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Cell surface antigens in normal and neoplastic human B lymphocyte differentiation : cellular distribution… Howard, Donald Raymond 1985

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CELL SURFACE ANTIGENS IN NORMAL AND NEOPLASTIC HUMAN B LYMPHOCYTE DIFFERENTIATION: CELLULAR DISTRIBUTION AND FUNCTIONAL  IMPLICATIONS  by  DONALD RAYMOND HOWARD  B.A., Boston U n i v e r s i t y , 1969 M.D., Albany M e d i c a l C o l l e g e , 1973  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 E x p e r i m e n t a l Pathology)  We accept t h i s t h e s i s as conforming to the r e q u i r e d s t a n d a r d  THE UNIVERSITY OF BRITISH COLUMBIA August, 1985 ®Donald Raymond Howard, 1985  In  presenting  degree  this  at the  thesis in  University of  partial  fulfilment  of  of  department publication  this or  thesis for by  his  or  scholarly purposes may her  permission.  Pathology  The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  DE-6(3/81)  representatives.  October 8,  1985  that the  for  an advanced  Library shall make  it  agree that permission for extensive be  It  of this thesis for financial gain shall not  Department of  requirements  British Columbia, I agree  freely available for reference and study. I further copying  the  is  granted  by the  understood  that  head of copying  my or  be allowed without my written  ii  ABSTRACT  D i f f e r e n t i a t i o n within are i n v o l v e d  i n a v a r i e t y of immune f u n c t i o n s .  p r o v i s i o n of help, cooperative is  the lymphoid system produces e f f e c t o r c e l l s which  suppression,  c y t o l y t i c a c t i v i t y and  cellular interactions.  the p r o d u c t i o n  For T c e l l s  The  of s p e c i f i c a n t i b o d y .  normal lymphocyte p r o l i f e r a t i o n and  these i n c l u d e  the  the r e g u l a t i o n of  primary f u n c t i o n of B l i n e a g e  cells  Understanding the r e g u l a t i o n of  d i f f e r e n t i a t i o n may  l e a d to a b e t t e r  a p p r e c i a t i o n of those f a c t o r s which r e s u l t i n the development of malignancy. The  non-Hodgkin's lymphomas are neoplasms of the immune system, the  of which are B c e l l biology,  little  The  advances i n immunology and  in B cell activation, their  transformation. advent of monoclonal a n t i b o d y technology a decade ago  antigens.  molecular  d i f f e r e n t i a t i o n or about those events l e a d i n g to  r e v o l u t i o n i z e d our a b i l i t y  already  Despite  i s known about the mechanisms i n v o l v e d  p r o l i f e r a t i o n and malignant  in origin.  majority  to i d e n t i f y and  Because the a c t i v a t i o n and  characterize c e l l  that might be  surface  c o n t r o l of p r o l i f e r a t i o n of B c e l l s  known to i n v o l v e s t r u c t u r e s at the c e l l s u r f a c e ,  u t i l i z e monoclonal a n t i b o d i e s  has  i t was  logical  to i d e n t i f y a d d i t i o n a l c e l l s u r f a c e  various  types of n e o p l a s t i c B c e l l s  to  to  molecules  important i n the f u n c t i o n of normal B lymphocytes and  might a l l o w normal and  was  that  be  distinguished. To a c h i e v e t h i s g o a l , we  developed monoclonal a n t i b o d i e s  that showed  d i f f e r e n t i a l r e a c t i v i t y between l a r g e a c t i v e l y d i v i d i n g lymphoma c e l l s small  i n a c t i v e (quiescent)  to i n h i b i t v a r i o u s T and  lymphocytes.  These were t e s t e d f o r t h e i r  B lymphocyte f u n c t i o n s  l i p o p o l y s a c c h a r i d e , phytohemagglutinin and  and  ability  ( i . e . responses to a n t i - u ,  the mixed lymphocyte response) as  iii well as for their reactivity with cell suspensions from a variety of malignant and nonmalignant hematopoietic tissues. From these studies emerged the following:  1) Cell surface molecules  other than Immunoglobulin are involved in regulating the activation of normal B cells.  This was shown by the discovery that monoclonal antibodies to both  lymphocyte function associated antigen (LFA-1) and certain HLA class II determinants were able to inhibit the activation of peripheral blood mononuclear cells by the B cell mitogens anti-p and LPS. This inhibition was shown to be mediated via effects of these antibodies on T cells and/or monocytes.  2) B lymphoma cells appear to express unique cell surface  antigens (defined by monoclonal antibodies LM-26 and LM-155) not detectable on cells of other lineages, and absent from normal resting or activated B lymphocytes. Future investigations will attempt to define the mechanisms by which the indirect involvement of LFA-1 and HLA class II molecules in B c e l l activation in vitro suggests new regulatory interactions not previously identified. Further studies will be required to define the mechanisms underlying these interactions and their significance in vivo.  Similarly, the structure and  function of the antigens detected by LM-26 and LM-155 remains to be determined.  Nevertheless, the expression of apparently unique molecules on B  lymphoma cells holds new promise for the diagnosis, classification and treatment of this group of diseases.  iv TABLE OF CONTENTS Page ABSTRACT  Chapter I  LIST OF TABLES  vi  LIST OF FIGURES  viii  ACKNOWLEDGEMENTS  xi  THE LYMPHOID SYSTEM 1) General Concepts of Lymphoid Differentiation A) The Cell Surface B) Monoclonal Antibodies C) Lymphocyte Ontogeny, Subpopulations and Differentiation Antigens D) Lymphocyte Cell Surface Receptors and Gene Rearrangement 2) Mechanisms of B Cell Activation A) Cell Surface Interactions B) Growth Factors 3) Cell Surface Antigens on Normal and Neoplastic Human B-Lymphocytes A) Definition Using Monoclonal Antibodies B) The Human Major Histocompatibility Complex (HLA System) C) Lymphocyte Function Associated Antigen (LFA) Family of Molecules 4) Neoplasms of the Immune System: The Non-Hodgkin's Lymphomas 5) Thesis Objectives References  Chapter II  i i  1 1 3 3 9 11 11 15 19 19 23 28 31 39 40  MATERIALS AND METHODS 1) Cells 2) Monoclonal Antibodies 3) Preparation of Ascites 4) Purification of Antibody 5) Binding Assays 6) Antibody Coupling Procedure 7) Antibody Labeling 8) Stimulation Assays 9) Inhibition Assays 10) Colony Assays 11) Purification of B Cells 12) FACS Analysis 13) Immunoprecipitations 14) Antibody Blocking Studies References  62 63 65 66 66 67 67 68 69 70 70 71 71 73 74  V  Chapter III  LYMPHOCYTE FUNCTION ASSOCIATED ANTIGEN (LFA-1) IS INVOLVED IN B CELL ACTIVATION 1) Introduction 2) Results A) Monoclonal Antibody NB-107 Defines a Distinct Epitope on the LFA-1 Molecule B) Expression of NB-107 on Peripheral Blood Mononuclear Cells, Neoplastic and Non-Neoplastic Cell Lines C) NB-107 (Anti-LFA-1) Inhibits B Cell Activation 3) Discussion References  Chapter IV  Chapter VI  77 79 83 89 96  MONOCLONAL ANTIBODIES TO HLA-CLASS II DETERMINANTS: FUNCTIONAL EFFECTS ON THE ACTIVATION AND PROLIFERATION OF NORMAL AND EBV TRANSFORMED B CELLS 1) Introduction 2) Results A) Antibody Specificity B) Inhibition of PBMC Stimulation C) Inhibition of Purified B Cells D) Inhibition of EBV Cell Lines 3) Discussion References  Chapter V  76 77  98 99 99 106 110 110 110 118  TWO MONOCLONAL ANTIBODIES THAT DEFINE UNIQUE ANTIGENIC DETERMINANTS ON B-LYMPHOMA CELLS 1) Introduction 2) Results A) Reactivity with Cell Lines B) Reactivity with Fresh Tissues C) Reactivity with Normal B-Blasts 3) Discussion References  120 121 121 123 129 129 136  SUMMARY AND CONCLUSIONS  139  vi LIST OF TABLES Page TABLE I TABLE II TABLE III  Monoclonal antibodies defining B cell and B cell related surface determinants A comparison of the proposed "Working Formulation" with classifications for non-Hodgkin's lymphomas  21 36  3  Competitive inhibition of H-lysine labeled NB-107 binding to DHL-4 cells  81  TABLE IV  Cell line reactivity of NB-107 (FACS analysis)  85  TABLE V  Inhibition of B cell activation by anti-LFA-1  86  TABLE VI  Inhibition of LPS stimulation:  Titration using  purified NB-107  87  TABLE VII  Inhibition of T cell proliferation by anti-LFA-1  88  TABLE VIII  Lack of inhibition of EBV c e l l line growth by anti-LFA-1.  90  TABLE IX  Inhibition of anti-y stimulation:  TABLE X  Effect of anti-LFA-1 on bone marrow progenitor cells  TABLE XI  Reactivity of anti-class II monoclonal antibodies with homozygous DR cell lines: FACS analysis  TABLE XII TABLE XIII TABLE XIV TABLE XV  Purified B cells  91 92 101  125  Cross blocking of I-labeled anti-class II antibodies: DHL-4 cells Inhibition of stimulation of normal PBMC by anti-class II monoclonal antibodies Inhibition of anti-u stimulation of normal PBMC: Titration using purified anti-HLA class II antibody Inhibition of mixed lymphocyte reaction by  104 108 109  anti-class II monoclonal antibodies  111  TABLE XVI  Inhibition of PHA stimulation of normal PBMC  112  TABLE XVII  Inhibition of anti-y stimulation of purified B cells  113  TABLE XVIII TABLE XIX  Inhibition of EBV cell line proliferation by Cell line reactivity of antilymphoma anti-class II monoclonal antibodies antibodies: FACS analysis  114 122  vii TABLE XX  Analysis of fresh tissues: positive  B c e l l malignancies -  TABLE XXI  Analysis of fresh tissues: malignancies - negative  B and T cell  TABLE XXII  Analysis of fresh tissues: proliferations - negative  Reactive lymphoid  TABLE XXIII  Analysis of fresh tissues:  Miscellaneous - negative  124 127 128 130  viii LIST OF FIGURES Page FIGURE 1  Schematic diagram of lymphocyte differentiation. Beginning with the pluripotent hematopoietic stem cell (PHSC) lymphocytes pass through a series of stages ending with functionally mature cells of B or T lineage (vertical arrows). For contrast, the transformation of mature B and T lymphocytes to large actively dividing immunoblasts is represented by the horizontal arrows. Immunoblasts may differentiate further to effector cells of T or B lineage (e.g. cytotoxic T cells, plasma cells). The mechanism of transformation i s illustrated in more detail in Figures 2 and 3.  FIGURE 2  Model of T lymphocyte transformation. Resting T cells must f i r s t be activated before they are able to respond to interleukin-2 (IL-2).  FIGURE 3  Simplified schematic diagram of B lymphocyte transformation. Figure shown illustrates the classical concepts of how normal B cells transform into B immunoblasts in response to antigen or mitogens. According to this model B cells must f i r s t be activated by antigen or mitogen before being capable of responding to B cell growth factor (BCGF). Recently, this concept has been questioned. Newer evidence suggests that BCGF (B c e l l stimulatory factor-1, BSF-1) may induce resting B cells to become more responsive to stimuli such as anti-immunoglobulin. See text for details.  12  Follicular center c e l l concept of lymphocyte transformation. According to this hypothesis, normal B cells pass through a series of morphologic stages within the follicular centers of lymph nodes. B c e l l lymphomas may be classified according to which subtype of cell predominates. The predominant cell type, within a given lymphoma, may correspond to one of the stages in normal B cell transformation illustrated.  35  The molecular weight of the antigen precipitated from DHL-4 cells by NB-107 is approximately 170 and 95 Kd under reducing conditions (R) and 170 and 115 Kd under non-reducing conditions (NR). . Negative control (antibody to Thy 1.2) and positive control (antibody to Transferrin receptor, TR) are included for comparison.  78  FIGURE 4  FIGURE 5  ix FIGURE 6  FIGURE 7  FIGURE 8  FIGURE 9  FIGURE 10  Sequential immunoprecipitation ("preclearing"): Antibody to transferrin receptor (NB-65), completely removes transferrin receptor from DHL-4 lysate. LFA-1 detected by TS1/18, TS1/22 and NB-107 remains (A). Preclearing with NB-107 (B) removes material reactive with TS1/18, TS1/22 and NB-107, while leaving transferrin receptor unaffected. Note that molecules immunoprecipitated by NB-107, TS1/18 and TS1/22 have an identical appearance and mobility.  80  FACS analysis of NB-107 tested against normal peripheral blood mononuclear cells. Cells with the greatest amount of light scatter (larger cells, predominantly monocytes) display the most intense staining by NB-107. Cells of intermediate size (lymphocytes) show a spectrum of reactivity from strong to weak.  82  Dual fluorescence of normal peripheral blood mononuclear cells using phycoerythrin labeled anti-DR and fluorescein labeled NB-107. Cells with the greatest intensity of DR staining (principally monocytes) also express the highest amounts of LFA-1 defined by NB-107.  84  125  Immunoprecipitation using I-labeled WALK (DR4) cells. NB 29 (anti-DQ), DH-224 (anti-DR) and DH-84 (anti-DQ+DR) immunoprecipitate bands of approximately 35,000 and 28,000 molecular weight. Shown for comparison are the known anti-DR monoclonals from Ortho (OKIa) and Becton-Dickinson (BD-DR). The amount of material precipitated by DH-84 and OKIa i s considerably less than that of the other antibodies. This probably relates to differences in antibody affinity. The molecular weight of each chain precipitated by the anti-DQ monoclonal NB-29 is 1 to 2 kd less than that of the anti-DR monoclonals (e.g. BD-DR).  102  125  Immunoprecipitation using I-labeled WALK (DR4) cells. Antibodies NB-29, DH-84 and DH-224 are shown for comparison with known anti-DQ monoclonal antibodies BT3.4 and Leu-10. NB-29 and BT3.4 immunoprecipitate identical bands, each of which has mobility slightly greater than those precipitated by BD-DR.  103  Sequential immunoprecipitation "preclearing" of I-labeled WALK (DR4) cell lysate. "A" is precleared with antibody of unrelated specificity. DH-84, DH-224, Leu-10 and BD-DR each precipitate bands of approximately 35,000 and 28,000 m.w. "B" is precleared using DH-84 which substantially reduces the amount of BD-DR and DH-224. The marked diminution in the amount of material precipitated by the known anti-DR monoclonal antibody (BD-DR) indicates DH-84 has specificity for DR molecules. The amount of Leu-10 (anti-DQ) precipitated material i s unaffected. "C" is precleared using DH-224. In addition to removing material reactive with itself, preclearing with DH-224 has markedly reduced the amount of DR precipitated by BD-DR while leaving DQ reactive material (precipitated by Leu-10) unchanged. 12S  Sequential immunoprecipitation "preclearing" of ^I-labeled WALK (DR4) cell lysate. "A" i s precleared with control antibody of unrelated specificity. NB-29 and DH-84 each precipitate bands of approximately 35,000 and 28,000 m.w. "B" is precleared with NB-29. NB-29 preclearing removes a l l material reactive with itself, while not quantitatively affecting the amount of material precipitated by DH-84. 12  FACS histogram of small cleaved cell lymphoma stained with A) negative control antibody, B) anti-kappa, C) anti-lambda, D) LM-26. A monoclonal lambda pattern of surface immunoglobulin i s identified. Staining intensity of LM-26 exceeds that of anti-lambda for some cells. FACS contour plot of small cleaved c e l l lymphoma stained with A) negative control antibody, B) anti-kappa, C) anti-lambda, D) LM-26. Cell number is reflected in the 'Z' axis. Both anti-lambda and LM-26 stain lymphoma cells of a l l sizes. This indicates LM-26 binding is not restricted to a particular subtype of c e l l based on size (e.g. large transformed lymphoid cells) within a given lymphoma. FACS histogram of purified LPS stimulated normal B c e l l blasts stained with A) negative control antibody, B) anti-polyvalent surface immunoglobulin, C) LM-26, D) 0KT11. Ninety per cent of cells are surface immunoglobulin positive B cells (B), which by light scatter and morphologic examination of stained cytospins, are predominantly blasts. These cells do not bind LM-26 (C). There i s only five per cent residual contamination with T cells (D).  xi  ACKNOWLEDGEMENTS  "There i s a t i d e i n the a f f a i r s of men which, taken a t the f l o o d , l e a d s on to f o r t u n e ; omitted, a l l the voyage of t h e i r l i f e i s bound i n shallows and i n m i s e r i e s . On such a f u l l sea are we now a f l o a t , and we must take the c u r r e n t when i t s e r v e s , or l o s e our v e n t u r e s . " W.  I wish  Shakespeare ( J u l i u s  Caesar)  to express my g r a t i t u d e  To Drs. A. EAVES, C. EAVES and F. TAKEI f o r p r o v i d i n g the o p p o r t u n i t y , f a c i l i t i e s and s t i m u l a t i o n n e c e s s a r y to complete t h i s work, To Drs. C. TAYLOR, J . BATSAKIS and B. MACPHERSON who, i n i t i a t e d me i n t o the world of academic pathology,  by t h e i r example,  To C. SMITH, D. NIPIUS, W. DRAGOWSKA, and the o t h e r t e c h n o l o g i s t s from whom I have l e a r n e d or who have a s s i s t e d w i t h t h i s p r o j e c t ,  this  To J . WAITE and M. COULOMBE who thesis, To T. FOX  who  inspired  To A. LANCASTER who To DENISE who  have had  supported  me,  s u f f e r e d w i t h me,  NERY who  to type  me,  and To M.V.  the p a t i e n c e and s k i l l  started  it all.  MARISSA and TREVOR who  made me  happy,  1 C H A P T E R  THE  I  LYMPHOID SYSTEM  "There are two k i n d s of c o n f i d e n c e which a r e a d e r may have i n h i s author... there i s a c o n f i d e n c e i n f a c t s and a c o n f i d e n c e i n v i s i o n . . . T h e former r e q u i r e s simple f a i t h . The l a t t e r c a l l s upon you to judge f o r y o u r s e l f and form your own conclusions." Anthony T r o l l o p e  1) GENERAL CONCEPTS OF (A) The  Cell  LYMPHOCYTE DIFFERENTIATION  Surface  Many important p h y s i o l o g i c a l f u n c t i o n s are mediated at the s u r f a c e s cells.  These i n c l u d e :  cell-cell  s e l e c t i v e transport  of s m a l l molecules and  of  ions,  i n t e r a c t i o n s , c e l l adhesion, c e l l a c t i v a t i o n by hormones, growth  f a c t o r s and  mitogens, phagocytosis,  regulation (1).  The  lipid  b i o l o g i c a l membranes. f u n c t i o n s and  serve  exocytosis  and  endocytosis  and  metabolic  b i l a y e r determines the b a s i c s t r u c t u r e of  However, p r o t e i n s are r e s p o n s i b l e  as s p e c i f i c r e c e p t o r s ,  f o r most membrane  enzymes or t r a n s p o r t e r s .  The  m a t r i x of c e l l membranes i s a b i l a y e r composed predominantly of phospholipids, and  g l y c o l i p i d s and  cholesterol.  amphipathic  a s s o c i a t e w i t h t h e i r more hydrophobic p o r t i o n s o r i e n t e d i n t e r n a l l y  t h e i r h y d r o p h i l i c ends p r o t r u d i n g the  These molecules are  externally.  i n t e g r a l p r o t e i n s of the membrane.  p r o t e i n s ; most are g l y c o p r o t e i n s .  components u n l e s s  Embedded i n the b i l a y e r are  Many of these are  transmembrane  At p h y s i o l o g i c a l temperatures,  membrane e x i s t s as a two-dimensional f l u i d ,  i n which p r o t e i n and  s p e c i f i c a l l y r e s t r i c t e d , may  and  move f r e e l y .  The  the lipid cell  2 membrane does not exist i n i s o l a t i o n .  In contrast to integral proteins,  peripheral proteins are not associated with the l i p i d bilayer but exist within the aqueous phase of the c e l l membrane, non-covalently attached to protruding regions of integral proteins.  The concept of peripheral proteins  may help to explain how c e l l membranes relate to their external  (exoskeleton)  and i n t e r n a l (cytoskeleton) environments (e.g. microfilaments, intermediate filaments and microtubules i n the cytoskeleton; fibronectin and collagen i n the exoskeleton (1, 2). Recent evidence has implicated a quantitatively minor group of membrane phospholipids (the polyphosphoinositides) i n signal transmission for a wide variety of growth factors, neurotransmitters and hormones.  A c t i v a t i o n of the  polyphosphoinositide system results i n the release of products which act as "second messengers" i n evoking the c e l l ' s responses.  Two products released  from polyphosphoinositide as a consequence of receptor activation are i n o s i t o l triphosphate and d i a c y l g l y c e r o l .  I n o s i t o l triphosphate causes an  increase i n i n t r a c e l l u l a r calcium ions which modulates further reactions within the c e l l .  Diacylglycerol appears to act independently by stimulating  a protein phosphokinase (3). The possible involvement beginning to be explored.  of this system i n lymphocyte activation i s just  However, recent evidence suggests that  perturbations of the T3-antigen receptor complex by monoclonal antibodies r e s u l t s i n the release of i n o s i t o l triphosphate.  This i n turn causes the  release of calcium ions from i n t r a c e l l u l a r stores; an effect which i s thought to be important  i n the activation of the human T c e l l l i n e , Jurkat, to  produce interleukin 2 (4).  Furthermore, some evidence supports the  hypothesis that cross-linking of B c e l l surface immunoglobulin leads to subsequent activation through a series of events including phosphatidyl  3 i n o s i t o l hydrolysis, followed by the generation of d i a c y l g l y c e r o l and protein kinase C a c t i v a t i o n (5, 6). (B) Monoclonal Antibodies Recently, technology has become available which has revolutionized the a b i l i t y to study c e l l surface molecules.  F i r s t developed by Kohler and  M i l s t e i n i n 1975, monoclonal antibodies promise to be of major value i n furthering the understanding of c e l l interactions and function (7).  By  combining an antibody producing c e l l with a neoplastic plasma c e l l , a hybrid (or hybridoma) can be generated with the desired c h a r a c t e r i s t i c s of each. Usually, a mouse of the same genetic background as the myeloma c e l l s i s immunized with an antigen.  The spleen of the mouse i s removed and teased  apart to y i e l d a c e l l suspension.  Myeloma c e l l s and splenic B c e l l s are then  fused together to form a single c e l l or hybrid.  Fusion i s f a c i l i t a t e d using  polyethyleneglycol, an e l e c t r i c a l pulse or Sendai virus.  Hybrids are then  selected, screened f o r antibody production, cloned, rescreened, recloned and f i n a l l y the antibodies are characterized.  Inherited from the parent myeloma  c e l l i s the property of immortality i n culture; from the immune lymphocyte the production of s p e c i f i c antibody.  Monoclonal antibodies, because of their  exquisite s p e c i f i c i t y can be used to detect d i s t i n c t c e l l surface molecules or epitopes.  Furthermore, monoclonal antibodies may be used to purify  antigens or to assess biologic functions i n i n v i t r o assays (8).  To a great  degree, monoclonal antibodies have been responsible f o r the characterization and increased understanding of lymphocyte d i f f e r e n t i a t i o n that has occurred in the last decade. (C) Lymphocyte Ontogeny, Subpopulations and D i f f e r e n t i a t i o n Antigens It i s now clear that there are two classes of lymphocytes each mediating distinct  functions (Figure 1).  B c e l l s secrete antibody; T c e l l s  subserve  4  Differentiation  vs  Transformation  PHSC Hemopoiesis Lymphopoiesis Lymphoid Progenitor Cell Pre-B cell B-lmmunoblast*—mature B cell Plasma cell  FIGURE 1  Memory B lymphocyte  Pre-T cell mature T c e l l * * * T-lmmunoblast Effector T lymphocyte  Memory T lymphocyte  Schematic diagram o f lymphocyte d i f f e r e n t i a t i o n B e g i n n i n g with the p l u r i p o t e n t h e m a t o p o i e t i c stem c e l l (PHSC) lymphocytes pass through a s e r i e s o f stages ending with f u n c t i o n a l l y mature c e l l s o f B or T l i n e a g e ( v e r t i c a l a r r o w s ) . For c o n t r a s t , the t r a n s f o r m a t i o n of mature B and T lymphocytes to l a r g e a c t i v e l y d i v i d i n g immunoblasts i s r e p r e s e n t e d by the h o r i z o n t a l arrows. Immunoblasts may d i f f e r e n t i a t e f u r t h e r to e f f e c t o r c e l l s o f T o r B l i n e a g e (e.g. c y t o t o x i c T c e l l s , plasma c e l l s ) . The mechanism of t r a n s f o r m a t i o n i s i l l u s t r a t e d i n more d e t a i l i n F i g u r e s 2 and 3.  5 r e g u l a t o r y and e f f e c t o r f u n c t i o n s . lymphoid  organ c a l l e d  In b i r d s , B c e l l s a r e generated i n the  the bursa o f F a b r i c i u s .  In mammals, h e m a t o p o i e t i c stem  c e l l s m i g r a t e from the y o l k sac to the f e t a l l i v e r where they d i f f e r e n t i a t e i n t o e r y t h r o i d , myeloid and B c e l l s .  Stem c e l l s  then populate the bone  marrow which becomes the major organ o f hematopoiesis. and o t h e r c e l l s are c o n t i n u o u s l y produced B lymphocytes  i n marrow throughout  and  IgA,  IgD and I g E ) .  (9).  life  they s y n t h e s i z e .  t h e i r t e r m i n a l l y d i f f e r e n t i a t e d progeny  s y n t h e s i z e and/or  s e c r e t e a l l c l a s s e s o f immunoglobulin Evidence now  plasma  B  cells  molecules  (IgM,  More mature B c e l l s express both c e l l s u r f a c e IgM and IgD.  called  result  lymphocytes  are thought  Further  may  to have IgM on t h e i r c e l l s u r f a c e s .  a n t i b o d y responses upon subsequent  Memory B  a n t i g e n i c exposure  (10).  I s o t y p e s w i t c h i n g may  heavy c h a i n  be observed d u r i n g  c l o n a l p r o l i f e r a t i o n o f B c e l l s i n response to c e r t a i n a n t i g e n s or Constant heavy c h a i n (CH) r e g i o n i s o t y p e s w i t c h i n g may  o c c u r d u r i n g the pre-B  to B c e l l  transition.  f u n c t i o n o f these c e l l s ,  s w i t c h i n g may  of a n t i g e n and T  switched at e a r l y s t a g e s , i s unknown.  be e i t h e r s e q u e n t i a l , from IgM  mitogens also  At t h i s stage o f development  the s w i t c h i n g process i s most probably independent The  cells  (anamnestic)  I t i s c l e a r that B c e l l s undergo a process of immunoglobulin switching during d i f f e r e n t i a t i o n ,  So-  express s u r f a c e IgG, w h i l e " v i r g i n " B  a r e f u n c t i o n a l l y important f o r development o f r a p i d secondary  such as LPS.  class.  i n the e x p r e s s i o n of IgG w i t h or without IgD.  "memory" B lymphocytes  IgG,  supports the h y p o t h e s i s that the e a r l i e s t  p r o g e n i t o r s o f a n t i g e n - s p e c i f i c B c e l l s possess r e c e p t o r s o f the IgM  m a t u r a t i o n may  cells  are c a t e g o r i z e d i n t o f u n c t i o n a l s u b p o p u l a t i o n s on the  b a s i s o f the d i f f e r e n t c l a s s e s of immunoglobulin lymphocytes  Thereafter, B  to o t h e r immunoglobulin  i n o r d e r o f the CH genes on the chromosome, or d i r e c t , from IgM  cells. Isotype isotypes  to any o f the  6 other isotypes encoded by downstream CH genes * Evidence suggests that the most commonly used pathways are d i r e c t isotype switches from IgM (9, 11-13). A number of reports have recently been published describing monoclonal antibodies to c e l l surface antigens present on B lymphocytes (14-18). Although these show promise i n d e l i n e a t i n g d i f f e r e n t subpopulations of B c e l l s based on surface antigens, a f u n c t i o n a l c o r r e l a t i o n analogous to that i n the T lymphocyte system has yet to be shown.  However, evidence does e x i s t  which suggests that there are at least two subsets of human B c e l l s which d i f f e r with regard to t h e i r r e l a t i v e s u s c e p t i b i l i t y to p r o l i f e r a t i v e s i g n a l s d e l i v e r e d by B c e l l growth factor (BCGF) (19). B c e l l heterogeneity i s a l s o evident from studies of t h e i r p h y s i c a l properties (e.g. s i z e , t i s s u e d i s t r i b u t i o n and charge) and from t h e i r f u n c t i o n a l c h a r a c t e r i s t i c s ( r e a c t i v i t y to d i f f e r e n t mitogens, antigens, genetic requirements f o r a c t i v a t i o n by T helper c e l l s , and s u s c e p t i b i l i t y to tolerance i n d u c t i o n ) .  It  i s s t i l l not e n t i r e l y c l e a r whether t h i s heterogeneity r e f l e c t s changes associated with the c l o n a l expansion of a s i n g l e l i n e of B c e l l s or whether i t a r i s e s as a r e s u l t of the generation of d i s t i n c t sublines of B c e l l s (2022).  Support f o r the concept of d i s t i n c t subpopulations of B c e l l s has  emerged from the study of mice with the immune d e f i c i e n c y determined by the X chromosomal gene X i d . Xid mice appear to lack a subpopulation of B c e l l s that express the c e l l surface determinants Lyb3, Lyb5 and Lyb7, which are important i n the response to type 2 antigens (e.g. soluble polysaccharides) (23). In contrast to B c e l l s , T c e l l s mature i n the microenvironment of the thymus.  Although i t i s not known with c e r t a i n t y how T c e l l s mature, various  theories have been proposed.  