s " phase when few non-  adherent c e l l s can be d e t e c t e d i n the c u l t u r e s .  Then, patches of s m a l l non-  adherent c e l l s reappear i n the adherent c e l l l a y e r and these s u b s e q u e n t l y  35  expand i n number.  Most o f these c e l l s resemble s m a l l lymphocytes,  some appear t o be b l a s t s . adherent  The f i r s t e v i d e n c e t h a t B c e l l s a r e among the non-  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  These shoved a l a r g e amount o f H and L c h a i n s y n t h e s i s ( 3 3 ) . of  ^H-thymidine  although  experiments.  Autoradiography  l a b e l l e d c e l l s i n d i c a t e d t h a t a l a r g e p r o p o r t i o n o f 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 t h a t the c u l t u r e s  i n the f i r s t t h r e e months c o n t a i n e d 10-20% membrane I g - b e a r i n g c e l l s . However, a t 15-20 weeks, s u b p o p u l a t i o n s o f e i t h e r pre-B o r B c e l l s tend t o dominate the c u l t u r e s , and the p a t t e r n o f H and L c h a i n s s y n t h e s i z e d s u g g e s t s t h a t the lymphoid c e l l s have become p a u c i c l o n a l ( 3 3 ) . e x p r e s s a low l e v e l o f B220. h i g h amounts o f TdT (106).  10-30% o f the c e l l s  These s y n t h e s i z e few i f any H c h a i n s but produce A n a l y s i s o f rearrangements  a t the I g H c h a i n l o c u s  has shown t h a t 10-30% o f the DNA c o n t a i n e d H c h a i n sequences i n an unrearranged, germline s t a t e .  These d a t a i n d i c a t e t h a t the n u l l c e l l  lymphoid  p o p u l a t i o n present represent very e a r l y B-lineage c e l l s . Thus, lymphoid LTC's a l l o w 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 o f v e r y e a r l y B c e l l p r e c u r s o r s to I g - b e a r i n g B c e l l s i n the presence o f a s t r o m a l c e l l l a y e r .  The i n t r o d u c t i o n o f t h i s c u l t u r e system has  p r o v i d e d a p o w e r f u l t o o l t o study the r e g u l a t i o n o f pre-B c e l l l y m p h o p o i e s i s . At l e a s t t h r e e d i f f e r e n t a s p e c t s o f 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 o f the m o l e c u l a r changes t h a t 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 , a r e +  s e n s i t i v e t a r g e t s f o r A-MuLV ( 1 6 0 ) , and A-MuLV transformed c e l l  lines  r e p r e s e n t i n g d i f f e r e n t s t a g e s of e a r l y B c e l l ontogeny can be e a s i l y o b t a i n e d 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 o f I g gene l o c i  i n these  c e l l s a f t e r c o n t i n u e d maintenance has shown t h a t some A-MuLV t r a n s f o r m e d s u b c l o n e s can undergo e x t e n s i v e I g gene rearrangement  shortly after  viral  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 a n t i g e n s ( 9 1 ) .  However, T c e l l s , a c c e s s o r y  and s t r o m a l c e l l s a r e normal i n these x i d mice (88,138).  Such CBA/N mice have  been used by K u r l a n d (164) i n o r d e r t o demonstrate the presence p r e c u r s o r s u b p o p u l a t i o n i n lymphoid from lymphoid  LTC's.  of a B - c e l l  He showed t h a t i n j e c t i o n o f c e l l s  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  (CBA/NxBALB/c)  cells  irradiated  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 o f the  B-lymphocyte compartment o f the i n j e c t e d mice. (ii) determined  SCID Mice.  s e v e r e combined immune d e f i c i e n c y d i s e a s e (SCID) (165,166).  mouse s t r a i n almost  Another animal model i s t h a t b e a r i n g a g e n e t i c a l l y  i s homozygous f o r an autosomal m u t a t i o n which r e s u l t s i n the  complete absence o f normal B and T lymphocytes and serum I g .  marrow o f SCID mice have no d e t e c t a b l e pre-B o r B c e l l s . hemopoietic  Bone  However, o t h e r  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 , m y e l o i d c e l l s , and  a n t i g e n - p r e s e n t i n g c e l l s appear t o be u n a f f e c t e d by the d e f e c t SCID marrow c e l l s a r e not a b l e t o r e c o n s t i t u t e the lymphoid i r r a d i a t e d normal h o s t s (168). from normal lymphoid (169).  This  (165-167).  systems o f  I n c o n t r a s t , normal bone marrow o r even c e l l s  LTC's a r e a b l e t o f u l l y c o r r e c t the d e f e c t i n SCID mice  T h i s i n d i c a t e s t h a t 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 t o a  lymphoid  p r o g e n i t o r p o p u l a t i o n and i s not due t o a d e f e c t i v e microenvironment p r o v i d e d by n o n - t r a n s p l a n t a b l e s t r o m a l c e l l s (169). s u c c e s s f u l l y i n i t i a t e lymphoid  LTC's (170).  SCID marrow can be used t o T h i s shows t h a t SCID mice have  p r o g e n i t o r s o f B c e l l s t h a t a r e a b l e t o p r o l i f e r a t e i n v i t r o , a l t h o u g h they do not g i v e r i s e t o c u c e l l s . +  Southern  b l o t a n a l y s i s o f the c e l l s produced i n  these SCID LTC's demonstrated t h a t a b e r r a n t o r n o n p r o d u c t i v e  H gene  rearrangements a r e u n u s u a l l y common (170).  f o r the absence  of pre-B and B c e l l s i n SCID mice.  T h i s may account  38  R e c e n t l y , SCID mice have a l s o been used to s t u d y 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 a r e composed  p r e d o m i n a n t l y of pre-pre-B lymphocytes which have not undergone I g gene rearrangement and do not c o n t a i n s I g M of  f e t a l l i v e r LTC c e l l s  c e l l s and serum IgM.  cells.  +  Nevertheless,  transplantation  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  However, T lymphocyte r e c o n s t i t u t i o n was not o b s e r v e d  and serum IgG l e v e l s were v e r y low i n d i c a t i n g i m p a i r e d 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 w i t h these f e t a l  liver  LTC c e l l s , the B lymphocytes became a b l e to undergo c l a s s s w i t c h i n g and responded n o r m a l l y to T-dependent a n t i g e n s (171).  Thus, the use of LTC and  SCID r e c i p i e n t s has a l l o w e d the s e p a r a t i o n of B lymphocyte development  into 3  s t a g e s : 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 , p r o g r e s s i o n 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 . (iii)  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 m u t a t i o n , motheaten are  (me/me) or v i a b l e motheaten  (me/me ), v  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 d e v e l o p  autoimmune d i s e a s e c h a r a c t e r i z e d by hyperimmunoglobulinemia, e x p r e s s i o n o f m u l t i p l e a u t o a n t i b o d i e s 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). die  Because of the s e v e r e immunologic d i s t u r b a n c e s i n these mice, they  a t an e a r l y age.  S e v e r a l s t u d i e s have shown t h a t the development  prothymocytes, pre-B c e l l s , and T d T  +  of  lymphoid p r e c u r s o r s i n the bone marrow o f  motheaten mice i s d e f e c t i v e because of a l a c k of an a p p r o p r i a t e microenvironment f o r the g e n e r a t i o n of T d T  +  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 r e p o r t e d t h a t the s t r o m a l l a y e r of motheaten c u l t u r e s i s overgrown by a m a c r o p h a g e - l i k e c e l l p o p u l a t i o n and me/me c e l l s suppress the growth o f normal v  p r o g e n i t o r s i n lymphoid LTC's (176).  Thus, motheaten  (me/me) o r v i a b l e  39  motheaten (me/me ) mice represent an interesting model in which the severe v  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 c e l 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).  T a b l e 2.  B C e l l Growth F a c t o r s  E f f e c t s on B C e l l s  Name  Interleukin-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 proliferation & differentiation of A g - s t i m u l a t e d B c e l l s m a t u r a t i o n of pre-B c e l l s  Interleukin-2 (IL-2)  Induces: p r o l i f e r a t i o n w i t h a n t i - I g + LPS proliferation & differentiation of A g - s t i m u l a t e d B c e l l s d i f f e r e n t i a t i o n with anti-Ig + IL-4  Interleukin-3 (IL-3)  Supports growth of pre-B c e l l  Interleukin-4 (IL-4)  Induces p r o l i f e r a t i o n w i t h 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 e x p r e s s i o n 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» g 2 b ' S g 3 s e c r e t i o n by L P S - a c t i v a t e d B c e l l s x  G  J  M  &  x  lines  G  Interleukin-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 L P S - s t i m u l a t e d B c e l l s T IL-2 r e c e p t o r e x p r e s s i o n  Interleukin-6 (IL-6)  Induces growth of B c e l l hybridomas & plasmacytomas  Interferon Y  Induces: I g s e c r e t i o n w i t h IL-2 I g s e c r e t i o n by r e s t i n g B c e l l s t IgG , I IgG Gi G by LPS s t i m u l a t e d B c e l l s 4- IL-4 a c t i v i t y  (IFN-Y)  2 a  From R e f e r e n c e 51.  2 b  3  41  F a c t o r s t h a t r e g u l a t e e a r l y s t a g e s o f B c e l l development most p a r t not been w e l l c h a r a c t e r i z e d .  have f o r the  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 s t r o m a l c e l l l i n e s s u p p o r t pre-B c e l l growth and/or d i f f e r e n t i a t i o n  that  (60,124,139,140,142).  D o r s h k i n d e t a l (142) r e c e n t l y d e s c r i b e d the c h a r a c t e r i s t i c s o f a l i n e (S17) d e r i v e d from the adherent l a y e r of Dexter LTC which can s u p p o r t B c e l l differentiation.  W h i t l o c k e t a l (124) have s i m i l a r l y i s o l a t e d c l o n e d s t r o m a l  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 o f e a r l y B p r o g e n i t o r s i n t o B220+ and c y  +  c e l l s but w i t h o u t 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 s u p p o r t s the i n v i t r o growth o f e a r l y lymphoid and m y e l o i d c e l l s has been d e s c r i b e d by Hunt e t a l ( 1 4 0 ) , and Quesenberry and co-workers have d e s c r i b e d an adherent marrow  cell  l i n e (TC-1) t h a t 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 s u p p o r t s pre-B c e l l growth ( 6 0 ) . Hunt e t a l (140) a l s o showed t h a t ALC produce a f a c t o r t h a t s t i m u l a t e s the p r o l i f e r a t i o n o f pre-B c e l l s .  cells  Using a  c l o n a l pre-B c e l l l i n e they showed t h a t 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 m o l e c u l e .  A pre-B s t i m u l a t i n g f a c t o r o f 25 kD has a l s o been r e c e n t l y  d e s c r i b e d , p u r i f i e d and c l o n e d by Namen (178,179).  The TC-1 a c t i v i t y  reported  by Quesenberry appears to be a h i g h m o l e c u l a r weight g l y c o p r o t e i n ( 6 0 ) . L a n d r e t h (142) p u r i f i e d two f a c t o r s from the c o n d i t i o n e d medium o f S17 c e l l s , a 50-60 kD m o l e c u l e and a 10 kD m o l e c u l e .  Both induce B220 and cy p r o t e i n  e x p r e s s i o n i n pre-B c e l l s that have not y e t begun to e x p r e s s these markers. I n t e r e s t i n g l y , none o f 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 s t r o m a l c e l l s i s o l a t e d to date have been found t o s e c r e t e d e t e c t a b l e I L - 3 and none o f 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 h e m o p o i e t i c growth f a c t o r s .  I t thus appears t h a t the r e g u l a t i o n o f  the  may be complex i n v o l v i n g s e v e r a l new  e a r l y e v e n t s o f B c e l l development  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 c e l l s . 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 c e l l 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 l e is known about the role  or mechanisms by which stromal cells and B c e l l progenitors might interact directly.  Bernard! (38) have recently shown that precursor B lymphoid c e l l  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 c e l l 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 c e 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 c e l l maturation and that these multiple receptors may play a role in normal lymphoid precursor c e l 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 s u g g e s t i n g requires signals  t h a t o p t i m a l pre-B c e l l s t i m u l a t i o n  from s t r o m a l c e l l s that are not a c h i e v e d w i t h o u t c e l l  contact.  4)  THESIS OBJECTIVES D u r i n g the l a s t  ten y e a r s ,  numerous advances have been made i n the  development of new t e c h n o l o g i e 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 o f B c e l l development.  The g e n e r a t i o n of MoAb a g a i n s t B c e l l  lines  has a l l o w e d 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 progenitors.  M o l e c u l a r a n a l y s i s of I g gene s t r u c t u r e and I g gene  rearrangement  i n i s o l a t e d c l o n e s has l e d to an u n d e r s t a n d i n g o f the  g e n e t i c m o d i f i c a t i o n s that l e a d to the e x p r e s s i o n of cy and s l g .  successive  The  d i s c o v e r y of a system f o r s u p p o r t i n g the l o n g - t e r m p r o l i f e r a t i o n of normal p r e - 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 r e p r e s e n t an i n v i t r o  model o f B c e l l development and have a l l o w e d mechanisms of s t r o m a l mediated c o n t r o l of p r e - 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 b e g i n to be defined. However, a number of major q u e s t i o n s s t i l l  remain u n s o l v e d .  FACS  a n a l y s i s has shown t h a t p l u r i p o t e n t and B-committed stem c e l l s e x p r e s s different  phenotypes  (121), and  a comparison between m y e l o i d and lymphoid  L T C ' 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 t h a t  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 s u b j e c t  to e x t r i n s i c i n f l u e n c e s .  44 A second q u e s t i o n concerns the d e f i n i t i o n of how feedback, c o n t r o l o f B c e l l production i s maintained i n v i v o . c e l l s give r i s e i n a step-wise  I t has been shown t h a t B - l i n e a g e  f a s h i o n a s s o c i a t e d 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 l y m p h o c y t e s .  Somehow, the p r o d u c t i o n and death of  p e r i p h e r a l b l o o d lymphocytes i s kept c o n s t a n t  throughout a d u l t l i f e .  s u g g e s t s 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 between these two parameters.  elucidated. exist,  Indeed,  exert  This  balance  A l t h o u g h i t has been shown v e r y r e c e n t l y t h a t  s t r o m a l c e l l s can s e c r e t e s e v e r a l pre-B growth f a c t o r s , by which these f a c t o r s  stem  the a c t u a l mechanism  t h e i r e f f e c t s on pre-B c e l l s has not y e t been  i t i s not yet c l e a r how many d i f f e r e n t p r e - B f a c t o r s  may  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  p o p u l a t i o n s been  established.  The major g o a l of the work undertaken f o r t h i s t h e s i s was to d e f i n e at c e l l u l a r and m o l e c u l a r l e v e l mechanisms t h a t r e g u l a t e normal and p r e - B lymphoid c e l l s by s t r o m a l c e l l s . y e a r s ago,  v e r y l i t t l e was known about  the work were t h e r e f o r e as  transformed  When t h i s p r o j e c t was s t a r t e d the n a t u r e of the i n t e r a c t i o n s  o c c u r between s t r o m a l c e l l s and pre-B c e l l s ( 1 0 6 ) .  three that  The s p e c i f i c o b j e c t i v e s  f o l l o w s : to determine whether s t r o m a l c e l l s  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 ) ;  a  of  secrete  to determine whether a d i r e c t  i n t e r a c t i o n between s t r o m a l c e l l s and pre-B c e l l s p l a y s a r o l e i n s t r o m a l mediated support of pre-B c e l l g r o w t h ; and f i n a l l y  to determine whether e ' i t h e r  o f these mechanisms are a l t e r e d i n transformed pre-B c e l l p o p u l a t i o n s  that  have become m a l i g n a n t and c a p a b l e of autonomous growth i n v i t r o . I n o r d e r to f u l f i l l  the o b j e c t i v e s  o p e r a t i v e i n lymphoid L T C ' s appeared However, the f a c t  c i t e d above,  analysis  of  mechanisms  to be the most u s e f u l s t a r t i n g p o i n t .  t h a t such L T C ' 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  cells  45 at d i f f e r e n t s t a g e s of B c e l l development, cloned regulatory (stromal) would f a c i l i t a t e  and responder  suggested t h a t the i s o l a t i o n o f (pre-B)  the s t u d i e s to be i n i t i a t e d .  c e l l constituent  populations  Thus, the f i r s t o b j e c t i v e was  to g e n e r a t e and c h a r a c t e r i z e such l i n e s and then to d e f i n e 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 p r e - B growth i s  regulated.  The f i r s t q u e s t i o n to be asked was whether s t r o m a l c e l l s s t i m u l a t e cells  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 ) o r whether d i r e c t  contact  is required.  pre-B  cell  To address t h i s q u e s t i o n r e q u i r e d the use of an  e x p e r i m e n t a l system where i t would be p o s s i b l e (if  cell  to measure the s t i m u l a t o r y r o l e  any) of s t r o m a l 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 c o n t a c t between these two c e l l  types.  The next q u e s t i o n was  to  d e t e r m i n e whether any ECM component a n d / o r m e t a b o l i c a l l y i n a c t i v e ( i . e . with glutaraldehyde)  fixed  s t r o m a l c e l l s c o u l d a l o n e 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 m a l i g n a n t of p r e - B c e l l s a l t e r s  transformation  t h e i r i n t e r a c t i o n s w i t h s t r o m a l c e l l s and the n a t u r e o r  b a s i s o f any changes f o u n d .  A l t h o u g h 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 d e r i v e d 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 f e a t u r e s 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 l o n e normal pre-B c e l l l i n e s and then d e r i v e cloned transformants  from these c e l l s u s i n g A-MuLV.  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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 s u p p o r t s growth of mouse pre-B c e l l c l o n e s 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 c l o n e s t h a t e x p r e s s B-220 s u r f a c e a n t i g e n , c o n t a i n I g genes i n g e r m - l i n e c o n f i g u r a t i o n , and g e n e r a t e 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, N e r i 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 a n t i b o d y 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 d e f i n e s two major p o p u l a t i o n s of B c e l l p r e c u r s o r s i n the bone marrow. Immunol Rev 93:125, 1986.  155.  Schrader JW, Schrader S. I n v i t r o s t u d i e s on lymphocyte differentiation. 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.  D o r s h k i n d 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 p r e c u r s o r s p r e s e n t i n l o n g - t e r m bone marrow c u l t u r e s . J Immunol 131:2240, 1983.  157.  W h i t l o c k CA, Robertson D, W i t t e ON. Murine B c e l l l y m p h o p o i e s i s i n l o n g term c u l t u r e . J Immunol Meth 67:353, 1984.  158.  D o r s h k i n d K, W i t t e ON. Long-term murine h e m o p o i e t i c 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, W i t t e ON. I n v i t r o development of B lymphocytes from l o n g term c u t l u r e d p r e c u r s o r c e l l s . Proc N a t l Acad S c i USA 83:441, 1986.  160.  S i d e n E J , B a l t i m o r e 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 A b e l s o n murine l e u k e m i a v i r u s . C e l l 1979:16, 389.  161.  W h i t l o c k CA, Z i e g l e r SF, Treiman L J , S t a f f o r d J l , W i t t e ON. . D i f f e r e n t i a t i o n of c l o n e d p o p u l a t i o n s of immature B c e l l s a f t e r t r a n s f o r m a t i o n w i t h A b e l s o n murine leukemia v i r u s . C e l l 32:903,  1983.  162.  W h i t l o c k CA, Z i e g l e r SF, W i t t e ON. P r o g r e s s i o n of the t r a n s f o r m e d phenotype i n c l o n a l l i n e s of A b e l s o n 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 , W i t t e 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 v e r y e a r l y B lymphoid phenotype. J Exp Med 160:1087, 1984.  57  164.  K u r l a n d J I , Z i e g l e r SF, W i t t e ON. Long-term c u t l u r e d B lymphocytes and t h e i r p r e c u r s o r s r e c o n s t i t u t e the B-lymphocyte l i n e a g e i n v i t r o . Proc N a t l Acad S c i USA 81:7554, 1984.  165.  Bosma GC, C u s t e r RP, Bosma MJ. A severe combined m u t a t i o n i n the mouse. Nature 301:527, 1983.  166.  C u s t e r RP, Bosma GC, Bosma MJ. Severe combined immunodeficiency (SCID) i n the mouse: p a t h o l o g y , 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.  D o r s h k i n d K, P o l l a c k SB, Bosma MJ, P h i l l i p s RA. Natural k i l l e r c e l l s a r e p r e s e n t i n mice w i t h severe combined immunodeficiency (scid). J Immunol 134:3798, 1985.  168.  O ' t o o l e M, Bosma MJ. F u n c t i o n a l s t a t u s of c e l l s from lymphoid and m y e l o i d t i s s u e s i n mice w i t h severe combined immunodeficiency d i s e a s e . J Immunol 132:1804, 1984.  169.  F u l o p 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 w i t h normal stem c e l l s r e q u i r e s low-dose i r r a d i a t i o n o f the r e c i p i e n t s . J Immunol 136:4438, 1986.  170.  W i t t e 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 l i n e a g e p r o g e n i t o r c e l l s from mice w i t h severe combined immune d e f i c i e n c y d i s e a s e (SCID) made p o s s i b l e by l o n g term c u l t u r e . J Immunol 138:2698, 1987.  171.  Denis KA, D o r s k h i n d K, W i t t e ON. R e g u l a t e d p r o g r e s s i o n 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.  S c h u l t z LD. P l e i o t r o p i c m u t a t i o n s c a u s i n g a b n o r m a l i t i e s i n the murine immune system and the s k i n . C u r r P r o b l Dermatol 17:236, 1987.  173.  K i n c a d e PW. formation.  174.  G r e i n e r DL, G o l d s c h n e i d e r I , K o m s c h l i e s KL, Medlock ES, B o l l u m F J , S c h u l t z L. D e f e c t i v e l y m p h o p o i e s i s i n bone marrow of motheaten (me/me) and v i a b l e motheaten (me /me ) mutant mice. I . A n a l y s i s of development of prothymocytes. E a r l y B l i n e a g e c e l l s , and 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 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.  E x p e r i m e n t a l models f o r u n d e r s t a n d i n g B Adv Immunol 41:181, 1987.  v  175.  lymphocyte  v  Medlock ES, G o l d s c h n e i d e r I , G r e i n e r DL, S c h u l t z L. D e f e c t i v e l y m p h o p o i e s i s i n the bone marrow of motheaten (me/me) and v i a b l e motheathen (me /me ) mutant mice. I I . D e s c r i p t i o n of a m i c r o e n v i r o n m e n t a l d e f e c t f o r the g e n e r a t i o n of 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 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. v  176.  immunodeficiency  v  Hayashi S-I, W i t t e PL, S h u l t z 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 w i t h 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, C h e r w i n s k i HM, S a v e l k o u l HFJ, Finkelman FD, Bond MW, Mosmann TR. The r o l e o f h e l p e r 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 C J , O v e r e l l RW, Park LS, U r d a l DL, . M o c h i z u k i DY. B c e l l p r e c u r s o r 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, W i g n a l l J , M o c h i z u k i DY, Schmierer A, Mosley B, March C J , U r d a l DL, G i l l i s S, Cosman D, Goodwin RG. S t i m u l a t i o n o f B - c e l l p r o g e n i t o r s by c l o n e d murine i n t e r l e u k i n - 7 . N a t u r e 333:571, 1988.  59  C H A P T E R  II  MATERIALS AND METHODS  1)  ESTABLISHED CELL LINES  B6SUtA c e l l s (1) were m a i n t a i n e d  i n RPMI 1640 supplemented w i t h 20% FCS  and medium c o n d i t i o n e d by pokeweed mitogen s t i m u l a t e d mouse s p l e e n (PWM-SCCM) a t a f i n a l  cells  c o n c e n t r a t i o n o f 5% ( 2 ) .  32D c l o n e 3 c e l l s ( 1 ) , were s i m i l a r l y maintained  i n RPMI 1640  supplemented w i t h 20% FCS, but 15% WEHI 3B c o n d i t i o n e d medium (CM) i n s t e a d o f PWM-SCCM was used as a source o f I L - 3 . NIH 3T3 f i b r o b l a s t s were grown i n DMEM supplemented w i t h 10% FCS.  2)  ANIMALS  B a l b / c J - +/+ and B a l b / c J - nu/nu mice were o r i g i n a l l y purchased e i t h e r from J a c k s o n L a b o r a t o r i e s (Bar Harbour,ME) o r 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 m a i n t a i n e d (C57B1/6J x C3H/HeJ) ? were bred and m a i n t a i n e d  1  i n our f a c i l i t y .  h y b r i d mice and (WB/Re x C57B1/6J) F  i n our f a c i l i t y ,  1  - +/+ mice  s t a r t i n g from p a r e n t a l s t o c k s  o r i g i n a l l y purchased from J a c k s o n L a b o r a t o r i e s .  60  3)  GROWTH FACTORS  Recombinant human IL-1|3, recombinant murine I L - 3 and recombinant  murine  GM-CSF were k i n d l y p r o v i d e d as h i g h l y p u r i f i e d r e a g e n t s a f t e r e x p r e s s i o n i n E. c o l i by Biogen ( B o s t o n , MA). IL-2  Highly p u r i f i e d  forms o f recombinant human  ( 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 I L - 4 were purchased from Genzyme ( B o s t o n , MA). purified  Highly  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 N e t h e r l a n d s ) . H i g h l y recombinant human  IFN-Y  was o b t a i n e d from Dr.  J.  Gutterman  purified  (M.D. Anderson  H o s p i t a l and Tumor I n s t i t u t e , Houston, T X ) . P u r i f i e d r a t TGF-a was purchased from Bachem I n c . ( T o r r a n c e , CA) and p u r i f i e d p o r c i n e TGF-P^ was purchased from R. and D. Systems, I n c . ( M i n n e a p o l i s , MN).  4)  ANTIBODIES  R a b b i t 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 o b t a i n e d from Dr. H. Furthmayr ( Y a l e U n i v e r s i t y , New Haven, CT) and had been g e n e r a t e d by i m m u n i z a t i o n o f r a b b i t s w i t h the c o r r e s p o n d i n g antigen p u r i f i e d  from the ECM ( 4 , 5 ) .  R a b b i t a n t i - F N r e c e p t o r (FN-R) and a n t i - V N r e c e p t o r (VN-R) a n t i b o d i e s were k i n d l y p r o v i d e d by Dr.M. P i e r s c h b a c h e r ( L a J o l l a Cancer R e s e a r c h F o u n d a t i o n , La J o l l a , CA). They had been g e n e r a t e d by i m m u n i z a t i o n o f r a b b i t s w i t h the c o r r e s p o n d i n g r e c e p t o r 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 p l a c e n t a as d e s c r i b e d ( 6 , 7 ) . chromatography  Immune s e r a were then p u r i f i e d by a f f i n i t y  on r e c e p t o r Sepharose  columns.  61  R a b b i t 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 o b t a i n e d from Dako C o r p o r a t i o n ( S a n t a B a r b a r a , CA). A l i s t and d e s c r i p t i o n o f the s p e c i f i c i t y and s o u r c e o f the r a t and mouse MoAb's used i s g i v e n i n T a b l e 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 ( a b ' ) 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 a n t i b o d y (from ATCC) was l a b e l l e d w i t h FITC and k i n d l y p r o v i d e d by Dr. P. Lansdorp ( T e r r y Fox L a b o r a t o r y , Vancouver, BC). FITC l a b e l e d goat anti-mouse IgM (y c h a i n s p e c i f i c ) was o b t a i n e d from C e d a r l a n e L a b o r a t o r i e s (Hornby, ONT).  5)  PROBES  To d e t e c t I g H c h a i n gene rearrangement a 0.770 kb X b a l - E c o R I i n s e r t c o r r e s p o n d i n g t o a r e g i o n 3' o f J g ^ and 5' o f Cu was i s o l a t e d from the pJ  H  12/23 p l a s m i d (from F. A l t , Columbia U n i v e r s i t y , New York, NY - 2 0 ) . For T i p gene rearrangements a p l a s m i d 86T5 c o n t a i n i n g a 0.650 kb EcoRI  cDNA fragment (from M. D a v i s , S t a n f o r d U n i v e r s i t y , S t a n f o r d , CA - 21) encompassing v a r i a b l e , j o i n i n g and c o n s t a n t r e g i o n s o f the T i p gene was used. To d e t e c t v - a b l sequences i n c l o n a l c e l l l i n e s , a p l a s m i d pABlsub9 (from D . B a l t i m o r e , M a s s a c h u s e t t s I n s t i t u t e o f Technology, Cambridge, MA -22) was used.  6)  PROTEINS AND PEPTIDES  FN and VN were k i n d l y p r o v i d e d by Dr. M.D. P i e r s c h b a c h e r 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, S t . L o u i s , M0)  62  T a b l e 3.  L i s t and d e s c r i p t i o n o f the monoclonal a n t i b o d i e s used i n t h i s s t u d y .  S p e c i f i c A n t i b o d i e s Used f o r A n a l y s i s Reagent  RA3-3A /6.1 1  Origin  (B220)  Reference  Specificity  Rat  pre-B & B lymphocytes  9  YE1/30  (Thy 1)  Rat  thymocytes & p r i m i t i v e h e m o p o i e t i c cells  10  YE1/21  (T200)  Rat  hemopoietic c e l l s (except e r y t h r o i d c e l l s )  10  YE1/9  Rat  t r a n s f e r r i n receptor  11  Ml/69  Rat  h e m o p o i e t i c c e l l s (except p e r i p h e r a l T lymphocytes)  12  YE4/20  Rat  IgM a l l o t y p e  13  ThB  Rat  thymocytes and B lymphocytes  14  YE6/26  Rat  Mo-MuLV gp70  13  Ml/70 (Mac-1)  Rat  monocytes  12  10-4-22  Mouse  IgD o f a,c,d, o r f a l l o t y p e  15  BP-1  Mouse  e a r l y pre-B, newly formed B lymphocytes  16  34-5-8S  Mouse  H2-D  17  Mouse  H2-D  MK-D6  Mouse  I-A  14-4-4S  Mouse  I-E /C  Anti-mouse IgM (u c h a i n s p e c i f i c )  Goat  l a t e pre-B lymphocyte  TdT  Rabbit  T lymphocytes e a r l y pre-B lymphocytes  15-5-5S  a  a  and g r a n u l o c y t e s  d  18  k  19  d  k  18  k  T h i s a n t i b o d y c r o s s - r e a c t s w i t h H2-K. and the f h a p l o t y p e . d  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 R e s e a r c h  ( L e x i n g t o n , MA). The s y n t h e t i c p e p t i d e G l y - A r g - G l y - A s p - S e r - P r o (GRGDSP) was o b t a i n e d from La J o l l a Cancer Research F o u n d a t i o n ( L a J o l l a , CA).  The s y n t h e t i c p e p t i d e  G l y - A r g - A l a - A s p - S e r - P r o (GRADSP) was purchased from the Department  of  B i o c h e m i s t r y , U n i v e r s i t y o f 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 s t r o m a l l i n e s ( e . g . M2-10B4 and M l - B l ) were i s o l a t e d from p r i m a r y c u l t u r e s o f marrow c e l l s from (C57Bl/6JxC3H/HeJ)  F^ h y b 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 m a i n t a i n e d a c c o r d i n g to s t a n d a r d p r o c e d u r e s f o r a c h i e v i n g l o n g - t e r m m y e l o p o i e s i s (23, 24) except t h a t h y d r o c o r t i s o n e ( S o l u c o r t e f , Upjohn Co.) was o m i t t e d from the growth medium and the c u l t u r e s were kept a t 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 s u b c u l t u r e d i n  RPMI 1640 p l u s 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 e v i d e n c e o f m y e l o p o i e s i s . A f t e r 1 o r 2 f u r t h e r passages (1 and 2 months l a t e r ) , c l o n e s o f 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 a t 300 c e l l s per 60 mm d i s h ) u s i n g c l o n i n g wells.  C l o n e s were then expanded,  r e c l o n e d and propagated by weekly  s u b c u l t u r i n g a t 5 x 10^ c e l l s per 25 cm  2  f l a s k i n RPMI 1640 p l u s 15% FCS.  P r i m a r y c u l t u r e s of s p l e e n 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 o f s p l e e n to adhere to p l a s t i c c u l t u r e d i s h e s o r f l a s k s i n a s m a l l volume o f RPMI 1640 p l u s 15% FCS.  When s i g n i f i c a n t outgrowth o f  f i b r o b l a s t s had o c c u r r e d (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 , s u b c u l t u r e d , c l o n e d and m a i n t a i n e d as f o r marrow c e l l l i n e s . l i n e i s o l a t e d from a (WB/Re x C57B1/6J) F^ - +/+  mouse.  S5-2 i s such a  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 o f 3-4 week-old B a l b / c J o r (C57B1/6J x C3H/HeJ) F  1  h y b r i d mice and m a i n t a i n e d  i n RPMI 1640 p l u s 5% FCS  and 50 uM 2-mercaptoethanol (ME) as d e s c r i b e d by W h i t l o c k and W i t t e ( 2 5 ) , w i t h the m o d i f i c a t i o n t h a t marrow c e l l s were o f t e n seeded onto p r e - e s t a b l i s h e d s y n g e n e i c adherent reliability In adherent  marrow c e l l l a y e r s , as we found t h i s improved the  of o b t a i n i n g productive c u l t u r e s .  o r d e r 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 , nonc 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 v a r i o u s i r r a d i a t e d (80 Gy) s t r o m a l 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 d i l u t i o n as d e s c r i b e d below.  LTC's and then c l o n e d by l i m i t i n g  Cloned c e l l l i n e s were then expanded and  m a i n t a i n e d on i r r a d i a t e d M2-10B4 c e l l s l i k e s t a n d a r d LTC's.  Bn and Bp  r e p r e s e n t 2 normal c l o n a l pre-B c e l l l i n e s o b t a i n e d from a C57B1/6J x C3H/HeJ c u l t u r e i n t h i s way. In  one o f these secondary  lymphoid  LTC's o r i g i n a l l y o f B a l b / c J o r i g i n , a  s u b p o p u l a t i o n o f non-adherent c e l l s e v o l v e d 6 months l a t e r t h a t c o u l d be maintained cells.  i n c o n t i n u o u s s u s p e n s i o n c u l t u r e i n the absence o f s t r o m a l  Both "feeder-dependent"  and "feeder-independent"  feeder  c l o n e d 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 o f c u l t u r e a t 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 ( f l a t - b o t t o m e d ) m i c r o t i t r e p l a t e s seeded the p r e v i o u s day w i t h 6000 M2-10B4 c e l l s t h a t had been i r r a d i a t e d w i t h 80 Gy 250 Kvp X - r a y s .  C e l l s f o r c l o n i n g were f i r s t s o r t e d w i t h a FACS 440  (Bectori D i c k i n s o n , Sunnyvale,  CA) to e x c l u d e s t r o m a l c e l l s , d e b r i s and dead  c e l l s by a p p r o p r i a t e g a t i n g o f c e l l s by s i z e , forward s c a t t e r and p r o p i d i u m iodide-positivity.  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  c l o n e s i n them a m p l i f i e d and then t e s t e d f o r t h e i r a b i l i t y p l a s t i c (feeder-independent feeders  c l o n e s , e.g. H9 c e l l s ) o r o n l y on p r e - e s t a b l i s h e d  (feeder-dependent c l o n e s , e.g. A8 c e l l s ) .  procedure was repeated these s t u d i e s . and 50 mM 2-ME.  t o grow d i r e c t l y on  T h i s c l o n i n g and s c r e e n i n g  a t l e a s t once f o r each c l o n e d l i n e s u b s e q u e n t l y  I n i t i a l l y a l l l i n e s were m a i n t a i n e d  used i n  i n RPMI 1640 p l u s 5% FCS  However, l a t e r s t u d i e s r e v e a l e d t h a t A8 c e l l s grew o p t i m a l l y  i n 15% FCS ( s e e R e s u l t s ) .  T h e r e a f t e r , the growth medium used f o r maintenance  o f t h i s l i n e and a l l experiments w i t h A8 c e l l s was RPMI 1640 p l u s 15% FCS and 50 mM 2-ME u n l e s s o t h e r w i s e 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  line  d e r i v e d 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 B a l b / c J marrow p o p u l a t i o n seeded onto a M2-10B4 f e e d e r 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 t o A-MuLV by i n c u b a t i n g 10^ c e l l s a t room temperature i n 0.5 ml o f f r e s h RPMI c o n t a i n i n g 5% FCS, 50 uM ME and A-MuLV ( p l 6 0 - o r i g i n a l l y o b t a i n e d  from Dr. 0. W i t t e ) a t a  f i n a l v i r u s c o n c e n t r a t i o n o f 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 a t 1200 rpm, resuspended i n f r e s h medium and  then p l a t e d onto a p r e - e s t a b l i s h e d i r r a d i a t e d M2-10B4 f e e d e r l a y e r  (4 x I O M2-10B4 c e l l s / w e l l ) i n 24 w e l l ( f l a t - b o t t o m e d ) L i n b r o 7603305 p l a t e s 4  at a c o n c e n t r a t i o n o f 5 x 10^ c e l l s / m l . A-MuLV t r a n s f o r m a n t s  Thus, ABn and ABp c e l l l i n e s a r e the  o f 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 c l o n e s were i n c u b a t e d  under  the same c o n d i t i o n s w i t h a s t o c k o f Moloney h e l p e r v i r u s (Mo-MuLV) o r medium alone.  These c e l l s were maintained  u s i n g the same p r o t o c o l as f o r s t a n d a r d  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 . c e l l s t h a t had  At t h i s time,  the  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 m a i n t a i n e d  t h e r e a f t e r as  suspension  cultures.  10)  IMMUNOFLUORESCENCE ANALYSES  For the s t a i n i n g of s t r o m a l 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 w i t h phosphate b u f f e r e d s a l i n e (PBS) appropriate f i r s t  c o n t a i n i n g 2% FCS,  stained with  the  ( r a b b i t or r a t ) a n t i b o d i e s f o l l o w e d by FITC c o n j u g a t e d  a n t i - r a b b i t (or r a t ) Ig antibody.  goat  C o n t r o l s i n c l u d e d 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 c l o n e s f o r cu, c y t o s p i n p r e p a r a t i o n s 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 w i t h FITC l a b e l e d goat anti-mouse (y  IgM  c h a i n s p e c i f i c ) u s i n g 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 To s t a i n f o r TdT, antibody  T - c e l l l i n e - 26) as a n e g a t i v e  control.  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 a n t i - T d T  (Gibco/BRL, G a i t h e r s b u r g , MD)  l a b e l e d goat a n t i - r a b b i t I g was  was  used as the f i r s t reagent and  used as the second a n t i b o d y .  FITC  All  immunofluorescent s l i d e p r e p a r a t i o n s were examined under a Z e i s s e p i s c o p e  and  at l e a s t 100 c e l l s were e v a l u a t e d . To d e t e c t v a r i o u s s u r f a c e markers, f r e s h c e l l s were s t a i n e d u s i n g  the  a p p r o p r i a t e 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 p r o p i d i u m (2 ug/ml) and suspensions  then a n a l y z e d w i t h a FACS 440.  