One of these suggests that bone marrow stem  7  c e l l s migrate to the thymic cortex during ontogeny and as these c e l l s move to the medulla they undergo a s e r i e s of d i f f e r e n t i a t i o n stages induced by thymic stromal c e l l s .  Monoclonal antibodies define a number of T c e l l surface  antigens which are thought to c o r r e l a t e with thymic d i f f e r e n t i a t i o n and the a c q u i s i t i o n of T c e l l functions.  Approximately  10% of the t o t a l human  thymocyte pool d i s p l a y markers of e a r l y c o r t i c a l thymocytes: T9.  T i l , T10 and  Late c o r t i c a l thymocytes (80%) maintain T i l and T10, lose T9 but gain  T8, T6,- T4 and T l . At the c o r t i c a l stage, thymocytes do not demonstrate p e r i p h e r a l T c e l l functions.  As thymocytes migrate from the cortex to the  medulla, they d i f f e r e n t i a t e i n t o two d i s t i n c t lineages; one d i s p l a y i n g T4, the other T8. T i l , T10 and T3 are retained i n both lineages.  After further  medullary d i f f e r e n t i a t i o n i n which T10 i s l o s t , c e l l s begin to acquire functions of mature T c e l l s .  Once exported to the periphery, T c e l l s are  f u n c t i o n a l l y mature and can be distinguished on the basis of T4 and T8 expression.  T8+ c e l l s p r i m a r i l y mediate T c e l l c y t o t o x i c i t y .  T4+ c e l l s a c t  as e f f e c t o r c e l l s f o r delayed h y p e r s e n s i t i v i t y , provide "help" f o r B c e l l and c y t o t o x i c i t y functions and are involved i n the induction of suppression. The function of most of these T c e l l surface molecules has not been w e l l defined.  However, T4 and T8 appear to be involved r e s p e c t i v e l y i n the  r e c o g n i t i o n of MHC c l a s s I I and c l a s s I antigens. with the T c e l l receptor.  T3 i s c l o s e l y associated  T9 i s i d e n t i c a l with the receptor f o r t r a n s f e r r i n .  T i l (sheep red blood c e l l receptor) may be an a c t i v a t i o n s t r u c t u r e through which the e f f e c t s of phytohemagglutinin  (PHA) are mediated (9, 24-29).  Postulated c e l l u l a r i n t e r a c t i o n s involved i n T 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 are i l l u s t r a t e d i n Figure 2. Recently, t h i s c l a s s i c a l concept of thymic T c e l l d i f f e r e n t i a t i o n has been challenged and two possible independent pathways of T c e l l  maturation  8  Model of T Lymphocyte Transformation Ag  +  ®  ^  ~0 IL-2 IL-2R  Proliferation Differentiation Factors (?) T-mitogens Effector T cell  FIGURE 2  Model o f T lymphocyte t r a n s f o r m a t i o n . Resting T c e l l s must f i r s t be a c t i v a t e d b e f o r e they a r e a b l e to respond to i n t e r l e u k i n - 2 ( I L - 2 ) .  9 have been proposed.  In the first of these, superficial thymic cortical  lymphoblasts divide and differentiate  to give rise to small deep cortical  thymic lymphocytes, medullary lymphocytes and cells leaving the thymus.  The  second hypothesis suggests that the medulla contains an independent selfrenewing c e l l population that contains the precursors of the peripheral Tc e l l pool (30,31).  These issues remain to be resolved.  (D) Lymphocyte Cell Surface Receptors and Gene Rearrangement The B lymphocyte surface receptor involved in antigen recognition is immunoglobulin (Ig). The T c e l l receptor for antigen has been characterized as a class of c e l l surface heterodimers,  termed T i , that are membrane  associated with the T3 glycoprotein complex.  Ti molecules are present on a l l  mature T cells and confer exquisite specificity to these cells in terms of their ability to recognize antigen (32, 33).  Genes coding for both B c e l l  receptors (Ig) and T c e l l receptors (Ti) have been cloned (34-36) and found to exhibit considerable homology (37). During the process of B and T cell differentiation rearranged.  Ig and Ti genes are  Rearrangement precedes the c e l l surface expression of the  respective receptors (34-39).  Using appropriate DNA probes, a distinction  can be readily made between monoclonal and polyclonal populations of B and T cells based on the pattern of Ig or Ti gene rearrangement.  This technology  has recently been applied to determine the cell of origin of lymphoid malignancies of uncertain histogenesis, such as acute lymphoblastic leukemia and hairy c e l l leukemia.  Moreover, immunoglobulin and T c e l l receptor gene  rearrangement can be used as a diagnostic criterion for malignancies of B and T c e l l type that lack characteristic  cell surface markers (40-45).  The earliest stage of B cell development involves the commitment of pluripotent hematopoietic stem cells to the B cell pathway, rearrangement of  10 immunoglobulin (Ig) genes, and expression of cell^surface Ig which serves as the antigen receptor.  Cells must migrate to appropriate peripheral lymphoid  tissues where they can be activated by antigen in concert with secondary factors.  Finally, selected and activated B cells must expand in number and  be induced to secrete immunoglobulin (46). Along the differentiation pathway from pluripotent hematopoietic stem c e l l (PHSC) to functionally mature B c e l l , a series of changes occur which may be conveniently delineated based upon detectable cellular events.  The  PHSC remains a hypothetical cell whose characteristics have yet to be determined.  Upon commitment to the B lineage, the committed lymphoid stem  c e l l undergoes a process of immunoglobulin gene rearrangement, detectable by in vitro molecular hybridization.  Studies comparing Ig gene arrangement in B  cells with nonlymphoid embryonic tissue have provided insight into the mechanism by which immunoglobulin diversity is generated.  These studies have  shown that light chain genes are organized in a discontinuous system of germ line variable (VL) regions, joining (JL) segments and constant (CL) regions. Heavy chain genes are organized similarly (VH, JH, CH) but appear to have an additional diversity (DH) segment located between their VH and JH regions. At some point early in B cell differentiation a cell rearranges i t s genome to form the sequences that encode the heavy (VH/DH/JH) and light (VL/JL) chain variable regions.  Further differentiation, transcription and RNA  splicing  results in the linking of variable sequences with heavy (CH) or light (CL) chain constant region sequences.  It appears that immunoglobulin heavy chain  variable region gene formation precedes that of light chain, and kappa light chain gene formation precedes that of lambda (47, 48). Cells at an early stage of B cell commitment lack detectable immunoglobulin.  First to appear is cytoplasmic IgM which defines the pre-B  11 cell.  Subsequently, IgM appears on the cell surface (immature B cell) often  with IgD (mature B c e l l ) .  These stages in normal B lymphocyte  differentiation have their counterpart in leukemic B cells.  Acute  lymphoblastic leukemia (ALL) may be subdivided into prognostically significant subtypes based on the expression of c e l l surface markers and the presence of Ig gene rearrangement.  These include the common ALL antigen  (CALLA) positive subtype (Ig genes rearranged, lack of detectable Ig), pre-B ALL (cytoplasmic IgM positive) and B ALL (surface Ig positive) subtypes (49).  2) MECHANISMS OF B CELL ACTIVATION (A) Cell Surface Interactions Human B cells, like those of the mouse, can be activated by a number of triggering signals acting at the cell surface.  Under appropriate influences,  B cells once activated, proliferate and differentiate to immunoglobulin secreting plasma cells.  The prevailing view of how this process occurs may  be summarized as follows (19, 50-73, Figure 3): Step 1. Helper T cells interact with antigen presented by accessory cells in the context of MHC class II determinants.  These T cells are then  activated to produce diffusible molecules (lymphokines) that can influence the growth and differentiation of various hematopoietic c e l l lineages. During the activation process both T cells and accessory cells produce lymphokines to which B lymphocytes respond.  T cells produce B c e l l growth  factor (BCGF), interleukin 2 (IL-2), B c e l l maturation factors, interferons and various erythropoietic and myelopoietic growth factors.  Accessory cells  produce interleukin 1 (IL-1) and other less well characterized factors. Lymphokines act on essentially a l l lymphocytes, of appropriate lineage and differentiation state, without regard to antigen specificity (29, 50, 53, 54, 56-59, 69).  12  Model of B Lymphocyte Transformation  (W)+(^)  BCGF  Ag BCGF-R I Activated]  B BCGF-R T-'independent' mitogens  FIGURE 3  Proliferation BCDF TRF Plasma cell  S i m p l i f i e d schematic diagram o f B lymphocyte transformation. F i g u r e shown i l l u s t r a t e s the c l a s s i c a l concepts o f how normal B c e l l s transform i n t o B immunoblasts i n response to a n t i g e n o r mitogens. A c c o r d i n g to t h i s model B c e l l s must f i r s t be a c t i v a t e d by a n t i g e n or mitogen b e f o r e being capable o f responding to B c e l l growth f a c t o r (BCGF). R e c e n t l y , t h i s concept has been q u e s t i o n e d . Newer evidence suggests that BCGF (B c e l l s t i m u l a t o r y f a c t o r - 1 , BSF-1) may induce r e s t i n g B c e l l s to become more r e s p o n s i v e to s t i m u l i such as anti-immunoglobulin. See text f o r d e t a i l s .  13 Step 2.  The second step in B cell activation involves the transition of  the B c e l l from a resting GO state to an activated Gl state.  B cells may be  activated from GO to Gl by a number of mechanisms including: (1) activation by specific antigen, (2) activation by polyclonal activators (e.g. lipopolysaccharide, anti-^i), (3) selective activation by antigen-specific, Ia restricted helper T cells, (4) activation by alloreactive T cells, (5) activation by T-independent antigens (e.g. bacterial polysaccharides and Ficoll) (19, 50, 52, 55, 58, 60, 61, 63, 66-68, 70).  B cells may also be stimulated by anti-idiotype antibodies (72). B cells require T cell help to produce specific antibody.  Classically,  this has been thought to occur through a mechanism by which antigen-specific helper T cells interact with antigen-specific B cells via an antigen bridge. By this concept B cells bind to one determinant on an antigen molecule (the hapten), while the T cells recognize simultaneously another determinant (the carrier).  T helper cells may also bind specifically to antigen presenting  cells (APC) which have picked up and processed the appropriate antigen (Figure 3).  This interaction, like the interaction of T-helper cells with  specific B cells is restricted by products encoded by the major histocompatibility complex (MHC class II products). suggests this model may be oversimplified.  Recent work, however,  It appears that antigen must  f i r s t be internalized and processed by specific B cells before i t is presented to T cells in an MHC-restricted manner, analogous to presentation by conventional APC (e.g. macrophages) (29, 58, 59, 61). Step 3.  This step involves the control of subsequent B c e l l  proliferation and maturation events.  Various T cell and accessory c e l l  derived soluble factors act on responsive B cells to induce their proliferation and differentiation into clones of immunoglobulin secreting plasma cells (50, 62, 64, 65, 70 and also see below).  14 Binding of specific antigen to immunoglobulin receptors on the surface of B cells initiates the activation process leading to proliferation and immunoglobulin production.  Anti-immunoglobulin antibodies (especially anti-  u) have been employed as polyclonal B cell activators on the assumption that they mimic the action of multivalent antigens on B cells, by cross-linking c e l l surface immunoglobulin (74).  Lipopolysaccharide (LPS) likewise induces  B c e l l activation by cross-linking surface receptors.  However, LPS receptors  and surface IgM appear to be distinct molecules, not physically linked to each other in the plane of the cell membrane (69). Anti-u antibody binds to and cross links surface membrane IgM on B cells resulting in cellular activation.  Depending on the concentration of anti-ju  used, two B c e l l responses may occur (75).  At low concentrations of anti-ju,  B cells are induced to enlarge and increase RNA synthesis but do not progress into S phase without the addition of T cell derived stimulatory factors. At high concentrations, anti-p is thought to initiate a direct proliferative effect on B cells independent of T cells, monocytes or their products (13, 63, 75-78).  Accessory cells appear able to play a synergistic role in in  vitro stimulation of B cells (79-83) but the mechanism(s) involved are not clear. The biochemical events which occur at the membrane and within the c e l l after cross-linking of B cell surface immunoglobulin, also remain to be fully elucidated.  However, recent evidence suggests that F(ab')2 fragments of  antibodies specific for IgM are able to induce changes in B c e l l physiology that are indicative of cell activation.  These include membrane  depolarization, followed by increased I-A expression, GO to Gl transition and thymidine uptake (84).  Furthermore, evidence has been provided which  supports the hypothesis that cross-linking of B c e l l surface immunoglobulin  15 leads to subsequent activation through a series of events including phosphatidylinositol hydrolysis, followed by the generation of diacylglycerol and protein kinase C activation (5, 6).  These findings, suggesting that  protein kinase C is involved in the regulation of normal B cell activation and proliferation, are intriguing since a number of oncogene products are protein kinases (85).  It is tempting to speculate that lymphomatous  transformation of B cells may be somehow linked to abnormal regulatory control of these enzymes. (B) Growth Factors Growth factors (interleukins, lymphokines), as applied to the lymphoid system, are genetically unrestricted peptides that nonspecifically modulate immunologic and inflammatory responses by regulating the growth and differentiation of a wide variety of cell types (86). produced primarily by lymphocytes and monocytes.  These factors are  Those factors most relevant  to B c e l l growth and differentiation include interleukin 1 (IL-1), interleukin 2 (IL-2, T-cell growth factor), B cell stimulatory factor (BSF, B-cell growth factor) and a number of less well characterized B c e l l differentiation factors. Interleukin 1 The principal sources of IL-1 production are blood monocytes, phagocytic cells that line the liver and spleen, and other tissue macrophages (87).  IL-  1 has diverse effects on multiple organ systems including the activation of T and B lymphocytes and neutrophils, the induction of the acute phase response and fever.  The production of IL-1 may be initiated by microorganisms or  their products, antigen-antibody complexes, toxins, injury and inflammatory processes. lymphocytes.  various  IL-1 enhances the production of lymphokines by T  Its effects on B lymphocytes are less clear.  However, i t  16 appears that IL-1 can directly augment B lymphoproliferative and antibodyproducing responses as well as indirectly augmenting these responses via effects on T cells (86-88). Interleukin 2 Interleukin 2 (formerly T cell growth factor) is produced by mature helper T lymphocytes appropriately stimulated by antigens or mitogens. has been purified and i t s gene cloned. approximately  IL-2  The molecular weight of IL-2 is  15,000 as judged by SDS-polyacrylaiiiide gel electrophoresis.  The major direct function of this T-cell derived factor is to stimulate the proliferation of activated T cells.  Using IL-2 i t has been possible to  continuously propagate normal and neoplastic T cells in vitro (86, 89, 90). In order to respond to IL-2, T cells must be induced to express receptors specific for this product.  This occurs through an i n i t i a l signal supplied by  a lectin, or antigen in conjunction with accessory cells (90-92).  A  monoclonal antibody (anti-Tac) has been raised to the IL-2 receptor (93) and the gene coding for this molecule has been cloned and sequenced (94, 95).  It  appears that the IL-2 receptor may coincidentally be the receptor for human T-cell leukemia/lymphoma virus (HTLV-1) (96). Recently, activated normal B cells as well as some B c e l l lines have been reported to express receptors for IL-2 (97).  B cells have significantly  fewer sites and lower affinity receptors compared to mitogen stimulated normal T c e l l blasts (98).  However, IL-2 can promote the growth and  differentiation of normal B cells in vitro (99-100).  A physiologic role for  IL-2 in B c e l l responses has not yet been demonstrated in vivo. Interleukin 3 Interleukin 3 (burst promoting activity, BPA), a factor whose predominant effects are on the differentiation of cells of myeloid and  17 erythroid lineages, has been suggested to have a role in the regulation of lymphocyte differentiation and growth (101).  However, the significance and  relative importance of these observations is as yet unclear.  Murine IL-3 has  recently been purified to homogeneity and i t s gene cloned also (102). B Cell Stimulatory Factors B c e l l growth and stimulatory factors have not been as well characterized as the other interleukins.  Recently, a committee met and  revised the nomenclature related to B cell factors (103).  This group  proposed that factors that had been well characterized functionally and chemically be given the designation B cell stimulatory factor (BSF) followed by a consecutive number (i.e. 1, 2...n).  Unless the factor had been purified  to homogeneity or i t s structure determined from gene cloning and sequencing a "p" for provisional would precede this number.  It was decided that the  factor previously referred to as B cell growth factor (BCGF) had been sufficiently well characterized to warrant the designation BSF-pl. factor (human) has a molecular weight of approximately  This  12,000 by SDS-PAGE.  When i t s purification is complete or i t s gene cloned, i t would be given the designation BSF-1  (103).  BCGF or BSF-pl has been defined as a T cell derived lymphokine that acts as a co-stimulator of polyclonal B-cell growth in B cells cultured with antiimmunoglobulin (e.g. anti-u) (104).  B cell stimulatory factors have been  derived from mitogen stimulated normal T cells, T hybridomas, T leukemia c e l l lines and HTLV-1 transformed T cell lines and are distinct from IL-2 110).  (104-  Most early studies supported the concept that anti-u induces c e l l  enlargement, transition from GO to Gl of the cell cycle, and expression of receptors for BSF-pl. (50, 52, 62-64, 74-78).  BSF-pl then induces entry of the cells into S phase  18 This concept has recently been challenged. which suggests that BSF-pl may  Evidence has been provided  be a d i f f e r e n t i a t i o n or a c t i v a t i o n factor  instead of, or i n addition to, a growth factor.  These studies have shown  that BSF-pl acts upon resting B c e l l s i n the absence of anti-immunoglobulin (Ig) antibodies,  to prepare these c e l l s to respond to anti-Ig.  possibly i n conjunction phase (111-114).  Anti-Ig,  with BSF-pl then induces entry of the c e l l s into S  The differences between e a r l i e r and more recent  apparently relate to methodological considerations. conditions, c e l l washing, and  studies  These include  timing of the addition of the various  and antisera to p u r i f i e d populations of B c e l l s . of these findings remains to be elucidated. the i n a b i l i t y to clone and reproducibly  culture factors  The complete s i g n i f i c a n c e  They may,  however, account for  propagate, normal factor dependent B  c e l l s i n long term culture, as have been T c e l l s (115-118). Recently, a monoclonal antibody to BSF-pl has been prepared.  This  antibody has allowed characterization of this B - c e l l factor and c l e a r l y shown that i t i s d i s t i n c t from IL-1, IL-2 and IL-3. 'p' for provisional be removed and as BSF-1  It was  recommended that  the  this lymphokine be henceforth referred to  (119).  B C e l l D i f f e r e n t i a t i o n Factors (BCDF) B c e l l d i f f e r e n t i a t i o n factors act on post-activated d i f f e r e n t i a t i o n to immunoglobulin secreting c e l l s .  B c e l l s to induce  These factors have  received a variety of names and may  be a family of molecules rather than a  single lymphokine.  B c e l l d i f f e r e n t i a t i o n factor (BCDF), T  These include:  c e l l replacing factor (TRF), B maturation factor (BMF) enhancing factor (BEF)  (106,  120-131).  and B c e l l - d e r i v e d  These factors, i n general, are not  well characterized and appear to be heterogeneous.  They may  be capable of  inducing varied responses depending on the B c e l l subpopulation affected (108).  19 Autostimulatory B Cell Factors The concept of autocrine secretion of growth factors in the control of c e l l proliferation and differentiation has recently gained popularity. B cell growth factors and differentiation factors have been reported to be products of Epstein Barr virus (EBV) transformed and neoplastic B cells (122, 132-136).  It has been hypothesized that autocrine growth factors may be  important in neoplastic transformation and autogenous growth of a variety of c e l l types (137, 138). Whether normal adult or embryonic B cells are capable of producing their own autostimulatory factors is not known. A recent report has shown that mycoplasmal contamination of cell lines may also result in a "lymphokine-like" soluble product that induces proliferation and maturation of B cells (139).  Therefore, these and other reports claiming to show growth  factor activity from c e l l line supernatants, should be viewed with some skepticism until confirmed free of mycoplasma.  3) CELL SURFACE ANTIGENS ON NORMAL AND NEOPLASTIC HUMAN B-LYMPHOCYTES (A) Definition Using Monoclonal Antibodies Monoclonal antibodies have shown considerable application to the study of hematologic diseases. In conjunction with flow cytometry they have provided useful clinical information with regard to: immunodeficiency disorders. (ALL).  1. Diagnosis of  2. Subtyping of acute lymphoblastic leukemia  3. Distinguishing between ALL and acute myelogenous leukemia (AML)  and between lymphoid and myeloid blast crises of chronic myelogenous leukemia (CML).  4. Immunologic phenotyping of T and B chronic lymphocytic leukemia  and differentiating these entities from reactive lymphocytoses. 5. Immunologic phenotyping of the non-Hodgkin's lymphomas and the separation of reactive from malignant, tissue based lymphoproliferative diseases.  20 6. Monitoring treatment and detecting residual disease post-treatment for leukemias and lymphomas (140-146).  In addition, monoclonal antibodies, in  conjunction with immunotoxins or complement, have shown promise in the treatment of hematologic neoplasia using both in vivo and ex vivo treatment protocols (147-149). Despite major improvements in the objectivity of classifying hematologic neoplasia with monoclonal antibodies, this approach has contributed relatively l i t t l e to our understanding of the biology of these diseases (150, 151).  This i s especially true of the B lymphoid system.  Recently, a number  of monoclonal antibodies have been raised toward B lymphoma cells.  These  have been evaluated as diagnostic reagents, for their ability to subclassify the lymphomas and to determine i f they are able to improve our understanding of this heterogeneous group of diseases. Most of these monoclonal antibodies f a l l into one of the following categories:  B c e l l restricted (react only  with cells of B lymphocyte lineage), B c e l l associated (react with B cells but also cells of other lineages), blast associated (define antigens present on normal B-blasts but absent from resting B cells), and antibodies whose principal reactivity is with Burkitt's lymphoma cells or EBV transformed c e l l lines (14-18, 152-170) (see Table I). The function of the antigens defined by these antibodies i s largely unknown with few exceptions. These include:  monoclonal antibody B2 which  defines the membrane receptor for the complement fragment C3d (CR2) (171, 172); BI which binds to a 35 Kd B c e l l activation antigen (173); and AB1 which may react with a receptor for BCGF (161).  TABLE I Monoclonal Antibodies Defining B Cell and B Cell Related Cell Surface Determinants  Antibody  Specificity  Reference  AA4.1, GF1.2  B cell subset  McKearn et a l , 1984 (14)  LN-1, LN-2  B cell associated  Epstein et a l , 1984 (15)  BI, B2, B4, PC-1 Ia, CALLA, TI, T10 PCA-1, PCA-2  B cell restricted B cell associated  Anderson et a l , 1984 (16) Anderson et a l , 1984 (152)  OKB1 0KB2  B cell subset B cell associated B cell restricted  Mittler et a l , 1983 (17) Knowles et a l , 1984 (18) Nadler et a l , 1981 (157)  SIg positive B cells  Jephthah et a l , 1984 (153)  B cell subset  Zola et a l , 1984 (154) Brooks et a l , 1981 (155)  HK-9, HK-19, HK-20  Mature B cells (DR related)  Shipp et a l , 1983 (156)  B220  B cells, some bone marrow cells  Sarmiento et a l , 1982 (170)  ABl  Activated B cells (BSF receptor)  Jung and Fu, 1984 (161)  0KB4, OKB7 CB2 FMC7  TABLE I (continued) Monoclonal Antibodies Defining B Cell and B Cell Related Cell Surface Determinants  Antibody  Specificity  Reference  Tac  Activated B cells (IL-2 receptor)  Tsudo et a l , 1982 (158)  33.1  Activated B cells  Marti et a l , 1982 (159)  BB-1 LB-1  B cell blasts B and T cell blasts  Yokochi et a l , 1982 (160)  4F2  Activated lymphocytes, monocytes, embryonic fibroblasts Dividing cells (transferrin receptor)  Kehrl et a l , 1984 (163) Sutherland et a l , 1981 (164)  B532  Activated B cells  Frisman et a l , 1983 (162)  GB 1,2,3,5,6 8,10,11,13,14  Some B cell lymphomas variable reactivity with B cells  Funderud et a l , 1983 (165)  38.13  Burkitt lymphoma cells, some B lymphoma/leukemia cells some EBV transformed cell lines  Klein et a l , 1983 (166) Lipinski et a l , 1982 (167)  41H.16  Normal and EBV transformed B cells, B-CLL  Zipf et a l , 1983 (168)  AB89  B cell lymphomas (10%)  Nadler et a l , 1980 (169)  5E9  K)  23 (B) The Human Major Histocompatibility Complex (HLA System) Nomenclature and Genetic Organization Understanding the human major histocompatibility complex (MHC) began in the 1950's when i t was observed that sera from multiply transfused patients and multiparous women contained antibodies reactive with leukocytes of nonidentical donors.  Since that time our appreciation of the complexity of the  HLA system has increased enormously. short arm of chromosome 6.  The human HLA complex is located on the  Genes located in the MHC region encode at least  three families of molecules.  Class III molecules are elements of the  complement system and will not be further discussed.  Class I molecules  (HLA-  A, B and C antigens) and class II molecules (HLA-D or D-related antigens) comprise what is commonly referred to as the HLA system (174-177). Class I antigens are located on a l l nucleated cells and are composed of a 44 Kd transmembrane glycoprotein noncovalently associated with the 12 Kd protein P2~ i 6l°bulin (encoded on human chromosome 15). m  cro  Class I antigens  are principally detected in vitro by complement mediated antibody dependent cytotoxicity (174-177).  Using serologic techniques HLA class I antigens have  been shown to be extremely polymorphic with at least 20 distinct alleles at the A locus, over 40 distinct alleles at the B locus and approximately 8 alleles at the C locus (177).  They are the principal antigens recognized by  the host during graft rejection.  The physiological role of class I antigens  appears to involve the restriction of recognition of cell surface antigens (such as viral antigens on infected cells) by cytotoxic T lymphocytes; i.e. T cells which are exposed to virus infected cells must see those cells in the context of both the virus and identical class I antigens in order to generate cytotoxicity.  Evidence has suggested the cell surface antigen defined by  monoclonal antibodies 0KT8/Leu-2 may be involved in the recognition of class I molecules (26, 178, 179).  24 The HLA c l a s s I I a n t i g e n system appears t o be even more complex.  Class  I I a n t i g e n s a r e h e t e r o d i m e r i c transmembrane g l y c o p r o t e i n s composed o f a heavy  (a) c h a i n o f 33-35 Kd and a l i g h t ( 3 ) c h a i n o f 27-29 Kd. C l a s s I I a n t i g e n s are found  p r i n c i p a l l y on B lymphocytes, a c t i v a t e d T lymphocytes and a n t i g e n  presenting c e l l s  (macrophages, monocytes, d e n d r i t i c c e l l s , e t c ) .  a l s o present on some myeloid  c e l l s and t h e i r p r e c u r s o r s .  They a r e  Low l e v e l s o f c l a s s  I I a n t i g e n s have been r e p o r t e d on r e n a l tubule c e l l s and on e n d o t h e l i a l cells.  C l a s s I I a n t i g e n s have a l s o been r e p o r t e d on tumor c e l l s  melanomas and gliomas  (174,  180,  181).  F a c t o r s present  including  i n mitogen a c t i v a t e d  as w e l l as pure y - i n t e r f e r o n a r e capable o f i n d u c i n g c l a s s  T c e l l supernatant  I I a n t i g e n e x p r e s s i o n on c l a s s I I n e g a t i v e macrophages, e n d o t h e l i a l c e l l s and melanoma c e l l s  (174,  182,  183).  B i o c h e m i c a l and molecular g e n e t i c a n a l y s e s o f the genes and gene p r o d u c t s a s s o c i a t e d w i t h the HLA-D r e g i o n i n d i c a t e the e x i s t e n c e o f a t l e a s t t h r e e groups o f products recently revised  (177).  (180).  The nomenclature f o r these products was  The d e s i g n a t i o n s now i n c l u d e HLA-DR, HLA-DQ  ( f o r m e r l y DC, DS, MB) and HLA-DP ( f o r m e r l y SB).  The genes c o d i n g f o r many o f  the c l a s s I I products have been cloned and amino a c i d sequences o f t h e i r p r o t e i n products determined. molecular  and  o f s e r o l o g y , b i o c h e m i s t r y and  b i o l o g y i n d i c a t e s there a r e m u l t i p l e c l a s s I I genes c o d i n g f o r a t  l e a s t seven l i g h t region  A combination  (184).  (B)  chains and a t l e a s t s i x heavy (a)  Recent evidence  supports  c h a i n s w i t h i n the D  the e x i s t e n c e o f a t l e a s t one a l p h a  two o r three beta c h a i n genes w i t h i n HLA-DR, two a l p h a and two beta c h a i n  genes w i t h i n DQ, two a l p h a and two beta c h a i n genes w i t h i n DP, as w e l l as an a d d i t i o n a l a l p h a c h a i n which has p r e v i o u s l y been termed DZ-alpha (180, 188).  I t appears that the same heavy c h a i n may a s s o c i a t e w i t h l i g h t  from more than one l o c u s and that determinants  may be shared  between  185chains  25 different chains (180).  The extensive polymorphism of class II antigens  detected by serological, functional and structural studies appears to be mainly restricted to the $ subunits (187). Due to random matings, the frequency of association of one HLA allele with another located at a different locus should simply be the product of the frequencies of each allele in the population.  However, certain combinations  of alleles are found with a frequency much greater than expected'. phenomenon is termed "linkage disequilibrium".  This  Several hypotheses have been  proposed to explain the occurrence of linkage disequilibrium, although none of these is entirely satisfactory.  These include:  (1) a selective advantage  of a given haplotype, (2) migration and admixture of different populations, (3) inbreeding, and (4) random drift (174). Part of the difficulty in sorting out the individual products of the HLA-D region has been due to the occurrence of DR and DQ antigens in close linkage disequilibrium (i.e. an unexpected association of linked genes in a population). DR (180).  DP does not appear to be in linkage disequilibrium with DQ or  By biochemical criteria, human DR antigens appear homologous to  murine I-E antigens; DQ antigens appear homologous to I-A.  DP antigens  appear intermediate in homology, although a murine counterpart for DP has not yet been identified (180, 181). The HLA-D region was originally defined by cellular typing using the mixed lymphocyte response (MLR). The major determinants inducing the MLR probably reside on HLA-DR molecules.  However, class II antigens other than  HLA-DR may also induce an MLR. Therefore, what has previously been termed HLA-D may actually be the sum total of responses to several different class II antigens and not, by itself, a distinct entity (174).  26 DP (SB) antigens were originally defined by the secondary MLR lymphocyte typing, PLT).  Now,  (primed  at least some DP antigens may be detected by  monoclonal antibodies (184, 189). The functions of class II antigens remain to be fully elucidated. However, they appear to be key elements in the control of immune responses and determine several immunologic phenomena including:  control of the level  of the immune response, delayed type hypersensitivity, susceptibility to certain diseases and primary (MLR) and secondary (PLT) allogeneic T c e l l proliferation (174, 180, 181, 190).  HLA class II antigens appear  particularly important in the recognition of antigens by regulatory T lymphocytes.  In order to respond, T helper cells must recognize antigens in  the context of appropriate class II molecules (i.e. they show MHC class II restriction) (191).  This ability to recognize class II determinants may be  related to the T lymphocyte molecule defined by 0KT4/Leu-3 monoclonal antibodies (192). Monoclonal Antibodies Directed Against HLA Determinants A number of monoclonal antibodies directed against HLA class I and class II antigens have been described. monomorphic determinants. determinants (193-203).  Most of these have been directed against  Some detect polymorphic class I and class II Most early reports of monoclonal antibodies directed  against class II antigens reported them as "anti-DR"*  With the increased  appreciation of the complexity of the MHC i t is preferable to refer to these as anti-class II monoclonal antibodies until i t is known whether these react with DP, DQ or DR antigens.  This can be determined by sequential  immunoprecipitation, cross-blocking studies and by testing these antibodies against appropriate panels of DR homozygous c e l l lines or c e l l lines transfected with DP, DQ or DR genes (189, 198).  27 Three anti-HLA-DQ monoclonal antibodies have been described and characterized in detail (198).  These are Genox 3.53, BT3/4 and anti-Leu-10.  These antibodies were shown to react with a different population of molecules (DQ) than did the HLA-DR specific monoclonal L243. When tested against DR homozygous c e l l lines Genox 3.53 reactivity correlated with DR1,2,6; BT3/4 reactivity correlated with DR1,2,4,6,8 and anti-Leu-10 reactivity correlated with DR1,2,4,5,6,8 and 9.  These antibodies most likely define different  polymorphisms of DQ molecules (197, 204). Monoclonal antibodies against HLA class II antigens have been very useful in delineating expression of these antigens on various c e l l subpopulations, as well as in increasing our understanding of the structure and function of these molecules.  Different roles for DP, DQ and DR molecules  in the immune response have not been definitely established (205-214). However, these differences may be important since studies of DQ antigen expression on monocytes and macrophages have revealed functional differences between DQ  +  and DQ~ subpopulations.  DQ positive monocyte subpopulations are  involved in antigen presentation and stimulation in the autologous mixed lymphocyte reaction, while DQ negative subpopulations are not (196, 215, 216).  In addition, a unique pattern of expression of class II antigens on  myeloid progenitor cells has been reported. positive but DQ negative (217-220).  These cells appear to be DR  The physiological significance of this  latter observation is not yet clear. In summary, the MHC codes for two classes of c e l l surface molecules which are involved in a variety of crucial immunologic responses.  The  inherent complexity of this system is just now beginning to be appreciated. Recent advances have allowed the determination of the genetic and molecular structure of these antigens and their genes.  Monoclonal antibodies have been  28 very useful in delineating subpopulations of HLA class I and class II molecules and correlating these with various immune functions.  (C) Lymphocyte Function Associated Antigen (LFA) Family of Molecules Lymphocyte function-associated antigen 1 (LFA-1), Mac-1 and pl50,95 c e l l surface molecules constitute a novel family of structurally and functionally related glycoproteins. Each molecule contains a common beta chain (mw = 95 Kd) noncovalently associated with an alpha chain.  The alpha subunits have  molecular weights of 177 Kd (LFA-1), 165 Kd (Mac-1) and 150 Kd (pl50,95), different isoelectric points, and are immunologically non-cross-reactive. The a and 3 subunits appear to be synthesized intracellularly as distinct a 1 and 6 precursors.  11 These then associate into a 3 complexes, are processed,  and transported to the cell surface in the mature aB form.  The a subunits  have been suggested to bear determinants that govern the specificity of c e l l interactions, while the identical beta subunits may mediate a common function such as signal transduction (221-223). LFA-1 is expressed on lymphocytes, monocytes, large granular lymphocytes, weakly on granulocytes and on approximately 35% of bone marrow cells.  Mac-1 (Mo-1, 0KM-1) is found on monocytes, granulocytes and large  granular lymphocytes.  P150,95 is expressed on monocytes, lymphocytes and  strongly on granulocytes.  Human LFA-1, like mouse LFA-1 is present on both B  and T lymphocytes, although quantitatively greater amounts are found on T cells.  Also, there is a 3 to 4-fold difference in the quantitative  expression of LFA-1 on lymphocytes.  Whether there are functional  implications to these differences is not known (224-226). The Mac-1 molecule (also identified by 0KM-1 and Mo-1 monoclonal antibodies) appears to be identical to the complement receptor type 3 (CR3).  29 The i d e n t i t i e s of LFA-1 and pl50,95 have not been established.  However, the  c e l l u l a r d i s t r i b u t i o n of these molecules suggest they may be important i n a wide v a r i e t y of T c e l l , B c e l l , granulocyte and monocyte functions  (221).  Monoclonal antibodies directed against determinants on the LFA-1 molecule i n h i b i t a number of i n v i t r o immune functions.  These include:  c y t o t o x i c T lymphocyte (CTL) k i l l i n g , natural k i l l e r (NK) c e l l a c t i v i t y , T c e l l p r o l i f e r a t i v e responses to antigen, mitogens and a l l o g e n e i c c e l l s , and antibody dependent c e l l u l a r c y t o t o x i c i t y (ADCC). function has not been w e l l characterized.  The r o l e of LFA-1 i n B c e l l  However, monoclonal anti-LFA-1  antibodies have been shown to i n h i b i t T c e l l dependent plaque forming c e l l responses, but not T c e l l independent responses.  Also, i n the mouse these  antibodies d i d not i n h i b i t lipopolysaccharide (LPS) induced B c e l l p r o l i f e r a t i o n (227-236). Most d e t a i l e d studies on the r o l e of LFA-1 i n immune function have centered on the r e l a t i o n s h i p of t h i s molecule to CTL mediated k i l l i n g .  Anti-  LFA-1 monoclonal antibodies appear to block CTL mediated k i l l i n g by i n h i b i t i n g adhesion between the CTL and the target c e l l .  I t has been  +2 hypothesized  that LFA-1 may p a r t i c i p a t e i n the Mg  -dependent adhesion step  of CTL mediated k i l l i n g and that the LFA-1 c e l l surface s t r u c t u r e i s involved i n strengthening e f f e c t o r - t a r g e t c e l l adhesion.  Quantitative d i f f e r e n c e s i n  the a b i l i t y of d i f f e r e n t anti-LFA-1 monoclonal antibodies to block c y t o l y s i s i n d i c a t e d i s t i n c t f u n c t i o n a l and antigenic epitopes e x i s t on the LFA-1 molecule.  With respect to CTL function anti-LFA-1 blocks k i l l i n g by binding  to e f f e c t o r c e l l s rather than target c e l l s (227-236). Recently, the structure of the a subunit of LFA-1 has been p a r t i a l l y determined by N-terminal amino acid sequencing.  Sequence homology shows that  the a subunits of a l l members of the family are r e l a t e d and suggests t h e i r  30 evolution occurred by gene duplication. A further unexpected homology was found between LFA-1 and leukocyte (a) interferon. The significance of this homology is not known (237). Clinical Implications Recently a hereditary disorder in which patients manifest multiple recurrent bacterial infections, progressive periodontitis and impaired wound healing has been described.  These patients have an inherited deficiency of  the Mac-1, LFA-1, pl50,95 glycoprotein family on their c e l l surfaces. Patients have severe impairment of adherence and adhesion dependent c e l l functions.  Immune abnormalities described include:  defective antibody  dependent cellular cytotoxicity (ADCC), natural k i l l e r (NK) c e l l function, phagocytosis, neutrophil migration and mitogen stimulation (238-244).  In  some studies the defect in phagocyte function has been more profound than lymphocyte function (238).  However, increasing evidence supports the  c l i n i c a l pathologic importance of lymphoid c e l l function in this disorder (221). Monoclonal antibodies directed against this family of molecules have been able to reproduce in vitro many of these defects when co-cultured with normal cells (238-242).  Family studies suggest this disorder is inherited as  an autosomal recessive.  Biosynthetic experiments have shown the presence of  I  normal amounts of a  (LFA-1) intracellular precursor.  This in conjunction  with the absence of a l l three members of the family, each with a different a chain but a common 3 chain suggests the primary deficiency is of the 3 subunit (221). The importance of this family of molecules, is further emphasized by the recent observation that granulocytes may markedly increase their expression of Mo-1  after appropriate stimulation.  During degranulation Mo-la , located  31 in specific neutrophilic granules, is translocated to the plasma membrane. This results in a 5-10 fold increase in surface expression of this glycoprotein.  Clinically, patients may show a 5 fold increase in Mo-1 expression  within minutes after beginning renal dialysis.  This enhanced expression of  Mo-1 may provide a mechanism for initiating leukocyte aggregation and sequestration and explain the neutropenia of dialysis (243, 244). 4) NEOPLASMS OF THE IMMUNE SYSTEM: THE NON-HODGKIN'S LYMPHOMAS Classification The concept of a pluripotential progenitor cell (the reticulum cell) was invoked in the earlier part of this century to attempt to conceptually explain morphologically and clinically diverse types of lymphoid malignancies (245, 246).  The distinction between the various types of lymphomas was based  primarily on cell size.  The terms "reticulum c e l l sarcoma", "lymphosarcoma"  and "giant follicular lymphoma" were in popular usage.  Malignancies of small  lymphocytes were termed lymphosarcoma; those composed of larger cells were designated as reticulum c e l l sarcoma (247).  There was considerable d i f f i -  culty correlating the various subtypes of lymphoma with patient survival. In addition, these subcategories each included biologically unrelated disease entities. In 1956, Rappaport proposed a classification of the non-Hodgkin's lymphomas which was prognostically relevant and made pathologic subtyping relatively easy (248).  The reasoning behind this revision of the older  classification was that the "reticulum c e l l " could not be identified as a precise entity and that previous classifications failed to provide sufficient prognostically and therapeutically useful information. Rappaport's classification was based on morphology. The degree of presumed differentiation and the similarity of the malignant cells of the various lymphomas to what was thought to be their normal cellular counter-  32 parts were the criteria by which these malignancies were subtyped.  Individu-  al cells of large cell lymphomas were thought to resemble histiocytes; "histiocytic lymphoma" replaced "reticulum cell sarcoma" as a diagnostic category.  Lymphomas composed of small normal appearing lymphocytes were  termed "well-differentiated lymphoma" replacing the older term "lymphosarcoma".  Lymphomas were classified as either well differentiated or  poorly differentiated depending on whether the cell size and nuclear configuration more or less resembled that of normal lymphocytes.  Further  categor-  ies were created to include lymphomas that appeared to be composed of more than one c e l l type (mixed lymphocytic-histiocytic lymphoma) and those that appeared especially primitive or "undifferentiated". Rappaport also demonstrated that a nodular (follicular) pattern of growth within a given subgroup of lymphoma was a prognostically favorable feature (248). Subsequent clinicopathologic studies demonstrated that the histopathologic classification proposed by Rappaport was relevant prognostically and useful in the clinical management of patients with non-Hodgkin's lymphomas (249-251).  Generally, lymphomas composed of larger, more "poorly  differentiated" cells carried the worst prognosis.  Well-differentiated  lymphocytic lymphoma carried the best prognosis, histiocytic or undifferentiated lymphomas the poorest, poorly differentiated and mixed lymphomas were intermediate in their clinical outcome. Advances in immunology have markedly changed older concepts of the malignant lymphomas. These neoplasms are now known to involve T and B lymphocytes (252-254).  The Rappaport classification was proposed before T  and B cells were defined as distinct functional subpopulations and the phenomenon of lymphocyte transformation recognized.  These older classifica-  tion schemes became obsolete in light of the modern appreciation of the functional complexity of the immune system.  33  Lymphocytes may be.functionally divided into cells of T and B lineage. These exist as small cells with dense nuclear chromatin, round nuclei and barely discernable nucleoli until stimulated to transform by antigen or mitogen.  Transformed cells develop characteristics of "blasts" (large size, fine  nuclear chromatin, prominent nucleoli).  Individually, transformed lympho-  cytes appear similar or identical by light and electron microscopy to cells of various non-Hodgkin's lymphomas. the clonal proliferation of cells.  Lymphoid neoplasms are characterized by In contrast, reactive c e l l populations  tend to be morphologically and functionally heterogeneous (252-254). In the past decade, major advances have taken place in the understanding of the biology of normal lymphocytes.  These changes have taken place pari  passu with advances in immunologic techniques that have permitted the dissection of the immune system into i t s functional components.  Newer classifica-  tions of the lymphomas have evolved in parallel with greater appreciation of the functional complexities of normal lymphocytes (252-261). remain hypotheses based on data indicating:  These proposals  (1) that morphologically homoge-  neous populations of lymphoid cells are functionally heterogeneous (B cells, T cells and their subsets) and, (2) that lymphocytes during the course of their immunoregulatory and effector cell functions may undergo a variety of morphologic changes reflecting their state of activation and differentiation (262-267).  Recent reports suggest that immunologic studies may assist in  recognizing clinically relevant subgroups of non-Hodgkin's lymphomas (268274). The revolution in the terminology of lymphomas began with the work of Lukes and Collins in the United States and Lennert in West Germany in the early 1970s (252-254, 259).  Since that time many others (Dorfman, Rappaport  etc) have contributed significantly toward increasing the understanding of the biology of the lymphomas.  However, in North America the classification  34 of Lukes and Collins has gained the most widespread popularity. They proposed a new concept that related the malignant lymphomas to the T and B lymphocytic systems and alterations in lymphocyte transformation.  The in  vivo counterpart of B lymphocyte transformation in vitro was hypothesized to occur in the follicular centers of lymph nodes - from which is derived the term follicular center cell lymphoma. T-cell transformation occurs outside of the f o l l i c l e s (Figure 4).  According to this new reasoning, c e l l size and  nuclear configuration are not necessarily related to the degree of differentiation of lymphoma cells, but instead reflect the point along the lymphoid transformation continuum that malignant change occurs.  A "block" or "switch-  on" of cells at discrete stages of normal B lymphocyte differentiation by this concept, result in the morphologic expression of malignancy.  may, Thus,  histiocytic lymphomas are not composed of histiocytes but rather are the neoplastic counterpart of the large transformed lymphocyte.  Immunologic  marker studies and other techniques have supported these observations (254, 264,  266). The Lukes' classification attempts to synthesize morphology and  function.  Using immunologic markers, lymphomas are separated into those of T  c e l l , B c e l l and true histiocytic (macrophage) types.  A small proportion are  undefined or unclassifiable by currently available techniques.  The T and B  c e l l lymphomas are further subdivided based upon their morphologic appearance and relationship to lymphocyte transformation (252-254, 259, 265).  A  comparison of the major classifications is found in Table II. Recently, a prognostically relevant synthesis of the leading classifications (based on morphology alone), has been proposed.  This was termed the "international  working formulation" and was not intended to be a classification scheme, but rather a vehicle by which different classifications may be compared (256, 274).  35  FIGURE 4  F o l l i c u l a r c e n t e r c e l l concept o f lymphocyte t r a n s f o r m a t i o n . A c c o r d i n g to t h i s h y p o t h e s i s , normal B c e l l s pass through a s e r i e s o f morphologic stages w i t h i n the f o l l i c u l a r c e n t e r s o f lymph nodes. B c e l l lymphomas may be c l a s s i f i e d a c c o r d i n g to which subtype of c e l l predominates. The predominant c e l l type, w i t h i n a g i v e n lymphoma, may correspond to one o f the stages i n normal B c e l l t r a n s f o r m a t i o n i l l u s t r a t e d (252-254).  TABLE I I A Comparison o f the Proposed  "Working F o r m u l a t i o n " w i t h C l a s s i f i c a t i o n s f o r Non-Hodgkin's Lymphomas  WORKING FORMULATION  RAPPAPORT  Low Grade  A. W e l l d i f f e r e n t i a t e d  LUKES-COLLINS lymphocytic  A. Small l y m p h o c y t i c  C. F o l l i c u l a r , mixed s m a l l c l e a v e d and l a r g e c e l l  Intermediate Grade large  Poorly d i f f e r e n t i a t e d B. nodular E. d i f f u s e  Mixed h i s t i o c y t i c - l y m p h o c y t i c C. nodular F. d i f f u s e  cell  F. D i f f u s e , mixed, s m a l l and cell Diffuse, large  large  cell  High Grade H. Large c e l l , I . Lymphoblastic nonconvoluted) J.  immunoblastic (convoluted or  Small noncleaved  lymphocytic  cell  Others H a i r y C e l l , Cutaneous T - C e l l , e t c  A. Small Lymphocytic A. P l a s m a c y t o i d Lymphocytic F o l l i c u l a r Center C e l l Types ( f o l l i c u l a r or d i f f u s e )  E. D i f f u s e , s m a l l cleaved  G.  ? U C e l l (Undefined) B Cell  B. F o l l i c u l a r , s m a l l c l e a v e d  D. F o l l i c u l a r ,  (274)  (B or E) Small c l e a v e d (D or G) Large c l e a v e d (D or G) Large noncleaved (J) Small noncleaved  Histiocytic D. nodular G. , H. d i f f u s e  H. Immunoblastic  I.  T Cell  Lymphoblastic  Undifferentiated J. Burkitt's J . pleomorphic  Sarcoma  A. Small l y m p h o c y t i c I . Convoluted lymphocytic ? C e r e b r i f o r m (cutaneous) F.G. L y m p h o e p i t h e l i o i d c e l l H. Immunoblastic sarcoma  Histiocytic  37 Etiology Very l i t t l e is known about the etiology of the non-Hodgkin's lymphomas (275).  Epstein-Barr virus (EBV) has been shown to be present within the  tumor cells of patients with the endemic form of Burkitt's lymphoma; a causal relationship is suspected but not proven (276). Also, the majority of Burkitt's lymphomas are associated with a characteristic chromosomal abnormality t(8;14) in which the myc oncogene, located on chromosome 8, is translocated to the region of the immunoglobulin heavy chain gene on chromosome 14.  Variants occur in which myc is translocated to the region of  the kappa t(2;8) or lambda t(8;22) light chain genes (277-283).  These three  different chromosomal rearrangements result in a deregulation of c-myc so that i t is expressed at high levels, while the normal c-myc oncogene on the uninvolved chromosome 8 is transcriptionally silent (284-286).  The existence  of enhancer elements has been postulated, within the three immunoglobulin loci genes.  These enhancers are thought capable of activating transcription  of the translocated c-myc which then may result in neoplastic transformation (284).  Other oncogenes (e.g. ras) and less well characterized transforming  sequences may also play a role in certain lymphoid malignancies (287, 288). A number of characteristic chromosomal abnormalities have been described in B c e l l lymphomas and leukemias. Some of these show a tendency to associate with specific histologic subtypes (289-292). associations are not invariant.  However, these  Recently, recombinant DNA probes were  utilized to detect DNA rearrangements in cases of follicular (B cell) lymphoma.  These probes detected a gene (bcl-2 gene) which seems to be  interrupted in most cases of follicular lymphomas carrying the t(14;18)  38 chromosomal translocation.  It was speculated  that the bcl-2 gene may have a  role in the pathogenesis of this subtype of lymphoma (293).  Whether there  are analogous chromosomal defects in other lymphomas remains to be determined. The concept of autocrine secretion of growth factors as a major contributor to the evolution of neoplastic cell populations was postulated by Todaro (294).  first  More recently, B c e l l growth factors and  differentiation factors have been reported to be products of neoplastic and EBV transformed c e l l lines (122, 132-136). Whether autocrine secretion of growth factors is important in the pathogenesis of B c e l l malignancy is not yet known. The putative causative agent of some human T c e l l leukemias and lymphomas is the human T lymphocytic virus (HTLV-1)., Infection with this retrovirus does not invariably result in malignancy.  The mechanism by which  HTLV-1 might induce neoplasia is not known (295-297). In summary, the non-Hodgkin's lymphomas are a pathologically, c l i n i c a l l y and immunologically diverse group of diseases.  There is very l i t t l e known  about the mechanisms involved in the neoplastic transformation process.  The  growth requirements of the non-Hodgkin's lymphomas are also unknown. Whether lymphoma cells secrete autostimulatory  growth factors or are factor  independent remains to be determined.  Since many cellular, growth factor and  v i r a l interactions occur at the cell surface, identification and characterization of molecules characteristic of or unique to lymphoma cells is of obvious biologic interest.  39  5) THESIS OBJECTIVES The c e l l surface i s involved i n many events which are c r u c i a l to the function of normal B lymphocytes.  These include:  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 , c e l l - c e l l interactions, growth factor-receptor binding and regulation of metabolic processes.  the  A role for c e l l surface molecules i n the  neoplastic transformation of B lymphocytes has been postulated but not documented.  Monoclonal antibodies are powerful tools for delineating c e l l  surface determinants due to their exquisite s p e c i f i c i t y . The purpose of this research i s to: 1. Characterize c e l l surface antigens on normal, activated and neoplastic lymphocytes u t i l i z i n g monoclonal antibodies as probes. 2 . Determine the functional importance of these molecules i n B lymphocyte a c t i v a t i o n . 3.  Raise monoclonal antibodies s p e c i f i c for neoplastic or blast  associated lymphocyte antigens, with the object of defining those events underlying neoplastic  transformation.  4. Ultimately u t i l i z e these monoclonal antibodies i n the c l a s s i f i c a t i o n and therapy of the non-Hodgkin's lymphomas.  40 REFERENCES  1.  Singer SJ: Structure of the c e l l surface. 25-30, 1982.  Natl Cancer Inst Monogr 60:  2.  Alberts B, Bray D, Levis J , Raff M, Roberts K, Watson J . : The plasma membrane. 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Popovic M, Reitz J r MS, Sarngadharan MG, Robert-Guroff M, Kalyanaraman VS, Nakao Y, Miyoshi I, Minowada J , Yoshida M, Ito Y, Gallo RC: The v i r u s of Japanese adult T - c e l l leukaemia i s a member of the human Tc e l l leukaemia virus group. Nature 300: 63-66, 1982.  JE, Marquardt H, Bryant ML, Sherwin SA, S l i s k e AH: factors produced by tumor c e l l s and v i r u s A possible growth advantage f o r the producer c e l l s . Conf C e l l P r o l i f e r a t i o n 6: 113-127, 1979.  62 C H A P T E R  II  MATERIALS AND METHODS  "Beware that you do not lose the substance by grasping at the shadow." Aesop, 550 B.C. 1) CELLS Light density mononuclear c e l l s were obtained  from peripheral blood from  2 normal volunteers a f t e r separation on 1.077 gm/cm L i t t o n Bionetics, Bethesda, MD).  