As p o s i t i v e c o n t r o l s , c e l l  from normal B a l b / c J thymus, s p l e e n and bone marrow were a l s o  iodide  67  s t a i n e d and a n a l y z e d .  As n e g a t i v e c o n t r o l s , c e l l s were l a b e l e d w i t h the  second FITC l a b e l e d reagent o n l y , o r w i t h an i r r e l e v a n t mouse a n t i b o d y as the first  reagent.  To e s t i m a t e the p r o p o r t i o n o f p o s i t i v e c e l l s ,  the c h a n n e l  number where the n e g a t i v e c o n t r o l and the t e s t sample curves c r o s s e d each o t h e r was f i r s t determined. channel in  The number o f n e g a t i v e c e l l s t o the r i g h t o f t h i s  ( h i g h e r f l u o r e s c e n c e v a l u e s ) was s u b t r a c t e d from the number o f c e l l s  the t e s t sample t h a t f e l l t o the r i g h t o f the c r o s s o v e r p o i n t .  The  percentage  o f p o s i t i v e c e l l s r e l a t i v e t o n e g a t i v e c o n t r o l a n t i b o d y was  determined  on 10^ c e l l s .  11)  HISTOCHEMISTRY  S t r o m a l c e l l s were prepared as d e s c r i b e d f o r immunofluorescence s t a i n i n g , air  d r i e d , and then f i x e d and s t a i n e d f o r a c i d phosphatase, c h l o r o a c e t a t e  e s t e r a s e , a l p h a - n a p h t h y l - a c e t a t e e s t e r a s e , p e r o x i d a s e , and a l k a l i n e phosphatase u s i n g s t a n d a r d  12)  techniques.  VIRUS ASSAYS  F r e s h l y o r r a p i d l y f r o z e n c e l l l i n e s u p e r n a t a n t s were t e s t e d 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 d e s c r i b e d by Scher and S i e g l e r ( 2 7 ) .  Briefly,  early  passage NIH 3T3 c e l l s were seeded i n 60 mm p l a s t i c F a l c o n d i s h e s ( 1 0 ^ / d i s h ) i n medium w i t h 10 ug/ml o f p o l y b r e n e .  The next day the medium was r e p l a c e d w i t h  0.5 ml of v i r a l s t o c k o r a l i q u o t s o f t e s t s u p e r n a t a n t s and i n c u b a t e d a t 37°C for  1 hour w i t h o c c a s i o n a l r o c k i n g o f the d i s h e s .  a f t e r 5 days and transformed microscope.  Medium was changed a g a i n  f o c i s c o r e d on the 10th day u s i n g an i n v e r t e d  68  13)  ASSAYS FOR  10 -10 5  6  TUMORIGENICITY  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  s y n g e n e i c , B a l b / c J - n u / n u mice and/or i n t o syngeneic-+/+ mice t h a t had immunocompromised by treatment w i t h 7 Gy of 270 Kvp 3.6  x 10^ s y n g e n e i c marrow c e l l s a week p r e v i o u s l y .  m o n i t o r e d f o r up  14)  X-rays and  Tumor development  population  doubling  times were determined e i t h e r exclusion)  of ^ r l - t h y m i d i n e 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  P r e l i m i n a r y experiments e s t a b l i s h e d seen at t h i s time.  3596, Cambridge,  that maximum ^H-thymidine  added to each 100 y l of c u l t u r e and C e l l u l a r DNA  was  MA).  incorporation  To measure - ^ - t h y m i d i n e i n c o r p o r a t i o n , 1 y C i  3 H ~ t h y m i d i n e (20 mCi/mmole, Amersham, O a k v i l l e , ONT)  a d d i t i o n a l 4 hours.  or from measurements  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 C o s t a r m i c r o t i t r e p l a t e s ( C o s t a r  and  was  CELL PROLIFERATION ASSAYS  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  was  a g r a f t of  to 6 months.  Pre-B c e l l growth and  was  been  of  i n 20 y l of growth medium  the c e l l s were then i n c u b a t e d  f o r an  h a r v e s t e d onto g l a s s f i b e r f i l t e r  the amount of ^H-thymidine i n c o r p o r a t i o n determined by  paper  scintillation  counting. I n e x p e r i m e n t s where the e f f e c t of a 0.35% i n t e r l a y e r was incubated  examined (Chapter I I I ) , 0.7  i n 24 w e l l ( f l a t - b o t t o m e d )  i n i t i a t e d by p l a t i n g 2.1  x 10  the w e l l s or onto a c o n f l u e n t  4  A8  ml pre-B c e l l c u l t u r e s were f i r s t  L i n b r o 7603305 p l a t e s .  c e l l s or 2100  80 Gy  agarose (Sigma, S t . L o u i s ,  Cultures  were  H9 c e l l s e i t h e r d i r e c t l y i n  i r r a d i a t e d M2-10B4 f e e d e r l a y e r  (4 x 10^ M2-10B4 c e l l s / w e l l ) , i n each case w i t h or w i t h o u t an  intermediate  M0)  69  0.2 ml i n t e r l a y e r o f 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 - ^ - t h y m i d i n e , the c e l l s on top o f the agarose i n each w e l l were resuspended w i t h o u t 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 t o the w e l l s o f a 96 f l 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 h a r v e s t i n g  the DNA.  For some o f the c o - c u l t u r e experiments d e s c r i b e d 3 x 10  6  i n Chapter I V ,  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. 24 w e l l ( f l a t - b o t t o m e d )  0.2 ml o f t h i s m i x t u r e were then p l a t e d i n  L i n b r o 7603305 p l a t e s ( i . e . 6 x 1 0  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 w i t h o u t c e l l s was then poured on top o f 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 o v e r l a y e d c o n t a i n i n g 2 x 10^ c e l l s / m l .  w i t h 0.7 ml o f medium  A f t e r 3 days o f c u l t u r e , 3 - t h y m i d i n e uptake H  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  M i c r o w e l l p l a t e s (96 w e l l , C o s t a r  3596, Cambridge, MA) were coated  the d e s i g n a t e d  p r o t e i n s by i n c u b a t i n g the p r o t e i n s o l u t i o n s a t v a r i o u s  concentrations  i n the p l a t e s o v e r n i g h t  from the p l a t e s by washing w i t h PBS.  a t 4°C.  with  Unbound p r o t e i n s were removed  The p l a t e s were then i n c u b a t e d  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 a t 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) a t 5 x 10^ c e l l s / m l . s u s p e n s i o n was added t o the p r o t e i n - c o a t e d incubated  One hundred u l o f each c e l l  w e l l s , and the p l a t e s were  a t 37°C f o r 1 h r . A f t e r washing g e n t l y w i t h PBS, a t t a c h e d  cells  were f i x e d w i t h 3% paraformaldhyde (100 u l ) f o r 30 minutes a t room temperature and  then s t a i n e d w i t h 0.5% t o l u i d i n e b l u e i n 3.7% paraformaldhyde (100 u l )  70  overnight.  The number o f a t t a c h e d  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 o r H9 c e l l s were p l a t e d i n 35 mm t i s s u e concentration  c u l t u r e dishes at a f i n a l  o f 300 c e l l s p e r ml i n 1.1 ml volumes o f 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 ) c o n t a i n i n g 5% FCS and 50 uM 2-ME. d i s h e s had been seeded the p r e v i o u s  I n some c a s e s , t h e  day w i t h 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 t o g i v e a c o n f l u e n t  monolayer.  The  m e t h y l c e l l u l o s e medium c o n t a i n i n g A8 o r 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 o f the feeder of t h e same medium ( w i t h o u t agarose.  l a y e r , o r on t o p o f an i n t e r m e d i a t e  0.5 ml l a y e r  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%  A f t e r 7 days i n c u b a t i o n a t 37°C, c o l 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 d i s h e s u s i n g an i n v e r t e d microscope.  17)  MYELOID COLONY ASSAYS  F r e s h l y suspended (C57B1/6J x C3H/HeHJ) F^ at a f i n a l c o n c e n t r a t i o n  bone marrow c e l l s were p l a t e d  o f 3 x 10^ c e l l s i n 1.1 ml o f 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% d e i o n i z e d BSA, 100 uM 2-ME, 3 u n i t s o f Epo (28) p e r m l , and e i t h e r 10% M2-10B4 CM ( o r S5-2CM) o r 10% H9 CM, o r 1% PWM-SCCM i n a l p h a medium ( 2 ) .  C u l t u r e s were then i n c u b a t e d  days a t 37°C and c o l o n i e s c o n t a i n i n g more than 20 g r a n u l o c y t e s ,  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 o r i n v a r y i n g scored  d i r e c t l y i n the d i s h e s .  Under these c o n d i t i o n s , c u l t u r e s  f o r 14  combinations containing  71  o n l y 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 s m a l l pure e r y t h r o i d or s m a l l macrophage c o l o n i e s .  18)  ANALYSIS OF DNA  REARRANGEMENTS  H i g h m o l e c u l a r weight DNA was i s o l a t e d by p r o t e i n a s e f o l l o w e d by phenol and c h l o r o f o r m  K digestion of c e l l s  e x t r a c t i o n ( 2 9 ) . DNA was d i g e s t e d  with  EcoRI f o r v - a b l sequences and f o r I g heavy c h a i n gene a n a l y s i s , and w i t h Hind III  f o r T c e l l receptor  0 c h a i n ( T i g ) gene a n a l y s i s .  were s e p a r a t e d by e l e c t r o p h o r e s i s  The d i g e s t e d  f o r 16 hours a t 2 v o l t s / c m  fragments  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 t o 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) u s i n g s t a n d a r d methods (29) o r t o n y l o n f i l t e r s  (Zeta-probe,  B i o r a d ) by the a l k a l i n e t r a n s f e r t e c h n i q u e ( 3 0 ) . N i t r o c e l l u l o s e f i l t e r s were p r e h y b r i d i z e d ,  h y b r i d i z e d , washed  using  s t a n d a r d t e c h n i q u e s ( 2 9 ) . F o r n y l o n 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 N a C l , 10% formamide (BRL), 1% SDS, 2 mM EDTA, 2% n o n - f a t d r i e d m i l k and 0,5 mg/ml d e n a t u r a t e d salmon sperm DNA. H y b r i d i z a t i o n included  buffer  the same components except f o r formamide ( 2 0 % ) .  The probes used to d e t e c t 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 o r T i g gene rearrangements were ^ 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 ( N i c k 2  T r a n s l a t i o n K i t , Bethesda Research L a b o r a t o r y , G a i t h e r s b u r g ,  MD).  The probe used f o r I g heavy c h a i n gene rearrangement, was ^ P - l a b e l l e d by 2  the hexamer primer method (Pharmacia, o l i g o l a b e l l i n g k i t ) ( 3 1 ) . A f t e r h y b r i d i z a t i o n f o r 18-24 h o u r s , f i l t e r s were washed 3 times i n 0.1% SDS, 0.1% SSC and 0.1% sodium pyrophosphate a t 60°C f o r 30 minutes w i t h the exception (i.e.  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  0.1% SSC and 0.1% SDS a t 68°C) was used f o r the J  H  conditions  probe i n o r d e r t o  72  e l i m i n a t e the h y b r i d i z a t i o n o f r e p e t i t i v e sequences ( 3 2 ) . A u t o r a d i o g r a p h y  was  performed a t -70°C f o r 1-4 days u s i n g 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 o r S5-2 c e l l s were seeded i n 75 cm 10 ml o f RPMI 1640 supplemented w i t h 15% FCS. subconfluent  2  Corning  flasks containing  When the c e l l s were  still  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 w i t h 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 o n l y , o r w i t h 50 uM 2-ME o n l y , was added. CM was h a r v e s t e d  a f t e r 24 hours and s t o r e d a t 4°C p r i o r t o 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 H9 and A-MuLV transformed  specified.  pre-B c e l l c l o n e s i n l o g phase were washed  t w i c e w i t h serum-free RPMI 1640 supplemented o n l y w i t h 50 uM 2-ME.  They were  then resuspended i n 10 ml o f the same f r e s h medium a t a c o n c e n t r a t i o n o f 5 x 10^ c e l l s / m l and p l a c e d i n 75 cm  2  f l a s k s f o r 24-36 hours.  CM was  c e n t r i f u g e d t o 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 t o 56°C f o r 1 hour t o i n a c t i v a t e any v i r u s p r e s e n t .  CM was s t o r e d a t 4°C p r i o r t o 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 , otherwise  unless  specified.  Medium c o n d i t i o n e d by human p e r i p h e r a l b l o o d l e u k o c y t e s i n c u b a t e d (agar-LCM, 33) o r a c t i v a t e d i n s u s p e n s i o n PWM-SCCM were prepared  i n agar  c u l t u r e by PHA (PHA-LCM, 3 4 ) , and  i n our l a b o r a t o r y u s i n g s t a n d a r d  procedures ( 2 ) .  73  20)  GEL PERMEATION CHROMATOGRAPHY  F r e s h l y harvested  M2-10B4 CM was f i l t e r e d through a 0.22 y  (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  d i s t i l l e d water t o o b t a i n a 1 0 - f o l d c o n c e n t r a t i o n .  filter double  One ml o f t h e c o n c e n t r a t e d  sample was r u n 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, M i s s i s s a u g a ,  ONT) and e l u t e d w i t h PBS  (pH + 7.4) a t a f l o w r a t e o f 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 a t 4°C and t e s t e d 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) D e x t r a n B l u e ( v o i d volume), 2) Ovalbumin  (MW = 45,000), 3) Cytochrome  C  (MV = 12,384), 4) Phenol r e d (MW = 354). Freshly harvested  H9 CM was l y o p h i l i z e d o v e r n i g h t  and then resuspended i n  s t e r i l e double d i s t i l l e d water to o b t a i n a 5 0 - f o l d c o n c e n t r a t i o n . the c o n c e n t r a t e d  One ml o f  sample was r u n 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, M i s s i s s a u g a ,  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) a t a f l o w r a t e o f 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 a t 4°C and t e s t e d f o r b i o l o g i c a l a c t i v i t y . calibrated with: 3) Cytochrome (MW = 3 5 4 ) .  The column was  1) Dextran b l u e ( v o i d volume), 2) Ovalbumin  (MW = 45,000),  C (MW = 12,384), 4) V i t a m i n B12 (MW = 1,355), 5) Phenol r e d  74  REFERENCES 1.  Greenberger J S , Eckner RJ, Sakakeeny M, Marks P, R e i d 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 h e m a t o p o i e t i c progenitor c e l l l i n e s . F e d e r a t i o n Proceedings 42:2762, 1983.  2.  Eaves C J , K r y s t a l G, Eaves AC. Erythropoietic cells. In: "Bibliotheca Haematologica, No 48 - C u r r e n t Methodology i n E x p e r i m e n t a l Hematology", (ed. SJ Baum), S. K r a g e r , B a s e l , pp 81, 1984.  3.  Aarden LA, de Groot ER, Schaap OL, growth f a c t o r by human monocytes.  4.  Madri JA, Dryer B, P i t l i c k F, Furthmayr H. The c o l l a g e n o u s 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 I n v e s t 43:303, 1980.  5.  Madri JA, W i l l i a m s SK, Wyatt T, Mezzio C. 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C e l l u l a r RNA homologous t o the A b e l s o n murine leukemia v i r u s t r a n s f o r m i n g gene e x p r e s s i o n and r e l a t i o n s h i p t o the v i r a l sequence. Mol C e l l B i o l 3:773, 1983.  23.  Greenberger J S . S e n s i t i v i t y o f c o r t i c o s t e r o i d - d e p e n d e n t 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 p r e a d i p o c y t e s o f o b e s e - d i a b e t i c (db/db) mice. N a t u r e 275:752, 1978.  24.  Eaves C, Coulombel L, Eaves A. A n a l y s i s o f hemopoiesis i n l o n g - t e r m human marrow c u l t u r e s . I n : "Haemopoietic Stem C e l l s " . ( e d s . Sv-Aa K i l l m a n n , EP C r o n k i t e , CN M u l l e r - B e r a t ) , Munksgaard, Copenhagen, pp 287, 1983.  25.  W h i t l o c k CA, W i t t e ON. Long-term c u l t u r e o f B lymphocytes and t h e i r p r e c u r s o r s from murine bone marrow. Proc N a t l Acad S c i USA 79:3608, 1982.  26.  Chan P-Y, T a k e i F. 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Blood 61:876, 1983.  77  CHAPTER  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 p r e c u r s o r s i n the marrow t h a t possess a broader range o f 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 . of  These i n c l u d e a p o p u l a t i o n  p l u r i p o t e n t h e m o p o i e t i c stem c e l l s which p e r s i s t throughout a d u l t  life  (1,2) and which a r e c a p a b l e o f g e n e r a t i n g v a r i o u s m y e l o i d daughter c e l l as w e l l as T and B c e l l s .  types  The e x t e n t t o which B lymphocyte numbers a r e  n o r m a l l y m a i n t a i n e d by p l u r i p o t e n t h e m o p o i e t i c stem c e l l s o r 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 p r e c u r s o r s i s not known. D e l i n e a t i o n o f the sequence o f I g H and L c h a i n gene rearrangement and e x p r e s s i o n i n d e v e l o p i n g B c e l l s has a l l o w e d the d e f i n i t i o n o f s e v e r a l s t a g e s of  d i f f e r e n t i a t i o n a l o n g 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 o f f a c t o r s t h a t r e g u l a t e the s i z e o f these v a r i o u s pre-B populations. of  The l o c a l i z a t i o n o f B c e l l development  cell  within s p e c i f i c regions  h e m o p o i e t i c and lymphoid t i s s u e s s u g g e s t s t h a t i n v i v o the s o u r c e s o f such  f a c t o r s may i n c l u d e f i x e d elements o f non-hemopoietic o r i g i n .  R e c e n t l y , an i n  v i t r o system t h a t s u p p o r t s 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 o f v e r y e a r l y B c e l l p r e c u r s o r s f o r s e v e r a l months has been d e s c r i b e d ( 4 ) .  A key f e a t u r e of  these pre-B lymphoid LTC's, l i k e the m y e l o i d LTC's from which they were  78  adapted (5) and from which they can be d e r i v e d 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 a c c e s s o r y c e l l s . F u r t h e r a n a l y s i s of the mechanisms i n v o l v e d 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 s h o u l d 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 o f a v a r i e t y of c l o n e d c e l l l i n e s r e p r e s e n t i n g the d i f f e r e n t components o f the l o n g - t e r m pre-B lymphoid c u l t u r e system. have been d e s c r i b e d ( 7 - 1 2 ) .  R e c e n t l y , a number of such l i n e s  D u r i n g 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 e s t a b l i s h m e n t and maintenance o f pre-B LTC's from mouse marrow, a s p o n t a n e o u s l y i m m o r t a l i z e d l i n e a r o s e t h a t  cell  was  i n i t i a l l y feeder-dependent but over time g e n e r a t e d f e e d e r - i n d e p e n d e n t variants.  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 c l o n e s o f  each o f these phenotypes as w e l l as s e v e r a l c l o n e d s t r o m a l c e l l l i n e s  that  s u p p o r t e d t h e i r growth i s d e s c r i b e d below t o g e t h e r w i t h t h e i r use i n s t u d i e s of  2)  the mechanism of s t r o m a l support of pre-B  cells.  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 S u p p o r t i v e S t r o m a l C e l l L i n e s  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 s p l e e n c e l l c u l t u r e s and c l o n e d as d e s c r i b e d i n the M a t e r i a l s and Methods. T a b l e 4 shows the growth s u p p o r t i n g c a p a c i t y of 3 of these when t e s t e d as f e e d e r s 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  d e s c r i b e d by W h i t l o c k and W i t t e ( 4 ) .  I n the p a r t i c u l a r experiment p r e s e n t e d  i n T a b l e 4, a s i m i l a r growth s u p p o r t i n g r o l e of 3T3 c e l l s i s a l s o demonstrated. c e l l s per cm  2  A d d i t i o n a l experiments of t h i s type showed t h a t 2 x 10^ f e e d e r was s u f f i c i e n t to g i v e o p t i m a l s t i m u l a t i o n of such nonadherent,  79  T a b l e 4.  Comparison of the a b i l i t y of v a r i o u s i r r a d i a t e d (80 Gy) f e e d e r c e l l lines  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 Feeder C e l l  a  a  Non-Adherent  10 ) 5  Total  Control  0.8  0.8  M2-10B4  53  107  Ml-Bl  20  66  S5-2  91  94  3T3  54  58  T r y p s i n i z e d f e e d e r c e l l s were i r r a d i a t e d i n s u s p e n s i o n w i t h 80 Gy to p r e v e n t t h e i r subsequent growth and were then p l a t e d i n d u p l i c a t e a t 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 l o n g t e r m 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 d i s h e s w i t h o u t f e e d e r s , i . e . d i r e c t l y on p l a s t i c , s e r v e d 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 o r not the feeder p o p u l a t i o n 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 ( d a t a not shown, see a l s o F i g u r e 7 below). A l l 4 o f the adherent c e l l l i n e s t e s t e d i n T a b l e 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 t o 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 ) , s u g g e s t i v e o f 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 ( 1 3 ) .  On the o t h e r hand, no  e v i d e n c e o f F a c t o r V I I I a s s o c i a t e d a n t i g e n , a common marker o f mature e n d o t h e l i a l c e l l s , was d e t e c t e d .  Some c e l l s i n a l l l i n e s c o u l d be c o n v e r t e d  i n t o o i l red 0 - p o s i t i v e l i p i d laden adipocytes hydrocortisone  f o l l o w i n g a d d i t i o n of  t o the c u l t u r e medium as d e s c r i b e d by o t h e r s ( 7 , 1 4 ) .  the s t r o m a l l i n e s were p o s i t i v e f o r any o f the h e m o p o i e t i c  None o f  c e l l markers t e s t e d  (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 o f Pre-B C e l l  Lines  A c l o n e d l i n e o f feeder-dependent and f e e d e r - i n d e p e n d e n t 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 o b t a i n e d as d e s c r i b e d i n t h e  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 f e e d e r - i n d e p e n d e n t f o r  more than 2 y e a r s , but feeder-independent v a r i a n t s were found t o a r i s e c o n t i n u o u s l y from A8 c e l l s a t a frequency  of approximately  10~^.  Thus t o  m a i n t a i n A8 c e l l s as a homogeneous feeder-dependent p o p u l a t i o n t h i s l i n e was r e c l o n e d every 2-3 months. Both A8 and H9 c e l l s resemble lymphoblasts  morphologically.  Additional  c h a r a c t e r i z a t i o n s t u d i e s showed t h a t no A8 o r H9 c e l l s a r e p o s i t i v e f o r s u r f a c e (IgM o r IgD) o r c y t o p l a s m i c the m y e l o i d ThB,  ( y c h a i n ) I g , B220 a n t i g e n , TdT, o r any o f  o r T c e l l markers t e s t e d .  and Mac-1.  The l a t t e r i n c l u d e Thy-1, T200, Ml/69,  However, >90% o f c e l l s i n both l i n e s s t r o n g l y express BP-1,  T a b l e 5.  Immunological and h i s t o 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 s t r o m a l c e l l used i n t h i s  lines  study.  M2-10B4  Ml-Bl  S5-2  Laminin  +  +  +  C o l l a g e n Type IV  +  +  -  C o l l a g e n Type I  -  -  Factor V I I I  -  -  -  -  -  -  A c i d Phosphatase  -  -  Chloroacetate esterase  +  +  -  -  Myeloperoxidase  -  -  A l k a l i n e Phosphatase  -  -  Ml/70 ( M a c - l ) YE1/21 ( T 2 0 0 )  a  a  3T3 + +/-  +++ -  -  ND^  ND  « naphthyl-acetate esterase  a  b  M o n o c l o n a l 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 g r a n u l o c y t e s , YE1/21 (T200) r e a c t s g e n e r a l l y w i t h hemopoietic c e l l s , except erythroid cells. ND = not done.  82  an a n t i g e n c h a r a c t e r i s t i c  o f pre-B c e l l s ( 1 5 ) .  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 o f C l a s s I MHC a n t i g e n s and the t r a n s f e r r i n receptor. A n a l y s i s o f A8 and H9 c e l l DNA r e v e a l e d that both a l l e l e s o f J H , r e g i o n appear t o be i d e n t i c a l l y rearranged  i n both l i n e s ( F i g u r e 5 a ) .  In contrast,  a n a l y s i s w i t h a Tg probe r e v e a l e d no evidence o f rearrangement o f t h i s gene ( F i g u r e 5b).  These d a t a a r e c o n s i s t e n t w i t h a pre-B c e l l o r i g i n o f both A8  and H9 c e l l s , and suggest that they r e p r e s e n t subclones from the same i m m o r t a l i z e d  C)  o r i g i n a l l y derived  cell.  I n V i t r o Growth o f H9 and A8 C e l l s  We f i r s t a n a l y z e d  the FCS requirement  o f A8 and H9 c e l l s and found t h a t  o p t i m a l growth o f A8 c e l l s r e q u i r e s 15% FCS, but that growth o f H9 c e l l s i s not improved w i t h FCS c o n c e n t r a t i o n s above 5% ( d a t a not shown). these c o n d i t i o n s were used i n a l l subsequent experiments.  Therefore,  However, even when  the FCS c o n c e n t r a t i o n i s o p t i m i z e d , c e l l p r o l i f e r a t i o n i n both cases c e l l concentration-dependent, above*10^ c e l l s / m l , 2-ME independent.  as i l l u s t r a t e d i n F i g u r e 6a.  At c o n c e n t r a t i o n s  H9 c e l l p r o l i f e r a t i o n appears to be c o m p l e t e l y  f e e d e r and  However, a t c o n c e n t r a t i o n s 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 supplementation  i s highly  o f the medium w i t h 2-ME.  on s u i t a b l e  f e e d e r s and by  T h i s can be seen by the i n c r e a s e d  s l o p e o f the curve f o r H9 c e l l s a l o n e by comparison t o H9 c e l l s c u l t u r e d on M2-10B4 c e l l s . ceases.  At c o n c e n t r a t i o n s below 3,000 H9 c e l l s / m l  proliferation  Thus H9 c e l l s show some f e e d e r and 2-ME dependence, but o n l y a t c e l l  c o n c e n t r a t i o n s much lower than those t y p i c a l l y used f o r l i n e maintenance (>10^  cells/ml).  T h i s 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-  6J6-U 43-  232  23  •  — 6 . 2 Kb  || 4.3  2.3 2 -  -—9.4 Kb  — 3.1 Kb  Kb  Kb  F i g u r e 5. P a n e l a. Southern b l o t a n a l y s i s o f I g H c h a i n 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 w i t h EcoRI and h y b r i d i z a t i o n t o a J probe. Lane 1, B a l b / c J s p l e e n ; l a n e 2, A20 B c e l l s ( f r o m ATCC, R o c k v i l l e , MD); l a n e 3, M2-10B4 c e l l s ; l a n e 4, H9 c e l l s ; and l a n e 5, A8 c e l l s . An arrow marks the expected p o s i t i o n o f the 6.2 Kb germ l i n e fragment. Rearrangement i s e v i d e n t 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 cells. H  P a n e l b. Southern b l o t a n a l y s i s o f the T c e l l r e c e p t o r 3 c h a i n 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 w i t h Hind I I I and h y b r i d i z a t i o n t o a Tp cDNA probe. Lane 1, B a l b / c J s p l e e n ; l a n e 2, A20 B c e l l s ; l a n e 3, A - K l T c e l l s ( 1 6 ) ; l a n e 4, B a l b / c J thymus; l a n e 5, M2-10B4 c e l l s ; l a n e 6, H9 c e l l s ; and l a n e 7, A8 c e l l s . Arrows mark the expected p o s i t i o n s o f the 9.4 and 3.1 Kb germ l i n e fragments. Rearrangement i s e v i d e n t i n A - K l 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 Number  15000 ol  150000  0 150  FI-H9 c e l l s p e r m l  1500 N  Panel a  u  m  b  e  r  15000 °'  F  D  "  A  8  c  e  l  l  s  150000 P  e r  m  l  Panel b  F i g u r e 6. P r o l i f e r a t i o n o f H9 c e l l s (panel a) and A8 c e l l s ( p a n e l b) seeded a t d i f f e r e n t c e l l c o n c e n t r a t i o n s under d i f f e r e n t c o n d i t i o n s . on p r e - e s t a b l i s h e d i r r a d i a t e d M2-10B4 s t r o m a l c e l l s (at 6 x 1 0 cells/ml or 2 x 1 0 cells/cm ); on p l a s t i c ; - - - on p l a s t i c i n t h e absence o f 2-ME. I I I I I background due to M2-10B4 c e l l s alone (6 x 1 0 c e l l s / m l o r 4  4  2  4  2 x 1 0 cells/cm ). 4  2  85  f e e d e r - i n d e p e n d e n t c e l l s c o u l d not be c l o n e d i n the absence o f a f e e d e r .  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 ( o r even remain v i a b l e ) on p l a s t i c at c o n c e n t r a t i o n s below 3 x 1 0 6 x 10  4  4  c e l l s / m l ( F i g u r e 6b).  Above  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 d e t e c t e d but t h i s i s  s h o r t - l i v e d and o c c u r s o n l y i n the presence of 50 yM 2-ME.  Under o p t i m a l  c o n d i t i o n s the p o p u l a t i o n d o u b l i n g times of H9 and A8 c e l l s a l s o d i f f e r are approximately  and  15 and 43 h o u r s , r e s p e c t i v e l y .  F i g u r e 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 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 . 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  the  The  was  3,000 c e l l s / m l , i . e . s u f f i c i e n t to g i v e s u b s t a n t i a l counts under o p t i m a l c o n d i t i o n s , but low enough to a l l o w the response to M2-10B4 f e e d e r s to a l s o be revealed. achieved ( i . e . 10  I t can be seen t h a t o p t i m a l s t i m u l a t i o n of 3,000 H9 c e l l s / m l i s w i t h a f e e d e r c e l l c o n c e n t r a t i o n of 3,000 M2-10B4 c e l l s / m l  4  c e l l s / cm^)  ( F i g u r e 7a).  At M2-10B4 c o n c e n t r a t i o n s 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 a p p a r e n t .  I t should a l s  be noted t h a t 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 c o n c e n t r a t i o n s of 3 x 1 0  4  cells/ml.  F i g u r e 7b shows the r e s u l t s f o r A8 c e l l s i n an experiment of design.  similar  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 h i g h e r  c o n c e n t r a t i o n (3 x 1 0  4  c e l l s / m l ) to g i v e a comparable feeder-dependent  p r o l i f e r a t i v e response ( i . e . to a c h i e v e time - 4 days - o f  approximately  the p r o d u c t i o n i n the same p e r i o d of  the same number of c e l l s ) .  The  d a t a 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 d a t a f o r H9 c e l l s shown i n F i g u r e 7a but w i t h a 2 0 - f o l d s h i f t ( i n c r e a s e ) i n the M2-10B4 c e l l requirement of A8  cells.  relative  E a  o  ~i 0  150  1500  r  1 15000  1500C0  Number ol M2-10B4 cells per ml  ~i 0  150  1500  r 15000  Number of M 2 - 1 0 B 4 cells per  150000 ml  Panel b  Panel a  F i g u r e 7. P r o l i f e r a t i o n o f 3,000 B9 c e l l s / m l (panel a) and 3 x 1 0 A8 c e l l s / m l ( p a n e l b) when p l a t e d on i r r a d i a t e d M2-10B4 c e l l s a t d i f f e r e n t M2-10B4 c e l l c o n c e n t r a t i o n s . The s o l i d l i n e shows the r e s u l t s f o r c u l t u r e s c o n t a i n i n g H9 o r A8 c e l l s . The dashed l i n e shows the background due t o the i r r a d i a t e d M2-10B4 feeder c e l l s o n l y . 4  87  D)  E v i d e n c e f o r a S o l u b l e Pre-B C e l l S t i m u l a t i n g  Factor  To d e t e r m i n e whether the s t r o m a l 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 t o the r e l e a s e o f a s o l u b l e growth f a c t o r , the e f f e c t o f separating  the t a r g e t A8 o r H9 c e l l s from M2-10B4 c e l l s  i n t e r l a y e r was examined.  by an agarose  I n these experiments A8 o r H9 c e l l s were seeded a t  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 o f growth by c o l o n y formation,  o r i n l i q u i d medium f o r assessment o f growth by ^H-thymidine  incorporation.  R e s u l t s o f a t y p i c a l experiment a r e shown i n T a b l e 6.  The  presence o f an agarose i n t e r l a y e r d i d not a l t e r the number o f 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 o f  M2-10B4 c e l l s t o 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 s e p a r a t e d 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.  cultures As f u r t h e r  shown i n T a b l e 6, M2-10B4 c e l l s were a l s o a b l e t o s t i m u l a t e normal pre-B (obtained  cells  from a r e c e n t l y e s t a b l i s h e d l o n g - t e r m pre-B lymphoid c u l t u r e ) when  s e p a r a t e d 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 o b t a i n e d  when non-  i r r a d i a t e d M2-10B4 f e e d e r s were used ( d a t a 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 t o s t i m u l a t e A8 o r H9 c e l l p r o l i f e r a t i o n i n low density cultures.  P r e l i m i n a r y experiments e s t a b l i s h e d t h a t a p r o l i f e r a t i v e  response o f A8 c e l l s , and a l s o o f normal pre-B c e l l s , c o u l d not be r e a d i l y detected  w i t h M2-10B4 CM.  response ( F i g u r e 8 a ) .  However, H9 c e l l s gave a c l e a r  CM h a r v e s t e d  a f t e r 1-2 days were c o n s i s t e n t l y found t o  c o n 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 . f a c t o r s c o u l d no l o n g e r be d e t e c t e d . S5-2 CM.  dose-dependent  A f t e r 3 days  stimulatory  S i m i l a r r e s u l t s have been o b t a i n e d  with  As shown i n F i g u r e 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 c o m p l e t e l y  T a b l e 6.  Demonstration o f a s o l u b l e f a c t o r d e r i v e d from M2-10B4 c e l l s s t i m u l a t i n g i m m o r t a l i z e d pre-B c e l l l i n e s and normal pre-B c e l l s .  Colony A s s a y Conditions  H9  C e l l P r o l i f e r a t i o n Assay  3  A8  H9  on p l a s t i c  0  0  129 + 16  on agarose  0  0  71 + 1 4  on i r r a d i a t e d M2-10B4  47,43  43,33  1,788 + 1 0 8  on i r r a d i a t e d M2-10B4 + agarose  48,47  22,9  1,179 + 100  c  0  A8  Normal Pre-B C e l l s  238 + 33  387 + 39  76 + 21  134 + 13  11,561 + 8 1 0  16,511 + l , 7 5 0  c  2,504 + 93  d  3,468 + 187  R e s u l t s r e p r e s e n t d u p l i c a t e measurements o f c o l o n y numbers (>10 c e l l s / c o l o n y ) per 1.1 ml m e t h y l c e l l u l o s e c u l t u r e from 2 r e p r e s e n t a t i v e experiments. C e l l s were p l a t e d a t 300 c e l l s per 1.1 ml c u l t u r e and c o l o n i e s were s c o r e d on Day 7. R e s u l t s r e p r e s e n t the mean cpm + SEM from t r i p l i c a t e assays o f 2 r e 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 e x p e r i m e n t s . I n one, H9 c e l l s were c u l t u r e d a t a c o n c e n t r a t i o n o f 3,000 c e l l s / m l ( o r 2100 c e l l s / w e l l ) and A8 c e l l s were c u l t u r e d a t a c o n c e n t r a t i o n o f 3 x 1 0 c e l l s / m l ( or 2.1 x 1 0 c e l l s / w e l l ) . I n the o t h e r , B220-negative pre-B c e l l s from the non-adherent f r a c t i o n o f a 4-month o l d long-term lymphoid marrow c e l l c u l t u r e were o b t a i n e d by FACS and c u l t u r e d a t a concentration of 2 x 1 0 cells/ml. »^Level o f ^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 a l o n e , which was 874 (+ 121) ( c ) , and 498 + 46 ( d ) , i s i n c l u d e d i n these r e s u l t s . a  D  4  4  4  Panel a  Panel b  F i g u r e 8. Panel a. P r o l i f e r a t i o n o f H9 c e l l s (3000 c e l l s / m l ) seeded i n the presence o f v a r i o u s c o n c e n t r a t i o n s o f M2-10B4 CM h a r v e s t e d a t 1 • , 2 A , 3 • o r 5 O days a f t e r a d d i n g f r e s h medium t o the M2-10B4 c e l l s . |||| H9 c e l l s (3000 c e l l s / m l ) seeded w i t h medium a l o n e . Panel b: P r o l i f e r a t i o n o f H9 c e l l s (3000 c e l l s / m l ) seeded i n the presence o f v a r i o u s c o n c e n t r a t i o n s o f M2-10B4 Day 1 CM a f t e r h e a t i n g t o 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 r e p r e s e n t s 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 t o DI curve from p a n e l A. |||| H9 c e l l s (3000 c e l l s / m l ) seeded w i t h medium a l o n e .  90  r e s i s t a n t t o h e a t i n g a t 56°C f o r 1 hour.  F i g u r e 9 shows a r e p r e s e n t a t i v e  p r o f i l e o f 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 t h a t a l l o f 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, w i t h an apparent m o l e c u l a r weight o f a p p r o x i m a t e l y 10,000.  E)  S p e c i f i c i t y o f the A c t i v i t y Produced by S t r o m a l C e l l  Lines  To determine whether the a c t i v i t y i n M2-10B4 CM c o u l d be a t t r i b u t e d t o a known growth f a c t o r , the a b i l i t y o f t h i s CM t o s t i m u l a t e the p r o l i f e r a t i o n o f B6SUtA (17) and 32D c l o n e 3 (17) c e l l s was t e s t e d by Dr. G. K r y s t a l u s i n g 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 d e s c r i b e d the t a r g e t responder c e l l s were h a r v e s t e d days.  that  a f t e r 24 hours r a t h e r than a f t e r 3-4  B6SUtA c e l l s which respond t o I L - 3 ( 1 7 ) , GM-CSF and I L - 4  (Dr. G. K r y s t a l , p e r s o n a l  communcation), and 32D c l o n e 3 c e l l s which a r e  e x c l u s i v e l y IL-3 responsive M2-10B4 CM ( d a t a not shown).  ( 1 7 ) , were n e i t h e r s t i m u l a t e d nor i n h i b i t e d by T h i s CM was a l s o t e s t e d 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 o g y , P a l o A l t o , CA) and not found  to c o n t a i n d e t e c t a b l e I L - 4 a c t i v i t y . ability  f o r H9 c e l l s , except  M2-10B4 CM (Day 1) when t e s t e d f o r i t s  to s t i m u l a t e m y e l o i d o r e r y t h r o i d colony  mouse marrow c e l l s p l a t e d i n standard  formation  by 3 x 1 0  However, M2-10B4 CM h a r v e s t e d  a f t e r longer  containing  periods  s t i m u l a t e d l a r g e r numbers o f c o l o n i e s c o n t a i n i n g up to 500 macrophages suggesting  normal  methylcellulose cultures containing 3  u n i t s / m l o f Epo (18) supported the growth o f o n l y a few c o l o n i e s <20 macrophages.  4  t h a t M2-10B4 c e l l s may produce some M-CSF.  (only)  91  Phenol red  60 Fraction  number  F i g u r e 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 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 o f 8X c o n c e n t r a t e d serum-free M2-10B4 CM. Each p o i n t r e p r e s e n t s the mean o f 3 r e p l i c a t e s + SEM o f the a c t i v i t y p r e s e n t i n f r a c t i o n s assayed a t a f i n a l c o n c e n t r a t i o n 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 o f d e x t r a n b l u e (V0), ovalbumin (OVA), cytochrome C ( C y t . C) and phenol r e d .  92  F)  Response of Feeder-Independent H9 C e l l s to D e f i n e d Growth F a c t o r s  T a b l e 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 t e s t e d 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 o f crude CM o r d e f i n e d 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 o r i n h i b i t v a r i o u s hemopoietic c o n c e n t r a t i o n s used.  3)  and/or lymphoid  c e l l types a t the  None of these s t i m u l a t e d H9  intermediate  cells.  DISCUSSION  We have d e s c r i b e d 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 o f  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 d e r i v e d 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 o f 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 l i n e a g e f o r c o n t i n u e d s u r v i v a l and  growth.  