Ficoll-Hypaque (LSM,  C e l l s were obtained  from tissues (lymph  nodes, spleens etc) by gently teasing these apart, using forceps, into RPMI 1640 + 10% FCS under aseptic conditions. gently pressed  through a f i n e wire mesh to further release c e l l s and remove  larger pieces of tissue.  Bone marrow specimens were obtained  the mononuclear f r a c t i o n separated bone marrows were obtained evaluation.  The larger fragments were then  as above on Ficoll-Hypaque.  i n Heparin and Tissues and  from patients as part of their routine diagnostic  C e l l s were then washed i n RPMI 1640 v i t h 10% FCS and prepared  for c e l l culture or FACS analysis (see belov). B-blasts vere prepared by culturing splenic lymphocytes from a kidney donor i n Falcon flasks at 10 c e l l s / m l i n RPMI 10% FCS v i t h 100 ug/ml of 6  lipopolysaccharide (LPS, E. c o l i , Sigma, St. Louis, M0) f o r four days.  T  c e l l s vere depleted by E-rosette sedimentation using aminoisothiouronium bromide (AET) treated sheep red blood c e l l s (SRBC) (1).  The remaining c e l l s  vere 90% surface immunoglobulin p o s i t i v e B c e l l s and vere large i n s i z e as  63 judged by forward scatter on the FACS.  Morphologic examination of stained  cytospins showed that these c e l l s were predominantly large lymphoblasts. C e l l s prepared i n this way were < 5% T c e l l s and monocytes as shown by s t a i n i n g with Leu-5 and Leu-M3 (Becton Dickinson, Mountain View, CA). 9 EBV c e l l l i n e s were the generous g i f t s of Drs. John Hansen and Paolo A n t o n e l l i (Genetic Systems, Seattle, VA) and the 8 lymphoma l i n e s the kind g i f t s of Drs. Alan Epstein (Univ. S. C a l i f o r n i a , Los Angeles, CA), Jun Minowada (VA Medical Center, Hines, IL) and the late Dr. Henry Kaplan (Stanford Univ., Stanford, CA) (2-5).  A l l c e l l l i n e s were maintained i n RPMI  1640 medium plus 10-15% FCS and subcultured twice weekly or as required.  2) MONOCLONAL ANTIBODIES (C3HXBALB/c) F l (bred i n our colony) or BALB/c (Charles River Laboratories) mice were immunized with DHL-10 c e l l s (DH-84, DH-224), DHL-10 membranes (LM-26, LM-155) or LPS stimulated spleen c e l l s from a patient with B c e l l lymphoma and splenic involvement (NB-29, NB-65). (10  7  C e l l s or membranes  c e l l s or equivalent membranes) were homogenized i n Freund's  complete  adjuvant (Difco Labs, Detroit, MI) and injected i n t r a p e r i t o n e a l l y (IP) into mice.  Two IP injections were given at least 3-4 weeks apart.  C e l l s (2 x  10 ) or equivalent membranes i n saline were administered intravenously (IV) 7  3-4 weeks a f t e r the l a s t IP i n j e c t i o n .  C e l l fusion took place the fourth day  a f t e r IV boost according to established procedures (6). 9 C e l l membranes were prepared by washing 2 x 10  DHL-10 c e l l s with  phosphate buffered saline (PBS) then resuspending these c e l l s i n 10 mM  Tris  HCL pH 8.0 plus PMSF (phenylmethylsulfonyl f l u o r i d e , Sigma, St. Louis, M0) (7).  C e l l s were homogenized v i t h a syringe and 21 gauge needle, spun at  2,000 RPM  for 15 minutes to remove nuclei and intact c e l l s .  The supernatant  64 was overlayed on 40% sucrose i n dH^O  and spun at 25,000 RPM  for 60 minutes.  Membranes were removed from the interface and washed with T r i s HCL.  The  protein content of the p e l l e t was measured at 280 nm, and the crude membrane material aliquoted and frozen at -70°C u n t i l used. P r i o r to fusion, NS-1 medium (DMM)  c e l l s were grown i n Dulbecco's minimal e s s e n t i a l  plus 5% f e t a l c a l f serum (PCS) i n spinner f l a s k s .  washed 3 times i n DMM  immediately prior to fusion.  a s e p t i c a l l y from an immune mouse.  C e l l s were  The spleen was removed  C e l l s were gently teased into  f i l t e r e d to remove large clumps and washed.  DMM,  Spleen c e l l s and NS-1  mixed at a r a t i o of 10:1, d i l u t e d to 50 ml i n DMM  and spun.  After  c e l l s were removing  the supernate, 1 ml of 50% polyethylene g l y c o l 1500 (PEG)/DMM was added slowly over one minute with constant a g i t a t i o n . added slowly over the next f i v e minutes.  i n 100 ml DMM  was  then  Fused c e l l s were spun at 1,200  for 5 minutes and resuspended slowly i n 5 ml DMM resuspended  Nine ml of DMM  + 15% FCS.  RPM  C e l l s were then  + 15% FCS and 1 ml aliquoted into each well of  p l a s t i c multiwell (24) plates (Flow Labs, McLean, VA).  One ml of normal  syngeneic spleen c e l l s used as feeders were dispensed into each well at a concentration of 2 x 10^ c e l l s / m l . at 5% CO2 and 100% humidity.  C e l l s were cultured i n a 37°C incubator  On day one post fusion approximately 1 ml of  supernate was removed from each well and replaced with hypoxanthineaminopterin-thymidine (HAT) media. 6.  This procedure was repeated on days 3 and  On day 8 or 9 post fusion c e l l s were switched by a s i m i l a r procedure to  hypoxanthine-thymidine  (H-T) media.  Reagents for HAT and H-T media were  obtained from Sigma, St. Louis, M0.  Stock HAT was made by d i s s o l v i n g 0.65  hypoxanthine, 0.0095 g aminopterin and 0.195 NaOH dH 0. 2  g thymidine i n 500 ml of 0.01  Stock H-T was prepared by dissolving 0.135  0.039 g thymidine i n 100 ml of NaOH.  v  g hypoxanthine  g N  and  Stock solutions were d i l u t e d 1:100  with  65 media p r i o r to use. F i n a l concentrations i n culture of reagents vere hypoxanthine  (13 ug/ml), aminopterin (0.19 jig/ml) and thymidine (3.9 pg/ml).  Wells vere observed d a i l y for hybridoma grovth.  When hybrids appeared  veil  established, supernatants vere screened for antibody a c t i v i t y using the binding assay described belov.  Antibody positive cups vere transferred to  DMM 15% FCS i n i n d i v i d u a l flasks and cups and rescreened p r i o r to cloning. Hybridomas vere plated i n methylcellulose media (40 ml 2% methylcellulose i n alpha media + 1 ml glutamine + 1 ml 2 mercaptoethanol + 10 ml FCS + 30 ml DMM).  V i s i b l e colonies vere detected i n approximately one  veek, plucked and transferred to i n d i v i d u a l m u l t i v e l l plates.  When grovn,  cultures vere rescreened f o r antibody a c t i v i t y and the cloning process repeated. FCS to 5%.  Hybrids vere grovn i n DMM gradually reducing the concentration of C e l l s vere frozen at appropriate stages of the cloning process  and a f t e r double cloning, thaved and v i a b i l i t y checked.  Antibody subclasses  vere determined by Ouchterlony immunodiffusion i n 1.2% agar using goat a n t i mouse IgG subclass s p e c i f i c antisera (Tago Inc., Burlingame, CA). Antibodies vhich p r e f e r e n t i a l l y bound to large B-lymphoma c e l l s (DHL-10) compared to small B-lymphocytes (CLL c e l l s ) by binding assay or FACS analysis (see belov) vere selected f o r further characterization.  3) PREPARATION OF ASCITES Hybrid clones vere thaved and grovn i n DMM + 5% FCS.  C e l l s vere vashed  four times v i t h normal saline, resuspended i n saline and then injected IP into pristane (2, 6, 10, 14-Tetramethylpentadecane,  A l d r i c h Chem. Co.,  Milvaukee, Wl) primed mice (2 x 10 c e l l s per mouse). 7  Mice vere primed v i t h  0.5 cc of pristane IP 1 veek p r i o r to being injected v i t h hybridomas.  After  1 to 2 veeks ascites vere c o l l e c t e d , spun at 2,000 RPM f o r 10 minutes and stored at -20°C.  66  4) PURIFICATION OF ANTIBODY Equal volumes of immune ascites and saturated ammonium sulfate were mixed and s t i r r e d for one hour at room temperature. 10,000 RPM  The mixture vas spun at  for 15 minutes and the supernate removed.  The p r e c i p i t a t e  was  then dissolved i n 20 mM phosphate buffer pH 8.0 and dialyzed overnight against the same buffer.  A DEAE A f f i - G e l Blue column (Bio-Rad, Richmond, CA)  vas prepared and washed sequentially with 0.5 M phosphate buffer pH 8.0 20 mM phosphate buffer pH 8.0.  and  Dialyzed antibody vas loaded onto the column  followed by 20 mM phosphate buffer.  Samples vere collected i n a f r a c t i o n  c o l l e c t o r and each f r a c t i o n i n d i v i d u a l l y analyzed for protein concentration at 0D 280 nm, binding a c t i v i t y to appropriate c e l l l i n e s , and SDS-PAGE g e l electrophoresis. 20°C.  Aliquots containing antibody vere pooled and frozen at -  Preparation of rabbit anti-mouse  immunoglobulin  a f f i n i t y p u r i f i c a t i o n using mouse immunoglobulin  F(ab')  2  (RaMIg) was  by  conjugated to sepharose  beads (8).  5) BINDING ASSAYS Target c e l l s (e.g. DHL-10, CLL) were washed in Earl's balanced s a l t s o l u t i o n (EBSS) + 0.5% BSA + 10 mM HEPES + 0.1% azide (binding assay media, BAM).  C e l l s were resuspended at 10  7  per ml i n the above media.  10^ c e l l s  per well vere aliquoted into microtiter wells, spun, and the supernate removed.  50 |ul of hybridoma supernate was added per well i n duplicate.  A f t e r a 1 hour incubation at 4°C, c e l l s were washed twice i n BAM.  10^ cpm of  125 I labeled F(ab')2 rabbit anti-mouse added to each well.  immunoglobulin  i n 50 u l of BAM  was  After a one hour incubation at 4°C, c e l l s vere vashed  three times and resuspended i n 100 u l of BAM.  They vere then transferred to  tubes and counted on a Beckman model 5500 gamma counter.  Supernates shoving  67 > 1,000 cpm were considered positive r e l a t i v e to negative control values of < 300 cpm.  6) ANTIBODY COUPLING PROCEDURE One ml of Affi-Gel-10 beads (Bio-Rad, Richmond, CA) were packed into a column per 5 mg of antibody available for conjugation.  Beads were washed  with 3 bed volumes of isopropyl alcohol, followed by 3 bed volumes of cold deionized water and 0.1 M NaHCO^ pH 8.0.  P u r i f i e d antibody and beads were  mixed and incubated overnight at 4°C with continuous shaking.  Beads were  then washed with at least 10 volumes of coupling buffer, resuspended i n binding assay media and stored at 4°C.  7) ANTIBODY LABELING 125 I l a b e l i n g was performed  according to established procedures (9).  A  p30 column was prepared by placing a small piece of glass wool i n a pasteur pipette which was then f i l l e d with a p30 s i z i n g g e l (Bio-Rad, Richmond, VA). The g e l was washed with BAM to saturate protein binding s i t e s , followed by phosphate buffered s a l i n e (PBS).  25-50 ug of antibody i n 25-50 u l was added  to 10 u l of chloramine T solution (0.5 mg/ml i n dB^O, Sigma) i n a small 125 microfuge  tube.  To this was added 1 mC:  I (as Nal, Amersham Int. Ltd.,  Amersham, U.K.) followed by a 15 minute incubation at room temperature.  50  u l of sodium b i s u l p h i t e (20 pg/ml i n PBS, Sigma) was added to stop the reaction.  The mixture was added to the column of p30 followed by PBS.  Fractions were c o l l e c t e d i n tubes and a gross estimate of the r a d i o a c t i v i t y made using a Geiger counter.  Fractions containing labeled antibody were  pooled d i l u t e d with BAM and stored at 4°C.  68 3  H-lysine l a b e l i n g :  5 x 10  6  hybridoma c e l l s vere vashed i n l y s i n e free  DMEM media containing glutamine + 10% dialyzed FCS. 3 in 5 ml of the same media containing 1 mCi Boston, MA)  C e l l s vere  H-lysine (Nev England Nuclear,  and incubated 6-8 hours at 37°C.  An additional 5 x 10^ c e l l s  vere then added and the mixture incubated overnight at 37°C. removed by centrifugation.  resuspended  C e l l s vere  To the supernatant an equal volume of saturated  ammonium s u l f a t e vas added, the mixture microfuged and the precipitate dissolved i n s a l i n e .  This procedure vas repeated 3 times.  p r e c i p i t a t e vas redissolved i n BAM  F i n a l l y , the 3 and dialyzed to remove unbound H-lysine.  8) STIMULATION ASSAYS Mitogen stimulation assays vere performed by modifying existing procedures (10, 11).  C e l l s vere separated as above and resuspended  i n RPMI  1640 + 2 mM glutamine + Na pyruvate (110 mg/liter) + 10 mM HEPES buffer + 5% FCS at a concentration of 2 x 10** c e l l s / m l .  0.1 ml (2 x 10  5  c e l l s ) vere  placed i n v e i l s of f l a t bottom microtiter plates (Linbro, Flov Labs, McLean, VA).  LPS (150 jug/ml) (E. c o l i , Sigma, St. Louis, M0) PHA  (2%)  (Gibco,  Chagrin F a l l s , OH) or anti-p (150 pg/ml) (Cappel Labs, Cochranville, PA) vere d i l u t e d i n the above media.  0.1 ml of mitogen vas added to each of  t r i p l i c a t e v e i l s to give a f i n a l volume of 0.2 ml. c e l l s vithout mitogen i n the same volume.  Control v e i l s contained  Cultures vere incubated for 3 days  (PHA) or 4 days (LPS, anti-p) at 37°C, 5% C0 3  2  and 100% humidity.  One luCi of  H-thymidine (Amersham Int. Ltd., Amersham, UK, s p e c i f i c a c t i v i t y 2.0 Ci/mM) vas added to each v e i l for 4 hours.  C e l l s vere harvested onto glass f i l t e r  disks using a multiple automated sample harvester, dried, dissolved i n s c i n t i l l a t i o n f l u i d and counted on a Beckman l i q u i d s c i n t i l l a t i o n counter.  69  Stimulator c e l l s for one-way mixed lymphocyte cultures (MLC) vere prepared by suspending  PBMC at a concentration of 10 c e l l s / m l i n the above 7  media and adding 0.1 ml stock, mitomycin C (Sigma, St. Louis, MO) per ml of c e l l suspension. media.  C e l l s vere incubated 1 hour at 37°C and vashed 3 times v i t h  5 5 1.5 x 10 responder c e l l s (untreated, vashed PBMC) and 1.5 x 10  mitomycin treated stimulator c e l l s vere added to each v e i l of a m i c r o t i t e r plate i n t r i p l i c a t e folloved by 50 u l of antibody or media as appropriate. 3 C e l l s vere cultured 4 days, pulsed v i t h  H-thymidine, harvested and counted,  as above. 9 ) INHIBITION ASSAYS Anti-p, LPS, PHA or MLR cultures vere set up as described under stimulation assays.  Each v e i l of the microtiter plate contained c e l l s i n  media or c e l l s i n media plus mitogen.  To each of these vas added 50 p i of  hybridoma supernate or p u r i f i e d antibody.  P u r i f i e d antibodies vere t i t r a t e d  for t h e i r a b i l i t y to i n h i b i t stimulation over a concentration ranging from 0.3 ug/ml to 50 pg/ml.  A l l tests vere set up i n t r i p l i c a t e and included  negative controls ( c e l l s + media, c e l l s + test monoclonal antibody), p o s i t i v e controls ( c e l l s + mitogen) and test samples ( c e l l s + mitogen) and test samples ( c e l l s + mitogen + antibody).  Results are expressed as mean counts  per minute (cpm) v i t h standard error of the mean (SEM) of t r i p l i c a t e v e i l s . Percent i n h i b i t i o n vas calculated by d i v i d i n g mean test cpm by p o s i t i v e control cpm a f t e r subtracting background counts according to the f o l l o v i n g equation:  fl\  (Test) - (antibody control)  j  (Positive control) - (Negative c o n t r o l ) / X 100. Those  instances i n vhich the numerator of this equation exceeds the denominator are reported as 0 percent i n h i b i t i o n .  70 C e l l l i n e s used for i n h i b i t i o n assays were cultured at an i n i t i a l density of 2 x 10  c e l l s per ml i n RPMI 1640 + 10% FCS either with or without  50 u l of antibody supernatant  or 50 p i of p u r i f i e d antibody at various  concentrations (1 to 20 jug/ml f i n a l concentration).  C e l l s were pulsed with  t r i t i a t e d thymidine, harvested on day three and counted as above.  10) COLONY ASSAYS E r y t h r o p o i e t i c (CFU-E and BFU-E), granulopoietic (CFU-GM), and pluripotent (CFU-G/E) progenitors were assayed  i n 0.8% methylcellulose i n  Iscove's medium, supplemented with 30% FCS, 1% deionized BSA, 10"^ M 2mercaptoethanol, 200 mM L-glutamine,  3 units/ml of human urinary  e r y t h r o p o i e t i n ( p u r i f i e d to a s p e c i f i c a c t i v i t y of > 100 u/mg) (12) and agarstimulated human leucocyte conditioned medium (13) with or without the addition of p u r i f i e d NB-107 at a f i n a l concentration of 5 pg/ml. H i s t o l o g i c a l l y normal fresh marrow c e l l s from two d i f f e r e n t donors were 5 assayed  by p l a t i n g 2 x 10  washed buffy coat c e l l s per 1.1 ml of culture and  mature e r y t h r o i d , granulocyte-macrophage, and mixed colonies i d e n t i f i e d and scored according to standard c r i t e r i a (13). 11) PURIFICATION OF B CELLS PBMC from one unit (500 ml) of freshly drawn blood were separated Ficoll-Hypaque.  T c e l l depletion was then accomplished  these c e l l s with aminoethylisothiouronium  by incubation of  bromide (AET) treated sheep red  blood c e l l s (S-RBC) followed by Ficoll-Hypaque separation (1).  Monocytes  were depleted by adherence to glass p e t r i dishes overnight at 37°C. these two procedures  over  Using  B c e l l s were enriched to 60-70% as determined by  p o s i t i v i t y f o r surface immunoglobulin.  P a r t i a l l y p u r i f i e d B c e l l s were then  71 stained using 0KT11-FITC (Ortho Diagnostics, Raritan, NJ) which detects the sheep-RBC receptor on T c e l l s .  Sort gates on a fluorescence-activated  cell  sorter (FACS 440, Becton Dickinson, Sunnyvale, CA) were set to exclude T c e l l s (fluorescein p o s i t i v e ) and monocytes (on the basis of l i g h t s c a t t e r ) . The f i n a l B c e l l enrichment resulted i n 85-90% B c e l l s , ~5% monocytes and < 1% T c e l l s as determined by FACS analysis and staining for surface immunoglobulin,  Leu-M3 and 0KT11  respectively.  P u r i f i e d B c e l l s were then  set up i n culture as described above.  12) FACS ANALYSIS C e l l s (1 x 10^) were washed with RPMI 1640 containing 0.5% bovine serum albumin, 0.1% NaN^ and 10 mM HEPES buffer and vere resuspended i n 50 (ul of undiluted culture supernatant or p u r i f i e d antibody.  After a 1 hour  incubation at 4°C, the c e l l s were washed twice and resuspended i n 50 u l of p r e t i t r a t e d FITC conjugated goat anti-mouse IgG F(ab')2 (Tago, CA).  Burlingame,  After an additional incubation at 4°C for 1 hour, c e l l s were washed  three times with PBS containing azide, fixed i n formalin and analyzed on a fluorescence-activated c e l l sorter (FACS 440, Becton Dickinson, Sunnyvale, CA).  Appropriate positive and negative antibody controls vere performed  each experimental run.  These included antibodies of irrelevant  vith  specificity  (e.g. anti-Thy 1.2) as negative controls, and antibodies knovn to react v i t h hematopoietic c e l l populations of interest (e.g. anti-leukocyte, Becton Dickinson, Mountain Viev, CA) as p o s i t i v e controls.  13) IMMUN0PRECIPITATI0NS At least 2 X 10 v i t h PBS.  7  viable DHL-4, DHL-10 or WALK c e l l s vere vashed 3 times  The c e l l s vere counted, resuspended i n 0.5 ml PBS and transferred  72 to an iodogen (Pierce, Rockford, IL) v i a l .  10 pCi of  125  I were added and the  mixture incubated on a rocker at room temperature for 1 hour with constant shaking.  C e l l s were then washed 3 times with PBS, once with BAM  resuspended i n 1.5 ml of 50 mM T r i s - s a l i n e containing 0.5% BSA.  and 0.5 ml of 2%  NP40 i n 50 mM T r i s - s a l i n e was added and the c e l l s observed for l y s i s .  Lysed  c e l l s were microfuged for 10 minutes at 4°C, the supernate removed and 10 u l counted i n a gamma counter. c e l l lysate was u t i l i z e d .  A minimum of 2 x 10^ counts per tube of labeled  To the lysate was added 30 p i of antibody  supernatant followed by a one hour incubation at 4°C.  Rabbit anti-mouse IgG  (RaMIg) conjugated to beads was washed 3 times with T r i s - s a l i n e buffer containing 0.5% BSA and 0.5% NP40. was  40 p i of 50% suspension of washed beads  transferred to each antibody-lysate containing tube.  This mixture was  rocked at 4°C 4 hours to overnight. Beads were then washed 4 times with 0.5% NP40 T r i s - s a l i n e and 150 p i of non-reducing sample buffer added to each tube. Samples were boiled 5 minutes and the supernate removed. supernate was  Half of each  transferred to a microfuge tube and 5 p i of mercaptoethanol  added and the sample boiled again for 5 minutes.  Reducing and non-reducing  samples were stored at 4°C u n t i l ready for g e l electrophoresis. Sequential immunoprecipitations were performed with c e l l s labeled as above.  C e l l lysate was divided into an appropriate number of tubes.  50 p i  of negative control (Thy 1.2, NHL 30.5) (14), positive control (NB65, a n t i t r a n s f e r r i n receptor) (15), or test antibodies were added to each tube followed by a 1 hour incubation at 4°C.  100 p i of 50% RaMIg beads were added  to each tube which were shaken for 1 hour, spun and the supernatant removed. This process was repeated three times with the last bead incubation l a s t i n g overnight.  Precleared supernates were then processed as above for ordinary  immunoprecipitations.  In some instances (the a n t i - c l a s s II antibodies)  73 complete removal of immunoreactive material could not be accomplished this i n d i r e c t technique. conjugated  using  Therefore, these antibodies were p u r i f i e d ,  to beads and preclearing performed  using conjugated beads.  Under  these circumstances 100 |ul of 50% suspension of beads was added to labeled c e l l lysate followed by a 1 hour incubation and removal of supernate a f t e r centrifugation.  This process was repeated up to 5 times, with the l a s t  incubation l a s t i n g overnight.  Following this d i r e c t preclearing,  immunoprecipitation was performed  i n the usual way as described above.  Reduced and nonreduced samples were run i n a 10% polyacrylamide g e l using a slab gel electrophoresis apparatus (Bio-Rad, Richmond, CA).  14) ANTIBODY BLOCKING STUDIES 125 Antibodies were labeled as above with  3 I or  H-lysine.  Labeled  antibodies were t i t r a t e d and saturating l e v e l s u t i l i z e d for blocking experiments  according to modifications of previously described methods (16).  Unlabeled ascites of the test antibodies were t i t r a t e d and each shown to have s i g n i f i c a n t r e a c t i v i t y with DHL-4, DHL-10 or WALK c e l l s at t i t e r s i n excess of  1:30,000.  10  m i c r o t i t e r wells.  c e l l s per well (e.g. DHL-4) were placed into round bottom Labeled antibody was mixed with an equal volume of  s e r i a l l y d i l u t e d ascites.  50 ^ul of labeled-unlabeled antibody mixture  added to each well and incubated 1 hour at 4°C.  C e l l s were washed 3 times  with binding assay media, then counted i n a Beckman l i q u i d counter.  was  scintillation  Controls for non-specific i n h i b i t i o n of binding included ascites  f l u i d containing antibody against unrelated determinants present on test cells.  The percentage i n h i b i t i o n was calculated as a r a t i o of counts per  well, containing cold antibody, to control wells without cold antibody.  74 REFERENCES  1.  Madsen H , Johnsen H, Hansen P, Christiansen S: I s o l a t i o n of human T and B lymphocytes by E-rosette gradient centrifugation. Characterization of the i s o l a t e d subpopulations. J Immunol Meth 33: 323-336, 1980.  2.  Nilsson K, Sundstrom C: Establishment and c h a r a c t e r i s t i c s of two unique c e l l l i n e s from patients with lymphosarcoma. Int J Cancer 13: 808-823, 1974.  3.  Winter J , V a r i a k o j i s D, Epstein A: Phenotypic analysis of established d i f f u s e h i s t i o c y t i c lymphoma c e l l l i n e s u t i l i z i n g monoclonal antibodies and cytochemical techniques. Blood 63: 140-146, 1984.  4.  Epstein AL, Kaplan HS: Feeder layer and n u t r i t i o n a l requirements f o r the establishment and cloning of human malignant lymphoma c e l l l i n e s . Cancer Res 39: 1748-1759, 1979.  5.  Epstein AL, Levy R, Kim H, Henle W, Henle G, Kaplan HS: Biology of the human malignant lymphomas. IV. Functional characterization of ten d i f f u s e h i s t i o c y t i c lymphoma c e l l l i n e s . Cancer 42: 2379-2385, 1978.  6.  Kohler G, M i l s t e i n C: Continuous cultures of fused c e l l s secreting antibody of predefined s p e c i f i c i t y . Nature 256: 495-498, 1975.  7.  Takei F: MALA-1: a surface antigen expressed on activated murine T and B lymphocytes. J Immunol 133: 345-349, 1984.  8.  Stanworth DR, Turner NW: Immunochemical analysis of immunoglobulins and their subunits. In: Handbook of Experimental Immunology, 3rd E d i t i o n , Weir DM (ed), Blackwell S c i e n t i f i c Publications, Oxford, Chapter 6, pp 6.1-6.102, 1978.  9.  Markwell MAK, Fox CF: Surface s p e c i f i c iodination of membrane proteins of viruses and eukaryotic c e l l s using l,3,4,6-tetrachloro-3a, 6ad i p h e n y l g l y c o u r i l . Biochemistry 17: 4807-4851, 1978.  10.  Geha RS, Merler E: Response of human thymus-derived (T) and non-thymus derived (B) lymphocytes to mitogenic stimulation i n v i t r o . Eur J Immunol 4: 193-199, 1974.  11.  M i l l e r R, Gartner S, Kaplan H: Stimulation of mitogenic responses i n human peripheral blood lymphocytes by lipopolysaccharide: Serum and T helper c e l l requirements. J Immunol 121: 2160-2164, 1978.  12.  K r y s t a l G, Eaves CJ, Eaves AC: CM A f f i - G e l Blue chromatography of human urine: a simple one-step procedure for obtaining erythropoietin s u i t a b l e ^ i n v i t r o erythropoietic progenitor assays. Br J Haematol 58: 533546, 1984. o r  13.  Eaves CJ, Eaves AC: Erythropoietin (Ep) dose-response curves f o r three classes of erythroid progenitors i n normal human marrow and i n patients with polycythemia vera. Blood 52: 1196-1210, 1978.  75 14.  Askew DS, Eaves AC, Takei P: NHL-30.5: A monoclonal antibody r e a c t i v e with an acute myeloid leukemia (AML)-associated antigen. Leuk Res 9: 135-145, 1985.  15.  Howard DR, Eaves AC, Takei F: Monoclonal antibody defined c e l l surface molecules regulate lymphocyte a c t i v a t i o n . In: Leukocyte Typing I I , Reinherz EL, Haynes BF, Nadler LM, Bernstein ID (eds), Springer-Verlag New York Inc, New York (In Press).  16.  Springer T, Galfre G, Secher D, M i l s t e i n C: Monoclonal xenogeneic antibodies to murine c e l l surface antigens: i d e n t i f i c a t i o n of novel leukocyte d i f f e r e n t i a t i o n antigens. Eur J Immunol 8: 539-544, 1978.  76 C H A P T E R  III  LYMPHOCYTE FUNCTION ASSOCIATED ANTIGEN (LFA-1) IS INVOLVED IN B CELL ACTIVATION  "Whatever you do w i l l be i n s i g n i f i c a n t , but i t i s very important that you do i t . " Gandhi 1) INTRODUCTION An i n t e r r e l a t e d family of three d i f f e r e n t c e l l surface molecules, expressed on hematopoietic c e l l s of diverse types, has recently been identified.  These molecules, termed LFA-1, Mac-1  (0KM1, Mo-1), and  are defined by d i f f e r e n t monoclonal antibodies (1-3).  pl50,95,  Each possess a common,  apparently i d e n t i c a l , 6 subunit of Mr 95,000 and variable a subunits of approximately Mr 177,000, 165,000 and 150,000 respectively. defined by 0KM1  The antigen  appears to be the complement receptor type three (CR3) (1).  The s p e c i f i c functions of LFA-1  and pl50,95 are unknown.  LFA-1  i s a widely  expressed human leukocyte antigen present on lymphocytes, monocytes, thymocytes, granulocytes and some bone marrow c e l l s (2).  LFA-1 has been  shown to play an important role i n a variety of T c e l l interactions. Monoclonal antibodies to LFA-1 functions including: killing,  have been shown to i n h i b i t various T c e l l  antigen-specific cytotoxic T lymphocyte (CTL) mediated  natural k i l l e r (NK) c y t o l y s i s , T c e l l p r o l i f e r a t i v e responses  antigen, mitogen and allogeneic c e l l s , and T c e l l dependent c e l l responses  (1-6).  to  plaque-forming  The mechanism of action of monoclonal anti-LFA-1 i n  77 i n h i b i t i n g the diverse c e l l u l a r immune processes i s largely unexplored, although evidence that anti-LFA-1 monoclonal antibodies block. CTL mediated k i l l i n g by i n h i b i t i n g adhesion between the CTL and the target c e l l has been +2 reported.  According to these studies LFA-1 may participate i n the Mg  dependent adhesion step of CTL mediated k i l l i n g (2).  -  Despite the  demonstrated widespread importance of LFA-1 i n T c e l l responses a role f o r LFA-1  i n B c e l l function has not been documented. B c e l l s may be induced to p r o l i f e r a t e i n response to a variety of  stimuli including s p e c i f i c antigens as well as polyclonal mitogens such as lipopolysaccharide (LPS) and anti-IgM (|u) antibodies.  The molecular  interactions underlying B c e l l a c t i v a t i o n are largely unknown. At high concentrations, anti-u i s thought to i n i t i a t e a direct p r o l i f e r a t i v e e f f e c t on B c e l l s independent of T c e l l s , monocytes or their products ( 7 , 8 ) .  In  this chapter the i n h i b i t i o n of normal B c e l l activation by a monoclonal antibody to LFA-1 i s reported, and evidence provided that this effect i s mediated v i a action of the antibody on accessory c e l l s or T lymphocytes. These findings document a previously unrecognized role for LFA-1 i n the regulation of B c e l l p r o l i f e r a t i o n and suggest a more generalized role f o r LFA-1  i n the regulation of immune function.  