Such a p r o p e r t y i s a f e a t u r e 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 o f progeny generated term lymphoid  the  their  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 -  marrow c u l t u r e s ( 4 ) .  E x t e n s i v e 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 t h a t they l a c k a l l B - c e l l p h e n o t y p i c markers examined ( i . e . B220, s u r f a c e and c y t o p l a s m i c I g , ThB, expressed  and TdT), w i t h the e x c e p t i o n of BP-1  a t r e l a t i v e l y h i g h l e v e l s on a l l A8 c e l l s .  t h a t a s i m i l a r h i g h e x p r e s s i o n of BP-1  which i s  I t i s perhaps noteworthy  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  t r a n s f o r m a t i o n of murine B - l i n e a g e c e l l s i n c o n t r a s t to normal B - l i n e a g e which a r e o n l y weakly BP-1  p o s i t i v e (15, and d a t a not shown).  the  cells  Interestingly,  more r e c e n t s t u d i e s have demonstrated that A8 c e l l s are t u m o r i g e n i c i n syngeneic  nude mice a l t h o u g h  they do not r e l e a s e t r a n s f o r m i n g v i r u s a c t i v e i n  a f o c u s f o r m i n g assay on 3T3 c e l l s , and do not express  the Gp70 a n t i g e n o f  93  T a b l e 7.  Lack o f r e s p o n s i v e n e s s o f H9 c e l l s t o d e f i n e d growth  H9 c e l l s (300 c / w e l l ) Growth F a c t o r s '  positive  control^  negative c o n t r o l r h IL-1 0  a  cpm  3  e  factors.  Concentration  28729 + 875 7905 + 303  0  H9 c e l l s + M2-10B4 c e l l s M2-10B4 CM 25% (1-50%)  818 ± 125  medium a l o n e  978 + 145  120 U/ml (12 - 1200 U/ml)  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 IL-2  1030 +  r h G-CSF r h IFNy  98  100 U/ml (10 - 1000 U/ml)  894 + 489  100 U/ml (10 - 1000 U/ml)  1044 +  58  100 U/ml (1 - 1000 U/ml)  76 +  16  5 ng/ml (0.5 - 50 ng/ml)  TGF a  569 + 110  50 ng/ml (5 - 500 ng/ml)  EGF  807 +  0.5 ng/ml (0.5 - 5 ng/ml)  r h HGF TGF p  x  EMT-6 CM  f  PWM - SCCM  92  653 + . 47  5%  649 + 108  0.1% ( 0 . 1 % - 10%)  (1% - 10%)  Agar-LCM  1155 +  61  1% (1 - 10%)  PHA-LCM  820 +  93  5% (1 - 10%)  :  R e s u l t s r e p r e s e n t the mean o f t r i p l i c a t e s and a r e e x p r e s s e d i n cpm + SEM I L - l , 3 and GM-CSF were o b t a i n e d as p u r i f i e d f a c t o r s from B i o g e n . I L - 2 , G-CSF and EGF were purchased from Amgen (Thousand Oaks, CA); n a t u r a l GM-CSF and I L - 4 were purchased from Genzyme ( B o s t o n , MA); HGF, which i s the same as interferon 0 I L - 6 was o b t a i n e d from Dr. L.A. Aarden ( 1 9 ) ; i n t e r f e r o n y (IFNy) was o b t a i n e d from Dr. J . Gutterman (Houston, TX); p o r c i n e TGF-P^ was purchased from R. & D. Systems, I n c . ( M i n n e a p o l i s , MN); and r a t TGF-a was purchased from Bachem I n c . ( T o r r a n c e , CA); r = recombinant, h = human, m = murine. ( ) c o n c e n t r a t i o n range t e s t e d . ^Feeder a l o n e 335 + 13. C e l l s c u l t u r e d i n medium a l o n e on p l a s t i c . ^EMT-6 CM c o n t a i n s M-CSF a c t i v i t y and was prepared as d e s c r i b e d p r e v i o u s l y (20). a b  a  2  c  e  n  d  94  murine Moloney v i r u s (Chapter I V ) .  The pre-B phenotype o f A8 c e l l s was  c o n f i r m e d by Southern a n a l y s i s which i n d i c a t e d t h a t both J J J a l l e l e s were rearranged.  An u n p r o d u c t i v e rearrangement o f both J J J a l l e l e s c o u l d e x p l a i n  the observed  f a i l u r e o f these c e l l s to generate more mature phenotypes ( 2 2 ) .  The  second c l o n e d l i n e o f pre-B c e l l s (H9) has v e r y s i m i l a r f e a t u r e s t o  A8 c e l l s and was d e r i v e d from the same o r i g i n a l c u l t u r e . appear t o have i d e n t i c a l  Both H9 and A8 c e l l s  gene rearrangements and t h e r e f o r e p r o b a b l y d e r i v e  from the same o r i g i n a l l y i m m o r t a l i z e d pre-B c e l l .  The H9 c e l l l i n e appears t o  d i f f e r o n l y w i t h r e g a r d t o i t s much lower (>20-fold) requirement  for a stromal  c e l l f e e d e r l a y e r and a h i g h e r s e n s i t i v i t y t o a pre-B s t i m u l a t o r y f a c t o r produced by such  cells.  To f a c i l i t a t e a n a l y s i s o f the pre-B c e l l s t i m u l a t o r y mechanism mediated by s t r o m a l 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 o f adherent c e l l l i n e s o f marrow and s p l e e n o r i g i n t h a t a r e a b l e t o support pre-B growth. which,  cell  A l l t h r e e such l i n e s d e s c r i b e d i n t h i s s t u d y share a common phenotype l i k e 3T3 c e l l s , appears t o be i n t e r m e d i a t e between f i b r o b l a s t s and  endothelial cells.  P h e n o t y p i c a l l y s i m i l a r s t r o m a l c e l l s have a l s o r e c e n t l y  been d e s c r i b e d by o t h e r s (7-12).  I n s p i t e o f some p h e n o t y p i c  amongst d i f f e r e n t s t r o m a l c e l l l i n e s , evidence  homogeneity  for variations of functional  p o t e n t i a l has been r e p o r t e d (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 t o promote m y e l o p o i e s i s and/or l y m p h o p o i e s i s 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 w i t h c a u t i o n 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 t h e 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 under d i f f e r e n t c o n d i t i o n s .  o f these may a l s o v a r y  N e v e r t h e l e s s , 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 o r  i n d u c i b l e p r o d u c t i o n o f v a r i o u s h e m o p o i e t i c 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 s t r o m a l R.K. Humphries, p e r s o n a l  l i n e s has a l s o been documented ( 1 1 , and  communication).  The s i g n i f i c a n c e o f these l a t t e r d i f f e r e n c e s to the mechanism o f pre-B c e l l s t i m u l a t i o n i s not y e t c l e a r . stromal  We have shown that a t l e a s t two o f our  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 . stromal  c e l l CM was o b t a i n e d  harvested  Time course s t u d i e s showed t h a t the most p o t e n t when serum-free medium c o n t a i n i n g 2-ME  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 .  to h e a t i n g  to 56°C but u n u s u a l l y  The a c t i v i t y i s r e s i s t a n t  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 e x p e r i m e n t s showed t h a t our s t r o m a l  c e l l l i n e s do not make  d e t e c t a b l e I L - 3 , GM-CSF o r I L - 4 . Moreover, s i n c e H9 c e l l s a r e not to any o f these growth f a c t o r s , nor to I L - 1 , I L - 2 , and IFN02> 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 m o l e c u l e . s t i m u l a t i o n by I L - 3 has been r e p o r t e d derived  was  responsive  the pre-B c e l l  A l t h o u g h pre-B  cell  ( 2 6 ) , o t h e r s have not found pre-B c e l l s  from l o n g - t e r m lymphoid marrow c u l t u r e to be I L - 3 r e s p o n s i v e ( 1 2 ) .  T h i s appears to be the case f o r A8 and H9 c e l l s a l s o . groups (12,27) have r e p o r t e d  Recently  two o t h e r  a n o v e l a c t i v i t y produced by d i f f e r e n t s t r o m a l  c e l l l i n e s and a c t i v e on B - l i n e a g e  cells.  However, the apparent m o l e c u l a r  w e i g h t s o f 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 e t a l (12) (~35,000) a r e q u i t e d i f f e r e n t . !  Further  i n f o r m a t i o n on both  f a c t o r s w i l l be r e q u i r e d to e s t a b l i s h whether these a r e 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 s u b c l o n e s of i m m o r t a l i z e d  pre-B c e l l s  that  show marked and c o 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 o f pre-B  c e l l growth f a c t o r s and t h e i r mechanism o f a c t i o n .  Preliminary data i n d i c a t e  96  t h a t these l i n e s w i l l a l s o be u s e f u l f o r s t u d i e s of changes t h a t accompany pre-B c e l l  transformation.  97  REFERENCES WU AM, S i m i n o v i t c h L, T i l l J E , M c C u l l o c h EA. Evidence f o r a r e l a t i o n s h i p between mouse h e m o p o i e t i c stem c e l l s and c e l l s f o r m i n g 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. J E , M a g l i 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 o f 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 c a p a b l e of l o n g term r e c o n s t i t u t i o n of the h e m o p o i e t i c system of w/w mice. C e l l 42:71, 1985. v  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 d u r i n g lymphocyte d i f f e r e n t i a t i o n . Immunol Rev 89:5, 1986. W h i t l o c k CA, W i t t e ON. Long-term c u l t u r e of B lymphocytes and t h e i r p r e c u r s o r s from murine bone marrow. Proc N a t l Acad S c i USA 79:3608, 1982. D e x t e r TM, A l l e n TD, L a j t h a LG. C o n d i t i o n s 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 h e m o p o i e t i c stem c e l l s i n v i t r o . J Cell Physiol 91:355, 1977. D o r s h k i n d K. I n 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 h e m o p o i e t i c p r e c u r s o r s p r e s e n t i n l o n g - t e r m 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, K i t a m u r a Y. MC3T3-G2/PA6 p r e a d i p o c y t e s support i n v i t r o p r o l i f e r a t i o n of h e m o p o i e t i c stem c e l l s through a mechanism d i f f e r e n t from t h a t 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. W h i t l o c k CA, Robertson D, W i t t e ON. Murine B c e l l l y m p h o p o i e s i s i n l o n g term c u l t u r e . J Immunol Meth 67:353, 1984. C o l l i n s LS, D o r s h k i n d K. A s t r o m a l c e l l l i n e from m y e l o i d l o n g - t e r m bone marrow c u l t u r e s can support m y e l o p o i e s i s and B l y m p h o p o i e s i s . 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. Multilineage synergistic 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. R e n n i c k D, Yang G, Gemmell L, Lee F. C o n t r o l of hemopoiesis by a bone marrow s t r o m a l c e l l c l o n e : 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 p r o d u c t i o n 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 . B l o o d 69:682, 1987. Hunt P, R o b e r t s o n D, Weiss D, Rennick D, Lee F, W i t t e ON. A s i n g l e bone marrow-derived s t r o m a l c e l l type s u p p o r t s i n v i t r o growth of e a r l y lymphoid and m y e l o i d 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 m a t r i x p r o d u c t i o n by the adherent c e l l s of l o n g - t e r m murine bone marrow c u l t u r e s . B l o o d 61:540, 1983.  98  14.  Greenberger J S . S e n s i t i v i t y o f c o r t i c o s t e r o i d - d e p e n d e n t 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 p r e a d i p o c y t e s o f o b e s e - d i a b e t i c (db/db) mice. Nature 275:752, 1978.  15.  Cooper MD, Mulvaney D, Coutinho A, Cazenave P-A. A n o v e l c e l l molecule on e a r l y B - l i n e a g e c e l l s . Nature 321:616, 1986.  16.  T a k e i F. Murine T lymphoma c e l l s express a n o v e l membrane-associated a n t i g e n w i t h unique f e a t u r e s . J Immunol 139:649, 1987.  17.  Greenberger J S , Eckner R J , Sakakeeny M, Marks P, R e i d 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 h e m a t o p o i e t i c progenitor c e l l l i n e s . F e d e r a t i o n Proceedings 42:2762, 1983.  18.  Eaves C J , 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: "Bibliotheca H a e m a t o l o g i c a , No 48 - C u r r e n t Methodology i n E x p e r i m e n t a l Hematology", (ed. SJ Baum ), S. K r a g e r , B a s e l , pp 81, 1984.  19.  Aarden LA, de Groot ER, Schaap OL, Lansdorp PM. P r o d u c t i o n o f hybridoma growth f a c t o r by human monocytes. Eur J Immunol 17:1411, 1987.  20.  Gregory C J , Eaves AC. I n v i t r o s t u d i e s o f e r y t h r o p o i e t i c p r o g e n i t o r cell differentiation. I n : " D i f f e r e n t i a t i o n o f Normal and N e o p l a s t i c H e m a t o p o i e t i c C e l l s " , (eds. B C l a r k s o n , PA Marks, J E T i l l ) , C o l d S p r i n g Harbor L a b o r a t o r y , New York, pp 179, 1978.  21.  M u l l e r - S i e b u r g CE, W h i t l o c k CA, Weissman I L . I s o l a t i o n o f two e a r l y B lymphocyte p r o g e n i t o r s from mouse marrow: a committed pre-pre-B c e l l and a c l o n o g e n i c Thy-1-'- h e m a t o p o i e t i c stem c e l l . C e l l 44:653, 1986.  surface  0  22.  A l t F, Rosenberg N, Lewis S, Thomas E, B a l t i m o r e D. O r g a n i z a t i o n and r e o r g a n i z a t i o n o f immunoglobulin genes i n A-MuLV-transformed c e l l s : rearrangement o f heavy but not l i g h t c h a i n genes. C e l l 27:381, 1981.  23.  Roberts RA, Spooncer E, P a r k i n s o n EK, Lord B I , 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 s t r o m a l c e l l s to promote the p r o l i f e r a t i o n and development o f m u l t i p o t e n t h a e m o p o i e t i c 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. P h e n o t y p i c h e t e r o g e n e i t y among s t r o m a l 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 m a t r i x c o m p o s i t i o n 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, K l a s s e n V, Sakakeeny MA, F i t z G e r a l d T J , H a r r i s o n D, Rybak ME, Greenberger J S . 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 o f c l o n e d permanent s t r o m a l c e l l l i n e s from anemic S l / S l mice and +/+ l i t t e r m a t e s . Exp Hematol 15:636, 1987. d  26.  P a l a c i o s R, Henson G, Steinmetz M, McKearn J P . I n t e r l e u k i n - 3 s u p p o r t s growth o f 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.  W h i t l o c k CA, Tidmarsh GF, M u l l e r - S i e b u r g C, Weissman I L . Bone marrow s t r o m a l c e l l l i n e s w i t h l y m p h o p o i e t i c a c t i v i t y express h i g h l e v e l s o f a pre-B n e 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.  99  CHAPTER  IV  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 o f p r i m i t i v e B - l i n e a g e  cells  appears t o be r e g u l a t e d by i n t e r a c t i o n s w i t h mesenchymally d e r i v e d elements ( 1 ) .  Although  "stromal"  d i r e c t c o n t a c t between the pre-B c e l l and the s t r o m a l  c e l l may be i n v o l v e d ( 2 ) , there i s a l s o e v i d e n c e that s u p p o r t i v e mesenchymal c e l l s can r e l e a s 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 ( 3 - 5 ) .  Decreased  dependence on exogenously p r o v i d e d growth r e g u l a t o r s i s now a w e l l documented f e a t u r e o f many m a l i g n a n t c e l l types i n c l u d i n g examples i n the h e m o p o i e t i c system.  I n some e x p e r i m e n t a l  s i t u a t i o n s , the e x p l a n a t i o n f o r the autonomous  growth c a p a c i t y o f 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 t o t h e  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). transformed clear.  hematopoietic  I n spontaneously  c e l l s the r o l e o f a u t o c r i n e growth f a c t o r s i s l e s s  A number o f s t e p s i n the s i g n a l t r a n s d u c t i o n pathway c l e a r l y  p o t e n t i a l t a r g e t s f o r malignant t r a n s f o r m a t i o n .  Nevertheless,  represent  activation of  a u t o c r i n e growth f a c t o r p r o d u c t i o n appears t o 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 o f e v i d e n c e suggested t h a t a u t o c r i n e mechanisms might p l a y a r o l e i n the e v o l u t i o n o f malignant pre-B c e l l p o p u l a t i o n s .  The f i r s t  from s t u d i e s o f A-MuLV-induced t r a n s f o r m a t i o n o f pre-B c e l l s i n v i t r o .  derived These  100-  demonstrated t h a t 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  o f 1-2 months b e f o r e they e v o l v e the c a p a c i t y f o r autonomous, i . e . s t r o m a l c e l l - i n d e p e n d e n t , growth ( 1 1 ) .  The second d e r i v e d from the s t u d i e s d e s c r i b e d  i n Chapter I I I o f s e v e r a l s p o n t a n e o u s l y transformed murine pre-B c e l l which were found t o e x h i b i t d i f f e r e n t degrees o f autonomy i n v i t r o ,  lines  depending  on the c o n c e n t r a t i o n o f the c e l l s i n the c u l t u r e s used t o t e 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 o b t a i n e d from the s p o n t a n e o u s l y t r a n s f o r m e d 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 t o s t i m u l a t e the p r o l i f e r a t i o n o f H9 c e l l s i n low d e n s i t y s u s p e n s i o n c u l t u r e s .  F i g u r e 10 shows the r e s u l t s o f a t y p i c a l  t i t r a t i o n o f H9 CM by comparison t o CM from M2-10B4 c e l l s .  I t can be seen  t h a t 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 , a l t h o u g h c o n s i d e 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 t o 10 f o l d l e s s p o t e n t , even though the c o n c e n t r a t i o n o f H9 c e l l s used t o p r e p a r e the CM was almost 10 times h i g h e r . T h i s d i f f e r e n c e i n the H9 c e l l a c t i v i t y o f H9 and M2-10B4 CM was a r e p r o d u c i b l e f i n d i n g .  stimulating  Interestingly,  a l t h o u g h 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 t o be f r e e o f the i n h i b i t o r s r o u t i n e l y d e t e c t a b l e i n M2-10B4 CM when t h i s was t e s t e d a t c o n c e n t r a t i o n s above 25%.  I n o r d e r t o determine whether H9 c e l l s produced a  f a c t o r t h a t c o u l d 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 i n c u b a t e d  i n the presence o r absence o f H9 c e l l s s e p a r a t e d by an agar i n t e r l a y e r .  As a  s o u r c e o f 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 e x p e r i m e n t s .  The r e s u l t s o f a t y p i c a l experiment a r e shown  101  12 -  Concentration of Conditioned Medium  (%)  F i g u r e 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. ( ), o r M2-10B4 CM ( ). (Ill) background H - t h y m i d i n e i n c o r p o r a t i o n by H9 c e l l s a t 3,000 c e l l s / m l and c u l t u r e d i n medium a l o n e . 3  102  i n T a b l e 8.  They demonstrate that H9 c e l l s s e c r e t e 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)  P r o d u c t i o n o f 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 L i n e s  To i n v e s t i g a t e whether o t h e r t r a n s f o r m e d 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 o f a u t o c r i n e growth f a c t o r p r o d u c t i o n , two p h 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) g e n e r a t e d i n d e p e n d e n t l y u s i n g A-MuLV as the t r a n s f o r m i n g 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  T a b l e 9, both o f t h e s e , l i k e H9 c e l l s , produced tumors i n immunocompromised mice.  I n c o n t r a s t , the two p h e n o t y p i c a l l y normal pre-B c e l l c l o n e s (Bn and  Bp) from which the r e s p e c t i v e A-MuLV t r a n s f o r m a n t s were d e r i v e d , o r which were exposed o n l y t o Mo-MuLV, were not t u m o r i g e n i c , as e x p e c t e d .  Assessment o f the  presence o f Mo-MuLV Gp70 a n t i g e n c o n f i r m e d the e f f e c t i v e n e s s o f i n f e c t i o n s w i t h both A-MuLV and Mo-MuLV s t o c k s and showed that t h i s a n t i g e n was absent i n b o t h normal pre-B c e l l c l o n e s and i n H9 c e l l s .  When s u p e r n a t a n t s from each o f  the  c e l l l i n e s were assayed f o r f o c u s - f o r m i n g a c t i v i t y on NIH-3T3 c e l l s ,  the  two A-MuLV t r a n s f o r m a n t s were p o s i t i v e .  of  only  Southern a n a l y s i s o f DNA from a l l  the l i n e s c o n f i r m e d the presence o f v - a b l bands e x c l u s i v e l y i n the A-MuLV  t r a n s f o r m a n t s ( d a t a not shown).  The r e s u l t s o f 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 o f the A-MuLV d e r i v e d t r a n s f o r m a n t s a r e shown i n T a b l e 10.  Both A-MuLV l i n e s e x p r e s s B220, T200 and TdT.  One l i n e  e x p r e s s e s BP-1 ( 1 5 ) . T h i s g e n e r a l p a t t e r n i s d i f f e r e n t from t h a t t y p i c a l o f e i t h e r the normal pre-B c e l l c l o n e s o r the H9 c e l l l i n e .  I g gene  rearrangement a n a l y s i s ( F i g u r e 11) showed the same rearrangement o f 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 t r a n s f o r m e d c e l l  lines  103  Table  Culture  8.  Demonstration that  Condition  Underlayer  H9 a u t o c r i n e  activity  H9 c e l l s bottom agar  No  in layer  stimulates  Target liquid  normal  pre-B  cells in overlay  1  M2-10B4 monolayer  Normal p r e - B cells  2  -  Yes  3  -  No  Normal p r e - B cells  4  -  Yes  Normal p r e - B cells  -  cells.  H-thymidine incorporation ( cpm)  10,097 + 441  147+14 67+9 3,728 + 397  A l l c u l t u r e s c o n t a i n e d 2 a g a r l a y e r s : t h e l o w e r one w i t h o r w i t h o u t H9 c e l l s as i n d i c a t e d , t h e u p p e r one w i t h o u t any c e l l s t o s e r v e as a s p a c e r p r e v e n t i n g any c o n t a c t b e t w e e n t h e H9 or M2-10B4 c e l l s a n d t h e n o r m a l p r e - B t a r g e t c e l l s w h i c h 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 t o p o f t h e u p p e r a g a r l a y e r . F o r 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 M e t h o d s .  104  Table  Cell  9.  lines*  Transformed  Tumor  10^  H9 line)  cells/mouse  phenotype  of c l o n a l  incidence  lymphoid  cell  lines.  Mo-MuLV Gp70  10^ c e l l s / m o u s e  (% p o s i t i v e  cells)  A-MuLV  production  (FFU/ml)  3/3  2/2  0  0  ND  0/2  0  0  Bn  (original  Bn  + MoMuLV  ND  0/2  31  0  Bn  + A-MuLV  2/2  2/2  95  4 x  Bp  (original  ND  0/2  0  0  Bp + MoMuLV  ND  0/2  43  0  Bp + A-MuLV  2/2  2/2  98  8 x  line)  d  A l l c e l l l i n e s were t e s t e d more t h a n 4weeks a f t e r e x p o s u r e t o v i r u s (Bn, B p - s e r i e s ) ,(H9 ) . Gp70 a n t i g e n e n c o d e d by Mo-MuLV was d e t e c t e d by u s i n g YE6/26 ( 1 2 ) i n an i n d i r e c t i m m u n o f l u o r e s c e n t s t a i n i n g p r o c e d u r e and FACS | n a l y s i s . jPositive control ( v i r u s s t o c k s u p e r n a t a n t ) : 10 focus-forming u n i t s (FFU/ml). ND = n o t d o n e .  10  3  10  3  or c l o n i n g  0  105  Table  Antibodies  Reagent  10.  used  Origin  Phenotype  data  of  normal  and  transformed  for analysis  Cell  Specificity  /6.1  Rat  Pre-B,  B  lines  (%  clones.  positive  Normal Bn  RA3-3A  lymphoid  cells)  Transformed  Bp  H9 (spontaneous)  lymphocytes  ABn (A-MuLV)  ABp (A-MuLV)  61  99  99  100  (B220T YE1/30 (Thy-1 )  Thymocytes, hemopoietic  primitive cells  YE1/21 (T200 )  Rat  Hemopoietic cells (except erythroid cells)  99  98  YE1/9  Rat  Transferrin  receptor  76  51  98  95  95  BP-1  Hous<  Early pre-B, newly formed B lymphocytes  0  0  97  0  14  Rabbit  Surface  0  0  0  Rabbit  Intracytoplasmic //-chain protein  0  10  10  Rabbit  T Lymphocytes early pre-B lymphocytes  15  15  Anti  IgM  Anti-//  TdT  chain  IgM  B 2 2 0 was o b t a i n e d f r o m A m e r i c a n 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). YE1/30 ( 1 3 ) , YE1/21 ( 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 Fox L a b o r a t o r y , B.C. Cancer Research Centre, Vancouver, B.C.). BP-1 ( 1 5 ) was a g i f t f r o m D r . M.D. Cooper (University of Alabama, Birmingham, A L ) .  106  1  23 9.6 6.6 4.3 2.3 ~  Kb  i  2 I  3 4 I  I  5  6  I  M  7 I  | ;  •+  ii M  I  M  6.2 Kb  M  ) t  F i g u r e 1 1 . Southern b l o t a n a l y s i s o f I g H c h a i n gene rearrangements i n normal pre-B c e l l c l o n e s and d e r i v a t i v e A-MuLV t r a n s f o r m a n t s . C e l l u l a r DNA was d i g e s t e d w i t h EcoRI and h y b r i d i z e d t o a probe as d e s c r i b e d 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); l a n e 2, thymus; l a n e 3, A - K l T c e l l s ( 1 3 ) ; l a n e 4, Bn c e l l s ; l a n e 5, A-MuLV Bn c e l l s ; l a n e 6, Bp c e l l s ; l a n e 7, A-MuLV Bp c e l l s . An arrow marks the expected p o s i t i o n o f the 6.2 Kb germ l i n e fragment. A l l l a n e s were loaded w i t h 10 ug o f DNA.  107  tested.  However, i n both A-MuLV transformed  l i n e s the i n t e n s i t y o f the  germ l i n e a l l e l e was decreased ( l a n e s 5 and 7 ) , and new bands were apparent suggesting  that secondary rearrangements had o c c u r r e d  transformation.  f o l l o w i n g A-MuLV  A n a l y s i s w i t h a Tp probe r e v e a l e d no e v i d e n c e o f  rearrangements o f t h i s gene i n these c e l l s ( d a t a not shown). were d e r i v e d i n d e p e n d e n t l y  from a d i f f e r e n t long-term  H9 c e l l s  that  pre-B c e l l c u l t u r e a l s o  show rearrangement o f both a l l e l e s o f the Jrj gene and no rearrangement o f t h e i r Tp genes ( 5 ) .  C y t o s p i n p r e p a r a t i o n s showed t h a t 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 CM were prepared  morphology.  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 a t v a r i o u s  times a f t e r exposure t o A-MuLV, and from c o n t r o l c u l t u r e s a c c o r d i n g t o the p r o t o c o l d e s c r i b e d 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  t e s t e d f o r t h e i r a b i l i t y t o 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 . F i g u r e 12, normal pre-B c e l l s d i d not produce d e t e c t a b l e activity.  Weak a c t i v i t y was present  As shown i n  H9-stimulating  i n media c o n d i t i o n e d by A-MuLV i n f e c t e d  c e l l s t h a t were s t i l l h i g h l y s t r o m a l 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 i n c r e a s e d (on a per c e l l b a s i s ) when prepared o f the same l i n e t h a t were more advanced and capable  from c e l l s  o f autonomous growth i n  the absence o f a s t r o m a l 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 a u t o c r i n e e f f e c t , the same A-MuLV c e l l CM were t e s t e d for  t h e i r a b i l i t y t o s t i m u l a t e the A-MuLV transformed  A-MuLV i n f e c t e d c u l t u r e s used to g e n e r a t e the CM.  c e l l s d e r i v e d from the  A s i m i l a r increase i n  a u t o c r i n e f a c t o r c o n c e n t r a t i o n was apparent between 4 and 8 weeks post A-MuLV i n f e c t i o n ( d a t a riot shown).  108  0  1  10  100  Concentration of Conditioned Medium (%)  F i g u r e 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 c o n d i t i o n e d 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 o b t a i n e d from A-MuLV i n f e c t e d Bp c e l l s o b t a i n e d 4 weeks a f t e r i n f e c t i o n , and (-•- _^ ) CM o b t a i n e d 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 o b t a i n e d 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 o f the A u t o c r i n e 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 s t e p towards the i d e n t i f i c a t i o n o f the a c t i v i t y produced by t r a n s f o r m e d pre-B c e l l s , t h e i r CM's were t e s t e d by Dr. G. K r y s t a l u s i n g a number o f s t a n d a r d b i o a s s a y s . A d d i t i o n o f up to 20% CM from any o f the normal or  t r a n s f o r m e d 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 t r a n s f o r m a n t s ,  to B6SUtA o r 32D c l o n e 23 c e l l c u l t u r e s f a i l e d t o 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 ( d a t a not shown).  T h i s suggests that  n e i t h e r the normal nor the transformed pre-B c e l l s produce I L - 3 , GM-CSF o r IL-4 t o which B6SUtA c e l l s ( a l l t h r e e ) and 32D c e l l s ( I L - 3 o n l y ) respond. Pre-B c e l l CM's a t c o n c e n t r a t i o n s o f up t o 10% a l s o f a i l e d t o s u p p o r t the growth o f any c o l o n i e s i n s t a n d a r d m e t h y l c e l l u l o s e assays f o r m y e l o i d p r o g e n i t o r s ( d a t a not shown).  We have p r e v i o u s l y demonstrated t h a t H9 c e l l s  do not respond t o GM-CSF, G-CSF, I L - 1 , I L - 2 , I L - 3 , I L - 4 , I L - 6 (= I F N - 0 2 ) , IFN-Y,  EGF, PWM-SCCM, agar-LCM o r PHA-LCM ( 5 ) .  The s e l f - s t i m u l a t i n g  activity  produced by H9 c e l l s , l i k e t h a t produced by A-MuLV t r a n s f o r m a n t s c e l l s i s t h e r e f o r e l i k e l y t o be d i f f e r e n t from any o f these w e l l c h a r a c t e r i z e d  growth  factors. To f u r t h e r c h a r a c t e r i z e the a u t o c r i n e growth f a c t o r a c t i v e on H9 c e l l s , H9 CM was c o n c e n t r a t e d and s u b j e c t e d t o chromatography  on Sephadex G50.  F i g u r e 13 shows the p r o f i l e o f the H9 s t i m u l a t i n g a c t i v i t y which was r e p r o d u c i b l y e l u t e d as a s i n g l e peak w i t h an apparent m o l e c u l a r weight o f a p p r o x i m a t e l y 3,000.  T h i s s u g g e s t s a d i f f e r e n c e from the pre-B c e l l  produced by M2-10B4 c e l l s (5) o r d e s c r i b e d by o t h e r s ( 3 , 1 6 ) .  factors  However,  sequence d a t a may be r e q u i r e d b e f o r e 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  F i g u r e 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 p r e s e n t i n a 1 ml sample of 50X c o n c e n t r a t e d serum-free H9 CM. Arrows i n d i c a t e the p o s i t i o n a t which ovalbumin (Ova), cytochrome C (Cyt C ) , v i t a m i n B^2 ( 12)> * phenol red were e l u t e d from t h i s column. Each p o i n t 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 p r e s e n t i n each f r a c t i o n was assayed at a f i n a l c o n c e n t r a t i o n of 10%. v i t  B  an(  Ill  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 p r e s e n t i n l o n g - t e r m lymphoid marrow c u l t u r e s has been i n s t r u m e n t a l i n i d e n t i f y i n g mesenchymal s t r o m a l 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 for  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 ability may  to grow autonomously i n v i t r o , a l t h o u g h a c q u i s i t i o n o f t h i s p r o p e r t y  not r e p r e s e n t the f i r s t change to o c c u r .  T h i s has been w e l l documented i n  the case of transformed pre-B c e l l s d e v e l o p i n g a f t e r A-MuLV i n f e c t i o n of mouse marrow c e l l s ( 1 1 ) .  More r e c e n t l y , a s i m i l a r d e l a y i n the time r e q u i r e d f o r  A-MuLV induced mast c e l l t r a n s f o r m a n t s 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 t h a t both A-MuLV and spontaneous mechanisms of pre-B c e l l t r a n s f o r m a t i o n are a s s o c i a t e d w i t h a c t i v a t i o n of a u t o c r i n e growth production.  factor  I n t e r e s t i n g l y , we found t h a t 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 e x p r e s s i o n of s e v e r a l h e m o p o i e t i c 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 I L - 3 and GM-CSF, the l a t t e r two b e i n g f a c t o r s t h a t a r e r e q u i r e d f o r the p r o d u c t i o n and s u r v i v a l i n v i t r o of normal mast c e l l p r e c u r s o r s (19,20).  On the o t h e r hand, A-MuLV transformed mast c e l l s  g e n e r a t e d 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 i n c r e a s e d p r o d u c t i o n of a d i f f e r e n t spectrum of growth f a c t o r s ( 2 1 ) . e x p e r i m e n t s , a c t i v a t i o n of m y e l o i d growth  I n the p r e s e n t  f a c t o r s was a l s o not d e t e c t e d .  Thus, t h e r e i s c l e a r l y h e t e r o g e n e i t y i n the growth  factor activation pattern  o b t a i n e d when the same t r a n s f o r m a t i o n 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  t a r g e t c e l l s (B c e l l and mast c e l l p r e c u r s o r s )  o r when d i f f e r e n t c o n d i t i o n s  are used t o i s o l a t e t r a n f o r m a n t s o f the same l i n e a g e . may r e l a t e t o the phenotype o f the t a r g e t c e l l , and  How ( o r whether) t h i s  the i n i t i a l  transforming  the c o n d i t i o n s i n which the c e l l s u b s e q u e n t l y i s s t i m u l a t e d  event  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 t o r e s o l v e . These s t u d i e s a l s o r a i s e q u e s t i o n s abnormal growth f a c t o r p r o d u c t i o n disease  (10,22).  about the p o t e n t i a l s i g n i f i c a n c e o f  by malignant B l i n e a g e c e l l s i n human  The mechanism(s) u n d e r l y i n g a c t i v a t i o n o f growth f a c t o r  genes i n m a l i g n a n t h e m o p o i e t i c and lymphoid c e l l s a r e , i n most i n s t a n c e s , a complete mystery.  Enhancer i n s e r t i o n , as appears to have o c c u r r e d  i n the  development o f the I L - 3 p r o d u c i n g WEHI-3B c e l l l i n e , i s one model ( 2 3 ) , but t h i s i s u n l i k e l y t o e x p l a i n the a c t i v a t i o n o f 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 t r a n s f o r m e d mast c e l l s Further  (19-21).  i n v e s t i g a t i o n o f the mechanisms i n v o l v e d w i l l depend on the  a v a i l a b i l i t y o f more s p e c i f i c reagents t o i d e n t i f y the pre-B c e l l  activity  i t s e l f and the gene t h a t 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 s p o n t a n e o u s l y transformed pre-B c e l l l i n e s t u d i e d t o d a t e 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 s t r o m a l  cell  l i n e ( 5 ) s u g g e s t s t h a t d i f f e r e n t , o r d i f f e r e n t l y p r o c e s s e d , f a c t o r s may be involved.  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 w i t h 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 r e p o r t e d  recently  (3,16).  None o f the transformed pre-B c e l l s showed as h i g h a l e v e l o f 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 p r o d u c t i o n c e l l s , a r e p r e s e n t a t i v e cloned marrow s t r o m a l  when compared t o M2-10B4  cell line.  I t i s possible  that  l e s s f a c t o r may be r e q u i r e d i f produced by the r e s p o n d i n g c e l l i t s e l f , and t h a t some s t i m u l a t i o n might a l s o o c c u r w i t h o u t the n e c e s s i t y o f e x t r a c e l l u l a r  113  secretion.  A u t o c r i n e 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 o t h e r transformed c e l l t y p e s , i n c l u d i n g examples r e s p o n d i n g t o b o t h PDGF ( 6 ) and GM-CSF ( 7 ) . studied  I n a d d i t i o n , the transformed pre-B c e l l s  here may w e l l have undergone o t h e r changes c o n t r i b u t i n g  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  N e v e r t h e l e s s , i t seems l i k e l y that a c t i v a t i o n o f an a u t o c r i n e  to a decreased i n vivo.  growth f a c t o r  mechanism may p l a y a r o l e i n A-MuLV t r a n s f o r m a t i o n o f murine pre-B c e l l s , since  f a c t o r production increased  concomitant w i t h  autonomous growth p o t e n t i a l i n v i t r o .  the a c q u i s i t i o n o f  The f a c t that a s i m i l a r mechanism was  a l s o found i n a s p o n t a n e o u s l y transformed pre-B c e l l l i n e f u r t h e r that a u t o c r i n e  suggests  growth f a c t o r p r o d u c t i o n may be a r e l a t i v e l y common event i n  the development o f transformed pre-B c e l l s .  114  REFERENCES 1.  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O s t e r W, Lindemann A, Horn S, Mertelsmann R, Herrmann F. Constitutive e x p r e s s i o n of genes f o r h e m a t o p o i e t i c growth f a c t o r s i n a c u t e m y e l o b l a s t i c l e u k e m i a . Blood 70 ( S u p p l 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 t h a t 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 s t r o m a l c e l l s may mechanisms than s i m p l e growth f a c t o r p r o d u c t i o n , c e l l u l a r i n t e r a c t i o n s was differentiation  suggested  by the f a c t  i n v o l v e more complex  A mechanism i n v o l v i n g  direct  that pre-B c e l l  requires 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 w i t h s t r o m a l c e l l CM or by s t r o m a l 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 .  about the types of i n t e r a c t i o n s that may c e l l s and  t h e i r ECM  A l t h o u g h v e r y l i t t l e i s known  occur between pre-B c e l l s and  p r o t e i n s , B e r n a r d i et a l (4) u s i n g an i n v i t r o  assay r e c e n t l y showed t h a t p r e c u r s o r lymphoid  c e l l l i n e s blocked at  s t a g e s of d i f f e r e n t i a t i o n a t t a c h s p e c i f i c a l l y to FN. i n f a c i l i t a t i n g s t r o m a l c e l l - m e d i a t e d support 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  i n i t i a t e d to address t h i s q u e s t i o n . become capable of stromal-independent  adhesion specific  Whether FN p l a y s a  role  of normal pre-B c e l l  present s t u d i e s were t h e r e f o r e  Because most transformed  pre-B c e l l  growth i n v i t r o , and a number of  r e l a t e d c l o n e d normal and d e r i v a t i v e transformed been generated  stromal  pre-B c e l l l i n e s had  lines closely  already  i n t h i s study (see Chapters I I I and I V ) , p a r a l l e l e x p e r i m e n t s  117  were u n d e r t a k e n w i t h both t o l o o k f o r changes t h a t might be r e l a t e d t o t h e transformation  2)  process.  RESULTS  A)  D i f f e r e n t i a l Attachment o f Normal and M a l i g n a n t  Pre-B C e l l L i n e s t o  FN  The  a b i l i t y o f normal pre-B c e l l s to adhere t o v a r i o u s d e f i n e d ECM  components was t e s t e d under serum f r e e c o n d i t i o n s u s i n g the c l o n e d , dependent l i n e s d e s c r i b e d i n T a b l e 10.  stromal-  F i g u r e 14 shows t h a t normal pre-B  c e l l s a t t a c h to FN i n a dose-dependent manner, and under t h e c o n d i t i o n s used a p l a t e a u i s reached a t a FN c o n c e n t r a t i o n o f 10 yg/ml.  