2) RESULTS (A) Monoclonal Antibody NB-107 Defines a Distinct Epitope on the LFA-1 Molecule Monoclonal antibody NB-107 i s an IgG^ monoclonal antibody which immunoprecipitates a noncovalently linked heterodimer (170 and 95 Kd) from 125 the c e l l surface of  I labeled DHL-4 B-lymphoma c e l l s ( F i g . 5). The  molecules immunoprecipitated by NB-107 appear i d e n t i c a l to the two subunits  78  FIGURE 5  The molecular weight of the antigen precipitated from DHL-4 cells by NB-107 is approximately 170 and 95 Kd under reducing conditions (R) and 170 and 115 Kd under non-reducing conditions (NR). Negative control (antibody to Thy 1.2) and positive control (antibody to Transferrin receptor, TR) are included for comparison.  79 immunoprecipitated  by monoclonal antibodies TS1/18 and TS1/22, which react  p r e f e r e n t i a l l y with the B and a chains respectively of the LFA-1 molecule (1).  Removal of material i n DHL-4 c e l l lysates reactive with NB-107 by  preclearing with this antibody, completely eliminated lysate r e a c t i v i t y with TS1/18 and TS1/22 indicating these antibodies react with i d e n t i c a l molecules. In contrast, binding of antibody NB-65 to the t r a n s f e r r i n receptor vas not s i g n i f i c a n t l y decreased by preclearing v i t h NB-107. Preclearing v i t h the same antibody to the t r a n s f e r r i n receptor, hovever, d i d remove a l l detectable transferrin-receptor and did not a f f e c t immunoprecipitation using the a n t i LFA-1  monoclonal antibodies ( F i g . 6). That NB-107 reacts v i t h a d i s t i n c t epitope on the LFA-1 molecule vas  demonstrated by the antibody i n h i b i t i o n studies shovn i n Table I I I . The binding of radioactively labeled NB-107 to B lymphoma c e l l s vas nearly completely i n h i b i t e d by cold p u r i f i e d NB-107 even at very high d i l u t i o n s of cold antibody.  In contrast, no s i g n i f i c a n t i n h i b i t i o n of labeled NB-107  binding vas observed by TS1/18 or TS1/22 even at high concentrations of these antibodies.  These findings demonstrate that NB-107 reacts v i t h an epitope on  the LFA-1 molecule that i s d i s t i n c t from those defined by TS1/18 and TSl/22. (B) Expression of NB-107 on Peripheral Blood Mononuclear C e l l s , Neoplastic and Non-Neoplastic C e l l Lines FACS analysis of NB-107 binding to peripheral blood mononuclear c e l l s from 27 normal individuals revealed a mean + SEM r e a c t i v i t y of 83 + 13 percent.  Mean r e a c t i v i t y v i t h bone marrov c e l l s from 7 patients (3 CML, 1  AML, 1 myelodysplasia, 1 monocytosis, (range 21-72 percent). against normal PBMC.  1 hairy c e l l leukemia) was 45 percent  Figure 7 shows a FACS analysis of NB-107 tested  C e l l s with the greatest amount of l i g h t scatter (larger  c e l l s , predominantly monocytes) displayed the most intense staining by  80  FIGURE 6  Sequential immunoprecipitation ("preclearing"): Antibody to transferrin receptor (NB-65), completely removes transferrin receptor from DHL-4 lysate. LFA-1 detected by TS1/18, TS1/22 and NB-107 remains (A). Preclearing with NB-107 (B) removes material reactive with TS1/18, TS1/22 and NB-107, while leaving transferrin receptor unaffected. Note that molecules immunoprecipitated by NB-107, TS1/18 and TS1/22 have an identical appearance and mobility.  81  TABLE I I I Competitive I n h i b i t i o n of  Antibody NB-107  TS1-18  TS1-22  Control  3  H-lysine Labeled NB-107 Binding to DHL-4 C e l l s  Ascites Dilution  NB-107 Binding (CPM)  % Inhibition  200  201  94  400  240  93  800  372  90  1600  621  87  3200  1003  72  200  3208  9  400  3583  0  800  3692  0  1600  3549  0  3200  3500  1  200  3529  0  400  3616  0  800  3681  0  1600  3706  0  3200  3171  10  none  3542  82  FIGURE 7  FACS analysis of NB-107 tested against normal peripheral blood mononuclear cells. Cells with the greatest amount of light scatter (larger cells, predominantly monocytes) display the most intense staining by NB-107. Cells of intermediate size (lymphocytes) show a spectrum of reactivity from strong to weak.  83 NB-107.  C e l l s of intermediate size (lymphocytes) showed a spectrum of  r e a c t i v i t y from strong to weak.  Dual labeling of PBMC with phycoerythrin  anti-DR and fluoresceinated NB-107 revealed that a major fraction of both DR p o s i t i v e c e l l s (B c e l l s and monocytes) and DR negative c e l l s ( p r i n c i p a l l y T c e l l s ) were positive for NB-107 ( F i g . 8). Whether there i s any functional s i g n i f i c a n c e to the quantitative heterogeneity of LFA-1 expression on lymphoid c e l l s i s not known. We also measured the expression of this antigen on neoplastic and EBV transformed B c e l l l i n e s .  Table IV shows that LFA-1 defined by NB-107 was  present on a l l 9 EBV transformed DR-homozygous c e l l l i n e s examined and 3 of 7 B lymphoma c e l l l i n e s . c e l l lines  Noteworthy was i t s absence on the other 4 B lymphoma  tested.  (C) NB-107 (Anti-LFA-1) Inhibits B C e l l Activation NB-107 profoundly inhibited both LPS and anti-p induced p r o l i f e r a t i v e responses of PBMC (Table V). p u r i f i e d antibody.  This was true for both hybridoma supernate and  Significant  i n h i b i t i o n was observed over a wide range of  antibody concentrations (Table VI).  In a l l experiments isotype i d e n t i c a l  negative controls were performed and showed no s i g n i f i c a n t Antibody to t r a n s f e r r i n receptor, a known i n h i b i t o r of c e l l served as a positive control (10).  NB-107 inhibited  reported for anti-LFA-1 monoclonal antibodies (11).  inhibition. proliferation  the MLR, as previously In contrast to other  studies, we d i d not observe i n h i b i t i o n of PHA stimulation by anti-LFA-1 monoclonal antibody (Table VII) (6). In order to determine whether anti-LFA-1 has a direct effect on B c e l l s , we attempted  to i n h i b i t the growth of EBV transformed B c e l l l i n e s by  addition of p u r i f i e d NB-107 to cultures of these c e l l s .  Two d i f f e r e n t EBV  c e l l l i n e s were tested and i n neither case was evidence of s i g n i f i c a n t  8 4  NB-107 FLUORESCEIN  FIGURE 8  Dual f l u o r e s c e n c e o f normal p e r i p h e r a l b l o o d mononuclear c e l l s u s i n g p h y c o e r y t h r i n l a b e l e d anti-DR and f l u o r e s c e i n l a b e l e d NB-107. C e l l s w i t h the g r e a t e s t i n t e n s i t y o f DR s t a i n i n g ( p r i n c i p a l l y monocytes) a l s o express the h i g h e s t amounts o f LFA-1 d e f i n e d by NB-107.  85  TABLE IV C e l l Line Reactivity of NB-107 (FACS Analysis)  C e l l Line  Type  JREE  EBV (DR1)  CMG  EBV (DR2)  RMG  EBV (DR3)  WAIK  EBV (DRA)  SWEI  EBV (DR5)  ELD  EBV (DR6)  BN  EBV (DR7)  MAD  EBV (DRW8)  KOZ  EBV (DRW9)  SU-DHL-1  B lymphoma  SU-DHL-4  B lymphoma  SU-DHL-6  B lymphoma  SU-DHL-8  B lymphoma  SU-DHL-10  B lymphoma  BALM-5  B lymphoma  U-698-M  B lymphoma  Jurkat  T leukemia  HL-60  Myeloid  K562  Erythroleukemia  leukemia  Reactivity  86  TABLE V I n h i b i t i o n of B C e l l A c t i v a t i o n by A n t i - L F A - l  Mitogen  A »-w A Antibody  ^H-Thymidine Incorporation (Mean CPM + SEM) ' Control  Anti-u  LPS  d  None  1167  0  Test  „ . % Inhibition T  c  192  3248 + 402  NB-65e  586 + 92  1735 + 129  69 + 21  NB-107  566 + 173  1456  71 + 21  None NHL 30.5  a  a  f  +  +  119  1946  +  752  4028 + 426  1338  +  190  3150 + 475  12 + 7  901 + 125  68 + 16  NB-65  491 + 21  NB-107  725 + 320  1349 + 83  80 + 14  R e s u l t s from a representative experiment are expressed as mean counts per  minute (CPM) + standard error of the mean (SEM) of t r i p l i c a t e w e l l s containing 2 x 10^ p e r i p h e r a l blood mononuclear c e l l s (PBMC). i n h i b i t i o n was c a l c u l a t e d as described  Per cent  i n materials and methods and i s the  mean + SEM of s i x i n d i v i d u a l experiments. D  C u l t u r e s without mitogens.  c  C u l t u r e s with mitogens.  ^ A f f i n i t y p u r i f i e d p o l y c l o n a l goat anti-human IgM. e  A n t i b o d y to t r a n s f e r r i n receptor used as a p o s i t i v e c o n t r o l .  ^NHL 30.5 i s a subclass i d e n t i c a l monoclonal antibody d i r e c t e d against a myeloid d i f f e r e n t i a t i o n antigen ( 9 ) ; one of several monoclonals used as negative c o n t r o l s .  87  TABLE VI I n h i b i t i o n of LPS S t i m u l a t i o n : T i t r a t i o n Using P u r i f i e d NB-107  3  Antibody  3  H-Thymidine Incorporation ± ( M e a n  C P M  S E M )  Control  None  310 + 27  4469 + 839  NHL 30.5 (10)  387 + 85  4390 + 1528  4  NB-107 (20)  137 + 25  1109 + 375  77  NB-107 (10)  157 + 31  1159 + 111  76  NB-107 (5)  228 + 66  1959 + 307  68  NB-107 (2.5)  180 + 31  2071 + 227  55  NB-107 (supernate)  212 + 40  1407 + 374  71  NB-65 (supernate)  246 + 25  997 + 97  82  /ml  See Table V f o r d e s c r i p t i o n of antibodies. 'Without mitogen. With mitogen.  Test  % Inhibition  <r 8 > l  C  88  TABLE VII I n h i b i t i o n of T C e l l P r o l i f e r a t i o n  Mean + SEM Antibody  a  Control  Test  X Inhibition'  0  MLR None  459 + 204  17,998 +  797  NHL 30.5  679 + 141  19,810 +  1941  NB-65  343 +  93  4,028 +  502  83 + 6  NB-107  219 + 104  3,811 +  826  85 + 7  0  PHA None  519 +  62  85,767 +  1,665  NHL 30.5  272 +  48  87,022 +  3,951  0  NB-65  305 +  82  48,373 +  3,083  43 + 1  NB-107  164 +  60  98,442 + 12,123  3 + 3  See Table V f o r description of antibodies. Culture without stimulator c e l l s or mitogen. Cultures with stimulator c e l l s or mitogen. Wan  + SEM of two i n d i v i d u a l experiments.  89 i n h i b i t i o n obtained (Table VIII).  This was i n contrast to the marked  i n h i b i t i o n achieved by the addition of a n t i - t r a n s f e r r i n receptor antibody. The p r o l i f e r a t i v e response of highly p u r i f i e d normal B c e l l s to anti-^u was, likewise, not i n h i b i t e d by NB-107 (Table IX) nor did the presence of NB-107 have any i n h i b i t o r y e f f e c t on colony formation by bone marrow CFU-E, BFU-E, CFU-GM or CFU-G/E assayed i n standard methyl c e l l u l o s e cultures (Table X).  3) DISCUSSION We have demonstrated that LFA-1 may be important i n the regulation of B c e l l p r o l i f e r a t i o n . Monoclonal antibody to LFA-1 (NB-107) profoundly i n h i b i t e d stimulation of human peripheral blood mononuclear c e l l s (PBMC) by LPS and anti-p.  In order to further investigate whether NB-107 i n h i b i t s B  lymphocyte mitogen induced a c t i v a t i o n v i a a d i r e c t action on B c e l l s or by i n h i b i t i n g an e s s e n t i a l accessory c e l l function or T-B c e l l i n t e r a c t i o n , the e f f e c t of NB-107 on a c t i v a t i o n of highly p u r i f i e d B c e l l s was tested.  NB-107  antibody d i d not i n h i b i t the anti-p stimulation of the B enriched populations,  which contained  less than 1% T c e l l s (0KT4 ), approximately 5% +  monocytes (0KM1 ) and 85-90% surface immunoglobulin p o s i t i v e B c e l l s . 3 +  Furthermore, NB-107 also did not i n h i b i t transformed B c e l l l i n e s .  Therefore,  H-thymidine uptake of several EBV  anti-LFA-1 does not seem to i n h i b i t B  cells directly. These findings r a i s e the question of how the B c e l l response to anti-p involves other c e l l s (such as T c e l l s or monocytes) and how anti-LFA-1 i n h i b i t s the B c e l l response. populations  Because anti-p stimulated highly  enriched  of B c e l l s , either this stimulation does not require T c e l l s  and/or monocytes, or those c e l l s s t i l l contaminating the B enriched population  may be s u f f i c i e n t and also necessary for the stimulation of B  90 TABLE VIII Lack of I n h i b i t i o n of EBV C e l l Line Growth  WALK (DR4) C e l l s Antibody  3  (pg/ml)  None NB-65  Mean CPM + SEM  X Inhibition  1339 +  289  494 +  19  69  NHL-30.5  (20)  1563 +  881  0  NHL-30.5  (5)  1663 +  152  0  NHL-30.5  (1.25)  1602 +  152  0  NHL-30.5  (0.3)  1581 +  256  0  NB-107  (20)  1226 +  48  9  NB-107  (5)  1067 +  947  22  NB-107  (1.25)  1071 +  149  22  NB-107  (0.3)  1293 +  359  4  ELD (DR6) C e l l s None NB-65  7492 +  270  671 +  62  90  NHL-30.5  (20)  5644 +  279  25  NHL-30.5  (5)  8036 +  823  0  NHL-30.5  (1.25)  7244 + 2282  3  NHL-30.5  (0.3)  9075 + 1826  0  384  22  5829 +  NB-107  (20)  NB-107  (5)  6341 + 1520  15  NB-107  (1.25)  5858 + 1734  22  NB-107  (0.3)  6702 +  11  a  109  See Table V for description of antibodies.  TABLE IX I n h i b i t i o n of Anti-p Stimulation: Purified B C e l l s  Antibody (10 pg/ml)  969 +  None None  Mean CPM + SEM  0  X Inhibition  85  13,225 + 156  NHL 30.5  11,379 + 724  15  NB-107  12,661 + 550  5  a  C e l l s without anti-p  D  C e l l s with anti-p  92  TABLE X Effect of Anti-LFA-1 on Bone Marrow Progenitor C e l l s  Colonies* Progenitor  NB-107  CFU-E ( l a t e erythroid)  +  BFU-E ( p r i m i t i v e erythroid)  +  CFU-GM (granulopoietic)  +  CFU-G/E (pluripotent)  +  Exp 1  Exp 2  409  107  399  110  204  169  149  156  286  128  254  109  1.5  1  2  0  Number of colonies per 2 x 10^ normal human marrow buffy coat c e l l s .  a  Values are means of counts from 2 r e p l i c a t e 1.1 ml methylcellulose cultures  5 each i n i t i a l l y containing 2 x 10  cells.  93 cells.  I f anti-LFA-1 i n h i b i t s B c e l l a c t i v a t i o n by a f f e c t i n g T c e l l s and/or  monocytes, and i f the contaminating T c e l l s and monocytes are involved i n the stimulation of B c e l l s , the antibody should also i n h i b i t the stimulation of purified B cells.  However, the present  study c l e a r l y showed that anti-LFA-1  does not i n h i b i t the stimulation of p u r i f i e d B c e l l s by anti-p. apparent paradox may be explained as follows:  This  At low c e l l d e n s i t i e s  (unfractionated PBMC) B c e l l stimulation by anti-^u requires factors produced by T c e l l s and/or monocytes.  These l a t t e r c e l l s are i n h i b i t e d by anti-LFA-1  which r e s u l t s i n i n h i b i t i o n of PBMC stimulation by anti-p.  At high  cell  d e n s i t i e s ( p u r i f i e d B c e l l s ) anti-p stimulates B c e l l s i n the absence of accessory  c e l l s and/or monocytes perhaps due to a c r i t i c a l c e l l density which  i s achieved  or required for endogenous release of BCGF and autostimulation  (see below). The mechanisms involved i n B c e l l a c t i v a t i o n are currently being a c t i v e l y researched.  LPS stimulation of B c e l l s appears to be macrophage but  not T c e l l dependent (13).  Anti-p antibody i s thought to exert i t s e f f e c t s  on B c e l l s by two concentration dependent mechanisms.  At low concentrations,  anti-p induces c e l l enlargement, RNA synthesis and c e l l surface expression of B c e l l growth factor receptors.  P r o l i f e r a t i o n of B c e l l s i n this case then  requires a second s i g n a l , mediated by BCGF secreted by activated T helper cells.  At high concentrations  (such as used i n this study), anti-p has been  thought to i n i t i a t e a d i r e c t p r o l i f e r a t i v e e f f e c t on B c e l l s independent of T c e l l s , monocytes or their products (7,8,14). EBV  Recent studies have shown that  transformed B c e l l s require either a c r i t i c a l c e l l density or  supplementation with exogenous factors for p r o l i f e r a t i o n . c e l l s appear to secrete their own autostimulatory (17,18).  Furthermore these  B c e l l growth factor  Whether normal stimulated B c e l l s at high c e l l densities are  capable of autostimulation  i s not yet known.  94 Previous  studies have not shown an i n h i b i t o r y e f f e c t of anti-LFA-1  monoclonal antibodies on LPS stimulation of mouse lymphocytes (12). differences between these and our findings may  be related to species  differences between mouse and human or r e l a t i v e concentrations Human LFA-1  expresses multiple unique antigenic epitopes  varying degrees of s p a t i a l proximity molecule (19).  The  of B c e l l s .  that reside with  to the functional region(s) of the  NB-107 c l e a r l y reacts with a d i f f e r e n t epitope on the  molecule than does TS1/18 and TS1/22 as shown by competitive studies (Table I I I ) .  LFA-1 LFA-1  inhibition  Monoclonal antibodies to d i f f e r e n t LFA-1  epitopes  appear to vary i n their a b i l i t y to i n h i b i t T lymphocyte function (20). region defined by NB-107 may  be uniquely  involved i n the  i n t e r a c t i o n of B c e l l s with monocytes and/or T c e l l s and  The  cooperative thus susceptible to  i n h i b i t i o n by NB-107 but not other anti-LFA-1 monoclonal antibodies. I n h i b i t i o n of the MLR described  by NB-107 (Table VII) i s s i m i l a r to that previously  for other anti-LFA-1 monoclonal antibodies.  stimulation of T lymphocytes was  In contrast,  PHA  not inhibited by this antibody (6).  Our findings of the expression of LFA-1  (defined by NB-107) on B and T  lymphocytes and most intensely on monocytes of normal peripheral blood i s consistent with previous EBV  reports (Fig. 7,8)  (6,21).  LFA-1  was  present on a l l  c e l l l i n e s examined (9/9), but absent on 4 of 7 neoplastic B lymphoma  lines.  The l a t t e r f i n d i n g implies the loss of LFA-1  associated with the development of B c e l l malignancy. growth regulatory c e l l surface molecules may autonomous (neoplastic) c e l l growth. of LFA-1  on B lymphoma c e l l s may  product modification. c e l l s are not known.  The  may  be somehow  Loss of normal B c e l l  be a step i n the development of  A l t e r n a t i v e l y , the surface  expression  simply be lost by altered gene expression  functional implications of the loss of LFA-1  or  on B  95 Although approximately  h a l f of bone marrow c e l l s are p o s i t i v e f o r LFA-1  (2) the i d e n t i t y of these c e l l s has not been e s t a b l i s h e d . whether LFA-1 i s important  i n hematopoietic  I n order to t e s t  stem c e l l d i f f e r e n t i a t i o n ,  p u r i f i e d NB-107 was added to cultures of normal human marrow buffy coat cells.  No i n h i b i t o r y e f f e c t of NB-107 on the growth of CFU-E, BFU-E, CFU-GM  or CFU-G/E was i d e n t i f i e d suggesting that LFA-1 i s not important  i n the  formation o f colonies of diverse myeloid c e l l lineages. Recently, the family of glycoproteins LFA-1, 0KM1, pl50,95 have been found to be d e f i c i e n t on the c e l l s of patients with chronic b a c t e r i a l i n f e c t i o n s and m u l t i p l e immune abnormalities (22-24).  I t has been postulated  that these abnormalities r e s u l t from the abnormal f u n c t i o n of granulocytes, monocytes and/or T c e l l s and that these d e f e c t i v e functions may be a t t r i b u t a b l e to the l i m i t e d expression of LFA-1, 0KM1 and pl50,95 on these cells. antibody  We report the i n h i b i t i o n of B lymphocyte a c t i v a t i o n by a monoclonal to LFA-1, and provide evidence that t h i s i s due to e f f e c t s on  monocytes and/or T c e l l s .  These f i n d i n g s suggest that t h i s c l a s s of c e l l  surface molecules has a more diverse f u n c t i o n a l r o l e i n the r e g u l a t i o n of the immune response than previously r e a l i z e d .  We are c u r r e n t l y i n v e s t i g a t i n g B  c e l l f u n c t i o n and LFA-1 expression i n patients with hereditary immune d e f i c i e n c y and chronic b a c t e r i a l i n f e c t i o n s .  96 REFERENCES  1.  Sanchez-Madrid F, Nagy J , Robbins E, Simon P, Springer TA: A human leukocyte d i f f e r e n t i a t i o n antigen family with d i s t i n c t a-subunits and a common 3-subunit. J Exp Med 158: 1785-1803, 1983.  2.  Sanchez-Madrid F, Simon P, Thompson S, Springer TA: Mapping of antigenic and functional epitopes on the a - and 8-subunits of two related mouse glycoproteins involved i n c e l l interactions LFA-1 and Mac-1. J Exp Med 158: 586-602, 1983.  3.  Beatty PG, Ledbetter J , Martin PJ, Price T, Hansen JA: D e f i n i t i o n of a common leukocyte c e l l - s u r f a c e antigen (Lp95-150) associated with diverse cell-mediated immune functions. J Immunol 131: 2913-2918, 1983.  4.  Miedema F, Tetleroo P, Hesselink W, Werner G, Spits H, Melief C: Both Fc receptors and lymphocyte-function-associated antigen 1 on human Ty lymphocytes are required for antibody-dependent c e l l u l a r c y t o t o x i c i t y ( k i l l e r c e l l a c t i v i t y ) . Eur J Immunol 14: 518-522, 1984.  5.  Krensky AM, Robbins E, Springer TA, Burakoff S: LFA-1, LFA-2, and LFA-3 antigens are involved i n CTL-target conjugation. J Immunol 132: 2180-2182, 1984.  6.  Krensky AM, Sanchez-Madrid F, Robbins E, Nagy J , Springer TA, Burakoff S: The functional s i g n i f i c a n c e , d i s t r i b u t i o n , and structure of LFA-1, LFA-2 and LFA-3: C e l l surface antigens associated with CTL-target i n t e r a c t i o n s . J Immunol 131: 611-616, 1983.  7.  Kehrl JH, Muraguchi A, Butler JL, Falkoff RJM, Fauci AS: Human B c e l l a c t i v a t i o n 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 . Immunol Rev 78: 75-96, 1984.  8.  Howard M, Nakanishi K, Paul WE: B c e l l growth and d i f f e r e n t i a t i o n f a c t o r s . Immunol Rev 78: 185-210, 1984.  9.  Askew DS, Eaves AC, Takei F: NHL-30.5: A monoclonal antibody reactive with an acute myeloid leukemia (AML)-associated antigen. Leuk Res 9: 135-145, 1985.  10.  Trowbridge I, Lopez F: Monoclonal antibody to t r a n s f e r r i n receptor blocks t r a n s f e r r i n binding and i n h i b i t s human tumor c e l l growth i n v i t r o . Proc Natl Acad Sci USA 79: 1175-1179, 1982.  11.  Kvirzinger K, Reynolds T, Germain R, Davignon D, Martz E, Springer TA: A novel lymphocyte function-associated antigen (LFA-1): C e l l u l a r d i s t r i b u t i o n , quantitative expression, and structure. J Immunol 127: 596-601, 1981.  12.  Davignon D, Martz E, Reynolds T, Kurzinger K, Springer TA: Monoclonal antibody to a novel lymphocyte function-associated antigen (LFA-1): Mechanism of blockade of T lymphocyte-mediated k i l l i n g and e f f e c t s on other T and B lymphocyte functions. J Immunol 127: 590-595, 1981.  97 13.  Melchers F, Corbel C: Studies on B - c e l l a c t i v a t i o n i n v i t r o . Immunol 134: 63-73, 1983.  14.  Melchers F, Andersson J : B c e l l a c t i v a t i o n : Three steps and v a r i a t i o n s . C e l l 37: 715-720, 1984.  15.  Forni L, Coutinho A: Receptor interactions on the membrane of r e s t i n g and activated B c e l l s . Nature 273: 304-306, 1978.  16.  Howard DR, Eaves AC, Takei F: Monoclonal antibody defined c e l l surface molecules regulate lymphocyte a c t i v a t i o n . In Leukocyte Typing I I , Reinherz EL, Haynes BF, Nadler LM, Bernstein ID (eds), Springer-Verlag New York Inc, New York (In Press).  17.  Gordon J , Ley SC, Melamed MD, English LS, Hughes-Jones N: Immortalized lymphocytes produce B - c e l l growth factor. Nature 310: 145-147, 1984.  18.  Blazar BA, Sutton LM, Strome M: Self-stimulating growth factor production by B c e l l l i n e s derived from Burkitt's lymphomas and other l i n e s transformed i n v i t r o by Epstein-Barr v i r u s . Cancer Res 43: 4562-4568, 1983.  19.  Ware CF, Sanchez-Madrid F, Krensky A, Burakoff S, Strominger J , Springer TA: Human lymphocyte function associated antigen-1 (LFA-1): I d e n t i f i c a t i o n of multiple antigenic epitopes and their r e l a t i o n s h i p to CTL-mediated c y t o t o x i c i t y . J Immunol 131: 1182-1188, 1983.  20.  Springer TA: Analysis of macrophage d i f f e r e n t i a t i o n and function with monoclonal antibodies. Contemp Top Immunobiol 13: 1-15, 1984.  21.  Sanchez-Madrid F, Krensky A, Ware C, Robbins E, Strominger JL, Burakoff S, Springer TA: Three d i s t i n c t antigens associated with human Tlymphocyte mediated c y t o l y s i s : LFA-1, LFA-2 and LFA-3. J Immunol 79: 7489-7493, 1982.  22.  Dana N, Todd RF, P i t t J, Springer TA, Arnaout MA: Deficiency of a surface membrane glycoprotein (Mol) i n man. J C l i n Invest 73: 153-159, 1984.  23.  Anderson DC, Schmalstieg F, Arnaout M, Kohl S, Tosi M, Dana N, Buffone G, Hughes B, Brinkley B, Dickey W, Abramson J, Springer T, Boxer L, Hollers J , Smith CW: Abnormalities of polymorphonuclear leukocyte function associated with a heritable deficiency of high molecular weight surface glycoproteins (GP 138): Common relationship to diminished c e l l adherence. J C l i n Invest 74: 536-551, 1984.  24.  Beatty PG, Harlan J, Rosen H, Hansen J, Ochs H, Price T, Taylor R, Klebanoff S: Absence of monoclonal-antibody-defined protein complex i n boy with abnormal leukocyte function. The Lancet March 10: 535-537, 1984.  v  Ann their  B  98 C H A P T E R  IV  MONOCLONAL ANTIBODIES TO HLA-CLASS I I DETERMINANTS: FUNCTIONAL EFFECTS ON THE OF NORMAL AND  ACTIVATION AND  EBV  PROLIFERATION  TRANSFORMED B CELLS  "Probable i m p o s s i b i l i t i e s are to be p r e f e r r e d to improbable p o s s i b i l i t i e s . "  A r i s t o t l e 384-322 B.C. 1) INTRODUCTION HLA  c l a s s I I molecules a r e h e t e r o d i m e r i c transmembrane p r o t e i n s  c o n s i s t i n g o f a heavy (a) c h a i n of 33-35,000 MW  28,000 MW  s t r u c t u r a l l y homologous to murine I-E. murine I-A,  and DP  DQ  (DS, MB,  (SB) has been d e f i n e d by primed  i s e v i d e n c e f o r a t l e a s t s i x a l p h a and  the D r e g i o n (4). to  express at l e a s t  m o l e c u l e s , and  one  c l a s s I I genes and responses, behavior  chain of  25-  I I (D) r e g i o n have r e v e a l e d the e x i s t e n c e  t h r e e groups o f molecules w i t h s i m i l a r s t r u c t u r e s  There  (3)  (1). G e n e t i c and b i o c h e m i c a l a n a l y s e s o f the genes and gene  p r o d u c t s encoded i n the HLA-class of  and a l i g h t  DC)  DR i s  shows homology to  lymphocyte t y p i n g  f i v e c l a s s I I molecules: SB molecule  (5).  Although  (4).  seven b e t a c h a i n genes w i t h i n  At the p r o t e i n l e v e l , HLA-DR homozygous c e l l two DR  molecules,  lines two  appear  DS  i t i s well established  that  t h e i r p r o d u c t s are i n v o l v e d i n a v a r i e t y o f immune  very l i t t l e  (2,3).  (1-3).  i s known about  how  these molecules modulate b i o l o g i c  99 Evidence i n both mouse and man indicates that class I I genes determine several immunologic phenomena.  These include:  control of the l e v e l of  immune responses, delayed type h y p e r s e n s i t i v i t y , s u s c e p t i b i l i t y to c e r t a i n diseases, and primary (mixed lymphocyte lymphocyte  culture) and secondary (primed  typing) allogeneic T c e l l p r o l i f e r a t i o n .  In addition, HLA c l a s s  II molecules are involved i n T c e l l a c t i v a t i o n , apparently by playing an associative role i n the recognition of antigen ( 3 ) . The r e l a t i v e contribution of DQ vs DR molecules to these processes i s unknovn.  A role f o r  HLA class II molecules i n B c e l l a c t i v a t i o n has not been documented. However, accessory c e l l s (which express class I I antigens) and T c e l l s are thought to have a regulatory role i n the a c t i v a t i o n of B c e l l s ( 6 ) . B c e l l s are induced to p r o l i f e r a t e i n response to a variety of s t i m u l i including s p e c i f i c antigens as well as polyclonal mitogens such as lipopolysaccharide (LPS) and anti-IgM (u) antibodies (6,7).  The molecular  events underlying B c e l l a c t i v a t i o n by these mitogens i s not understood, although both c r o s s - l i n k c e l l surface molecules (6).  In order to investigate  the functional role of HLA class I I antigens i n B c e l l a c t i v a t i o n we have generated a panel of a n t i - c l a s s I I monoclonal antibodies. are  In t h i s chapter  described the c h a r a c t e r i s t i c s of three of these, that d i f f e r i n t h e i r  s p e c i f i c i t i e s f o r DQ and DR determinants as well as i n their e f f e c t s on the p r o l i f e r a t i o n of normal and EBV transformed B c e l l s .  2) RESULTS (A) Antibody S p e c i f i c i t y Monoclonal antibodies to HLA class II molecules were produced using the B lymphoma c e l l l i n e DHL-10 (DH-84, DH-224) or LPS stimulated s p l e n i c B lymphoma c e l l s (NB-29) as immunogens.  Monoclonal antibodies DH-84 and DH-224  100 reacted consistently by FACS analysis with a subpopulation of PBMC from each of 30 normal donors (14 and 11 percent of PBMC, r e s p e c t i v e l y ) .  In addition,  these antibodies reacted with a panel of DR homozygous c e l l s encompassing DR1 through DRw9 s p e c i f i c i t i e s (Table XI).  Both immunoprecipitated  of approximately 35,000 and 28,000 m.w.  (Fig.9).  