I n c o n t r a s t , i n the  same e x p e r i m e n t s these normal pre-B c e l l s d i d not adhere t o VN, c o l l a g e n I  (COL I ) ( d a t a not shown), o r LM even a t h i g h p r o t e i n c o n c e n t r a t i o n s  type  (up t o  20 ug/ml). I n o r d e r t o determine which b i n d i n g s i t e s o f the FN m o l e c u l e i n t e r a c t w i t h pre-B c e l l s , m i c r o w e l l s were coated w i t h a f i x e d c o n c e n t r a t i o n o f FN (5 yg/ml) and then pre-B c e l l s t e s t e d f o r t h e i r c a p a c i t y t o adhere i n the presence o f i n c r e a s i n g c o n c e n t r a t i o n s o f the p e p t i d e GRGDSP ( a sequence t h a t recognizes  the c e l l b i n d i n g s i t e o f FN). F i g u r e 15 shows t h a t the attachment  o f pre-B c e l l s t o FN can be i n h i b i t e d i n a dose dependent manner by GRGDSP. The  GRADSP p e p t i d e , which s e r v e s as a n e g a t i v e c o n t r o l , has no e f f e c t on t h e  b i n d i n g o f pre-B c e l l s t o FN. I n a next s e r i e s o f e x p e r i m e n t s , a n A-MuLV transformed :  derivative  s t r o m a l - i n d e p e n d e n t pre-B c e l l l i n e (Table 10, Chapter I V ) and a transformed  spontaneously  pre-B c e l l l i n e (H9) w i t h s i m i l a r p r o p e r t i e s (see C h a p t e r s I I I and  118  F i g u r e 14. — - -# ^ . , . O"  : : •  :  Attachment of normal pre-B c e l l s to FN.  Normal pre-B c e l l s (Bp) added to FN p r e - c o a t e d w e l l s Mo-MuLV pre-B c e l l s (MoBp) added to FN p r e - c o a t e d 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 o f s t r o m a l c e l l s f o r growth, see Chapter IV.) Normal pre-B c e l l s (Bp) added to LM p r e - c o a t e d w e l l s  25  -  F i g u r e 15.  _  : :  E f f e c t o f s y n t h e t i c p e p t i d e s on t h e attachment of normal pre-B c e l l s (Bp) t o 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 p e p t i d e Normal pre-B c e l l s (Bp) + GRADSP s y n t h e t i c p e p t i d e used as n e g a t i v e c o n t r o l  120  I V ) were t e s t e d i n the same way.  As shown i n F i g u r e 16 even a t 20 ug/ml o f  FN, which i s t w i c e the c o n c e n t r a t i o n  r e q u i r e d f o r maximum attachment o f the  normal pre-B c e l l s , both t r a n s f o r m e d pre-B c e l l l i n e s d i d not adhere t o FN.  B)  E f f e c t o f FN on the P r o l i f e r a t i o n o f Normal and Transformed Pre-B C e l l s i n the Presence o f S t r o m a l CM  To t e s t the r o l e o f 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 p r e - c o a t e d w i t h FN and t h y m i d i n e uptake was then measured shown i n T a b l e 11.  3 days l a t e r .  The r e s u l t s o f one such experiment a r e  FN by i t s e l f was unable t o s u p p o r t the p r o l i f e r a t i o n o f  normal pre-B c e l l s . However, when M2-10B4 CM was added t o FN-coated w e l l s , the p r o l i f e r a t i o n o f normal pre-B c e l l s was s l i g h t l y , a l t h o u g h s i g n i f i c a n t l y enhanced compared  t o the e f f e c t o f M2-10B4 CM a l o n e (p<0.05, by t - t e s t ) .  This  s u g g e s t s t h a t the s t i m u l a t i o n o f 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 t o be p r e s e n t i n M2-10B4 CM can be s y n e r g i z e d  by FN.  Transformed  pre-B c e l l s were a l s o t e s t e d i n t h i s type o f experiment ( T a b l e 12).  As  p r e v i o u s l y shown i n F i g u r e 10 (Chapter I V ) , M2-10B4 has a d e t e c t a b l e s t i m u l a t o r y e f f e c t on t r a n s f o r m e d pre-B c e l l s when these a r e c u l t u r e d a t low c e l l density.  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 f a c t t h a t the p r o l i f e r a t i o n o f the t r a n s f o r m e d pre-B  c e l l s was the same on p l a s t i c as on FN p r e - c o a t e d w e l l s a l s o shows t h a t the FN p r e p a r a t i o n d i d not c o n t a i n any n o n - s p e c i f i c  inhibitors.  121  20eg  i  16-  X E f  12-  o  84-  [FNj^g/ml  F i g u r e 16. : :  Attachment o f t r a n s f o r m e d pre-B c e l l s t o FN.  A-MuLV t r a n s f o r m e d pre-B c e l l s (ABp) added t o FN p r e - c o a t e d wells H9 ( s p o n t a n e o u s l y t r a n s f o r m e d pre-B c e l l s ) added t o FN pre-coated w e l l s (These c e l l s do not r e q u i r e the presence o f s t r o m a l c e l l s f o r growth when c u l t u r e d a t c o n c e n t r a t i o n s >2 x 1 0 c e l l s / m l , see F i g u r e 6 and Chapter I V . ) Attachment o f normal (Bp) pre-B c e l l s ( d a t a from F i g u r e 14) 4  :  122  T a b l e 11.  E f f e c t o f FN on normal pre-B c e l l  proliferation  induced by s t r o m a l pre-B s t i m u l a t i n g a c t i v i t y (M2-10B4 CM).  Cell Proliferation Conditions  Plastic  Bp (20,000 c e l l s / m l )  103 + 17  0  FN  79 + 9  M2-10B4 CM  912 + 7  FN + M2-10B4 CM  1,174 + 63  (cpm)  a  L+VII A 8 (50,000 c e l l s / m l ) b  179 + 27 169 + 8 1,489  + 50  2,064 + 67  a  R e s u l t s r e p r e s e n t 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 a r e c u s l g M pre-B c e l l s d e r i v e d from a lymphoid LTC. They can be m a i n t a i n e d e i t h e r i n the presence o f M2-10B4 c e l l s or w i t h 50% M2-10B4 CM a l o n e . I n c o n t r a s t t o the u s u a l pre-B a s s a y s (e.g. Bp c e l l s ) where - ^ - t h y m i d i n e i n c o r p o r a t i o n was measured a f t e r 3 days o f c u l t u r e , the L V I I A8 c e l l assay was t e r m i n a t e d a f t e r 1 day o f 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  T a b l e 12.  Effect  o f FN on transformed pre-B c e l l  proliferation  induced by s t r o m a l pre-B s t i m u l a t i n g a c t i v i t y (M2-10B4 CM).  Cell Proliferation Conditions  Plastic  5  (cpm)  H9  a  ABp  3,000 c e l l s / m l  20,000 c e l l s / m l  20,000  cells/ml  131 + 4  915 + 55  21,920 + 126  FN  118 + 2  1,012 + 32  24,264 + 719  M2-10B4 CM  189 + 2 4  2,106 + 38  24,330 + 108  FN + M2-10B4 CM  235 + 10  2,225 + 62  24,319 + 22  R e s u l t s r e p r e s e n t the mean cpm + SEM from t r i p l i c a t e a s s a y s . Cell 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 d e s c r i b e d i n Chapter II. a  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 n o r 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 ) . D  124  C)  D i f f e r e n t i a l E f f e c t of FN-R  A n t i b o d i e s 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  a s t r o m a l - i n d e p e n d e n t phenotype may  of t r a n s f o r m a t i o n l e a d i n g to  be commonly a s s o c i a t e d w i t h an impairment  of the a b i l i t y of normal pre-B c e l l s to b i n d to FN and hence a reduced to be s t i m u l a t e d by s t r o m a l c e l l s . FN-R  a n t i b o d i e s on normal and  investigated.  To t e s t t h i s f u r t h e r , the e f f e c t o f  transformed  pre-B c e l l p r o l i f e r a t i o n  anti-  was  As shown i n F i g u r e 17 anti-FN-R a n t i b o d i e s were a b l e to i n h i b i t  up to 70% of the thymidine  i n c o r p o r a t e d 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 n e g a t i v e c o n t r o l ) had no e f f e c t . 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 by the same F N - R - a n t i b o d i e s ,  3)  ability  In  c o n t r a s t , the  pre-B c e l l l i n e s was  by a f a c t o r of  less inhibited  two.  DISCUSSION  FN,  l i k e VN,  i s w e l l known f o r i t s s t r o n g adhesive  c a p a b i l i t i e s and  been g e n e r a l l y assumed to p l a y 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 demonstration  has  recent  t h a t 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 t r a n s p l a n t p a t i e n t s l e d to the suggestion  t h a t FN may  S u b s e q u e n t l y the FN-R  enhance t h e i r b i n d i n g to a c c e s s o r y was  demonstrated on the s u r f a c e of normal p e r i p h e r a l  b l o o d lymphocytes (6) and monocytes ( 7 , 8 ) . r e g u l a t i o n of h e m o p o i e t i c demonstration  c e l l s (5).  FN may  cell proliferation.  that hemopoietic  a l s o p l a y a r o l e i n the  T h i s i s suggested by the  recent  p r o g e n i t o r s adhere s p e c i f i c a l l y to FN (9)  by the f a c t t h a t o p t i m a l growth of hemopoietic  progenitors i n myeloid  and  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 w i t h s t r o m a l c e l l s (10) which l i k e 3T3  cells  ANTIBODY  CONCENTRATION  (*j/ml,  ANTIBODY  CONCENTRATION  Panel a F i g u r e 17.  Panel a.  E f f e c t o f FN-R and VN-R a n t i b o d i e s on the p r o l i f e r a t i o n  (j-Q/ml)  Panel b o f normal pre-B c e l l s .  : Normal pre-B c e l l s (Bp) + FN-R a n t i b o d i e s : Normal pre-B c e l l s (Bp) + VN-R a n t i b o d i e s Panel b. R o l e o f FN-R and VN-R a n t i b o d i e s on the p r o l i f e r a t i o n  A  o f transformed pre-B c e l l s .  A-MuLV transformed pre-B c e l l s + FN-R a n t i b o d i e s H9 spontaneously transformed pre-B c e l l s + FN-R a n t i b o d i e s Transformed pre-B c e l l s + VN-R a n t i b o d i e s : Normal pre-B c e l l s (Bp) + FN-R a n t i b o d i e s (see panel a ) A n t i b o d i e s were mixed at i n c r e a s i n g c o n c e n t r a t i o n s w i t h e i t h e r normal o r 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 p r e - e s t a b l i s h e d M2-10B4 s t r o m a l c e l l l a y e r (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 d e s c r i b e d i n Chapter I I .  126  produce FN ( 1 1 ) .  I n t h i s s t u d y , we f o c u s s e d on the i n t e r a c t i o n o f pre-B c e l l s  w i t h FN and o b t a i n e d evidence  t h a t FN p l a y s 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 s t i m u l a t e d by i n t e r a c t i o n s between s t r o m a l c e l l s . Normal pre-B c e l l s were shown to a t t a c h s p e c i f i c a l l y to FN, and  although  n e i t h e r FN a l o n e ( T a b l e 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 - ) s t r o m a l ( d a t a not shown) c o u l d support pre-B c e l l p r o l i f e r a t i o n , FN was enhance the p r o l i f e r a t i v e response s t r o m a l c e l l CM.  of normal pre-B c e l l s to f a c t o r s p r e s e n t i n pre-B  by d i f f e r e n t mechanisms, showed the same i m p a i r e d  a b i l i t y to a t t a c h to FN. transformed  a b l e to  I n c o n t r a s t two i n d e p e n d e n t l y i s o l a t e d t r a n s f o r m e d  c e l l l i n e s transformed  cells  I n 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  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.  T h i s l a t t e r f i n d i n g i n d i c a t e s t h a t pre-B c e l l  transformation  i s commonly a s 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 s t r o m a l c e l l s t h a t are n o r m a l l y mediated by FN. to,  o r r e s u l t s from, the m a l i g n a n t , stromal-independent  c h a r a c t e r i s t i c of these c e l l s i s not y e t c l e a r . p r o p e r t i e s i s common to many transformed in  Whether t h i s c o n t r i b u t e s  t h i s a r e a w i t h r e g a r d to hemopoietic  i n the i n t e r a c t i o n of CML  phenotype  Changes i n a d h e s i v e  c e l l types, although l i t t l e cells.  i s known  R e c e n t l y , however, a l t e r a t i o n s  p r o g e n i t o r s w i t h marrow s t r o m a l c e l l s was  reported  (12). To d a t e , v e r y l i t t l e has been r e p o r t e d about the r o l e of FN i n B lymphopoiesis. transformed  B e r n a r d i e t a l has suggested  t h a t the attachment o f  pre-B c e l l l i n e s to FN i n v o l v e s both the FN c e l l b i n d i n g s i t e  and  the h e p a r i n b i n d i n g s i t e , whereas t r a n s f o r m a n t s a r r e s t e d a t a more mature s t a g e 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). lymphoid  S t . John e t a l a l s o r e c e n t l y r e p o r t e d ( i n an a b s t r a c t , 13) t h a t c e l l l i n e s adhere to the h e p a r i n - b i n d i n g domain of FN.  At  first  127  g l a n c e , these r e s u l t s appear to d i f f e r from those d e s c r i b e d h e r e , where t r a n s f o r m a t i o n 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 FN.  However, i t i s important  to note t h a t serum was  p r e s e n t i n the  performed by B e r n a r d i e t a l (4) whereas those presented performed i n the absence of serum. i t s presence  to_adhere_to assays  i n F i g u r e 16 were  Serum c o n t a i n s s e v e r a l ECM  components and  t h e r e f o r e c o m p l i c a t e s i n t e r p r e t a t i o n of the f i n d i n g s .  example, i t i s p o s s i b l e t h a t the attachment of transformed  For  pre-B c e l l s to the  h e p a r i n domain of FN might be u n d e t e c t a b l e 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 t h r e e s t u d i e s a r e c o n s i s t e n t w i t h the i d e a t h a t  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, a l t h o u g h t h i s does appear to be a p r o p e r t y of normal pre-B c e l l s . The p r e s e n t s t u d i e s p r o v i d e the f i r s t r e p o r t 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 .  T h i s 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 o b t a i n e d e i t h e r i n the presence c e l l CM.  Whether FN s i m p l y p l a y s a mechanical  of s t r o m a l c e l l s o r s t r o m a l  r o l e by i m p r o v i n g the c o n t a c t  between growth f a c t o r p r o d u c i n g s t r o m a l c e l l s and  the pre-B t a r g e t c e l l s ,  or  whether FN c o n c e n t r a t e s 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 t h a t FN b i n d i n g a l t e r s  r e s p o n s i v e n e s s of the pre-B c e l l r a t h e r than p l a y i n g a r o l e p r i m a r i l y  the by  i n c r e a s i n g the c o n c e n t r a t i o n of growth f a c t o r the c e l l i s exposed t o . a n a l y s i s of both normal and  Further  transformed pre-B c e l l s s h o u l d 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 h e l p to e s t a b l i s h the s i g n i f i c a n c e o f the a l t e r e d a b i l i t y of transformed  c e l l s to respond  t o , or b i n d t o , FN.  128  REFERENCES I-.  K i e r n e y PC, D o r s h k i n d K. B lymphocyte p r e c u r s o r s and m y e l o i d 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 w i t h s t r o m a l 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. Partial c h a r a c t e r i z a t i o n of a n o v e l s t r o m a l 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 i m m o r t a l i z e d 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. A u t o c r i n e p r o d u c t i o n o f 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 lines. Cancer Res, i n p r e s s .  4.  B e r n a r d i P, P a t e l VP, L o d i s h HF. 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 .  5.  Klingemann H-G, T s o i M-S, S t o r b R. Fibronectin restores defective 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. Fibronectin increases lymphocyte p r o l i f e r a t i o n by m e d i a t i n g adhesion between immunoreactive cells. J C e l l Biochem ( S u p p l 12E):174, 1988.  7.  Hosein B, Bianco C. Monocyte r e c e p t o r s 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 a n t i b o d y that i n t e r f e r e s w i t h r e c e p t o r 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 r e c e p t o r of human macrophages r e c o g n i z e s 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. F i b r o n e c t i n - p l a s m a 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 p r o d u c t s 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 g r a n u l o c y t e development. J Supramolec S t r u c t u r e 13:513, 1980.  11.  Z i p o r i D, Toledo J , von der Mark K. Phenotypic h e t e r o g e n e i t y among s t r o m a l 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 m a t r i x c o m p o s i t i o n 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. Altered a d h e s i v e i n t e r a c t i o n s w i t h marrow stroma of h a e m a t o p o i e t i c p r o g e n i t o r c e l l s i n c h r o n i c myeloid leukaemia. Nature 328:342, 1987.  13.  S t . John J , A p p l e g r e e n RA, L i a o N-S, Cheung HT. Adherence o f lymphoid c e l l l i n e s to the c a r b o x y - t e r m i n a l h e p a r i n b i n d i n g domain o f f i b r o n e c t i n . J C e l l B i o l 105:45a, 1987.  Lymphoid p r e c u r s o r c e l l s adhere to J C e l l B i o l 105:489, 1987.  two  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 o f many types o f h e m o p o i e t i c  t h a t the p r o l i f e r a t i o n  c e l l s i n c l u d i n g pre-B c e l l s i s r e g u l a t e d by t h e i r  i n t e r a c t i o n s w i t h marrow s t r o m a l c e l l s .  I t i s c l e a r t h a t s t r o m a l c e l l s can be  a c t i v a t e d to s e c r e t e a number o f 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 t h a t a c t e i t h e r on m y e l o i d o r a c t i v a t e d mature B c e l l s ( T a b l e s 1 and 2 ) . m o l e c u l a r mechanisms by which s t r o m a l c e l l s may r e g u l a t e B c e l l  The  production  from t h e i r p r e c u r s o r s i n the marrow a r e now a l s o b e g i n n i n g to be d e f i n e d . The lymphoid  LTC was chosen as a b a s i s f o r i n i t i a t i n g t h i s  of stromal-mediated  c o n t r o l o f 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 o f  d e l i n e a t i n g mechanisms thought malignant  transformation.  both an adherent  investigation  to be r e l e v a n t i n v i v o and a f f e c t e d by  However, because lymphoid  LTC's a r e composed o f  heterogenous s t r o m a l c e l l l a y e r and a non-adherent  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 a t d i f f e r e n t s t a g e s o f pre-B  lymphoid  cell  d i f f e r e n t i a t i o n , i t seemed t h a t a c r i t i c a l f i r s t s t e p to f u r t h e r a n a l y s i s would be t o i s o l a t e c l o n a l p o p u l a t i o n s c o r r e s p o n d i n g of c e l l p o p u l a t i o n s . a b i l i t y t o support  to each o f the two types  Stromal c e l l l i n e s were t h e r e f o r e screened  for their  the growth o f pre-B c e l l s d e r i v e d from the non-adherent  f r a c t i o n o f lymphoid  LTC's and then c l o n e d .  l i n e s o f pre-B c e l l s were a l s o i s o l a t e d .  Permanent s t r o m a l  cell-dependent  From these, c l o n e d l i n e s of  130  spontaneous or A-MuLV induced t r a n s f o r m a n t s were d e r i v e d .  The  availability  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 a n a l y z e the n a t u r e of the i n t e r a c t i o n s t h a t occur between pre-B c e l l s and  stromal  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  A)  One  c e l l s , and  how  transformation.  Factors  of the s t r o m a l  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  s e c r e t e a s o l u b l e f a c t o r a b l e 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 s p o n t a n e o u s l y transformed but s t i l l s t r o m a l (H9).  Further  c e l l - r e s p o n s i v e pre-B c e l l  line  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  M2-10B4 c e l l s i n d i c a t e d that i t r e p r e s e n t s  a p r e v i o u s l y u n d e s c r i b e d HGF  an apparent m o l e c u l a r weight of 10,000 d a l t o n s . produced by d i f f e r e n t s t r o m a l  c e l l s t i m u l a t i n g stromal  with  Very r e c e n t l y s e v e r a l f a c t o r s  c e l l l i n e s and a c t i n g on B c e l l p r e c u r s o r s  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 ) .  by  We  have  a l s o found t h a t a pre-B  c e l l l i n e d e r i v e d from the s p l e e n  (S5-2, d e s c r i b e d  in  Chapter I I I ) s e c r e t e s 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 d a l t o n s )  from t h a t produced by M2-10B4 c e l l s ( d a t a not shown).  i t i s p o s s i b l e that d i f f e r e n t stromal  Thus,  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 a c t 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. represent Therefore, great  On  the o t h e r hand, some of these f a c t o r s  may  d i f f e r e n t l y g l y c o s y l a t e d v e r s i o n s of the same gene p r o d u c t . 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  importance to e v a l u a t e  s p e c i f i c i t i e s i n v i t r o and  t h e i r degree of o v e r l a p and  i n vivo.  functional  of  131  B)  Cell-Cell Interactions  When pre-B c e l l s were s t i m u l a t e d by s t r o m a l c e l l s through an i n t e r l a y e r o f agar, o r by s t r o m a l c e l l c o n d i t i o n e d medium (CM), proliferation  was never as good as when pre-B c e l l s were p l a t e d d i r e c t l y on  stromal c e l l s .  T h i s suggests  t h a t c e l l c o n t a c t p l a y s 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 o f pre-B c e l l growth by s t r o m a l c e l l s . lines  the e x t e n t o f  had been shown t o b i n d s p e c i f i c a l l y  S i n c e o t h e r pre-B  cell  to FN, and o t h e r s t r o m a l c e l l s ,  like  3T3 c e l l s make FN ( 5 ) , FN was c o n s i d e r e d as a p o s s i b l e b i n d i n g i n t e r m e d i a t e between pre-B c e l l s and s t r o m a l c e l l s . FN not o n l y adheres s p e c i f i c a l l y their  proliferation  proliferation  The s t u d i e s performed here showed t h a t  t o normal pre-B c e l l s , i t a l s o s t i m u l a t e s  i n c o n c e r t w i t h a s t r o m a l pre-B f a c t o r .  This increased  c o u l d be e x p l a i n e d by one o r more o f a number o f p o s s i b i l i t i e s .  B i n d i n g c o u l d permit a h i g h e r l o c a l exposure o f pre-B c e l l s t o s t r o m a l d e r i v e d growth f a c t o r s by f i x i n g them i n c l o s e r p r o x i m i t y t o the producer  cells.  A l t e r n a t i v e l y , FN might i t s e l f b i n d and hence c o n c e n t r a t e pre-B growth f a c t o r ( s ) and p r o t e c t them from enzymatic suggested  d e g r a d a t i o n , as r e c e n t l y was  f o r heparan s u l f a t e w i t h r e s p e c t to I L - 3 and GM-CSF ( 6 ) .  experiments  a r e r e q u i r e d to determine  whether FN a c t s a l o n e o r j o i n t l y w i t h  o t h e r ECM components, such as heparan s u l f a t e . i n t e r e s t i n g t o note t h a t FN possesses  Further  I n t h i s regard, i t i s  a heparin binding s i t e .  I t i salso  p o s s i b l e t h a t FN can s e r v e as a second s y n e r g i z i n g s i g n a l , o r may cause s u r f a c e o r c o n f o r m a t i o n a l changes i n pre-B c e l l s t h a t f a c i l i t a t e s response  t o growth f a c t o r s .  cell  their  The a v a i l a b i l i t y o f a s i m p l e model i n which the  s t i m u l a t i n g ( s t r o m a l c e l l ) and r e s p o n d i n g  (pre-B c e l l ) components can be  r e a d i l y o b t a i n e d as s e p a r a t e homogeneous c l o n e d p o p u l a t i o n s s h o u l d be u s e f u l for  f u r t h e r a n a l y s i s o f the p r e c i s e r o l e p l a y e d by FN.  Such s t u d i e s may  132  p r o v i d e i m p o r t a n t i n s i g h t s not o n l y 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 o f 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 o t h e r c e l l  2)  types.  ALTERED MECHANISMS IN TRANSFORMED PRE-B CELLS  Recent o b s e r v a t i o n s o f a u t o c r i n e h e m o p o i e t i c growth f a c t o r p r o d u c t i o n i n human AML ( 7 ) and some lymphoid m a l i g n a n c i e s ( 8 , 9 ) , p l u s the d e m o n s t r a b l e a c t i v a t i o n o f growth f a c t o r gene e x p r e s s i o n i n a v a r i e t y o f t r a n s f o r m e d h e m o p o i e t i c 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 r e l e v a n c e o f such changes t o the a c q u i s i t i o n o f autonomous growth p o t e n t i a l by t r a n s f o r m e d B-lineage  cells.  The p r e s e n t s t u d i e s have y i e l d e d d a t a t h a t bear on t h i s .  Both  spontaneous and A-MuLV t r a n s f o r m a t i o n o f pre-B c e l l s were found t o l e a d t o the a c q u i s i t i o n o f a u t o c r i n e growth f a c t o r p r o d u c t i o n .  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 t r a n s f o r m e d mast c e l l l i n e s i n which m u l t i p l e h e m o p o i e t i c 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). the to  Thus, i t i s p o s s i b l e that e x p r e s s i o n o f v - a b l can d e r e g u l a t e  e x p r e s s i o n o f s e v e r a l d i f f e r e n t h e m o p o i e t i c growth f a c t o r genes a c c o r d i n g the type o f c e l l s i n i t i a l l y  infected.  I t i s i n t e r e s t i n g that the a u t o c r i n e f a c t o r produced by the spontaneous t r a n s f o r m e d 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 . a physiologic role f o r this factor.  This suggests  One might s p e c u l a t e as t o 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 c i r c u m s t a n c e s i n the presence o f s t r o m a l CM ( 1 ) , may do so because they a l s o a r e programmed t o produce low l e v e l s o f 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 m o l e c u l a r  weight between a l l o f the d e s c r i b e d 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 t h a t these a r e d i f f e r e n t ; however, as mentioned sequence d a t a w i l l be r e q u i r e d b e f o r e t h i s i s s u e can be c o n c l u s i v e l y  above  resolved.  The f a c t t h a t the a b i l i t y o f normal pre-B c e l l s t o adhere t o FN i s g r e a t l y d e c r e a s e d a f t e r t r a n s f o r m a t i o n can be compared independence observed i n t r a n s f o r m e d f i b r o b l a s t s ( 1 2 ) .  t o the anchorageI n the l a t t e r  cells,  t r a n s f o r m a t i o n has been shown t o cause an a l t e r a t i o n o f the FN-R e x p r e s s i o n . 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 o f a u t o c r i n e s e c r e t i o n d u r i n g the oncogenic p r o g r e s s i o n o f t r a n s f o r m e d pre-B c e l l s remains to be answered.  I n f a c t , t h i s can be viewed as p a r t o f the l a r g e r q u e s t i o n o f  whether t h e r e i s a r e l a t i o n s h i p between a u t o c r i n e s e c r e t i o n , a l t e r a t i o n o f FN-R and the e x p r e s s i o n o f oncogenes B-lineage malignancies (13-16).  that a r e known t o be i n v o l v e d i n  The myc f a m i l y o f genes, i . e . c-myc, N-myc,  L-myc a r e d i f f e r e n t i a l l y e x p r e s s e d d u r i n g murine B c e l l development  (13) and  these as w e l l as c-myb have been found t o be e i t h e r o v e r e x p r e s s e d o r a m p l i f i e d i n b o t h A-MuLV and s p o n t a n e o u s l y t r a n s f o r m e d B lymphoid c e l l l i n e s ( 1 3 - 1 5 ) . I t has a l s o been shown t h a t c o n s t i t u t i v e c-myc e x p r e s s i o n enhanced the anchorage-independent growth o f 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 ( 1 7 ) .  3)  PROPOSED MODEL  The i n f o r m a t i o n o b t a i n e d from the r e s u l t s p r e s e n t e d i n t h i s  thesis  t o g e t h e r w i t h the f i n d i n g s o f o t h e r s suggest t h a t the r e g u l a t i o n o f normal and t r a n s f o r m e d pre-B c e l l growth by s t r o m a l c e l l s might be d e s c r i b e d by the f o l l o w i n g model.  S t r o m a l 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 o f normal pre-B c e l l p r e c u r s o r s by s e c r e t i n g d i f f e r e n t pre-B f a c t o r s .  soluble  D i r e c t c o n t a c t between s t r o m a l c e l l s and pre-B c e l l s mediated  134  by FN can i n c r e a s e the response to s e c r e t e d 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 o f pre-B c e l l s .  Even normal pre-B c e l l s may  have the c a p a c i t y f o r p r o d u c i n g some a u t o c r i n e growth f a c t o r s a l t h o u g h a r e not d e t e c t a b l e i n the supernatant  o f normal pre-B c e l l c u l t u r e s .  these I n pre-B  m a l i g n a n c i e s , one o r 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 t h a t the c e l l s become independent o f s t r o m a l c e l l s i n o r d e r t o 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 o v e r e x p r e s s i o n o r a m p l i f i c a t i o n o f c-myc which i n t u r n l e a d s t o an a l t e r a t i o n i n FN-R e x p r e s s i o n and l o s s o f the normal a b i l i t y to i n t e r a c t w i t h s t r o m a l c e l l s . p l a c e a s t r o n g s e l e c t i v e p r e s s u r e on a u t o c r i n e v a r i a n t s .  T h i s would  Alternatively,  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 t r a n s f o r m a t i o n p r o c e s s 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 t o  block, s t r o m a l induced q u i e s c e n c e o r d i f f e r e n t i a t i o n .  The g o a l o f t h i s t h e s i s was t o o b t a i n f u r t h e r i n f o r m a t i o n about mechanisms r e g u l a t i n g normal and transformed  pre-B c e l l growth.  q u e s t i o n s have been answered, many more have been r a i s e d .  A l t h o u g h some  In particular i t i s  hoped t h a t the f i n d i n g s w i l l be u s e f u l as a b a s i s f o r i m p r o v i n g our knowledge of the mechanisms t h a t a r e a l t e r e d i n human B - l i n e a g e m a l i g n a n c i e s . s u c c e s s f u l development f o r human c e l l s o f a lymphoid  The  LTC system analogous t o  t h a t 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 s t e p i n t h a t direction.  135  REFERENCES 1.  Hunt P, R o b e r t s o n D, Weiss D, Rennick D, Lee F, W i t t e ON. A s i n g l e bone marrow-derived s t r o m a l c e l l type s u p p o r t s i n v i t r o growth of e a r l y lymphoid and m y e l o i d c e l l s . C e l l 48:997, 1987.  2.  L a n d r e t h KS, D o r s h k i n d K. Pre-B c e l l g e n e r a t i o n 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 s t r o m a l c e l l l i n e . J Immunol 140:845, 1988.  3.  Namen AE, Schmierer AE, March C J , O v e r e l l RW, Park LS, U r d a l DL, M o c h i z u k i DY. B c e l l p r e c u r s o r growth-promoting a c t i v i t y . J Exp 167:988, 1988.  4.  Lemoine FM, Humphries RK, Abraham SDM, K r y s t a l G, Eaves C J . Partial c h a r a c t e r i z a t i o n of a n o v e l s t r o m a l 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 i m m o r t a l i z e d 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. P h e n o t y p i c h e t e r o g e n e i t y among s t r o m a l 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 m a t r i x c o m p o s i t i o n 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 . B l o o d 66: 447, 1985.  6.  R o b e r t s R, G a l l a g h e r J , Spooncer E, A l l e n TD, B l o o m f i e l d F, D e x t e r TM. Heparan s u l p h a t e bound growth f a c t o r s : A mechanism f o r s t r o m a l c e l l mediated h a e m o p o i e s i s . Nature 332:376, 1988.  7.  Young DC, G r i f f i n JD. A u t o c r i n e s e c r e t i o n of GM-CSF i n a c u t e m y e l o b l a s t i c l e u k e m i a . B l o o d 68:1178, 1986.  8.  Gordon I , Ley SC, Melamed MD, E n g l i s h LS, Hughes-Jones NC. I m m o r t a l i z e d 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, R o n c e l l a S, C o z z o l i n o F, Zupo S, F e r r a r i n i M. 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Zimmerman KA, Yancopoulos GD, Collum RG, Smith RK, K o h l NE, Denis KA, Nau MM, W i t t e ON, T o r a n - A l l e r a n d D, Gee CE, Minna JD, A l t FW. Differential e x p r e s s i o n of myc f a m i l y genes d u r i n g murine development. Nature 319:780, 1986.  136  14.  C i t r i Y, Braun J , B a l t i m o r e D. E l e v a t e d myc e x p r e s s i o n and c-myc a m p l i f i c a t i o n i n s p o n t a n e o u s l y 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, K u e h l WM. D i f f e r e n t i a l e x p r e s s i o n o f 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 T J , T r o n i c k SR, Robbins KC. f g r proto-oncogene mRNA induced i n B lymphocytes by E p s t e i n - B a r r v i r u s i n f e c t i o n . N a t u r e 319:238, 1986.  17.  S o r r e n t i n o V, D r o z d o f f 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 o f growth f a c t o r a c t i v i t y by exogenous c-myc e x p r e s s i o n . P r o c N a t l Acad S c i USA 83:8167, 1986.  PUBLICATIONS  F r a n c o i s M. Lemoine  1.  Lemoine F & Dao C. 1981.  D i a g n o s i s o f purpuras.  Rev Med P a r i s 22: 2449-2457,  2.  Metman EH, Danquechin-Dorval E, Lemoine F & B e r t r a n d J . F i b e r endoscopy and treatment o f 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 e t thrombocytoses. E n c y c l Med C h i r P a r i s CP H e m a t o l o g i c 4.6.10: 2399, 1982.  4.  Khayat D, Ricome J L , R i c h a r d C, Lemoine F, R i m a i l h o A & Auzepy P. I n t o x i c a t i o n a i q u e aux b a r b i t u r i q u e s l e n t s e t incompetence c a r d i a q u e t r a n s i t o i r e . Nouv P r e s s e Med 45: 3354, 1982.  5.  Lemoine F, B e n a t r e A, Metman EH, D e l s o l AL, L h u i n t r e J P , 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.  R i c h a r d C, Ricome J L , Lemoine F, R i m a i l h o A, L a n t z 0 & Auzepy P. Interet du C a p t o p r i l dans l e t r a i t e m e n t d'une h y p e r t e n s i o n a r t e r i e l l e systemique et pulmonaire avec a c t i v i t e r e n i n e p l a s m a t i q u e augmentee au cours d'une s c l e r o d e r m i c . Rev Med I n t 4: 125-129, 1983.  7.  Lemoine F, Najman A, L a p o r t e J P , G o r i n NC & Duhamel G. V i n d e s i n e P r e d n i s o n e i n the treatment of b l a s t c r i s i s o f c h r o n i c m y e l o i d l e u k e m i a . Cancer T r e a t Rep 69: 203-204, 1985.  8.  Lemoine F, Najman A, B a i l l o u C, Stachowiak J , B o f f a G, A e g e r t e r P, Douay L, L a p o r t e J P , G o r i n NC & Duhamel G. A p r o s p e c t i v e s t u d y o f the v a l u e o f bone marrow e r y t h r o i d p r o g e n i t o r c u l t u r e s i n p o l y c y t h e m i a . B l o o d 68: 996-1002, 1986. —  9.  V i l l e v a l J L , Cramer P, Lemoine F, H e n r i 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, B r e t o n - G o r i u s J & V a i n c h e n k e r W. Phenotype o f e a r l y e r y t h r o b l a s t i c leukemias. B l o o d 68: 1167-1174, 1986.  10.  Lemoine FM, Humphries RK, Abraham SDM, K r y s t a l G & Eaves C J . P a r t i a l c h a r a c t e r i z a t i o n o f a n o v e l s t r o m a l 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 i m m o r t a l i z e d 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 C J . P a r t i a l c h a r a c t e r i z a t i o n o f a n o v e l s t r o m a l 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 i m m o r t a l i z e d 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 C J . A c t i v a t i o n o f m u l t i p l e hemopoietic growth f a c t o r genes i n A b e l s o n v i r u s t r a n s f o r m e d m y e l o i d c e l l s . Exp Hematol ( i n p r e s s )  13.  Lemoine FM, K r y s t a l G, Humphries RK & Eaves CJ. A u t o c r i n e p r o d u c t i o n o f 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 o f transformed pre-B c e l l lines. Cancer Res ( i n p r e s s )  REVISED:  September 15, 1988  ABSTRACTS  F r a n c o i s M.  Lemoine  1.  Lemoine F, Ricome J L , R i c h a r d C, Khayat D, R i m a i l h o A & Auzepy P. C o l e c t a s i e s a i g u e s sous v e n t i l a t i o n a s s i s t e e : R o l e de l a P h e n o p e r i d i n e . Congres de Medecine I n t e r n e , Geneve, J u i n 1983.  2.  Lemoine F, T a b i l i o A, B r e t o n - G o r i u s J , Najman A & Duhamel G. Leucemie a i g u e c h i m i o - i n d u i t e survenant au cours d'une leucemie lymphoide c h r o n i q u e : I n t e r e t de l a c y t o c h i m i e u l t r a s t r u c t u r a l e et des t e c h n i q u e s de marquage a l ' a i d e d ' a n t i c o r p s monoclonaux dans l a c a r a c t e r i s a t i o n des cellules blastiques. S o c i e t e F r a n c a i s e d'Hematologic, P a r i s , Mars 1984. Nouv Rev F r Hemat 26: 108, 1984.  3.  V i l l e v a l J L , Matamis H, Lemoine F, Bernaudin F, Cramer P, Rochant H, V a i n c h e n k e r W & B r e t o n - G o r i u s J . D i f f e r e n t phenotypes of b l a s t s i n e r y t h r o l e u k e m i a : I d e n t i f i c a t i o n of e a r l y s t a g e s o f G l y c o p h o r i n A n e g a t i v e 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. B l o o d 64 ( S u p p l 1 ) : 199a, 1984.  4.  Lemoine F, Najman A, B a i l l o u C, Stachowiak J , L a p o r t e J P , G o r i n NC & Duhamel G. V a l e u r d i a g n o s t i q u e de l a c u l t u r e de p r o g e n i t e u r s 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 F r a n c a i s d'Hematologic, Mai 1985. Nouv Rev F r Hematol 27: 71, 1985.  5.  Lemoine F, Najman A, B a i l l o u C, Stachowiak J , L a p o r t e J P , G o r i n 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 p o l y c y t h e m i a . Blood 66 ( S u p p l 1 ) : 178a, 1985.  6.  V i l l e v a l J L , Cramer P, H e n r i A, Lemoine F, Berger R, B r e t o n - G o r i u s J & V a i n c h e n k e r W. Phenotypes of e a r l y e r y t h r o b l a s t i c l e u k e m i a . B l o o d 66 ( S u p p l 1 ) : 184a, 1985.  7.  Lemoine FM, Humphries RK & Eaves CJ. I s o l a t i o n of c l o n e d l i n e s of e a r l y pre-B c e l l s t h a t show v a r i a b l e degrees of autonomy but r e t a i n r e s p o n s i v e n e s s to a f a c t o r r e l e a s e d by a c l o n e d mesenchymal c e l l l i n e . B l o o d 68 ( S u p p l 1 ) : 97a, 1986.  8.  Humphries RK, Abraham S, Lemoine F, Lansdorp P & Eaves C J . A c t i v a t i o n of m u l t i p l e h e m o p o i e t i c growth f a c t o r genes i n A b e l s o n v i r u s t r a n s f o r m e d murine m y e l o i d c e l l s . Exp Hematol 15 ( S u p p l 5 ) : 499, 1987.  9.  Lemoine FM, K r y s t a l G, Humphries RK & Eaves CJ. A u t o c r i n e 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 lines. B l o o d 70 ( S u p p l 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 t r a n s f o r m e d 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 ( S u p p l 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 r e c e p t o r s on murine pre-B c e l l s are a s s o c i a t e d w i t h t r a n s f o r m a t i o n and a c q u i s i t i o n of an a u t o c r i n e phenotype. Blood ( i n p r e s s )  the  

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