These values are s i m i l a r to  the molecular weights of the heterodimers immunoprecipitated DR.  heterodimers  by OKIa and BD-  NB-29 monoclonal antibody reacted with homozygous c e l l l i n e s (HCL) DR  l,2,4,6,8,w9.  This pattern of r e a c t i v i t y i s the same as that obtained with  BT 3.4, a known and well characterized anti-DQ monoclonal antibody (8). 29 also immunoprecipitated  NB-  a c e l l surface heterodimer s i m i l a r i n molecular  weight to that precipitated by BT 3.4 and Leu-10, which i s s l i g h t l y l e s s f o r both chains than that precipitated by the known anti-DR monoclonals (Figs.9,10). NB-29 reacted with the same class I I epitope as d i d BT 3.4.  This was  demonstrated by the a b i l i t y of BT 3.4 to completely i n h i b i t the binding of 125  I labeled NB-29 (Table XII).  NB-29 binding was also s i g n i f i c a n t l y  i n h i b i t e d by the known anti-DQ monoclonal antibody, Leu-10.  Thus, on the  basis of the molecular weight of i t s target antigen, i t s r e a c t i v i t y with DR homozygous c e l l l i n e s and by cross-blocking studies, NB-29 appears monoclonal antibody to a polymorphic determinant  to be a  present on DQ molecules,  that i s s i m i l a r or i d e n t i c a l to that defined by BT 3.4. As shown i n Table XII, DH-224 binding was blocked markedly by BD-DR but only weakly by OKIa. NB-29 binding.  On the other hand DH-224 d i d not block either DH-84 or  Removal of nearly a l l material reactive with DH-224 by  "preclearing", substantially reduced the amount of DR p r e c i p i t a t e d by BD-DR. However, the amount of Leu-10 reactive material (DQ) was not affected (Fig.11).  Based upon i t s r e a c t i v i t y with normal PBMC and DR-HCL, and from  TABLE XI Reactivity of Anti-class I I Monoclonal Antibodies with Homozygous DR C e l l Lines: FACS A n a l y s i s  C e l l Line  D/DR  3  Specificity HLA-ABC  NB-29  BT3.4  Antibody Leu-10 1DH-84  DH-224  BD-DR  JREE  DR1, DW1  A2, BW44 (W4)  +  +  +  +  +  +  CMG  DR2, DW2  A3, B7  +  +  +  +  +  +  RMG  DR3, DW3  A l , B8  -  -  -  +  +  +  WALK  DR4, DW4  A2, BW44, CW5  +  +  +  +  +  +  SWEI  DRS, DW5  A29, B40  -  -  +  +  +  +  ELD  DR6, DW6 (DW "6.1")  A2, B40  +  +  +  +  +  +  BD  DR7, DW7  A2, B13  -  -  -  +  +  +  MAD  DR8, DV8 (LD "8.1")  A2, B40, CW3  +  +  +  +  +  +  KOZ  DRV9,, LD "4x7"  AW24, 26 BVS4, 40  +  +  +  +  +  +  C e l l lines were considered positive i f greater than 10% of c e l l s were reactive with the test antibody compared to negative control antibody of i r r e l e v a n t s p e c i f i c i t y .  102  — 90 —  FIGURE 9  67  Immunoprecipitation using I - l a b e l e d WALK (DR4) cells. NB 29 ( a n t i - D Q ) , DH-224 ( a n t i - D R ) and DH-84 (anti-DQ+DR) immunoprecipitate bands o f a p p r o x i m a t e l y 35,000 and 28,000 m o l e c u l a r weight. Shown f o r comparison a r e the known anti-DR monoclonals from Ortho (OKIa) and B e c t o n - D i c k i n s o n (BD-DR). The amount o f m a t e r i a l p r e c i p i t a t e d by DH-84 and OKIa i s c o n s i d e r a b l y l e s s than that o f the o t h e r a n t i b o d i e s . This probably r e l a t e s to d i f f e r e n c e s i n a n t i b o d y a f f i n i t y . The molecular weight of each c h a i n p r e c i p i t a t e d by the anti-DQ monoclonal NB-29 i s 1 to 2 k d l e s s than that of the anti-DR monoclonals ( e . g . BD-DR). 1 2 5  103  FIGURE 10  Immunoprecipitation u s i n g I - l a b e l e d WALK (DR4) cells. A n t i b o d i e s NB-29, DH-84 and DH-224 a r e shown f o r comparison w i t h known anti-DQ monoclonal a n t i b o d i e s BT3.4 and Leu-10. NB-29 and BT3.4 immunoprecipitate i d e n t i c a l bands, each o f which has m o b i l i t y s l i g h t l y g r e a t e r than those p r e c i p i t a t e d by BD-DR. 1 2 5  104  TABLE XII Cross Blocking of  125  I-labeled A n t i - c l a s s I I Antibodies: DHL-4 C e l l s  Labeled Antibody NB-29  X Inhibition  Blocking Antibody  DH-224  DH-84 0  NHL-30.5  0  0  0  NB-65  0  0  0  4  3  0  NB-29  99  0  0  DH-84  78  91  0  7  0  94  BT3.4  99  0  0  Leu-10  50  0  0  BD-DR  ND  ND  72  OKIa  ND  ND  18  C  Thyl.2  c  DH-224  a  ND = not done °Calculated as described i n materials and methods. °Hybridoma supernate  105  FIGURE 11  Sequential immunoprecipitation " p r e c l e a r i n g " of I - l a b e l e d WALK (DR4) c e l l l y s a t e . "A" i s precleared with antibody of unrelated specificity. DH-84, DH-224, Leu-10 and BD-DR each p r e c i p i t a t e bands o f a p p r o x i m a t e l y 35,000 and 28,000 a.w. "B" i s p r e c l e a r e d u s i n g DH-84 which s u b s t a n t i a l l y reduces the amount o f BD-DR and DH-224. The marked d i m i n u t i o n i n the amount o f m a t e r i a l p r e c i p i t a t e d by the known anti-DR monoclonal a n t i b o d y (BD-DR) i n d i c a t e s DH-84 has s p e c i f i c i t y f o r DR m o l e c u l e s . The amount o f Leu-10 (anti-DQ) p r e c i p i t a t e d m a t e r i a l i s u n a f f e c t e d . "C" i s p r e c l e a r e d u s i n g DH-224. I n a d d i t i o n to removing m a t e r i a l r e a c t i v e w i t h i t s e l f , p r e c l e a r i n g with DH-224 has markedly reduced the amount of DR p r e c i p i t a t e d by BD-DR w h i l e l e a v i n g DQ r e a c t i v e m a t e r i a l ( p r e c i p i t a t e d by Leu-10) unchanged.  1 2 5  106 the cross-blocking and sequential immunoprecipitation data, DH-224 appears to react with a monomorphic DR determinant s i m i l a r , but not i d e n t i c a l i n s p e c i f i c i t y , to BD-DR. The s p e c i f i c i t y of DH-84 was more d i f f i c u l t with a l l HCL and normal control PBMC tested. with DH-84 s u b s t a n t i a l l y diminished  to e s t a b l i s h .  DH-84 reacted  Preclearing of c e l l lysates  the amount of material r e a c t i v e with BD-  DR, but did not remove i t completely; Leu-10 reactive material was not diminished  (Fig.11).  Preclearing of material reactive with NB-29 d i d not  s u b s t a n t i a l l y remove DH-84 reactive molecules (Fig.12).  These findings  suggest that DH-84 reacts with a monomorphic DR determinant.  However, DH-84  125 substantially and reproducibly blocked cells.  I labeled NB-29 binding to DHL-4  As described above, NB-29 i s s p e c i f i c for a polymorphic determinant  on HLA-DQ molecules.  Therefore,  DH-84 appears to have s p e c i f i c i t y  predominantly for DR, but also reacts with a subpopulation  of DQ molecules.  Whether these antibodies show any cross r e a c t i v i t y with DP molecules has not been determined. (B) I n h i b i t i o n of PBMC Stimulation Both DH-84 and DH-224 profoundly  i n h i b i t e d PBMC stimulation by a n t i s e r a  against IgM (mean i n h i b i t i o n of 70-90%), and LPS (mean i n h i b i t i o n of 80-90%) (Tables XIII and XIV).  T i t r a t i o n of the i n h i b i t o r y e f f e c t of DH-84 on anti-u  induced stimulation showed that p u r i f i e d DH-84 was e f f e c t i v e over a wide concentration  range (from 4 jug/ml to 0.16 |ug/ml).  Similar i n h i b i t i o n was not  seen i n cultures containing any of a number of monoclonal antibodies of unrelated s p e c i f i c i t y (e.g. NHL-30.5 Table XIV).  In contrast to DH-84 and  DH-224 which define monomorphic DR determinants, NB-29 (DQ polymorphic) did not show s i g n i f i c a n t i n h i b i t i o n of LPS or anti-u stimulation (Table XIII). Likewise DH-84 and DH-224 markedly i n h i b i t e d the mixed lymphocyte response,  t  OL  OL  107  CM 00 H e  00  O CQ X O DQ CJ Z Q CJ  FIGURE  12  2  Q  Sequential immunoprecipitation " p r e c l e a r i n g " of I - l a b e l e d WALK (DR4) c e l l l y s a t e . "A" i s precleared with c o n t r o l antibody of u n r e l a t e d specificity. NB-29 and DH-84 each p r e c i p i t a t e bands o f a p p r o x i m a t e l y 35,000 and 28,000 m.w. "B" i s p r e c l e a r e d w i t h NB-29. NB-29 p r e c l e a r i n g removes a l l m a t e r i a l r e a c t i v e w i t h i t s e l f , w h i l e not q u a n t i t a t i v e l y a f f e c t i n g the amount o f m a t e r i a l p r e c i p i t a t e d by DH-84.  12 5  108  TABLE XIII Inhibition of Stimulation of Normal PBMC'  Mean cpm + SEM Antibody (5 ug/ml)  Control  Test  Anti-u media NB-29 DH-84 DH-224  232 445 197 247  + 124 + 159 + 46 + 75  3,436 2,421 298 939  + + + +  X Inhibition  0  d  637 198 54 397  17 + 10 83 + 22 70 + 29  LPS media NB-29 DH-84 DH-224 NB-65 e  1,520 1,541 222 455 523  + 339 + 296 + 38 + 109 + 53  10,951 7,969 1,332 2,207 2,914  + 1 ,323 237 + + 256 + 249 484 +  Results  18 83 84 64  + + + +  13 11 11 17  are expressed as mean counts per minute (tjpm) + standard error of the mean (SEM) of t r i p l i c a t e wells containing 2 x 10 p e r i p h e r a l blood mononuclear c e l l s (PBMC). Per cent i n h i b i t i o n was calculated as described i n Materials and Methods and i s the mean + SEM of values from four separate experiments. ^Cultures without mitogens. °Cultures with mitogens. ^ A f f i n i t y p u r i f i e d polyclonal goat anti-human IgM. e  Antibody  to t r a n s f e r r i n receptor (hybridoma supernate).  109  TABLE XIV I n h i b i t i o n of Anti-u Stimulation of Normal PBMC:  T i t r a t i o n Using  Purified  Anti-HLA Class I I Antibody  Mean cpm + SEM Antibody (ug/ml)  Control  737 +  media  X Inhibition  Test  12  4,586 + 252  DH-84  (4.0)  360 + 113  654 +  91  92  DH-84  (0.8)  404 +  70  892 + 104  87  DH-84  (0.16)  348 +  72  1,416 +  30  71  NHL-30.5 (4.0)  1,241 + 232  4,669 + 510  9  NHL-30.5  (0.8)  1,033 + 156  3,984 + 771  22  NHL-30.5  (0.16)  1,027 + 137  4,013 + 795  21  a  NHL-30.5 i s a monoclonal antibody directed against a myeloid d i f f e r e n t i a t i o n antigen, one of several monoclonals used as negative controls.  110 while NB-29 did not, even when tested against reactive i n d i v i d u a l s (Table XV).  None of these anti-class II monoclonal antibodies i n h i b i t e d PHA  stimulation of PBMC i n culture (Table XVI). (C) I n h i b i t i o n of Purified B C e l l s To test whether the i n h i b i t o r y e f f e c t s of these monoclonal antibodies were mediated v i a direct effects on B c e l l s or through an action on accessory c e l l s , B c e l l s were extensively enriched by a combination of E-rosette depletion of T c e l l s , monocyte adherence to glass and c e l l s o r t i n g .  These  extensively p u r i f i e d populations of B c e l l s showed an excellent stimulatory response to anti-u, and this was not affected by the addition of any of the monoclonal a n t i - c l a s s II antibodies studied here (Table  XVII).  (D) I n h i b i t i o n of EBV C e l l Lines To assess further whether the i n h i b i t o r y e f f e c t s of DH-84 and DH-224 on PBMC mitogenesis were d i r e c t or i n d i r e c t , the a b i l i t y of these antibodies to i n h i b i t the growth of EBV transformed B c e l l l i n e s was tested.  As shown i n  Table XVIII, both p u r i f i e d antibody and hybridoma supernate s u b s t a n t i a l l y inhibited the p r o l i f e r a t i o n of EBV c e l l l i n e s .  NB-29 also i n h i b i t e d these  l i n e s , but the e f f e c t was less than that obtained with the monomorphic antibodies.  Control antibodies, including both one that did (NB-107) and one  that did not (NHL-30.5) react with the c e l l l i n e s , d i d not i n h i b i t growth (Table  their  XVIII).  3) DISCUSSION To determine whether HLA class I I molecules are involved i n B lymphocyte a c t i v a t i o n and p r o l i f e r a t i o n , we tested several new a n t i - c l a s s I I monoclonal antibodies f o r their a b i l i t y to i n h i b i t various lymphocyte responses. Consistent  i n h i b i t i o n of LPS and anti-^u stimulation of PBMC by two  TABLE XV I n h i b i t i o n of Mixed Lymphocyte Reaction  Mean cpm + SEM Antibody (5 ug/ml)  Control  3  Test  % Inhibition 797  media  459 + 204  17,998 +  NB-29  591 + 118  16,117 + 1,175  DH-84  111 +  20  4,748 +  286  69 + 8  DH-224  139 +  69  6,654 +  644  61 + 3  NHL-30.5  679 + 141  NB-65  343 +  d  93  Cultures without stimulator c e l l s . Cultures with stimulator c e l l s . Mean + SEM of two separate experiments. 'Hybridoma supernate.  19,810 + 1,941 4,028 +  502  13 + 3  0 83 + 6  0  TABLE XVI I n h i b i t i o n of PHA Stimulation of Normal PBMC  Mean cpm + SEM Antibody  Control  X Inhibition'  Test  media  519 +  62  85,767 + 1,665  NB-29  332 + 101  84,530 + 8,938  5 + 7  DH-84  132 +  20  89,735 + 5,416  0.5 + 1  DH-224  113 +  17  83,102 + 1,850  9 + 9  NHL-30.5  327 +  79  85,280 + 6,105  0  NB-65  305 +  82  48,373 + 3,083  43 + 1  b  Mean + SEM of two i n d i v i d u a l experiments. Antibody to t r a n s f e r r i n receptor consistently PHA stimulation.  showed 40-50% i n h i b i t i o n of  113  TABLE XVII I n h i b i t i o n of Anti-p Stimulation of P u r i f i e d B C e l l s  Antibody (10 ug/ml) Media Control  Mean cpm + SEM  3  X Inhibition  969 +  85  13,225 +  156  NHL-30.5  11,379 +  724  15  NB-107  12,661 +  550  5  NB-29  17,191 + 2,532  0  DH-84  11,558 +  538  14  DH-224  12,764 +  275  4  Anti-p  alone  Data i s representative of three separate experiments a l l showing equivalent r e s u l t s .  114  TABLE XVIII I n h i b i t i o n of EBV C e l l Line P r o l i f e r a t i o n :  Antibody (5 yg/ml)  Mean cpm + SEM  ELD (DR6) C e l l s  X Inhibition  Media control  7,492 +  270  NHL-30.5  8,036 +  823  0  NB-107  6,341 + 1,520  15  DH-84  3,710 +  647  51  NB-29  4,406 +  751  41  DH-224  1,578 +  155  79  671 +  62  90  NB-65  b  Data i s representative of four separate experiments with each of two EBV transformed c e l l l i n e s (ELD and WALK). a  ^Hybridoma supernate.  3  115 a n t i - c l a s s I I antibodies, DH-224 and DH-84, was observed.  These are  antibodies directed against monomorphic DR (DH-224) and DQ + DR (DH-84) determinants.  In order to investigate the r e l a t i v e roles of DQ and DR i n  this phenomenon, these antibodies and an antibody to a polymorphic DQ determinant (NB-29) were evaluated further for their a b i l i t y to i n h i b i t mitogen stimulation of PBMC and p u r i f i e d normal B c e l l s , and the p r o l i f e r a t i o n of EBV transformed B c e l l l i n e s . Monoclonal antibodies DH-84 and DH-224 profoundly i n h i b i t e d the anti-u and LPS stimulation of PBMC; NB-29 did not.  This observation suggests that  DR and DQ molecules d i f f e r i n terms of their functional involvement i n B c e l l activation.  However, these studies could not d i s t i n g u i s h which e f f e c t s  result from d i r e c t binding of the antibodies to B c e l l s and which might result from antibody binding to accessory c e l l s .  To answer t h i s question, B  c e l l s were extensively enriched from PBMC using a combination of E-rosette separation of T c e l l s , glass adherence removal of monocytes, and "negative" c e l l sorting.  The resultant highly p u r i f i e d population of B c e l l s , while  showing an excellent response to anti-u, was not i n h i b i t e d by any of the a n t i - c l a s s I I monoclonal antibodies studied here.  These observations suggest  that the a n t i - j j and LPS activation of PBMC depends on a DR s e n s i t i v e step i n which accessory c e l l s are involved.  Since DQ molecules are expressed only on  the subpopulation of monocytes capable of presenting antigen (9,10), lack of i n h i b i t i o n by NB-29 might be due to the i n a b i l i t y of this monoclonal to i n h i b i t the monocyte subpopulation which i s necessary for PBMC responses to anti-u and LPS. Anti-HLA class II polyclonal or monoclonal antisera have been shown to i n h i b i t a variety of c e l l u l a r responses i n human and mouse systems including: p r o l i f e r a t i v e and plaque forming responses to Con A and pokeveed mitogen  116 (11-13), primary and secondary MLR  (14-16), and antigen s p e c i f i c  lymphoproliferative responses (17-19).  A n t i - l a has also been shown to  i n h i b i t LPS stimulation of mouse lymphocytes (20).  An inhibitory e f f e c t of  anti-HLA class II monoclonal antibodies on anti-^u, LPS and PHA  induced  p r o l i f e r a t i o n of human c e l l s has not been previously reported.  Anti-p  antibody appears to exert i t s e f f e c t s on B c e l l s by two dependent mechanisms (6). enlargement, RNA  concentration  At low concentrations anti-p induces  cell  synthesis and c e l l surface expression of B c e l l growth  factor (BCGF) receptors.  P r o l i f e r a t i o n of B c e l l s i n this case then requires  a second s i g n a l , mediated by BCGF secreted by activated helper T c e l l s .  At  high concentrations (as used i n this study), anti-ju i s thought to i n i t i a t e a d i r e c t p r o l i f e r a t i v e effect on B c e l l s independent of T c e l l s , monocytes or their products.  Surface receptors for LPS on B c e l l s appear to be d i s t i n c t  from surface IgM  (21).  I n h i b i t i o n of both LPS and anti-p induced  p r o l i f e r a t i o n of human B c e l l s suggests a fundamental role for HLA  class II  molecules i n the regulation of B c e l l growth at physiological concentrations of B c e l l s as seen i n unfractionated PBMC. are present at low c e l l density and may  As a component of PBMC, B c e l l s  be dependent upon i n t e r a c t i o n with  monocytes or a monocyte derived factor i n order to p r o l i f e r a t e i n response to anti-u.  At higher c e l l densities B c e l l s may  not require this factor or  produce s u f f i c i e n t endogenous factor to be independent of accessory analogous to EBV  transformed  I n h i b i t i o n of MLR  may  cells,  B c e l l s (see below).  by anti-HLA c l a s s II antibodies as shovn i n this study  confirms previous reports (13).  The complete lack, of i n h i b i t i o n of  PHA  stimulation suggests that a c t i v a t i o n by this mitogen, which i s p r i m a r i l y stimulatory to T c e l l s , may  not involve HLA  class II antigens.  117 A l l three anti-HLA class II monoclonal antibodies exerted a d i r e c t a n t i p r o l i f e r a t i v e e f f e c t on the growth of EBV c e l l l i n e s , although i n h i b i t i o n was consistently  greater for DH-84 and DH-224 than NB-29.  Why EBV transformed  c e l l l i n e s should behave d i f f e r e n t l y from p u r i f i e d normal B c e l l s i s of interest.  At low c e l l concentrations EBV c e l l l i n e s have been shown to be  dependent upon exogenous growth factor, while at higher concentrations they appear to secrete a self-stimulating  factor analogous to BCGF (22-25). In  order to avoid overgrowth of these l i n e s by the day three harvest, they were seeded at a concentration of 5 x 10 cells/ml or l e s s . 4  At t h i s r e l a t i v e l y  low concentration, EBV transformed c e l l s appear to be susceptible to i n h i b i t i o n by anti-HLA class II monoclonal antibodies. i n h i b i t o r y e f f e c t can be overcome by the addition remains to be determined.  Whether this  of exogenous growth factor  However since EBV transformed B c e l l s are not  normal, the d i f f e r e n t responses of these c e l l l i n e s compared to normal p u r i f i e d B c e l l s may also be due to constitutive  effects r e s u l t i n g from EBV  transformation. In summary, these findings  document a d i f f e r e n t i a l i n h i b i t o r y e f f e c t of  anti-DR and anti-DQ monoclonal antibodies on the a c t i v a t i o n of PBMC by the B c e l l mitogens anti-p and LPS.  Failure to i n h i b i t the anti-^u induced  stimulation of p u r i f i e d B c e l l s suggests an i n d i r e c t e f f e c t on accessory c e l l s , that at higher B c e l l densities HLA  may be overcome.  Furthermore, a n t i -  class I I monoclonal antibodies (anti-DQ, anti-DR and anti-DQ + DR) were  shown to exert a d i r e c t a n t i p r o l i f e r a t i v e e f f e c t on the growth of EBV transformed c e l l l i n e s .  118 REFERENCES  1.  Shackelford DA, Kaufman JF, Korman AJ, Strominger JL: HLA-HLA Class I I Antigens: structure, separation of subpopulations, gene cloning and function. Immunol Rev 66: 133-187, 1982.  2.  Auffray C, L i l l i e JW, Arnot D, Grossberger D, Kappes D, Strominger JL: Isotypic and a l l o t y p i c v a r i a t i o n of human class II h i s t o c o m p a t i b i l i t y antigen a - c h a i n genes. Nature 308: 327-329, 1984.  3.  Kaufman JF, Auffray C, Korman AJ, Shackelford DA, Strominger J : The class II molecules of the human and murine major h i s t o c o m p a t i b i l i t y complex. C e l l 36: 1-13, 1984.  4.  Hunter J, Noreen H, Mickelson E, Reinsmoen N: IX I n t e r n a t i o n a l workshop: a b r i e f summary of the c e l l u l a r and serologic findings. Am Soc Histocompatibility and Immunogenetics Quarterly. Summer: 5-8, 1984.  5.  Karr RW, Alber C, Goyert S, S i l v e r J, Duquesnoy R: The complexity of HLA-DS molecules. J Exp Med 159: 1512-1531, 1984.  6.  Kehrl JH, Muraguchi A, Butler JL, Falkoff RJM, a c t i v a t i o n 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 . 1984.  7.  Howard M, Nakanishi K, Paul WE: B c e l l growth and d i f f e r e n t i a t i o n factors. Immunol Rev 78: 185-210, 1984.  8.  Brodsky FM: A matrix approach to human class II h i s t o c o m p a t i b i l i t y antigens: reactions of four monoclonal antibodies with the products of nine haplotypes. Immunogenetics 19: 179-194, 1984.  9.  Gonwa T, Picker L, Raff H, Goyert S, S i l v e r J, Stobo J : Antigenpresenting c a p a b i l i t i e s of human monocytes correlates with their expression of HLA-DS, an Ia determinant d i s t i n c t from HLA-DR. J Immunol 130: 706-711, 1983.  Fauci AS: Human B c e l l Immunol Rev 78: 75-96,  10.  Nunez G, G i l e s R, B a l l E, Hurley C, Capra J , Stastny P: Expression of HLA-DR, MB, MT and SB antigens on human mononuclear c e l l s : i d e n t i f i c a t i o n of two phenotypically d i s t i n c t monocyte populations. J Immunol 133: 1300-1306, 1984.  11.  Friedman Chess L: by human antisera 1977.  12.  Broder S, Mann DL, Waldmann TA: P a r t i c i p a t i o n of suppressor T c e l l s i n the immunosuppressive a c t i v i t y of a heteroantiserum to human l a - l i k e antigens. J Exp Med 151: 257-262, 1980.  SM, Breard JM, Humphries RE, Strominger JL, Schlossman SF, I n h i b i t i o n of p r o l i f e r a t i v e and plaque-forming c e l l responses bone-marrow-derived lymphocytes from peripheral blood by to the p 23,30 antigen. Proc Natl Acad Sci USA 74: 711-715,  119 13.  Mizouchi T, Yamashita T, Hamaoka T, Morinaki K: The role of Ia antigens i n the a c t i v a t i o n of T c e l l s by Con A: an evidence for the species r e s t r i c t i o n between T c e l l s and accessory c e l l s . C e l l Immunol 57: 28-41, 1981.  14.  Pawelec GP, Shaw S, Ziegler A, Muller C, Wernet P: D i f f e r e n t i a l i n h i b i t i o n of HLA-D or SB-directed secondary lymphoproliferative responses with monoclonal antibodies detecting human l a - l i k e determinants. J Immunol 129: 1070-1075, 1982.  15.  Eckels DD, Woody JN, Hartzman RJ: Monoclonal and xenoantibodies s p e c i f i c for HLA-HLA Class II i n h i b i t primary responses to HLA-D but f a i l to i n h i b i t secondary p r o l i f e r a t i v e (PLT) responses to allogeneic c e l l s . Hum Immunol 3: 133-142, 1981.  16.  Dubreuil PC, C a i l l o l DH, Lemonnier FA: Analysis of unexpected i n h i b i t i o n s of T lymphocyte p r o l i f e r a t i o n to soluble antigen, alloantigen and mitogen by unfragmented anti I-A or anti-I-E/C monoclonal antibodies. Immunogenetics 9: 11-24, 1982.  17.  T r i e b e l F, Missenard-Leblond V, Couty M, Charron DJ, Debre P: D i f f e r e n t i a l i n h i b i t i o n of human antigen-specific T c e l l clone p r o l i f e r a t i v e responses by d i s t i n c t monoclonal anti-HLA-HLA Class I I antibodies. J Immunol 132: 1773-1778, 1984.  18.  Sterkers G, Henin Y, K a l i l J , Bagot M, Levy J: Influence of HLA class I and class II s p e c i f i c monoclonal antibodies on HLA Class I I r e s t r i c t e d lymphoproliferative responses. J Immunol 131: 2735-2740, 1983.  19.  Marrack P, Kappler JW: A n t i - l a i n h i b i t s the a c t i v i t y of B c e l l s but not a T c e l l - d e r i v e d helper mediator. Immunogenetics 4: 541-555, 1977.  20.  Niederhuber JE, Frelinger JA, Dugan E, Coutinho A, S h r e f f l e r DC: E f f e c t s of a n t i - l a serum on mitogen responses. J Immunol 115: 1672-1677, 1975.  21.  Forni L, Coutinho A: Receptor interactions on the membrane of r e s t i n g and activated B c e l l s . Nature 273: 304-306, 1978.  22.  Maizel AL, Morgan JE, Mehta SR, Kouttab NM, Bator JM, Sahasrabuddhe CG: Long-term growth of human B c e l l s and their use i n a microassay for B c e l l growth factor. Proc Natl Acad Sci USA 80: 5047-5050, 1983.  23.  Blazar B, Sutton L, Strome M: Self-stimulating growth factor production by B - c e l l l i n e s derived from Burkitt's lymphomas and other l i n e s transformed i n v i t r o by Epstein-Barr virus. Cancer Res 43: 4562-4567, 1983.  24.  Gordon J , Ley S, Melamed M, Aman P, Hughes-Jones N: Soluble factor requirements for the autostimulatory growth of B lymphoblasts immortalized by Epstein-Barr v i r u s . J Exp Med 159: 1554-1559, 1984.  25.  Gordon J , Ley S, Melamed M, English L, Hughes-Jones N: Immortalized B lymphocytes produce B - c e l l growth factor. Nature 310: 145-147, 1984.  120 C H A P T E R  V  TWO MONOCLONAL ANTIBODIES THAT DEFINE UNIQUE ANTIGENIC DETERMINANTS ON B-LYMPHOMA CELLS  "The thing that hath been, i t i s that which s h a l l be; and that which i s done i s that which s h a l l be done: and there i s no new thing under the sun." Ecclesiastes 1:9 1) INTRODUCTION Normal B lymphocytes can be induced by s p e c i f i c  antigens or mitogens to  reenter the c e l l cycle, enlarge, divide and further d i f f e r e n t i a t e into immunoglobulin producing plasma c e l l s i n the presence of appropriate regulatory factors (1-3).  Activated B lymphocytes subsequently develop into  immunoglobulin producing plasma c e l l s .  Most non-Hodgkin's lymphomas are  neoplasms that a r i s e i n c e l l s of B-lymphocyte lineage. present  i n a given lymphoma represent  The neoplastic c e l l s  the c l o n a l expansion of a single c e l l  and exhibit considerable morphologic homogeneity (4,5), although the predominant c e l l type varies considerably  from one lymphoma to another.  On  an i n d i v i d u a l c e l l basis, non-Hodgkin's lymphoma c e l l s are indistinguishable from the various stages of activated normal B lymphocytes when examined by l i g h t and electron microscopy.  This i s the basis of the popular concept that  most of the non-Hodgkin's lymphomas are neoplasms of B lineage c e l l s i n which the large c l o n a l population produced consists of c e l l s that are phenotypically "frozen" or "switched-on" at some point along the B lymphocyte transformation  continuum (4-8).  Nevertheless,  there i s l i t t l e known about  121 the c e l l type i n i t i a l l y transformed, the p o s s i b i l i t y that some lymphoma c e l l s can be induced to d i f f e r e n t i a t e  further, and the role of abnormal gene  expression i n these malignancies. Recently a number of monoclonal antibodies have been evaluated as diagnostic reagents f o r the c l a s s i f i c a t i o n of B c e l l lymphomas.  Most of  these f a l l into one of the following categories: B c e l l s p e c i f i c (react only with c e l l s of B lymphocyte lineage), B c e l l associated (react with B c e l l s but also c e l l s of other lineages), blast associated (define antigens present on normal B-blasts but absent from resting B c e l l s ) , principal  reactivity  l i n e s (9-24). cells.  and antibodies whose  i s with Burkitt's lymphoma c e l l s or EBV transformed  cell  To date, none of these have shown s p e c i f i c i t y for B lymphoma  In this chapter, two monoclonal antibodies are described which have  thus f a r shown remarkable s p e c i f i c i t y for human B lymphoma c e l l s .  From these  i n i t i a l findings i t appears that these antibodies may be useful i n the future diagnosis, c l a s s i f i c a t i o n and treatment of the non-Hodgkin's lymphomas.  2) RESULTS (A) Reactivity with C e l l Lines Two monoclonal antibodies, LM-26 and LM-155, reactive with the immunizing  DHL-10 lymphoma c e l l s but not with CLL c e l l s , were i n i t i a l l y  tested f o r their r e a c t i v i t i e s with various c e l l lines by FACS analysis.  LM-  26 reacted with 4/7 B lymphoma c e l l l i n e s ; LM-155 reacted with 7/7 (Table XIX).  These l i n e s were o r i g i n a l l y derived from patients with a  diagnosis of d i f f u s e " h i s t i o c y t i c " lymphoma (DHL-1, DHL-4, DHL-8, DHL-10), d i f f u s e mixed h i s t i o c y t i c and lymphocytic lymphoma (DHL-6), lymphosarcoma (U698-M) and an unspecified B lymphoma (BALM-5) (25-29).  In contrast, 9/10  EBV transformed "normal" B c e l l l i n e s were unreactive with these antibodies.  TABLE XIX C e l l Line Reactivity  Cell line  of Antilymphoma Antibodies:  FACS A n a l y s i s  Type  LM-26  B-lymphoma B-lymphoma B-lymphoma B-lymphoma B-lymphoma B-lymphoma B-lymphoma  -  +  + + + + + + +  JREE CMG RMG WALK StfEI ELD BN MAD KOZ WAY  EBV EBV EBV EBV EBV EBV EBV EBV EBV EBV  _  _  -  -  --  -  U937 1937  Histiocytic Histiocytic  _  _  -  -  _  —  -  -  DHL-1 DHL-4 DHL-6 DHL-8 DHL-10 U698-M BALM-5  Jurkat  T leukemia  HL-60 K562  Myeloid Erythroid  + +  +  -  --  W+  -  LM-155  w -+ D  -  -  C e l l l i n e s were considered positive i f >10% of c e l l s reacted with the test antibody compared to negative control antibody of irrelevant s p e c i f i c i t y . a  W=weak (10-15% of c e l l s showed low intensity fluorescence). D  3  123 Neoplastic c e l l l i n e s of monocyte/macrophage (U937, 1937), T leukemia (Jurkat), myeloid (HL-60) and erythroid (K.562) o r i g i n also showed no r e a c t i v i t y with either antibody. Cytogenetic analysis of the Giemsa banded metaphases from non-reactive EBV c e l l l i n e WAY-1  revealed a normal 46 X,Y karyotype.  DHL-10 c e l l s ,  were p o s i t i v e with both antibodies, had a highly abnormal karyotype: +7,-8, lOq", l l q , +  14q , +  which 47,  XY,  +M.  (B) R e a c t i v i t y with Fresh Tissues Eighteen of 23 non-Hodgkin's B c e l l lymphomas were p o s i t i v e by FACS analysis f o r the antigen defined by LM-26 (Table XX).  This included both  small and large c e l l lymphomas, both with and without nuclear cleavages. Figures 13 and 14 show histogram and contour plot p r o f i l e s respectively of c e l l s from a small cleaved c e l l lymphoma.  Noteworthy i s the same broad  pattern of staining (small, intermediate and large c e l l s ) with  anti-lambda  antibodies, which establishes the presence of the neoplastic clone, and 26.  LM-  LM-155 reacted with only 5 of the 23 B c e l l lymphomas tested thus f a r .  Examples of B lymphoma c e l l s with either kappa or lambda surface immunoglobulin l i g h t chains and with d i f f e r i n g heavy chain isotypes were found to be reactive with LM-26 and LM-155.  One patient with CALLA p o s i t i v e  ALL (a known early B c e l l malignancy) reacted with LM-155. Five B c e l l lymphomas, three T c e l l lymphoma/leukemias and one lymphoma of uncertain immunologic subtype were negative with both LM-26 and LM-155 (Table XXI).  Tissues containing reactive lymphoid  i n f i l t r a t e s (lymph  nodes 8, spleen 2, lung 1) also did not react with these antibodies (Table XXII).  C e l l s i n peripheral blood and bone marrow samples from normal  controls, as well as patients with reactive lymphocytosis, chronic lymphocytic leukemia (CLL) and a variety of other lymphoid and nonlymphoid  TABLE  Analysis of Fresh Tlsauesi  Patient  Age/Sex  Tissue 8  Pathologic13 Diagnosis  19P  LN PB  SCCL-D LSCL  XX  B Call Malignancies - Positive % Positive PACS LM-155  LM-26 79 24  5 37  K  X  79 81  1 1  78 78  Leu 12  Leu 5  Leu-M3 2 2  2  84P  LN  SCCL  39  14  74  3  66  26 7 32  3  60P  LN  SCCL-P  25  1  59  <1  60  42  2  4  40P  LN  SCCL-D  21  <1  54  6  51  22  <1  1  9  5  78P  LN  SCCL-D  13  12  58  6  66  43  22  6  72M  LN  SCCL-P  49  2  4  74  87  14  <1  7  66M  Spleen  SCCL  37  2  54  4  68  25  4  8  56M  BM  SCCL  23  <1  71  6  58  8  6  9  65M  PB  LSCL  23  1  54  6  76  7  2  10  40P  I AT  MCL-D  62  1  72  <1  72  24  2  11  18M  PB  SNC  31  <1  78  10  50  14  15  12  66M  LN  LCC-N  26  <1  2  28  36  38  <1  13  60H  LN  LCC  30  <1  50  <1  32  13  1  14  85P  LN  LCC-N  23  3  <1  34  37  41  1  15  69M  RT  HL  49  29  81  6  ND  6  ND  16  1»H  BM  ALL  3  13  ND  ND  89  5  <1  17  49P  LN  NCL-D  20  1  4  55  56  21  1  18  26M  Spleen  SCCL  37  2  54  4  68  25  4  19  52F  PP  71  32  52°  68°  71  24  2  LCL/B-IBS  a LN, lymph node; PB, peripheral blood; IAT, Intra-abdominal tumor; RT, retroperitoneal tumor; BM, bone marrow; PF, pleural fluid. b SCCL, small cleaved cell lymphoma; P, follicular; D, diffuse; LSCL, lymphosarcoma c e l l leukemia; MCL mixed small and large c e l l lymphoma; SNC, small non-cleaved lymphoma; LCC, large cleaved c e l l lymphoma; HL, histiocytic lymphoma; ALL acute lymphoblastic leukemia; LCL, large c e l l lymphoma; IBS, immunoblastic sarcoma; NCL, non-cleaved c e l l lymphoma.  ° C l o n a l i t y not determined.  125  FIGURE 13  FACS histogram of small cleaved cell lymphoma stained with A) negative control antibody, B) anti-kappa, C) anti-lambda, D) LM-26. A monoclonal lambda pattern of surface immunoglobulin is identified. Staining intensity of LM-26 exceeds that of anti-lambda for some cells.  126  FIGURE 14  FACS contour plot of small cleaved cell lymphoma stained with A) negative control antibody, B) anti-kappa, C) anti-lambda, D) LM-26. Cell number i s reflected in the 'Z' axis. Both anti-lambda and LM-26 stain lymphoma cells of a l l sizes. This indicates LM-26 binding is not restricted to a particular subtype of c e l l based on size (e.g. large transformed lymphoid cells) within a given lymphoma.  TABLE XXI Analysis of Fresh Tissues:  B and T Cell Malignancies - Negative % Positive FACS  Age/Sex  TiBsue8  Patnologicb Diagnosis  1  6 OH  LM  SCC-F  5  2  44H  LN  SCC-F  3  76M  LN  4  74M  5  K  X  Leu 12  Leu 5  <1  41  3  45  52  4  <1  1  3  52  79  28  <1  SCC-D  4  4  4  2  60  15  3  LN  SLL  3  7  61  5  72  34  6  63M  LN  IBS  3  <1  52  <1  49  10  3  6  53M  LN  SNC-D  <1  <1  <1  48  57  7  <1  7  84F  LN  MCL-F  <1  <1  1  1  42  44  <1  8  60M  LN  T-LL  <1  <1  2  <1  2  91  6  9  79M  LN  PTCL  2  2  19  14  28  47  4  10  48M  PB  TCL  <1  <1  IB  10  1  62  17  Patient  LM-26  LM-155  See Table XX; T-LL, T-lymphoblastic lymphoma; PTCL, peripheral T c e l l lymphoma; TCL, T c e l l leukemia.  Leu-M3  TABLE XXII Analysis of Freeh Tissues:  Reactive Lymphoid Proliferations - Negative  Age/Sex  Tissue  Pathologic Diagnosis  1  12F  LN  Lipogranulomata  2  52M  LN  FH with KS  3  76M  LN  RLH  4  77M  LN  MCa  5  10F  LN  FH with SH  6  67F  7  29F  LN  8  56F  9 10  Patient  a  % Positive FACS X  Leu 12  8  4  25  66  1  <1  13  5  24  26  4  7  5  30  24  44  35  5  <1  <1  26  27  <1  <1  <1  4  1  14  8  24  62  <1  7  8  50'  34  52  47  6  GL  <1  <1  15  8  25  39  2  LN Spleen  SH CC  4 2  2 1  14 23  6 11  25 28  59 54  2 7  50M  Spleen  MCHD  75M  Lung  LN, submax.  BLPL  LP I  LM-26  LM-155  2  <1  <1  K  Leu 5  Leu-M3  <1  <1  16  12  8  16  12  4  <1  37  28  41  48  <1  See Table XX.  FH, follicular hyperplasia; KS, Kaposi's sarcoma; RLH, reactive lymphoid hyperplasia; MCa, metastatic carcinoma; SH, sinus histiocytosis; BLPL, benign lymphoproliferative lesion of salivary gland; GL, granulomatous lymphadenitis; CC, chronic congestion; MCHD, mixed cellularlty Hodgkin's disease; LPI, lymphoplasmacytoid infiltrate. b  129 hematologic malignancies vere a l l unreactive with LM-26 and LM-155 (Table XXIII). (C) Reactivity with Normal B-blasts In order to determine whether these antibodies reacted with lymphocyte activation-associated c e l l surface antigens, enriched populations of B c e l l s were analyzed a f t e r their s e l e c t i v e a c t i v a t i o n i n v i t r o .  For this study,  normal spleen c e l l s were stimulated i n culture with lipopolysaccharide (LPS), and 4 days l a t e r the c e l l s harvested and T c e l l s removed by rosette sedimentation with AET treated SRBC.  Remaining c e l l s consisted of 90%  surface immunoglobulin positive B c e l l blasts as determined of  by FACS analysis  fluorescence and l i g h t scatter and morphologic observation of stained  cytospins.  However no evidence of any r e a c t i v i t y of LM-26 and LM-155 with  these normal B c e l l blasts was obtained (Figure 15).  3) DISCUSSION Attempts to raise monoclonal antibodies to lymphoma c e l l s have i n most instances resulted i n antibodies which detect normal B c e l l s p e c i f i c or B c e l l associated antigens (15-24).  A few antibodies detecting antigens on  normal B c e l l blasts or showing s p e c i f i c i t y for Burkitt's lymphoma c e l l s have also been described (9,10,16,17).  We have recently isolated two monoclonal  antibodies, LM-26 and LM-155, that appear to show a high degree of s p e c i f i c i t y for non-Hodgkin's B lymphoma c e l l s .  To our knowledge this  represents the f i r s t report of antibodies with this type of r e a c t i v i t y . LM-26 reacted with some but not a l l B lymphoma c e l l l i n e s , only weakly with one of ten EBV transformed B c e l l lines and was negative with a l l other c e l l l i n e s tested.  These included various c e l l lines with features of  h i s t i o c y t i c , T leukemia, myeloid and erythroid o r i g i n .  By FACS analysis,  TABLE XXIII Analysis of Fresh Tissues:  Miscellaneous - Negative'  , Diagnosis  Tissue  0  FACS analysis # cases negative  Normal  PB  30  Normal  BM  3  PB BM PF  4 1 1  PB  10  PB  1  ALL  PB BM  1 1  MF/Sezary's  PB  2  CML  PB BM  10 6  CML-BC  PB  3  AML  BM  7  AMML  PB BM  HCL  PB BM  A p l a s t i c anemia  PB  Myelodysplasia  BM  Reactive lymphocytosis  CLL •prolymphocyte leukemia  L e s s than 3% of c e l l s reacted with test antibodies (LM-26, LM-155) compared to isotype i d e n t i c a l negative control antibody of irrelevant s p e c i f i c i t y . a  ^CLL, chronic lymphocytic leukemia; ALL, acute lymphoblastic leukemia; MF, mycosis fungoides; CML, chronic myelogenous leukemia, BC, blast c r i s i s ; AML, acute myelogenous leukemia; AMML, acute myelomonocytic leukemia; HCL, hairy c e l l leukemia. °PB, peripheral blood; BM, bone marrow; PF, p l e u r a l  fluid.  131  FIGURE 15  FACS histogram of p u r i f i e d LPS s t i m u l a t e d normal B c e l l b l a s t s s t a i n e d w i t h A) n e g a t i v e c o n t r o l a n t i b o d y , B) a n t i - p o l y v a l e n t s u r f a c e immunoglobulin, C) LM-26, D) 0KT11. N i n e t y per cent of c e l l s a r e s u r f a c e immunoglobulin p o s i t i v e B c e l l s ( B ) , which by l i g h t s c a t t e r and morphologic examination o f s t a i n e d c y t o s p i n s , a r e predominantly b l a s t s . These c e l l s do not bind LM-26 ( C ) . There i s o n l y f i v e per cent r e s i d u a l c o n t a m i n a t i o n with T c e l l s ( D ) .  132 LM-26 reacted v i t h 80% of B c e l l lymphomas freshly explanted from patients. No r e a c t i v i t y vas observed v i t h T c e l l lymphomas or leukcmias,  CLL,  hyperplastic lymph nodes, reactive peripheral blood lymphocytes, normal peripheral blood and marrov c e l l s , or blood and marrov c e l l s from a variety of non-lymphoid hematologic  malignancies.  LM-155, i n contrast, reacted v i t h a l l B lymphoma c e l l l i n e s tested, but only veakly v i t h one EBV  transformed B c e l l l i n e .  negative v i t h this antibody.  A l l other c e l l l i n e s vere  LM-155 reacted v i t h 20% of freshly explanted B  lymphomas and one CALLA positive ALL (expressing B c e l l surface markers). A l l other normal and neoplastic tissues examined vere negative for the antigenic determinant  defined by this antibody.  These findings suggest  that these monoclonal antibodies are detecting  lymphoma s p e c i f i c or lymphoma associated antigens. antibodies vhich vere i n i t i a l l y thought a f t e r extensive examination  In the past, monoclonal  to be tumor s p e c i f i c have turned out  to be normal d i f f e r e n t i a t i o n antigens  expressed  at lov l e v e l s or on highly r e s t r i c t e d subpopulations of c e l l s i n normal lymphoid malignant  or myeloid tissue samples (9-24).  Why  large populations of  c e l l s accumulate that express surface antigens found only  transiently  on normal c e l l s i s not v e i l understood.  Deregulation or  svitching-on of genes normally expressed only during embryogenesis has been documented for some tumor types (e.g. those expressing CEA or AFP).  Evidence  of other mechanisms, including the expression of activated, altered c-onc genes has a l s o been reported (30).  Since the non-Hodgkin's lymphomas are  regarded conceptually as neoplasms of mature but activated lymphocytes, ve tested the p o s s i b i l i t y that the antigens detected by LM-26 and LM-155 might also be expressed on their normal lymphoid  counterparts.  Hovever, FACS  analysis of highly p u r i f i e d populations of 4 day old LPS stimulated human  133 splenic B lymphocytes f a i l e d to reveal the presence of detectable numbers of LM-26 or LM-155 positive c e l l s .  Lack, of expression of antigens reactive with  LM-26 and LM-155 on normal c e l l s was  further supported by the negative  results obtained with a variety of reactive lymphoid hyperplasias from lymph node, p e r i p h e r a l blood, spleen and lung.  These reactive c e l l populations  contained s i g n i f i c a n t proportions of i n vivo activated lymphoblasts. However, ve cannot yet rule out the p o s s i b i l i t y these antibodies may  that the antigens detected by  s t i l l be present at low l e v e l s on minor  of normal reactive B c e l l s .  subpopulations  Since a small proportion (up to 7-8%)  of  reactive lymph node c e l l s from some patients were positive by FACS a n a l y s i s , i t i s conceivable that these p o s i t i v e c e l l s may subpopulation expressing these antigens.  represent a unique B c e l l  A l t e r n a t i v e l y , the detection of  small numbers of positive c e l l s i n reactive lymphoid p r o l i f e r a t i o n s may  be a  sign of i n c i p i e n t neoplasia. Most lymphomas are clonal neoplasms and the c e l l s present i n many tumors show considerable morphologic homogeneity. d i s s i m i l a r c e l l types may  However, morphologically  be admixed and are generally assumed to be part of  the same neoplastic clone (4,5).  It was of interest therefore to determine  i f LM-26 and LM-155 detected determinants  expressed on a p a r t i c u l a r  morphologic subpopulation of c e l l s i n a given specimen.  As shown i n Figure  14, c e l l s from a small cleaved c e l l lymphoma that stained i n a monoclonal pattern with anti-lambda  l i g h t chain antisera varied i n size over a  considerable range including intermediate and large c e l l s as well as small cells. size.  S i m i l a r l y LM-26 also stained c e l l s of small, intermediate and larger Therefore i t seems u n l i k e l y that staining patterns with this antibody  are related to parameters that control c e l l s i z e .  Variations i n antigen  expression i n d i f f e r e n t phases of the c e l l cycle i s also u n l i k e l y , since i n  134 most small c e l l lymphomas fever than 10% of c e l l s are i n cycle (31).  Finally  i t i s of interest that the antigens detected by both LM-26 and LM-155 could be found on c e l l s from B lymphomas of d i f f e r e n t morphologic subtypes, including examples composed predominantly of small or large and cleaved or noncleaved c e l l s . or  At present l i t t l e information about the molecular nature  function of the antigens defined by these antibodies i s a v a i l a b l e .  Attempts  to immunoprecipitate the antigens detected by LM-26 and LM-155 using 125  standard successful.  I c e l l surface l a b e l i n g techniques (32) have to date not been Hovever, i t seems u n l i k e l y that LM-26 and LM-155 detect a  determinant associated v i t h immunoglobulin  since both of these antibodies  reacted v i t h neoplastic c e l l s possessing d i f f e r e n t heavy and l i g h t chain isotypes and neither reacted v i t h normal resting or transformed immunoglobulin  positive B c e l l s .  The s i g n i f i c a n c e of veak r e a c t i v i t y of each of these antibodies v i t h 1 of  10 EBV transformed B c e l l l i n e s i s uncertain. These c e l l l i n e s (except  for WAY-1) have been passed i n culture for many years.  Given the knovn  genetic i n s t a b i l i t y of EBV transformed l i n e s and the tendency f o r these l i n e s to develop karyotypic abnormalities v i t h time (33), i t vould not be s u r p r i s i n g i f this r e a c t i v i t y proved to be associated v i t h a genetic change that occurred a f t e r immortalization i n v i t r o .  The EBV l i n e WAY-1 vhich vas  i s o l a t e d from a patient less than s i x months prior to testing and shovn to be k a r y o t y p i c a l l y normal vas negative for LM-26 and LM-155 binding.  On the  other hand, the B-lymphoma c e l l l i n e DHL-10 vhich reacted v i t h both LM-26 and LM-155 shoved multiple karyotypic abnormalities as has been reported f o r many other B lymphoma l i n e s (34-36).  Burkitt's lymphoma c e l l s appear to express  d i f f e r e n t c e l l surface antigens depending on vhether they possess the usual t(8;14) or variant t(8;2 or 8;22) translocations (14).  Other c h a r a c t e r i s t i c  135 chromosomal abnormalities have been described i n lymphoma c e l l s f r e s h l y removed from p a t i e n t s (37). This r a i s e s the question as to whether there might be a p a r t i c u l a r genetic abnormality present i n some lymphoma c e l l s that confers LM-26 or LM-155 r e a c t i v i t y . These f i n d i n g s suggest that many B c e l l lymphomas d i s p l a y on t h e i r surface common epitopes not found on the majority of r e s t i n g or a c t i v a t e d normal B c e l l s or c e l l s of other hemopoietic  lineages.  Whether these  antigens are stage s p e c i f i c d i f f e r e n t i a t i o n antigens, v i r a l or oncogene products, or r e s u l t from de novo a l t e r a t i o n s of surface molecules due to other processes associated with n e o p l a s t i c transformation remains to be determined.  Regardless of the explanation, i t i s noteworthy that  heterogeneity of expression of such antigens i n a given population of lymphoma c e l l s was observed.  I t may be a n t i c i p a t e d that antibodies such as  LM-26 and LM-155 w i l l be u s e f u l i n analyzing f u r t h e r the biology of the nonHodgkin's lymphomas.  In a d d i t i o n , they are p o t e n t i a l l y valuable reagents f o r  d i a g n o s i s , d e t e c t i o n of r e s i d u a l disease, and p o s s i b l y treatment therapeutic s t r a t e g i e s .  i n future  136 REFERENCES  1.  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Knowles D, T o l i d j i a n B, Marboe C, M i t t l e r R, T a l l e M, Goldstein G: D i s t r i b u t i o n of antigens defined by 0KB monoclonal antibodies on benign and malignant lymphoid c e l l s and on nonlymphoid tissues. Blood 63: 886-896, 1984.  23.  Anderson K, Bates M, Slaughenhoupt B, Pinkus G, Schlossman S, Nadler L: Expression of human B cell-associated antigens on leukemias and lymphomas: a model of human B c e l l d i f f e r e n t i a t i o n . Blood 63: 1424-1433, 1984.  24.  Epstein A, Morder R, Winter J , Fox R: Two new monoclonal antibodies (LN1, LN-2) reactive i n B5 formalin-fixed, paraffin-embedded tissues with f o l l i c u l a r center and mantle zone human B lymphocytes and derived tumors. J Immunol 133: 1028-1036, 1984.  138 25.  Epstein AL, Kaplan HS: Feeder layer and n u t r i t i o n a l requirements f o r the establishment and cloning of human malignant lymphoma c e l l l i n e s . Cancer Res 39: 1748-1759, 1979.  26.  Srivastava B, Rossowski W, Minowada J : Cytochemical comparison of immunologically characterized human leukaemia/lymphoma c e l l l i n e s representing different levels of maturation. Br J Cancer 47: 771-779, 1983.  27.  G i t t e r B, Finn 0, Mitzgar R: Cyto-fluorometric i s o l a t i o n of 1937, an Ia antigen-bearing variant of the la-negative human monocytic c e l l l i n e U937. J Immunol 134: 280-283, 1985.  28.  N i l s s o n K, Sundstrom C: Establishment and c h a r a c t e r i s t i c s of two unique c e l l l i n e s from patients with lymphosarcoma. Int J Cancer 13: 808-823, 1974.  29.  Winter J , Variakojis D, Epstein A: Phenotypic analysis of established d i f f u s e h i s t i o c y t i c lymphoma c e l l l i n e s u t i l i z i n g monoclonal antibodies and cytochemical techniques. Blood 63: 140-146, 1984.  30.  Drebin J , Stern D, Link V, Weinberg R, Greene M: Monoclonal antibodies i d e n t i f y a cell-surface antigen associated with an activated c e l l u l a r oncogene. Nature 312: 545-547, 1984.  31.  Shackney SE, Skramstad K: A dynamic interpretation of multiparameter studies i n the lymphomas. Am J C l i n Path 72: 756-764, 1979.  32.  Markwell M, Fox CF: Surface s p e c i f i c iodination of membrane proteins of viruses and eukaryotic c e l l s using 1,3,4,6 tetrachloro-3 , 6 d i p h e n y l g l y c o u r i l . Biochemistry 17: 4807-4811, 1978.  33.  Steel C, Woodward M, Davidson C, Philipson J , Arthur R: Non-random chromosome gains i n human lymphoblastoid c e l l l i n e s . Nature 270: 349-351, 1977.  34.  Dillman R, Handley H, Royston I: Establishment and characterization of an Epstein-Barr virus-negative lymphoma B c e l l l i n e from a patient with a d i f f u s e large c e l l lymphoma. Cancer Res 42: 1368-1373, 1982.  35.  Watanabe S, Kuroki M, Sato Y, Shimosato Y, Hasegawa T: The establishment of a c e l l l i n e (NH-AR) from a human nodular lymphoma and a comparison with lymphoblastoid c e l l l i n e . Cancer 46: 2438-2445, 1980.  36.  Miyoshi I, Kubonishi I, Yoshimoto S, H i k i t a T, Dabasaki H, Tanaka T, Kimura I, Tabuchi K, Nishimoto A: C h a r a c t e r i s t i c s of a brain lymphoma c e l l l i n e derived from primary i n t r a c r a n i a l lymphoma. Cancer 49: 456-459, 1982.  37.  Yunis J , Oken M, Kaplan M, Ensrud K, Howe R, Theologides A: D i s t i n c t i v e chromosomal abnormalities i n h i s t o l o g i c subtypes of non-Hodgkin's lymphoma. N Engl J Med 307: 1231-1235, 1982.  139  C H A P T E R  SUMMARY AND  VI  CONCLUSIONS  "The youth, a t t r a c t e d by nature and a r t , t r u s t s i n h i s v i v i d d e s i r e , soon to e n t e r i n t o the innermost sanctuary. The man r e a l i z e s , a f t e r a l o n g p e r e g r i n a t i o n that he went no f u r t h e r than i n t o the p r o p y l a e a . " Goethe  The  molecular  events  lymphocytes are unknown. including:  u n d e r l y i n g the n e o p l a s t i c t r a n s f o r m a t i o n of normal A v a r i e t y of mechanisms have been p o s t u l a t e d  g e n e t i c mutation,  chromosomal rearrangement, v i r a l  i n a p p r o p r i a t e oncogene e x p r e s s i o n . growth and  The  cell  i n t e r a c t i o n of r e c e p t o r s with growth f a c t o r s p r o v i d e differentiate.  r e g u l a t o r y c o n t r o l s may  result  to  The  of  c o n t r o l those events  Why  w h i l e o t h e r s progress to  that r e s u l t  heterogeneous group of d i s e a s e s .  B-lymphocytes.  Clearly, may  be b e t t e r a b l e  i n neoplasia. morphologically  and  Most of these a r e neoplasms  only s l o w l y i s unknown, but may t i s s u e tropism and  to  through improved  some B c e l l lymphomas have a r a p i d c l i n i c a l  the p r o l i f e r a t i v e p o t e n t i a l ,  for B-cells  i n autonomous p r o l i f e r a t i o n which l e a d s  non-Hodgkin's lymphomas are a c l i n i c a l l y ,  immunologically  the  the s t i m u l u s  of normal c e l l u l a r r e g u l a t o r y processes we  a p p r e c i a t e and  and  cells.  P e r t u r b a t i o n s i n normal growth  c l o n a l s e l e c t i o n , c l o n a l dominance and malignancy. understanding  surface  or s u r f a c e components of o t h e r  C r o s s - l i n k i n g of s u r f a c e immunoglobulin or o t h e r molecules,  to e n l a r g e , d i v i d e and  and  r e g u l a t i o n of normal B-lymphocyte  d i f f e r e n t i a t i o n i n v o l v e s the i n t e r a c t i o n of key  components w i t h e x t r a c e l l u l a r molecules  infection  course  r e l a t e at l e a s t  i n part  c e l l u l a r morphology of  the  140  dominant c l o n e w i t h i n a g i v e n tumor.  Attempts to p r e d i c t  the  clinical  b e h a v i o r of the lymphomas has l a r g e l y i n v o l v e d s u b t y p i n g based on criteria.  Very  from another  little  i s known about how  one morphologic  r e s e a r c h was and  subtype d i f f e r s  i n terms of c e l l 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  to growth f a c t o r s and o t h e r r e g u l a t o r y c o n t r o l s .  The  morphologic  responsiveness  long-term  goal of  to c h a r a c t e r i z e the c e l l s u r f a c e a n t i g e n s of B lymphoma  to s e a r c h f o r c o r r e l a t i o n s between t h e i r e x p r e s s i o n and o t h e r  this  cells  functional  d i f f e r e n c e s d i s t i n g u i s h i n g normal and n e o p l a s t i c lymphocytes. To a c h i e v e One  was  t h i s g o a l a number of t e c h n i c a l o b s t a c l e s had  to be overcome.  o b t a i n i n g s u f f i c i e n t numbers of lymphoma c e l l s f o r immunization  mice p r i o r to the p r o d u c t i o n of monoclonal a n t i b o d i e s .  A second was  of  the  development of a s c r e e n i n g method f o r monoclonal a n t i b o d i e s t h a t would a l l o w the s e l e c t i o n of a n t i b o d i e s r e a c t i v e w i t h malignant lymphocytes but not r e s t i n g or normal c e l l s . by  the use of l a r g e c e l l  lymphoma c e l l  their a b i l i t y  how  transformed  These problems were surmounted  l i n e s and  c e l l s as p r o t o t y p e s , r e s p e c t i v e l y , of transformed A t h i r d problem was  or  c h r o n i c lymphocytic and  resting  to d e t e c t f u n c t i o n a l l y r e l e v a n t molecules.  to s c r e e n monoclonal a n t i b o d i e s , r a i s e d a g a i n s t c e l l  for  their a b i l i t y  l i n e s and  The  B-lymphocytes.  to d e v i s e methods of t e s t i n g these a n t i b o d i e s f o r  was  lymphocytes.  to i n h i b i t  The  approach  taken  surface antigens,  i n v i t r o mitogen s t i m u l a t i o n of normal B and  e x p r e s s i o n of a n t i g e n s d e t e c t e d by these a n t i b o d i e s on  f r e s h l y explanted n e o p l a s t i c and n o n - n e o p l a s t i c  examined u s i n g the f l u o r e s c e n c e a c t i v a t e d c e l l s o r t e r  t i s s u e s was  normal and n e o p l a s t i c B c e l l these d e s c r i b e c e l l  f u n c t i o n and d i f f e r e n t i a t i o n .  s u r f a c e determinants  T cell  then  (FACS).  Based upon these approaches, t h i s t h e s i s focuses on three areas  an important  leukemia  (LFA-1, HLA-class  The  first  of two  I I ) which may  r e g u l a t o r y f u n c t i o n i n normal B c e l l a c t i v a t i o n  and  of play  141 proliferation.  The third area, and perhaps the most c l i n i c a l l y promising,  centers on the characterization of c e l l surface antigens which appear uniquely expressed on B lymphoma c e l l s .  This work, i s summarized as follows:  Lymphocyte Function Associated Antigen (LFA-1) i s Involved i n B C e l l Activation Human LFA-1  i s a widely expressed leukocyte antigen present on c e l l s of  myeloid and lymphoid lineage. Monoclonal antibodies to LFA-1 have been shown to i n h i b i t i n v i t r o T c e l l immune functions. c e l l a c t i v a t i o n has not been documented.  However, a role for LFA-1  in B  To investigate this p o s s i b i l i t y ,  examined the d i s t r i b u t i o n of LFA-1 on normal, neoplastic and EBV  we  transformed  B c e l l s as well as the a b i l i t y of a monoclonal anti-LFA-1 antibody (NB-107) to i n h i b i t B c e l l mitogenesis.  NB-107 immunoprecipitates a noncovalently  linked heterodimer of approximately 170 and 95 Kd.  Sequential  immunoprecipitation and cross-blocking studies showed that NB-107 i d e n t i f i e d a d i s t i n c t epitope on the LFA-1 molecule.  NB-107 defined LFA-1 was present  on PBMC from a l l normal individuals (N=27) and EBV transformed c e l l l i n e s (N=9), but was absent from 4 of 7 neoplastic B lymphoma l i n e s .  NB-107 was  observed to profoundly i n h i b i t the response of peripheral blood mononuclear c e l l s (PBMC) to the B c e l l mitogens anti-IgM ( u ) (mean 71% i n h i b i t i o n ) and lipopolysaccharide (LPS) (mean 80% i n h i b i t i o n ) .  In order to investigate the  mechanism of i n h i b i t i o n , B c e l l s were sequentially p u r i f i e d from PBMC using a combination of E-rosette depletion of T c e l l s , monocyte removal by glass adherence and f i n a l l y c e l l sorting.  These extensively enriched populations  of B c e l l s , while s t i l l responding to anti-p, showed no evidence of i n h i b i t i o n by NB-107.  Growth of EBV transformed c e l l l i n e s , cultured i n the  presence of NB-107, also were not inhibited by this antibody.  When tested i n  assays for T c e l l function, NB-107 was shown to i n h i b i t the mixed lymphocyte  142 response (MLR), but had no effect on PHA stimulation of PBMC, nor on the clonal growth and differentiation of granulopoietic, erythropoietic and pluripotent progenitor cells.  We conclude that anti-LFA-1 monoclonal  antibody inhibits B c e l l mitogens via indirect effects on monocytes and/or T cells, rather than by a direct antiproliferative effect on B cells. Monoclonal Antibodies to HLA-Class II Determinants:  Functional Effects  on the Activation and Proliferation of EBV Transformed B Cells Three new anti-HLA class II monoclonal antibodies were generated with differing specificities for DQ and DR determinants.  Each of these antibodies  (NB-29, DH-84 and DH-224) immunoprecipitates a heterodimer of approximately 125 35,000 and 28,000 MW from  I surface labeled B lymphoma cells as shown by  SDS-PAGE. NB-29 (IgGl) detects a polymorphic DQ determinant, while DH-224 (IgGl) is reactive with monomorphic DR determinants, and DH-84 (IgG2a) has specificity for both DQ and DR. To investigate the function of HLA Class II molecules in B c e l l activation these were tested for their ability to inhibit various B and T lymphocyte responses.  Both DH-224 and DH-84, but not NB-29,  were found to inhibit significantly the stimulation of peripheral blood mononuclear cells (PBMC) by  anti-p  (70-90% inhibition) and by  lipopolysaccharide (80-90% inhibition), as measured by incorporation of tritiated thymidine.  When added to highly purified populations of peripheral  blood B cells, none of these anti-class II monoclonal antibodies inhibited  anti-p  induced stimulation.  This suggests that the inhibitory effect that  DH-224 and DH-84 have on the stimulation of unfractionated PBMC may be due to their ability to interfere with the action of accessory cells. EBV transformed B c e l l lines, in contrast, showed substantial inhibition of growth when cultured in the presence of any of the three antibodies.  With  respect to T cells, DH-84 and DH-224 strongly inhibited the mixed lymphocyte  143 response (MLR); NB-29 did not. stimulation of PBMC by PHA.  None of these antibodies inhibited  These findings suggest that DQ and DR HLA class  II molecules have differing roles in B cell activation and document a direct antiproliferative effect of anti-HLA class II monoclonal antibodies on the growth of EBV transformed cell lines. Monoclonal Antibodies Define Unique Antigenic Determinants Expressed on B-Lymphoma Cells The non-Hodgkin's lymphomas are a clinically, morphologically and immunologically heterogeneous group of diseases. Why lymphoma cells are unresponsive to normal regulatory growth controls and how they differ from normal lymphocytes is not well understood.  In order to begin to address  these questions we have developed monoclonal antibodies with specificity for neoplastic B cells.  Two were found, LM-26 and LM-155, that showed a high  degree of specificity for B c e l l lymphomas. When tested by FACS analysis, LM-26 reacted with 80 per cent (18/23) of B cell lymphomas freshly explanted from patients and LM-155 reacted with 20 per cent (5/23).  The antigenic  determinant detected by LM-26 was also found to be present on 4 of 7 neoplastic large cell B-lymphoma lines. present on a l l 7 of these lines.  LM-155 detected a determinant  For neither monoclonal was there any  association between antibody reactivity and the morphologic subtype of lymphoma examined or the type of cell surface immunoglobulin expressed. 155 reacted with one case of B cell-ALL.  LM-  Neither antibody reacted with  normal B c e l l blasts, normal peripheral blood mononuclear or marrow cells, T c e l l leukemias or lymphomas, CLL cells, or lymphocytes from reactive lymph nodes, spleen, peripheral blood and lung.  Both monoclonals were also  unreactive with non-B lymphoid neoplastic cell lines, 9 of 10 EBV transformed B c e l l lines and cells freshly explanted from patients with malignancies of  144 diverse cellular origins.  FACS analysis of the expression of the antigens  defined by LM-26 and LM-155 on lymphoma cells and normal B c e l l blasts suggests that they are not normal differentiation antigens associated with lymphocyte activation or proliferation.  The highly restricted expression of  detectable levels of antigens reactive with monoclonal antibodies LM-26 and LM-155 on non-Hodgkin's lymphoma cells suggests a possible relation to their neoplastic properties.  From a practical viewpoint these monoclonals may also  prove useful in the diagnosis, classification, detection of residual disease and treatment. General Comments The significance of this work is two-fold.  First, i t demonstrates for  the f i r s t time a functional role for LFA-1 and HLA-class II molecules in B c e l l activation and proliferation.  Second, two monoclonal antibodies are  described which detect unique antigenic determinants highly specific for B lymphoma cells.  These findings prompt additional questions that may  answered by future research:  be  1) Although i t was demonstrated that monoclonal  antibodies to LFA-1 and HLA-DR determinants inhibit B c e l l activation by acting on non-B cells, what is the exact mechanism? primarily on monocytes or T cells? important in the  anti-p  not purified B cells?  Do these antibodies act  Why are accessory cells or T cells  activation of peripheral blood mononuclear cells, but  What is the significance of the differential ability  of HLA-DQ and DR monoclonal antibodies to inhibit B-cell activation?  What is  the mechanism by which anti-class II monoclonal antibodies inhibit the growth of EBV-transformed cell lines?  Although technically d i f f i c u l t , i t may  be  possible to determine whether LFA-1 and HLA-DR antibodies inhibit monocytes and/or T cells by performing reconstitution experiments in which antibody treated monocytes and T cells are individually "added back" to purified  145 populations of B cells.  With the increasing availability of purified growth  factors, these substances may be used in co-culture experiments of purified c e l l populations to determine i f the mechanism of inhibition involves soluble factors.  Sorting out the individual contributions of HLA-DQ and DR molecules  in B cell activation and proliferation may be more d i f f i c u l t .  However,  transfection of the individual genes coding for these products may allow delineation of their separate functions. 2) Are the lymphoma antigens detected by LM-26 and LM-155 specific for malignant B cells or are they expressed on a minor subpopulation of cells at some stage of differentiation? What i s the function of these antigens? lymphoma stem cell?  Are they expressed on the putative  Further attempts at immunoprecipitating the antigens  defined by LM-26 and LM-155 w i l l be necessary before these molecules can be characterized in detail.  If LM-26 and LM-155 detect a determinant present on  some normal cells, these are present either at very low antigen density or only on a small subpopulation of cells.  Purification of minor subpopulations  of cells using a combination of techniques (e.g. E-rosette depletion of T cells, monocyte adherence and c e l l sorting) may be necessary.  Finally, i t  may be possible to clone the gene coding for the LM-26 and LM-155 defined antigens.  So doing will certainly expedite evaluation of the role of these  antigens in the development of B cell neoplasia. LM-26 and LM-155 define cell surface antigens present on neoplastic B lymphoma cells which are not detectable on normal B cells or cells of other lineages, as tested by flow cytometric analysis.  The question arises as to  whether these antigens are expressed uniquely on neoplastic B lymphocytes, are found on a small subset of normal B cells, or are expressed during a discreet stage of B c e l l differentiation but are lost terminally. The absence of detectable reactivity with normal B c e l l blasts suggests that  146 these antibodies do not react with normal transformation associated or blast antigens.  If LM-26 and LM-155 are uniquely expressed on malignant B cells,  then this observation would appear restricted to lymphomas and unlike other human tumor systems yet studied. Such expression might be a consequence of infection by a putative virus, oncogene activation or alterations of c e l l surface molecules which occur in association with neoplastic transformation. In contrast, i f these antibodies also detect determinants present on a minor population of normal B cells, then i t would be necessary to postulate that this subpopulation is the preferential target for those events which result in the development of lymphomas.  An additional possibility is that LM-26 and  LM-155 detect antigens normally present on B lineage cells during embryogenesis but are later lost on normal mature B cells (oncofetal antigens).  By analogy with other tumor systems studied in more detail (e.g.  the acute leukemias) i t is most likely that these antibodies detect antigens present at some discreet stage of B cell differentiation which are lost on normal mature resting or activated B cells, only to be reexpressed again in association with neoplastic transformation. Further work is necessary to distinguish between these possibilities. The clinical importance of these antilymphoma monoclonal antibodies w i l l also require additional studies. Clearly, however, the potential exists for these antibodies to be used to detect residual disease, monitor therapy, classify and treat the non-Hodgkin's lymphomas.  For example, i t may be  possible to conjugate these antibodies to various immunotoxins such as ricin or use them in conjunction with complement in "purging" neoplastic cells from bone marrow in vitro to facilitate autologous marrow transplantation.  147  Future Prospects The non-Hodgkin's lymphomas are clonal neoplasms of lymphoid origin. That tumors are clones has been shown conclusively by many different lines of investigation.  These include:  cytogenetics, glucose-6-phosphate  dehydrogenase analysis, the demonstration of monoclonal surface immunoglobulin, B and T cell gene rearrangement and restriction fragment length polymorphisms (1-5).  As tumors progress there tends to be the  acquisition of a number of heritable characteristics such as:  an increase in  growth fraction, loss of differentiation, decreased antigenicity, increasing cytogenetic abnormality, the acquisition of drug resistance, elaboration of products and altered response to hormones or growth factors (2,3).  How  these  changes are initiated and how they progress remain central unanswered questions in tumor biology. Oncogene activation has been postulated as a possible mechanism in the genesis of many human malignancies.  An i n i t i a l insult or perturbation to a  c e l l may result in the activation of a particular oncogene.  This may or may  not be sufficient to allow the phenotypic expression of malignancy.  In fact,  the concept of cooperation between two or more oncogenes has been invoked by some investigators to help explain the multistep development of human cancers (6-9).  Weinberg has suggested that the reason why carcinogenesis is  multistep is a requirement for activating sequentially multiple genes. is some support for this hypothesis in animal models.  There  For example, when  either the myc or ras oncogene was introduced into normal rat embryo fibroblasts, neither caused tumorigenic transformation.  When introduced  together, myc and ras were able to do what neither could do alone.  Cells co-  transfected with these two oncogenes expanded into vigorously growing cultures and produced rapidly growing tumors in nude mice (6). Whether  148 oncogenes are involved in the multistage development of human lymphomas i s not known. Certainly myc appears to play a role in Burkitt's lymphoma. Many Burkitt's lymphoma cells have also been reported to carry a B-lym transforming gene (7). The significance of these observations remains to be determined. One of the more conceptually attractive theories concerning the development of neoplasia is that of clonal selection.  By this concept, an  i n i t i a l insult to or predisposition within an apparently normal c e l l , confers to i t an heritable growth advantage and/or increased genetic instability. This combination of an unstable genome and a relative growth advantage allows the selection of progressively more abnormal c e l l types.  The net result is  the emergence of a dominant clone of cells with features characteristic of malignancy (4). There are c l i n i c a l examples to support this concept.  Philadelphia  chromosome (Ph^) positive CML tends to be a slowly progressive form of leukemia for a period of several years; until the development of blast crisis.  Blast crisis is characterized by the emergence of a dominant clone  with characteristics of primitive myeloid or lymphoid cells.  These cells may  i  express other cytogenic markers in addition to Ph . Once blast transformation develops in CML the disease is rapidly fatal. Chronic lymphocytic leukemia (CLL) is most often a slowly progressive disease occurring in the elderly.  Occasionally, this form of leukemia, in  which the cells are small and relatively quiescent, may terminate in the development of large cell lymphoma/leukemia (Richter's syndrome).  In one  well documented case a patient with CLL during the indolent phase of her disease had a normal diploid karyotype gradually replaced by a pseudodiploid lymphocyte population.  Upon the development of Richter's syndrome the  149 neoplastic cells became increasingly cytogenetically abnormal (hypertriploid) but retained the original pseudodiploid marker chromosomes (5). These findings demonstrated the evolution of an aggressive form of leukemia/lymphoma from one which was relatively benign. Other examples are well documented. The non-Hodgkin's lymphomas may progress from small cell to large cell types or from a nodular to a diffuse pattern during the course of disease. rapidly progressive clinical course.  The latter are associated with a more EBV infected normal B cells become  immortalized in in vitro culture. Initially these cells are cytogenetically normal and polyclonal. After a period of months to years in culture these cells become monoclonal (i.e. a dominant clone emerges) and develop cytogenetic abnormalities.  Rous sarcoma virus when introduced into rats  produces a tumor which in i t s early stages is cytogenetically normal.  As the  tumor progresses, however, increasing karyotypic abnormalities develop (2, 4, 10). There are a number of mechanisms possible for the development of genetic instability in tumor cells (i.e. the increased tendency to develop genetic structural abnormalities compared to normal cells).  These include inherited  defects (chromosomal breakage syndromes, subclinical gene defects, constitutional chromosomal abnormalities), acquired defects (gene mutations, acquired chromosomal alterations) and extracellular factors (viruses, radiation, chemical agents etc). In addition, there may be a central role for host factors in clonal evolution. These might include: immune surveillance mechanisms, nutritional status, the microenvironment, regulatory substances, exposure to infectious agents and treatment (4). It should be emphasized that the concepts of genetic instability and clonal evolution, in the progression of most tumors, remain attractive but as  150 yet unproven hypotheses.  However, using these concepts i t "may be possible to  view the older notions of chemical carcinogenesis relating to initiators and promoters in a new way.  An initiated cell may be one which is genetically  unstable and which has a heritable growth advantage.  Promotion would then be  the proliferative stimulus that allows these characteristics to be expressed. These concepts may be applied to the lymphomas.  For example, Burkitt's  lymphoma as discussed previously is characterized by the translocation of the myc oncogene, located on chromosome 8, to an active site of genes coding for immunoglobulin heavy (chromosome 14) or light (chromosomes 2, 22) chains.  It  has been postulated that this change in the regulatory environment of myc results in i t s abnormal expression which then leads to neoplastic transformation.  There are two types of Burkitt's lymphoma; the endemic type  occurring primarily in Africa and the nonendemic or sporadic type which occurs elsewhere.  Both types of lymphoma are similar morphologically, both  have the characteristic chromosomal translocation, but only in the endemic form is cellular infection with EB virus characteristic.  It would therefore  seem reasonable to postulate that there may be several "causes" of Burkitt's lymphoma.  One cause may be EB virus.  The others, through the interaction  with constitutional or environmental factors may lead to the development of the characteristic chromosomal abnormality and the phenotypic expression of malignancy (11-14). Much less information is presently available to suggest possible mechanisms for the development of the other subtypes of non-Hodgkin's lymphomas.  In some of these, characteristic but not invariant chromosomal  abnormalities may be found (15, 16).  Putative oncogenes, responsible for  malignant transformation in these lymphomas, remain to be identified.  151 It is apparent that most or a l l malignancies have phenotypic characteristics similar or identical to a normal cellular counterpart at some stage in differentiation.  For example, CALLA ALL appears to have as i t s +  normal cellular counterpart an early committed B lymphoid stem c e l l ; one in which immunoglobulin gene rearrangement has occurred and which expresses B c e l l surface markers but lacks detectable surface or cytoplasmic immunoglobulin.  Likewise, the normal cellular counterpart of pre-B ALL is  the pre-B c e l l .  This c e l l , in addition to expressing B c e l l surface  antigens, contains cytoplasmic but not surface IgM.  B c e l l ALL is probably a  leukemic phase of Burkitt's lymphoma. These cells express, in addition to other markers, surface Ig.  B cell type of CLL appears to have as i t s normal  cellular counterpart the small resting (mature) B cell which expresses surface IgM with or without IgD. The normal cellular counterparts of the non-Hodgkin's lymphomas have not been definitively established. It has been postulated that during the course of lymphocyte transformation, B cells go through a series of morphologic stages recognizable in the germinal centers of lymph nodes.  These stages in  normal lymphocyte development are thought to have as a neoplastic counterpart, tumors in which one particular morphologic c e l l type dominates (see Chapter I). One way to prove or disprove this hypothesis is by characterizing the cell surface antigens on the various subtypes of lymphomas and correlating these with subpopulations of normal cells.  This is one  application of monoclonal antibodies such as LM-26 and LM-155 described in this thesis. What are the central questions relative to understanding the biology of the non-Hodgkin's lymphomas and, given existing technology, may answers be realistically expected?  Clearly, i t is important to understand normal B c e l l  152  function.  What f a c t o r s r e g u l a t e the growth, t r a n s f o r m a t i o n and  d i f f e r e n t i a t i o n o f normal B c e l l s ? cells?  V i a surface receptors?  they p e r t u r b e d i n malignant independent?  How do these f a c t o r s i n t e r a c t w i t h B  What i s the nature o f these r e c e p t o r s ?  B cells?  Are  Are lymphoma c e l l s growth f a c t o r  Do they r e l y f o r t h e i r growth advantage on a u t o s t i m u l a t o r y  f a c t o r s o r a r e they e x q u i s i t e l y s e n s i t i v e to low l e v e l s o f these  factors?  With r e s p e c t to T c e l l s and T c e l l neoplasms some o f these q u e s t i o n s a r e b e i n g answered. cell  I t i s not unreasonable  to assume that  s t i m u l a t o r y f a c t o r s and t h e i r r e c e p t o r s w i l l  the genes c o d i n g f o r B  i n the next  few y e a r s be  c l o n e d and t h e i r products c h a r a c t e r i z e d . Do s p e c i f i c subtypes o f lymphomas c o r r e l a t e w i t h a p a r t i c u l a r chromosomal a b n o r m a l i t y o r w i t h the a c t i v a t i o n o f a p a r t i c u l a r oncogene? example, i f B u r k i t t ' s lymphoma i s caused oncogene then perhaps  o t h e r morphologic  For  by the a c t i v a t i o n o f the myc subtypes o f lymphomas r e s u l t  from the  abnormal e x p r e s s i o n o f d i f f e r e n t oncogenes o r e x p r e s s i o n o f the same oncogene at  v a r y i n g times i n the c e l l  cycle.  What i s the n a t u r e o f the r e p l i c a t i n g c e l l  i n the non-Hodgkin's  lymphomas, i . e . the p u t a t i v e lymphoma "stem c e l l " ? unique perhaps  characteristics  have  that a l l o w i t to be d i s t i n g u i s h e d from a s s o c i a t e d ,  more d i f f e r e n t i a t e d , progeny?  normal growth r e g u l a t o r y c o n t r o l s ? morphologic  Does t h i s c e l l  Why i s t h i s c e l l u n r e s p o n s i v e to  Does t h i s c e l l d i f f e r ,  depending  subtype o f lymphoma or i s i t the same i n a l l B c e l l  on the  lymphomas,  w i t h o t h e r e x t r i n s i c f a c t o r s c o n t r i b u t i n g to the morphology o f i n d i v i d u a l tumors?  Nodular  lymphomas have a l e s s a g g r e s s i v e c l i n i c a l  course than do  d i f f u s e lymphomas.  What f a c t o r s c o n t r i b u t e to the maintenance o f t h i s  p a t t e r n o f growth?  Why i s t h i s p a t t e r n l o s t d u r i n g the p r o g r e s s i o n o f many  tumors?  153 One of the difficulties in trying to study the non-Hodgkin's lymphomas has been the inability to culture cells from these tumors in vitro over long periods.  Long term culture of lymphoma cells is feasible with existing  technology.  To do so will require identifying those nutrient, growth factor  and environmental substances which are crucial to the maintenance of c e l l viability and growth.  This approach has already been successfully applied to  the study of bone marrow myeloid and erythroid progenitors (17). Finally, what are the causative factors which initiate malignant transformation?  Viral infection?  environmental factors?  Spontaneous mutation?  Hereditary or  How can these factors be eliminated or controlled?  Although much has been accomplished, much remains to be done.  154 REFERENCES  1.  Robinson MA, Kindt TJ: Segregation of polymorphic T-cell receptor genes in human families. Proc Natl Acad Sci USA 82: 3804-3808, 1985.  2.  Pitot HC: Fundamentals of Oncology 2nd Edition. York, 1981.  3.  Vogelstein B, Fearon ER, Hamilton SR, Feinberg AP: Use of restriction fragment length polymorphisms to determine the clonal origin of human tumors. Science 227: 642-645, 1985.  4.  Nowell PC: Tumor progression and clonal evolution: The role of genetic instability. In: Chromosome Mutation and Neoplasia, Alan R Liss, Inc, New York, pp 413-432, 1983.  5.  Nowell P, Finan J, Glover D, Guerry D: Cytogenetic evidence for the clonal nature of Richter's syndrome. Blood 58, 183-186, 1981.  6.  Land H, Parada LF, Weinberg RA: Cellular oncogenes and multistep carcinogenesis. Science 222: 771-778, 1983.  7.  Marx JL: Cooperation between oncogenes.  8.  Robertson M: Oncogenes and multistep carcinogenesis. Nature 287: 1084-1086, 1983.  9.  Parada LF, Land H, Weinberg RA, Wolf D, Rotter V: Cooperation between gene encoding p53 tumor antigen and ras in cellular transformation. Nature 312: 649-651, 1984.  10.  McCaffrey R, Bell, R: The lymphomas: Etiologic considerations. In: Neoplastic Diseases of the Blood, Wiernik PH, Canellos GP, Kyle RA, Schiffer CA (eds), Churchill Livingstone, New York, Volume 2, pp 687692, 1985.  11.  Nichols WW: Viral interactions with the mammalian genome relevant to neoplasia. In: Chromosome Mutation and Neoplasia, Alan R Liss, Inc, New York, pp 317-332, 1983.  12.  Cooper MD, Kubagawa H: B-cell malignancies: Origin and extent of clonal involvement. In: Modern Trends in Human Leukemia V, Neth, Gallo, Greaves, Moore, Winkler (eds), Springer-Verlag, Berlin, Volume 28, pp 425-433, 1983.  13.  Klein G: Lymphoma development in mice and humans: Diversity of initiation is followed by convergent cytogenetic evolution. Proc Natl Acad Sci USA 76: 2442-2446, 1979.  14.  Klein G: Specific chromosomal translocations and the genesis of B-cellderived tumors in mice and men. Cell 32: 311-315, 1983.  Marcel Dekker Inc., New  Science 222: 602-603, 1983.  155 15.  Bloomfield CD: Cytogenetics of malignant lymphoma. In: Neoplastic Diseases of the Blood, Wiernik. PH, Canellos GP, Kyle RA, Schiffer CA (eds), Churchill Livingstone, New York, Volume 2, pp 773-787, 1985.  16.  Yunis JJ, Oken MM, Kaplan ME, Ensrud KM, Howe RR, Theologides A: Distinctive chromosomal abnormalities in histologic subtypes of nonHodgkin's lymphoma. N Engl J Med 307: 1231-1236, 1982.  17.  Coulombel L, Kalousek DK, Eaves CJ, Gupta CM, Eaves AC: Long-term marrow culture reveals chromosomally normal hemopoietic progenitor cells in patients with Philadelphia chromosome-positive chronic myelogenous leukemia. N Engl J Med 308: 1493-1498, 1983.  156  And ever, as the s t o r e y The w e l l s o f fancy And f a i n t l y s t r o v e that To put the s u b j e c t "The r e s t next t i m e — "  drained dry, weary one by, " I t i s the next  time!"  Thus grew the t a l e of Wonderland Thus s l o w l y , one by one, I t s q u a i n t events were hammered o u t — And now the t a l e i s done, And home we s t e e r , a merry crew Beneath the s e t t i n g sun. Lewis C a r r o l l A l i c e ' s Adventures i n Wonderland  PUBLICATIONS  Donald R. Howard  1.  Howard DR & Taylor CR. Immunohistological d i s t i n c t i o n of benign and malignant breast lesions u t i l i z i n g antibody present i n normal human sera. IRCS Med Sci 6: 267, 1978.  2.  Howard DR & Taylor CR. A method for distinguishing benign from malignant breast lesions u t i l i z i n g antibody present i n normal human sera. Cancer A3: 2279-2287, 1979.  3.  Howard DR. Expression of T-antigen on polyagglutinable erythrocytes and carcinoma c e l l s : Preparation of T-activated erythrocytes, anti-T l e c t i n , anti-T absorbed human serum and p u r i f i e d anti-T antibody. Vox Sang 37: 107-110, 1979.  A.  Howard DR & Taylor CR. An anti-tumor antibody i n normal human serum: Reaction of anti-T with breast carcinoma c e l l s . Oncology 37: 1A2-1A8, 1980.  5.  Howard DR & Taylor CR. The s i g n i f i c a n c e of myoepithelial c e l l s t a i n i n g by a tumor associated antibody. Breast, Diseases of the Breast 6: 10-15, 1980.  6.  Howard DR & Batsakis JG. Cytostructural l o c a l i z a t i o n of a tumor associated antigen. Science 210: 201-203, 1980.  7.  Howard DR. I n f e r t i l i t y associated with antisperm antibody. N Engl J Med 30A: 301, 1981.  8.  Harness J , Geelhoed G, Thompson N, Nishiyama R, Fajans S, Kraft R, Howard D & Clark K. Nesidioblastosis i n adults: A s u r g i c a l dilemma. Arch Surg 116: 575-580, 1981.  9.  Howard DR, Ferguson P & Batsakis JG. Carcinoma associated membrane antigenic a l t e r a t i o n s : Detection by l e c t i n binding. Cancer A7: 28722877, 1981. Pathology and pathogenesis.  (Letter)  10.  Howard DR. Hodgkin's disease: Lab Sci IA: 109-131, 1981.  C r i t Rev C l i n  11.  Batsakis JG & Howard DR. Sjogren's syndrome: An immune response associated disorder. C l i n Lab Ann 1: 171-188, 1982.  12.  Howard DR, Rundell C & Batsakis JG. Vitamin E does not modify HDL c h o l e s t e r o l . Am J C l i n Pathol 77: 86-89, 1982.  13.  Howard D, Bagley C & Batsakis JG. Warthin's tumor: A functional immunologic study. Am J Otolaryngol 3: 15-19, 1982.  IA.  Howard DR & Batsakis JG. Peanut agglutinin: A new marker f o r tissue h i s t i o c y t e s . Am J C l i n Pathol 77: A01-A08, 1982.  15.  Batsakis JG & Howard DR. Sjogren's syndrome: Lymphoepithelial s a l i v a r y gland. ASCP Check Sample 6(7), 1982.  l e s i o n of  PUBLICATIONS  Donald R. Howard  16.  Batsakis JG, Rice DH & Howard DR. The pathology of head and neck tumors: Spindle c e l l lesions (sarcomatoid carcinomas, nodular f a s c i i t i s , and fibrosarcomas) of the aerodigestive tracts. Head and Neck Surg 4: 499-513, 1982.  17.  Howard DR. T antigen does not induce c e l l mediated immunity i n patients with breast cancer. Cancer 51: 2053-2056, 1983.  18.  Howard DR, Wicken J & Nishiyama R. D i f f e r e n t i a t i o n of chronic lymphocytic leukemia from reactive lymphocytosis Using the mouse red c e l l rosette assay. Int Arch Allergy Immunol 73: 352-356, 1984.  19.  Springer GF, Taylor CR, Howard DR et a l . Tn, A carcinoma associated antigen reacts with anti-Tn of normal human sera. Cancer 55: 561-569, 1985.  20.  Howard DR & Batsakis JG. Hodgkin's disease: Contemporary c l a s s i f i c a t i o n and c o r r e l a t e s . Ann Otol Rhinol Laryngol 94: 220-221, 1985.  21.  Howard DR & Batsakis JG. Non-Hodgkin's lymphomas: Contemporary c l a s s i f i c a t i o n and c o r r e l a t e s . Ann Otol Rhinol Laryngol 94: 326-328, 1985.  22.  Howard DR, Eaves AC & Takei F. Monoclonal antibody defined c e l l surface molecules regulate lymphocyte a c t i v a t i o n . In: "Leukocyte Typing I I " , (eds. EL Reinherz, BF Haynes, LM Nadler & ID Bernstein), Springer-Verlag, New York ( i n press)  23.  Howard DR, Eaves AC & Takei F. Two monoclonal antibodies that define unique antigenic determinants expressed on human B-lymphoma c e l l s . Cancer Research ( i n press)  

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